Synergistic effects of dimethyloxallyl glycine and recombinant human bone morphogenetic protein-2 on repair of critical-sized bone defects in rats

NASA Astrophysics Data System (ADS)

Qi, Xin; Liu, Yang; Ding, Zhen-Yu; Cao, Jia-Qing; Huang, Jing-Huan; Zhang, Jie-Yuan; Jia, Wei-Tao; Wang, Jing; Liu, Chang-Sheng; Li, Xiao-Lin

2017-02-01

In bone remodeling, osteogenesis is closely coupled to angiogenesis. Bone tissue engineering using multifunctional bioactive materials is a promising technique which has the ability to simultaneously stimulate osteogenesis and angiogenesis for repair of bone defects. We developed mesoporous bioactive glass (MBG)-doped poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) composite scaffolds as delivery vehicle. Two bioactive molecules, dimethyloxalylglycine (DMOG), a small-molecule angiogenic drug, and recombinant human bone morphogenetic protein-2 (rhBMP-2), an osteoinductive growth factor, were co-incorporated into the scaffold. The synergistic effects of DMOG and rhBMP-2 released in the composite scaffolds on osteogenic and angiogenic differentiation of hBMSCs were investigated using real-time quantitative polymerase chain reaction and western blotting. Moreover, in vivo studies were conducted to observe bone regeneration and vascular formation of critical-sized bone defects in rats using micro-computed tomography, histological analyses, Microfil® perfusion, fluorescence labeling, and immunohistochemical analysis. The results showed that DMOG and rhBMP-2 released in the MBG-PHBHHx scaffolds did exert synergistic effects on the osteogenic and angiogenic differentiation of hBMSCs. Moreover, DMOG and rhBMP-2 produced significant increases in newly-formed bone and neovascularization of calvarial bone defects in rats. It is concluded that the co-delivery strategy of both rhBMP-2 and DMOG can significantly improve the critical-sized bone regeneration.

Synergistic effects of fibronectin and bone morphogenetic protein on the bioactivity of titanium metal.

PubMed

Biao, M N; Chen, Y M; Xiong, S B; Wu, B Y; Yang, B C

2017-09-01

To improve the biological properties of bioactive titanium metal, recombinant human bone morphogenetic protein 2(rhBMP-2) and fibronectin (Fn) were adsorbed on its surface solely or contiguously to modify the anodic oxidized titanium (AO-Ti), acid-alkali-treated titanium (AA-Ti), and polished titanium (P-Ti). It is found that the different bioactive titanium surface structures had great influence on protein adsorption. The adsorption amounts of BMP adsorbed solely and Fn/BMP adsorbed contiguously were AA-Ti > P-Ti > AO-Ti, and that for Fn adsorbed solely was AA-Ti ≈ P-Ti > AO-Ti. The conformation of proteins was changed remarkably after the adsorption. For BMP, the α-helix decreased on AA-Ti and stabilized on P-Ti and AO-Ti. For Fn, the β-sheet on PT-Ti and AA-Ti increased significantly. For Fn/BMP, the percentage of β-sheet on AA-Ti increased, and that of α-helix on all samples was stable. MSCs showed greater adhesion and spreading on Fn/BMP groups. MTT and Elisa tests showed that the synergistic effects of proteins made the cells proliferate and differentiate faster. It indicated both the surface structure and the synergistic effects of proteins could influence the biological properties of titanium metals. It provides research foundation for improving the biological properties of bioactive titanium metals by simultaneous application of several proteins. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2485-2498, 2017. © 2017 Wiley Periodicals, Inc.

Influence of Mussel-Derived Bioactive BMP-2-Decorated PLA on MSC Behavior in Vitro and Verification with Osteogenicity at Ectopic Sites in Vivo.

PubMed

Chen, Zhuoyue; Zhang, Zhen; Feng, Juantao; Guo, Yayuan; Yu, Yuan; Cui, Jihong; Li, Hongmin; Shang, Lijun

2018-04-11

Osteoinductive activity of the implant in bone healing and regeneration is still a challenging research topic. Therapeutic application of recombinant human bone morphogenetic protein-2 (BMP-2) is a promising approach to enhance osteogenesis. However, high dose and uncontrolled burst release of BMP-2 may introduce edema, bone overgrowth, cystlike bone formation, and inflammation. In this study, low-dose BMP-2 of 1 μg was used to design PLA-PD-BMP for functionalization of polylactic acid (PLA) implants via mussel-inspired polydopamine (PD) assist. For the first time, the binding property and efficiency of the PD coating with BMP-2 were directly demonstrated and analyzed using an antigen-antibody reaction. The obtained PLA-PD-BMP surface immobilized with this low BMP-2 dose can endow the implants with abilities of introducing strong stem cell adhesion and enhanced osteogenicity. Furthermore, in vivo osteoinduction of the PLA-PD-BMP-2 scaffolds was confirmed by a rat ectopic bone model, which is marked as the "gold standard" for the evidence of osteoinductive activity. The microcomputed tomography, Young's modulus, and histology analyses were also employed to demonstrate that PLA-PD-BMP grafted with 1 μg of BMP-2 can induce bone formation. Therefore, the method in this study can be used as a model system to immobilize other growth factors onto various different types of polymer substrates. The highly biomimetic mussel-derived strategy can therefore improve the clinical outcome of polymer-based medical implants in a facile, safe, and effective way.

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo.

PubMed

Neugebauer, Judith M; Kwon, Sunjong; Kim, Hyung-Seok; Donley, Nathan; Tilak, Anup; Sopory, Shailaja; Christian, Jan L

2015-05-05

Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease.

In vivo bioactivity of rhBMP-2 delivered with novel polyelectrolyte complexation shells assembled on an alginate microbead core template.

PubMed

Abbah, Sunny-Akogwu; Liu, Jing; Lam, Raymond W M; Goh, James C H; Wong, Hee-Kit

2012-09-10

Electrostatic interactions between polycations and polyanions are being explored to fabricate polyelectrolyte complexes (PEC) that could entrap and regulate the release of a wide range of biomolecules. Here, we report the in vivo application of PEC shells fabricated from three different polycations: poly-l-ornithine (PLO), poly-l-arginine (PLA) and DEAE-dextran (DEAE-D) to condense heparin on the surface of alginate microbeads and further control the delivery of recombinant human bone morphogenetic protein 2 (rhBMP-2) in spinal fusion application. We observed large differences in the behavior of PEC shells fabricated from the cationic polyamino acids (PLO and PLA) when compared to the cationic polysaccharide, DEAE-D. Whereas DEAE-D-based PEC shells eroded and released rhBMP-2 over 2 days in vitro, PLO- and PLA-based shells retained at least 60% of loaded rhBMP-2 after 3 weeks of incubation in phosphate-buffered saline. In vivo implantation in a rat model of posterolateral spinal fusion revealed robust bone formation in the PLO- and PLA-based PEC shell groups. This resulted in a significantly enhanced mechanical stability of the fused segments. However, bone induction and biomechanical stability of spine segments implanted with DEAE-D-based carriers were significantly inferior to both PLO- and PLA-based PEC shell groups (p<0.01). From these results, we conclude that PEC shells incorporating native heparin could be used for growth factor delivery in functional bone tissue engineering application and that PLA- and PLO-based complexes could represent superior options to DEAE-D for loading and in vivo delivery of bioactive BMP-2 in this approach. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of implants composed of bioactive materials for bone repair

NASA Astrophysics Data System (ADS)

Xiao, Wei

The purpose of this Ph.D. research was to address the clinical need for synthetic bioactive materials to heal defects in non-loaded and loaded bone. Hollow hydroxyapatite (HA) microspheres created in a previous study were evaluated as a carrier for controlled release of bone morphogenetic protein-2 (BMP2) in bone regeneration. New bone formation in rat calvarial defects implanted with BMP2-loaded microspheres (43%) was significantly higher than microspheres without BMP2 (17%) at 6 weeks postimplantation. Then hollow HA microspheres with a carbonate-substituted composition were prepared to improve their resorption rate. Hollow HA microspheres with 12 wt. % of carbonate showed significantly higher new bone formation (73 +/- 8%) and lower residual HA (7 +/- 2%) than stoichiometric HA microspheres (59 +/- 2% new bone formation; 21 +/- 3% residual HA). The combination of carbonate-substituted hollow HA microspheres and clinically-safe doses of BMP2 could provide promising implants for healing non-loaded bone defects. Strong porous scaffolds of bioactive silicate (13-93) glass were designed with the aid of finite-element modeling, created by robocasting and evaluated for loaded bone repair. Scaffolds with a porosity gradient to mimic human cortical bone showed a compressive strength of 88 +/- 20 MPa, a flexural strength of 34 +/- 5 MPa and the ability to support bone infiltration in vivo. The addition of a biodegradable polylactic acid (PLA) layer to the external surface of these scaffolds increased their load-bearing capacity in four-point bending by 50% and dramatically enhanced their work of fracture, resulting in a "ductile" mechanical response. These bioactive glass-PLA composites, combining bioactivity, high strength, high work of fracture and an internal architecture conducive to bone infiltration, could provide optimal implants for structural bone repair.

Short communication: Tryptic β-casein hydrolysate modulates enteric nervous system development in primary culture.

PubMed

Cossais, F; Clawin-Rädecker, I; Lorenzen, P C; Klempt, M

2017-05-01

The intestinal tract of the newborn is particularly sensitive to gastrointestinal disorders, such as infantile diarrhea or necrotizing colitis. Perinatal development of the gut also encompasses the maturation of the enteric nervous system (ENS), a main regulator of intestinal motility and barrier functions. It was recently shown that ENS maturation can be enhanced by nutritional factors to improve intestinal maturation. Bioactivity of milk proteins is often latent, requiring the release of bioactive peptides from inactive native proteins. Several casein-derived hydrolysates presenting immunomodulatory properties have been described recently. Furthermore, accumulating data indicate that milk-derived hydrolysate can enhance gut maturation and enrichment of milk formula with such hydrolysates has recently been proposed. However, the capability of milk-derived bioactive hydrolysate to target ENS maturation has not been analyzed so far. We, therefore, investigated the potential of a recently described tryptic β-casein hydrolysate to modulate ENS growth parameters in an in vitro model of rat primary culture of ENS. Rat primary cultures of ENS were incubated with a bioactive tryptic β-casein hydrolysate and compared with untreated controls or to cultures treated with native β-casein or a Prolyve β-casein hydrolysate (Lyven, Colombelles, France). Differentiation of enteric neurons and enteric glial cells, and establishment of enteric neural network were analyzed using immunohistochemistry and quantitative PCR. Effect of tryptic β-casein hydrolysate on bone morphogenetic proteins (BMP)/Smad pathway, an essential regulator of ENS development, was further assessed using quantitative PCR and immunochemistry. Tryptic β-casein hydrolysate stimulated neurite outgrowth and simultaneously modulated the formation of enteric ganglia-like structures, whereas native β-casein or Prolyve β-casein hydrolysate did not. Additionally, treatment with tryptic bioactive β-casein hydrolysate increased the expression of the glial marker glial fibrillary acidic protein and induced profound modifications of enteric glial cells morphology. Finally, expression of BMP2 and BMP4 and activation of Smad1/5 was altered after treatment with tryptic bioactive β-casein hydrolysate. Our data suggests that this milk-derived bioactive hydrolysate modulates ENS maturation through the regulation of BMP/Smad-signaling pathway. This study supports the need for further investigation on the influence of milk-derived bioactive peptides on ENS and intestinal maturation in vivo. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

In vitro and in vivo evaluation of bone morphogenetic protein-2 (BMP-2) immobilized collagen-coated polyetheretherketone (PEEK)

NASA Astrophysics Data System (ADS)

Du, Ya-Wei; Zhang, Li-Nan; Ye, Xin; Nie, He-Min; Hou, Zeng-Tao; Zeng, Teng-Hui; Yan, Guo-Ping; Shang, Peng

2015-03-01

Polyetheretherketone (PEEK) is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydrophobic interaction, collagen was cross-linked with N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC/NHS system also contributed to the immobilization of rhBMP-2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

[Preparation of vanilline cross-linked rhBMP-2/chitosan microspheres and its effect on mesenchymal stem cells].

PubMed

Wu, Gui; Wang, Hai; Qiu, Guixing; Yu, Xin; Su, Xinlin; Ma, Pei; Yin, Bo; Wu, Zhihong

2015-06-02

To prepare rhBMP-2/chitosan microspheres (rhBMP-2 CMs) with vanilline as a cross-linking reagent and study the biocompatibility and drug release characteristic of microspheres in vitro. Emulsion cross-linking method was utilized to prepare rhBMP-2 CMs, Scanning electron microscope (SEM) was used to observe the microstructure of microspheres.Leaching solution of microspheres and blank culture medium were designated as experimental and control groups respectively. Both groups were cultured with human mesenchymal stem cells (hMSCs) to determine its cytotoxicity and its effect on the proliferation of hMSCs. Dynamic immersion method was used to examine the in vitro release characteristic of rhBMP-2. And the alkaline phosphatase (ALP) activity of hMSCs was determined to reveal the bioactivity of released rhBMP-2. The rhBMP-2 CMs were spherical under SEM.After treating with leaching solution for 24 and 48 h, there was no inter-group statistical difference in optical density (OD) values at both timepoints (24 h:0.72 ± 0.07 vs 0.73 ± 0.05, P > 0.05; 48 h:1.19 ± 0.11 vs 1.27 ± 0.06, P > 0.05). After culturing with leaching solution for 1, 3 and 7 days, the number of cells increased with time for both groups. And the OD values were not statistically different at each timepoint. Five milligram rhBMP-2 CMs soaked for 19 days with a gradual release of rhBMP-2. The concentration of rhBMP-2 was 216.1 ± 20.0 ng/ml at Day 19. At Days 3 and 7, the ALP activities of hMSCs were (0.50 ± 0.07) and (0.68 ± 0.06) µmol pNPP·min⁻¹·mg⁻¹ protein respectively and both were higher than that of blank culture medium group (0.14 ± 0.01) (P < 0.05). With an excellent biocompatibility, rhBMP-2 CMs may be an ideal carrier for control-released rhBMP-2 and encapsulated rhBMP-2 remains bioactive.

BMP-2 Derived Peptide and Dexamethasone Incorporated Mesoporous Silica Nanoparticles for Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells.

PubMed

Zhou, Xiaojun; Feng, Wei; Qiu, Kexin; Chen, Liang; Wang, Weizhong; Nie, Wei; Mo, Xiumei; He, Chuanglong

2015-07-29

Bone morphogenetic protein-2 (BMP-2), a growth factor that induces osteoblast differentiation and promotes bone regeneration, has been extensively investigated in bone tissue engineering. The peptides of bioactive domains, corresponding to residues 73-92 of BMP-2 become an alternative to reduce adverse side effects caused by the use of high doses of BMP-2 protein. In this study, BMP-2 peptide functionalized mesoporous silica nanoparticles (MSNs-pep) were synthesized by covalently grafting BMP-2 peptide on the surface of nanoparticles via an aminosilane linker, and dexamethasone (DEX) was then loaded into the channel of MSNs to construct nanoparticulate osteogenic delivery systems (DEX@MSNs-pep). The in vitro cell viability of MSNs-pep was tested with bone mesenchymal stem cells (BMSCs) exposure to different particle concentrations, revealing that the functionalized MSNs had better cytocompatibility than their bare counterparts, and the cellular uptake efficiency of MSNs-pep was remarkably larger than that of bare MSNs. The in vitro results also show that the MSNs-pep promoted osteogenic differentiation of BMSCs in terms of the levels of alkaline phosphatase (ALP) activity, calcium deposition, and expression of bone-related protein. Moreover, the osteogenic differentiation of BMSCs can be further enhanced by incorporating of DEX into MSNs-pep. After intramuscular implantation in rats for 3 weeks, the computed tomography (CT) images and histological examination indicate that this nanoparticulate osteogenic delivery system induces effective osteoblast differentiation and bone regeneration in vivo. Collectively, the BMP-2 peptide and DEX incorporated MSNs can act synergistically to enhance osteogenic differentiation of BMSCs, which have potential applications in bone tissue engineering.

Effect of grafting RGD and BMP-2 protein-derived peptides to a hydrogel substrate on osteogenic differentiation of marrow stromal cells.

PubMed

He, Xuezhong; Ma, Junyu; Jabbari, Esmaiel

2008-11-04

Osteogenic differentiation and mineralization of bone marrow stromal (BMS) cells depends on the cells' interactions with bioactive peptides associated with the matrix proteins. The RGD peptides of ECM proteins interact with BMS cells through integrin surface receptors to facilitate cell spreading and adhesion. The BMP peptide corresponding to residues 73-92 of bone morphogenetic protein-2 promotes differentiation and mineralization of BMS cells. The objective of this work was to investigate the effects of RGD and BMP peptides, grafted to a hydrogel substrate, on osteogenic differentiation and mineralization of BMS cells. RGD peptide was acrylamide-terminated by reacting acrylic acid with the N-terminal amine group of the peptide to produce the functionalized Ac-GRGD peptide. The PEGylated BMP peptide was reacted with 4-carboxybenzenesulfonazide to produce an azide functionalized Az-mPEG-BMP peptide. Poly (lactide-co-ethylene oxide- co-fumarate) (PLEOF) macromer was cross-linked with Ac-GRGD peptide and propargyl acrylate to produce an RGD conjugated hydrogel. Az-mPEG-BMP peptide was grafted to the hydrogel by "click chemistry". The RGD and BMP peptide density on the hydrogel surface was 1.62+/-0.37 and 5.2+/-0.6 pmol/cm2, respectively. BMS cells were seeded on the hydrogels and the effect of RGD and BMP peptides on osteogenesis was evaluated by measuring ALPase activity and calcium content with incubation time. BMS cells cultured on RGD conjugated, BMP peptide grafted, and RGD+BMP peptide modified hydrogels showed 3, 2.5, and 5-fold increase in ALPase activity after 14 days incubation. BMS cells seeded on RGD+BMP peptides modified hydrogel showed 4.9- and 11.8-fold increase in calcium content after 14 and 21 days, respectively, which was significantly higher than RGD conjugated or BMP grafted hydrogels. These results demonstrate that RGD and BMP peptides, grafted to a hydrogel substrate, act synergistically to enhance osteogenic differentiation and mineralization of BMS cells. These findings are potentially useful in developing engineered scaffolds for bone regeneration.

Plasma Surface Modification for Immobilization of Bone Morphogenic Protein-2 on Polycaprolactone Scaffolds

NASA Astrophysics Data System (ADS)

Kim, Byung Hoon; Myung, Sung Woon; Jung, Sang Chul; Ko, Yeong Mu

2013-11-01

The immobilization of recombinant human bone formation protein-2 (rhBMP-2) on polycaprolactone (PCL) scaffolds was performed by plasma polymerization. RhBMP-2, which induces osteoblast differentiation in various cell types, is a growth factor that plays an important role in bone formation and repair. The surface of the PCL scaffold was functionalized with the carboxyl groups of plasma-polymerized acrylic acid (PPAA) thin films. Plasma polymerization was carried out at a discharge power of 60 W at an acrylic acid flow rate of 7 sccm for 5 min. The PPAA thin film exhibited moderate hydrophilic properties and possessed a high density of carboxyl groups. Carboxyl groups and rhBMP-2 on the PCL scaffolds surface were identified by attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The alkaline phosphatase activity assay showed that the rhBMP-2 immobilized PCL scaffold increased the level of MG-63 cell differentiation. Plasma surface modification for the preparation of biomaterials, such as biofunctionalized polymer scaffolds, can be used for the binding of bioactive molecules in tissue engineering.

Citrus bergamia juice: phytochemical and technological studies.

PubMed

Picerno, Patrizia; Sansone, Francesca; Mencherini, Teresa; Prota, Lucia; Aquino, Rita Patrizia; Rastrelli, Luca; Lauro, Maria Rosaria

2011-07-01

Fresh juice from bergamot (Citrus bergamia Risso) has been studied to evaluate the polyphenolic composition by HPLC-DAD analysis and total polyphenols content by UV method. The main constituent, Naringin, has been selected as analytical and biological marker of the juice. Juice has been loaded onto maltodextrin matrix by spray-drying. The produced maltodextrin/juice powder (BMP) showed neither significant change in total polyphenols content nor decrease in antioxidant properties with respect to fresh juice. Moreover, BMP displayed high in vitro dissolution rate of the bioactive constituents in water and in simulated biological fluids. BMP appears as promising functional raw material for food, nutraceutical and pharmaceutical products. With this aim, a formulation study to develop tablets (BMT) for oral administration has been also performed. The produced solid oral dosage form preserved high polyphenols content, showed complete disaggregation in few minutes and satisfying dissolution rate of the bioactive constituents in simulated biological fluids.

pHEMA-nHA encapsulation and delivery of vancomycin and rhBMP-2 enhances its role as a bone graft substitute.

PubMed

Li, Xinning; Xu, Jianwen; Filion, Tera M; Ayers, David C; Song, Jie

2013-08-01

Bone grafts are widely used in orthopaedic procedures. Autografts are limited by donor site morbidity while allografts are known for considerable infection and failure rates. A synthetic composite bone graft substitute poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) was previously developed to stably press-fit in and functionally repair critical-sized rat femoral segmental defects when it was preabsorbed with a single low dose of 300 ng recombinant human bone morphogenetic protein-2/7 (rhBMP-2/7). To facilitate clinical translation of pHEMA-nHA as a synthetic structural bone graft substitute, we examined its ability to encapsulate and release rhBMP-2 and the antibiotic vancomycin. We analyzed the compressive behavior and microstructure of pHEMA-nHA as a function of vancomycin incorporation doses using a dynamic mechanical analyzer and a scanning electron microscope. In vitro release of vancomycin was monitored by ultraviolet-visible spectroscopy. Release of rhBMP-2 from pHEMA-nHA-vancomycin was determined by ELISA. Bioactivity of the released vancomycin and rhBMP-2 was examined by bacterial inhibition and osteogenic transdifferentiation capabilities in cell culture, respectively. Up to 4.8 wt% of vancomycin was incorporated into pHEMA-nHA without compromising its structural integrity and compressive modulus. Encapsulated vancomycin was released in a dose-dependent and sustained manner in phosphate-buffered saline over 2 weeks, and the released vancomycin inhibited Escherichia coli culture. The pHEMA-nHA-vancomycin composite released preabsorbed rhBMP-2 in a sustained manner over 8 days and locally induced osteogenic transdifferentiation of C2C12 cells in culture. pHEMA-nHA can encapsulate and deliver vancomycin and rhBMP-2 in a sustained and localized manner with reduced loading doses. The elasticity, osteoconductivity, and rhBMP-2/vancomycin delivery characteristics of pHEMA-nHA may benefit orthopaedic reconstructions or fusions with enhanced safety and efficiency and reduced infection risk.

Acceleration of Alveolar Ridge Augmentation Using a Low Dose of Recombinant Human Bone Morphogenetic Protein-2 Loaded on a Resorbable Bioactive Ceramic.

PubMed

Fahmy, Rania A; Mahmoud, Naguiba; Soliman, Samia; Nouh, Samir R; Cunningham, Larry; El-Ghannam, Ahmed

2015-12-01

The aim of the present study was to evaluate the effect of a porous silica-calcium phosphate composite (SCPC50) loaded with and without recombinant human bone morphogenetic protein-2 (rhBMP-2) on alveolar ridge augmentation in saddle-type defects. Micro-granules of SCPC50 resorbable bioactive ceramic were coated with rhBMP-2 10 mg and then implanted into a saddle-type defect (12 × 7 mm) in a dog mandible and covered with a collagen membrane. Control groups included defects grafted with SCPC50 granules without rhBMP-2 and un-grafted defects. Bone healing was evaluated at 8 and 16 weeks using histologic and histomorphometric techniques. The increase in bone height and total defect fill were assessed for each specimen using the ImageJ 1.46 program. The release kinetics of rhBMP-2 was determined in vitro. The height of the bone in the grafted defects and the total defect fill were statistically analyzed. SCPC50 enhanced alveolar ridge augmentation as indicated by the increased vertical bone height, bone surface area, and bone volume after 16 weeks. SCPC50-rhBMP-2 provided a sustained release profile of a low effective dose (BMP-2 4.6 ± 1.34 pg/mL per hour) during the 1- to 21-day period. The slow rate of release of rhBMP-2 from SCPC50 accelerated synchronized complete bone regeneration and graft material resorption in 8 weeks. Successful rapid reconstruction of the alveolar ridge by SCPC50 and SCPC50-rhBMP-2 occurred without any adverse excessive bone formation, inflammation, or fluid-filled voids. Results of this study suggest that SCPC50 is an effective graft material to preserve the alveolar ridge after tooth extraction. Coating SCPC50-rhBMP-2 further accelerated bone regeneration and a considerable increase in vertical bone height. These findings make SCPC50 the primary choice as a carrier for rhBMP-2. SCPC50-rhBMP-2 can serve as an alternative to autologous bone grafting. Published by Elsevier Inc.

Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

PubMed Central

Sudiman, Jaqueline; Sutton-McDowall, Melanie L.; Ritter, Lesley J.; White, Melissa A.; Mottershead, David G.; Thompson, Jeremy G.; Gilchrist, Robert B.

2014-01-01

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies. PMID:25058588

Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

PubMed

Sudiman, Jaqueline; Sutton-McDowall, Melanie L; Ritter, Lesley J; White, Melissa A; Mottershead, David G; Thompson, Jeremy G; Gilchrist, Robert B

2014-01-01

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

Multifunctional Thin Film Biomatrice Biosensor in a Degradable Scaffold Containing Bone Morphogenetic Protein-2 (BMP-2) for Controlled Release in Skeletal Tissue Engineering

NASA Astrophysics Data System (ADS)

McDaniel, Harvey; Lomax, Linda

2001-03-01

Bone morphonogenetic proteins (BMP-2) have been under investigation for three decades. Deminerialized bone and extracts of deminerialized bone are o steoinductive with a temporal sequence of bone induction. Native and recombi nant BMP's have shown the ability, thru growth and differentiative factors t o induce de novo bone formation both invitro and invivo. Their principle fun ction is to induce transformation of undifferentiated mesenchymal cells into osteoblasts. Native and recombinant BMP's, when purified and used without carrier disp erse after implantation and exert no effect on bone induction. The delivery system provides the missing component to successsfully applying osteogenic p roteins for clinical need. Biological and physio-chemical properties are str ictly adhered tofor a successful delivery system. The BMP delivery system ca rrier for osteo inductive payload provided; 1)non tumorgenic genecity, 2) no n immunogenecity, 3) water insoluble, 4) biosorbability with predictable enz ymatic degradation, and 5) an optimized surface for compatibility, cell migr ation and attachment with a negative surface change that encouraged target c ell attachment. Being a controlled Release System, it binded the proteins wi th predictible BMP released kinetics. Porosity with interconnecting voids pr otected the BMP from noon specific proteolysis and promoted rapid vascular a nd mesenchymal invasion. Far wide ranging clinical applications of mechanica l and biofunctional requirements were met with the BMP delivery system. Cohe sion and malleability were reqiured forcontour augmentation, and reconstruct ion of the discontinuity defects, prevented dislocation and retained the sha pe and bone replaced the system. Biological systems have elastic activity associated with them. The activi ty was current associated with a time dependant biological/biochemical react ion (enzymic activity). Bioelectric phoenomena associated with charged molec ules in a biologic structure caused changes in distribution resulting from s pecific processes altering the local anatomy. Electron conductivity mechanisms in the body involved ions as charge carr iers. Bioelectric signal detection involved interacting ionic charge carrier s and transducing ionic current into electric current required by wires (fib eroptic electrolets impregnated on a thin filmed-collagen biomatrix and elec tronic instrumentation(invitro test setup)). The transducing function of the invitro testing was carried out by the electrodes consisting of electrical conduction (thin film biomatrix) in contact with the aqueous solution of the bone. Performance parameters of the system were given special consideration because of the interaction between electrons in the electrodes and ions in the body. The surgical protocol consisted of minimally invasive surgical pro tocol with an out patient procedure.

Tissue engineering skeletal muscle for orthopaedic applications

NASA Technical Reports Server (NTRS)

Payumo, Francis C.; Kim, Hyun D.; Sherling, Michael A.; Smith, Lee P.; Powell, Courtney; Wang, Xiao; Keeping, Hugh S.; Valentini, Robert F.; Vandenburgh, Herman H.

2002-01-01

With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.

Surface modification of 3D-printed porous scaffolds via mussel-inspired polydopamine and effective immobilization of rhBMP-2 to promote osteogenic differentiation for bone tissue engineering.

PubMed

Lee, Sang Jin; Lee, Donghyun; Yoon, Taek Rim; Kim, Hyung Keun; Jo, Ha Hyeon; Park, Ji Sun; Lee, Jun Hee; Kim, Wan Doo; Kwon, Il Keun; Park, Su A

2016-08-01

For tissue engineering, a bio-porous scaffold which is applied to bone-tissue regeneration should provide the hydrophilicity for cell attachment as well as provide for the capability to bind a bioactive molecule such as a growth factor in order to improve cell differentiation. In this work, we prepared a three-dimensional (3D) printed polycaprolactone scaffold (PCLS) grafted with recombinant human bone morphogenic protein-2 (rhBMP2) attached via polydopamine (DOPA) chemistry. The DOPA coated PCL scaffold was characterized by contact angle, water uptake, and X-ray photoelectron spectroscopy (XPS) in order to certify that the surface was successfully coated with DOPA. In order to test the loading and release of rhBMP2, we examined the release rate for 28days. For the In vitro cell study, pre-osteoblast MC3T3-E1 cells were seeded onto PCL scaffolds (PCLSs), DOPA coated PCL scaffold (PCLSD), and scaffolds with varying concentrations of rhBMP2 grafted onto the PCLSD 100 and PCLSD 500 (100 and 500ng/ml loaded), respectively. These scaffolds were evaluated by cell proliferation, alkaline phosphatase activity, and real time polymerase chain reaction with immunochemistry in order to verify their osteogenic activity. Through these studies, we demonstrated that our fabricated scaffolds were well coated with DOPA as well as grafted with rhBMP2 at a quantity of 22.7±5ng when treatment with 100ng/ml rhBMP2 and 153.3±2.4ng when treated with 500ng/ml rhBMP2. This grafting enables rhBMP2 to be released in a sustained pattern. In the in vitro results, the cell proliferation and an osteoconductivity of PCLSD 500 groups was greater than any other group. All of these results suggest that our manufactured 3D printed porous scaffold would be a useful construct for application to the bone tissue engineering field. Tissue-engineered scaffolds are not only extremely complex and cumbersome, but also use organic solvents which can negatively influence cellular function. Thus, a rapid, solvent-free method is necessary to improve scaffold generation. Recently, 3D printing such as a rapid prototyping technique has several benefits in that manufacturing is a simple process using computer aided design and scaffolds can be generated without using solvents. In this study, we designed a bio-active scaffold using a very simple and direct method to manufacture DOPA coated 3D PCL porous scaffold grafted with rhBMP2 as a means to create bone-tissue regenerative scaffolds. To our knowledge, our approach can allow for the generation of scaffolds which possessed good properties for use as bone-tissue scaffolds. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The Influence of Peptide Modifications of Bioactive Glass on Human Mesenchymal Stem Cell Growth and Function

NASA Astrophysics Data System (ADS)

Ammar, Mohamed

2011-12-01

Bioactive glass is known for its potential as a bone scaffold due to its ability to stimulate osteogenesis and induce bone formation. Broadening this potential to include the differentiation of human mesenchymal stem cells (hMSCs) to bone cells will enhance the healing process in bone defects. The surface of bioactive glass made by the sol-gel technique with the composition of 70% SiO2-30% CaO (mol %) was grafted with 3 peptides sequences in different combinations from proteins (fibronectin BMP-2 and BMP-9) that are known to promote the adhesion, differentiation and osteogenesis process. The experiment was done in two forms, a 2D non-porous thin film and a 3D nano-macroporous structure. hMSCs were grown on the materials for a total of five weeks. The 2D materials were tested for the expression of 3 osteogenic markers (osteopontin, osteocalcin and osteonectin) through immunocytochemistry. The 3D forms were monitored for cell's adhesion, morphology, spreading and proliferation by scanning electron microscopy, in addition to proliferation assay and alkaline phosphatase activity measurement. Results showed that hMSCs poorly adhered to the 2D thin films, but the few cells survived showed enhanced expression of the osteogenic markers. On the 3D form, cells showed enhanced proliferation at week one and more survival of the cells on the materials grafted with the adhesion peptide for the successive weeks in comparison to the positive control samples. Enhanced alkaline phosphatase activity was also detected compared to the negative control samples but were still below the positive control samples. In conclusion, the peptide grafting could increase the effect of bioactive glass but more peptide combinations should be examined to improve the effects on the differentiation and osteogenic activity of the hMSCs.

Nanotemplated polyelectrolyte films as porous biomolecular delivery systems. Application to the growth factor BMP-2.

PubMed

Gand, Adeline; Hindié, Mathilde; Chacon, Diane; Van Tassel, Paul R; Pauthe, Emmanuel

2014-01-01

Biomaterials capable of delivering controlled quantities of bioactive agents, while maintaining mechanical integrity, are needed for a variety of cell contacting applications. We describe here a nanotemplating strategy toward porous, polyelectrolyte-based thin films capable of controlled biomolecular loading and release. Films are formed via the layer-by-layer assembly of charged polymers and nanoparticles (NP), then chemically cross-linked to increase mechanical rigidity and stability, and finally exposed to tetrahydrofuran to dissolve the NP and create an intra-film porous network. We report here on the loading and release of the growth factor bone morphogenetic protein 2 (BMP-2), and the influence of BMP-2 loaded films on contacting murine C2C12 myoblasts. We observe nanotemplating to enable stable BMP-2 loading throughout the thickness of the film, and find the nanotemplated film to exhibit comparable cell adhesion, and enhanced cell differentiation, compared with a non-porous cross-linked film (where BMP-2 loading is mainly confined to the film surface).

MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

PubMed Central

2014-01-01

Background The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detegent. These results add to the current understanding of producing recombinant TGF-β superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-β chimeras for activity screen is also discussed. PMID:24559319

Efficient delivery of recombinant human bone morphogenetic protein (rhBMP-2) with dextran sulfate-chitosan microspheres.

PubMed

Xia, Yuan-Jun; Xia, Hong; Chen, Ling; Ying, Qing-Shui; Yu, Xiang; Li, Li-Hua; Wang, Jian-Hua; Zhang, Ying

2018-04-01

Bone morphogenetic protein-2 (BMP-2) serves an important role in the development of bone and cartilage. However, administration of BMP-2 protein alone by intravenous delivery is not very effective. Sustained delivery of stabilized BMP-2 by carriers has been proven necessary to improve the osteogenesis effect of BMP-2. The present study constructed a novel drug delivery system using dextran sulfate (DS)-chitosan (CS) microspheres and investigated the efficiency of the delivery system on recombinant human bone morphogenetic protein (rhBMP-2). The microsphere morphology, optimal ratio of DS/CS/rhBMP-2, and drug loading rate and entrapment efficiency of rhBMP-2 CS nanoparticles were determined. L929 cells were used to evaluate the cytotoxicity and effect of DS/CS/rhBMP-2 microspheres on cell proliferation. Differentiation study was conducted using bone marrow mesenchymal stem cells (BMSCs-C57) cells treated with DS/CS/rhBMP-2 microspheres or the control microspheres. The DS/CS/rhBMP-2 microspheres delivery system was successfully established. Subsequent complexation of rhBMP-2-bound DS with polycations afforded well defined microspheres with a diameter of ~250 nm. High protein entrapment efficiency (85.6%) and loading ratio (47.245) µg/mg were achieved. Release of rhBMP-2 from resultant microspheres persisted for over 20 days as determined by ELISA assay. The bioactivity of rhBMP-2 encapsulated in the CS/DS microsphere was observed to be well preserved as evidenced by the alkaline phosphatase activity assay and calcium nodule formation of BMSCs-C57 incubated with rhBMP-2-loaded microspheres. The results demonstrated that microspheres based on CS-DS polyion complexes were a highly efficient vehicle for delivery of rhBMP-2 protein. The present study may provide novel orientation for bone tissue engineering for repairing and regenerating bone defects.

Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole

PubMed Central

Jeong, Byung-Chul; Choi, Hyuck; Hur, Sung-Woong; Kim, Jung-Woo; Oh, Sin-Hye; Kim, Hyun-Seung; Song, Soo-Chang; Lee, Keun-Bae; Park, Kwang-Bum; Koh, Jeong-Tae

2015-01-01

Recently a submicron particle of biphasic calcium phosphate ceramic (BCP) with through-hole (donut-shaped BCP (d-BCP)) was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing. PMID:26491693

Repair of Cranial Bone Defects Using rhBMP2 and Submicron Particle of Biphasic Calcium Phosphate Ceramics with Through-Hole.

PubMed

Jeong, Byung-Chul; Choi, Hyuck; Hur, Sung-Woong; Kim, Jung-Woo; Oh, Sin-Hye; Kim, Hyun-Seung; Song, Soo-Chang; Lee, Keun-Bae; Park, Kwang-Bum; Koh, Jeong-Tae

2015-01-01

Recently a submicron particle of biphasic calcium phosphate ceramic (BCP) with through-hole (donut-shaped BCP (d-BCP)) was developed for improving the osteoconductivity. This study was performed to examine the usefulness of d-BCP for the delivery of osteoinductive rhBMP2 and the effectiveness on cranial bone regeneration. The d-BCP was soaked in rhBMP2 solution and then freeze-dried. Scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), and Raman spectroscopy analyses confirmed that rhBMP2 was well delivered onto the d-BCP surface and the through-hole. The bioactivity of the rhBMP2/d-BCP composite was validated in MC3T3-E1 cells as an in vitro model and in critical-sized cranial defects in C57BL/6 mice. When freeze-dried d-BCPs with rhBMP2 were placed in transwell inserts and suspended above MC3T3-E1, alkaline phosphatase activity and osteoblast-specific gene expression were increased compared to non-rhBMP2-containing d-BCPs. For evaluating in vivo effectiveness, freeze-dried d-BCPs with or without rhBMP2 were implanted into critical-sized cranial defects. Microcomputed tomography and histologic analysis showed that rhBMP2-containing d-BCPs significantly enhanced cranial bone regeneration compared to non-rhBMP2-containing control. These results suggest that a combination of d-BCP and rhBMP2 can accelerate bone regeneration, and this could be used to develop therapeutic strategies in hard tissue healing.

Ginger Stimulates Hematopoiesis via Bmp Pathway in Zebrafish

PubMed Central

Ferri-Lagneau, Karine F.; Moshal, Karni S.; Grimes, Matthew; Zahora, Braden; Lv, Lishuang; Sang, Shengmin; Leung, TinChung

2012-01-01

Background Anemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis. Methodology/Principal Findings Here, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1. Conclusions/Significance Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents. PMID:22761764

Synergistic effects of BMP-2, BMP-6 or BMP-7 with human plasma fibronectin onto hydroxyapatite coatings: A comparative study.

PubMed

Brigaud, Isabelle; Agniel, Rémy; Leroy-Dudal, Johanne; Kellouche, Sabrina; Ponche, Arnaud; Bouceba, Tahar; Mihailescu, Natalia; Sopronyi, Mihai; Viguier, Eric; Ristoscu, Carmen; Sima, Felix; Mihailescu, Ion N; Carreira, Ana Claudia O; Sogayar, Mari Cleide; Gallet, Olivier; Anselme, Karine

2017-06-01

Design of new osteoinductive biomaterials to reproduce an optimized physiological environment capable of recruiting stem cells and instructing their fate towards the osteoblastic lineage has become a priority in orthopaedic surgery. This work aims at evaluating the bioactivity of BMP combined with human plasma fibronectin (FN/BMP) delivered in solution or coated onto titanium-hydroxyapatite (TiHA) surfaces. Herein, we focus on the comparison of in vitro osteogenic efficacy in mouse C2C12 pre-osteoblasts of three BMP members, namely: BMP-2, BMP-6 and BMP-7. In parallel, we evaluated the molecular binding strength between each BMP with FN using the Surface Plasmon Resonance (SPR) technology. The affinity of BMPs for FN was found totally different and dependent on BMP type. Indeed, the combination of FN with BMP-2 on TiHA surfaces potentiates the burst of gene-mediated osteogenic induction, while it prolongs the osteogenic activity of BMP-6 and surprisingly annihilates the BMP-7 one. These results correlate with FN/BMP affinity for TiHA, since BMP-6>BMP-2>BMP-7. In addition, by analyzing the osteogenic activity in the peri-implant environment, we showed that osteoinductive paracrine effects were significantly decreased upon (FN/BMP-6), as opposed to (FN/BMP-2) coatings. Altogether, our results support the use of FN/BMP-6 to develop a biomimetic microenvironment capable to induce osteogenic activity under physiological conditions, with minimum paracrine signalization. The originality of our paper relies on the first direct comparison of the in vitro osteogenic potential of three osteogenic BMPs (BMP-2, -6 and -7) combined with native human plasma fibronectin delivered in solution or coated by laser transfer onto titanium hydroxyapatite surfaces. We confirm that BMP association with fibronectin enhances the osteogenic activity of BMP-2, -6 and -7, but with essential discrepancies, depending on the BMP member, and in agreement with the affinity of BMPs for fibronectin. Moreover, we bring elements to explain the origin of the BMP-2 medical life-threatening side-effects by analyzing in vitro paracrine effects. Finally, this work supports the alternative use of FN/BMP-6 to induce osteogenic activity under physiological conditions, with minimum side effects. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Release and bioactivity of bone morphogenetic protein-2 are affected by scaffold binding techniques in vitro and in vivo.

PubMed

Suliman, Salwa; Xing, Zhe; Wu, Xujun; Xue, Ying; Pedersen, Torbjorn O; Sun, Yang; Døskeland, Anne P; Nickel, Joachim; Waag, Thilo; Lygre, Henning; Finne-Wistrand, Anna; Steinmüller-Nethl, Doris; Krueger, Anke; Mustafa, Kamal

2015-01-10

A low dose of 1μg rhBMP-2 was immobilised by four different functionalising techniques on recently developed poly(l-lactide)-co-(ε-caprolactone) [(poly(LLA-co-CL)] scaffolds. It was either (i) physisorbed on unmodified scaffolds [PHY], (ii) physisorbed onto scaffolds modified with nanodiamond particles [nDP-PHY], (iii) covalently linked onto nDPs that were used to modify the scaffolds [nDP-COV] or (iv) encapsulated in microspheres distributed on the scaffolds [MICS]. Release kinetics of BMP-2 from the different scaffolds was quantified using targeted mass spectrometry for up to 70days. PHY scaffolds had an initial burst of release while MICS showed a gradual and sustained increase in release. In contrast, NDP-PHY and nDP-COV scaffolds showed no significant release, although nDP-PHY scaffolds maintained bioactivity of BMP-2. Human mesenchymal stem cells cultured in vitro showed upregulated BMP-2 and osteocalcin gene expression at both week 1 and week 3 in the MICS and nDP-PHY scaffold groups. These groups also demonstrated the highest BMP-2 extracellular protein levels as assessed by ELISA, and mineralization confirmed by Alizarin red. Cells grown on the PHY scaffolds in vitro expressed collagen type 1 alpha 2 early but the scaffold could not sustain rhBMP-2 release to express mineralization. After 4weeks post-implantation using a rat mandible critical-sized defect model, micro-CT and Masson trichrome results showed accelerated bone regeneration in the PHY, nDP-PHY and MICS groups. The results demonstrate that PHY scaffolds may not be desirable for clinical use, since similar osteogenic potential was not seen under both in vitro and in vivo conditions, in contrast to nDP-PHY and MICS groups, where continuous low doses of BMP-2 induced satisfactory bone regeneration in both conditions. The nDP-PHY scaffolds used here in critical-sized bone defects for the first time appear to have promise compared to growth factors adsorbed onto a polymer alone and the short distance effect prevents adverse systemic side effects. Copyright © 2014. Published by Elsevier B.V.

Dual Delivery of EPO and BMP2 from a Novel Modular Poly-ɛ-Caprolactone Construct to Increase the Bone Formation in Prefabricated Bone Flaps

PubMed Central

Patel, Janki Jayesh; Modes, Jane E.; Flanagan, Colleen L.; Krebsbach, Paul H.; Edwards, Sean P.

2015-01-01

Poly-ɛ-caprolactone (PCL) is a biocompatible polymer that has mechanical properties suitable for bone tissue engineering; however, it must be integrated with biologics to stimulate bone formation. Bone morphogenetic protein-2 (BMP2) delivered from PCL produces bone when implanted subcutaneously, and erythropoietin (EPO) works synergistically with BMP2. In this study, EPO and BMP2 are adsorbed separately on two 3D-printed PCL scaffold modules that are assembled for codelivery on a single scaffold structure. This assembled modular PCL scaffold with dual BMP2 and EPO delivery was shown to increase bone growth in an ectopic location when compared with BMP2 delivery along a replicate scaffold structure. EPO (200 IU/mL) and BMP2 (65 μg/mL) were adsorbed onto the outer and inner portions of a modular scaffold, respectively. Protein binding and release studies were first quantified. Subsequently, EPO+BMP2 and BMP2 scaffolds were implanted subcutaneously in mice for 4 and 8 weeks, and the regenerated bone was analyzed with microcomputed tomography and histology; 8.6±1.4 μg BMP2 (22%) and 140±29 IU EPO (69.8%) bound to the scaffold and <1% BMP2 and 83% EPO was released in 7 days. Increased endothelial cell proliferation on EPO-adsorbed PCL discs indicated protein bioactivity. At 4 and 8 weeks, dual BMP2 and EPO delivery regenerated more bone (5.1±1.1 and 5.5±1.6 mm3) than BMP2 alone (3.8±1.1 and 4.3±1.7 mm3). BMP2 and EPO scaffolds had more ingrowth (1.4%±0.6%) in the outer module when compared with BMP2 (0.8%±0.3%) at 4 weeks. Dual delivery produced more dense cellular marrow, while BMP2 had more fatty marrow. Dual EPO and BMP2 delivery is a potential method to regenerate bone faster for prefabricated flaps. PMID:25809081

Dual Delivery of EPO and BMP2 from a Novel Modular Poly-ɛ-Caprolactone Construct to Increase the Bone Formation in Prefabricated Bone Flaps.

PubMed

Patel, Janki Jayesh; Modes, Jane E; Flanagan, Colleen L; Krebsbach, Paul H; Edwards, Sean P; Hollister, Scott J

2015-09-01

Poly-ɛ-caprolactone (PCL) is a biocompatible polymer that has mechanical properties suitable for bone tissue engineering; however, it must be integrated with biologics to stimulate bone formation. Bone morphogenetic protein-2 (BMP2) delivered from PCL produces bone when implanted subcutaneously, and erythropoietin (EPO) works synergistically with BMP2. In this study, EPO and BMP2 are adsorbed separately on two 3D-printed PCL scaffold modules that are assembled for codelivery on a single scaffold structure. This assembled modular PCL scaffold with dual BMP2 and EPO delivery was shown to increase bone growth in an ectopic location when compared with BMP2 delivery along a replicate scaffold structure. EPO (200 IU/mL) and BMP2 (65 μg/mL) were adsorbed onto the outer and inner portions of a modular scaffold, respectively. Protein binding and release studies were first quantified. Subsequently, EPO+BMP2 and BMP2 scaffolds were implanted subcutaneously in mice for 4 and 8 weeks, and the regenerated bone was analyzed with microcomputed tomography and histology; 8.6±1.4 μg BMP2 (22%) and 140±29 IU EPO (69.8%) bound to the scaffold and <1% BMP2 and 83% EPO was released in 7 days. Increased endothelial cell proliferation on EPO-adsorbed PCL discs indicated protein bioactivity. At 4 and 8 weeks, dual BMP2 and EPO delivery regenerated more bone (5.1±1.1 and 5.5±1.6 mm(3)) than BMP2 alone (3.8±1.1 and 4.3±1.7 mm(3)). BMP2 and EPO scaffolds had more ingrowth (1.4%±0.6%) in the outer module when compared with BMP2 (0.8%±0.3%) at 4 weeks. Dual delivery produced more dense cellular marrow, while BMP2 had more fatty marrow. Dual EPO and BMP2 delivery is a potential method to regenerate bone faster for prefabricated flaps.

Efficacy of rhBMP-2 Loaded PCL/β-TCP/bdECM Scaffold Fabricated by 3D Printing Technology on Bone Regeneration.

PubMed

Bae, Eun-Bin; Park, Keun-Ho; Shim, Jin-Hyung; Chung, Ho-Yun; Choi, Jae-Won; Lee, Jin-Ju; Kim, Chang-Hwan; Jeon, Ho-Jun; Kang, Seong-Soo; Huh, Jung-Bo

2018-01-01

This study was undertaken to evaluate the effect of 3D printed polycaprolactone (PCL)/ β -tricalcium phosphate ( β -TCP) scaffold containing bone demineralized and decellularized extracellular matrix (bdECM) and human recombinant bone morphogenetic protein-2 (rhBMP-2) on bone regeneration. Scaffolds were divided into PCL/ β -TCP, PCL/ β -TCP/bdECM, and PCL/ β -TCP/bdECM/BMP groups. In vitro release kinetics of rhBMP-2 were determined with respect to cell proliferation and osteogenic differentiation. These three reconstructive materials were implanted into 8 mm diameter calvarial bone defect in male Sprague-Dawley rats. Animals were sacrificed four weeks after implantation for micro-CT, histologic, and histomorphometric analyses. The findings obtained were used to calculate new bone volumes (mm 3 ) and new bone areas (%). Excellent cell bioactivity was observed in the PCL/ β -TCP/bdECM and PCL/ β -TCP/bdECM/BMP groups, and new bone volume and area were significantly higher in the PCL/ β -TCP/bdECM/BMP group than in the other groups ( p < .05). Within the limitations of this study, bdECM printed PCL/ β -TCP scaffolds can reproduce microenvironment for cells and promote adhering and proliferating the cells onto scaffolds. Furthermore, in the rat calvarial defect model, the scaffold which printed rhBMP-2 loaded bdECM stably carries rhBMP-2 and enhances bone regeneration confirming the possibility of bdECM as rhBMP-2 carrier.

Collagen I derived recombinant protein microspheres as novel delivery vehicles for bone morphogenetic protein-2.

PubMed

Mumcuoglu, Didem; de Miguel, Laura; Jekhmane, Shehrazade; Siverino, Claudia; Nickel, Joachim; Mueller, Thomas D; van Leeuwen, Johannes P; van Osch, Gerjo J; Kluijtmans, Sebastiaan G

2018-03-01

Bone morphogenetic protein-2 (BMP-2) is a powerful osteoinductive protein; however, there is a need for the development of a safe and efficient BMP-2 release system for bone regeneration therapies. Recombinant extracellular matrix proteins are promising next generation biomaterials since the proteins are well-defined, reproducible and can be tailored for specific applications. In this study, we have developed a novel and versatile BMP-2 delivery system using microspheres from a recombinant protein based on human collagen I (RCP). In general, a two-phase release pattern was observed while the majority of BMP-2 was retained in the microspheres for at least two weeks. Among different parameters studied, the crosslinking and the size of the RCP microspheres changed the in vitro BMP-2 release kinetics significantly. Increasing the chemical crosslinking (hexamethylene diisocyanide) degree decreased the amount of initial burst release (24h) from 23% to 17%. Crosslinking by dehydrothermal treatment further decreased the burst release to 11%. Interestingly, the 50 and 72μm-sized spheres showed a significant decrease in the burst release compared to 207-μm sized spheres. Very importantly, using a reporter cell line, the released BMP-2 was shown to be bioactive. SPR data showed that N-terminal sequence of BMP-2 was important for the binding and retention of BMP-2 and suggested the presence of a specific binding epitope on RCP (K D : 1.2nM). This study demonstrated that the presented RCP microspheres are promising versatile BMP-2 delivery vehicles. Copyright © 2017 Elsevier B.V. All rights reserved.

Osseous regeneration in the rat calvarium using novel delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2).

PubMed

Kenley, R; Marden, L; Turek, T; Jin, L; Ron, E; Hollinger, J O

1994-10-01

In the current investigation, we report osseous regeneration in critical-size rat calvarial defects using recombinant human bone morphogenetic protein-2 (rhBMP-2) and novel delivery systems based on biomaterials. The novel systems combine rhBMP-2 with dry powder microparticles of poly(D,L-lactide-co-glycolide) (PLGA). The mixture of rhBMP-2 with PLGA microparticles is added to an aqueous solution of biopolymer to yield a semisolid paste. The biopolymers tested include autologous blood clot, hydroxypropyl methylcellulose, and sodium alginate cross-linked with calcium ion. Insoluble collageneous bone matrix was also studied as a control. Test articles were made at 0-, 10-, and 30-micrograms doses of rhBMP-2 and imiplanted in 8-mm-diameter rat calvarial defects (which will not heal if left untreated). The animals were examined 21 days after implantation by radiography, radiomorphometry, histology, and histomorphometry. All tested materials containing rhBMP-2 restored radiopacity and normal contouring to the calvarial defects. Samples without added rhBMP-2 yielded only soft tissue within the defects. Histology showed restoration of inner and outer bone tables plus marrow constituents. The PLGA microparticles were significantly resorbed at the 21-day time point. Although small differences between delivery systems were evident at 0- and 10-micrograms rhBMP-2 doses, all test articles performed essentially equivalently at the 30-micrograms dose. Thus, novel delivery systems for rhBMP-2 offer the promise of combining the intrinsic bioactivity of the osteoinductive protein with pharmaceutically acceptable biomaterials.

The role of the micro-pattern and nano-topography of hydroxyapatite bioceramics on stimulating osteogenic differentiation of mesenchymal stem cells.

PubMed

Zhao, Cancan; Wang, Xiaoya; Gao, Long; Jing, Linguo; Zhou, Quan; Chang, Jiang

2018-06-01

The micro/nano hybrid structure is considered to be a biomaterial characteristic to stimulate osteogenesis by mimicking the three-dimensional structure of the bone matrix. However, the mechanism of the hybrid structure induced osteogenic differentiation of stem cells is still unknown. For elucidating the mechanisms, one of the challenge is to directly fabricate micro/nano hybrid structure on bioceramics because of its brittleness. In this study, hydroxyapatite (HA) bioceramics with the micro/nano hybrid structure were firstly fabricated via a hydrothermal treatment and template method, and the effect of the different surface structures on the expression of integrins, BMP2 signaling pathways and cell-cell communication was investigated. Interestingly, the results suggested that the osteogenic differentiation induced by micro/nano structures was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, while activated BMP2 could in turn activate integrins and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect. The micro/nano hybrid structure has been found to have synergistic bioactivity on osteogenesis. However, it is still a challenge to fabricate the hybrid structure directly on the bioceramics, and the role of micro- and nano-structure, in particular the mechanism of the micro/nano-hybrid structure induced stem cell differentiation is still unknown. In this study, we firstly fabricated hydroxyapatite bioceramics with the micro/nano hybrid structure, and then investigated the effect of different surface structure on expression of integrins, BMP2 signaling pathways and cell-cell communication. Interestingly, we found that the osteogenic differentiation induced by structure was modulated first through activating integrins and then further activating BMP2 signaling pathway and cell-cell communication, and activated BMP2 could in turn activate some integrin subunits and Cx43-related cell-cell communication. Furthermore, differences in activation of integrins, BMP2 signaling pathway, and gap junction-mediated cell-cell communication were observed, in which nanorod and micropattern structures activated different integrin subunits, BMP downstream receptors and Cx43. This finding may explain the synergistic effect of the micro/nano hybrid structure on the activation of osteogenic differentiation of BMSCs. Based on our study, we concluded that the different activation mechanisms of micro- and nano-structures led to the synergistic stimulatory effect on integrin activation and osteogenesis, in which not only the direct contact of cells on micro/nano structure played an important role, but also other surface characteristics such as protein adsorption might contribute to the bioactive effect. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Assessment of a polyelectrolyte multilayer film coating loaded with BMP-2 on titanium and PEEK implants in the rabbit femoral condyle

PubMed Central

Guillot, R.; Pignot-Paintrand, I.; Lavaud, J.; Decambron, A.; Bourgeois, E.; Josserand, V.; Logeart-Avramoglou, D.; Viguier, E.; Picart, C.

2016-01-01

The aim of this study was to evaluate the osseointegration of titanium implants (Ti-6Al-4V, noted here TA6V) and poly(etheretherketone) PEEK implants induced by a BMP-2-delivering surface coating made of polyelectrolyte multilayer films. The in vitro bioactivity of the polyelectrolyte film-coated implants was assessed using the alkaline phosphatase assay. BMP-2-coated TA6V and PEEK implants with a total dose of 9.3 µg of BMP-2 were inserted into the femoral condyles of New Zealand white rabbits and compared to uncoated implants. Rabbits were sacrificed 4 and 8 weeks after implantation. Histomorphometric analyses on TA6V and PEEK implants and microcomputed tomography on PEEK implants revealed that the bone-to-implant contact and bone area around the implants were significantly lower for the BMP-2-coated implants than for the bare implants. This was confirmed by scanning electron microscopy imaging. This difference was more pronounced at 4 weeks in comparison to the 8-week time point. However, bone growth inside the hexagonal upper hollow cavity of the screws was higher in the case of the BMP-2 coated implants. Overall, this study shows that a high dose of BMP-2 leads to localized and temporary bone impairment, and that the dose of BMP-2 delivered at the surface of an implant needs to be carefully optimized. PMID:26965394

Self-Assembling Peptide Amphiphiles for Therapeutic Delivery of Proteins, Drugs, and Stem Cells

NASA Astrophysics Data System (ADS)

Lee, Sungsoo Seth

Biomaterials are used to help regenerate or replace the structure and function of damaged tissues. In order to elicit desired therapeutic responses in vivo, biomaterials are often functionalized with bioactive agents, such as growth factors, small molecule drugs, or even stem cells. Therefore, the strategies used to incorporate these bioactive agents in the microstructures and nanostructures of biomaterials can strongly influence the their therapeutic efficacy. Using self-assembling peptide amphiphiles (PAs), this work has investigated supramolecular nanostructures with improved interaction with three types of therapeutic agents: bone morphogenetic protein 2 (BMP-2) which promotes osteogenic differentiation and bone growth, anti-inflammatory drug naproxen which is used to treat osteo- and rheumatoid arthritis, and neural stem cells that could differentiate into neurons to treat neurodegenerative diseases. For BMP-2 delivery, two specific systems were investigated with affinity for BMP-2: 1) heparin-binding nanofibers that display the natural ligand of the osteogenic protein, and 2) nanofibers that display a synthetic peptide ligand discovered in our laboratory through phage display to directly bind BMP-2. Both systems promoted enhanced osteoblast differentiation of pluripotent C2C12 cells and augmented bone regeneration in two in vivo models, a rat critical-size femur defect model and spinal arthrodesis model. The thesis also describes the use of PA nanofibers to improve the delivery of the anti-inflammatory drug naproxen. To promote a controlled release, naproxen was chemically conjugated to the nanofiber surface via an ester bond that would only be cleaved by esterases, which are enzymes found naturally in the body. In the absence of esterases, the naproxen remained conjugated to the nanofibers and was non-bioactive. On the other hand, in the presence of esterases, naproxen was slowly released and inhibited cyclooxygenase-2 (COX-2) activity, an enzyme responsible for inflammation. Finally, PA nanofibers were utilized as synthetic extracellular matrices (ECM) to encapsulate neural stem cells and promote neuron differentiation. Here, the influence of ECM nanostructures on neuron differentiation was assessed by comparing wide and narrow nanoribbons that were crafted from identical PA molecules. The PAs self-assembled into wide nanoribbons at pH 6.6, but raising the pH to 7.2 promoted increased side-chain ionization and electrostatic repulsion, thus resulting in a structural transformation into thin nanoribbons. In contrast, introducing divalent Ca2+ counterions at pH 6.6 stabilized the intermolecular cohesion of the PA molecules and the wide nanoribbon shape was preserved upon increasing pH. Interestingly, the wide nanoribbon gel exhibited a higher stiffness than the narrow nanoribbon gel. When neural stem cells were encapsulated, the wide nanoribbon matrix was able to promote neuron differentiation, while the thin nanoribbon matrix contracted and prevented neuron differentiation. Mechanistically, the collapse of the thin ribbons likely increased the local density and cell-cell contact of the stem cells, which has been found previously to prevent differentiation into neuronal lineage. Taken together, these findings demonstrate the significance of protein-material or cell-material interactions in achieving optimal therapeutic effects and provide future strategies for developing functional supramolecular biomaterials.

Nanotemplated polyelectrolyte films as porous biomolecular delivery systems

PubMed Central

Gand, Adeline; Hindié, Mathilde; Chacon, Diane; van Tassel, Paul R; Pauthe, Emmanuel

2014-01-01

Biomaterials capable of delivering controlled quantities of bioactive agents, while maintaining mechanical integrity, are needed for a variety of cell contacting applications. We describe here a nanotemplating strategy toward porous, polyelectrolyte-based thin films capable of controlled biomolecular loading and release. Films are formed via the layer-by-layer assembly of charged polymers and nanoparticles (NP), then chemically cross-linked to increase mechanical rigidity and stability, and finally exposed to tetrahydrofuran to dissolve the NP and create an intra-film porous network. We report here on the loading and release of the growth factor bone morphogenetic protein 2 (BMP-2), and the influence of BMP-2 loaded films on contacting murine C2C12 myoblasts. We observe nanotemplating to enable stable BMP-2 loading throughout the thickness of the film, and find the nanotemplated film to exhibit comparable cell adhesion, and enhanced cell differentiation, compared with a non-porous cross-linked film (where BMP-2 loading is mainly confined to the film surface). PMID:25482416

Fabrication of a biomimetic ZeinPDA nanofibrous scaffold impregnated with BMP-2 peptide conjugated TiO2 nanoparticle for bone tissue engineering.

PubMed

Babitha, S; Annamalai, Meenakshi; Dykas, Michal Marcin; Saha, Surajit; Poddar, Kingshuk; Venugopal, Jayarama Reddy; Ramakrishna, Seeram; Venkatesan, Thirumalai; Korrapati, Purna Sai

2018-04-01

A biomimetic Zein polydopamine based nanofiber scaffold was fabricated to deliver bone morphogenic protein-2 (BMP-2) peptide conjugated titanium dioxide nanoparticles in a sustained manner for investigating its osteogenic differentiation potential. To prolong its retention time at the target site, BMP-2 peptide has been conjugated to titanium dioxide nanoparticles owing to its high surface to volume ratio. The effect of biochemical cues from BMP-2 peptide and nanotopographical stimulation of electrospun Zein polydopamine nanofiber were examined for its enhanced osteogenic expression of human fetal osteoblast cells. The sustained delivery of bioactive signals, improved cell adhesion, mineralization, and differentiation could be attributed to its highly interconnected nanofibrous matrix with unique material composition. Further, the expression of osteogenic markers revealed that the fabricated nanofibrous scaffold possess better cell-biomaterial interactions. These promising results demonstrate the potential of the composite nanofibrous scaffold as an effective biomaterial substrate for bone regeneration. Copyright © 2017 John Wiley & Sons, Ltd.

Fabrication of polycaprolactone-silanated β-tricalcium phosphate-heparan sulfate scaffolds for spinal fusion applications.

PubMed

Bhakta, Gajadhar; Ekaputra, Andrew K; Rai, Bina; Abbah, Sunny A; Tan, Tuan Chun; Le, Bach Quang; Chatterjea, Anindita; Hu, Tao; Lin, Tingxuan; Arafat, M Tarik; van Wijnen, Andre J; Goh, James; Nurcombe, Victor; Bhakoo, Kishore; Birch, William; Xu, Li; Gibson, Ian; Wong, Hee-Kit; Cool, Simon M

2018-05-01

Interbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site. We recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device. A combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds. Freshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120-150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology. Progenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants. The combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery. Copyright © 2017 Elsevier Inc. All rights reserved.

In vivo bone regeneration using a novel porous bioactive composite

NASA Astrophysics Data System (ADS)

Xie, En; Hu, Yunyu; Chen, Xiaofeng; Bai, Xuedong; Li, Dan; Ren, Li; Zhang, Ziru

2008-11-01

Many commercial bone graft substitutes (BGS) and experimental bone tissue engineering scaffolds have been developed for bone repair and regeneration. This study reports the in vivo bone regeneration using a newly developed porous bioactive and resorbable composite that is composed of bioactive glass (BG), collagen (COL), hyaluronic acid (HYA) and phosphatidylserine (PS), BG-COL-HYA-PS. The composite was prepared by a combination of sol-gel and freeze-drying methods. A rabbit radius defect model was used to evaluate bone regeneration at time points of 2, 4 and 8 weeks. Techniques including radiography, histology, and micro-CT were applied to characterize the new bone formation. 8 weeks results showed that (1) nearly complete bone regeneration was achieved for the BG-COL-HYA-PS composite that was combined with a bovine bone morphogenetic protein (BMP); (2) partial bone regeneration was achieved for the BG-COL-HYA-PS composites alone; and (3) control remained empty. This study demonstrated that the novel BG-COL-HYA-PS, with or without the grafting of BMP incorporation, is a promising BGS or a tissue engineering scaffold for non-load bearing orthopaedic applications.

Novel osteoinductive photo-cross-linkable chitosan-lactide-fibrinogen hydrogels enhance bone regeneration in critical size segmental bone defects

PubMed Central

Kim, Sungwoo; Bedigrew, Katherine; Guda, Teja; Maloney, William J.; Park, Sangwon; Wenke, Joseph C.; Yang, Yunzhi Peter

2014-01-01

The purpose of this study was to develop and characterize a novel photo-cross-linkable chitosan-lactide-fibrinogen (CLF) hydrogel and evaluate the efficacy of bone morphogenetic protein-2 (BMP-2) containing CLF hydrogel for osteogenesis in vitro and in vivo. We synthesized the CLF hydrogels and characterized their chemical structure, degradation rate, compressive modulus, and in vitro BMP-2 release kinetics. We evaluated bioactivities of the BMP-2 containing CLF hydrogels (0, 50, 100, and 500 ng/ml) in vitro using W-20-17 preosteoblast mouse bone marrow stromal cells and C2C12 mouse myoblast cells. The effect of BMP-2 containing CLF gels (0, 0.5, 1, 2, and 5μg) on bone formation was evaluated using rat critical size segmental bone defects for 4 weeks. FTIR spectra and SEM images showed chemical and structural changes by addition of fibrinogen into chitosan-lactide copolymer. Incorporation of fibrinogen molecules significantly increased compressive modulus of the hydrogels. In vitro BMP-2 release study showed initial burst releases from the CLF hydrogels followed by sustained releases, regardless of the concentration of the BMP-2 over 4 weeks. Cells in all groups were viable in the presence of the hydrogels regardless of BMP-2 doses, indicating non-cytotoxicity of hydrogels. Alkaline phosphate activity and mineralization of cells exhibited dose dependence on BMP-2 containing CLF hydrogels. Radiographs, microcomputed tomography, and histology confirmed that the BMP-2 containing CLF hydrogels prompted neo-osteogenesis and accelerated healing of the defects in a dose-dependent manner. Thus the CLF hydrogel is a promising delivery system of growth factors for bone regeneration. PMID:25174669

[Experimental study of the effect of new bone formation on new type artificial bone composed of bioactive ceramics].

PubMed

Zhu, Minghua; Zeng, Yi; Sun, Tao; Peng, Qiang

2005-03-15

To investigate the osteogenic potential of four kinds of new bioactive ceramics combined with bovine bone morphogenetic proteins (BMP) and to explore the feasibility of using compounds as bone substitute material. Ninety-six rats were divided into 4 groups (24 in each group). BMP was combined with hydroxyapatite (HA), tricalcium phosphate (TCP), fluoridated-HA (FHA), and collagen-HA(CHA) respectively. The left thighs of the rats implanted with HA/BMP, TCP/BMP, FHA/BMP, and CHA/BMP were used as experimental groups. The right thighs of the rats implanted with HA, TCP, CHA, and decalcified dentin matrix (DDM) were used as control groups. The rats were sacrificed 1, 3, 5 and 7 weeks after implantation and bone induction was estimated by alkaline phosphatase (ALP), phosphorus (P), and total protein (TP) measurement. The histological observation and electronic microscope scanning of the implants were also made. The cartilage growth in the 4 experimental groups and the control group implanted with DDM was observed 1 week after operation and fibrous connective tissues were observed in the other 3 control groups. 3 weeks after implantation, lamellar bone with bone marrow and positive reaction in ALP stain were observed in the 4 experimental groups. No bone formation or positive reaction in ALP stain were observed in the control groups. The amount of ALP activity, P value, and new bone formation in the experimental groups were higher than those in the control group(P < 0.05). The amount of ALP activity, P value, and new bone formation in TCP/BMP group were higher than those in HA/BMP, CHA/BMP and FHA/BMP groups (P < 0.05). There was no significant difference in TP between the BMP treatment group and the control groups. From 5th to 7th week, new bone formation, histochemistry evaluation, and the level of ALP, P, TP value were as high as those in the 3rd week. New composite artificial bone of TCP/BMP, HA/BMP, CHA/BMP, and FHA/BMP all prove to be effective, but TCP/BMP is the most effective so that it is the most suitable biomaterial replacement of tissue.

Bisphosphonate-coated BSA nanoparticles lack bone targeting after systemic administration.

PubMed

Wang, Guilin; Kucharski, Cezary; Lin, Xiaoyue; Uludağ, Hasan

2010-09-01

A polymeric conjugate of polyethyleneimine-graft-poly(ethylene glycol) and 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (PEI-PEG-thiolBP) was prepared and used for surface coating of bovine serum albumin (BSA) nanoparticles (NPs) designed for bone-specific delivery of bone morphogenetic protein-2 (BMP-2). The NP coating was achieved with a dialysis and an evaporation method, and the obtained NPs were characterized by particle size, zeta-potential, morphology, and cytotoxicity in vitro. The particle size and surface charge of the NPs could be effectively tuned by the PEG and thiolBP substitution ratios of the conjugate, the coating method, and the polymer concentration used for coating. The PEG modification on PEI reduced the toxicity of PEI and the coated NPs, based on in vitro assessment with human C2C12 cells and rat bone marrow stromal cells. On the basis of an alkaline phosphatase (ALP) induction assay, the NP-encapsulated BMP-2 displayed full retention of its bioactivity, except for BMP-2 in PEI-coated NPs. By encapsulating (125)I-labeled BMP-2, the polymer-coated NPs were assessed for hydroxyapatite (HA) affinity; all NP-encapsulated BMP-2 showed significant affinity to HA as compared with free BMP-2 in vitro, and the PEI-PEG-thiolBP coated NPs improved the in vivo retention of BMP-2 compared with uncoated NPs. However, the biodistribution of NPs after intravenous injection in a rat model indicated no beneficial effects of thiolBP-coated NPs for bone targeting. Our results suggested that the BP-conjugated NPs are useful for localized delivery of BMP-2 in bone repair and regeneration, but they are not effective for bone targeting after intravenous administration.

Development of collagen-hydroxyapatite scaffolds incorporating PLGA and alginate microparticles for the controlled delivery of rhBMP-2 for bone tissue engineering.

PubMed

Quinlan, Elaine; López-Noriega, Adolfo; Thompson, Emmet; Kelly, Helena M; Cryan, Sally Ann; O'Brien, Fergal J

2015-01-28

The spatiotemporally controlled delivery of the pro-osteogenic factor rhBMP-2 would overcome most of the severe secondary effects linked to the products delivering this protein for bone regeneration. With this in mind, the aim of the present work was to develop a controlled rhBMP-2 release system using collagen-hydroxyapatite (CHA) scaffolds, which had been previously optimized for bone regeneration, as delivery platforms to produce a device with enhanced capacity for bone repair. Spray-drying and emulsion techniques were used to encapsulate bioactive rhBMP-2 in alginate and PLGA microparticles, with a high encapsulation efficiency. After incorporation of these microparticles into the scaffolds, rhBMP-2 was delivered in a sustained fashion for up to 28days. When tested in vitro with osteoblasts, these eluting materials showed an enhanced pro-osteogenic effect. From these results, an optimal rhBMP-2 eluting scaffold composition was selected and implanted in critical-sized calvarial defects in a rat model, where it demonstrated an excellent healing capacity in vivo. These platforms have an immense potential in the field of tissue regeneration; by tuning the specific therapeutic molecule to the tissue of interest and by utilizing different collagen-based scaffolds, similar systems can be developed for enhancing the healing of a diverse range of tissues and organs. Copyright © 2014 Elsevier B.V. All rights reserved.

Osthole Promotes Bone Fracture Healing through Activation of BMP Signaling in Chondrocytes.

PubMed

Wang, Pinger; Ying, Jun; Luo, Cheng; Jin, Xing; Zhang, Shanxing; Xu, Taotao; Zhang, Lei; Mi, Meng; Chen, Di; Tong, Peijian; Jin, Hongting

2017-01-01

Osthole is a bioactive coumarin derivative and has been reported to be able to enhance bone formation and improve fracture healing. However, the molecular mechanism of Osthole in bone fracture healing has not been fully defined. In this study we determined if Osthole enhances bone fracture healing through activation of BMP2 signaling in mice. We performed unilateral open transverse tibial fracture procedure in 10-week-old C57BL/6 mice which were treated with or without Osthole. Our previous studies demonstrated that chondrocyte BMP signaling is required for bone fracture healing, in this study we also performed tibial fracture procedure in Cre-negative and Col2-Cre;Bmp2 flox/flox conditional knockout (KO) mice ( Bmp2 Col2Cre ) to determine if Osthole enhances fracture healing in a BMP2-dependent manner. Fracture callus tissues were collected and analyzed by X-ray, micro-CT (μCT), histology, histomorphometry, immunohistochemistry (IHC), biomechanical testing and quantitative gene expression analysis. In addition, mouse chondrogenic ATDC5 cells were cultured with or without Osthole and the expression levels of chondrogenic marker genes were examined. The results demonstrated that Osthole promotes bone fracture healing in wild-type (WT) or Cre - control mice. In contrast, Osthole failed to promote bone fracture healing in Bmp2 Col2Cre conditional KO mice. In the mice receiving Osthole treatment, expression of cartilage marker genes was significantly increased. We conclude that Osthole could promote bone strength and enhance fracture healing by activation of BMP2 signaling. Osthole may be used as an alternative approach in the orthopaedic clinic for the treatment of fracture healing.

Osthole Promotes Bone Fracture Healing through Activation of BMP Signaling in Chondrocytes

PubMed Central

Wang, Pinger; Ying, Jun; Luo, Cheng; Jin, Xing; Zhang, Shanxing; Xu, Taotao; Zhang, Lei; Mi, Meng; Chen, Di; Tong, Peijian; Jin, Hongting

2017-01-01

Osthole is a bioactive coumarin derivative and has been reported to be able to enhance bone formation and improve fracture healing. However, the molecular mechanism of Osthole in bone fracture healing has not been fully defined. In this study we determined if Osthole enhances bone fracture healing through activation of BMP2 signaling in mice. We performed unilateral open transverse tibial fracture procedure in 10-week-old C57BL/6 mice which were treated with or without Osthole. Our previous studies demonstrated that chondrocyte BMP signaling is required for bone fracture healing, in this study we also performed tibial fracture procedure in Cre-negative and Col2-Cre;Bmp2flox/flox conditional knockout (KO) mice (Bmp2Col2Cre) to determine if Osthole enhances fracture healing in a BMP2-dependent manner. Fracture callus tissues were collected and analyzed by X-ray, micro-CT (μCT), histology, histomorphometry, immunohistochemistry (IHC), biomechanical testing and quantitative gene expression analysis. In addition, mouse chondrogenic ATDC5 cells were cultured with or without Osthole and the expression levels of chondrogenic marker genes were examined. The results demonstrated that Osthole promotes bone fracture healing in wild-type (WT) or Cre- control mice. In contrast, Osthole failed to promote bone fracture healing in Bmp2Col2Creconditional KO mice. In the mice receiving Osthole treatment, expression of cartilage marker genes was significantly increased. We conclude that Osthole could promote bone strength and enhance fracture healing by activation of BMP2 signaling. Osthole may be used as an alternative approach in the orthopaedic clinic for the treatment of fracture healing. PMID:28924381

Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation.

PubMed

Basic-Jukic, Nikolina; Gulin, Marijana; Hudolin, Tvrtko; Kastelan, Zeljko; Katalinic, Lea; Coric, Marijana; Veda, Marija Varnai; Ivkovic, Vanja; Kes, Petar; Jelakovic, Bojan

2016-01-01

Delayed graft function (DGF) is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2) is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF) and DGF. 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients) and DGF group (15 patients). BMP-2 expression in intima media (BMP2m) and endothelium (BMP2e) of epigastric artery was assessed by immunohistochemistry. Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001) (P<0.001 for no expression and P = 0.015 for 1st grade expression). Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477]) and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]). Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF. © 2016 The Author(s) Published by S. Karger AG, Basel.

Influence of rhBMP-2 on Guided Bone Regeneration for Placement and Functional Loading of Dental Implants: A Radiographic and Histologic Study in Dogs.

PubMed

Ben Amara, Heithem; Lee, Jung-Won; Kim, Jung-Ju; Kang, Yun-Mi; Kang, Eun-Jung; Koo, Ki-Tae

Evidence on the outcomes of functional loading placed in recombinant human bone morphogenetic protein 2 (rhBMP-2)/acellular collagen sponge (ACS)-induced bone is lacking. The aim of this study was to verify whether guided bone regeneration (GBR) with rhBMP-2/ACS enhances regeneration of missing bone and osseointegration of dental implants subject to functional loading. Two bilateral standardized large saddle-type defects (≈10 × 10 × 6 mm) were surgically created in each mandible of seven beagle dogs 2 months after tooth extraction. Defects were immediately reconstructed randomly using rhBMP-2 (O-BMP or InFuse) soaked in ACS, deproteinized bovine bone mineral (DBBM) granules, or ACS alone as surgical control and subsequently covered with collagen membrane. Screw-type sand-blasted, acid-etched dental implants were placed 3 months later into the reconstructed defects and into adjacent bone. Osseointegration was allowed to progress for 3 months before functional loading of 3 months until sacrifice. Significantly more bone fill was radiographically observed for GBR with rhBMP-2/ACS (O-BMP: 92.5%, InFuse: 79%) in comparison to the DBBM (52%) and ACS alone groups (56.6%). Osseointegration was achieved and maintained in all experimental defects challenged by prostheses-driven functional load. The bone density ranged from 37.49% in the ACS group to 64.9% in the rhBMP-2/ACS (InFuse) group with no significance. The highest mean percentage of BIC was found in rhBMP-2/ACS (InFuse: 52.98%) with no statistical difference. Crestal bone resorption was observed around implants placed in reconstructed areas without any significant difference. GBR with rhBMP-2/ACS provided the greatest bone fill among the three treatment procedures. GBR with rhBMP-2/ACS showed efficacy for placement, osseointegration, and functional loading of titanium implants in alveolar ridge defects.

Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

PubMed Central

Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

2010-01-01

BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

BMP2 and BMP7 play antagonistic roles in feather induction

PubMed Central

Michon, Frédéric; Forest, Loïc; Collomb, Elodie; Demongeot, Jacques; Dhouailly, Danielle

2008-01-01

Summary During embryonic development, feathers first appear as primordia consisting of an epidermal placode associated with a dermal condensation. In most previous studies, the BMPs have been proposed to function as inhibitors of the formation of cutaneous appendages. We showed that the function of BMPs is quite nuanced: BMP-2 and BMP-7, which are expressed in both skin components, act antagonistically and yet are both involved in the dermal condensations formation. BMP-7, the first to be expressed, is implicated in chemotaxis which leads to cell recruitment to the condensation, whereas BMP-2, which is expressed later, leads to an arrest of cell migration, likely via its modulation of EIIIA Fibronectin domain and α4-Integrin expression. We also propose a mathematical model, a reaction-diffusion system, based on cell proliferation, chemotaxis and the timing of BMP-2 and BMP-7 expression, which simulates the endogenous situation and reproduces the negative effects of excess BMP-2 or BMP-7 on feather patterning. PMID:18635609

Locally applied simvastatin improves fracture healing at late period in osteoporotic rat

NASA Astrophysics Data System (ADS)

Tian, Faming; Zhang, Liu; Kang, Yuchuan; Zhang, Junshan; Ao, Jiao; Yang, Fang

effect of simvastatin locally applied from a bioactive polymer coating of implants on osteoporotic fracture healing at late period. Methods:Femur fracture model was established on normal or osteotoporotic mature female SD rats, intramedullary stabilization was achieved with uncoated titanium Kirschnerwires in normal rats(group A),with polymer-only coated vs. polymer plus simvastatin coated titanium Kirschner wires in osteoporotic rats(group B and C, respectively).Femurs were harvested after 12 weeks, and underwent radiographic and histologic analysis, as well as immunohistochemical evaluation for BMP-2 expression. Results:Radiographic results demonstrated progressed callus in the simvastatin-treated groups compared to the uncoated group.The histologic analysis revealed a significantly processed callus with irregular-shaped newly formed bone trabeculae in simvastatin-treated group. Immunohistochemical evaluation showed markedly higher expression levels of B:MP-2 in simvastatin-treated group.Conclusions: The present study revealed a improved fracture healing under local application of simvastatin in osteoporotic rat,which might partially from upregulation of the B:MP-2 expression at fractured site.

Mesoporous bioactive glass nanolayer-functionalized 3D-printed scaffolds for accelerating osteogenesis and angiogenesis

NASA Astrophysics Data System (ADS)

Zhang, Yali; Xia, Lunguo; Zhai, Dong; Shi, Mengchao; Luo, Yongxiang; Feng, Chun; Fang, Bing; Yin, Jingbo; Chang, Jiang; Wu, Chengtie

2015-11-01

The hierarchical microstructure, surface and interface of biomaterials are important factors influencing their bioactivity. Porous bioceramic scaffolds have been widely used for bone tissue engineering by optimizing their chemical composition and large-pore structure. However, the surface and interface of struts in bioceramic scaffolds are often ignored. The aim of this study is to incorporate hierarchical pores and bioactive components into the bioceramic scaffolds by constructing nanopores and bioactive elements on the struts of scaffolds and further improve their bone-forming activity. Mesoporous bioactive glass (MBG) modified β-tricalcium phosphate (MBG-β-TCP) scaffolds with a hierarchical pore structure and a functional strut surface (~100 nm of MBG nanolayer) were successfully prepared via 3D printing and spin coating. The compressive strength and apatite-mineralization ability of MBG-β-TCP scaffolds were significantly enhanced as compared to β-TCP scaffolds without the MBG nanolayer. The attachment, viability, alkaline phosphatase (ALP) activity, osteogenic gene expression (Runx2, BMP2, OPN and Col I) and protein expression (OPN, Col I, VEGF, HIF-1α) of rabbit bone marrow stromal cells (rBMSCs) as well as the attachment, viability and angiogenic gene expression (VEGF and HIF-1α) of human umbilical vein endothelial cells (HUVECs) in MBG-β-TCP scaffolds were significantly upregulated compared with conventional bioactive glass (BG)-modified β-TCP (BG-β-TCP) and pure β-TCP scaffolds. Furthermore, MBG-β-TCP scaffolds significantly enhanced the formation of new bone in vivo as compared to BG-β-TCP and β-TCP scaffolds. The results suggest that application of the MBG nanolayer to modify 3D-printed bioceramic scaffolds offers a new strategy to construct hierarchically porous scaffolds with significantly improved physicochemical and biological properties, such as mechanical properties, osteogenesis, angiogenesis and protein expression for bone tissue engineering applications, in which the incorporation of nanostructures and bioactive components into the scaffold struts synergistically play a key role in the improved bone formation.

Bone Morphogenetic Protein 15 (BMP15) Acts as a BMP and Wnt Inhibitor during Early Embryogenesis*

PubMed Central

Di Pasquale, Elisa; Brivanlou, Ali H.

2009-01-01

Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor β (TGFβ) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of β-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure. PMID:19553676

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

PubMed

Lee, Heon-Woo; Chong, Diana C; Ola, Roxana; Dunworth, William P; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L; Eichmann, Anne; Jin, Suk-Won

2017-04-01

Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2 , and Alk3 in mouse retinal vessels. Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2 . Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2 / acvr1 or Alk3 / Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels. © 2017 The Authors.

Bmp signaling regulates a dose-dependent transcriptional program to control facial skeletal development.

PubMed

Bonilla-Claudio, Margarita; Wang, Jun; Bai, Yan; Klysik, Elzbieta; Selever, Jennifer; Martin, James F

2012-02-01

We performed an in depth analysis of Bmp4, a critical regulator of development, disease, and evolution, in cranial neural crest (CNC). Conditional Bmp4 overexpression, using a tetracycline-regulated Bmp4 gain-of-function allele, resulted in facial skeletal changes that were most dramatic after an E10.5 Bmp4 induction. Expression profiling uncovered a signature of Bmp4-induced genes (BIG) composed predominantly of transcriptional regulators that control self-renewal, osteoblast differentiation and negative Bmp autoregulation. The complimentary experiment, CNC inactivation of Bmp2, Bmp4 and Bmp7, resulted in complete or partial loss of multiple CNC-derived skeletal elements, revealing a crucial requirement for Bmp signaling in membranous bone and cartilage development. Importantly, the BIG signature was reduced in Bmp loss-of-function mutants, indicating Bmp-regulated target genes are modulated by Bmp dose. Chromatin immunoprecipitation (ChIP) revealed a subset of the BIG signature, including Satb2, Smad6, Hand1, Gadd45γ and Gata3, that was bound by Smad1/5 in the developing mandible, revealing direct Smad-mediated regulation. These data support the hypothesis that Bmp signaling regulates craniofacial skeletal development by balancing self-renewal and differentiation pathways in CNC progenitors.

Select polyphenolic fractions from dried plum enhance osteoblast activity through BMP-2 signaling.

PubMed

Graef, Jennifer L; Rendina-Ruedy, Elizabeth; Crockett, Erica K; Ouyang, Ping; King, Jarrod B; Cichewicz, Robert H; Lucas, Edralin A; Smith, Brenda J

2018-05-01

Dried plum supplementation has been shown to enhance bone formation while suppressing bone resorption. Evidence from previous studies has demonstrated that these responses can be attributed in part to the fruit's polyphenolic compounds. The purpose of this study was to identify the most bioactive polyphenolic fractions of dried plum with a focus on their osteogenic activity and to investigate their mechanisms of action under normal and inflammatory conditions. Utilizing chromatographic techniques, six fractions of polyphenolic compounds were prepared from a crude extract of dried plum. Initial screening assays revealed that two fractions (DP-FrA and DP-FrB) had the greatest osteogenic potential. Subsequent experiments using primary bone-marrow-derived osteoblast cultures demonstrated these two fractions enhanced extracellular alkaline phosphatase (ALP), an indicator of osteoblast activity, and mineralized nodule formation under normal conditions. Both fractions enhanced bone morphogenetic protein (BMP) signaling, as indicated by increased Bmp2 and Runx2 gene expression and protein levels of phosphorylated Smad1/5. DP-FrB was most effective at up-regulating Tak1 and Smad1, as well as protein levels of phospho-p38. Under inflammatory conditions, TNF-α suppressed ALP and tended to decrease nodule formation (P=.0674). This response coincided with suppressed gene expression of Bmp2 and the up-regulation of Smad6, an inhibitor of BMP signaling. DP-FrA and DP-FrB partially normalized these responses. Our results show that certain fractions of polyphenolic compounds in dried plum up-regulate osteoblast activity by enhancing BMP signaling, and when this pathway is inhibited by TNF-α, the osteogenic response is attenuated. Copyright © 2017 Elsevier Inc. All rights reserved.

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein Induced Retinal Angiogenesis

PubMed Central

Lee, Heon-Woo; Chong, Diana C.; Ola, Roxana; Dunworth, William P.; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M.; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L.; Eichmann, Anne; Jin, Suk-Won

2017-01-01

Objective Increasing evidence suggests that Bone Morphogenetic Protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early post-natal angiogenesis by analysis of inducible, endothelial specific deletion of the BMP receptor components Bmpr2, Alk1, Alk2 and Alk3 in mouse retinal vessels. Approach and Results Expression analysis of several BMP ligands showed that pro-angiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of BMP Type 2 receptor (Bmpr2). Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branchpoints behind the front, leading to attenuated radial expansion. To identify critical BMPR1s associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of three BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for pro-angiogenic BMP signaling in retinal vessels. Conclusions Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential pro-angiogenic cue for retinal vessels. PMID:28232325

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains*

PubMed Central

Kienast, Yvonne; Jucknischke, Ute; Scheiblich, Stefan; Thier, Martina; de Wouters, Mariana; Haas, Alexander; Lehmann, Christian; Brand, Verena; Bernicke, Dirk; Honold, Konrad; Lorenz, Stefan

2016-01-01

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants. PMID:26677222

Effect of dual delivery of antibiotics (vancomycin and cefazolin) and BMP-7 from chitosan microparticles on Staphylococcus epidermidis and pre-osteoblasts in vitro

PubMed Central

Mantripragada, Venkata P.; Jayasuriya, Ambalangodage C.

2016-01-01

The main aims of this manuscript are to: i) determine the effect of commonly used antibiotics to treat osteoarticular infections on osteoblast viability, ii) study the dual release of the growth factor (BMP-7) and antibiotics (vancomycin and cefazolin) from chitosan microparticles iii) demonstrate the bioactivity of the antibiotics released in vitro on Staphylococcus epidermidis. The novelty of this work is dual delivery of growth factor and antibiotic from the chitosan microparticles in a controlled manner without affecting their bioactivity. Cefazolin and vancomycin have different therapeutic concentrations for their action in vivo and therefore, two different concentrations of the drugs were used. Osteoblast cytotoxicity test concluded that cefazolin concentrations of 50 and 100 μg/ml were found to have positive influence on osteoblast proliferation. A significant increase in osteoblast proliferation was observed in the presence of cefazolin and BMP-7 in comparison with BMP-7 alone group; indicating cefazolin might play a role in osteoblast proliferation. On the other hand, vancomycin concentration of 1000 μg/ml was found to significantly reduce (p < 0.01) osteoblast proliferation in comparison with controls. The microbial study indicated that cefazolin at a minimum concentration of 21.5 μg/ml could inhibit ~85% growth of S. epidermidis, whereas vancomycin at a concentration of 30 μg/ml was found to inhibit ~80% bacterial growth. PMID:27287137

Delivery of bioactive lipids from composite microgel-microsphere injectable scaffolds enhances stem cell recruitment and skeletal repair.

PubMed

Das, Anusuya; Barker, Daniel A; Wang, Tiffany; Lau, Cheryl M; Lin, Yong; Botchwey, Edward A

2014-01-01

In this study, a microgel composed of chitosan and inorganic phosphates was used to deliver poly(lactic-co-glycolic acid) (PLAGA) microspheres loaded with sphingolipid growth factor FTY720 to critical size cranial defects in Sprague Dawley rats. We show that sustained release of FTY720 from injected microspheres used alone or in combination with recombinant human bone morphogenic protein-2 (rhBMP2) improves defect vascularization and bone formation in the presence and absence of rhBMP2 as evaluated by quantitative microCT and histological measurements. Moreover, sustained delivery of FTY720 from PLAGA and local targeting of sphingosine 1-phosphate (S1P) receptors reduces CD45+ inflammatory cell infiltration, promotes endogenous recruitment of CD29+CD90+ bone progenitor cells and enhances the efficacy of rhBMP2 from chitosan microgels. Companion in vitro studies suggest that selective activation of sphingosine receptor subtype-3 (S1P3) via FTY720 treatment induces smad-1 phosphorylation in bone-marrow stromal cells. Additionally, FTY720 enhances stromal cell-derived factor-1 (SDF-1) mediated chemotaxis of CD90+CD11B-CD45- bone progenitor cells in vitro after stimulation with rhBMP2. We believe that use of such small molecule delivery formulations to recruit endogenous bone progenitors may be an attractive alternative to exogenous cell-based therapy.

Delivery of Bioactive Lipids from Composite Microgel-Microsphere Injectable Scaffolds Enhances Stem Cell Recruitment and Skeletal Repair

PubMed Central

Das, Anusuya; Barker, Daniel A.; Wang, Tiffany; Lau, Cheryl M.; Lin, Yong; Botchwey, Edward A.

2014-01-01

In this study, a microgel composed of chitosan and inorganic phosphates was used to deliver poly(lactic-co-glycolic acid) (PLAGA) microspheres loaded with sphingolipid growth factor FTY720 to critical size cranial defects in Sprague Dawley rats. We show that sustained release of FTY720 from injected microspheres used alone or in combination with recombinant human bone morphogenic protein-2 (rhBMP2) improves defect vascularization and bone formation in the presence and absence of rhBMP2 as evaluated by quantitative microCT and histological measurements. Moreover, sustained delivery of FTY720 from PLAGA and local targeting of sphingosine 1-phosphate (S1P) receptors reduces CD45+ inflammatory cell infiltration, promotes endogenous recruitment of CD29+CD90+ bone progenitor cells and enhances the efficacy of rhBMP2 from chitosan microgels. Companion in vitro studies suggest that selective activation of sphingosine receptor subtype-3 (S1P3) via FTY720 treatment induces smad-1 phosphorylation in bone-marrow stromal cells. Additionally, FTY720 enhances stromal cell-derived factor-1 (SDF-1) mediated chemotaxis of CD90+CD11B-CD45- bone progenitor cells in vitro after stimulation with rhBMP2. We believe that use of such small molecule delivery formulations to recruit endogenous bone progenitors may be an attractive alternative to exogenous cell-based therapy. PMID:25077607

Rebamipide delivered by brushite cement enhances osteoblast and macrophage proliferation.

PubMed

Pujari-Palmer, Michael; Pujari-Palmer, Shiuli; Engqvist, Håkan; Karlsson Ott, Marjam

2015-01-01

Many of the bioactive agents capable of stimulating osseous regeneration, such as bone morphogenetic protein-2 (BMP-2) or prostaglandin E2 (PGE2), are limited by rapid degradation, a short bioactive half-life at the target site in vivo, or are prohibitively expensive to obtain in large quantities. Rebamipide, an amino acid modified hydroxylquinoline, can alter the expression of key mediators of bone anabolism, cyclo-oxygenase 2 (COX-2), BMP-2 and vascular endothelial growth factor (VEGF), in diverse cell types such as mucosal and endothelial cells or chondrocytes. The present study investigates whether Rebamipide enhances proliferation and differentiation of osteoblasts when delivered from brushite cement. The reactive oxygen species (ROS) quenching ability of Rebampide was tested in macrophages as a measure of bioactivity following drug release incubation times, up to 14 days. Rebamipide release from brushite occurs via non-fickian diffusion, with a rapid linear release of 9.70% ± 0.37% of drug per day for the first 5 days, and an average of 0.5%-1% per day thereafter for 30 days. Rebamipide slows the initial and final cement setting time by up to 3 and 1 minute, respectively, but does not significantly reduce the mechanical strength below 4% (weight percentage). Pre-osteoblast proliferation increases by 24% upon exposure to 0.4 uM Rebamipide, and by up to 73% when Rebamipide is delivered via brushite cement. Low doses of Rebamipide do not adversely affect peak alkaline phosphatase activity in differentiating pre-osteoblasts. Rebamipide weakly stimulates proliferation in macrophages at low concentrations (118 ± 7.4% at 1 uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts.

Rebamipide Delivered by Brushite Cement Enhances Osteoblast and Macrophage Proliferation

PubMed Central

Pujari-Palmer, Michael; Pujari-Palmer, Shiuli; Engqvist, Håkan; Karlsson Ott, Marjam

2015-01-01

Many of the bioactive agents capable of stimulating osseous regeneration, such as bone morphogenetic protein-2 (BMP-2) or prostaglandin E2 (PGE2), are limited by rapid degradation, a short bioactive half-life at the target site in vivo, or are prohibitively expensive to obtain in large quantities. Rebamipide, an amino acid modified hydroxylquinoline, can alter the expression of key mediators of bone anabolism, cyclo-oxygenase 2 (COX-2), BMP-2 and vascular endothelial growth factor (VEGF), in diverse cell types such as mucosal and endothelial cells or chondrocytes. The present study investigates whether Rebamipide enhances proliferation and differentiation of osteoblasts when delivered from brushite cement. The reactive oxygen species (ROS) quenching ability of Rebampide was tested in macrophages as a measure of bioactivity following drug release incubation times, up to 14 days. Rebamipide release from brushite occurrs via non-fickian diffusion, with a rapid linear release of 9.70% ±0.37% of drug per day for the first 5 days, and an average of 0.5%-1% per day thereafter for 30 days. Rebamipide slows the initial and final cement setting time by up to 3 and 1 minute, respectively, but does not significantly reduce the mechanical strength below 4% (weight percentage). Pre-osteoblast proliferation increases by 24% upon exposure to 0.4uM Rebamipide, and by up to 73% when Rebamipide is delivered via brushite cement. Low doses of Rebamipide do not adversely affect peak alkaline phosphatase activity in differentiating pre-osteoblasts. Rebamipide weakly stimulates proliferation in macrophages at low concentrations (118 ±7.4% at 1uM), and quenches ROS by 40-60%. This is the first investigation of Rebamipide in osteoblasts. PMID:26023912

Influence of Poly(L-Lactic Acid) Nanofibers and BMP-2–Containing Poly(L-Lactic Acid) Nanofibers on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Schofer, Markus D.; Fuchs-Winkelmann, Susanne; Gräbedünkel, Christian; Wack, Christina; Dersch, Roland; Rudisile, Markus; Wendorff, Joachim H.; Greiner, Andreas; Paletta, Jürgen R. J.; Boudriot, Ulrich

2008-01-01

The aim of this study was to characterize synthetic poly-(L-lactic acid) (PLLA) nanofibers concerning their ability to promote growth and osteogenic differentiation of stem cells in vitro, as well as to test their suitability as a carrier system for growth factors. Fiber matrices composed of PLLA or BMP-2–incorporated PLLA were seeded with human mesenchymal stem cells and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of alkaline phosphatase (ALP), osteocalcin (OC), and collagen I (COL-I). Furthermore, COL-I and OC deposition, as well as cell densities and proliferation, were analyzed using fluorescence microscopy. Although the presence of nanofibers diminished the dexamethasone-induced proliferation, there were no differences in cell densities or deposition of either COL-I or OC after 22 days of culture. The gene expression of ALP, OC, and COL-I decreased in the initial phase of cell cultivation on PLLA nanofibers as compared to cover slip control, but normalized during the course of cultivation. The initial down-regulation was not observed when BMP-2 was directly incorporated into PLLA nanofibers by electrospinning, indicating that growth factors like BMP-2 might survive the spinning process in a bioactive form. PMID:19112539

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry.

PubMed

Siverino, Claudia; Tabisz, Barbara; Lühmann, Tessa; Meinel, Lorenz; Müller, Thomas; Walles, Heike; Nickel, Joachim

2018-03-29

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Interplay between self-assembled structure of bone morphogenetic protein-2 (BMP-2) and osteoblast functions in three-dimensional titanium alloy scaffolds: Stimulation of osteogenic activity.

PubMed

Nune, K C; Kumar, A; Murr, L E; Misra, R D K

2016-02-01

Three-dimensional cellular scaffolds are receiving significant attention in bone tissue engineering to treat segmental bone defects. However, there are indications of lack of significant osteoinductive ability of three-dimensional cellular scaffolds. In this regard, the objective of the study is to elucidate the interplay between bone morphogenetic protein (BMP-2) and osteoblast functions on 3D mesh structures with different porosities and pore size that were fabricated by electron beam melting. Self-assembled dendritic microstructure with interconnected cellular-type morphology of BMP-2 on 3D scaffolds stimulated osteoblast functions including adhesion, proliferation, and mineralization, with prominent effect on 2-mm mesh. Furthermore, immunofluorescence studies demonstrated higher density and viability of osteoblasts on lower porosity mesh structure (2 mm) as compared to 3- and 4-mm mesh structures. Enhanced filopodia cellular extensions with extensive cell spreading was observed on BMP-2 treated mesh structures, a behavior that is attributed to the unique self-assembled structure of BMP-2 that effectively communicates with the cells. The study underscores the potential of BMP-2 in imparting osteoinductive capability to the 3D printed scaffolds. © 2015 Wiley Periodicals, Inc.

Derivation and application of a mathematical model for long bone growth.

PubMed

Seetharam, Suneil; Bhatia, Sujata K

2012-01-01

The objective of this work was to develop a mathematical model of long bone growth and to gain insights regarding growth disorders. A cell balance (mass balance of moving cells) assessment was performed on the three regions of the growth plate, to determine the variables (including number of proliferating cells, and division rate of proliferating cells) that influence tibia growth rate. Once this relationship was established, clinical data were used to understand how tibia growth rate and number of proliferating cells change with time. These equations were then inserted into the model to determine how cell division rate changes with time. The model was utilized to determine the influence of growth time, and to measure changes in vitamin C deficiency, Indian hedgehog (IHH) expression, and bone morphogenetic protein-2 (BMP-2) implants on tibia length. According to the model, a 10-month discrepancy in growth time between the two tibias is required to produce clinically significant leg asymmetry. In addition, vitamin C deficiency, IHH overexpression, and BMP-2 implants can all affect tibia length. These bioactive molecules have the greatest effect on tibia growth rate when these perturbations occur early in life for extended periods of time. The results are significant for modeling and predicting the effects of perturbations, including bioactive implants, on long bone growth.

Bioactive borate glass promotes the repair of radius segmental bone defects by enhancing the osteogenic differentiation of BMSCs.

PubMed

Zhang, Jieyuan; Guan, Junjie; Zhang, Changqing; Wang, Hui; Huang, Wenhai; Guo, Shangchun; Niu, Xin; Xie, Zongping; Wang, Yang

2015-11-20

Bioactive borate glass (BG) has emerged as a promising alternative for bone regeneration due to its high osteoinductivity, osteoconductivity, compressive strength, and biocompatibility. However, the role of BG in large segmental bone repair is unclear and little is known about the underlying mechanism of BG's osteoinductivity. In this study, we demonstrated that BG possessed pro-osteogenic effects in an experimental model of critical-sized radius defects. Transplanting BG to radius defects resulted in better repair of bone defects as compared to widely used β-TCP. Histological and morphological analysis indicated that BG significantly enhanced new bone formation. Furthermore, the degradation rate of the BG was faster than that of β-TCP, which matched the higher bone regeneration rate. In addition, ions from BG enhanced cell viability, ALP activity, and osteogenic-related genes expression. Mechanistically, the critical genes Smad1/5 and Dlx5 in the BMP pathway and p-Smad1/5 proteins were significantly elevated after BG transplantation, and these effects could be blocked by the BMP/Smad specific inhibitor. Taken together, our findings suggest that BG could repair large segmental bone defects through activating the BMP/Smad pathway and osteogenic differentiation in BMSCs.

Inactivation of bone morphogenetic protein 2 may predict clinical outcome and poor overall survival for renal cell carcinoma through epigenetic pathways

PubMed Central

Mitsui, Yozo; Hirata, Hiroshi; Arichi, Naoko; Hiraki, Miho; Yasumoto, Hiroaki; Chang, Inik; Fukuhara, Shinichiro; Yamamura, Soichiro; Shahryari, Varahram; Deng, Guoren; Saini, Sharanjot; Majid, Shahana; Dahiya, Rajvir; Tanaka, Yuichiro; Shiina, Hiroaki

2015-01-01

We investigated whether impaired regulation of bone morphogenetic protein-2 (BMP-2) via epigenetic pathways is associated with renal cell carcinoma (RCC) pathogenesis. Expression and CpG methylation of the BMP-2 gene were analyzed using RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. A significant association of BMP-2 mRNA expression was also found with advanced tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression induced p21WAF1/CIP1 and p27KIP1 expression, and cellular apoptosis in RCC cells. BMP-2 mRNA expression was significantly enhanced in RCC cells by 5-aza-2′-deoxycitidine treatment. The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC. PMID:25797254

Enhanced angiogenesis and osteogenesis in critical bone defects by the controlled release of BMP-2 and VEGF: implantation of electron beam melting-fabricated porous Ti6Al4V scaffolds incorporating growth factor-doped fibrin glue.

PubMed

Lv, Jia; Xiu, Peng; Tan, Jie; Jia, Zhaojun; Cai, Hong; Liu, Zhongjun

2015-06-24

Electron beam melting (EBM)-fabricated porous titanium implants possessing low elastic moduli and tailored structures are promising biomaterials for orthopedic applications. However, the bio-inert nature of porous titanium makes reinforcement with growth factors (GFs) a promising method to enhance implant in vivo performance. Bone-morphogenic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) are key factors of angiogenesis and osteogenesis. Therefore, the present study is aimed at evaluating EBM-fabricated porous titanium implants incorporating GF-doped fibrin glue (FG) as composite scaffolds providing GFs for improvement of angiogenesis and osteogenesis in rabbit femoral condyle defects. BMP-2 and VEGF were added into the constituent compounds of FG, and then this GF-doped FG was subsequently injected into the porous scaffolds. In five groups of implants, angiogenesis and osteogenesis were evaluated at 4 weeks post-implantation using Microfil perfusion and histological analysis: eTi (empty scaffolds), cTi (containing undoped FG), BMP/cTi (containing 50 μg rhBMP-2), VEGF/cTi (containing 0.5 μg VEGF) and Dual/cTi (containing 50 μg rhBMP-2 and 0.5 μg VEGF). The results demonstrate that these composite implants are biocompatible and provide the desired gradual release of the bioactive growth factors. Incorporation of GF delivery, whether a single factor or dual factors, significantly enhanced both angiogenesis and osteogenesis inside the porous scaffolds. However, the synergistic effect of the dual factors combination was observable on angiogenesis but absent on osteogenesis. In conclusion, fibrin glue is a biocompatible material that could be employed as a delivery vehicle in EBM-fabricated porous titanium for controlled release of BMP-2 and VEGF. Application of this method for loading a porous titanium scaffold to incorporate growth factors is a convenient and promising strategy for improving osteogenesis of critical-sized bone defects.

Effect of dual delivery of antibiotics (vancomycin and cefazolin) and BMP-7 from chitosan microparticles on Staphylococcus epidermidis and pre-osteoblasts in vitro.

PubMed

Mantripragada, Venkata P; Jayasuriya, Ambalangodage C

2016-10-01

The main aims of this manuscript are to: i) determine the effect of commonly used antibiotics to treat osteoarticular infections on osteoblast viability, ii) study the dual release of the growth factor (BMP-7) and antibiotics (vancomycin and cefazolin) from chitosan microparticles iii) demonstrate the bioactivity of the antibiotics released in vitro on Staphylococcus epidermidis. The novelty of this work is dual delivery of growth factor and antibiotic from the chitosan microparticles in a controlled manner without affecting their bioactivity. Cefazolin and vancomycin have different therapeutic concentrations for their action in vivo and therefore, two different concentrations of the drugs were used. Osteoblast cytotoxicity test concluded that cefazolin concentrations of 50 and 100μg/ml were found to have positive influence on osteoblast proliferation. A significant increase in osteoblast proliferation was observed in the presence of cefazolin and BMP-7 in comparison with BMP-7 alone group; indicating cefazolin might play a role in osteoblast proliferation. On the other hand, vancomycin concentration of 1000μg/ml was found to significantly reduce (p<0.01) osteoblast proliferation in comparison with controls. The microbial study indicated that cefazolin at a minimum concentration of 21.5μg/ml could inhibit ~85% growth of S. epidermidis, whereas vancomycin at a concentration of 30μg/ml was found to inhibit ~80% bacterial growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Gene Therapy of Bone Morphogenetic Protein for Periodontal Tissue Engineering

PubMed Central

Jin, Q-M.; Anusaksathien, O.; Webb, S.A.; Rutherford, R.B.; Giannobile, W.V.

2009-01-01

Background The reconstruction of lost periodontal support including bone, ligament, and cementum is a major goal of therapy. Bone morphogenetic proteins (BMPs) have shown much potential in the regeneration of the periodontium. Limitations of BMP administration to periodontal lesions include need for high-dose bolus delivery, BMP transient biological activity, and low bioavailability of factors at the wound site. Gene transfer offers promise as an alternative treatment strategy to deliver BMPs to periodontal tissues. Methods This study utilized ex vivo BMP-7 gene transfer to stimulate tissue engineering of alveolar bone wounds. Syngeneic dermal fibroblasts (SDFs) were transduced ex vivo with adenoviruses encoding either green fluorescent protein (Ad-GFP or control virus), BMP-7 (Ad-BMP-7), or an antagonist of BMP bioactivity, noggin (Ad-noggin). Transduced cells were seeded onto gelatin carriers and then transplanted to large mandibular alveolar bone defects in a rat wound repair model. Results Ad-noggin treatment tended to inhibit osteogenesis as compared to the control-treated and Ad-BMP-7-treated specimens. The osseous lesions treated by Ad-BMP-7 gene delivery demonstrated rapid chrondrogenesis, with subsequent osteogenesis, cementogenesis and predictable bridging of the periodontal bone defects. Conclusion These results demonstrate the first successful evidence of periodontal tissue engineering using ex vivo gene transfer of BMPs and offers a new approach for repairing periodontal defects. PMID:12666709

Bone Morphogenetic Proteins and myostatin pathways: key mediator of human sarcopenia.

PubMed

Scimeca, Manuel; Piccirilli, Eleonora; Mastrangeli, Francesca; Rao, Cecilia; Feola, Maurizio; Orlandi, Augusto; Gasbarra, Elena; Bonanno, Elena; Tarantino, Umberto

2017-02-15

Sarcopenia, osteoporosis and osteoarthritis are the most frequent musculoskeletal disorders affecting older people. The main aim of this study was to test the hypothesis that the balance between BMPs and myostatin pathways regulates the age-related muscle degeneration in OP and OA patients. To this end, we investigated the relationship among the expression of BMP-2/4-7, myostatin and phosphorylated Smads1-5-8 and the muscle quality, evaluated in term of fibers atrophy and satellite cells activity. In this retrospective study, we collected 123 biopsies of vastus lateralis: 48 biopsies from patients who underwent hip arthroplasty for subcapital fractures of the femur (OP), 55 biopsies from patients who underwent hip arthroplasty for osteoarthritis (OA) and 20 biopsies from patients who underwent hip arthroplasty for high-energy hip fractures (CTRL). Muscle biopsies were fixed in 4% paraformaldehyde and paraffin embedded. Serial sections were used for morphometrical and immunohistochemical analysis (BMP/2/4-7, myostatin, Smads1-5-8, Pax7 and myogenin). In addition, 1 mm 3 of muscle tissue of each patient was embedded in epon for ultrastructural study. Morphometric data indicated an increase of the number of atrophic fibers in OP patients compare to OA. In line with these data, we found an high regenerative potential in muscle tissues of OA patients due to the significant amount of both Pax7 and myogenin positive satellite cells detected in OA group. In addition, our data showed the decrease of BMP2/4 and -7 expression in OP patients compared to both OA group and CTRL. Conversely, OP patients were characterized by high levels of myostatin expression. A different expression profile was also found for phosphorylated Smad1-5-8 between OP and OA patients. In particular, OP patients showed a low number of positive phosphorylated Smad1-5-8 nuclei. The identification of molecular pathways involved in the pathogenesis of sarcopenia open new prospective for the development of drugs able to prevent/treat the muscle impairment that occur in elderly. Results here reported, highlighting the role of BMPs and myostatin pathways in physio-pathogenesis of human sarcopenia, allow us to propose human recombinant BMP-2/7 and anti-myostatin antibodies as a possible therapeutic option for the sarcopenia.

A conserved post-transcriptional BMP2 switch in lung cells.

PubMed

Jiang, Shan; Fritz, David T; Rogers, Melissa B

2010-05-15

An ultra-conserved sequence in the bone morphogenetic protein 2 (BMP2) 3' untranslated region (UTR) markedly represses BMP2 expression in non-transformed lung cells. In contrast, the ultra-conserved sequence stimulates BMP2 expression in transformed lung cells. The ultra-conserved sequence functions as a post-transcriptional cis-regulatory switch. A common single-nucleotide polymorphism (SNP, rs15705, +A1123C), which has been shown to influence human morphology, disrupts a conserved element within the ultra-conserved sequence and altered reporter gene activity in non-transformed lung cells. This polymorphism changed the affinity of the BMP2 RNA for several proteins including nucleolin, which has an increased affinity for the C allele. Elevated BMP2 synthesis is associated with increased malignancy in mouse models of lung cancer and poor lung cancer patient prognosis. Understanding the cis- and trans-regulatory factors that control BMP2 synthesis is relevant to the initiation or progression of pathologies associated with abnormal BMP2 levels. (c) 2010 Wiley-Liss, Inc.

The effect of recombinant bone morphogenetic protein-2 in revision tibiotalocalcaneal arthrodesis: utilization of the Retrograde Arthrodesis Intramedullary Nail database.

PubMed

DeVries, J George; Nguyen, Minh; Berlet, Gregory C; Hyer, Christopher F

2012-01-01

The use of bone morphogenetic protein-2 (BMP-2) has been recommended for high-risk fusions and nonunion. Patients undergoing revisional tibiotalocalcaneal (TTC) arthrodesis via a retrograde arthrodesis nail to evaluate the influence of BMP-2 on rate of fusion in this high-risk population are presented. A retrospective chart and radiographic review were performed on 23 patients with failed prior fusion attempts at the ankle treated with retrograde intramedullary nailing. Sixteen patients were treated without BMP-2 (None group) and 7 were treated with BMP-2 (BMP group). The primary and secondary end-points were successful fusion, and time to fusion, respectively, with a variety of variables evaluated for influence. Other than the use of BMP-2, there were no statistical differences in the patient population. Overall, 11 of 16 ankles (68.8%) in the None group and 5 of 7 ankles (71.4%) in the BMP group resulted in a stable, functional limb. Rate of complication was similar between the 2 groups (p > .05). Time to radiographic ankle union was 115.2 and 184.0 days in the None and BMP groups (p > .05). The effect of BMP-2 on revisional TTC fusions with retrograde nails is reported here. The overall result as a stable, functional limb was 69.6%, which suggests that revision surgery in this high-risk population is a reasonable consideration. Even though this study was unable to demonstrate statistically significant differences, biologic augmentation with BMP-2 did not increase the complication rate and showed a slightly enhanced salvage rate for revision TTC fusions with an intramedullary nail. Copyright © 2012 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish.

PubMed

Goutel, C; Kishimoto, Y; Schulte-Merker, S; Rosa, F

2000-12-01

In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

An Autonomous BMP2 Regulatory Element in Mesenchymal Cells

PubMed Central

Kruithof, Boudewijn P.T.; Fritz, David T.; Liu, Yijun; Garsetti, Diane E.; Frank, David B.; Pregizer, Steven K.; Gaussin, Vinciane; Mortlock, Douglas P.; Rogers, Melissa B.

2014-01-01

BMP2 is a morphogen that controls mesenchymal cell differentiation and behavior. For example, BMP2 concentration controls the differentiation of mesenchymal precursors into myocytes, adipocytes, chondrocytes, and osteoblasts. Sequences within the 3′untranslated region (UTR) of the Bmp2 mRNA mediate a post-transcriptional block of protein synthesis. Interaction of cell and developmental stage-specific trans-regulatory factors with the 3′UTR is a nimble and versatile mechanism for modulating this potent morphogen in different cell types. We show here, that an ultra-conserved sequence in the 3′UTR functions independently of promoter, coding region, and 3′UTR context in primary and immortalized tissue culture cells and in transgenic mice. Our findings indicate that the ultra-conserved sequence is an autonomously functioning post-transcriptional element that may be used to modulate the level of BMP2 and other proteins while retaining tissue specific regulatory elements. PMID:21268088

Ectopic application of recombinant BMP-2 and BMP-4 can change patterning of developing chick facial primordia.

PubMed

Barlow, A J; Francis-West, P H

1997-01-01

The facial primordia initially consist of buds of undifferentiated mesenchyme, which give rise to a variety of tissues including cartilage, muscle and nerve. These must be arranged in a precise spatial order for correct function. The signals that control facial outgrowth and patterning are largely unknown. The bone morphogenetic proteins Bmp-2 and Bmp-4 are expressed in discrete regions at the distal tips of the early facial primordia suggesting possible roles for BMP-2 and BMP-4 during chick facial development. We show that expression of Bmp-4 and Bmp-2 is correlated with the expression of Msx-1 and Msx-2 and that ectopic application of BMP-2 and BMP-4 can activate Msx-1 and Msx-2 gene expression in the developing facial primordia. We correlate this activation of gene expression with changes in skeletal development. For example, activation of Msx-1 gene expression across the distal tip of the mandibular primordium is associated with an extension of Fgf-4 expression in the epithelium and bifurcation of Meckel's cartilage. In the maxillary primordium, extension of the normal domain of Msx-1 gene expression is correlated with extended epithelial expression of shh and bifurcation of the palatine bone. We also show that application of BMP-2 can increase cell proliferation of the mandibular primordia. Our data suggest that BMP-2 and BMP-4 are part of a signalling cascade that controls outgrowth and patterning of the facial primordia.

BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis.

PubMed

Gupta, Sunny; Zhu, Hao; Zon, Leonard I; Evans, Todd

2006-06-01

The bone morphogenetic protein (BMP) signaling pathway is essential during gastrulation for the generation of ventral mesoderm, which makes it a challenge to define functions for this pathway at later stages of development. We have established an approach to disrupt BMP signaling specifically in lateral mesoderm during somitogenesis, by targeting a dominant-negative BMP receptor to Lmo2+ cells in developing zebrafish embryos. This results in expansion of hematopoietic and endothelial cells, while restricting the expression domain of the pronephric marker pax2.1. Expression of a constitutively active receptor and transplantation experiments were used to confirm that BMP signaling in lateral mesoderm restricts subsequent hemato-vascular development. The results show that the BMP signaling pathway continues to function after cells are committed to a lateral mesoderm fate, and influences subsequent lineage decisions by restricting hemato-vascular fate in favor of pronephric development.

Human treated dentin matrices combined with Zn-doped, Mg-based bioceramic scaffolds and human dental pulp stem cells towards targeted dentin regeneration.

PubMed

Bakopoulou, Athina; Papachristou, Eleni; Bousnaki, Maria; Hadjichristou, Christina; Kontonasaki, Eleana; Theocharidou, Anna; Papadopoulou, Lambrini; Kantiranis, Nikolaos; Zachariadis, George; Leyhausen, Gabriele; Geurtsen, Werner; Koidis, Petros

2016-08-01

This study aimed to investigate the potential of Mg-based bioceramic scaffolds combined with human treated-dentin matrices (hTDMs) and dentinogenesis-related morphogens to promote odontogenic differentiation and dentin-like tissue formation by Dental Pulp Stem Cells-DPSCs. DPSC cultures were established and characterized by flow cytometry. Experimental cavities were prepared inside crowns of extracted teeth and demineralized by EDTA (hTDMs). Zn-doped, Mg-based bioceramic scaffolds, synthesized by the sol-gel technique, were hosted inside the hTDMs. DPSCs were spotted inside the hTDMs/scaffold constructs with/without additional exposure to DMP-1 or BMP-2 (100ng/ml, 24h). Scanning Electron Microscopy-SEM, live/dead fluorescence staining and MTT assay were used to evaluate cell attachment and viability; Real time PCR for expression of osteo/odontogenic markers; Inductively Coupled Plasma-Atomic Emission Spectrometry-ICP/AES for scaffold elemental release analysis; ELISA for hTDM growth factor release analysis; SEM and X-ray Diffraction-XRD for structural/chemical characterization of the regenerated tissues. Scaffolds constantly released low concentrations of Mg(2+), Ca(2+), Zn(2+) and Si(4+), while hTDMs growth factors, like DMP-1, BMP-2 and TGFβ-1. hTDMs/scaffold constructs supported DPSC viability, inducing their rapid odontogenic shift, indicated by upregulation of DSPP, BMP-2, osteocalcin and osterix expression. Newly-formed Ca-P tissue overspread the scaffolds partially transforming into bioapatite. Exposure to DMP-1 or BMP-2 pronouncedly enhanced odontogenic differentiation phenomena. This is the first study to validate that combining the bioactivity and ion releasing properties of bioceramic materials with growth factor release by treated natural dentin further supported by exogenous addition of key dentinogenesis-related morphogens (DMP-1, BMP-2) can be a promising strategy for targeted dentin regeneration. Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Functionalization of PCL-3D Electrospun Nanofibrous Scaffolds for Improved BMP2-Induced Bone Formation.

PubMed

Miszuk, Jacob M; Xu, Tao; Yao, Qingqing; Fang, Fang; Childs, Josh D; Hong, Zhongkui; Tao, Jianning; Fong, Hao; Sun, Hongli

2018-03-01

Bone morphogenic protein 2 (BMP2) is a key growth factor for bone regeneration, possessing FDA approval for orthopedic applications. BMP2 is often required in supratherapeutic doses clinically, yielding adverse side effects and substantial treatment costs. Considering the crucial role of materials for BMPs delivery and cell osteogenic differentiation, we devote to engineering an innovative bone-matrix mimicking niche to improve low dose of BMP2-induced bone formation. Our previous work describes a novel technique, named thermally induced nanofiber self-agglomeration (TISA), for generating 3D electrospun nanofibrous (NF) polycaprolactone (PCL) scaffolds. TISA process could readily blend PCL with PLA, leading to increased osteogenic capabilities in vitro , however, these bio-inert synthetic polymers produced limited BMP2-induced bone formation in vivo. We therefore hypothesize that functionalization of NF 3D PCL scaffolds with bone-like hydroxyapatite (HA) and BMP2 signaling activator phenamil will provide a favorable osteogenic niche for bone formation at low doses of BMP2. Compared to PCL-3D scaffolds, PCL/HA-3D scaffolds demonstrated synergistically enhanced osteogenic differentiation capabilities of C2C12 cells with phenamil. Importantly, in vivo studies showed this synergism was able to generate significantly increased new bone in an ectopic mouse model, suggesting PCL/HA-3D scaffolds act as a favorable synthetic extracellular matrix for bone regeneration.

Metastatic function of BMP-2 in gastric cancer cells: The role of PI3K/AKT, MAPK, the NF-{kappa}B pathway, and MMP-9 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Myoung Hee; Oh, Sang Cheul; Lee, Hyun Joo

2011-07-15

Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-{kappa}B, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of I{kappa}B{alpha} and the nuclear translocation/activation of NF-{kappa}B.more » Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-{kappa}B. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-{kappa}B inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-{kappa}B and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.« less

Functionalization of deproteinized bovine bone with a coating-incorporated depot of BMP-2 renders the material efficiently osteoinductive and suppresses foreign-body reactivity.

PubMed

Wu, Gang; Hunziker, Ernst B; Zheng, Yuanna; Wismeijer, Daniel; Liu, Yuelian

2011-12-01

The repair of critical-sized bony defects remains a challenge in the fields of implantology, maxillofacial surgery and orthopaedics. As an alternative bone-defect filler to autologous bone grafts, deproteinized bovine bone (DBB) is highly osteoconductive and clinically now widely used. However, this product suffers from the disadvantage of not being intrinsically osteoinductive. In the present study, this property was conferred by coating DBB with a layer of calcium phosphate into which bone morphogenetic protein 2 (BMP-2) was incorporated. Granules of DBB bearing a coating-incorporated depot of BMP-2--together with the appropriate controls (DBB bearing a coating but no BMP-2; uncoated DBB bearing adsorbed BMP-2; uncoated DBB bearing no BMP-2)--were implanted subcutaneously in rats. Five weeks later, the implants were withdrawn for a histomorphometric analysis of the volume densities of (i) bone, (ii) bone marrow, (iii) foreign-body giant cells and (iv) fibrous capsular tissue. Parameters (i) and (ii) were highest, whilst parameters (iii) and (iv) were lowest in association with DBB bearing a coating-incorporated depot of BMP-2. Hence, this mode of functionalization not only confers DBB with the property of osteoinductivity but also improves its biocompatibility--thus dually enhancing its clinical potential in the repair of bony defects. Copyright © 2011 Elsevier Inc. All rights reserved.

Targeted Knockdown of Bone Morphogenetic Protein Signaling within Neural Progenitors Protects the Brain and Improves Motor Function following Postnatal Hypoxia-Ischemia

PubMed Central

Dettman, Robert W.; Birch, Derin; Fernando, Augusta; Kessler, John A.; Dizon, Maria L.V.

2018-01-01

Hypoxic-ischemic injury (HI) to the neonatal human brain results in myelin loss that, in some children, can manifest as cerebral palsy. Previously, we had found that neuronal overexpression of the bone morphogenic protein (BMP) inhibitor noggin during development increased oligodendroglia and improved motor function in an experimental model of HI utilizing unilateral common carotid artery ligation followed by hypoxia. As BMPs are known to negatively regulate oligodendroglial fate specification of neural stem cells and alter differentiation of committed oligodendroglia, BMP signaling is likely an important mechanism leading to myelin loss. Here, we showed that BMP signaling is upregulated within oligodendroglia of the neonatal brain. We tested the hypothesis that inhibition of BMP signaling specifically within neural progenitor cells (NPCs) is sufficient to protect oligodendroglia. We conditionally deleted the BMP receptor 2 subtype (BMPR2) in NG2-expressing cells after HI. We found that BMPR2 deletion globally protects the brain as assessed by MRI and protects motor function as assessed by digital gait analysis, and that conditional deletion of BMPR2 maintains oligodendrocyte marker expression by immunofluorescence and Western blot and prevents loss of oligodendroglia. Finally, BMPR2 deletion after HI results in an increase in noncompacted myelin. Thus, our data indicate that inhibition of BMP signaling specifically in NPCs may be a tractable strategy to protect the newborn brain from HI. PMID:29324456

Deproteinized bovine bone functionalized with the slow delivery of BMP-2 for the repair of critical-sized bone defects in sheep.

PubMed

Liu, Tie; Wu, Gang; Wismeijer, Daniel; Gu, Zhiyuan; Liu, Yuelian

2013-09-01

As an alternative to an autologous bone graft, deproteinized bovine bone (DBB) is widely used in the clinical dentistry. Although DBB provides an osteoconductive scaffold, it is not capable of enhancing bone regeneration because it is not osteoinductive. In order to render DBB osteoinductive, bone morphogenetic protein 2 (BMP-2) has previously been incorporated into a three dimensional reservoir (a biomimetic calcium phosphate coating) on DBB, which effectively promoted the osteogenic response by the slow delivery of BMP-2. The aim of this study was to investigate the therapeutic effectiveness of such coating on the DBB granules in repairing a large cylindrical bone defect (8 mm diameter, 13 mm depth) in sheep. Eight groups were randomly assigned to the bone defects: (i) no graft material; (ii) autologous bone; (iii) DBB only; (iv) DBB mixed with autologous bone; (v) DBB bearing adsorbed BMP-2; (vi) DBB bearing a coating but no BMP-2; (vii) DBB bearing a coating with adsorbed BMP-2; and (viii) DBB bearing a coating-incorporated depot of BMP-2. 4 and 8 weeks after implantation, samples were withdrawn for a histological and a histomorphometric analysis. Histological results confirmed the excellent biocompatibility and osteoconductivity of all the grafts tested. At 4 weeks, DBB mixed with autologous bone or functionalized with coating-incorporated BMP-2 showed more newly-formed bone than the other groups with DBB. At 8 weeks, the volume of newly-formed bone around DBB that bore a coating-incorporated depot of BMP-2 was greatest among the groups with DBB, and was comparable to the autologous bone group. The use of autologous bone and BMP-2 resulted in more bone marrow formation. Multinucleated giant cells were observed in the resorption process around DBB, whereas histomorphometric analysis revealed no significant degradation of DBB. In conclusion, it was shown that incorporating BMP-2 into the calcium phosphate coating of DBB induced strong bone formation around DBB for repairing a critical-sized bone defect. Copyright © 2013 Elsevier Inc. All rights reserved.

PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation.

PubMed

Xu, Xiaolong; Qiu, Sujun; Zhang, Yuxian; Yin, Jie; Min, Shaoxiong

2017-03-01

Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine-chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine-chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.

Optimization of implant/bone attachment: The effects of implant surface porosity, bioactive ceramic coatings, and delivery of adsorbed growth factors

NASA Astrophysics Data System (ADS)

Melican, Mora Carolynne

Various surface treatments and coating materials have been tested for use on metal alloy orthopaedic implants. Their purpose has been to enhance the bioactivity of the implant surfaces, and thus to increase the rate and degree of bony attachment in vivo in an attempt to hasten recovery time, increase implant service lifetime, and lessen pain associated with loosened orthopaedic implants. A series of in vivo and in vitro studies were performed to determine the influence of different implant surfaces including porous metal surfaces with varied porosity with depth, resorbable and non-resorbable plasma-sprayed hydroxyapatite (HA) coatings, and finally HA coatings with an adsorbed layer of human recombinant bone morphogenetic protein (rhBMP-2), an osteoinductive protein. Textured as-cast metal surfaces produced by investment casting in three dimensionally printed ceramic molds have exhibited superior bony ingrowth and attachment. Plasma-sprayed HA coatings have been shown to be appropriate substrates for osteoblast proliferation (particularly on highly crystalline HA) and stem cell proliferation (particularly on less crystalline HA). Less crystalline HA coatings have shown promise as delivery systems for different levels of rhBMP-2. The osteoinductive protein has been shown to remain active after delivery to the system, and was most effective when delivered in concentrations ranging from 30 to 50 ng/ml. Combinations of these surface treatments for metal implant surfaces warrant further investigation.

The non-canonical BMP and Wnt/β-catenin signaling pathways orchestrate early tooth development

PubMed Central

Yuan, Guohua; Yang, Guobin; Zheng, Yuqian; Zhu, Xiaojing; Chen, Zhi; Zhang, Zunyi; Chen, YiPing

2015-01-01

BMP and Wnt signaling pathways play a crucial role in organogenesis, including tooth development. Despite extensive studies, the exact functions, as well as if and how these two pathways act coordinately in regulating early tooth development, remain elusive. In this study, we dissected regulatory functions of BMP and Wnt pathways in early tooth development using a transgenic noggin (Nog) overexpression model (K14Cre;pNog). It exhibits early arrested tooth development, accompanied by reduced cell proliferation and loss of odontogenic fate marker Pitx2 expression in the dental epithelium. We demonstrated that overexpression of Nog disrupted BMP non-canonical activity, which led to a dramatic reduction of cell proliferation rate but did not affect Pitx2 expression. We further identified a novel function of Nog by inhibiting Wnt/β-catenin signaling, causing loss of Pitx2 expression. Co-immunoprecipitation and TOPflash assays revealed direct binding of Nog to Wnts to functionally prevent Wnt/β-catenin signaling. In situ PLA and immunohistochemistry on Nog mutants confirmed in vivo interaction between endogenous Nog and Wnts and modulation of Wnt signaling by Nog in tooth germs. Genetic rescue experiments presented evidence that both BMP and Wnt signaling pathways contribute to cell proliferation regulation in the dental epithelium, with Wnt signaling also controlling the odontogenic fate. Reactivation of both BMP and Wnt signaling pathways, but not of only one of them, rescued tooth developmental defects in K14Cre;pNog mice, in which Wnt signaling can be substituted by transgenic activation of Pitx2. Our results reveal the orchestration of non-canonical BMP and Wnt/β-catenin signaling pathways in the regulation of early tooth development. PMID:25428587

Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

PubMed

Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

2016-06-01

Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Medium-Term Function of a 3D Printed TCP/HA Structure as a New Osteoconductive Scaffold for Vertical Bone Augmentation: A Simulation by BMP-2 Activation

PubMed Central

Moussa, Mira; Carrel, Jean-Pierre; Scherrer, Susanne; Cattani-Lorente, Maria; Wiskott, Anselm; Durual, Stéphane

2015-01-01

Introduction: A 3D-printed construct made of orthogonally layered strands of tricalcium phosphate (TCP) and hydroxyapatite has recently become available. The material provides excellent osteoconductivity. We simulated a medium-term experiment in a sheep calvarial model by priming the blocks with BMP-2. Vertical bone growth/maturation and material resorption were evaluated. Materials and methods: Titanium hemispherical caps were filled with either bare- or BMP-2 primed constructs and placed onto the calvaria of adult sheep (n = 8). Histomorphometry was performed after 8 and 16 weeks. Results: After 8 weeks, relative to bare constructs, BMP-2 stimulation led to a two-fold increase in bone volume (Bare: 22% ± 2.1%; BMP-2 primed: 50% ± 3%) and a 3-fold decrease in substitute volume (Bare: 47% ± 5%; BMP-2 primed: 18% ± 2%). These rates were still observed at 16 weeks. The new bone grew and matured to a haversian-like structure while the substitute material resorbed via cell- and chemical-mediation. Conclusion: By priming the 3D construct with BMP-2, bone metabolism was physiologically accelerated, that is, enhancing vertical bone growth and maturation as well as material bioresorption. The scaffolding function of the block was maintained, leaving time for the bone to grow and mature to a haversian-like structure. In parallel, the material resorbed via cell-mediated and chemical processes. These promising results must be confirmed in clinical tests.

Bone Morphogenic Protein 4-Smad-Induced Upregulation of Platelet-Derived Growth Factor AA Impairs Endothelial Function.

PubMed

Hu, Weining; Zhang, Yang; Wang, Li; Lau, Chi Wai; Xu, Jian; Luo, Jiang-Yun; Gou, Lingshan; Yao, Xiaoqiang; Chen, Zhen-Yu; Ma, Ronald Ching Wan; Tian, Xiao Yu; Huang, Yu

2016-03-01

Bone morphogenic protein 4 (BMP4) is an important mediator of endothelial dysfunction in cardio-metabolic diseases, whereas platelet-derived growth factors (PDGFs) are major angiogenic and proinflammatory mediator, although the functional link between these 2 factors is unknown. The present study investigated whether PDGF mediates BMP4-induced endothelial dysfunction in diabetes mellitus. We generated Ad-Bmp4 to overexpress Bmp4 and Ad-Pdgfa-shRNA to knockdown Pdgfa in mice through tail intravenous injection. SMAD4-shRNA lentivirus, SMAD1-shRNA, and SMAD5 shRNA adenovirus were used for knockdown in human and mouse endothelial cells. We found that PDGF-AA impaired endothelium-dependent vasodilation in aortas and mesenteric resistance arteries. BMP4 upregulated PDGF-AA in human and mouse endothelial cells, which was abolished by BMP4 antagonist noggin or knockdown of SMAD1/5 or SMAD4. BMP4-impared relaxation in mouse aorta was also ameliorated by PDGF-AA neutralizing antibody. Tail injection of Ad-Pdgfa-shRNA ameliorates endothelial dysfunction induced by Bmp4 overexpression (Ad-Bmp4) in vivo. Serum PDGF-AA was elevated in both diabetic patients and diabetic db/db mice compared with nondiabetic controls. Pdgfa-shRNA or Bmp4-shRNA adenovirus reduced serum PDGF-AA concentration in db/db mice. PDGF-AA neutralizing antibody or tail injection with Pdgfa-shRNA adenovirus improved endothelial function in aortas and mesenteric resistance arteries from db/db mice. The effect of PDGF-AA on endothelial function in mouse aorta was also inhibited by Ad-Pdgfra-shRNA to inhibit PDGFRα. The present study provides novel evidences to show that PDGF-AA impairs endothelium-dependent vasodilation and PDGF-AA mediates BMP4-induced adverse effect on endothelial cell function through SMAD1/5- and SMAD4-dependent mechanisms. Inhibition of PGDF-AA ameliorates vascular dysfunction in diabetic mice. © 2016 American Heart Association, Inc.

Essential roles of zebrafish bmp2a, fgf10, and fgf24 in the specification of the ventral pancreas

PubMed Central

Naye, François; Voz, Marianne L.; Detry, Nathalie; Hammerschmidt, Matthias; Peers, Bernard; Manfroid, Isabelle

2012-01-01

In vertebrates, pancreas and liver arise from bipotential progenitors located in the embryonic gut endoderm. Bone morphogenic protein (BMP) and fibroblast growth factor (FGF) signaling pathways have been shown to induce hepatic specification while repressing pancreatic fate. Here we show that BMP and FGF factors also play crucial function, at slightly later stages, in the specification of the ventral pancreas. By analyzing the pancreatic markers pdx1, ptf1a, and hlxb9la in different zebrafish models of BMP loss of function, we demonstrate that the BMP pathway is required between 20 and 24 h postfertilization to specify the ventral pancreatic bud. Knockdown experiments show that bmp2a, expressed in the lateral plate mesoderm at these stages, is essential for ventral pancreas specification. Bmp2a action is not restricted to the pancreatic domain and is also required for the proper expression of hepatic markers. By contrast, through the analysis of fgf10−/−; fgf24−/− embryos, we reveal the specific role of these two FGF ligands in the induction of the ventral pancreas and in the repression of the hepatic fate. These mutants display ventral pancreas agenesis and ectopic masses of hepatocytes. Overall, these data highlight the dynamic role of BMP and FGF in the patterning of the hepatopancreatic region. PMID:22219376

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling.

PubMed

Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

2017-01-30

In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

NASA Astrophysics Data System (ADS)

Fitzpatrick, Vincent; Fourel, Laure; Destaing, Olivier; Gilde, Flora; Albigès-Rizo, Corinne; Picart, Catherine; Boudou, Thomas

2017-01-01

In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

Colloid, adhesive and release properties of nanoparticular ternary complexes between cationic and anionic polysaccharides and basic proteins like bone morphogenetic protein BMP-2.

PubMed

Petzold, R; Vehlow, D; Urban, B; Grab, A L; Cavalcanti-Adam, E A; Alt, V; Müller, M

2017-03-01

Herein we describe an interfacial local drug delivery system for bone morphogenetic protein 2 (BMP-2) based on coatings of polyelectrolyte complex (PEC) nanoparticles (NP). The application horizon is the functionalization of bone substituting materials (BSM) used for the therapy of systemic bone diseases. Nanoparticular ternary complexes of cationic and anionic polysaccharides and BMP-2 or two further model proteins, respectively, were prepared in dependence of the molar mixing ratio, pH value and of the cationic polysaccharide. As further proteins chymotrypsin (CHY) and papain (PAP) were selected, which served as model proteins for BMP-2 due to similar isoelectric points and molecular weights. As charged polysaccharides ethylenediamine modified cellulose (EDAC) and trimethylammonium modified cellulose (PQ10) were combined with cellulose sulphatesulfate (CS). Mixing diluted cationic and anionic polysaccharide and protein solutions according to a slight either anionic or cationic excess charge colloidal ternary dispersions formed, which were cast onto germanium model substrates by water evaporation. Dynamic light scattering (DLS) demonstrated, that these dispersions were colloidally stable for at least one week. Fourier Transform Infrared (FTIR) showed, that the cast protein loaded PEC NP coatings were irreversibly adhesive at the model substrate in contact to HEPES buffer and solely CHY, PAP and BMP-2 were released within long-term time scale. Advantageously, out of the three proteins BMP-2 showed the smallest initial burst and the slowest release kinetics and around 25% of the initial BMP-2 content were released within 14days. Released BMP-2 showed significant activity in the myoblast cells indicating the ability to regulate the formation of new bone. Therefore, BMP-2 loaded PEC NP are suggested as novel promising tool for the functionalization of BSM used for the therapy of systemic bone diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells.

PubMed

Sugimoto, Asuna; Miyazaki, Aya; Kawarabayashi, Keita; Shono, Masayuki; Akazawa, Yuki; Hasegawa, Tomokazu; Ueda-Yamaguchi, Kimiko; Kitamura, Takamasa; Yoshizaki, Keigo; Fukumoto, Satoshi; Iwamoto, Tsutomu

2017-12-18

The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression.

Repulsive Guidance Molecule is a structural bridge between Neogenin and Bone Morphogenetic Protein

PubMed Central

Healey, Eleanor G.; Bishop, Benjamin; Elegheert, Jonathan; Bell, Christian H.; Padilla-Parra, Sergi; Siebold, Christian

2015-01-01

Repulsive guidance molecules (RGMs) control crucial processes spanning cell motility, adhesion, immune cell regulation and systemic iron metabolism. RGMs signal via two fundamental signaling cascades: the Neogenin (NEO1) and the Bone Morphogenetic Protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a novel protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the ternary BMP2–RGM–NEO1 complex crystal structure, which combined with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM–NEO1 complex. Our results show how RGM acts as the central hub linking BMP and NEO1 and physically connecting these fundamental signaling pathways. PMID:25938661

SMOC can act as both an antagonist and an expander of BMP signaling.

PubMed

Thomas, J Terrig; Eric Dollins, D; Andrykovich, Kristin R; Chu, Tehyen; Stultz, Brian G; Hursh, Deborah A; Moos, Malcolm

2017-03-21

The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone ( pent ), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation.

Expression of bone morphogenetic proteins and Msx genes during root formation.

PubMed

Yamashiro, T; Tummers, M; Thesleff, I

2003-03-01

Like crown development, root formation is also regulated by interactions between epithelial and mesenchymml tissues. Bone morphogenetic proteins (BMPs), together with the transcription factors Msx1 and Msx2, play important roles in these interactions during early tooth morphogenesis. To investigate the involvement of this signaling pathway in root development, we analyzed the expression patterns of Bmp2, Bmp3, Bmp4, and Bmp7 as well as Msx1 and Msx2 in the roots of mouse molars. Bmp4 was expressed in the apical mesenchyme and Msx2 in the root sheath. However, Bmps were not detected in the root sheath epithelium, and Msx transcripts were absent from the underlying mesenchyme. These findings indicate that this Bmp signaling pathway, required for tooth initiation, does not regulate root development, but we suggest that root shape may be regulated by a mechanism similar to that regulating crown shape in cap-stage tooth germs. Msx2 expression continued in the epithelial cell rests of Malassez, and the nearby cementoblasts intensely expressed Bmp3, which may regulate some functions of the fragmented epithelium.

Cellular Therapy to Obtain Rapid Endochondral Bone Formation

DTIC Science & Technology

2008-02-01

efficiency of the delivery cells for optimal BMP2 production is the key parameter in determining the ex- tent of bone formation (Olmsted et al., 2001...quan- titative bone analysis software provided with the MicroCT sys- tem. For this analysis, any tissue with a hydroxyapatite density greater than 0.26...2B. Continued. B duced cells do not interfere with the osteoinductive nature of BMP2. Using set parameters to obtain equivalent functional BMP2

Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring.

PubMed

Abadjieva, Desislava; Kistanova, Elena

2016-01-01

Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers' oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris.

Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring

PubMed Central

2016-01-01

Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers’ oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris. PMID:26928288

SIX2 and BMP4 mutations associate with anomalous kidney development.

PubMed

Weber, Stefanie; Taylor, Jaclyn C; Winyard, Paul; Baker, Kari F; Sullivan-Brown, Jessica; Schild, Raphael; Knüppel, Tanja; Zurowska, Aleksandra M; Caldas-Alfonso, Alberto; Litwin, Mieczyslaw; Emre, Sevinc; Ghiggeri, Gian Marco; Bakkaloglu, Aysin; Mehls, Otto; Antignac, Corinne; Network, Escape; Schaefer, Franz; Burdine, Rebecca D

2008-05-01

Renal hypodysplasia (RHD) is characterized by reduced kidney size and/or maldevelopment of the renal tissue following abnormal organogenesis. Mutations in renal developmental genes have been identified in a subset of affected individuals. Here, we report the first mutations in BMP4 and SIX2 identified in patients with RHD. We detected 3 BMP4 mutations in 5 RHD patients, and 3 SIX2 mutations in 5 different RHD patients. Overexpression assays in zebrafish demonstrated that these mutations affect the function of Bmp4 and Six2 in vivo. Overexpression of zebrafish six2.1 and bmp4 resulted in dorsalization and ventralization, respectively, suggesting opposing roles in mesendoderm formation. When mutant constructs containing the identified human mutations were overexpressed instead, these effects were attenuated. Morpholino knockdown of bmp4 and six2.1 affected glomerulogenesis, suggesting specific roles for these genes in the formation of the pronephros. In summary, these studies implicate conserved roles for Six2 and Bmp4 in the development of the renal system. Defects in these proteins could affect kidney development at multiple stages, leading to the congenital anomalies observed in patients with RHD.

Characterization of the post-translational modification of recombinant human BMP-15 mature protein

PubMed Central

Saito, Seiji; Yano, Keiichi; Sharma, Shweta; McMahon, Heather E.; Shimasaki, Shunichi

2008-01-01

Bone morphogenetic protein-15 (BMP-15) is an oocyte-secreted factor critical for the regulation of ovarian physiology. When recombinant human BMP-15 (rhBMP-15) produced in human embryonic kidney 293 cells was subjected to SDS-PAGE analysis, two mature protein forms corresponding to 16 kDa (P16) and 17 kDa (P17) were observed. Despite the physiological relevance and critical function of BMP-15 in female reproduction, little is known about the structure of rhBMP-15. Here, we have analyzed the structure of the rhBMP-15 mature proteins (P16 and P17) using state-of-the-art proteomics technology. Our findings are as follows: (1) the N-terminal amino acid of P16 and P17 is pyroglutamic acid; (2) the Ser residue at the sixth position of P16 is phosphorylated; (3) P17 is O-glycosylated at Thr10; and (4) the C-terminal amino acid of P16 and P17 is truncated. These findings are the first knowledge of the structure of rhBMP-15 mature protein toward understanding the molecular basis of BMP-15 function and could provide an important contribution to the rapidly progressing research area involving oocyte-specific growth factors in modulation of female fertility. PMID:18227435

BMP4 Signaling Is Able to Induce an Epithelial-Mesenchymal Transition-Like Phenotype in Barrett’s Esophagus and Esophageal Adenocarcinoma through Induction of SNAIL2

PubMed Central

Kestens, Christine; Siersema, Peter D.; Offerhaus, G. Johan A.; van Baal, Jantine W. P. M.

2016-01-01

Background Bone morphogenetic protein 4 (BMP4) signaling is involved in the development of Barrett’s esophagus (BE), a precursor of esophageal adenocarcinoma (EAC). In various cancers, BMP4 has been found to induce epithelial-mesenchymal transition (EMT) but its function in the development of EAC is currently unclear. Aim To investigate the expression of BMP4 and several members of the BMP4 pathway in EAC. Additionally, to determine the effect of BMP4 signaling in a human Barrett’s esophagus (BAR-T) and adenocarcinoma (OE33) cell line. Methods Expression of BMP4, its downstream target ID2 and members of the BMP4 pathway were determined by Q-RT-PCR, immunohistochemistry and Western blot analysis using biopsy samples from EAC patients. BAR-T and OE33 cells were incubated with BMP4 or the BMP4 antagonist, Noggin, and cell viability and migration assays were performed. In addition, expression of factors associated with EMT (SNAIL2, CDH1, CDH2 and Vimentin) was evaluated by Q-RT-PCR and Western blot analysis. Results Compared to squamous epithelium (SQ), BMP4 expression was significantly upregulated in EAC and BE. In addition, the expression of ID2 was significantly upregulated in EAC and BE compared to SQ. Western blot analysis confirmed our results, showing an upregulated expression of BMP4 and ID2 in both BE and EAC. In addition, more phosphorylation of SMAD1/5/8 was observed. BMP4 incubation inhibited cell viability, but induced cell migration in both BAR-T and OE33 cells. Upon BMP4 incubation, SNAIL2 expression was significantly upregulated in BAR-T and OE33 cells while CDH1 expression was significantly downregulated. These results were confirmed by Western blot analysis. Conclusion Our results indicate active BMP4 signaling in BE and EAC and suggest that this results in an invasive phenotype by inducing an EMT-like response through upregulation of SNAIL2 and subsequent downregulation of CDH1. PMID:27191723

BMP Sustains Embryonic Stem Cell Self-Renewal through Distinct Functions of Different Krüppel-like Factors.

PubMed

Morikawa, Masato; Koinuma, Daizo; Mizutani, Anna; Kawasaki, Natsumi; Holmborn, Katarina; Sundqvist, Anders; Tsutsumi, Shuichi; Watabe, Tetsuro; Aburatani, Hiroyuki; Heldin, Carl-Henrik; Miyazono, Kohei

2016-01-12

Bone morphogenetic protein (BMP) signaling exerts paradoxical roles in pluripotent stem cells (PSCs); it sustains self-renewal of mouse embryonic stem cells (ESCs), while it induces differentiation in other PSCs, including human ESCs. Here, we revisit the roles of BMP-4 using mouse ESCs (mESCs) in naive and primed states. SMAD1 and SMAD5, which transduce BMP signals, recognize enhancer regions together with KLF4 and KLF5 in naive mESCs. KLF4 physically interacts with SMAD1 and suppresses its activity. Consistently, a subpopulation of cells with active BMP-SMAD can be ablated without disturbing the naive state of the culture. Moreover, Smad1/5 double-knockout mESCs stay in the naive state, indicating that the BMP-SMAD pathway is dispensable for it. In contrast, the MEK5-ERK5 pathway mediates BMP-4-induced self-renewal of mESCs by inducing Klf2, a critical factor for the ground state pluripotency. Our study illustrates that BMP exerts its self-renewing effect through distinct functions of different Krüppel-like factors. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Trends and Cost of Posterior Cervical Fusions With and Without Recombinant Human Bone Morphogenetic Protein-2 in the US Medicare Population.

PubMed

Myhre, Sue Lynn; Buser, Zorica; Meisel, Hans-Joerg; Brodke, Darrel S; Yoon, S Tim; Wang, Jeffrey C; Park, Jong-Beom; Youssef, Jim A

2017-06-01

Retrospective database review. To analyze and report the trends and cost of posterior cervical fusions (PCFs) with and without off-label recombinant human bone morphogenetic protein-2 (rhBMP-2) in the Medicare population. Patient records from the PearlDiver database were retrospectively reviewed from January 1, 2005, to December 31, 2012, to distinguish individuals who underwent a PCF with or without rhBMP-2. Total numbers, incidence, age, gender, geographic region, reimbursement, and length of stay were analyzed and summarized. The combined total of non-rhBMP-2 (n = 39 479; 85.51%) and rhBMP-2 PCF (n = 6692; 14.49%) procedures performed between 2005 and 2012 was 46 171. In general, the number of PCFs without rhBMP-2 consistently increased over time, while the number of PCFs with rhBMP-2 had only a slight increase from 2005 to 2012. On average, PCFs without rhBMP-2 were associated with $1197 higher cost than those with rhBMP-2, but the average length of stay was similar (6 days). From 2005 to 2012, the average cost for procedures with and without rhBMP-2 increased by $12 605 and $7291, respectively. The percentage of rhBMP-2 use peaked in 2007 and dwindled until 2010, and declined an additional 2.84% from 2011 to 2012. Multiple age, region, and gender tendencies were observed. To our knowledge, this was the first study to use the PearlDiver database to report incidence and cost trends of PCF procedures. This article provides meaningful trend data on PCFs to surgeons and clinicians, researchers, and patients, as well as functions as a beacon for future research questions.

Bone Morphogenetic Protein-2 Promotes Human Mesenchymal Stem Cell Survival and Resultant Bone Formation When Entrapped in Photocrosslinked Alginate Hydrogels.

PubMed

Ho, Steve S; Vollmer, Nina L; Refaat, Motasem I; Jeon, Oju; Alsberg, Eben; Lee, Mark A; Leach, J Kent

2016-10-01

There is a substantial need to prolong cell persistence and enhance functionality in situ to enhance cell-based tissue repair. Bone morphogenetic protein-2 (BMP-2) is often used at high concentrations for osteogenic differentiation of mesenchymal stem cells (MSCs) but can induce apoptosis. Biomaterials facilitate the delivery of lower doses of BMP-2, reducing side effects and localizing materials at target sites. Photocrosslinked alginate hydrogels (PAHs) can deliver osteogenic materials to irregular-sized bone defects, providing improved control over material degradation compared to ionically cross-linked hydrogels. It is hypothesized that the delivery of MSCs and BMP-2 from a PAH increases cell persistence by reducing apoptosis, while promoting osteogenic differentiation and enhancing bone formation compared to MSCs in PAHs without BMP-2. BMP-2 significantly decreases apoptosis and enhances survival of photoencapsulated MSCs, while simultaneously promoting osteogenic differentiation in vitro. Bioluminescence imaging reveals increased MSC survival when implanted in BMP-2 PAHs. Bone defects treated with MSCs in BMP-2 PAHs demonstrate 100% union as early as 8 weeks and significantly higher bone volumes at 12 weeks, while defects with MSC-entrapped PAHs alone do not fully bridge. This study demonstrates that transplantation of MSCs with BMP-2 in PAHs achieves robust bone healing, providing a promising platform for bone repair. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

BMP signalling in human fetal ovary somatic cells is modulated in a gene-specific fashion by GREM1 and GREM2

PubMed Central

Bayne, Rosemary A.; Donnachie, Douglas J.; Kinnell, Hazel L.; Childs, Andrew J.; Anderson, Richard A.

2016-01-01

STUDY QUESTION Do changes in the expression of bone morphogenetic proteins (BMPs) 2 and 4, and their antagonists Gremlin1 (GREM1) and Gremlin2 (GREM2) during human fetal ovarian development impact on BMP pathway activity and lead to changes in gene expression that may influence the fate and/or function of ovarian somatic cells? STUDY FINDING BMPs 2 and 4 differentially regulate gene expression in cultured human fetal ovarian somatic cells. Expression of some, but not all BMP target genes is antagonised by GREM1 and GREM2, indicating the existence of a mechanism to fine-tune BMP signal intensity in the ovary. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker of immature ovarian somatic cells, is identified as a novel transcriptional target of BMP4. WHAT IS KNOWN ALREADY Extensive re-organisation of the germ and somatic cell populations in the feto-neonatal ovary culminates in the formation of primordial follicles, which provide the basis for a female's future fertility. BMP growth factors play important roles at many stages of ovarian development and function. GREM1, an extracellular antagonist of BMP signalling, regulates the timing of primordial follicle formation in the mouse ovary, and mRNA levels of BMP4 decrease while those of BMP2 increase prior to follicle formation in the human fetal ovary. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Expression of genes encoding BMP pathway components, BMP antagonists and markers of ovarian somatic cells were determined by quantitative (q)RT-PCR in human fetal ovaries (from 8 to 21 weeks gestation) and fetal ovary-derived somatic cell cultures. Ovarian expression of GREM1 protein was confirmed by immunoblotting. Primary human fetal ovarian somatic cell cultures were derived from disaggregated ovaries by differential adhesion and cultured in the presence of recombinant human BMP2 or BMP4, with or without the addition of GREM1 or GREM2. MAIN RESULTS AND THE ROLE OF CHANCE We demonstrate that the expression of BMP antagonists GREM1, GREM2 and CHRD  increases in the lead-up to primordial follicle formation in the human fetal ovary, and that the BMP pathway is active in cultured ovarian somatic cells. This leads to differential changes in the expression of a number of genes, some of which are further modulated by GREM1 and/or GREM2. The positive transcriptional regulation of LGR5 (a marker of less differentiated somatic cells) by BMP4 in vitro suggests that increasing levels of GREM1 and reduced levels of BMP4 as the ovary develops in vivo may act to reduce LGR5 levels and allow pre-granulosa cell differentiation. LIMITATIONS, REASONS FOR CAUTION While we have demonstrated that markers of different somatic cell types are expressed in the cultured ovarian somatic cells, their proportions may not represent the same cells in the intact ovary which also contains germ cells. WIDER IMPLICATIONS OF THE FINDINGS This study extends previous work identifying germ cells as targets of ovarian BMP signalling, and suggests BMPs may regulate the development of both germ and somatic cells in the developing ovary around the time of follicle formation. LARGE SCALE DATA Not applicable. STUDY FUNDING/COMPETING INTERESTS This work was supported by The UK Medical Research Council (Grant No.: G1100357 to RAA), and Medical Research Scotland (Grant No. 345FRG to AJC). The authors have no competing interests to declare. PMID:27385727

BMP signalling in human fetal ovary somatic cells is modulated in a gene-specific fashion by GREM1 and GREM2.

PubMed

Bayne, Rosemary A; Donnachie, Douglas J; Kinnell, Hazel L; Childs, Andrew J; Anderson, Richard A

2016-09-01

Do changes in the expression of bone morphogenetic proteins (BMPs) 2 and 4, and their antagonists Gremlin1 (GREM1) and Gremlin2 (GREM2) during human fetal ovarian development impact on BMP pathway activity and lead to changes in gene expression that may influence the fate and/or function of ovarian somatic cells? BMPs 2 and 4 differentially regulate gene expression in cultured human fetal ovarian somatic cells. Expression of some, but not all BMP target genes is antagonised by GREM1 and GREM2, indicating the existence of a mechanism to fine-tune BMP signal intensity in the ovary. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker of immature ovarian somatic cells, is identified as a novel transcriptional target of BMP4. Extensive re-organisation of the germ and somatic cell populations in the feto-neonatal ovary culminates in the formation of primordial follicles, which provide the basis for a female's future fertility. BMP growth factors play important roles at many stages of ovarian development and function. GREM1, an extracellular antagonist of BMP signalling, regulates the timing of primordial follicle formation in the mouse ovary, and mRNA levels of BMP4 decrease while those of BMP2 increase prior to follicle formation in the human fetal ovary. Expression of genes encoding BMP pathway components, BMP antagonists and markers of ovarian somatic cells were determined by quantitative (q)RT-PCR in human fetal ovaries (from 8 to 21 weeks gestation) and fetal ovary-derived somatic cell cultures. Ovarian expression of GREM1 protein was confirmed by immunoblotting. Primary human fetal ovarian somatic cell cultures were derived from disaggregated ovaries by differential adhesion and cultured in the presence of recombinant human BMP2 or BMP4, with or without the addition of GREM1 or GREM2. We demonstrate that the expression of BMP antagonists GREM1, GREM2 and CHRD  increases in the lead-up to primordial follicle formation in the human fetal ovary, and that the BMP pathway is active in cultured ovarian somatic cells. This leads to differential changes in the expression of a number of genes, some of which are further modulated by GREM1 and/or GREM2. The positive transcriptional regulation of LGR5 (a marker of less differentiated somatic cells) by BMP4 in vitro suggests that increasing levels of GREM1 and reduced levels of BMP4 as the ovary develops in vivo may act to reduce LGR5 levels and allow pre-granulosa cell differentiation. While we have demonstrated that markers of different somatic cell types are expressed in the cultured ovarian somatic cells, their proportions may not represent the same cells in the intact ovary which also contains germ cells. This study extends previous work identifying germ cells as targets of ovarian BMP signalling, and suggests BMPs may regulate the development of both germ and somatic cells in the developing ovary around the time of follicle formation. Not applicable. This work was supported by The UK Medical Research Council (Grant No.: G1100357 to RAA), and Medical Research Scotland (Grant No. 345FRG to AJC). The authors have no competing interests to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Collagen Sponge Functionalized with Chimeric Anti-BMP-2 Monoclonal Antibody Mediates Repair of Critical-Size Mandibular Continuity Defects in a Nonhuman Primate Model

PubMed Central

Xie, Yilin; Su, Yingying; Tang, Jianxia; Goh, Bee Tin; Saigo, Leonardo; Zhang, Chunmei; Wang, Jinsong; Khojasteh, Arash; Wang, Songlin

2017-01-01

Antibody-mediated osseous regeneration (AMOR) has been introduced by our research group as a tissue engineering approach to capture of endogenous growth factors through the application of specific monoclonal antibodies (mAbs) immobilized on a scaffold. Specifically, anti-Bone Morphogenetic Protein- (BMP-) 2 mAbs have been demonstrated to be efficacious in mediating bone repair in a number of bone defects. The present study sought to investigate the application of AMOR for repair of mandibular continuity defect in nonhuman primates. Critical-sized mandibular continuity defects were created in Macaca fascicularis locally implanted with absorbable collagen sponges (ACS) functionalized with chimeric anti-BMP-2 mAb or isotype control mAb. 2D and 3D analysis of cone beam computed tomography (CBCT) imaging demonstrated increased bone density and volume observed within mandibular continuity defects implanted with collagen scaffolds functionalized with anti-BMP-2 mAb, compared with isotype-matched control mAb. Both CBCT imaging and histologic examination demonstrated de novo bone formation that was in direct apposition to the margins of the resected bone. It is hypothesized that bone injury may be necessary for AMOR. This is evidenced by de novo bone formation adjacent to resected bone margins, which may be the source of endogenous BMPs captured by anti-BMP-2 mAb, in turn mediating bone repair. PMID:28401163

3D bioprinting of BMSC-laden methacrylamide gelatin scaffolds with CBD-BMP2-collagen microfibers.

PubMed

Du, Mingchun; Chen, Bing; Meng, Qingyuan; Liu, Sumei; Zheng, Xiongfei; Zhang, Cheng; Wang, Heran; Li, Hongyi; Wang, Nuo; Dai, Jianwu

2015-12-18

Three-dimensional (3D) bioprinting combines biomaterials, cells and functional components into complex living tissues. Herein, we assembled function-control modules into cell-laden scaffolds using 3D bioprinting. A customized 3D printer was able to tune the microstructure of printed bone mesenchymal stem cell (BMSC)-laden methacrylamide gelatin scaffolds at the micrometer scale. For example, the pore size was adjusted to 282 ± 32 μm and 363 ± 60 μm. To match the requirements of the printing nozzle, collagen microfibers with a length of 22 ± 13 μm were prepared with a high-speed crusher. Collagen microfibers bound bone morphogenetic protein 2 (BMP2) with a collagen binding domain (CBD) as differentiation-control module, from which BMP2 was able to be controllably released. The differentiation behaviors of BMSCs in the printed scaffolds were compared in three microenvironments: samples without CBD-BMP2-collagen microfibers in the growth medium, samples without microfibers in the osteogenic medium and samples with microfibers in the growth medium. The results indicated that BMSCs showed high cell viability (>90%) during printing; CBD-BMP2-collagen microfibers induced BMSC differentiation into osteocytes within 14 days more efficiently than the osteogenic medium. Our studies suggest that these function-control modules are attractive biomaterials and have potential applications in 3D bioprinting.

Diabetes mellitus affects the biomechanical function of the callus and the expression of TGF-beta1 and BMP2 in an early stage of fracture healing.

PubMed

Xu, M T; Sun, S; Zhang, L; Xu, F; Du, S L; Zhang, X D; Wang, D W

2016-01-01

Transforming growth factor beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) are important regulators of bone repair and regeneration. In this study, we examined whether TGF-β1 and BMP-2 expressions were delayed during bone healing in type 1 diabetes mellitus. Tibial fractures were created in 95 diabetic and 95 control adult male Wistar rats of 10 weeks of age. At 1, 2, 3, 4, and 5 weeks after fracture induction, five rats were sacrificed from each group. The expressions of TGF-β1 and BMP2 in the fractured tibias were measured by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction, weekly for the first 5 weeks post-fracture. Mechanical parameters (bending rigidity, torsional rigidity, destruction torque) of the healing bones were also assessed at 3, 4, and 5 weeks post-fracture, after the rats were sacrificed. The bending rigidity, torsional rigidity and destruction torque of the two groups increased continuously during the healing process. The diabetes group had lower mean values for bending rigidity, torsional rigidity and destruction torque compared with the control group (P<0.05). TGF-β1 and BMP-2 expression were significantly lower (P<0.05) in the control group than in the diabetes group at postoperative weeks 1, 2, and 3. Peak levels of TGF-β1 and BMP-2 expression were delayed by 1 week in the diabetes group compared with the control group. Our results demonstrate that there was a delayed recovery in the biomechanical function of the fractured bones in diabetic rats. This delay may be associated with a delayed expression of the growth factors TGF-β1 and BMP-2.

Multifunctional mesoporous bioactive glasses for effective delivery of therapeutic ions and drug/growth factors.

PubMed

Wu, Chengtie; Chang, Jiang

2014-11-10

Regeneration of large-size bone defects represents a significant challenge clinically, which requires the use of scaffolds with multifunction, such as anti-bacterial activity, and stimulation of osteogenesis and angiogenesis. It is known that functional ions or drug/growth factors play an important role to stimulate tissue regeneration. Mesoporous bioactive glasses (MBG) possess excellent bioactivity and drug-delivery ability as well as effective ionic release in the body fluids microenvironment due to its specific mesoporous structure and large surface area. For these reasons, functional ions (e.g. lithium (Li), strontium (Sr), Copper (Cu) and Boron (B)) and drug/growth factors (e.g. dexamethasone, vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)) have been incorporated into MBG, which shows high loading efficiency and effective release. The release of therapeutic ions and drug/growth factors from MBG offers it multifunctional properties, such as improved osteogenesis, angiogenesis, anti-bacterial/cancer activity. However, there is no a systematic review about delivery of therapeutic ions and drugs/growth factors from MBG for the functional effect on the tissue regeneration despite that significant progress has been achieved in the past five years. Therefore, in this review, we mainly focused on the new advances for the functional effect of delivering therapeutic ions and drugs/growth factors on the ostegeogenesis, angiogenesis and antibacterial activity. It is expected that the review will offer new concept to develop multifunctional biomaterials for bone regeneration by the synergistic effect of therapeutic ions and drug/growth factors. Copyright © 2014 Elsevier B.V. All rights reserved.

Notch regulates BMP responsiveness and lateral branching in vessel networks via SMAD6

PubMed Central

Mouillesseaux, Kevin P.; Wiley, David S.; Saunders, Lauren M.; Wylie, Lyndsay A.; Kushner, Erich J.; Chong, Diana C.; Citrin, Kathryn M.; Barber, Andrew T.; Park, Youngsook; Kim, Jun-Dae; Samsa, Leigh Ann; Kim, Jongmin; Liu, Jiandong; Jin, Suk-Won; Bautch, Victoria L.

2016-01-01

Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation. PMID:27834400

Gelatin- hydroxyapatite- calcium sulphate based biomaterial for long term sustained delivery of bone morphogenic protein-2 and zoledronic acid for increased bone formation: In-vitro and in-vivo carrier properties.

PubMed

Raina, Deepak Bushan; Larsson, David; Mrkonjic, Filip; Isaksson, Hanna; Kumar, Ashok; Lidgren, Lars; Tägil, Magnus

2018-02-28

In this study, a novel macroporous composite biomaterial consisting of gelatin-hydroxyapatite-calcium sulphate for delivery of bone morphogenic protein-2 (rhBMP-2) and zoledronic acid (ZA) has been developed. The biomaterial scaffold has a porous structure and functionalization of the scaffold with rhBMP-2 induces osteogenic differentiation of MC3T3-e1 cells seen by a significant increase in biochemical and genetic markers of osteoblastic differentiation. In-vivo muscle pouch experiments showed higher mineralization using scaffold+rhBMP-2 when compared to an approved absorbable collagen sponge (ACS)+rhBMP-2 as verified by micro-CT. Co-delivery of rhBMP-2+ZA via the novel scaffold enabled a reduction in the effective rhBMP-2 doses. The presence of tartrate resistant acid phosphatase staining in the rhBMP-2 group indicates osteoclastic resorption, which could be stalled by adding ZA, which by speculation could explain the net increase in mineralization. The new scaffold allowed for slow release of rhBMP-2 in-vitro (3.3±0.1%) after 4weeks. Using single photon emission computed tomography (SPECT), the release kinetics of 125 I-rhBMP-2 in-vivo was followed for 4weeks and a total of 65.3±15.2% 125 I-rhBMP-2 was released from the scaffolds. In-vitro 14 C-ZA release curve shows an initial burst release on day 1 (8.8±0.7%) followed by a slow release during the following 4weeks (13±0.1%). In-vivo, an initial release of 43.2±7.6% of 14 C-ZA was detected after 1day, after which the scaffold retained the remaining ZA during 4-weeks. Taken together, our results show that the developed biomaterial is an efficient carrier for spatio-temporal delivery of rhBMP-2 and ZA leading to increased bone formation compared to commercially available carrier for rhBMP-2. Copyright © 2018 Elsevier B.V. All rights reserved.

Smad7 mediates inhibition of Saos2 osteosarcoma cell differentiation by NF{kappa}B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eliseev, Roman A.; Schwarz, Edward M.; Zuscik, Michael J.

2006-01-01

The transcription factor NF{kappa}B is constitutively activated in various tumor cells where it promotes proliferation and represses apoptosis. The bone morphogenetic proteins (BMPs) delay cell proliferation and promote differentiation and apoptosis of bone cells through activation of Smad downstream effectors and via Smad-independent mechanisms. Thus, NF{kappa}B and BMP pathways play opposing roles in regulating osteoblastic cell fate. Here, we show that in osteosarcoma Saos2 osteoblasts, NF{kappa}B regulates the activity of the BMP/Smad signaling. Inhibition of NF{kappa}B by overexpression of mI{kappa}B leads to the induction of osteoblast differentiation. Saos2 cells overexpressing mI{kappa}B (Saos2-mI{kappa}B) exhibit higher expression of osteoblast phenotypic genes suchmore » as alkaline phosphatase, Runx2 and osteocalcin and are more responsive to BMP2 in comparison to wild-type cells (Saos2-wt) or empty vector infected controls (Saos2-EV). Furthermore, BMP-2 signaling and Smad phosphorylation are significantly increased in Saos2-mI{kappa}B cells in comparison to Saos2-EV cells. Inhibition of NF{kappa}B signaling in Saos2-mI{kappa}B cells is associated with decreased expression of the BMP signaling inhibitor Smad7. While gain of Smad7 function in Saos2-mI{kappa}B cells results in inhibition of BMP signaling, anti-sense knockdown of Smad7 in Saos2-EV cells leads to upregulation of BMP signaling. We therefore conclude that in osteosarcoma Saos2 cells, NF{kappa}B represses BMP/Smad signaling and BMP2-induced differentiation through Smad7.« less

Runx2 mediates epigenetic silencing of the bone morphogenetic protein-3B (BMP-3B/GDF10) in lung cancer cells

PubMed Central

2012-01-01

Background The Runt-related transcription factor Runx2 is essential for bone development but is also implicated in progression of several cancers of breast, prostate and bone, where it activates cancer-related genes and promotes invasive properties. The transforming growth factor β (TGF-β) family member bone morphogenetic protein-3B (BMP-3B/GDF10) is regarded as a tumor growth inhibitor and a gene silenced in lung cancers; however the regulatory mechanisms leading to its silencing have not been identified. Results Here we show that Runx2 is highly expressed in lung cancer cells and downregulates BMP-3B. This inverse relationship between Runx2 and BMP-3B expression is further supported by increased expression of BMP-3B in mesenchymal cells from Runx2 deficient mice. The ectopic expression of Runx2, but not DNA binding mutant Runx2, in normal lung fibroblast cells and lung cancer cells resulted in suppression of BMP-3B levels. The chromatin immunoprecipitation studies identified that the mechanism of Runx2-mediated suppression of BMP-3B is due to the recruitment of Runx2 and histone H3K9-specific methyltransferase Suv39h1 to BMP-3B proximal promoter and a concomitant increase in histone methylation (H3K9) status. The knockdown of Runx2 in H1299 cells resulted in decreased histone H3K9 methylation on BMP-3B promoter and increased BMP-3B expression levels. Furthermore, co-immunoprecipitation studies showed a direct interaction of Runx2 and Suv39h1 proteins. Phenotypically, Runx2 overexpression in H1299 cells increased wound healing response to TGFβ treatment. Conclusions Our studies identified BMP-3B as a new Runx2 target gene and revealed a novel function of Runx2 in silencing of BMP-3B in lung cancers. Our results suggest that Runx2 is a potential therapeutic target to block tumor suppressor gene silencing in lung cancer cells. PMID:22537242

The type I BMP receptors, Bmpr1a and Acvr1, activate multiple signaling pathways to regulate lens formation

PubMed Central

Rajagopal, Ramya; Huang, Jie; Dattilo, Lisa K.; Kaartinen, Vesa; Mishina, Yuji; Deng, Chu-Xia; Umans, Lieve; Zwijsen, An; Roberts, Anita B.; Beebe, David C.

2009-01-01

BMPs play multiple roles in development and BMP signaling is essential for lens formation. However, the mechanisms by which BMP receptors function in vertebrate development are incompletely understood. To determine the downstream effectors of BMP signaling and their functions in the ectoderm that will form the lens, we deleted the genes encoding the type I BMP receptors, Bmpr1a and Acvr1, and the canonical transducers of BMP signaling, Smad4, Smad1 and Smad5. Bmpr1a and Acvr1 regulated cell survival and proliferation, respectively. Absence of both receptors interfered with the expression of proteins involved in normal lens development and prevented lens formation, demonstrating that BMPs induce lens formation by acting directly on the prospective lens ectoderm. Remarkably, the canonical Smad signaling pathway was not needed for most of these processes. Lens formation, placode cell proliferation, the expression of FoxE3, a lens-specific transcription factor, and the lens protein, αA-crystallin were regulated by BMP receptors in a Smad-independent manner. Placode cell survival was promoted by R-Smad signaling, but in a manner that did not involve Smad4. Of the responses tested, only maintaining a high level of Sox2 protein, a transcription factor expressed early in placode formation, required the canonical Smad pathway. A key function of Smad-independent BMP receptor signaling may be reorganization of actin cytoskeleton to drive lens invagination. PMID:19733164

Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimada, Koichi, E-mail: shimada-ki@dent.nihon-u.ac.jp; Division of Advanced Dental Treatment, Dental Research Center, Nihon University School of Dentistry, Tokyo; Ikeda, Kyoko

2009-12-18

Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1.more » To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-{alpha} stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-{alpha}-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-{alpha} and BMP signaling pathways.« less

Homeobox protein MSX-1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer-binding factor 1 via Wnt/β-catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage.

PubMed

Feng, Xiao-Yu; Wu, Xiao-Shan; Wang, Jin-Song; Zhang, Chun-Mei; Wang, Song-Lin

2018-02-01

Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β-catenin signaling pathway. © 2017 Eur J Oral Sci.

Unique players in the BMP pathway: Small C-terminal domain phosphatases dephosphorylate Smad1 to attenuate BMP signaling

PubMed Central

Knockaert, Marie; Sapkota, Gopal; Alarcón, Claudio; Massagué, Joan; Brivanlou, Ali H.

2006-01-01

Smad transcription factors are key signal transducers for the TGF-β/bone morphogenetic protein (BMP) family of cytokines and morphogens. C-terminal serine phosphorylation by TGF-β and BMP membrane receptors drives Smads into the nucleus as transcriptional regulators. Dephosphorylation and recycling of activated Smads is an integral part of this process, which is critical for agonist sensing by the cell. However, the nuclear phosphatases involved have remained unknown. Here we provide functional, biochemical, and embryological evidence identifying the SCP (small C-terminal domain phosphatase) family of nuclear phosphatases as mediators of Smad1 dephosphorylation in the BMP signaling pathway in vertebrates. Xenopus SCP2/Os4 inhibits BMP activity in the presumptive ectoderm and leads to neuralization. In Xenopus embryos, SCP2/Os4 and human SCP1, 2, and 3 cause selective dephosphorylation of Smad1 compared with Smad2, inhibiting BMP- and Smad1-dependent transcription and leading to the induction of the secondary dorsal axis. In human cells, RNAi-mediated depletion of SCP1 and SCP2 increases the extent and duration of Smad1 phosphorylation in response to BMP, the transcriptional action of Smad1, and the strength of endogenous BMP gene responses. The present identification of the SCP family as Smad C-terminal phosphatases sheds light on the events that attenuate Smad signaling and reveals unexpected links to the essential phosphatases that control RNA polymerase II in eukaryotes. PMID:16882717

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

PubMed

Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

2012-03-01

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

Enhanced osteogenic activity and anti-inflammatory properties of Lenti-BMP-2-loaded TiO2 nanotube layers fabricated by lyophilization following trehalose addition

PubMed Central

Zhang, Xiaochen; Zhang, Zhiyuan; Shen, Gang; Zhao, Jun

2016-01-01

To enhance biocompatibility and osseointegration between titanium implants and surrounding bone tissue, numerous efforts have been made to modify the surface topography and composition of Ti implants. In this paper, Lenti-BMP-2-loaded TiO2 nanotube coatings were fabricated by lyophilization in the presence of trehalose to functionalize the surface. We characterized TiO2 nanotube layers in terms of the following: surface morphology; Lenti-BMP-2 and trehalose release; their ability to induce osteogenesis, proliferation, and anti-inflammation in vitro; and osseointegration in vivo. The anodized TiO2 nanotube surfaces exhibited an amorphous glassy matrix perpendicular to the Ti surface. Both Lenti-BMP-2 and trehalose showed sustained release over the course of 8 days. Results from real-time quantitative polymerase chain reaction studies demonstrated that lyophilized Lenti-BMP-2/TiO2 nanotubes constructed with trehalose (Lyo-Tre-Lenti-BMP-2) significantly promoted osteogenic differentiation of bone marrow stromal cells but not their proliferation. In addition, Lyo-Tre-Lenti-BMP-2 nanotubes effectively inhibited lipopolysaccharide-induced interleukin-1β and tumor necrosis factor-α production. In vivo, the formulation also promoted osseointegration. This study presents a promising new method for surface-modifying biomedical Ti-based implants to simultaneously enhance their osteogenic potential and anti-inflammatory properties, which can better satisfy clinical needs. PMID:26869786

Differences in fat and muscle mass associated with a functional human polymorphism in a post-transcriptional BMP2 gene regulatory element.

PubMed

Devaney, Joseph M; Tosi, Laura L; Fritz, David T; Gordish-Dressman, Heather A; Jiang, Shan; Orkunoglu-Suer, Funda E; Gordon, Andrew H; Harmon, Brennan T; Thompson, Paul D; Clarkson, Priscilla M; Angelopoulos, Theodore J; Gordon, Paul M; Moyna, Niall M; Pescatello, Linda S; Visich, Paul S; Zoeller, Robert F; Brandoli, Cinzia; Hoffman, Eric P; Rogers, Melissa B

2009-08-15

A classic morphogen, bone morphogenetic protein 2 (BMP2) regulates the differentiation of pluripotent mesenchymal cells. High BMP2 levels promote osteogenesis or chondrogenesis and low levels promote adipogenesis. BMP2 inhibits myogenesis. Thus, BMP2 synthesis is tightly controlled. Several hundred nucleotides within the 3' untranslated regions of BMP2 genes are conserved from mammals to fishes indicating that the region is under stringent selective pressure. Our analyses indicate that this region controls BMP2 synthesis by post-transcriptional mechanisms. A common A to C single nucleotide polymorphism (SNP) in the BMP2 gene (rs15705, +A1123C) disrupts a putative post-transcriptional regulatory motif within the human ultra-conserved sequence. In vitro studies indicate that RNAs bearing the A or C alleles have different protein binding characteristics in extracts from mesenchymal cells. Reporter genes with the C allele of the ultra-conserved sequence were differentially expressed in mesenchymal cells. Finally, we analyzed MRI data from the upper arm of 517 healthy individuals aged 18-41 years. Individuals with the C/C genotype were associated with lower baseline subcutaneous fat volumes (P = 0.0030) and an increased gain in skeletal muscle volume (P = 0.0060) following resistance training in a cohort of young males. The rs15705 SNP explained 2-4% of inter-individual variability in the measured parameters. The rs15705 variant is one of the first genetic markers that may be exploited to facilitate early diagnosis, treatment, and/or prevention of diseases associated with poor fitness. Furthermore, understanding the mechanisms by which regulatory polymorphisms influence BMP2 synthesis will reveal novel pharmaceutical targets for these disabling conditions. (c) 2009 Wiley-Liss, Inc.

Differences in Fat and Muscle Mass Associated With a Functional Human Polymorphism in a Post-Transcriptional BMP2 Gene Regulatory Element

PubMed Central

Devaney, Joseph M.; Tosi, Laura L.; Fritz, David T.; Gordish-Dressman, Heather A.; Jiang, Shan; Orkunoglu-Suer, Funda E.; Gordon, Andrew H.; Harmon, Brennan T.; Thompson, Paul D.; Clarkson, Priscilla M.; Angelopoulos, Theodore J.; Gordon, Paul M.; Moyna, Niall M.; Pescatello, Linda S.; Visich, Paul S.; Zoeller, Robert F.; Brandoli, Cinzia; Hoffman, Eric P.; Rogers, Melissa B.

2014-01-01

A classic morphogen, bone morphogenetic protein 2 (BMP2) regulates the differentiation of pluripotent mesenchymal cells. High BMP2 levels promote osteogenesis or chondrogenesis and low levels promote adipogenesis. BMP2 inhibits myogenesis. Thus, BMP2 synthesis is tightly controlled. Several hundred nucleotides within the 3′ untranslated regions of BMP2 genes are conserved from mammals to fishes indicating that the region is under stringent selective pressure. Our analyses indicate that this region controls BMP2 synthesis by post-transcriptional mechanisms. A common A to C single nucleotide polymorphism (SNP) in the BMP2 gene (rs15705, +A1123C) disrupts a putative post-transcriptional regulatory motif within the human ultra-conserved sequence. In vitro studies indicate that RNAs bearing the A or C alleles have different protein binding characteristics in extracts from mesenchymal cells. Reporter genes with the C allele of the ultra-conserved sequence were differentially expressed in mesenchymal cells. Finally, we analyzed MRI data from the upper arm of 517 healthy individuals aged 18–41 years. Individuals with the C/C genotype were associated with lower baseline subcutaneous fat volumes (P = 0.0030) and an increased gain in skeletal muscle volume (P = 0.0060) following resistance training in a cohort of young males. The rs15705 SNP explained 2–4% of inter-individual variability in the measured parameters. The rs15705 variant is one of the first genetic markers that maybe exploited to facilitate early diagnosis, treatment, and/or prevention of diseases associated with poor fitness. Furthermore, understanding the mechanisms by which regulatory polymorphisms influence BMP2 synthesis will reveal novel pharmaceutical targets for these disabling conditions. PMID:19492344

Enhanced osteogenic activity and anti-inflammatory properties of Lenti-BMP-2-loaded TiO₂ nanotube layers fabricated by lyophilization following trehalose addition.

PubMed

Zhang, Xiaochen; Zhang, Zhiyuan; Shen, Gang; Zhao, Jun

2016-01-01

To enhance biocompatibility and osseointegration between titanium implants and surrounding bone tissue, numerous efforts have been made to modify the surface topography and composition of Ti implants. In this paper, Lenti-BMP-2-loaded TiO2 nanotube coatings were fabricated by lyophilization in the presence of trehalose to functionalize the surface. We characterized TiO2 nanotube layers in terms of the following: surface morphology; Lenti-BMP-2 and trehalose release; their ability to induce osteogenesis, proliferation, and anti-inflammation in vitro; and osseointegration in vivo. The anodized TiO2 nanotube surfaces exhibited an amorphous glassy matrix perpendicular to the Ti surface. Both Lenti-BMP-2 and trehalose showed sustained release over the course of 8 days. Results from real-time quantitative polymerase chain reaction studies demonstrated that lyophilized Lenti-BMP-2/TiO2 nanotubes constructed with trehalose (Lyo-Tre-Lenti-BMP-2) significantly promoted osteogenic differentiation of bone marrow stromal cells but not their proliferation. In addition, Lyo-Tre-Lenti-BMP-2 nanotubes effectively inhibited lipopolysaccharide-induced interleukin-1β and tumor necrosis factor-α production. In vivo, the formulation also promoted osseointegration. This study presents a promising new method for surface-modifying biomedical Ti-based implants to simultaneously enhance their osteogenic potential and anti-inflammatory properties, which can better satisfy clinical needs.

Single bead-based electrochemical biosensor.

PubMed

Liu, Changchun; Schrlau, Michael G; Bau, Haim H

2009-12-15

A simple, robust, single bead-based electrochemical biosensor was fabricated and characterized. The sensor's working electrode consists of an electrochemically etched platinum wire, with a nominal diameter of 25 microm, hermetically heat-fusion sealed in a pulled glass capillary (micropipette). The sealing process does not require any epoxy or glue. A commercially available, densely functionalized agarose bead was mounted on the tip of the etched platinum wire. The use of a pre-functionalized bead eliminates the tedious and complicated surface functionalization process that is often the bottleneck in the development of electrochemical biosensors. We report on the use of a biotin agarose bead-based, micropipette, electrochemical (Bio-BMP) biosensor to monitor H(2)O(2) concentration and the use of a streptavidin bead-based, micropipette, electrochemical (SA-BMP) biosensor to detect DNA amplicons. The Bio-BMP biosensor's response increased linearly as the H(2)O(2) concentration increased in the range from 1 x 10(-6) to 1.2 x10(-4)M with a detection limit of 5 x 10(-7)M. The SA-BMP was able to detect the amplicons of 1pg DNA template of B. Cereus bacteria, thus providing better detection sensitivity than conventional gel-based electropherograms.

Bone morphogenetic protein (BMP)1-3 enhances bone repair.

PubMed

Grgurevic, Lovorka; Macek, Boris; Mercep, Mladen; Jelic, Mislav; Smoljanovic, Tomislav; Erjavec, Igor; Dumic-Cule, Ivo; Prgomet, Stefan; Durdevic, Dragan; Vnuk, Drazen; Lipar, Marija; Stejskal, Marko; Kufner, Vera; Brkljacic, Jelena; Maticic, Drazen; Vukicevic, Slobodan

2011-04-29

Members of the astacin family of metalloproteinases such as human bone morphogenetic protein 1 (BMP-1) regulate morphogenesis by processing precursors to mature functional extracellular matrix (ECM) proteins and several growth factors including TGFβ, BMP2, BMP4 and GFD8. We have recently discovered that BMP1-3 isoform of the Bmp-1 gene circulates in the human plasma and is significantly increased in patients with acute bone fracture. We hypothesized that circulating BMP1-3 might have an important role in bone repair and serve as a novel bone biomarker. When administered systemically to rats with a long bone fracture and locally to rabbits with a critical size defect of the ulna, recombinant human BMP1-3 enhanced bone healing. In contrast, neutralization of the endogenous BMP1-3 by a specific polyclonal antibody delayed the bone union. Invitro BMP1-3 increased the expression of collagen type I and osteocalcin in MC3T3-E(1) osteoblast like cells, and enhanced the formation of mineralized bone nodules from bone marrow mesenchymal stem cells. We suggest that BMP1-3 is a novel systemic regulator of bone repair. Copyright © 2011 Elsevier Inc. All rights reserved.

HapHop-Physio: a computer game to support cognitive therapies in children.

PubMed

Rico-Olarte, Carolina; López, Diego M; Narváez, Santiago; Farinango, Charic D; Pharow, Peter S

2017-01-01

Care and support of children with physical or mental disabilities are accompanied with serious concerns for parents, families, healthcare institutions, schools, and their communities. Recent studies and technological innovations have demonstrated the feasibility of providing therapy and rehabilitation services to children supported by computer games. The aim of this paper is to present HapHop-Physio, an innovative computer game that combines exercise with fun and learning, developed to support cognitive therapies in children. Conventional software engineering methods such as the Scrum methodology, a functionality test and a related usability test, were part of the comprehensive methodology adapted to develop HapHop-Physio. The game supports visual and auditory attention therapies, as well as visual and auditory memory activities. The game was developed by a multidisciplinary team, which was based on the Hopscotch ® platform provided by Fraunhofer Institute for Digital Media Technology IDMT Institute in Germany, and designed in collaboration with a rehabilitation clinic in Colombia. HapHop-Physio was tested and evaluated to probe its functionality and user satisfaction. The results show the development of an easy-to-use and funny game by a multidisciplinary team using state-of-the-art videogame technologies and software methodologies. Children testing the game concluded that they would like to play again while undergoing rehabilitation therapies.

PDGF-AA promotes osteogenic differentiation and migration of mesenchymal stem cell by down-regulating PDGFRα and derepressing BMP-Smad1/5/8 signaling.

PubMed

Li, Anna; Xia, Xuechun; Yeh, James; Kua, Huiyi; Liu, Huijuan; Mishina, Yuji; Hao, Aijun; Li, Baojie

2014-01-01

Platelet-derived growth factors (PDGFs) play important roles in skeletal development and bone fracture healing, yet how PDGFs execute their functions remains incompletely understood. Here we show that PDGF-AA, but not -AB or -BB, could activate the BMP-Smad1/5/8 pathway in mesenchymal stem cells (MSCs), which requires BMPRIA as well as PDGFRα. PDGF-AA promotes MSC osteogenic differentiation through the BMP-Smad1/5/8-Runx2/Osx axis and MSC migration via the BMP-Smad1/5/8-Twist1/Atf4 axis. Mechanistic studies show that PDGF-AA activates BMP-Smad1/5/8 signaling by feedback down-regulating PDGFRα, which frees BMPRI and allows for BMPRI-BMPRII complex formation to activate smad1/5/8, using BMP molecules in the microenvironment. This study unravels a physical and functional interaction between PDGFRα and BMPRI, which plays an important role in MSC differentiation and migration, and establishes a link between PDGF-AA and BMPs pathways, two essential regulators of embryonic development and tissue homeostasis.

Role of RGM coreceptors in bone morphogenetic protein signaling

PubMed Central

Halbrooks, Peter J; Ding, Ru; Wozney, John M; Bain, Gerard

2007-01-01

Background The repulsive guidance molecule (RGM) proteins, originally discovered for their roles in neuronal development, have been recently identified as co-receptors in the bone morphogenetic protein (BMP) signaling pathway. BMPs are members of the TGFβ superfamily of signaling cytokines, and serve to regulate many aspects of cellular growth and differentiation. Results Here, we investigate whether RGMa, RGMb, and RGMc play required roles in BMP and TGFβ signaling in the mouse myoblast C2C12 cell line. These cells are responsive to BMPs and are frequently used to study BMP/TGFβ signaling pathways. Using siRNA reagents to specifically knock down each RGM protein, we show that the RGM co-receptors are required for significant BMP signaling as reported by two cell-based BMP activity assays: endogenous alkaline phosphatase activity and a luciferase-based BMP reporter assay. Similar cell-based assays using a TGFβ-induced luciferase reporter show that the RGM co-receptors are not required for TGFβ signaling. The binding interaction of each RGM co-receptor to each of BMP2 and BMP12 is observed and quantified, and equilibrium dissociation constants in the low nanomolar range are reported. Conclusion Our results demonstrate that the RGMs play a significant role in BMP signaling and reveal that these molecules cannot functionally compensate for one another. PMID:17615080

BMP-induced reprogramming of the neural retina into retinal pigment epithelium requires Wnt signalling

PubMed Central

Steinfeld, Jörg; Steinfeld, Ichie; Bausch, Alexander; Coronato, Nicola; Hampel, Meggi-Lee; Depner, Heike; Layer, Paul G.

2017-01-01

ABSTRACT In vertebrates, the retinal pigment epithelium (RPE) and photoreceptors of the neural retina (NR) comprise a functional unit required for vision. During vertebrate eye development, a conversion of the RPE into NR can be induced by growth factors in vivo at optic cup stages, but the reverse process, the conversion of NR tissue into RPE, has not been reported. Here, we show that bone morphogenetic protein (BMP) signalling can reprogram the NR into RPE at optic cup stages in chick. Shortly after BMP application, expression of Microphthalmia-associated transcription factor (Mitf) is induced in the NR and selective cell death on the basal side of the NR induces an RPE-like morphology. The newly induced RPE differentiates and expresses Melanosomalmatrix protein 115 (Mmp115) and RPE65. BMP-induced Wnt2b expression is observed in regions of the NR that become pigmented. Loss of function studies show that conversion of the NR into RPE requires both BMP and Wnt signalling. Simultaneous to the appearance of ectopic RPE tissue, BMP application reprogrammed the proximal RPE into multi-layered retinal tissue. The newly induced NR expresses visual segment homeobox-containing gene (Vsx2), and the ganglion and photoreceptor cell markers Brn3α and Visinin are detected. Our results show that high BMP concentrations are required to induce the conversion of NR into RPE, while low BMP concentrations can still induce transdifferentiation of the RPE into NR. This knowledge may contribute to the development of efficient standardized protocols for RPE and NR generation for cell replacement therapies. PMID:28546339

Heterozygous Mutations in BMP6 Pro-peptide Lead to Inappropriate Hepcidin Synthesis and Moderate Iron Overload in Humans.

PubMed

Daher, Raed; Kannengiesser, Caroline; Houamel, Dounia; Lefebvre, Thibaud; Bardou-Jacquet, Edouard; Ducrot, Nicolas; de Kerguenec, Caroline; Jouanolle, Anne-Marie; Robreau, Anne-Marie; Oudin, Claire; Le Gac, Gerald; Moulouel, Boualem; Loustaud-Ratti, Veronique; Bedossa, Pierre; Valla, Dominique; Gouya, Laurent; Beaumont, Carole; Brissot, Pierre; Puy, Hervé; Karim, Zoubida; Tchernitchko, Dimitri

2016-03-01

Hereditary hemochromatosis is a heterogeneous group of genetic disorders characterized by parenchymal iron overload. It is caused by defective expression of liver hepcidin, the main regulator of iron homeostasis. Iron stimulates the gene encoding hepcidin (HAMP) via the bone morphogenetic protein (BMP)6 signaling to SMAD. Although several genetic factors have been found to cause late-onset hemochromatosis, many patients have unexplained signs of iron overload. We investigated BMP6 function in these individuals. We sequenced the BMP6 gene in 70 consecutive patients with a moderate increase in serum ferritin and liver iron levels who did not carry genetic variants associated with hemochromatosis. We searched for BMP6 mutations in relatives of 5 probands and in 200 healthy individuals (controls), as well as in 2 other independent cohorts of hyperferritinemia patients. We measured serum levels of hepcidin by liquid chromatography-tandem mass spectrometry and analyzed BMP6 in liver biopsy specimens from patients by immunohistochemistry. The functions of mutant and normal BMP6 were assessed in transfected cells using immunofluorescence, real-time quantitative polymerase chain reaction, and immunoblot analyses. We identified 3 heterozygous missense mutations in BMP6 (p.Pro95Ser, p.Leu96Pro, and p.Gln113Glu) in 6 unrelated patients with unexplained iron overload (9% of our cohort). These mutations were detected in less than 1% of controls. p.Leu96Pro also was found in 2 patients from the additional cohorts. Family studies indicated dominant transmission. Serum levels of hepcidin were inappropriately low in patients. A low level of BMP6, compared with controls, was found in a biopsy specimen from 1 patient. In cell lines, the mutated residues in the BMP6 propeptide resulted in defective secretion of BMP6; reduced signaling via SMAD1, SMAD5, and SMAD8; and loss of hepcidin production. We identified 3 heterozygous missense mutations in BMP6 in patients with unexplained iron overload. These mutations lead to loss of signaling to SMAD proteins and reduced hepcidin production. These mutations might increase susceptibility to mild-to-moderate late-onset iron overload. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Preliminary evaluation of a load-bearing BMP-2 carrier for segmental defect regeneration.

PubMed

Chu, Tien-Min G; Sargent, Peter; Warden, Stuart J; Turner, Charles H; Stewart, Rena L

2006-01-01

Large segmental defects in bones can result from tumor removal, massive trauma, congenital malformation, or non-union fractures. Such defects often are difficult to manage and require multiple-phase surgery to achieve adequate union and function. In this study, we propose a novel design of bone morphogenetic protein 2 (BMP-2) carrier for tissue engineering of segmental defect regeneration. The tube-shaped BMP-2 carrier was fabrication from a poly(propylene fumarate)/tricalcium phosphate (PPF/TCP) composite via casting technique developed in our laboratory. An in vitro evaluation showed that the compressive strength of the carrier decreased about 48% in 12 weeks while maintained a pH in the 6.8-7.4 range. In vivo study was conducted by implanting carriers loaded with 10 microg of BMP-2 in 5 mm rat femur gap model for 15 weeks. X-ray evidence of bridging was first found in the BMP group at 3 weeks. Bridging in all animals (N = 4) in the BMP group was found at 9 weeks. No x-ray evidence of bridging was found in the No BMP group (N = 3). pQCT analysis indicated that the bone mineral density of the callus in the BMP group has reached the level of native femur at 15 weeks after implantation, while the callus in the No BMP group has a bone mineral density at a lower level of 84% to the native femur. Histology analysis shows that a normal fatty bone marrow was restored and mineralized callus formed and bridged the segmental defect.

BMP6 down-regulates GDNF expression through SMAD1/5 and ERK1/2 signaling pathways in human granulosa-lutein cells.

PubMed

Zhang, Xin-Yue; Chang, Hsun-Ming; Taylor, Elizabeth L; Leung, Peter C K; Liu, Rui-Zhi

2018-05-09

Bone morphogenetic protein 6 (BMP6) is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells. Glial cell line-derived neurotrophic factor (GDNF) is an intraovarian neurotrophic factor that plays an essential role in regulating mammalian oocyte maturation. The aim of this study was to investigate the effect of BMP6 on the regulation of GDNF expression and the potential underlying mechanisms. We used an established immortalized human granulosa cell line (SVOG cells) and primary human granulosa-lutein cells as in vitro cell models. Our results showed that BMP6 significantly down-regulated the expression of GDNF in both SVOG and primary human granulosa-lutein cells. Using dual inhibition approaches (kinase receptor inhibitor and small interfering RNA knockdown), our results showed that both ALK2 and ALK3 are involved in BMP6-induced down-regulation of GDNF. In addition, BMP6 induced the phosphorylation of SMAD1/5/8 and ERK1/2 but not AKT or p38. Among three downstream mediators, both SMAD1 and SMAD5 are involved in BMP6-induced down-regulation of GDNF. Moreover, concomitant knockdown of endogenous SMAD4 and inhibition of ERK1/2 activity completely reversed BMP6-induced down-regulation of GDNF, indicating that both SMAD and ERK1/2 signaling pathways are required for the regulatory effect of BMP6 on GDNF expression. Our findings suggest an additional role for an intrafollicular growth factor in regulating follicular function through their paracrine interactions in human granulosa cells.

Experimental and Computational Investigation of the Effect of Hydrophobicity on Aggregation and Osteoinductive Potential of BMP-2-Derived Peptide in a Hydrogel Matrix

PubMed Central

Moeinzadeh, Seyedsina; Barati, Danial; Sarvestani, Samaneh K.; Karimi, Tahereh

2015-01-01

An attractive approach to reduce the undesired side effects of bone morphogenetic proteins (BMPs) in regenerative medicine is to use osteoinductive peptide sequences derived from BMPs. Although the structure and function of BMPs have been studied extensively, there is limited data on structure and activity of BMP-derived peptides immobilized in hydrogels. The objective of this work was to investigate the effect of concentration and hydrophobicity of the BMP-2 peptide, corresponding to residues 73–92 of the knuckle epitope of BMP-2 protein, on peptide aggregation and osteogenic differentiation of human mesenchymal stem cells encapsulated in a polyethylene glycol (PEG) hydrogel. The peptide hydrophobicity was varied by capping PEG chain ends with short lactide segments. The BMP-2 peptide with a positive index of hydrophobicity had a critical micelle concentration (CMC) and formed aggregates in aqueous solution. Based on simulation results, there was a slight increase in the concentration of free peptide in solution with 1000-fold increase in peptide concentration. The dose-osteogenic response curve of the BMP-2 peptide was in the 0.0005–0.005 mM range, and osteoinductive potential of the BMP-2 peptide was significantly less than that of BMP-2 protein even at 1000-fold higher concentrations, which was attributed to peptide aggregation. Further, the peptide or PEG-peptide aggregates had significantly higher interaction energy with the cell membrane compared with the free peptide, which led to a higher nonspecific interaction with the cell membrane and loss of osteoinductive potential. Conjugation of the BMP-2 peptide to PEG increased CMC and osteoinductive potential of the peptide whereas conjugation to lactide-capped PEG reduced CMC and osteoinductive potential of the peptide. Experimental and simulation results revealed that osteoinductive potential of the BMP-2 peptide is correlated with its CMC and the free peptide concentration in aqueous medium and not the total concentration. PMID:25051457

Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification.

PubMed

Dang, Phuong N; Dwivedi, Neha; Phillips, Lauren M; Yu, Xiaohua; Herberg, Samuel; Bowerman, Caitlin; Solorio, Loran D; Murphy, William L; Alsberg, Eben

2016-02-01

Bone tissue engineering via endochondral ossification has been explored by chondrogenically priming cells using soluble mediators for at least 3 weeks to produce a hypertrophic cartilage template. Although recapitulation of endochondral ossification has been achieved, long-term in vitro culture is required for priming cells through repeated supplementation of inductive factors in the media. To address this challenge, a microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Sequential exogenous presentation of soluble transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) at various defined time courses resulted in varying degrees of chondrogenesis and osteogenesis as demonstrated by glycosaminoglycan and calcium content. The time course that best induced endochondral ossification was used to guide the development of the microparticle-based controlled delivery system for TGF-β1 and BMP-2. Gelatin microparticles capable of relatively rapid release of TGF-β1 and mineral-coated hydroxyapatite microparticles permitting more sustained release of BMP-2 were then incorporated within hMSC aggregates and cultured for 5 weeks following the predetermined time course for sequential presentation of bioactive signals. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated TGF-β1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity at week 2 and a greater degree of mineralization by week 5. Staining for types I and II collagen, osteopontin, and osteocalcin revealed the presence of cartilage and bone. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming. Significance: This study demonstrates the regulation of chondrogenesis and osteogenesis with regard to endochondral bone formation in high-density stem cell systems through the controlled presentation of inductive factors from incorporated microparticles. This work lays the foundation for a rapidly implantable tissue engineering system that promotes bone repair via endochondral ossification, a pathway that can delay the need for a functional vascular network and has an intrinsic ability to promote angiogenesis. The modular nature of this system lends well to using different cell types and/or growth factors to induce endochondral bone formation, as well as the production of other tissue types. ©AlphaMed Press.

Investigating the effect of peptide agonists on the chondrogenic differentiation of human mesenchymal stem cells using design of experiments.

PubMed

Renner, Julie N; Liu, Julie C

2013-01-01

Human mesenchymal stem cells (MSCs) are attractive for use in cartilage tissue engineering. Cells are often seeded in a structural scaffold containing growth factors. Peptide mimics of full-length growth factors are a promising alternative because they are less expensive and easier to manufacture. We investigated four short peptides for their effect on the chondrogenesis of human MSCs. The peptides were originally designed to mimic bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta 1 (TGF-β1), and insulin, all of which have been shown to affect MSC chondrogenesis. Previous studies demonstrated that the peptides elicited bioactivity in other cell types, but the peptides have not been investigated for their effect on chondrogenesis in human MSCs. In a preliminary investigation, peptides were added to a pellet culture of human MSCs and assayed for their effect on glycosaminoglycan (GAG) production. These experiments determined peptide concentrations used in a full-factorial experiment to investigate any interactions. The experiment revealed the BMP peptide as a robust stimulant for GAG production. . © 2013 American Institute of Chemical Engineers.

In vitro BMP-2 peptide release from thiolated chitosan based hydrogel.

PubMed

Liu, Xujie; Yu, Bo; Huang, Qianli; Liu, Rui; Feng, Qingling; Cai, Qiang; Mi, Shengli

2016-12-01

Thiolated chitosan based thermo-sensitive hydrogel is a water soluble system and the existing thiol groups are beneficial for the delivery of cysteine-rich peptides. In the present study, a kind of thiolated chitosan, i.e. chitosan-4-thio-butylamidine (CS-TBA) conjugate was characterized and used to prepare CS-TBA/hydroxyapatite (HA)/beta-glycerophosphate disodium (β-GP) thermo-sensitive hydrogel. The cysteine terminated peptide 24 (P24) containing residues 73-92 of the knuckle epitope of BMP-2 (N→C: KIPKASSVPTELSAISTLYLSGGC) was synthesized and characterized. The release behavior of P24 from CS-TBA based hydrogel was investigated in vitro. The thiol groups in CS-TBA may react with thiol groups in P24, thus decreases the P24 release rate and maintains the peptide release for a longer time compared with unmodified chitosan based hydrogel. Moreover, the bioactivity of P24 is preserved during release process. These results indicate that P24 loaded CS-TBA based thermosensitive hydrogel is a potential material for minimally invasive surgery of bone repair. Copyright © 2016 Elsevier B.V. All rights reserved.

PhysioNet: physiologic signals, time series and related open source software for basic, clinical, and applied research.

PubMed

Moody, George B; Mark, Roger G; Goldberger, Ary L

2011-01-01

PhysioNet provides free web access to over 50 collections of recorded physiologic signals and time series, and related open-source software, in support of basic, clinical, and applied research in medicine, physiology, public health, biomedical engineering and computing, and medical instrument design and evaluation. Its three components (PhysioBank, the archive of signals; PhysioToolkit, the software library; and PhysioNetWorks, the virtual laboratory for collaborative development of future PhysioBank data collections and PhysioToolkit software components) connect researchers and students who need physiologic signals and relevant software with researchers who have data and software to share. PhysioNet's annual open engineering challenges stimulate rapid progress on unsolved or poorly solved questions of basic or clinical interest, by focusing attention on achievable solutions that can be evaluated and compared objectively using freely available reference data.

High resolution Physio-chemical Tissue Analysis: Towards Non-invasive In Vivo Biopsy

NASA Astrophysics Data System (ADS)

Xu, Guan; Meng, Zhuo-Xian; Lin, Jian-Die; Deng, Cheri X.; Carson, Paul L.; Fowlkes, J. Brian; Tao, Chao; Liu, Xiaojun; Wang, Xueding

2016-02-01

Conventional gold standard histopathologic diagnosis requires information of both high resolution structural and chemical changes in tissue. Providing optical information at ultrasonic resolution, photoacoustic (PA) technique could provide highly sensitive and highly accurate tissue characterization noninvasively in the authentic in vivo environment, offering a replacement for histopathology. A two-dimensional (2D) physio-chemical spectrogram (PCS) combining micrometer to centimeter morphology and chemical composition simultaneously can be generated for each biological sample with PA measurements at multiple optical wavelengths. This spectrogram presents a unique 2D “physio-chemical signature” for any specific type of tissue. Comprehensive analysis of PCS, termed PA physio-chemical analysis (PAPCA), can lead to very rich diagnostic information, including the contents of all relevant molecular and chemical components along with their corresponding histological microfeatures, comparable to those accessible by conventional histology. PAPCA could contribute to the diagnosis of many diseases involving diffusive patterns such as fatty liver.

Wnt/β-catenin pathway regulates Bmp2-mediated differentiation of dental follicle cells

PubMed Central

Silvério, Karina G.; Davidson, Kathryn C.; James, Richard G.; Adams, Allison M.; Foster, Brian L.; Nociti, Francisco H.; Somermam, Martha J.; Moon, Randall T.

2013-01-01

Background and Objectives Bmp2-induced osteogenic differentiation has been shown to occur through the canonical Wnt/β-catenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibited cell differentiation and promoted cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with Bmp2, would exhibit changes in genes/proteins associated with the Wnt/β-catenin pathway. Materials and Methods SVF4 cells were stimulated with Bmp2, and the following assays were carried out: 1) Wnt/β-catenin pathway activation assessed by western blot, β-catenin/TCF reporter assay, and gene expression of lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2, and 2) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp) by qPCR after Wnt3a treatment and knockdown of β-catenin. Results Wnt3a induced β-catenin nuclear translocation and upregulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with Wnt3a suppressed Bmp2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that Bmp2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-catenin. Wnt3a down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared to untreated cells. In contrast, Bmp2 induction of Bsp transcripts occurred independent of Wnt/β-catenin signaling. Conclusions These data suggest that stabilization of β-catenin by Wnt-3a treatment inhibits Bmp2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although Bmp2 requires endogenous Wnt/β-catenin signaling to promote cell maturation. PMID:22150562

Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3.

PubMed

Dwivedi, Prem P; Anderson, Peter J; Powell, Barry C

2012-08-03

Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.

Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation

PubMed Central

Levet, Sandrine; Ciais, Delphine; Merdzhanova, Galina; Mallet, Christine; Zimmers, Teresa A.; Lee, Se-Jin; Navarro, Fabrice P.; Texier, Isabelle; Feige, Jean-Jacques; Bailly, Sabine

2013-01-01

Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-β family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function. PMID:23741013

Reciprocal repression between Fgf8 and miR-133 regulates cardiac induction through Bmp2 signaling.

PubMed

Lopez-Sanchez, Carmen; Franco, Diego; Bonet, Fernando; Garcia-Lopez, Virginio; Aranega, Amelia; Garcia-Martinez, Virginio

2015-12-01

This data article contains complementary figures and results related to the research article entitled "Negative Fgf8-Bmp2 feed-back is controlled by miR-130 during early cardiac specification" [15], which reveals what specific role miR-130 plays during the cardiac induction process. This study evidenced miR-130 a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR- as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2. Thus, miR-130 regulates a negative Fgf8-Bmp2 feed-back loop responsible to achieve early cardiac specification. A significant aspect supporting our conclusions is given by the expression pattern of miR-130 during early cardiac specification, as well as by those results obtained after the designed experimental procedures. The data presented here reveal that miR-133 is also expressed within the precardiac areas during early cardiogenesis, pattern which is comparable to that of FGFR1, receptor involved in the Fgf8/ERK signaling pathway. Interestingly, our miR-133 overexpression experiments resulted in a decrease of Fgf8 expression, whereas we observed an increase of Bmp2 and subsequently of cardiac specific markers Nkx-2.5 and Gata4. Additionally, our loss-of-function experiments -through Fgf8 siRNA electroporation- showed an increase of miR-133 expression. Finally, after our Bmp2 experiments, we observed that miR-133 is upstream-regulated by Bmp2. All those results suggest that miR-133 also constitutes a crucial linkage in the crosstalk between Fgf8 and Bmp2 signaling by regulating the Fgf8/ERK pathway during cardiac induction.

TGF-β/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation

PubMed Central

Rahman, Md Shaifur; Akhtar, Naznin; Jamil, Hossen Mohammad; Banik, Rajat Suvra; Asaduzzaman, Sikder M

2015-01-01

Transforming growth factor-beta (TGF-β)/bone morphogenetic protein (BMP) plays a fundamental role in the regulation of bone organogenesis through the activation of receptor serine/threonine kinases. Perturbations of TGF-β/BMP activity are almost invariably linked to a wide variety of clinical outcomes, i.e., skeletal, extra skeletal anomalies, autoimmune, cancer, and cardiovascular diseases. Phosphorylation of TGF-β (I/II) or BMP receptors activates intracellular downstream Smads, the transducer of TGF-β/BMP signals. This signaling is modulated by various factors and pathways, including transcription factor Runx2. The signaling network in skeletal development and bone formation is overwhelmingly complex and highly time and space specific. Additive, positive, negative, or synergistic effects are observed when TGF-β/BMP interacts with the pathways of MAPK, Wnt, Hedgehog (Hh), Notch, Akt/mTOR, and miRNA to regulate the effects of BMP-induced signaling in bone dynamics. Accumulating evidence indicates that Runx2 is the key integrator, whereas Hh is a possible modulator, miRNAs are regulators, and β-catenin is a mediator/regulator within the extensive intracellular network. This review focuses on the activation of BMP signaling and interaction with other regulatory components and pathways highlighting the molecular mechanisms regarding TGF-β/BMP function and regulation that could allow understanding the complexity of bone tissue dynamics. PMID:26273537

TGF-β and BMP Signaling in Osteoblast Differentiation and Bone Formation

PubMed Central

Chen, Guiqian; Deng, Chuxia; Li, Yi-Ping

2012-01-01

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation. PMID:22298955

Intracellular co-delivery of Sr ion and phenamil drug through mesoporous bioglass nanocarriers synergizes BMP signaling and tissue mineralization.

PubMed

Lee, Jung-Hwan; Mandakhbayar, Nandin; El-Fiqi, Ahmed; Kim, Hae-Won

2017-09-15

Inducing differentiation and maturation of resident multipotent stem cells (MSCs) is an important strategy to regenerate hard tissues in mal-calcification conditions. Here we explore a co-delivery approach of therapeutic molecules comprised of ion and drug through a mesoporous bioglass nanoparticle (MBN) for this purpose. Recently, MBN has offered unique potential as a nanocarrier for hard tissues, in terms of high mesoporosity, bone bioactivity (and possibly degradability), tunable delivery of biomolecules, and ionic modification. Herein Sr ion is structurally doped to MBN while drug Phenamil is externally loaded as a small molecule activator of BMP signaling, for the stimulation of osteo/odontogenesis and mineralization of human MSCs derived from dental pulp. The Sr-doped MBN (85Si:10Ca:5Sr) sol-gel processed presents a high mesoporosity with a pore size of ∼6nm. In particular, Sr ion is released slowly at a daily rate of ∼3ppm per mg nanoparticles for up to 7days, a level therapeutically effective for cellular stimulation. The Sr-MBN is internalized to most MSCs via an ATP dependent macropinocytosis within hours, increasing the intracellular levels of Sr, Ca and Si ions. Phenamil is loaded maximally ∼30% into Sr-MBN and then released slowly for up to 7days. The co-delivered molecules (Sr ion and Phenamil drug) have profound effects on the differentiation and maturation of cells, i.e., significantly enhancing expression of osteo/odontogenic genes, alkaline phosphatase activity, and mineralization of cells. Of note, the stimulation is a result of a synergism of Sr and Phenamil, through a Trb3-dependent BMP signaling pathway. This biological synergism is further evidenced in vivo in a mal-calcification condition involving an extracted tooth implantation in dorsal subcutaneous tissues of rats. Six weeks post operation evidences the osseous-dentinal hard tissue formation, which is significantly stimulated by the Sr/Phenamil delivery, based on histomorphometric and micro-computed tomographic analyses. The bioactive nanoparticles releasing both Sr ion and Phenamil drug are considered to be a promising therapeutic nanocarrier platform for hard tissue regeneration. Furthermore, this novel ion/drug co-delivery concept through nanoparticles can be extensively used for other tissues that require different therapeutic treatment. This study reports a novel design concept in inorganic nanoparticle delivery system for hard tissues - the co-delivery of therapeutic molecules comprised of ion (Sr) and drug (Phenamil) through a unique nanoparticle of mesoporous bioactive glass (MBN). The physico-chemical and biological properties of MBN enabled an effective loading of both therapeutic molecules and a subsequently sustained/controlled release. The co-delivered Sr and Phenamil demonstrated significant stimulation of adult stem cell differentiation in vitro and osseous/dentinal regeneration in vivo, through BMP signaling pathways. We consider the current combination of Sr ion with Phenamil is suited for the osteo/odontogenesis of stem cells for hard tissue regeneration, and further, this ion/drug co-delivery concept can extend the applications to other areas that require specific cellular and tissue functions. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

HapHop-Physio: a computer game to support cognitive therapies in children

PubMed Central

Rico-Olarte, Carolina; López, Diego M; Narváez, Santiago; Farinango, Charic D; Pharow, Peter S

2017-01-01

Background Care and support of children with physical or mental disabilities are accompanied with serious concerns for parents, families, healthcare institutions, schools, and their communities. Recent studies and technological innovations have demonstrated the feasibility of providing therapy and rehabilitation services to children supported by computer games. Objective The aim of this paper is to present HapHop-Physio, an innovative computer game that combines exercise with fun and learning, developed to support cognitive therapies in children. Methods Conventional software engineering methods such as the Scrum methodology, a functionality test and a related usability test, were part of the comprehensive methodology adapted to develop HapHop-Physio. Results The game supports visual and auditory attention therapies, as well as visual and auditory memory activities. The game was developed by a multidisciplinary team, which was based on the Hopscotch® platform provided by Fraunhofer Institute for Digital Media Technology IDMT Institute in Germany, and designed in collaboration with a rehabilitation clinic in Colombia. HapHop-Physio was tested and evaluated to probe its functionality and user satisfaction. Conclusion The results show the development of an easy-to-use and funny game by a multidisciplinary team using state-of-the-art videogame technologies and software methodologies. Children testing the game concluded that they would like to play again while undergoing rehabilitation therapies. PMID:28740440

Bone morphogenetic protein 4 antagonizes hair cell regeneration in the avian auditory epithelium.

PubMed

Lewis, Rebecca M; Keller, Jesse J; Wan, Liangcai; Stone, Jennifer S

2018-07-01

Permanent hearing loss is often a result of damage to cochlear hair cells, which mammals are unable to regenerate. Non-mammalian vertebrates such as birds replace damaged hair cells and restore hearing function, but mechanisms controlling regeneration are not understood. The secreted protein bone morphogenetic protein 4 (BMP4) regulates inner ear morphogenesis and hair cell development. To investigate mechanisms controlling hair cell regeneration in birds, we examined expression and function of BMP4 in the auditory epithelia (basilar papillae) of chickens of either sex after hair cell destruction by ototoxic antibiotics. In mature basilar papillae, BMP4 mRNA is highly expressed in hair cells, but not in hair cell progenitors (supporting cells). Supporting cells transcribe genes encoding receptors for BMP4 (BMPR1A, BMPR1B, and BMPR2) and effectors of BMP4 signaling (ID transcription factors). Following hair cell destruction, BMP4 transcripts are lost from the sensory epithelium. Using organotypic cultures, we demonstrate that treatments with BMP4 during hair cell destruction prevent supporting cells from upregulating expression of the pro-hair cell transcription factor ATOH1, entering the cell cycle, and fully transdifferentiating into hair cells, but they do not induce cell death. By contrast, noggin, a BMP4 inhibitor, increases numbers of regenerated hair cells. These findings demonstrate that BMP4 antagonizes hair cell regeneration in the chicken basilar papilla, at least in part by preventing accumulation of ATOH1 in hair cell precursors. Copyright © 2018 Elsevier B.V. All rights reserved.

Attenuated BMP1 Function Compromises Osteogenesis, Leading to Bone Fragility in Humans and Zebrafish

PubMed Central

Asharani, P.V.; Keupp, Katharina; Semler, Oliver; Wang, Wenshen; Li, Yun; Thiele, Holger; Yigit, Gökhan; Pohl, Esther; Becker, Jutta; Frommolt, Peter; Sonntag, Carmen; Altmüller, Janine; Zimmermann, Katharina; Greenspan, Daniel S.; Akarsu, Nurten A.; Netzer, Christian; Schönau, Eckhard; Wirth, Radu; Hammerschmidt, Matthias; Nürnberg, Peter; Wollnik, Bernd; Carney, Thomas J.

2012-01-01

Bone morphogenetic protein 1 (BMP1) is an astacin metalloprotease with important cellular functions and diverse substrates, including extracellular-matrix proteins and antagonists of some TGFβ superfamily members. Combining whole-exome sequencing and filtering for homozygous stretches of identified variants, we found a homozygous causative BMP1 mutation, c.34G>C, in a consanguineous family affected by increased bone mineral density and multiple recurrent fractures. The mutation is located within the BMP1 signal peptide and leads to impaired secretion and an alteration in posttranslational modification. We also characterize a zebrafish bone mutant harboring lesions in bmp1a, demonstrating conservation of BMP1 function in osteogenesis across species. Genetic, biochemical, and histological analyses of this mutant and a comparison to a second, similar locus reveal that Bmp1a is critically required for mature-collagen generation, downstream of osteoblast maturation, in bone. We thus define the molecular and cellular bases of BMP1-dependent osteogenesis and show the importance of this protein for bone formation and stability. PMID:22482805

Cobalt chromium alloy with immobilized BMP peptide for enhanced bone growth.

PubMed

Poh, Chye Khoon; Shi, Zhilong; Tan, Xiao Wei; Liang, Zhen Chang; Foo, Xue Mei; Tan, Hark Chuan; Neoh, Koon Gee; Wang, Wilson

2011-09-01

Cobalt chromium (CoCr) alloys are widely used in orthopedic practice, however, lack of integration into the bone for long-term survival often occurs, leading to implant failure. Revision surgery to address such a failure involves increased risks, complications, and costs. Advances to enhancement of bone-implant interactions would improve implant longevity and long-term results. Therefore, we investigated the effects of BMP peptide covalently grafted to CoCr alloy on osteogenesis. The BMP peptide was derived from the knuckle epitope of bone morphogenic protein-2 (BMP-2) and was conjugated via a cysteine amino acid at the N-terminus. X-ray photoelectron spectroscopy and o-phthaldialdehyde were used to verify successful grafting at various stages of surface functionalization. Surface topography was evaluated from the surface profile determined by atomic force microscopy. Osteoblastic cells (MC3T3-E1) were seeded on the substrates, and the effects of BMP peptide on osteogenic differentiation were evaluated by measuring alkaline phosphatase (ALP) activity and calcium mineral deposition. The functionalized surfaces showed a twofold increase in ALP activity after 2 weeks incubation and a fourfold increase in calcium content after 3 weeks incubation compared to the pristine substrate. These findings are potentially useful in the development of improved CoCr implants for use in orthopedic applications. Copyright © 2011 Orthopaedic Research Society.

Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats.

PubMed

Hu, Tao; Abbah, Sunny Akogwu; Toh, Soo Yein; Wang, Ming; Lam, Raymond Wing Moon; Naidu, Mathanapriya; Bhakta, Gajadhar; Cool, Simon M; Bhakoo, Kishore; Li, Jun; Goh, James Cho-Hong; Wong, Hee-Kit

2015-12-01

The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. A prospective study using a rodent model of posterolateral spinal fusion was carried out. Thirty-six syngeneic Fischer rats comprised the patient sample. Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and β-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct. Copyright © 2015 Elsevier Inc. All rights reserved.

Correction of nonsense BMPR2 and SMAD9 mutations by ataluren in pulmonary arterial hypertension.

PubMed

Drake, Kylie M; Dunmore, Benjamin J; McNelly, Lauren N; Morrell, Nicholas W; Aldred, Micheala A

2013-09-01

Heritable pulmonary arterial hypertension (HPAH) is a serious lung vascular disease caused by heterozygous mutations in the bone morphogenetic protein (BMP) pathway genes, BMPR2 and SMAD9. One noncanonical function of BMP signaling regulates biogenesis of a subset of microRNAs. We have previously shown that this function is abrogated in patients with HPAH, making it a highly sensitive readout of BMP pathway integrity. Ataluren (PTC124) is an investigational drug that permits ribosomal readthrough of premature stop codons, resulting in a full-length protein. It exhibits oral bioavailability and limited toxicity in human trials. Here, we tested ataluren in lung- or blood-derived cells from patients with HPAH with nonsense mutations in BMPR2 (n = 6) or SMAD9 (n = 1). Ataluren significantly increased BMP-mediated microRNA processing in six of the seven cases. Moreover, rescue was achieved even for mutations exhibiting significant nonsense-mediated mRNA decay. Response to ataluren was dose dependent, and complete correction was achieved at therapeutic doses currently used in clinical trials for cystic fibrosis. BMP receptor (BMPR)-II protein levels were normalized and ligand-dependent phosphorylation of downstream target Smads was increased. Furthermore, the usually hyperproliferative phenotype of pulmonary artery endothelial and smooth muscle cells was reversed by ataluren. These results indicate that ataluren can effectively suppress a high proportion of BMPR2 and SMAD9 nonsense mutations and correct BMP signaling in vitro. Approximately 29% of all HPAH mutations are nonsense point mutations. In light of this, we propose ataluren as a potential new personalized therapy for this significant subgroup of patients with PAH.

Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2.

PubMed

Fukuda, Tomokazu; Scott, Gregory; Komatsu, Yoshihiro; Araya, Runa; Kawano, Masako; Ray, Manas K; Yamada, Masahisa; Mishina, Yuji

2006-04-01

BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines. Published 2006 Wiley-Liss, Inc.

Sensory Nerve Induced Inflammation Contributes to Heterotopic Ossification

PubMed Central

Salisbury, Elizabeth; Rodenberg, Eric; Sonnet, Corinne; Hipp, John; Gannon, Francis H.; Vadakkan, Tegy J.; Dickinson, Mary E.; Olmsted-Davis, Elizabeth A.; Davis, Alan R.

2012-01-01

Heterotopic ossification (HO), or bone formation in soft tissues, is often the result of traumatic injury. Much evidence has linked the release of BMPs (bone morphogenetic proteins) upon injury to this process. HO was once thought to be a rare occurrence, but recent statistics from the military suggest that as many as 60% of traumatic injuries, resulting from bomb blasts, have associated HO. In this study, we attempt to define the role of peripheral nerves in this process. Since BMP2 has been shown previously to induce release of the neuroinflammatory molecules, substance P (SP) and calcitonin gene related peptide (CGRP), from peripheral, sensory neurons, we examined this process in vivo. SP and CGRP are rapidly expressed upon delivery of BMP2 and remain elevated throughout bone formation. In animals lacking functional sensory neurons (TRPV1−/−), BMP2-mediated increases in SP and CGRP were suppressed as compared to the normal animals, and HO was dramatically inhibited in these deficient mice, suggesting that neuroinflammation plays a functional role. Mast cells, known to be recruited by SP and CGRP, were elevated after BMP2 induction. These mast cells were localized to the nerve structures and underwent degranulation. When degranulation was inhibited using cromolyn, HO was again reduced significantly. Immunohistochemical analysis revealed nerves expressing the stem cell markers nanog and Klf4, as well as the osteoblast marker osterix, after BMP2 induction, in mice treated with cromolyn. The data collectively suggest that BMP2 can act directly on sensory neurons to induce neurogenic inflammation, resulting in nerve remodeling and the migration/release of osteogenic and other stem cells from the nerve. Further, blocking this process significantly reduces HO, suggesting that the stem cell population contributes to bone formation. PMID:21678472

Vancomycin-bearing synthetic bone graft delivers rhBMP-2 and promotes healing of critical rat femoral segmental defects.

PubMed

Skelly, Jordan D; Lange, Jeffrey; Filion, Tera M; Li, Xinning; Ayers, David C; Song, Jie

2014-12-01

Bone grafts simultaneously delivering therapeutic proteins and antibiotics may be valuable in orthopaedic trauma care. Previously, we developed a poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) synthetic bone graft that, when preabsorbed with 400-ng rhBMP-2/7, facilitated the functional repair of critical-size rat femoral defects. Recently, we showed that pHEMA-nHA effectively retains/releases vancomycin and rhBMP-2 in vitro. The success of such a strategy requires that the incorporation of vancomycin does not compromise the structural integrity of the graft nor its ability to promote bone healing. (1) To evaluate the ability of pHEMA-nHA-vancomycin composites in combination with 3-µg rhBMP-2 to repair 5 mm rat femoral segmental defects, and (2) To determine if the encapsulated vancomycin impairs the graft/rhBMP-2-assisted bone repair. pHEMA-nHA-vancomycin, pHEMA-nHA, or collagen sponge control with/without 3-µg rhBMP-2 were press-fit in 5 mm femoral defects in SASCO-SD male rats (289-300 g). Histology, microcomputed tomography, and torsion testing were performed on 8- and 12-week explants to evaluate the extent and quality of repair. The effect of vancomycin on the temporal absorption of endogenous BMP-2 and stromal cell-derived factor-1 was evaluated by immunohistochemistry. These factors are important for bone healing initiation and stem cell recruitment, respectively. Partial bridging of the defect with bony callus by 12 weeks was observed with pHEMA-nHA-vancomycin without rhBMP-2 while full bridging with substantially mineralized callus and partial restoration of torsional strength was achieved with 3-µg rhBMP-2. The presence of vancomycin changed the absorption patterns of endogenous proteins on the grafts, but did not appear to substantially compromise graft healing. The composite pHEMA-nHA-vancomycin preabsorbed with 3-µg rhBMP-2 promoted repair of 5 mm rat femoral segmental defects. With the sample sizes applied, vancomycin encapsulation did not appear to have a negative effect on bone healing. pHEMA-nHA-vancomycin preabsorbed with rhBMP-2 may be useful in the repair of critical-size long bone defects prone to infections.

Bone Morphogenetic Proteins, Antagonists and Receptors in Prostate Cancer

DTIC Science & Technology

2005-01-01

expressed in prostate. This work investigates BMP receptors and BMP antagonists to understand the basic mechanisms to inhibit the BMP signaling in...during embryoge- nesis, and prostate cancer metastases to bone. BMP functions can be inhibited by antagonists such as Noggin or DAN. DAN is a protein...protein along with a constant 0-6 -1 10 100 1000 1O0ng/ml of BMP-6, we were able to show a ng/ml BMP-6 dose-dependent inhibition of BMP-6 activity in DU

Aggressive melanoma cells escape from BMP7-mediated autocrine growth inhibition through coordinated Noggin upregulation

PubMed Central

Hsu, Mei-Yu; Rovinsky, Sherry; Lai, Chiou-Yan; Qasem, Shadi; Liu, Xiaoming; How, Joan; Engelhardt, John F.; Murphy, George F.

2009-01-01

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily responsible for mediating a diverse array of cellular functions both during embryogenesis and in adult life. Previously, we reported that upregulation of BMP7 in human melanoma correlates with tumor progression. However, melanoma cells are either inhibited by or become resistant to BMP7 as a function of tumor progression, with normal melanocytes being most susceptible. Herein, real-time quantitative reverse transcriptase-polymerase chain reactions and Western blotting revealed that the expression of BMP antagonist, Noggin, correlates with resistance to BMP7 in advanced melanoma cells. To test the hypothesis that coordinated upregulation of Noggin protects advanced melanoma cells from autocrine inhibition by BMP7, functional expression of Noggin in susceptible melanoma cells was achieved by adenoviral gene transfer. The Noggin-overexpressing cells exhibited a growth advantage in response to subsequent BMP7 transduction in vitro under anchorage-dependent and -independent conditions, in three-dimensional skin reconstructs, as well as in vivo in severe combined immune-deficiency mice. In concordance, Noggin knockdown by lentiviral shRNA confers sensitivity to BMP7-induced growth inhibition in advanced melanoma cells. Our findings suggest that, like TGF-β, BMP7 acts as an autocrine growth inhibitor in melanocytic cells, and that advanced melanoma cells may escape from BMP7-induced inhibition through concomitant aberrant expression of Noggin. PMID:18560367

A pragmatic randomised controlled trial of the effectiveness and cost-effectiveness of 'PhysioDirect' telephone assessment and advice services for physiotherapy.

PubMed

Salisbury, C; Foster, N E; Hopper, C; Bishop, A; Hollinghurst, S; Coast, J; Kaur, S; Pearson, J; Franchini, A; Hall, J; Grove, S; Calnan, M; Busby, J; Montgomery, A A

2013-01-01

As a result of long delays for physiotherapy for musculoskeletal problems, several areas in the UK have introduced PhysioDirect services in which patients telephone a physiotherapist for initial assessment and treatment advice. However, there is no robust evidence about the effectiveness, cost-effectiveness or acceptability to patients of PhysioDirect. To investigate whether or not PhysioDirect is equally as clinically effective as and more cost-effective than usual care for patients with musculoskeletal (MSK) problems in primary care. Pragmatic randomised controlled trial to assess equivalence, incorporating economic evaluation and nested qualitative research. Patients were randomised in 2 : 1 ratio to PhysioDirect or usual care using a remote automated allocation system at the level of the individual, stratifying by physiotherapy site and minimising by sex, age group and site of MSK problem. For the economic analysis, cost consequences included NHS and patient costs, and the cost of lost production. Cost-effectiveness analysis was carried out from the perspective of the NHS. Interviews were conducted with patients, physiotherapists and their managers. Four community physiotherapy services in England. Adults referred by general practitioners or self-referred for physiotherapy for a MSK problem. Patients allocated to PhysioDirect were invited to telephone a senior physiotherapist for initial assessment and advice using a computerised template, followed by face-to-face care when necessary. Patients allocated to usual care were put on to a waiting list for face-to-face care. Primary outcome was the Short Form questionnaire-36 items, version 2 (SF-36v2) Physical Component Score (PCS) at 6 months after randomisation. Secondary outcomes included other measures of health outcome [Measure Yourself Medical Outcomes Profile, European Quality of Life-5 Dimensions (EuroQol health utility measure, EQ-5D), global improvement, response to treatment], wait for treatment, time lost from work and usual activities, patient satisfaction. Data were collected by postal questionnaires at baseline, 6 weeks and 6 months, and from routine records by researchers blind to allocation. A total of 1506 patients were allocated to PhysioDirect and 743 to usual care. Patients allocated to PhysioDirect had a shorter wait for treatment than those allocated to usual care [median 7 days vs 34 days; arm-time ratio 0.32, 95% confidence interval (CI) 0.29 to 0.35] and had fewer non-attended face-to-face appointments [incidence rate ratio 0.55 (95% CI 0.41 to 0.73)]. The primary outcome at 6 months' follow-up was equivalent between PhysioDirect and usual care [mean PCS 43.50 vs 44.18, adjusted difference in means -0.01 (95% CI -0.80 to 0.79)]. The secondary measures of health outcome all demonstrated equivalence at 6 months, with slightly greater improvement in the PhysioDirect arm at 6 weeks' follow-up. Patients were equally satisfied with access to care but slightly less satisfied overall with PhysioDirect compared with usual care. NHS costs (physiotherapy plus other relevant NHS costs) per patient were similar in the two arms [PhysioDirect £ 198.98 vs usual care £ 179.68, difference in means £ 19.30 (95% CI -£ 37.60 to £ 76.19)], while QALYs gained were also similar [difference in means 0.007 (95% CI -0.003 to 0.016)]. Incremental cost per QALY gained was £ 2889. The probability that PhysioDirect was cost-effective at a £ 20,000 willingness-to-pay threshold was 88%. These conclusions about cost-effectiveness were robust to sensitivity analyses. There was no evidence of difference between trial arms in cost to patients or value of lost production. No adverse events were detected. Providing physiotherapy via PhysioDirect is equally clinically effective compared with usual waiting list-based care, provides faster access to treatment, appears to be safe, and is broadly acceptable to patients. PhysioDirect is probably cost-effective compared with usual care.

Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation.

PubMed

Balint, Eva; Lapointe, David; Drissi, Hicham; van der Meijden, Caroline; Young, Daniel W; van Wijnen, Andre J; Stein, Janet L; Stein, Gary S; Lian, Jane B

2003-05-15

Understanding physiological control of osteoblast differentiation necessitates characterization of the regulatory signals that initiate the events directing a cell to lineage commitment and establishing competency for bone formation. The bone morphogenetic protein, BMP-2, a member of the TGFbeta superfamily, induces osteoblast differentiation and functions through the Smad signal transduction pathway during in vivo bone formation. However, the molecular targets of BMP-mediated gene transcription during the process of osteoblast differentiation have not been comprehensively identified. In the present study, BMP-2 responsive factors involved in the early stages of commitment and differentiation to the osteoblast phenotype were analyzed by microarray gene expression profiling in samples ranging from 1 to 24 h following BMP-2 dependent differentiation of C2C12 premyoblasts into the osteogenic lineage. A total of 1,800 genes were responsive to BMP-2 and expression was modulated from 3- to 14-fold for less than 100 genes during the time course. Approximately 50% of these 100 genes are either up- or downregulated. Major events associated with phenotypic changes towards the osteogenic lineage were identified from hierarchical and functional clustering analyses. BMP-2 immediately responsive genes (1-4 h), which exhibited either transient or sustained expression, reflect activation and repression of non-osseous BMP-2 developmental systems. This initial response was followed by waves of expression of nuclear proteins and developmental regulatory factors including inhibitors of DNA binding, Runx2, C/EBP, Zn finger binding proteins, forkhead, and numerous homeobox proteins (e.g., CDP/cut, paired, distaless, Hox) which are expressed at characterized stages during osteoblast differentiation. A sequential profile of genes mediating changes in cell morphology, cell growth, and basement membrane formation is observed as a secondary transient early response (2-8 h). Commitment to the osteogenic phenotype is recognized by 8 h, reflected by downregulation of most myogenic-related genes and induction of a spectrum of signaling proteins and enzymes facilitating synthesis and assembly of an extracellular skeletal environment. These genes included collagens Type I and VI and the small leucine rich repeat family of proteoglycans (e.g., decorin, biglycan, osteomodulin, fibromodulin, and osteoadherin/osteoglycin) that reached peak expression at 24 h. With extracellular matrix development, the bone phenotype was further established from 16 to 24 h by induction of genes for cell adhesion and communication and enzymes that organize the bone ECM. Our microarray analysis resulted in the discovery of a class of genes, initially described in relation to differentiation of astrocytes and oligodendrocytes that are functionally coupled to signals for cellular extensions. They include nexin, neuropilin, latexin, neuroglian, neuron specific gene 1, and Ulip; suggesting novel roles for these genes in the bone microenvironment. This global analysis identified a multistage molecular and cellular cascade that supports BMP-2-mediated osteoblast differentiation. Copyright 2003 Wiley-Liss, Inc.

Structure of Gremlin-2 in Complex with GDF5 Gives Insight into DAN-Family-Mediated BMP Antagonism.

PubMed

Nolan, Kristof; Kattamuri, Chandramohan; Rankin, Scott A; Read, Randy J; Zorn, Aaron M; Thompson, Thomas B

2016-08-23

The DAN family, including Gremlin-1 and Gremlin-2 (Grem1 and Grem2), represents a large family of secreted BMP (bone morphogenetic protein) antagonists. However, how DAN proteins specifically inhibit BMP signaling has remained elusive. Here, we report the structure of Grem2 bound to GDF5 at 2.9-Å resolution. The structure reveals two Grem2 dimers binding perpendicularly to each GDF5 monomer, resembling an H-like structure. Comparison to the unbound Grem2 structure reveals a dynamic N terminus that undergoes significant transition upon complex formation, leading to simultaneous interaction with the type I and type II receptor motifs on GDF5. Binding studies show that DAN-family members can interact with BMP-type I receptor complexes, whereas Noggin outcompetes the type I receptor for ligand binding. Interestingly, Grem2-GDF5 forms a stable aggregate-like structure in vitro that is not clearly observed for other antagonists, including Noggin and Follistatin. These findings exemplify the structural and functional diversity across the various BMP antagonist families. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Bmp2 and Notch cooperate to pattern the embryonic endocardium.

PubMed

Papoutsi, T; Luna-Zurita, L; Prados, B; Zaffran, S; de la Pompa, J L

2018-05-31

Signaling interactions between myocardium and endocardium pattern embryonic cardiac regions, instructing their development to fulfill specific functions in the mature heart. We show that ectopic Bmp2 expression in the mouse chamber myocardium changes the transcriptional signature of adjacent chamber endocardial cells into valve tissue, and enables them to undergo epithelial-mesenchyme transition. This induction is independent of valve myocardium specification and requires high levels of Notch1 activity. Biochemical experiments suggest that Bmp2-mediated Notch1 induction is achieved through transcriptional activation of the Notch ligand Jag1, and physical interaction of Smad1/5 with the intracellular domain of the Notch1 receptor. Thus, widespread myocardial Bmp2 and endocardial Notch signaling drive presumptive ventricular endocardium to differentiate into valve endocardium. Understanding the molecular basis of valve development is instrumental to designing therapeutic strategies for congenital heart valve defects. © 2018. Published by The Company of Biologists Ltd.

Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.

PubMed

Yan, C; Wang, P; DeMayo, J; DeMayo, F J; Elvin, J A; Carino, C; Prasad, S V; Skinner, S S; Dunbar, B S; Dube, J L; Celeste, A J; Matzuk, M M

2001-06-01

Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.

Cadherin juxtamembrane region derived peptides inhibit TGFβ1 induced gene expression

PubMed Central

Stavropoulos, Ilias; Golla, Kalyan; Moran, Niamh; Martin, Finian; Shields, Denis C

2014-01-01

Bioactive peptides in the juxtamembrane regions of proteins are involved in many signaling events. The juxtamembrane regions of cadherins were examined for the identification of bioactive regions. Several peptides spanning the cytoplasmic juxtamembrane regions of E- and N-cadherin were synthesized and assessed for the ability to influence TGFβ responses in epithelial cells at the gene expression and protein levels. Peptides from regions closer to the membrane appeared more potent inhibitors of TGFβ signaling, blocking Smad3 phosphorylation. Thus inhibiting nuclear translocation of phosphorylated Smad complexes and subsequent transcriptional activation of TGFβ signal propagating genes. The peptides demonstrated a peptide-specific potential to inhibit other TGFβ superfamily members, such as BMP4. PMID:25108297

Key role of the expression of bone morphogenetic proteins in increasing the osteogenic activity of osteoblast-like cells exposed to shock waves and seeded on bioactive glass-ceramic scaffolds for bone tissue engineering.

PubMed

Muzio, Giuliana; Martinasso, Germana; Baino, Francesco; Frairia, Roberto; Vitale-Brovarone, Chiara; Canuto, Rosa A

2014-11-01

In this work, the role of shock wave-induced increase of bone morphogenetic proteins in modulating the osteogenic properties of osteoblast-like cells seeded on a bioactive scaffold was investigated using gremlin as a bone morphogenetic protein antagonist. Bone-like glass-ceramic scaffolds, based on a silicate experimental bioactive glass developed at the Politecnico di Torino, were produced by the sponge replication method and used as porous substrates for cell culture. Human MG-63 cells, exposed to shock waves and seeded on the scaffolds, were treated with gremlin every two days and analysed after 20 days for the expression of osteoblast differentiation markers. Shock waves have been shown to induce osteogenic activity mediated by increased expression of alkaline phosphatase, osteocalcin, type I collagen, BMP-4 and BMP-7. Cells exposed to shock waves plus gremlin showed increased growth in comparison with cells treated with shock waves alone and, conversely, mRNA contents of alkaline phosphatase and osteocalcin were significantly lower. Therefore, the shock wave-mediated increased expression of bone morphogenetic protein in MG-63 cells seeded on the scaffolds is essential in improving osteogenic activity; blocking bone morphogenetic protein via gremlin completely prevents the increase of alkaline phosphatase and osteocalcin. The results confirmed that the combination of glass-ceramic scaffolds and shock waves exposure could be used to significantly improve osteogenesis opening new perspectives for bone regenerative medicine. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Effect of Escherichia coli-produced recombinant human bone morphogenetic protein 2 on the regeneration of canine segmental ulnar defects.

PubMed

Harada, Yasuji; Itoi, Takamasa; Wakitani, Shigeyuki; Irie, Hiroyuki; Sakamoto, Michiko; Zhao, Dongwei; Nezu, Yoshinori; Yogo, Takuya; Hara, Yasushi; Tagawa, Masahiro

2012-07-01

Because bone morphogenetic protein 2 gene transfected Escherichia coli (E-BMP-2) produce recombinant human BMP-2 (rhBMP-2) more efficiently than mammalian cells (Chinese hamster ovary [CHO]-BMP-2), they may be a more cost-effective source of rhBMP-2 for clinical use. However, use of E-BMP-2 for regenerating long bones in large animals has not been reported. In the current study, we evaluated the healing efficacy of E-BMP-2 in a canine model. We created 2.5-cm critical-size segmental ulnar defects in test animals, then implanted E-BMP-2 and 700 mg of artificial bone (beta-tricalcium phosphate; β-TCP) into the wounds. We examined the differential effects of 5 E-BMP-2 treatments (0, 35, 140, 560, and 2240 μg) across 5 experimental groups (control, BMP35, BMP140, BMP560, and BMP2240). Radiography and computed tomography were used to observe the regeneration process. The groups in which higher doses of E-BMP-2 were administered (BMP560 and BMP2240) displayed more pronounced bone regeneration; the regenerated tissues connected to the host bone, and the cross-sectional areas of the regenerated bone were larger than those of the originals. The groups in which lower doses of E-BMP-2 were administered (BMP35 and BMP140) experienced relatively less bone regeneration; furthermore, the regenerated tissues failed to connect to the host bone. In these groups, the cross-sectional areas of the regenerated bone were equal to or smaller than those of the originals. No regeneration was observed in the control group. These findings suggest that, like CHO-BMP-2, E-BMP-2 can be used for the regeneration of large defects in long bones and that its clinical use might decrease the cost of bone regeneration treatments.

Sizzled controls dorso-ventral polarity by repressing cleavage of the Chordin protein.

PubMed

Muraoka, Osamu; Shimizu, Takashi; Yabe, Taijiro; Nojima, Hideaki; Bae, Young-Ki; Hashimoto, Hisashi; Hibi, Masahiko

2006-04-01

The Bone morphogenetic protein (Bmp) signalling gradient has a major function in the formation of the dorso-ventral axis. The zebrafish ventralized mutant, ogon, encodes Secreted Frizzled (Sizzled). sizzled is ventrally expressed in a Bmp-dependent manner and is required for the suppression of Bmp signalling on the ventral side of zebrafish embryos. However, it remains unclear how Sizzled inhibits Bmp signalling and controls ventro-lateral cell fate. We found that Sizzled stabilizes Chordin, a Bmp antagonist, by binding and inhibiting the Tolloid-family metalloproteinase, Bmp1a, which cleaves and inactivates Chordin. The cysteine-rich domain of Sizzled is required for inhibition of Bmp1a activity. Loss of both Bmp1a and Tolloid-like1 (Tll1; another Tolloid-family metalloproteinase) function leads to a complete suppression and reversal of the ogon mutant phenotype. These results indicate that Sizzled represses the activities of Tolloid-family proteins, thereby creating the Chordin-Bmp activity gradient along the dorso-ventral axis. Here, we describe a previously unrecognized role for a secreted Frizzled-related protein.

Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

PubMed

Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

2016-01-01

Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

Receptor-selective retinoids implicate retinoic acid receptor alpha and gamma in the regulation of bmp-2 and bmp-4 in F9 embryonal carcinoma cells.

PubMed

Rogers, M B

1996-01-01

The effect of retinoids on malignant cells and embryos indicates that retinoids influence the expression of growth factors or alter the response of cells to growth factors. The bone morphogenetic proteins, Bmp-2 and Bmp-4, are candidates for such growth factors because retinoic acid (RA) treatment of F9 embryonal carcinoma cells induced Bmp-2 mRNA, while simultaneously repressing Bmp-4 levels. Also, recombinant Bmp-2 affected the growth and differentiation of these cells. Regulation of each gene was concentration dependent and required continuous RA treatment. The short half-lives of the Bmp-2 (75 +/- 11 min) and Bmp-4 (70 +/- 4 min) mRNAs suggest that their abundance is primarily controlled at the transcriptional level. To determine which RA receptor (RAR) controls bmp-2 and bmp-4 expression, F9 cells were exposed to various receptor-selective retinoids. RAR alpha- and gamma-selective retinoids induced Bmp-2 and repressed Bmp-4 equally as well as all-trans RA. In contrast, a RAR beta-selective retinoid had little effect on Bmp-2 induction but repressed Bmp-4. A RAR alpha-selective antagonist inhibited all-trans RA stimulation of Bmp-2, although not as dramatically as a RAR beta gamma-selective antagonist. No differences were observed between Bmp levels in all-trans RA and 9-cis RA-treated cells, indicating that the RXRs play little part in controlling these genes. The results are consistent with RAR alpha and gamma-controlled Bmp-2 and Bmp-4 regulation.

Regulatory role of melatonin and BMP-4 in prolactin production by rat pituitary lactotrope GH3 cells.

PubMed

Ogura-Ochi, Kanako; Fujisawa, Satoshi; Iwata, Nahoko; Komatsubara, Motoshi; Nishiyama, Yuki; Tsukamoto-Yamauchi, Naoko; Inagaki, Kenichi; Wada, Jun; Otsuka, Fumio

2017-08-01

The effects of melatonin on prolactin production and its regulatory mechanism remain uncertain. We investigated the regulatory role of melatonin in prolactin production using rat pituitary lactotrope GH3 cells by focusing on the bone morphogenetic protein (BMP) system. Melatonin receptor activation, induced by melatonin and its receptor agonist ramelteon, significantly suppressed basal and forskolin-induced prolactin secretion and prolactin mRNA expression in GH3 cells. The melatonin MT2 receptor was predominantly expressed in GH3 cells, and the inhibitory effects of melatonin on prolactin production were reversed by treatment with the receptor antagonist luzindole, suggesting functional involvement of MT2 action in the suppression of prolactin release. Melatonin receptor activation also suppressed BMP-4-induced prolactin expression by inhibiting phosphorylation of Smad and transcription of the BMP-target gene Id-1, while BMP-4 treatment upregulated MT2 expression. Melatonin receptor activation suppressed basal, BMP-4-induced and forskolin-induced cAMP synthesis; however, BtcAMP-induced prolactin mRNA expression was not affected by melatonin or ramelteon, suggesting that MT2 activation leads to inhibition of prolactin production through the suppression of Smad signaling and cAMP synthesis. Experiments using intracellular signal inhibitors revealed that the ERK pathway is, at least in part, involved in prolactin induction by GH3 cells. Thus, a new regulatory role of melatonin involving BMP-4 in prolactin secretion was uncovered in lactotrope GH3 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of wise protein and its molecular mechanism to interact with both Wnt and BMP signals.

PubMed

Lintern, Katherine B; Guidato, Sonia; Rowe, Alison; Saldanha, José W; Itasaki, Nobue

2009-08-21

Cross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals.

BMP signaling protects telencephalic fate by repressing eye identity and its Cxcr4-dependent morphogenesis.

PubMed

Bielen, Holger; Houart, Corinne

2012-10-16

Depletion of Wnt signaling is a major requirement for the induction of the anterior prosencephalon. However, the molecular events driving the differential regionalization of this area into eye-field and telencephalon fates are still unknown. Here we show that the BMP pathway is active in the anterior neural ectoderm during late blastula to early gastrula stage in zebrafish. Bmp2b mutants and mosaic loss-of-function experiments reveal that BMP acts as a repressor of eye-field fate through inhibition of its key transcription factor Rx3, thereby protecting the future telencephalon from acquiring eye identity. This BMP-driven mechanism initiates the establishment of the telencephalon prior to the involvement of Wnt antagonists from the anterior neural border. Furthermore, we demonstrate that Rx3 and BMP are respectively required to maintain and restrict the chemokine receptor cxcr4a, which in turn contributes to the morphogenetic separation of eye-field and telencephalic cells during early neurulation. Copyright © 2012 Elsevier Inc. All rights reserved.

Bone Morphogenetic Protein (BMP) signaling in animal reproductive system development and function.

PubMed

Lochab, Amaneet K; Extavour, Cassandra G

2017-07-15

In multicellular organisms, the specification, maintenance, and transmission of the germ cell lineage to subsequent generations are critical processes that ensure species survival. A number of studies suggest that the Bone Morphogenetic Protein (BMP) pathway plays multiple roles in this cell lineage. We wished to use a comparative framework to examine the role of BMP signaling in regulating these processes, to determine if patterns would emerge that might shed light on the evolution of molecular mechanisms that may play germ cell-specific or other reproductive roles across species. To this end, here we review evidence to date from the literature supporting a role for BMP signaling in reproductive processes across Metazoa. We focus on germ line-specific processes, and separately consider somatic reproductive processes. We find that from primordial germ cell (PGC) induction to maintenance of PGC identity and gametogenesis, BMP signaling regulates these processes throughout embryonic development and adult life in multiple deuterostome and protostome clades. In well-studied model organisms, functional genetic evidence suggests that BMP signaling is required in the germ line across all life stages, with the exception of PGC specification in species that do not use inductive signaling to induce germ cell formation. The current evidence is consistent with the hypothesis that BMP signaling is ancestral in bilaterian inductive PGC specification. While BMP4 appears to be the most broadly employed ligand for the reproductive processes considered herein, we also noted evidence for sex-specific usage of different BMP ligands. In gametogenesis, BMP6 and BMP15 seem to have roles restricted to oogenesis, while BMP8 is restricted to spermatogenesis. We hypothesize that a BMP-based mechanism may have been recruited early in metazoan evolution to specify the germ line, and was subsequently co-opted for use in other germ line-specific and somatic reproductive processes. We suggest that if future studies assessing the function of the BMP pathway across extant species were to include a reproductive focus, that we would be likely to find continued evidence in favor of an ancient association between BMP signaling and the reproductive cell lineage in animals. Copyright © 2017 Elsevier Inc. All rights reserved.

The Multifaceted Effects of Agmatine on Functional Recovery after Spinal Cord Injury through Modulations of BMP-2/4/7 Expressions in Neurons and Glial Cells

PubMed Central

Park, Yu Mi; Lee, Won Taek; Bokara, Kiran Kumar; Seo, Su Kyoung; Park, Seung Hwa; Kim, Jae Hwan; Yenari, Midori A.; Park, Kyung Ah; Lee, Jong Eun

2013-01-01

Presently, few treatments for spinal cord injury (SCI) are available and none have facilitated neural regeneration and/or significant functional improvement. Agmatine (Agm), a guanidinium compound formed from decarboxylation of L-arginine by arginine decarboxylase, is a neurotransmitter/neuromodulator and been reported to exert neuroprotective effects in central nervous system injury models including SCI. The purpose of this study was to demonstrate the multifaceted effects of Agm on functional recovery and remyelinating events following SCI. Compression SCI in mice was produced by placing a 15 g/mm2 weight for 1 min at thoracic vertebra (Th) 9 segment. Mice that received an intraperitoneal (i.p.) injection of Agm (100 mg/kg/day) within 1 hour after SCI until 35 days showed improvement in locomotor recovery and bladder function. Emphasis was made on the analysis of remyelination events, neuronal cell preservation and ablation of glial scar area following SCI. Agm treatment significantly inhibited the demyelination events, neuronal loss and glial scar around the lesion site. In light of recent findings that expressions of bone morphogenetic proteins (BMPs) are modulated in the neuronal and glial cell population after SCI, we hypothesized whether Agm could modulate BMP- 2/4/7 expressions in neurons, astrocytes, oligodendrocytes and play key role in promoting the neuronal and glial cell survival in the injured spinal cord. The results from computer assisted stereological toolbox analysis (CAST) demonstrate that Agm treatment dramatically increased BMP- 2/7 expressions in neurons and oligodendrocytes. On the other hand, BMP- 4 expressions were significantly decreased in astrocytes and oligodendrocytes around the lesion site. Together, our results reveal that Agm treatment improved neurological and histological outcomes, induced oligodendrogenesis, protected neurons, and decreased glial scar formation through modulating the BMP- 2/4/7 expressions following SCI. PMID:23349763

Bone morphogenetic protein 9 as a key regulator of liver progenitor cells in DDC-induced cholestatic liver injury.

PubMed

Addante, Annalisa; Roncero, Cesáreo; Almalé, Laura; Lazcanoiturburu, Nerea; García-Álvaro, María; Fernández, Margarita; Sanz, Julián; Hammad, Seddik; Nwosu, Zeribe C; Lee, Se-Jin; Fabregat, Isabel; Dooley, Steven; Ten Dijke, Peter; Herrera, Blanca; Sánchez, Aránzazu

2018-05-11

Bone morphogenetic protein 9 (BMP9) interferes with liver regeneration upon acute injury, while promoting fibrosis upon carbon tetrachloride-induced chronic injury. We have now addressed the role of BMP9 in 3,5 diethoxicarbonyl-1,4 dihydrocollidine (DDC)-induced cholestatic liver injury, a model of liver regeneration mediated by hepatic progenitor cell (known as oval cell), exemplified as ductular reaction and oval cell expansion. WT and BMP9KO mice were submitted to DDC diet. Livers were examined for liver injury, fibrosis, inflammation and oval cell expansion by serum biochemistry, histology, RT-qPCR and western blot. BMP9 signalling and effects in oval cells were studied in vitro using western blot and transcriptional assays, plus functional assays of DNA synthesis, cell viability and apoptosis. Crosslinking assays and short hairpin RNA approaches were used to identify the receptors mediating BMP9 effects. Deletion of BMP9 reduces liver damage and fibrosis, but enhances inflammation upon DDC feeding. Molecularly, absence of BMP9 results in overactivation of PI3K/AKT, ERK-MAPKs and c-Met signalling pathways, which together with an enhanced ductular reaction and oval cell expansion evidence an improved regenerative response and decreased damage in response to DDC feeding. Importantly, BMP9 directly targets oval cells, it activates SMAD1,5,8, decreases cell growth and promotes apoptosis, effects that are mediated by Activin Receptor-Like Kinase 2 (ALK2) type I receptor. We identify BMP9 as a negative regulator of oval cell expansion in cholestatic injury, its deletion enhancing liver regeneration. Likewise, our work further supports BMP9 as an attractive therapeutic target for chronic liver diseases. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

In Vivo Host Response and Degradation of Copolymer Scaffolds Functionalized with Nanodiamonds and Bone Morphogenetic Protein 2.

PubMed

Suliman, Salwa; Sun, Yang; Pedersen, Torbjorn O; Xue, Ying; Nickel, Joachim; Waag, Thilo; Finne-Wistrand, Anna; Steinmüller-Nethl, Doris; Krueger, Anke; Costea, Daniela E; Mustafa, Kamal

2016-03-01

The aim is to evaluate the effect of modifying poly[(l-lactide)-co-(ε-caprolactone)] scaffolds (PLCL) with nanodiamonds (nDP) or with nDP+physisorbed BMP-2 (nDP+BMP-2) on in vivo host tissue response and degradation. The scaffolds are implanted subcutaneously in Balb/c mice and retrieved after 1, 8, and 27 weeks. Molecular weight analysis shows that modified scaffolds degrade faster than the unmodified. Gene analysis at week 1 shows highest expression of proinflammatory markers around nDP scaffolds; although the presence of inflammatory cells and foreign body giant cells is more prominent around the PLCL. Tissue regeneration markers are highly expressed in the nDP+BMP-2 scaffolds at week 8. A fibrous capsule is detectable by week 8, thinnest around nDP scaffolds and at week 27 thickest around PLCL scaffolds. mRNA levels of ALP, COL1α2, and ANGPT1 are significantly upregulating in the nDP+BMP-2 scaffolds at week 1 with ectopic bone seen at week 8. Even when almost 90% of the scaffold is degraded at week 27, nDP are observable at implantation areas without adverse effects. In conclusion, modifying PLCL scaffolds with nDP does not aggravate the host response and physisorbed BMP-2 delivery attenuates inflammation while lowering the dose of BMP-2 to a relatively safe and economical level. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation of bone morphogenetic proteins in early embryonic development

NASA Astrophysics Data System (ADS)

Yamamoto, Yukiyo; Oelgeschläger, Michael

2004-11-01

Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

Periosteal BMP2 activity drives bone graft healing.

PubMed

Chappuis, Vivianne; Gamer, Laura; Cox, Karen; Lowery, Jonathan W; Bosshardt, Dieter D; Rosen, Vicki

2012-10-01

Bone graft incorporation depends on the orchestrated activation of numerous growth factors and cytokines in both the host and the graft. Prominent in this signaling cascade is BMP2. Although BMP2 is dispensable for bone formation, it is required for the initiation of bone repair; thus understanding the cellular mechanisms underlying bone regeneration driven by BMP2 is essential for improving bone graft therapies. In the present study, we assessed the role of Bmp2 in bone graft incorporation using mice in which Bmp2 has been removed from the limb prior to skeletal formation (Bmp2(cKO)). When autograft transplantations were performed in Bmp2cKO mice, callus formation and bone healing were absent. Transplantation of either a vital wild type (WT) bone graft into a Bmp2(cKO) host or a vital Bmp2(cKO) graft into a WT host also resulted in the inhibition of bone graft incorporation. Histological analyses of these transplants show that in the absence of BMP2, periosteal progenitors remain quiescent and healing is not initiated. When we analyzed the expression of Sox9, a marker of chondrogenesis, on the graft surface, we found it significantly reduced when BMP2 was absent in either the graft itself or the host, suggesting that local BMP2 levels drive periosteal cell condensation and subsequent callus cell differentiation. The lack of integrated healing in the absence of BMP2 was not due to the inability of periosteal cells to respond to BMP2. Healing was achieved when grafts were pre-soaked in rhBMP2 protein, indicating that periosteal progenitors remain responsive in the absence of BMP2. In contrast to the requirement for BMP2 in periosteal progenitor activation in vital bone grafts, we found that bone matrix-derived BMP2 does not significantly enhance bone graft incorporation. Taken together, our data show that BMP2 signaling is not essential for the maintenance of periosteal progenitors, but is required for the activation of these progenitors and their subsequent differentiation along the osteo-chondrogenic pathway. These results indicate that BMP2 will be among the signaling molecules whose presence will determine success or failure of new bone graft strategies. Copyright © 2012 Elsevier Inc. All rights reserved.

Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer.

PubMed

Irshad, Shazia; Bansal, Mukesh; Guarnieri, Paolo; Davis, Hayley; Al Haj Zen, Ayman; Baran, Brygida; Pinna, Claudia Maria Assunta; Rahman, Haseeb; Biswas, Sujata; Bardella, Chiara; Jeffery, Rosemary; Wang, Lai Mun; East, James Edward; Tomlinson, Ian; Lewis, Annabelle; Leedham, Simon John

2017-06-01

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial-mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP-Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP-Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Bone Morphogenetic Protein Regulation of Enteric Neuronal Phenotypic Diversity: Relationship to Timing of Cell Cycle Exit

PubMed Central

Chalazonitis, Alcmène; Pham, Tuan.D.; Li, Zhishan; Roman, Daniel; Guha, Udayan; Gomes, William; Kan, Lixin; Kessler, John A.; Gershon, Michael D.

2008-01-01

The effects of bone morphogenetic protein (BMP) signaling on enteric neuron development were examined in transgenic mice over expressing either the BMP inhibitor, noggin, or BMP4 under control of the neuron specific enolase (NSE) promoter. Noggin antagonism of BMP signaling increased total numbers of enteric neurons and those of subpopulations derived from precursors that exit the cell cycle early in neurogenesis (serotonin, calretinin, calbindin). In contrast, noggin overexpression decreased numbers of neurons derived from precursors that exit the cell cycle late (γ-aminobutyric acid, tyrosine hydroxylase [TH], dopamine transporter, calcitonin gene related peptide, TrkC). Numbers of TH- and TrkC-expressing neurons were increased by overexpression of BMP4. These observations are consistent with the idea that phenotypic expression in the enteric nervous system (ENS) is determined, in part, by the number of proliferative divisions neuronal precursors undergo before their terminal mitosis. BMP signaling may thus regulate enteric neuronal phenotypic diversity by promoting the exit of precursors from the cell cycle. BMP2 increased the numbers of TH- and TrkC-expressing neurons developing in vitro from immunoselected enteric crest-derived precursors; BMP signaling may thus also specify or promote the development of dopaminergic TrkC/NT-3-dependent neurons. The developmental defects in the ENS of noggin overexpressing mice caused a relatively mild disturbance of motility (irregular rapid transit and increased stool frequency, weight, and water content). Although the function of the gut thus displays a remarkable tolerance for ENS defects, subtle functional abnormalities in motility or secretion may arise when ENS defects short of aganglionosis occur during development. PMID:18537141

Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

PubMed

Wu, Lian; Wang, Feng; Donly, Kevin J; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

2015-11-01

Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. © 2015 Wiley Periodicals, Inc.

Preparation and bioactive properties of chitosan and casein phosphopeptides composite coatings for orthopedic implants.

PubMed

Qin, Liguo; Dong, Huanhuan; Mu, Ziqing; Zhang, Yali; Dong, Guangneng

2015-11-20

Using the layer-by-layer deposition method, functional chitosan/casein phospopeptides (CS/CPP) composite coatings were produced on Co-Cr-Mo alloy. The CS/CPP composite coatings had the dendritic topography, and were quite hydrophilic. Zeta potential measurements showed the composite coatings were negative charged at neural pH. XPS results indicated that the CS/CPP composite coatings were covalently bond to the substrate. When MC3T3-E1 cells were seeded on the CS/CPP composite coatings, no cytotoxicity was observed. The bone morphogenetic protein-2 (BMP-2) mRNA expression was significantly up-regulated in MC3T3-E1 cells cultured on the composite coatings and it was twice as much as that of cells cultured on the bare substrate. The expression of osteoprotegerin (OPG) mRNA and the ratio of OPG/receptor activator of nuclear factor-κB ligand (RNAKL) mRNA were increased 5-fold and 55-fold, respectively. These results suggested the CS/CPP composite coatings may have potential application in cobalt matrix orthopaedic implants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biological activity of a genetically modified BMP-2 variant with inhibitory activity

PubMed Central

Klammert, Uwe; Nickel, Joachim; Würzler, Kristian; Klingelhöffer, Christoph; Sebald, Walter; Kübler, Alexander C; Reuther, Tobias

2009-01-01

Background Alterations of the binding epitopes of bone morphogenetic protein-2 (BMP-2) lead to a modified interaction with the ectodomains of BMP receptors. In the present study the biological effect of a BMP-2 double mutant with antagonistic activity was evaluated in vivo. Methods Equine-derived collagenous carriers were loaded with recombinant human BMP-2 (rhBMP-2) in a well-known dose to provide an osteoinductive stimulus. The study was performed in a split animal design: carriers only coupled with rhBMP-2 (control) were implanted into prepared cavities of lower limb muscle of rats, specimens coupled with rhBMP-2 as well as BMP-2 double mutant were placed into the opposite limb in the same way. After 28 days the carriers were explanted, measured radiographically and characterized histologically. Results As expected, the BMP-2 loaded implants showed a typical heterotopic bone formation. The specimens coupled with both proteins showed a significant decreased bone formation in a dose dependent manner. Conclusion The antagonistic effect of a specific BMP-2 double mutant could be demonstrated in vivo. The dose dependent influence on heterotopic bone formation by preventing rhBMP-2 induced osteoinduction suggests a competitive receptor antagonism. PMID:19187528

A structured bowel management program for patients with severe functional constipation can help decrease emergency department visits, hospital admissions, and healthcare costs.

PubMed

Reck-Burneo, Carlos A; Vilanova-Sanchez, Alejandra; Gasior, Alessandra C; Dingemans, Alexander J M; Lane, Victoria A; Dyckes, Robert; Nash, Onnalisa; Weaver, Laura; Maloof, Tassiana; Wood, Richard J; Zobell, Sarah; Rollins, Michael D; Levitt, Marc A

2018-03-24

Published health-care costs related to constipation in children in the USA are estimated at $3.9 billion/year. We sought to assess the effect of a bowel management program (BMP) on health-care utilization and costs. At two collaborating centers, BMP involves an outpatient week during which a treatment plan is implemented and objective assessment of stool burden is performed with daily radiography. We reviewed all patients with severe functional constipation who participated in the program from March 2011 to June 2015 in center 1 and from April 2014 to April 2016 in center 2. ED visits, hospital admissions, and constipation-related morbidities (abdominal pain, fecal impaction, urinary retention, urinary tract infections) 12 months before and 12 months after completion of the BMP were recorded. One hundred eighty-four patients were included (center 1 = 96, center 2 = 88). Sixty-three (34.2%) patients had at least one unplanned visit to the ED before treatment. ED visits decreased to 23 (12.5%) or by 64% (p < 0.0005). Unplanned hospital admissions decreased from 65 to 28, i.e., a 56.9% reduction (p < 0.0005). In children with severe functional constipation, a structured BMP decreases unplanned visits to the ED, hospital admissions, and costs for constipation-related health care. 3. Copyright © 2018. Published by Elsevier Inc.

Functional expression of BMP7 receptors in oral epithelial cells. Interleukin-17F production in response to BMP7.

PubMed

Nishio, Kensuke; Ozawa, Yasumasa; Ito, Hisanori; Kifune, Takashi; Narita, Tatsuya; Iinuma, Toshimitsu; Gionhaku, Nobuhito; Asano, Masatake

2017-10-01

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Recently, BMP7 has been demonstrated to be produced by salivary glands and contribute to embryonic branching in mice. The BMP7 in saliva is thought to be delivered to the oral cavity and is expected to contact with stratified squamous epithelial cells which line the surface of oral mucosa. In this study, we attempted to investigate the effects of BMP7 on oral epithelial cells. The expression of BMP receptors was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). OSCCs were stimulated with human recombinant BMP7 (hrBMP7) and the phosphorylation status of Smad1/5/8 was examined by western blotting. For microarray analysis, Ca9-22 cells were stimulated with 100 ng/mL of hrBMP7 and total RNA was extracted and subjected to real-time PCR. The 5'-untranslated region (5'-UTR) of IL-17 F gene was cloned to pGL4-basic vector and used for luciferase assay. Ca9-22 cells were pre-incubated with DM3189, a specific inhibitor of Smad1/5/8, for inhibition assay. All isoforms of type I and type II BMP receptors were expressed in both Ca9-22 and HSC3 cells and BMP7 stimulation resulted in the phosphorylation of Smad1/5/8 in both cell lines. The microarray analysis revealed the induction of interleukin-17 F (IL-17 F), netrin G2 (NTNG2) and hyaluronan synthase 1 (HAS1). Luciferase assay using the 5'-UTR of the IL-17 F gene revealed transcriptional regulation. Induced IL-17 F production was further confirmed at the protein level by ELISA. Smad1/5/8 inhibitor pretreatment decreased IL-17 F expression levels in the cells.

Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

PubMed

Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

2016-06-01

Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor beta1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation.

PubMed

Lee, M H; Javed, A; Kim, H J; Shin, H I; Gutierrez, S; Choi, J Y; Rosen, V; Stein, J L; van Wijnen, A J; Stein, G S; Lian, J B; Ryoo, H M

1999-04-01

The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2alphaA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-beta1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfal may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype.

'PhysioDirect' telephone assessment and advice services for physiotherapy: protocol for a pragmatic randomised controlled trial

PubMed Central

Salisbury, Chris; Foster, Nadine E; Bishop, Annette; Calnan, Michael; Coast, Jo; Hall, Jeanette; Hay, Elaine; Hollinghurst, Sandra; Hopper, Cherida; Grove, Sean; Kaur, Surinder; Montgomery, Alan

2009-01-01

Background Providing timely access to physiotherapy has long been a problem for the National Health Service in the United Kingdom. In an attempt to improve access some physiotherapy services have introduced a new treatment pathway known as PhysioDirect. Physiotherapists offer initial assessment and advice by telephone, supported by computerised algorithms, and patients are sent written self-management and exercise advice by post. They are invited for face-to-face treatment only when necessary. Although several such services have been developed, there is no robust evidence regarding clinical and cost-effectiveness, nor the acceptability of PhysioDirect. Methods/Design This protocol describes a multi-centre pragmatic individually randomised trial, with nested qualitative research. The aim is to determine the effectiveness, cost-effectiveness, and acceptability of PhysioDirect compared with usual models of physiotherapy based on patients going onto a waiting list and receiving face-to-face care. PhysioDirect services will be established in four areas in England. Adult patients in these areas with musculoskeletal problems who refer themselves or are referred by a primary care practitioner for physiotherapy will be invited to participate in the trial. About 1875 consenting patients will be randomised in a 2:1 ratio to PhysioDirect or usual care. Data about outcome measures will be collected at baseline and 6 weeks and 6 months after randomisation. The primary outcome is clinical improvement at 6 months; secondary outcomes include cost, waiting times, time lost from work and usual activities, patient satisfaction and preference. The impact of PhysioDirect on patients in different age-groups and with different conditions will also be examined. Incremental cost-effectiveness will be assessed in terms of quality adjusted life years in relation to cost. Qualitative methods will be used to explore factors associated with the success or failure of the service, the acceptability of PhysioDirect to patients and staff, and ways in which the service could be improved. Discussion It is still relatively unusual to evaluate new forms of service delivery using randomised controlled trials. By combining rigorous trial methods with economic analysis of cost-effectiveness and qualitative research this study will provide robust evidence to inform decisions about the widespread introduction of PhysioDirect services. Trial registration Current Controlled Trials ISRCTN55666618 PMID:19650913

BMP15 c.-9C>G promoter sequence variant may contribute to the cause of non-syndromic premature ovarian failure.

PubMed

Fonseca, Dora Janeth; Ortega-Recalde, Oscar; Esteban-Perez, Clara; Moreno-Ortiz, Harold; Patiño, Liliana Catherine; Bermúdez, Olga María; Ortiz, Angela María; Restrepo, Carlos M; Lucena, Elkin; Laissue, Paul

2014-11-01

BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15 c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Spatial regulation of bone morphogenetic proteins (BMPs) in postnatal articular and growth plate cartilage

PubMed Central

Garrison, Presley; Yue, Shanna; Hanson, Jeffrey; Baron, Jeffrey; Lui, Julian C.

2017-01-01

Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers—the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may contribute to the spatial differentiation of chondrocytes in the postnatal endochondral skeleton. PMID:28467498

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

PubMed Central

Pohl, Theresa L. M.; Schwab, Elisabeth H.; Cavalcanti-Adam, Elisabetta A.

2013-01-01

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation. PMID:24021994

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

PubMed Central

El Gamal, Abrahim; Agarwal, Vinayak; Diethelm, Stefan; Rahman, Imran; Schorn, Michelle A.; Sneed, Jennifer M.; Louie, Gordon V.; Whalen, Kristen E.; Mincer, Tracy J.; Noel, Joseph P.; Paul, Valerie J.; Moore, Bradley S.

2016-01-01

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes. PMID:27001835

TNF-α Upregulates Expression of BMP-2 and BMP-3 Genes in the Rat Dental Follicle – Implications for Tooth Eruption

PubMed Central

Yao, Shaomian; Prpic, Veronica; Pan, Fenghui; Wise, Gary E.

2011-01-01

The dental follicle appears to regulate both the alveolar bone resorption and bone formation needed for tooth eruption. Tumor necrosis factor-alpha ( TNF-α) gene expression is maximally upregulated at postnatal day 9 in the rat dental follicle of the 1st mandibular molar, a time that correlates with rapid bone growth at the base of the tooth crypt, as well as a minor burst of osteoclastogenesis. TNF-α expression is correlated with the expression of bone morphogenetic protein-2 (BMP-2), a molecule expressed in the dental follicle that can promote bone formation. Because BMP-2 signaling may be augmented by bone morphogenetic protein-3 (BMP-3), it was the objective of this study to determine 1) if the dental follicle expresses BMP-3 and 2) if TNF-α stimulates the dental follicle cells to express BMP-2 and BMP-3. Dental follicles were collected from different postnatal ages of rat pups. Dental follicle cells were incubated with TNF-α to study its dosage and time-course effects on gene expression of BMP-2 and BMP-3, as determined by real-time RT-PCR. Next, immunostaining was conducted to confirm if the protein was synthesized and ELISA of the conditioned medium was conducted to determine if BMP-2 was secreted. We found that BMP-3 expression is correlated with the expression of TNF-α in the dental follicle and TNF-α significantly increased BMP-2 and BMP-3 expression in vitro. Immunostaining and ELISA showed that BMP-2 and BMP-3 were synthesized and secreted. This study suggests that TNF-α can upregulate the expression of bone formation genes that may be needed for tooth eruption. PMID:20067418

Bmp 2 and bmp 7 induce odonto- and osteogenesis of human tooth germ stem cells.

PubMed

Taşlı, P Neslihan; Aydın, Safa; Yalvaç, Mehmet Emir; Sahin, Fikrettin

2014-03-01

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain odontogenesis and osteogenesis. In this study, we studied the effect of bone morphogenic protein 2 (BMP 2) and bone morphogenic protein 7 (BMP 7) as differentiation inducers in tooth and bone regeneration. We compared the effect of BMP 2 and BMP 7 on odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs). Third molar-derived hTGSCs were characterized with mesenchymal stem cell surface markers by flow cytometry. BMP 2 and BMP 7 were transfected into hTGSCs and the cells were seeded onto six-well plates. One day after the transfection, hTGSCs were treated with odontogenic and osteogenic mediums for 14 days. For confirmation of odontogenic and osteogenic differentiation, mRNA levels of BMP2, BMP 7, collagen type 1 (COL1A), osteocalsin (OCN), and dentin sialophosphoprotein (DSPP) genes were measured by quantitative real-time PCR. In addition to this, immunocytochemistry was performed by odontogenic and osteogenic antibodies and mineralization obtained by von Kossa staining. Our results showed that the BMP 2 and BMP 7 both promoted odontogenic and osteogenic differentiation of hTGSCs. Data indicated that BMP 2 treatment and BMP 7 treatment induce odontogenic differentiation without affecting each other, whereas they induce osteogenic differentiation by triggering expression of each other. These findings provide a feasible tool for tooth and bone tissue engineering.

Protein-protein recognition control by modulating electrostatic interactions.

PubMed

Han, Song; Yin, Shijin; Yi, Hong; Mouhat, Stéphanie; Qiu, Su; Cao, Zhijian; Sabatier, Jean-Marc; Wu, Yingliang; Li, Wenxin

2010-06-04

Protein-protein control recognition remains a huge challenge, and its development depends on understanding the chemical and biological mechanisms by which these interactions occur. Here we describe a protein-protein control recognition technique based on the dominant electrostatic interactions occurring between the proteins. We designed a potassium channel inhibitor, BmP05-T, that was 90.32% identical to wild-type BmP05. Negatively charged residues were translocated from the nonbinding interface to the binding interface of BmP05 inhibitor, such that BmP05-T now used BmP05 nonbinding interface as the binding interface. This switch demonstrated that nonbinding interfaces were able to control the orientation of protein binding interfaces in the process of protein-protein recognition. The novel function findings of BmP05-T peptide suggested that the control recognition technique described here had the potential for use in designing and utilizing functional proteins in many biological scenarios.

Tricalcium phosphate and glutaraldehyde crosslinked gelatin incorporating bone morphogenetic protein--a viable scaffold for bone tissue engineering.

PubMed

Yang, Shu-Hua; Hsu, Chung-King; Wang, Kuo-Cheng; Hou, Sheng-Mou; Lin, Feng-Huei

2005-07-01

Bone defects caused by various etiologies must be filled with suitable substances to promote bone repair. Autogenous iliac crest graft is most frequently used, but is often associated with morbidities. Several bone graft substitutes have been developed to provide osteoconductive matrices as well as to enhance osteoinductivity. A tricalcium phosphate and glutaraldehyde crosslinked gelatin (GTG) scaffold, incorporated with bone morphogenetic proteins (BMPs), was developed to provide an alternative mean of bone tissue engineering. This study investigated differences between GTG and BMP-4 immobilized GTG (GTG-BMP) scaffolds on neonatal rat calvaria osteoblast activities. The GTG scaffold possessed an average pore size of 200 microm and a porosity of 75%. HE staining revealed uniform cell distribution throughout the scaffold 24 h post cell seeding. Alkaline phosphatase (ALP) activity of the GTG samples increased initially and then stabilized at 3 weeks postseeding. ALP activity of the GTG-BMP samples was similar to that of the GTG samples in the second and third weeks, but it continued increasing and became significantly greater than that of the GTG samples by the fourth week. Gla-type osteocalcin (Gla-OC) activity of the GTG-BMP samples was initially lower, but also became significantly greater than that of the GTG samples by the fourth week. An HE stain revealed greater numbers of attached cells and a richer matrix deposits in the GTG-BMP samples. A von Kossa stain showed larger mineralizing nodules, in greater numbers, after 4 weeks of in vitro cultivation. These findings suggest that the GTG scaffold provides an excellent porous structure, conductive to greater cell attachment and osteoblast differentiation, and that utility can be significantly enhanced by the inclusion of BMPs. A GTG-BMP scaffold holds promise as a superior bioactive material for bone tissue engineering. Copyright 2005 Wiley Periodicals, Inc.

BMP suppresses PTEN expression via RAS/ERK signaling.

PubMed

Beck, Stayce E; Carethers, John M

2007-08-01

Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

Comparison of the effects of recombinant human bone morphogenetic protein-2 and -9 on bone formation in rat calvarial critical-size defects.

PubMed

Nakamura, Toshiaki; Shirakata, Yoshinori; Shinohara, Yukiya; Miron, Richard J; Hasegawa-Nakamura, Kozue; Fujioka-Kobayashi, Masako; Noguchi, Kazuyuki

2017-12-01

Among bone morphogenetic protein (BMP) family members, BMP-2 and BMP-9 have demonstrated potent osteoinductive potential. However, in vivo differences in their potential for bone regeneration remain unclear. The present study aimed to compare the effects of recombinant human (rh) BMP-2 and rhBMP-9 on bone formation in rat calvarial critical-size defects (CSD). Twenty-eight Wistar rats surgically received two calvarial defects bilaterally in each parietal bone. Defects (n = 56) were allocated into four groups: absorbable collagen sponge (ACS) alone, rhBMP-2 with ACS (rhBMP-2/ACS), rhBMP-9/ACS, or sham surgery (control), on the condition that the treatments of rhBMP-2/ACS and rhBMP-9/ACS, or the same treatments were not included in the same animal. Animals were sacrificed at 2 and 8 weeks post-surgery. The calvarial defects were analyzed for bone volume (BV) by micro-computed tomography and for percentages of defect closure (DC/DL), newly formed bone area (NBA/TA), bone marrow area (BMA/NBA), adipose tissue area (ATA/NBA), central bone height (CBH), and marginal bone height (MBH) by histomorphometric analysis. The BV in the rhBMP-2/ACS group (5.44 ± 3.65 mm 3 , n = 7) was greater than the other groups at 2 weeks post-surgery, and the rhBMP-2/ACS and rhBMP-9/ACS groups (18.17 ± 2.51 and 16.30 ± 2.46 mm 3 , n = 7, respectively) demonstrated significantly greater amounts of BV compared with the control and ACS groups (6.02 ± 2.90 and 9.30 ± 2.75 mm 3 , n = 7, respectively) at 8 weeks post-surgery. The rhBMP-2/ACS and rhBMP-9/ACS groups significantly induced new bone formation compared to the control and ACS groups at 8 weeks post-surgery. However, there were no statistically significant differences found between the rhBMP-2/ACS and rhBMP-9/ACS groups in any of the histomorphometric parameters. The ATA/NBA in the rhBMP-2/ACS group (9.24 ± 3.72%, n = 7) was the highest among the treatment groups at 8 weeks post-surgery. Within the limits of this study, it can be concluded that rhBMP-2/ACS induced a slight early increase in new bone formation at 2 weeks and that rhBMP-9/ACS provided comparable new bone formation to rhBMP-2/ACS with less adipose tissues after a healing period of 8 weeks in rat CSD. RhBMP-9/ACS treatment provided new bone formation with less adipose tissues compared with rhBMP-2/ACS.

BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy

PubMed Central

Spagnolli, Ester; Ernande, Laura; Thoonen, Robrecht; Kolodziej, Starsha A.; Leyton, Patricio A.; Cheng, Juan; Tainsh, Robert E. T.; Mayeur, Claire; Rhee, David K.; Wu, Mei. X.; Scherrer-Crosbie, Marielle; Buys, Emmanuel S.; Zapol, Warren M.; Bloch, Kenneth D.; Bloch, Donald B.

2016-01-01

Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis. PMID:26873969

A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology.

PubMed

Tate, Courtney M; Mc Entire, Jacquelyn; Pallini, Roberto; Vakana, Eliza; Wyss, Lisa; Blosser, Wayne; Ricci-Vitiani, Lucia; D'Alessandris, Quintino Giorgio; Morgante, Liliana; Giannetti, Stefano; Larocca, Luigi Maria; Todaro, Matilde; Benfante, Antonina; Colorito, Maria Luisa; Stassi, Giorgio; De Maria, Ruggero; Rowlinson, Scott; Stancato, Louis

2015-01-01

Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.

Stiffness-dependent cellular internalization of matrix-bound BMP-2 and its relation to Smad and non-Smad signaling.

PubMed

Gilde, Flora; Fourel, Laure; Guillot, Raphael; Pignot-Paintrand, Isabelle; Okada, Takaharu; Fitzpatrick, Vincent; Boudou, Thomas; Albiges-Rizo, Corinne; Picart, Catherine

2016-12-01

Surface coatings delivering BMP are a promising approach to render biomaterials osteoinductive. In contrast to soluble BMPs which can interact with their receptors at the dorsal side of the cell, BMPs presented as an insoluble cue physically bound to a biomimetic matrix, called here matrix-bound (bBMP-2), are presented to cells by their ventral side. To date, BMP-2 internalization and signaling studies in cell biology have always been performed by adding soluble (sBMP-2) to cells adhered on cell culture plates or glass slides, which will be considered here as a "reference" condition. However, whether and how matrix-bound BMP-2 can be internalized by cells and its relation to canonical (SMAD) and non-canonical signaling (ALP) remain open questions. In this study, we investigated the uptake and processing of BMP-2 by C2C12 myoblasts. This BMP-2 was presented either embedded in polyelectrolyte multilayer films (matrix-bound presentation) or as soluble form. Using fluorescently labeled BMP-2, we showed that the amount of matrix-bound BMP-2 internalized is dependent on the level of crosslinking of the polyelectrolyte films. Cav-1-mediated internalization is related to both SMAD and ALP signaling, while clathrin-mediated is only related to ALP signaling. BMP-2 internalization was independent of the presentation mode (sBMP-2 versus bBMP-2) for low crosslinked films (soft, EDC10) in striking contrast with high crosslinked (stiff, EDC70) films where internalization was much lower and slower for bBMP-2. As anticipated, internalization of sBMP-2 barely depended on the underlying matrix. Taken together, these results indicate that BMP-2 internalization can be tuned by the underlying matrix and activates downstream BMP-2 signaling, which is key for the effective formation of bone tissue. The presentation of growth factors from material surfaces currently presents significant challenges in academic research, clinics and industry. Being able to deliver efficiently these growth factors by a biomaterial will open new perspectives for regenerative medicine. However, to date, very little is known about how matrix-bound growth factors are delivered to cells, especially whether they are internalized and how they are signaling to drive key differentiation events. These initial steps are crucial as they will guide the subsequent processes leading to tissue regeneration. In this work, we investigate the uptake and processing by cells of BMP-2 ligands embedded in polyelectrolyte multilayer films in comparison to soluble BMP-2. We show that BMP-2 responsive cells can internalize matrix-bound BMP-2 and that internalization is dependent on the cross-linking level of the polyelectrolyte films. In addition, we show that internalization is mediated by both clathrin- and caveolin-dependent pathways. While inhibiting clathrin-dependent endocytosis affects only non-canonical signaling, blocking caveolin-1-dependent endocytosis reduces both canonical and non-canonical BMP signaling. The signaling pathways found for matrix-bound BMP-2 are similar to those found for soluble BMP-2. These results highlight that BMP-2 presented by a biomaterial at the ventral side of the cell can trigger major endocytic and associated signaling pathways leading to bone regeneration. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

SMAD Signaling Is Required for Structural Integrity of the Female Reproductive Tract and Uterine Function During Early Pregnancy in Mice1

PubMed Central

Rodriguez, Amanda; Tripurani, Swamy K.; Burton, Jason C.; Clementi, Caterina; Larina, Irina; Pangas, Stephanie A.

2016-01-01

Pregnancy is a complex physiological process tightly controlled by the interplay among hormones, morphogens, transcription factors, and signaling pathways. Although recent studies using genetically engineered mouse models have revealed that ligands and receptors of transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) signaling pathways are essential for multiple reproductive events during pregnancy, the functional role of SMAD transcription factors, which serve as the canonical signaling platform for the TGFbeta/BMP pathways, in the oviduct and uterus is undefined. Here, we used a mouse model containing triple conditional deletion of the BMP receptor signaling Smads (Smad1 and Smad5) and Smad4, the central mediator of both TGFbeta and BMP signaling, to investigate the role of the SMADs in reproductive tract structure and function in cells from the Amhr2 lineage. Unlike the respective single- or double-knockouts, female Smad1flox/flox Smad5flox/flox Smad4flox/flox Amhr2cre/+conditional knockout (i.e., Smad1/5/4-Amhr2-cre KO) mice are sterile. We discovered that Smad1/5/4-Amhr2-cre KO females have malformed oviducts that subsequently develop oviductal diverticuli. These oviducts showed dysregulation of multiple genes essential for oviduct and smooth muscle development. In addition, uteri from Smad1/5/4-Amhr2-cre KO females exhibit multiple defects in stroma, epithelium, and smooth muscle layers and fail to assemble a closed uterine lumen upon embryo implantation, with defective uterine decidualization that led to pregnancy loss at early to mid-gestation. Taken together, our study uncovers a new role for the SMAD transcription factors in maintaining the structural and functional integrity of oviduct and uterus, required for establishment and maintenance of pregnancy. PMID:27335065

Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.

PubMed

Plikus, Maksim V; Mayer, Julie Ann; de la Cruz, Damon; Baker, Ruth E; Maini, Philip K; Maxson, Robert; Chuong, Cheng-Ming

2008-01-17

In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic beta-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/beta-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug delivery and stem cell engineering studies, because they highlight the acute need to differentiate supportive versus inhibitory regions in the host skin.

Overlapping and differential localization of Bmp-2, Bmp-4, Msx-2 and apoptosis in the endocardial cushion and adjacent tissues of the developing mouse heart.

PubMed

Abdelwahid, E; Rice, D; Pelliniemi, L J; Jokinen, E

2001-07-01

The bone morphogenetic proteins BMP-2 and BMP-4 and the homeobox gene MSX-2 are required for normal development of many embryonic tissues. To elucidate their possible roles during the remodeling of the tubular heart into a fully septated four-chambered heart, we have localized the mRNA of Bmp-2, Bmp-4, Msx-2 and apoptotic cells in the developing mouse heart from embryonic day (E)11 to E17. mRNA was localized by in situ hybridization, and apoptotic cells by TUNEL (TDT-mediated dUTP-biotin nick end-labeling) as well as by transmission electron microscopy. By analyzing adjacent serial sections, we demonstrated that the expression of Msx-2 and Bmp-2 strikingly overlapped in the atrioventricular canal myocardium, in the atrioventricular junctional myocardium, and in the maturing myocardium of the atrioventricular valves. Bmp-4 was expressed in the outflow tract myocardium and in the endocardial cushion of the outflow tract ridges from E12 to E14. Msx-2 appeared in the mesenchyme of the atrioventricular endocardial cushion from E11 to E14, while Bmp-2 and Bmp-4 were detected between E11 and E14. Apoptotic cells were also detected in the mesenchyme of the endocardial cushion between E12 and E14. Our results suggest that BMP-2 and MSX-2 are tightly linked to the formation of the atrioventricular junction and valves and that BMP-4 is involved in the development of the outflow tract myocardium and of the endocardial cushion. In addition, BMP-2, BMP-4 and MSX-2 and apoptosis seem to be associated with differentiation of the endocardial cushion.

The acceptability to patients of PhysioDirect telephone assessment and advice services; a qualitative interview study.

PubMed

Pearson, Jennifer; Richardson, Jane; Calnan, Michael; Salisbury, Chris; Foster, Nadine E

2016-03-28

In response to long waiting lists and problems with access to primary care physiotherapy, several Primary Care Trusts (PCTs) (now Clinical Commissioning Groups CCGs) developed physiotherapy-led telephone assessment and treatment services. The Medical Research Council (MRC) funded PhysioDirect trial was a randomised control trial (RCT) in four PCTs, with a total of 2252 patients that compared this approach with usual physiotherapy care. This nested qualitative study aimed to explore the acceptability of the PhysioDirect telephone assessment and advice service to patients with musculoskeletal conditions. We conducted 57 semi-structured interviews with adults from 4 PCTs who were referred from general practice to physiotherapy with musculoskeletal conditions and were participating in the PhysioDirect trial. The Framework method was used to analyse the qualitative data. The PhysioDirect service was largely viewed as acceptable although some saw it as a first step to subsequent face-to-face physiotherapy. Most participants found accessing the PhysioDirect service straightforward and smooth, and they valued the faster access to physiotherapy advice offered by the telephone service. Participants generally viewed both the PhysioDirect service and the physiotherapists providing the service as helpful. Participants' preferences and priorities for treatment defined the acceptable features of PhysioDirect but the acceptable features were traded off against less acceptable features. Some participants felt that the PhysioDirect service was impersonal and impaired the development of a good relationship with their physiotherapist, which made the service feel remote and less valuable. The PhysioDirect service was broadly acceptable to participants since it provided faster access to physiotherapy advice for their musculoskeletal conditions. Participants felt that it is best placed as one method of accessing physiotherapy services, in addition to, rather than as a replacement for, more traditional face-to-face physiotherapy assessment and treatment.

Zero-order controlled release of BMP2-derived peptide P24 from the chitosan scaffold by chemical grafting modification technique for promotion of osteogenesis in vitro and enhancement of bone repair in vivo.

PubMed

Chen, Yan; Liu, Xujie; Liu, Rui; Gong, Yong; Wang, Mingbo; Huang, Qianli; Feng, Qingling; Yu, Bo

2017-01-01

Combination of tissue-engineered bone scaffolds with cell-adhesive, osteoconductive, or osteoinductive biomolecules is a critical strategy to improve their properties that significantly influence cellular behaviors, such as adhesion, proliferation, and differentiation, which is beneficial for critical-sized bone defects repairing. However, the traditional surface modification techniques, such as physical adsorption, coating, and plasma treatment, et al, have great limitations for immobilization of bioactive molecules due to undesirable controlled delivery performance or overly complex multistep procedures. In this study, we functionalized the chitosan/hydroxyapatite (CS/HA) biomimetic composite scaffold for controlled delivery of BMP2-derived peptide (P24) by the chemical grafting modification technique: firstly, P24 was conjugated with a thiolated chitosan, chitosan-4-thiobutylamidine (CS-TBA); secondly, the resultant CS-P24 was then combined with HA to prepare CS-P24/HA scaffolds. The effect of CS-P24/HA scaffolds on bone regeneration was evaluated, along with the underlying biological mechanisms responsible in vitro and in vivo . In vitro , the controlled and sustained release of bioactive P24 could last up to 90 days, furthermore, the release profiles of CS-5%P24/HA and CS-10%P24/HA were linear and could be fitted according to zero-order kinetic model (R 2 =0.9929; R 2 =0.9757); P24 on the scaffold significantly promoted cell adhesion, proliferation, osteodifferentiation, and mineralization with synergistic effects. Bone marrow stromal cells (BMSCs) revealed spindle-shaped surface morphology, indicating the CS-P24/HA scaffolds supported cell adhesion and possessed a high proliferation rate that varied according to the P24 concentration levels. Furthermore, mRNA levels for OCN, Runx2, and collagen I were significantly up-regulated on CS-P24/HA scaffolds compared with cells grown on CS/HA scaffolds in vitro ( p < 0.05). Similarly, the BMSCs exhibited a higher ALP expression and calcium deposition level on CS-P24/HA scaffolds compared with CS/HA scaffolds ( p < 0.05). In vivo , osteoinductive studies revealed a significantly higher ectopic osteogenesis level of CS-10%P24/HA scaffolds in rat dorsal muscle pockets compared with that of CS/HA scaffolds. Finally, CS-P24/HA scaffolds showed superior performance in the reconstruction of rat calvarial bone defects. This novel CS-P24/HA scaffold is deemed a strong potential candidate for the repair of bone defects in human bone tissue engineering.

Zero-order controlled release of BMP2-derived peptide P24 from the chitosan scaffold by chemical grafting modification technique for promotion of osteogenesis in vitro and enhancement of bone repair in vivo

PubMed Central

Chen, Yan; Liu, Xujie; Liu, Rui; Gong, Yong; Wang, Mingbo; Huang, Qianli; Feng, Qingling; Yu, Bo

2017-01-01

Combination of tissue-engineered bone scaffolds with cell-adhesive, osteoconductive, or osteoinductive biomolecules is a critical strategy to improve their properties that significantly influence cellular behaviors, such as adhesion, proliferation, and differentiation, which is beneficial for critical-sized bone defects repairing. However, the traditional surface modification techniques, such as physical adsorption, coating, and plasma treatment, et al, have great limitations for immobilization of bioactive molecules due to undesirable controlled delivery performance or overly complex multistep procedures. In this study, we functionalized the chitosan/hydroxyapatite (CS/HA) biomimetic composite scaffold for controlled delivery of BMP2-derived peptide (P24) by the chemical grafting modification technique: firstly, P24 was conjugated with a thiolated chitosan, chitosan-4-thiobutylamidine (CS-TBA); secondly, the resultant CS-P24 was then combined with HA to prepare CS-P24/HA scaffolds. The effect of CS-P24/HA scaffolds on bone regeneration was evaluated, along with the underlying biological mechanisms responsible in vitro and in vivo. In vitro, the controlled and sustained release of bioactive P24 could last up to 90 days, furthermore, the release profiles of CS-5%P24/HA and CS-10%P24/HA were linear and could be fitted according to zero-order kinetic model (R2=0.9929; R2=0.9757); P24 on the scaffold significantly promoted cell adhesion, proliferation, osteodifferentiation, and mineralization with synergistic effects. Bone marrow stromal cells (BMSCs) revealed spindle-shaped surface morphology, indicating the CS-P24/HA scaffolds supported cell adhesion and possessed a high proliferation rate that varied according to the P24 concentration levels. Furthermore, mRNA levels for OCN, Runx2, and collagen I were significantly up-regulated on CS-P24/HA scaffolds compared with cells grown on CS/HA scaffolds in vitro (p < 0.05). Similarly, the BMSCs exhibited a higher ALP expression and calcium deposition level on CS-P24/HA scaffolds compared with CS/HA scaffolds (p < 0.05). In vivo, osteoinductive studies revealed a significantly higher ectopic osteogenesis level of CS-10%P24/HA scaffolds in rat dorsal muscle pockets compared with that of CS/HA scaffolds. Finally, CS-P24/HA scaffolds showed superior performance in the reconstruction of rat calvarial bone defects. This novel CS-P24/HA scaffold is deemed a strong potential candidate for the repair of bone defects in human bone tissue engineering. PMID:28435449

NF-kappaB specifically activates BMP-2 gene expression in growth plate chondrocytes in vivo and in a chondrocyte cell line in vitro.

PubMed

Feng, Jian Q; Xing, Lianping; Zhang, Jiang-Hong; Zhao, Ming; Horn, Diane; Chan, Jeannie; Boyce, Brendan F; Harris, Stephen E; Mundy, Gregory R; Chen, Di

2003-08-01

Bone morphogenetic protein-2 (BMP-2) regulates growth plate chondrogenesis during development and postnatal bone growth, but the control mechanisms of BMP-2 expression in growth plate chondrocytes are unknown. Here we have used both in vitro and in vivo approaches to demonstrate that transcription factor, NF-kappaB, regulates BMP-2 gene expression in chondrocytes. Two putative NF-kappaB response elements were found in the -2712/+165 region of the BMP-2 gene. Cotransfection of mutant I-kappaBalpha expression plasmids with BMP-2 promoter-luciferase reporters into TMC-23 chondrocyte cell line suppressed BMP-2 transcription. Mutations in NF-kappaB response elements in the BMP-2 gene lead to decreases in BMP-2 promoter activity. Electrophoretic mobility shift assay using nuclear extracts from TMC-23 chondrocytic cells revealed that the NF-kappaB subunits p50 and p65 bound to the NF-kappaB response elements of the BMP-2 gene. Thus, NF-kappaB may positively regulate BMP-2 gene transcription. Consistent with these findings, expression of BMP-2 mRNA was significantly reduced in growth plate chondrocytes in NF-kappaB p50/p52 dKO mice, which associated with decreased numbers of 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells in the proliferating zone of growth plate in these mice. Therefore, in postnatal growth plate chondrocytes, expression of BMP-2 is regulated by NF-kappaB, which may play an important role in chondrogenesis.

In vitro study on the osteogenesis enhancement effect of BMP-2 incorporated biomimetic apatite coating on titanium surfaces.

PubMed

Zhu, Xiaojing; Zhang, Hui; Zhang, Xinchun; Ning, Chengyun; Wang, Yan

2017-09-26

To fabricate a sustained-release delivery system of bone morphogenetic protein (BMP-2) on titanium surface, explore the effect of BMP-2 concentration on the loading/release behavior of BMP-2 and evaluate the cell compatibility of the system in vitro, pure titanium specimens were immersed into supersaturated calcium phosphate solutions (SCP) containing 4 different concentrations of BMP-2: 0, 50, 100, 200 and 400 ng/mL. Biomimetic calcium phosphate coating was formed on titanium surface and BMP-2 was incorporated into the coating through co-deposition. The release profile of BMP-2 suggested that BMP-2 were delivered sustainably up to 20 days. CCK-8 and ALP assay showed that 200 group and 400 ng/mL BMP-2 group have significant effect on promoting MC3T3-E1 cell proliferation and differentiation. The BMP-2 incorporated into the hybrid coating released in a sustained manner and significantly promoted the proliferation and differentiation of MC3T3-E1 on the titanium surface.

Bmp2 Deletion Causes an Amelogenesis Imperfecta Phenotype Via Regulating Enamel Gene Expression

PubMed Central

GUO, FENG; FENG, JUNSHENG; WANG, FENG; LI, WENTONG; GAO, QINGPING; CHEN, ZHUO; SHOFF, LISA; DONLY, KEVIN J.; GLUHAK-HEINRICH, JELICA; CHUN, YONG HEE PATRICIA; HARRIS, STEPHEN E.; MACDOUGALL, MARY; CHEN, SHUO

2015-01-01

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo. PMID:25545831

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

PubMed

Guo, Feng; Feng, Junsheng; Wang, Feng; Li, Wentong; Gao, Qingping; Chen, Zhuo; Shoff, Lisa; Donly, Kevin J; Gluhak-Heinrich, Jelica; Chun, Yong Hee Patricia; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

2015-08-01

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo. © 2015 Wiley Periodicals, Inc.

Low-dose rhBMP2/7 heterodimer to reconstruct peri-implant bone defects: a micro-CT evaluation.

PubMed

Wang, Jingxiao; Zheng, Yuanna; Zhao, Juan; Liu, Tie; Gao, Lixia; Gu, Zhiyuan; Wu, Gang

2012-01-01

To delineate the dynamic micro-architectures of bone induced by low-dose bone morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects compared to BMP2 and BMP7 homodimer. Peri-implant bone defects (8 mm in diameter, 4 mm in depth) were created surrounding SLA-treated titanium implants (3.1 mm in diameter, 10 mm in length) in minipig's calvaria. We administrated collagen sponges with adsorbed low-dose (30 ng/mm(3) ) BMP2/7 to treat the defects using BMP2, BMP7 or no BMP as controls.2, 3 and 6 weeks after implantation, we adopted micro-computer tomography to evaluate the micro-architectures of new bone using the following parameters: relative bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density, and structure mode index (SMI). Bone implant contact (BIC) was also revealed histologically. Consistent with 2 and 3 weeks, after 6 weeks post-operation, BMP2/7 resulted in significantly higher BV/TV (63.033 ± 2.055%) and significantly lower SMI (-4.405 ± 0.500) than BMP2 (BV/TV: 43.133 ± 2.001%; SMI: -0.086 ± 0.041) and BMP7 (BV/TV: 41.467 ± 1.850%; SMI: -0.044 ± 0.016) respectively. Significant differences were also found in Tb.N, Tb.Th and Tb.Sp at all time points. At 2 weeks, BMP2/7 resulted in significantly higher BIC than the controls. Low-dose BMP2/7 heterodimer facilitated more rapid bone regeneration in better quality in peri-implant bone defects than BMP2 and BMP7 homodimers. © 2011 John Wiley & Sons A/S.

Diversity, Novelty, and Antimicrobial Activity of Endophytic Actinobacteria From Mangrove Plants in Beilun Estuary National Nature Reserve of Guangxi, China.

PubMed

Jiang, Zhong-Ke; Tuo, Li; Huang, Da-Lin; Osterman, Ilya A; Tyurin, Anton P; Liu, Shao-Wei; Lukyanov, Dmitry A; Sergiev, Petr V; Dontsova, Olga A; Korshun, Vladimir A; Li, Fei-Na; Sun, Cheng-Hang

2018-01-01

Endophytic actinobacteria are one of the important pharmaceutical resources and well known for producing different types of bioactive substances. Nevertheless, detection of the novelty, diversity, and bioactivity on endophytic actinobacteria isolated from mangrove plants are scarce. In this study, five different mangrove plants, Avicennia marina, Aegiceras corniculatum, Kandelia obovota, Bruguiera gymnorrhiza , and Thespesia populnea , were collected from Beilun Estuary National Nature Reserve in Guangxi Zhuang Autonomous Region, China. A total of 101 endophytic actinobacteria strains were recovered by culture-based approaches. They distributed in 7 orders, 15 families, and 28 genera including Streptomyces, Curtobacterium, Mycobacterium, Micrococcus, Brevibacterium, Kocuria, Nocardioides, Kineococcus, Kytococcus, Marmoricola, Microbacterium, Micromonospora, Actinoplanes, Agrococcus, Amnibacterium, Brachybacterium, Citricoccus, Dermacoccus, Glutamicibacter, Gordonia, Isoptericola, Janibacter, Leucobacter, Nocardia, Nocardiopsis, Pseudokineococcus, Sanguibacter , and Verrucosispora . Among them, seven strains were potentially new species of genera Nocardioides, Streptomyces, Amnibacterium, Marmoricola , and Mycobacterium . Above all, strain 8BXZ-J1 has already been characterized as a new species of the genus Marmoricola . A total of 63 out of 101 strains were chosen to screen antibacterial activities by paper-disk diffusion method and inhibitors of ribosome and DNA biosynthesis by means of a double fluorescent protein reporter. A total of 31 strains exhibited positive results in at least one antibacterial assay. Notably, strain 8BXZ-J1 and three other potential novel species, 7BMP-1, 5BQP-J3, and 1BXZ-J1, all showed antibacterial bioactivity. In addition, 21 strains showed inhibitory activities against at least one "ESKAPE" resistant pathogens. We also found that Streptomyces strains 2BBP-J2 and 1BBP-1 produce bioactive compound with inhibitory activity on protein biosynthesis as result of translation stalling. Meanwhile, Streptomyces strain 3BQP-1 produces bioactive compound inducing SOS-response due to DNA damage. In conclusion, this study proved mangrove plants harbored a high diversity of cultivable endophytic actinobacteria, which can be a promising source for discovery of novel species and bioactive compounds.

Diversity, Novelty, and Antimicrobial Activity of Endophytic Actinobacteria From Mangrove Plants in Beilun Estuary National Nature Reserve of Guangxi, China

PubMed Central

Jiang, Zhong-ke; Tuo, Li; Huang, Da-lin; Osterman, Ilya A.; Tyurin, Anton P.; Liu, Shao-wei; Lukyanov, Dmitry A.; Sergiev, Petr V.; Dontsova, Olga A.; Korshun, Vladimir A.; Li, Fei-na; Sun, Cheng-hang

2018-01-01

Endophytic actinobacteria are one of the important pharmaceutical resources and well known for producing different types of bioactive substances. Nevertheless, detection of the novelty, diversity, and bioactivity on endophytic actinobacteria isolated from mangrove plants are scarce. In this study, five different mangrove plants, Avicennia marina, Aegiceras corniculatum, Kandelia obovota, Bruguiera gymnorrhiza, and Thespesia populnea, were collected from Beilun Estuary National Nature Reserve in Guangxi Zhuang Autonomous Region, China. A total of 101 endophytic actinobacteria strains were recovered by culture-based approaches. They distributed in 7 orders, 15 families, and 28 genera including Streptomyces, Curtobacterium, Mycobacterium, Micrococcus, Brevibacterium, Kocuria, Nocardioides, Kineococcus, Kytococcus, Marmoricola, Microbacterium, Micromonospora, Actinoplanes, Agrococcus, Amnibacterium, Brachybacterium, Citricoccus, Dermacoccus, Glutamicibacter, Gordonia, Isoptericola, Janibacter, Leucobacter, Nocardia, Nocardiopsis, Pseudokineococcus, Sanguibacter, and Verrucosispora. Among them, seven strains were potentially new species of genera Nocardioides, Streptomyces, Amnibacterium, Marmoricola, and Mycobacterium. Above all, strain 8BXZ-J1 has already been characterized as a new species of the genus Marmoricola. A total of 63 out of 101 strains were chosen to screen antibacterial activities by paper-disk diffusion method and inhibitors of ribosome and DNA biosynthesis by means of a double fluorescent protein reporter. A total of 31 strains exhibited positive results in at least one antibacterial assay. Notably, strain 8BXZ-J1 and three other potential novel species, 7BMP-1, 5BQP-J3, and 1BXZ-J1, all showed antibacterial bioactivity. In addition, 21 strains showed inhibitory activities against at least one “ESKAPE” resistant pathogens. We also found that Streptomyces strains 2BBP-J2 and 1BBP-1 produce bioactive compound with inhibitory activity on protein biosynthesis as result of translation stalling. Meanwhile, Streptomyces strain 3BQP-1 produces bioactive compound inducing SOS-response due to DNA damage. In conclusion, this study proved mangrove plants harbored a high diversity of cultivable endophytic actinobacteria, which can be a promising source for discovery of novel species and bioactive compounds. PMID:29780376

Comparative study suggests that human bone morphogenetic proteins have no influence on the outcome of operative treatment of aseptic clavicle non-unions.

PubMed

von Rüden, Christian; Morgenstern, Mario; Friederichs, Jan; Augat, Peter; Hackl, Simon; Woltmann, Alexander; Bühren, Volker; Hierholzer, Christian

2016-11-01

The purpose of this study was to evaluate the clinical and radiological outcome following compression plate fixation in combination with autologous bone grafting, with and without additional application of recombinant human bone morphogenetic protein (rhBMP) for treatment of aseptic clavicle non-union. Between April 2004 and April 2015, 82 patients were treated for clavicle fracture and had developed aseptic clavicle non-union. Seventy-three out of 82 patients were available for follow-up at least one year after revision surgery; among them, 27 women and 46 men, with a median age of 49 (range, 19-86) years. Forty-five patients received compression plate osteosynthesis with autologous bone grafting, and 28 patients obtained compression plate fixation with autologous bone grafting and additional application of rhBMP-2 (3/28 patients) or rhBMP-7 (25/28 patients). Seventy out of 73 non-unions (96 %) healed within 12 months after revision surgery. Functional outcome according to the DASH Outcome Measure (with rhBMP, 33.16 ± 1.17 points; without rhBMP, 30.58 ± 2.12 points [mean ± SEM]; p = 0.81), non-union healing (p = 0.86), time interval between revision surgery and bone healing (p = 0.37), as well as post-operative complications, did not demonstrate relevant differences between the treatment groups and were not age-dependent. Functional and radiological results demonstrate that successful healing of aseptic clavicle non-union is dependent on radical resection of non-union tissue, restoration of length of the shoulder girdle and application of stable locking-plate osteosynthesis in combination with autologous bone grafting, but not dependent on application of additional rhBMP.

Turning Bone Morphogenetic Protein 2 (BMP2) On and Off in Mesenchymal Cells†

PubMed Central

Rogers, Melissa B.; Shah, Tapan A.; Shaikh, Nadia N.

2016-01-01

The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. This article is protected by copyright. All rights reserved PMID:25776852

In vitro and in vivo protein release and anti-ischemia/reperfusion injury properties of bone morphogenetic protein-2-loaded glycyrrhetinic acid-poly(ethylene glycol)-b-poly(l-lysine) nanoparticles

PubMed Central

Shan, Fang; Liu, YuJuan; Jiang, Haiying; Tong, Fei

2017-01-01

Here, we describe a bone morphogenetic protein-2 (BMP-2) nanocarrier based on glycyrrhetinic acid (GA)-poly(ethylene glycol) (PEG)-b-poly(l-lysine) (PLL). A protein nanocarrier was synthesized, characterized and evaluated as a BMP-2 delivery system. The designed nanocarrier was synthesized based on the ring-opening polymerization of amino acid N-carboxyanhydride. The final product was measured with 1H nuclear magnetic resonance. GA-PEG-b-PLL nanocarrier could combine with BMP-2 through electrostatic interaction to form polyion complex (PIC) micelles. BMP-2 could be rapidly and efficiently encapsulated through the GA-PEG-b-PLL nanocarrier under physiological conditions, exhibiting efficient encapsulation and sustained release. In addition, the GA-PEG-b-PLL-mediated BMP-2 delivery system could target the liver against hepatic diseases as it has GA-binding receptors. The anti-hepatic ischemia/reperfusion injury (anti-HI/RI) effect of BMP-2/GA-PEG-b-PLL PIC micelles was investigated in rats using free BMP-2 and BMP-2/PEG-b-PLL PIC micelles as controls, and the results showed that BMP-2/GA-PEG-b-PLL PIC micelles indicated significantly enhanced anti-HI/RI property compared to BMP-2 and BMP-2/PEG-b-PLL. All results suggested that GA-PEG-b-PLL could be used as a potential BMP-2 nanocarrier. PMID:29089759

Continuous release of bone morphogenetic protein-2 through nano-graphene oxide-based delivery influences the activation of the NF-κB signal transduction pathway

PubMed Central

Zhong, Cheng; Feng, Jun; Lin, Xiangjin; Bao, Qi

2017-01-01

Graphene oxide (GO) has been used as a delivery vehicle for small molecule drugs and nucleotides. To further investigate GO as a smart biomaterial for the controlled release of cargo molecules, we hypothesized that GO may be an appropriate delivery vehicle because it releases bone morphogenetic protein 2 (BMP2). GO characterization indicated that the size distribution of the GO flakes ranged from 81.1 nm to 45,749.7 nm, with an approximate thickness of 2 nm. After BMP2 adsorption onto GO, Fourier-transformed infrared spectroscopy (FTIR) and thermal gravimetric analysis were performed. Compared to GO, BMP2-GO did not induce significant changes in the characteristics of the materials. GO continuously released BMP2 for at least 40 days. Bone marrow stem cells (BMSCs) and chondrocytes were treated with BMP2-GO in interleukin-1 media and assessed in terms of cell viability, flow cytometric characterization, and expression of particular mRNA. Compared to GO, BMP2-GO did not induce any significant changes in biocompatibility. We treated osteoarthritic rats with BMP2 and BMP2-GO, which showed significant differences in Osteoarthritis Research Society International (OARSI) scores (P<0.05). Quantitative assessment revealed significant differences compared to that using BMP2 and BMP2-GO (P<0.05). These findings indicate that GO may be potentially used to control the release of carrier materials. The combination of BMP2 and GO slowed the progression of NF-κB-activated degenerative changes in osteoarthritis. Therefore, we infer that our BMP2-GO strategy could alleviate the NF-κB pathway by inducing continuous BMP2 release. PMID:28243085

Developmental roles of the BMP1/TLD metalloproteinases.

PubMed

Ge, Gaoxiang; Greenspan, Daniel S

2006-03-01

The astacin family (M12A) of the metzincin subclan MA(M) of metalloproteinases has been detected in developing and mature individuals of species that range from hydra to humans. Functions of this family of metalloproteinase vary from digestive degradation of polypeptides, to biosynthetic processing of extracellular proteins, to activation of growth factors. This review will focus on a small subgroup of the astacin family; the bone morphogenetic protein 1 (BMP1)/Tolloid (TLD)-like metalloproteinases. In vertebrates, the BMP1/TLD-like metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating mineralization of the ECM of hard tissues. Roles in ECM formation include: processing of the C-propeptides of procollagens types I-III, to yield the major fibrous components of vertebrate ECM; proteolytic activation of the enzyme lysyl oxidase, necessary to formation of covalent cross-links in collagen and elastic fibers; processing of NH2-terminal globular domains and C-propeptides of types V and XI procollagen chains to yield monomers that are incorporated into and control the diameters of collagen type I and II fibrils, respectively; processing of precursors for laminin 5 and collagen type VII, both of which are involved in securing epidermis to underlying dermis; and maturation of small leucine-rich proteoglycans. The BMP1/TLD-related metalloproteinases are also capable of activating the vertebrate transforming growth factor-beta (TGF-beta)-like "chalones" growth differentiation factor 8 (GDF8, also known as myostatin), and GDF11 (also known as BMP11), involved in negative feedback inhibition of muscle and neural tissue growth, respectively; by freeing them from noncovalent latent complexes with their cleaved prodomains. BMP1/TLD-like proteinases also liberate the vertebrate TGF-beta-like morphogens BMP2 and 4 and their invertebrate ortholog decapentaplegic, from latent complexes with the vertebrate extracellular antagonist chordin and its invertebrate ortholog short gastrulation (SOG), respectively. The result is formation of the BMP signaling gradients that form the dorsal-ventral axis in embryogenesis. Thus, BMP1/TLD-like proteinases appear to be key to regulating and orchestrating formation of the ECM and signaling by various TGF-beta-like proteins in morphogenetic and homeostatic events. Copyright 2006 Wiley-Liss, Inc.

2,2'-Bis(monoacylglycero) PO4 (BMP), but Not 3,1'-BMP, increases membrane curvature stress to enhance α-tocopherol transfer protein binding to membranes.

PubMed

Baptist, Matilda; Panagabko, Candace; Nickels, Jonathan D; Katsaras, John; Atkinson, Jeffrey

2015-03-01

Previous work revealed that α-tocopherol transfer protein (α-TTP) co-localizes with bis(monoacylglycero)phosphate (BMP) in late endosomes. BMP is a lipid unique to late endosomes and is believed to induce membrane curvature and support the multivesicular nature of this organelle. We examined the effect of BMP on α-TTP binding to membranes using dual polarization interferometry and vesicle-binding assay. α-TTP binding to membranes is increased by the curvature-inducing lipid BMP. α-TTP binds to membranes with greater affinity when they contain the 2,2'-BMP versus 3,1'-BMP isomers.

2,2'-Bis(monoacylglycero) PO 4 (BMP), but Not 3,1'-BMP, Increases Membrane Curvature Stress to Enhance α-Tocopherol Transfer Protein Binding to Membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baptist, Matilda; Panagabko, Candace; Nickels, Jonathan D.

2015-01-21

Previous work revealed that α-tocopherol transfer protein (α-TTP) co-localizes with bis(monoacylglycero)phosphate (BMP) in late endosomes. BMP is a lipid unique to late endosomes and is believed to induce membrane curvature and support the multivesicular nature of this organelle. In this paper, we examined the effect of BMP on α-TTP binding to membranes using dual polarization interferometry and vesicle-binding assay. α-TTP binding to membranes is increased by the curvature-inducing lipid BMP. Finally, α-TTP binds to membranes with greater affinity when they contain the 2,2'-BMP versus 3,1'-BMP isomers.

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells.

PubMed

Cruz, Ariadne Cristiane Cabral; Silva, Mariana Lúcia; Caon, Thiago; Simões, Cláudia Maria Oliveira

2012-01-01

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

Crestal Sinus Augmentation with Recombinant Human Bone Morphogenetic Protein 2: Clinical and Radiographic Outcomes of 2-Year Pilot Trial.

PubMed

Kuchler, Ulrike; Rudelstorfer, Claudia M; Barth, Barbara; Tepper, Gabor; Lidinsky, Dominika; Heimel, Patrick; Watzek, Georg; Gruber, Reinhard

Recombinant human bone morphogenetic protein 2 (rhBMP-2) together with an absorbable collagen carrier (ACS) was approved for augmentation of the maxillary sinus prior to implant placement. The original registration trial was based on a lateral window approach. Clinical outcomes of crestal sinus augmentation with rhBMP-2 have not been reported so far. An uncontrolled pilot trial in which seven patients with a residual maxillary height below 5 mm were enrolled to receive crestal sinus augmentation with rhBMP-2/ACS was conducted. Elevation of the sinus mucosa was performed by gel pressure. Primary endpoints were the gain in augmentation height and volume measured by computed tomography after 6 months. Evaluation of bone quality at the time of implant placement was based on histology. Secondary endpoints were the clinical and radiologic evaluation of the implants and patient satisfaction by visual analog scale (VAS) at the 2-year follow-up. Median gain in augmentation height was 7.2 mm (range 0.0 to 17.5 mm). Five patients gained at least 5 mm of bone height. Two patients with a perforation of the sinus mucosa failed to respond to rhBMP-2/ACS and underwent lateral window augmentation. The median gain in augmentation volume of the five patients was 781.3 mm³ (range 426.9 to 1,242.8 mm³). Biopsy specimens showed a cancellous network consisting of primary plexiform bone with little secondary lamellar bone. After 2 years, implants were in function with no signs of inflammation or peri-implant bone loss. Patients were satisfied with the esthetic outcomes and chewing function. This pilot clinical trial supports the original concept that rhBMP-2/ACS supports bone formation, also in crestal sinus augmentation, and emphasizes the relevance of the integrity of the sinus mucosa to predict the bone gain.

Zygotic LvBMP5-8 is required for skeletal patterning and for left-right but not dorsal-ventral specification in the sea urchin embryo.

PubMed

Piacentino, Michael L; Chung, Oliver; Ramachandran, Janani; Zuch, Daniel T; Yu, Jia; Conaway, Evan A; Reyna, Arlene E; Bradham, Cynthia A

2016-04-01

Skeletal patterning in the sea urchin embryo requires coordinated signaling between the pattern-dictating ectoderm and the skeletogenic primary mesenchyme cells (PMCs); recent studies have begun to uncover the molecular basis for this process. Using an unbiased RNA-Seq-based screen, we have previously identified the TGF-ß superfamily ligand, LvBMP5-8, as a skeletal patterning gene in Lytechinus variegatus embryos. This result is surprising, since both BMP5-8 and BMP2/4 ligands have been implicated in sea urchin dorsal-ventral (DV) and left-right (LR) axis specification. Here, we demonstrate that zygotic LvBMP5-8 is required for normal skeletal patterning on the left side, as well as for normal PMC positioning during gastrulation. Zygotic LvBMP5-8 is required for expression of the left-side marker soxE, suggesting that LvBMP5-8 is required for left-side specification. Interestingly, we also find that LvBMP5-8 knockdown suppresses serotonergic neurogenesis on the left side. While LvBMP5-8 overexpression is sufficient to dorsalize embryos, we find that zygotic LvBMP5-8 is not required for normal DV specification or development. In addition, ectopic LvBMP5-8 does not dorsalize LvBMP2/4 morphant embryos, indicating that, in the absence of BMP2/4, BMP5-8 is insufficient to specify dorsal. Taken together, our data demonstrate that zygotic LvBMP5-8 signaling is essential for left-side specification, and for normal left-side skeletal and neural patterning, but not for DV specification. Thus, while both BMP2/4 and BMP5-8 regulate LR axis specification, BMP2/4 but not zygotic BMP5-8 regulates DV axis specification in sea urchin embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of Particle Size Influence on Proximate Composition, Physicochemical, Techno-Functional and Physio-Functional Properties of Flours Obtained from Persimmon (Diospyros kaki Trumb.) Coproducts.

PubMed

Lucas-González, Raquel; Viuda-Martos, Manuel; Pérez-Álvarez, José Ángel; Fernández-López, Juana

2017-03-01

The aim of the work was to study the influence of particle size in the composition, physicochemical, techno-functional and physio-functional properties of two flours obtained from persimmon (Diospyros kaki Trumb. cvs. 'Rojo Brillante' (RBF) and 'Triump' (THF) coproducts. The cultivar (RBF and THF) and particle size significantly affected all parameters under study, although depending on the evaluated property, only one of these effects predominated. Carbohydrates (38.07-46.98 g/100 g) and total dietary fiber (32.07-43.57 g/100 g) were the main components in both flours (RBF and THF). Furthermore, insoluble dietary fiber represented more than 68% of total dietary fiber content. All color properties studied were influenced by cultivar and particle size. For both cultivars, the lower particle size, the higher lightness and hue values. RBF flours showed high values for emulsifying activity (69.33-74.00 mL/mL), while THF presented high values for water holding capacity (WHC: 9.47-12.19 g water/g sample). The bile holding capacity (BHC) and fat/oil binding values were, in general, higher in RBF (19.61-12.19 g bile/g sample and 11.98-9.07, respectively) than THF (16.12-12.40 g bile/g sample and 9.78-7.96, respectively). The effect of particle size was really evident in both WHC and BHC. Due to their dietary fiber content, techno-functional and physio-functional properties, persimmon flours seem to have a good profile to be used as potential functional ingredient.

Dual delivery of rhPDGF-BB and bone marrow mesenchymal stromal cells expressing the BMP2 gene enhance bone formation in a critical-sized defect model.

PubMed

Park, Shin-Young; Kim, Kyoung-Hwa; Shin, Seung-Yun; Koo, Ki-Tae; Lee, Yong-Moo; Seol, Yang-Jo

2013-11-01

Bone tissue healing is a dynamic, orchestrated process that relies on multiple growth factors and cell types. Platelet-derived growth factor-BB (PDGF-BB) is released from platelets at wound sites and induces cellular migration and proliferation necessary for bone regeneration in the early healing process. Bone morphogenetic protein-2 (BMP-2), the most potent osteogenic differentiation inducer, directs new bone formation at the sites of bone defects. This study evaluated a combinatorial treatment protocol of PDGF-BB and BMP-2 on bone healing in a critical-sized defect model. To mimic the bone tissue healing process, a dual delivery approach was designed to deliver the rhPDGF-BB protein transiently during the early healing phase, whereas BMP-2 was supplied by rat bone marrow stromal cells (BMSCs) transfected with an adenoviral vector containing the BMP2 gene (AdBMP2) for prolonged release throughout the healing process. In in vitro experiments, the dual delivery of rhPDGF-BB and BMP2 significantly enhanced cell proliferation. However, the osteogenic differentiation of BMSCs was significantly suppressed even though the amount of BMP-2 secreted by the AdBMP2-transfected BMSCs was not significantly affected by the rhPDGF-BB treatment. In addition, dual delivery inhibited the mRNA expression of BMP receptor type II and Noggin in BMSCs. In in vivo experiments, critical-sized calvarial defects in rats showed enhanced bone regeneration by dual delivery of autologous AdBMP2-transfected BMSCs and rhPDGF-BB in both the amount of new bone formed and the bone mineral density. These enhancements in bone regeneration were greater than those observed in the group treated with AdBMP2-transfected BMSCs alone. In conclusion, the dual delivery of rhPDGF-BB and AdBMP2-transfected BMSCs improved the quality of the regenerated bone, possibly due to the modulation of PDGF-BB on BMP-2-induced osteogenesis.

BMP signaling in the development of the mouse esophagus and forestomach

PubMed Central

Rodriguez, Pavel; Da Silva, Susana; Oxburgh, Leif; Wang, Fan; Hogan, Brigid L. M.; Que, Jianwen

2010-01-01

The stratification and differentiation of the epidermis are known to involve the precise control of multiple signaling pathways. By contrast, little is known about the development of the mouse esophagus and forestomach, which are composed of a stratified squamous epithelium. Based on prior work in the skin, we hypothesized that bone morphogenetic protein (BMP) signaling is a central player. To test this hypothesis, we first used a BMP reporter mouse line harboring a BRE-lacZ allele, along with in situ hybridization to localize transcripts for BMP signaling components, including various antagonists. We then exploited a Shh-Cre allele that drives recombination in the embryonic foregut epithelium to generate gain- or loss-of-function models for the Bmpr1a (Alk3) receptor. In gain-of-function (Shh-Cre;Rosa26CAG-loxpstoploxp-caBmprIa) embryos, high levels of ectopic BMP signaling stall the transition from simple columnar to multilayered undifferentiated epithelium in the esophagus and forestomach. In loss-of-function experiments, conditional deletion of the BMP receptor in Shh-Cre;Bmpr1aflox/flox embryos allows the formation of a multilayered squamous epithelium but this fails to differentiate, as shown by the absence of expression of the suprabasal markers loricrin and involucrin. Together, these findings suggest multiple roles for BMP signaling in the developing esophagus and forestomach. PMID:21068065

Growth factor transgenes interactively regulate articular chondrocytes.

PubMed

Shi, Shuiliang; Mercer, Scott; Eckert, George J; Trippel, Stephen B

2013-04-01

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair. Copyright © 2012 Wiley Periodicals, Inc.

Regulation of Msx-1, Msx-2, Bmp-2 and Bmp-4 during foetal and postnatal mammary gland development.

PubMed

Phippard, D J; Weber-Hall, S J; Sharpe, P T; Naylor, M S; Jayatalake, H; Maas, R; Woo, I; Roberts-Clark, D; Francis-West, P H; Liu, Y H; Maxson, R; Hill, R E; Dale, T C

1996-09-01

Expression of the Msx-1 and Msx-2 homeobox genes have been shown to be coordinately regulated with the Bmp-2 and Bmp-4 ligands in a variety of developing tissues. Here we report that transcripts from all four genes are developmentally regulated during both foetal and postnatal mammary gland development. The location and time-course of the Bmp and Msx expression point to a role for Msx and Bmp gene products in the control of epithelial-mesenchymal interactions. Expression of Msx-2, but not Msx-1, Bmp-2 or Bmp-4 was decreased following ovariectomy, while expression of the human Msx-2 homologue was regulated by 17beta-oestradiol in the MCF-7 breast cancer cell line. The regulation of Msx-2 expression by oestrogen raises the possibility that hormonal regulation of mammary development is mediated through the control of epithelial-mesenchymal interactions.

Addition of BMP-2 or BMP-6 to dexamethasone, ascorbic acid, and β-glycerophosphate may not enhance osteogenic differentiation of human periodontal ligament cells.

PubMed

Khanna-Jain, Rashi; Agata, Hideki; Vuorinen, Annukka; Sándor, George K B; Suuronen, Riitta; Miettinen, Susanna

2010-12-01

This study was designed to investigate the potential merits of the combined use of bone morphogenetic protein (BMP)-2 or BMP-6 and osteogenic supplements (OS) [dexamethasone, ascorbic acid (AA), and β-glycerophosphate] on osteogenic differentiation of periodontal ligament cells (PDLCs). Osteogenic differentiation was evaluated by quantitative alkaline phosphatase (ALP) assay, alizarin red staining, quantitative calcium assay, and the qRT-PCR analysis for the expression of collagen type I, runt-related transcription factor-2, osteopontin (OPN), and osteocalcin in PDLCs. Culture with BMP-2 or BMP-6+AA increased ALP activity of PDLCs, suggesting their osteo-inductive effects. However, longer duration of culture showed neither of the BMPs induced in vitro mineralization. In contrast, OS were able to increase ALP activity and OPN expressions, and also induced in vitro mineralization. The mineralization ability was not enhanced by the addition of BMP-2 or BMP-6. These findings suggest that the addition of BMP-2 or BMP-6 to OS may not enhance an osteogenic differentiation of hPDLCs.

Postoperative Complications Associated With rhBMP2 Use in Posterior/Posterolateral Lumbar Fusion.

PubMed

Esmail, Nabil; Buser, Zorica; Cohen, Jeremiah R; Brodke, Darrel S; Meisel, Hans-Joerg; Park, Jong-Beom; Youssef, Jim A; Wang, Jeffrey C; Yoon, S Tim

2018-04-01

Retrospective database review. Posterior/posterolateral lumbar fusion (PLF) is an effective treatment for a variety of spinal disorders; however, variations in surgical technique have different complication profiles. The aim of our study was to quantify the frequency of various complications in patients undergoing PLF with and without human recombinant bone morphogenetic protein 2 (rhBMP2). We queried the orthopedic subset of the Medicare database (PearlDiver) between 2005 and 2011 for patients undergoing PLF procedures with and without rhBMP2. Complication and reoperation rates were analyzed within 1 year of the index procedure. Complications assessed include: acute renal failure, deep vein thrombosis, dural tear, hematoma, heterotopic ossification, incision and drainage, cardiac complications, nervous system complications, osteolysis, pneumonia, pseudarthrosis, pulmonary embolism, radiculopathy, respiratory complications, sepsis, urinary retention, urinary tract infection, mechanical, and wound complications. Chi-square analysis was used to calculate the complication differences between the groups. Our data revealed higher overall complication rates in patients undergoing PLF with rhBMP2 versus no_rhBMP2 (76.9% vs 68.8%, P < .05). Stratified by gender, rhBMP2 males had higher rates of mechanical complications, pseudarthrosis, and reoperations compared with no_rhBMP2 males ( P < .05), whereas rhBMP2 females had higher rates of pseudarthrosis, urinary tract infection, and urinary retention compared with no_rhBMP2 females ( P < .05). Our data revealed higher overall complication rates in PLF patients given rhBMP2 compared with no_rhBMP2. Furthermore, our data suggests that rhBMP2-associated complications may be gender specific.

fussel (fuss)--A negative regulator of BMP signaling in Drosophila melanogaster.

PubMed

Fischer, Susanne; Bayersdorfer, Florian; Harant, Eva; Reng, Renate; Arndt, Stephanie; Bosserhoff, Anja-Katrin; Schneuwly, Stephan

2012-01-01

The TGF-β/BMP signaling cascades control a wide range of developmental and physiological functions in vertebrates and invertebrates. In Drosophila melanogaster, members of this pathway can be divided into a Bone Morphogenic Protein (BMP) and an Activin-ß (Act-ß) branch, where Decapentaplegic (Dpp), a member of the BMP family has been most intensively studied. They differ in ligands, receptors and transmitting proteins, but also share some components, such as the Co-Smad Medea (Med). The essential role of Med is to form a complex with one of the two activating Smads, mothers against decapentaplegic (Mad) or dSmad, and to translocate together to the nucleus where they can function as transcriptional regulators of downstream target genes. This signaling cascade underlies different mechanisms of negative regulation, which can be exerted by inhibitory Smads, such as daughters against decapentaplegic (dad), but also by the Ski-Sno family. In this work we identified and functionally analyzed a new member of the Ski/Sno-family, fussel (fuss), the Drosophila homolog of the human functional suppressing element 15 (fussel-15). fuss codes for two differentially spliced transcripts with a neuronal expression pattern. The proteins are characterized by a Ski-Sno and a SAND homology domain. Overexpression studies and genetic interaction experiments clearly reveal an interaction of fuss with members of the BMP pathway, leading to a strong repression of BMP-signaling. The protein interacts directly with Medea and seems to reprogram the Smad pathway through its influence upon the formation of functional Mad/Medea complexes. This leads amongst others to a repression of downstream target genes of the Dpp pathway, such as optomotor blind (omb). Taken together we could show that fuss exerts a pivotal role as an antagonist of BMP signaling in Drosophila melanogaster.

BMP-2 and titanium particles synergistically activate osteoclast formation

PubMed Central

Sun, S.X.; Guo, H.H.; Zhang, J.; Yu, B.; Sun, K.N.; Jin, Q.H.

2014-01-01

A previous study showed that BMP-2 (bone morphogenetic protein-2) and wear debris can separately support osteoclast formation induced by the receptor activator of NF-κB ligand (RANKL). However, the effect of BMP-2 on wear debris-induced osteoclast formation is unclear. In this study, we show that neither titanium particles nor BMP-2 can induce osteoclast formation in RAW 264.7 mouse leukemic monocyte macrophage cells but that BMP-2 synergizes with titanium particles to enhance osteoclast formation in the presence of RANKL, and that at a low concentration, BMP-2 has an optimal effect to stimulate the size and number of multinuclear osteoclasts, expression of osteoclast genes, and resorption area. Our data also clarify that the effects caused by the increase in BMP-2 on phosphorylated SMAD levels such as c-Fos expression increased throughout the early stages of osteoclastogenesis. BMP-2 and titanium particles stimulate the expression of p-JNK, p-P38, p-IkB, and P50 compared with the titanium group. These data suggested that BMP-2 may be a crucial factor in titanium particle-mediated osteoclast formation. PMID:24820069

Insulin-like growth factor-1 (IGF-1) enhances the osteogenic activity of bone morphogenetic protein-6 (BMP-6) in vitro and in vivo, and together have a stronger osteogenic effect than when IGF-1 is combined with BMP-2.

PubMed

Rico-Llanos, Gustavo A; Becerra, Jose; Visser, Rick

2017-07-01

Bone morphogenetic protein-2 (BMP-2) is widely used in orthopedic surgery and bone tissue engineering because of its strong osteogenic activity. However, BMP-2 treatments have several drawbacks and many groups are actively exploring alternatives. Since BMP-6 has been demonstrated to be more osteoinductive, its use, either alone or together with other growth factors, might be an interesting option. In this work, we have compared the effect of BMP-2, BMP-6, or insulin-like growth factor-1 (IGF-1), either alone or in combination. Murine preosteoblasts were treated with 15 nM IGF-1 and/or 6 nM BMP-2 or -6 and the expression of osteogenic marker genes, proliferation, and alkaline phosphatase (ALP) activity in vitro were analyzed. The results showed that IGF-1 greatly enhanced the BMP-induced osteogenic differentiation of these cells in general and that the ALP activity in the cultures was higher when the combination was made with BMP-6 than with BMP-2. Furthermore, we tested the osteogenic potential of these treatments in vivo by loading 25 pmoles of IGF-1 and/or 10 pmoles of BMP-2 or -6 onto absorbable collagen sponges and implanting them into an ectopic bone formation model in rats. This study revealed that only BMP-6 was able to induce bone formation at the used dose and that the addition of IGF-1 contributed to an increase of the mineralization in the implants. Hence, the combination of BMP-6 with IGF-1 might be a better alternative than BMP-2 for orthopedic surgery or bone tissue engineering approaches. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1867-1875, 2017. © 2017 Wiley Periodicals, Inc.

The level of BMP4 signaling is critical for the regulation of distinct T-box gene expression domains and growth along the dorso-ventral axis of the optic cup

PubMed Central

Behesti, Hourinaz; Holt, James KL; Sowden, Jane C

2006-01-01

Background Polarised gene expression is thought to lead to the graded distribution of signaling molecules providing a patterning mechanism across the embryonic eye. Bone morphogenetic protein 4 (Bmp4) is expressed in the dorsal optic vesicle as it transforms into the optic cup. Bmp4 deletions in human and mouse result in failure of eye development, but little attempt has been made to investigate mammalian targets of BMP4 signaling. In chick, retroviral gene overexpression studies indicate that Bmp4 activates the dorsally expressed Tbx5 gene, which represses ventrally expressed cVax. It is not known whether the Tbx5 related genes, Tbx2 and Tbx3, are BMP4 targets in the mammalian retina and whether BMP4 acts at a distance from its site of expression. Although it is established that Drosophila Dpp (homologue of vertebrate Bmp4) acts as a morphogen, there is little evidence that BMP4 gradients are interpreted to create domains of BMP4 target gene expression in the mouse. Results Our data show that the level of BMP4 signaling is critical for the regulation of distinct Tbx2, Tbx3, Tbx5 and Vax2 gene expression domains along the dorso-ventral axis of the mouse optic cup. BMP4 signaling gradients were manipulated in whole mouse embryo cultures during optic cup development, by implantation of beads soaked in BMP4, or the BMP antagonist Noggin, to provide a local signaling source. Tbx2, Tbx3 and Tbx5, showed a differential response to alterations in the level of BMP4 along the entire dorso-ventral axis of the optic cup, suggesting that BMP4 acts across a distance. Increased levels of BMP4 caused expansion of Tbx2 and Tbx3, but not Tbx5, into the ventral retina and repression of the ventral marker Vax2. Conversely, Noggin abolished Tbx5 expression but only shifted Tbx2 expression dorsally. Increased levels of BMP4 signaling caused decreased proliferation, reduced retinal volume and altered the shape of the optic cup. Conclusion Our findings suggest the existence of a dorsal-high, ventral-low BMP4 signaling gradient across which distinct domains of Tbx2, Tbx3, Tbx5 and Vax2 transcription factor gene expression are set up. Furthermore we show that the correct level of BMP4 signaling is critical for normal growth of the mammalian embryonic eye. PMID:17173667

Bone Morphogenic Protein-2 (rhBMP2)-Loaded Silk Fibroin Scaffolds to Enhance the Osteoinductivity in Bone Tissue Engineering

NASA Astrophysics Data System (ADS)

Du, Guang-Yu; He, Sheng-Wei; Sun, Chuan-Xiu; Mi, Li-Dong

2017-10-01

There is an increasing demand for formulations of silk fibroin (SF) scaffolds in biomedical applications. SF was crosslinked via glutaraldehyde with osteoinductive recombinant human bone morphogenic protein-2 (rhBMP2) of different ratios viz. (i) 3% SF with no rhBMP2 (SF), (ii) 3% SF with equal amount of rhBMP2 (SF+BMP2), and (iii) 12% SF with 3% of rhBMP2 (4SF+BMP2), and these solutions were used in electrospinning-based fabrication of nanoscaffolds for evaluating increased osteoinductive potential of SF scaffolds with rhBMP2. Stress-strain relationship suggested there is no loss in mechanical strength of fibers with addition of rhBMP2, and mechanical strength of scaffold was improved with increase in concentration of SF. rhBMP2 association increased the water retention capacity of scaffold as evident from swelling studies. Viability of hMSCs was found to be higher in conjugated scaffolds, and scaffolds do not exhibit any cytotoxicity towards guest cells. Cells were found to have higher alkaline phosphatase activity in conjugated scaffolds under in vitro and in vivo conditions which establishes the increased osteoinductivity of the novel construct. The scaffolds were found to be effective for in vivo bone formation as well.

Reprogramming Antagonizes the Oncogenicity of HOXA13-Long Noncoding RNA HOTTIP Axis in Gastric Cancer Cells.

PubMed

Wu, Deng-Chyang; Wang, Sophie S W; Liu, Chung-Jung; Wuputra, Kenly; Kato, Kohsuke; Lee, Yen-Liang; Lin, Ying-Chu; Tsai, Ming-Ho; Ku, Chia-Chen; Lin, Wen-Hsin; Wang, Shin-Wei; Kishikawa, Shotaro; Noguchi, Michiya; Wu, Chu-Chieh; Chen, Yi-Ting; Chai, Chee-Yin; Lin, Chen-Lung Steve; Kuo, Kung-Kai; Yang, Ya-Han; Miyoshi, Hiroyuki; Nakamura, Yukio; Saito, Shigeo; Nagata, Kyosuke; Lin, Chang-Shen; Yokoyama, Kazunari K

2017-10-01

Reprogramming of cancer cells into induced pluripotent stem cells (iPSCs) is a compelling idea for inhibiting oncogenesis, especially through modulation of homeobox proteins in this reprogramming process. We examined the role of various long noncoding RNAs (lncRNAs)-homeobox protein HOXA13 axis on the switching of the oncogenic function of bone morphogenetic protein 7 (BMP7), which is significantly lost in the gastric cancer cell derived iPS-like cells (iPSLCs). BMP7 promoter activation occurred through the corecruitment of HOXA13, mixed-lineage leukemia 1 lysine N-methyltransferase, WD repeat-containing protein 5, and lncRNA HoxA transcript at the distal tip (HOTTIP) to commit the epigenetic changes to the trimethylation of lysine 4 on histone H3 in cancer cells. By contrast, HOXA13 inhibited BMP7 expression in iPSLCs via the corecruitment of HOXA13, enhancer of zeste homolog 2, Jumonji and AT rich interactive domain 2, and lncRNA HoxA transcript antisense RNA (HOTAIR) to various cis-element of the BMP7 promoter. Knockdown experiments demonstrated that HOTTIP contributed positively, but HOTAIR regulated negatively to HOXA13-mediated BMP7 expression in cancer cells and iPSLCs, respectively. These findings indicate that the recruitment of HOXA13-HOTTIP and HOXA13-HOTAIR to different sites in the BMP7 promoter is crucial for the oncogenic fate of human gastric cells. Reprogramming with octamer-binding protein 4 and Jun dimerization protein 2 can inhibit tumorigenesis by switching off BMP7. Stem Cells 2017;35:2115-2128. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Genetic Variants of BMP2 and Their Association with the Risk of Non-Syndromic Tooth Agenesis.

PubMed

Lu, Yun; Qian, Yajing; Zhang, Jinglu; Gong, Miao; Wang, Yuting; Gu, Ning; Ma, Lan; Xu, Min; Ma, Junqing; Zhang, Weibing; Pan, Yongchu; Wang, Lin

2016-01-01

Non-syndromic tooth agenesis (or non-syndromic congenitally missing tooth) is one of the most common congenital defects in humans affecting the craniofacial function and appearance. Single nucleotide polymorphisms (SNPs) have been associated with an individual's susceptibility to these anomalies. The aim of the present study was therefore to investigate the roles of the potentially functional SNPs of BMP2 in the occurrence of tooth agenesis. Overall, four potentially functional SNPs of BMP2 (rs15705, rs235768, rs235769 and rs3178250) were selected, and their associations with the susceptibility of tooth agenesis were evaluated in a case-control study of 335 non-syndromic tooth agenesis cases and 444 healthy controls. The SNPs rs15705 and rs3178250 were found to be associated with an individual's risk of tooth agenesis (P = 0.046 and P = 0.039, respectively). Both SNPs showed an increased risk of mandibular incisor agenesis (rs15705, AA/AC vs. CC = 1.58, 95% CI = [1.06-2.34], P = 0.024; rs3178250, TT/TC vs. CC = 1.60, 95% CI = [1.08-2.37], P = 0.020). Bioinformatics analysis indicated that these two SNPs located at the 3'-untranslated region (3'-UTR) of BMP2 might alter the binding ability of miR-1273d and miR-4639-5p, respectively, which was confirmed by luciferase activity assays in the 293A and COS7 cell lines (P < 0.001 in 293A and P < 0.01 in COS7 for miR-1273d; and P < 0.001 in both cells for miR-4639-5p). Furthermore, BMP2 mRNA expression decreased after transfecting either miR-1273d or miR-4639-5p into these two cell lines (P < 0.01 in 293A and P < 0.001 in COS7 for miR-1273d, and P < 0.01 in both cell lines for miR-4639-5p). Taken together, our findings indicate that rs15705 and rs317250 are associated with the susceptibility of non-syndromic tooth agenesis by possibly affecting miRNAs and mRNA interaction.

Different Requirements for Proteolytic Processing of Bone Morphogenetic Protein 5/6/7/8 Ligands in Drosophila melanogaster*

PubMed Central

Fritsch, Cornelia; Sawala, Annick; Harris, Robin; Maartens, Aidan; Sutcliffe, Catherine; Ashe, Hilary L.; Ray, Robert P.

2012-01-01

Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species. PMID:22199351

Comparison between psycho-acoustics and physio-acoustic measurement to determine optimum reverberation time of pentatonic angklung music concert hall

NASA Astrophysics Data System (ADS)

Sudarsono, Anugrah S.; Merthayasa, I. G. N.; Suprijanto

2015-09-01

This research tried to compare psycho-acoustics and Physio-acoustic measurement to find the optimum reverberation time of soundfield from angklung music. Psycho-acoustic measurement was conducted using a paired comparison method and Physio-acoustic measurement was conducted with EEG Measurement on T3, T4, FP1, and FP2 measurement points. EEG measurement was conducted with 5 persons. Pentatonic angklung music was used as a stimulus with reverberation time variation. The variation was between 0.8 s - 1.6 s with 0.2 s step. EEG signal was analysed using a Power Spectral Density method on Alpha Wave, High Alpha Wave, and Theta Wave. Psycho-acoustic measurement on 50 persons showed that reverberation time preference of pentatonic angklung music was 1.2 second. The result was similar to Theta Wave measurement on FP2 measurement point. High Alpha wave on T4 measurement gave different results, but had similar patterns with psycho-acoustic measurement

Complication Rates in Posterior Lumbar Interbody Fusion (PLIF) Surgery With Human Bone Morphogenetic Protein 2: Medicare Population.

PubMed

Alobaidaan, Raed; Cohen, Jeremiah R; Lord, Elizabeth L; Buser, Zorica; Yoon, S Tim; Youssef, Jim A; Park, Jong-Beom; Brodke, Darrel S; Wang, Jeffrey C; Meisel, Hans-Joerg

2017-12-01

Retrospective cohort study among Medicare beneficiaries who underwent posterior lumbar interbody fusion (PLIF) surgery. To identify the complication rates associated with the use of bone morphogenetic protein 2 (BMP2) in PLIF. Human BMP2 is commonly used in the "off-label" manner for various types of spine fusion procedures, including PLIF. However, recent studies have reported potential complications associated with the recombinant human BMP2 (rhBMP2) use in the posterior approach. Medicare records within the PearlDiver database were queried for patients undergoing PLIF procedure with and without rhBMP2 between 2005 and 2010. We evaluated complications within 1 year postoperatively. Chi-square was used to compare the complication rates between the 2 groups. A total of 8609 patients underwent PLIF procedure with or without rhBMP2. Individual complication rates in the rhBMP2 group ranged from 0.45% to 7.68% compared with 0.65% to 10.99 in the non-rhBMP2 group. Complication rates for cardiac, pulmonary, lumbosacral neuritis, infection, wound, and urinary tract (include acute kidney failure and post-operative complications) were significantly lower in the rhBMP2 group ( P < .05). There was no difference in the rates of central nervous system complications or radiculitis between the 2 groups. Our data showed that the patients who received rhBMP2 had lower complication rates compared to the non-rhBMP2 group. However, use of rhBMP2 was associated with a higher rate of pseudarthrosis. We did not observe any difference in radiculitis and central nervous system complications between the groups.

Complication Rates in Posterior Lumbar Interbody Fusion (PLIF) Surgery With Human Bone Morphogenetic Protein 2: Medicare Population

PubMed Central

Alobaidaan, Raed; Cohen, Jeremiah R.; Lord, Elizabeth L.; Yoon, S. Tim; Youssef, Jim A.; Park, Jong-Beom; Brodke, Darrel S.; Wang, Jeffrey C.; Meisel, Hans-Joerg

2017-01-01

Study Design: Retrospective cohort study among Medicare beneficiaries who underwent posterior lumbar interbody fusion (PLIF) surgery. Objective: To identify the complication rates associated with the use of bone morphogenetic protein 2 (BMP2) in PLIF. Human BMP2 is commonly used in the “off-label” manner for various types of spine fusion procedures, including PLIF. However, recent studies have reported potential complications associated with the recombinant human BMP2 (rhBMP2) use in the posterior approach. Methods: Medicare records within the PearlDiver database were queried for patients undergoing PLIF procedure with and without rhBMP2 between 2005 and 2010. We evaluated complications within 1 year postoperatively. Chi-square was used to compare the complication rates between the 2 groups. Results: A total of 8609 patients underwent PLIF procedure with or without rhBMP2. Individual complication rates in the rhBMP2 group ranged from 0.45% to 7.68% compared with 0.65% to 10.99 in the non-rhBMP2 group. Complication rates for cardiac, pulmonary, lumbosacral neuritis, infection, wound, and urinary tract (include acute kidney failure and post-operative complications) were significantly lower in the rhBMP2 group (P < .05). There was no difference in the rates of central nervous system complications or radiculitis between the 2 groups. Conclusion: Our data showed that the patients who received rhBMP2 had lower complication rates compared to the non-rhBMP2 group. However, use of rhBMP2 was associated with a higher rate of pseudarthrosis. We did not observe any difference in radiculitis and central nervous system complications between the groups. PMID:29238641

BMP15 regulates AMH expression via the p38 MAPK pathway in granulosa cells from goat.

PubMed

Zhao, Zhongquan; Guo, Fangyue; Sun, Xiaowei; He, Qijie; Dai, Zinuo; Chen, Xiaochuan; Zhao, Yongju; Wang, Jian

2018-05-31

Anti-Mullerian hormone (AMH), a member of the TGF-β superfamily, is produced by granulosa cells (GCs) of preantral and small antral follicles and plays a role in regulating the recruitment of primordial follicles and the FSH-dependent development of follicles. However, the regulation of AMH expression in follicles remains poorly understood. The objectives of this study were to determine the following: 1. the association between bone morphogenetic protein 15 (BMP15) and AMH; 2. whether BMP15 can regulate the expression of AMH by inhibiting the p38 MAPK pathway; and 3. whether SRY-related HMG box 9 (SOX9), a transcription factor for AMH, is involved in the regulation of AMH expression by BMP15. In this study, an inhibitor of p38 MAPK and an siRNA specific for p38 MAPK were used to prevent the function of the p38 MAPK signaling pathway. Then, AMH mRNA expression and AMH secretion were detected in goat GCs using an RT-PCR assay and ELISA, respectively, after treatment with BMP15. The results indicated that BMP15 up-regulates the transcription of AMH and that the inhibition of p38 MAPK decreases the BMP15-induced expression of AMH and SOX9, suggesting that BMP15 up-regulates the expression of AMH via the p38 MAPK signaling pathway, and this process involves the SOX9 transcription factor. Copyright © 2018 Elsevier Inc. All rights reserved.

Developmental patterning of the cardiac atrioventricular canal by Notch and Hairy-related transcription factors

PubMed Central

Rutenberg, Joshua B.; Fischer, Andreas; Jia, Haibo; Gessler, Manfred; Zhong, Tao P.; Mercola, Mark

2013-01-01

Mutations in Notch2, Jagged1 or homologs of the Hairy-related transcriptional repressor Hey2 cause congenital malformations involving the non-chamber atrioventricular canal (AVC) and inner curvature (IC) regions of the heart, but the underlying mechanisms have not been investigated. By manipulating signaling directly within the developing chick heart, we demonstrated that Notch2, Hey1 and Hey2 initiate a signaling cascade that delimits the non-chamber AVC and IC regions. Specifically, misactivation of Notch2 signaling, or misexpression of either Hey1 or Hey2, repressed Bmp2. Because Jagged (also known as Serrate in non-mammalian species) ligands were found to be present in prospective chamber myocardium, these data support the model that Notch2 and Hey proteins cause the progressive restriction of Bmp2 expression to within the developing AVC and IC, where it is essential for differentiation. Misactivation or inhibition of Notch2 specifically induced or inhibited Hey1, respectively, but these manipulations did not affect Hey2, implicating Hey1 as the direct mediator of Notch2. Bmp2 within the developing AVC and IC has been shown to induce Tbx2, and we found that Tbx2 misexpression inhibited the expression of both Hey1 and Hey2. Tbx2, therefore, is envisaged to constitute a feedback loop that sharpens the border with the developing AVC and IC by delimiting Hey gene expression to within prospective chamber regions. Analysis of the loss-of-function phenotype in mouse embryos homozygous for targeted disruption of Hey2 revealed an expanded AVC domain of Bmp2. Similarly, zebrafish gridlock (Hey2 homolog) mutant embryos showed ectopic expression of Bmp4, which normally marks AVC myocardium in this species. Thus, Hey pathway regulation of cardiac Bmp appears to be an evolutionarily conserved mechanism to delimit AVC and IC fate, and provides a potential mechanistic explanation for cardiac malformations caused by mutations in Serrate/Jagged1 and Notch signaling components. PMID:17021042

Improving excellence in scoliosis rehabilitation: a controlled study of matched pairs.

PubMed

Weiss, H-R; Klein, R

2006-01-01

Physiotherapy programmes so far mainly address the lateral deformity of scoliosis, a few aim at the correction of rotation and only very few address the sagittal profile. Meanwhile, there is evidence that correction forces applied in the sagittal plane are also able to correct the scoliotic deformity in the coronal and frontal planes. So it should be possible to improve excellence in scoliosis rehabilitation by the implementation of exercises to correct the sagittal deformity in scoliosis patients. An exercise programme (physio-logic exercises) aiming at a physiologic sagittal profile was developed to add to the programme applied at the centre or to replace certain exercises or exercising positions. To test the hypothesis that physio-logic exercises improve the outcome of Scoliosis Intensive Rehabilitation (SIR), the following study design was chosen: Prospective controlled trial of pairs of patients with idiopathic scoliosis matched by sex, age, Cobb angle and curve pattern. There were 18 patients in the treatment group (SIR + physio-logic exercises) and 18 patients in the control group (SIR only), all in matched pairs. Average Cobb angle in the treatment group was 34.5 degrees (SD 7.8) Cobb angle in the control group was 31.6 degrees (SD 5.8). Age in the treatment group was at average 15.3 years (SD 1.1) and in the control group 14.7 years (SD 1.3). Thirteen of the 18 patients in either group had a brace. Outcome parameter: average lateral deviation (mm), average surface rotation ( degrees ) and maximum Kyphosis angle ( degrees ) as evaluated with the help of surface topography (Formetric-system). Lateral deviation (mm) decreased significantly after the performance of the physio-logic programme and highly significantly in the physio-logic ADL posture; however, it was not significant after completion of the whole rehabilitation programme (2.3 vs 0.3 mm in the controls). Surface rotation improved at average 1.2 degrees in the treatment group and 0.8 degrees in the controls while Kyphosis angle did not improve in both groups. The physio-logic programme has to be regarded as a useful 'add on' to Scoliosis Rehabilitation with regards to the lateral deviation of the scoliotic trunk. A longitudinal controlled study is necessary to evaluate the long-term effect of the the physio-logic programme also with the help of X-rays.

BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration

PubMed Central

Inai, Kei; Burnside, Jessica L.; Hoffman, Stanley; Toole, Bryan P.; Sugi, Yukiko

2013-01-01

Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions. PMID:24147033

Comparison of Two Porcine Collagen Membranes Combined with rhBMP-2 and rhBMP-9 on Osteoblast Behavior In Vitro.

PubMed

Fujioka-Kobayashi, Masako; Schaler, Benoit; Shirakata, Yoshinori; Nakamura, Toshiaki; Noguchi, Kazuyuki; Zhang, Yufeng; Miron, Richard J

To investigate the bone-inducing properties of two types of collagen membranes in combination with recombinant human bone morphogenetic protein (rhBMP)-2 and rhBMP-9 on osteoblast behavior. Porcine pericardium collagen membranes (PPCM) and porcine dermis-derived collagen membranes (PDCM) were coated with either rhBMP-2 or rhBMP-9. The adsorption and release abilities were first investigated via enzyme-linked immunosorbent assay up to 10 days. Moreover, murine bone stromal ST2 cell adhesion, proliferation, and osteoblast differentiation were assessed by MTS assay; real-time polymerase chain reaction for genes encoding runt-related transcription factor 2 (Runx2); alkaline phosphatase (ALP); and osteocalcin, ALP assay, and alizarin red staining. Both rhBMP-2 and rhBMP-9 adsorbed to collagen membranes and were gradually released over time up to 10 days. PPCM showed significantly less cell attachment, whereas PDCM demonstrated comparable cell attachment with the control tissue culture plastic at 8 hours. While both rhBMPs were shown not to affect cell proliferation, collagen membranes combined with rhBMP-9 significantly increased ALP activity at 7 days and ALP mRNA levels at either 3 or 14 days compared with the control tissue culture plastic. Furthermore, rhBMP-9 increased osteocalcin mRNA levels and alizarin red staining at 14 days compared with the control tissue culture plastic. The results from this study suggest that both porcine-derived collagen membranes combined with rhBMP-9 accelerated the osteopromotive potential of ST2 cells. Interestingly, rhBMP-9 demonstrated additional osteogenic differentiation compared with rhBMP-2 and may serve as a suitable growth factor for future clinical use.

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

PubMed Central

CRUZ, Ariadne Cristiane Cabral; SILVA, Mariana Lúcia; CAON, Thiago; SIMÕES, Cláudia Maria Oliveira

2012-01-01

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. Objectives This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. Material and Methods Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. Conclusions We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs. PMID:23329244

Postoperative Complications Associated With rhBMP2 Use in Posterior/Posterolateral Lumbar Fusion

PubMed Central

Esmail, Nabil; Buser, Zorica; Cohen, Jeremiah R.; Brodke, Darrel S.; Meisel, Hans-Joerg; Park, Jong-Beom; Youssef, Jim A.; Wang, Jeffrey C.; Yoon, S. Tim

2017-01-01

Study Design: Retrospective database review. Objective: Posterior/posterolateral lumbar fusion (PLF) is an effective treatment for a variety of spinal disorders; however, variations in surgical technique have different complication profiles. The aim of our study was to quantify the frequency of various complications in patients undergoing PLF with and without human recombinant bone morphogenetic protein 2 (rhBMP2). Methods: We queried the orthopedic subset of the Medicare database (PearlDiver) between 2005 and 2011 for patients undergoing PLF procedures with and without rhBMP2. Complication and reoperation rates were analyzed within 1 year of the index procedure. Complications assessed include: acute renal failure, deep vein thrombosis, dural tear, hematoma, heterotopic ossification, incision and drainage, cardiac complications, nervous system complications, osteolysis, pneumonia, pseudarthrosis, pulmonary embolism, radiculopathy, respiratory complications, sepsis, urinary retention, urinary tract infection, mechanical, and wound complications. Chi-square analysis was used to calculate the complication differences between the groups. Results: Our data revealed higher overall complication rates in patients undergoing PLF with rhBMP2 versus no_rhBMP2 (76.9% vs 68.8%, P < .05). Stratified by gender, rhBMP2 males had higher rates of mechanical complications, pseudarthrosis, and reoperations compared with no_rhBMP2 males (P < .05), whereas rhBMP2 females had higher rates of pseudarthrosis, urinary tract infection, and urinary retention compared with no_rhBMP2 females (P < .05). Conclusion: Our data revealed higher overall complication rates in PLF patients given rhBMP2 compared with no_rhBMP2. Furthermore, our data suggests that rhBMP2-associated complications may be gender specific. PMID:29662744

Hyaline cartilage regeneration by combined therapy of microfracture and long-term bone morphogenetic protein-2 delivery.

PubMed

Yang, Hee Seok; La, Wan-Geun; Bhang, Suk Ho; Kim, Hak-Jun; Im, Gun-Il; Lee, Haeshin; Park, Jung-Ho; Kim, Byung-Soo

2011-07-01

Microfracture of cartilage induces migration of bone-marrow-derived mesenchymal stem cells. However, this treatment often results in fibrocartilage regeneration. Growth factors such as bone morphogenetic protein (BMP)-2 induce the differentiation of bone-marrow-derived mesenchymal stem cells into chondrocytes, which can be used for hyaline cartilage regeneration. Here, we tested the hypothesis that long-term delivery of BMP-2 to cartilage defects subjected to microfracture results in regeneration of high-quality hyaline-like cartilage, as opposed to short-term delivery of BMP-2 or no BMP-2 delivery. Heparin-conjugated fibrin (HCF) and normal fibrin were used as carriers for the long- and short-term delivery of BMP-2, respectively. Rabbit articular cartilage defects were treated with microfracture combined with one of the following: no treatment, fibrin, short-term delivery of BMP-2, HCF, or long-term delivery of BMP-2. Eight weeks after treatment, histological analysis revealed that the long-term delivery of BMP-2 group (microfracture + HCF + BMP-2) showed the most staining with alcian blue. A biochemical assay, real-time polymerase chain reaction assay and Western blot analysis all revealed that the long-term delivery of BMP-2 group had the highest glucosaminoglycan content as well as the highest expression level of collagen type II. Taken together, the long-term delivery of BMP-2 to cartilage defects subjected to microfracture resulted in regeneration of hyaline-like cartilage, as opposed to short-term delivery or no BMP-2 delivery. Therefore, this method could be more convenient for hyaline cartilage regeneration than autologous chondrocyte implantation due to its less invasive nature and lack of cell implantation.

Promising efficacy of Escherichia coli recombinant human bone morphogenetic protein-2 in collagen sponge for ectopic and orthotopic bone formation and comparison with mammalian cell recombinant human bone morphogenetic protein-2.

PubMed

Kim, In Sook; Lee, Eui Nam; Cho, Tae Hyung; Song, Yun Mi; Hwang, Soon Jung; Oh, Ji Hye; Park, Eun Kyung; Koo, Tai Young; Seo, Young-Kwon

2011-02-01

Nonglycosylated recombinant human bone morphogenetic protein (rhBMP)-2 prepared in Escherichia coli (E. coli rhBMP-2) has recently been considered as an alternative to mammalian cell rhBMP-2. However, its clinical use is still limited owing to lack of evidence for osteogenic activity comparable with that of mammalian cell rhBMP-2 via microcomputed tomography-based analysis. Therefore, this study aimed to evaluate the ability of E. coli rhBMP-2 in absorbable collagen sponge to form ectopic and orthotopic bone and to compare it to that of mammalian rhBMP-2. In vitro investigation was performed to study osteoblast differentiation of human mesenchymal stromal cells. Both types of rhBMP-2 enhanced proliferation, alkaline phosphatase activity, and matrix mineralization of human mesenchymal stromal cells at similar levels. Similar tendencies were observed in microcomputed tomography analysis, which determined bone volume, fractional bone volume, trabecular thickness, trabecular separation, bone mineral density, and other characteristics. Histology from an in vivo osteoinductivity test and from a rat calvarial defect model demonstrated a dose-dependent increase in local bone formation. The E. coli rhBMP-2 group (5 μg) not only induced complete regeneration of an 8-mm critical-sized defect at 4 weeks, but also led to new bone with the same bone mineral density as normal bone at 8 weeks, with the same efficiency as that of mammalian cell rhBMP-2 (5 μg). These uniformly favorable results provide evidence that the osteogenic activity of E. coli rhBMP-2 is not inferior to that of mammalian cell rhBMP-2 despite its low solubility and lack of gylcosylation. These results suggest that the application of E. coli rhBMP-2 in absorbable collagen sponge may be a promising equivalent to mammalian cell rhBMP-2 in bone tissue engineering.

In vitro and in vivo evaluation of osteoinductivity and bone fusion ability of an activin a/BMP2 chimera (AB204): a comparison study between AB204 and rhBMP-2.

PubMed

Zheng, Guang Bin; Lee, Jae Hyup; Jin, Yuan-Zhe

2017-12-01

This study compared osteoinductivity and osteogenic capacity between AB204 and rhBMP-2 using hMSCs in vitro and a beagle's posterolateral spinal fusion model. Cultured hMSCs were treated with AB204 or rhBMP-2 with low to high doses. Three male beagles were performed posterolateral spinal fusion with biphasic calcium phosphate (2 ml) + AB204 or rhBMP-2 (20, 50 or 200 µg). They were euthanized after 8 weeks. The fusion rate and bone formation of spine samples were examined. AB204 had higher alkaline phosphatase activity, mineralization and osteogenic-related gene expression than rhBMP-2. Fusion rates in all rhBMP-2 groups were 0. They were 100% for 50 μg and 200 μg AB204 groups. Therefore, AB204 showed higher osteogenicity than rhBMP-2. It could be a better bone graft substitute.

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice

PubMed Central

McKenzie, Jennifer A.; Buettmann, Evan G.; Gardner, Michael J.; Silva, Matthew J.

2015-01-01

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10–24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2. PMID:26344756

Combined effects of dentin sialoprotein and bone morphogenetic protein-2 on differentiation in human cementoblasts.

PubMed

Lee, So-Youn; Auh, Q-Schick; Kang, Soo-Kyung; Kim, Hyung-Joon; Lee, Jung-Woo; Noh, Kwantae; Jang, Jun-Hyeog; Kim, Eun-Cheol

2014-07-01

The aim of this study is to determine the effects of the combination of recombinant human BMP-2 (rh-BMP-2) and dentin sialoprotein (rh-DSP) on growth and differentiation in human cementoblasts and determine the underlying signal transduction mechanism. Compared to treatment of cementoblasts with either rh-BMP-2 or rh-DSP alone, the combination of rh-BMP-2 and rh-DSP synergistically increased cell growth, ALP activity, nodule formation and expression of differentiation markers. The differentiation-promoting effect was also observed in periodontal ligament cells and an osteoblastic cell line. Likewise, combination of rh-DSP and rh-BMP-2 increased BMP-2 mRNA expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. The expression levels of α2β1 integrin and RhoA, as well as the phosphorylation status of FAK and Akt, were increased by the combination of rh-BMP-2 and rh-DSP in a time-dependent manner. In addition, rh-BMP-2 and rh-DSP enhanced expression of Wnt ligands, β-catenin activation and GSK-3β phosphorylation, all of which were inhibited by the Wnt receptor antagonist DKK1. Furthermore, treatment with rh-DSP plus rh-BMP-2 resulted in phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 and also induced the nuclear translocation of the NF-κB p65 subunit, which was blocked by noggin. This study demonstrates for the first time that rh-DSP and rh-BMP-2 act synergistically, enhancing each other's ability to stimulate cementoblastic cell growth and differentiation in vitro via autocrine BMP, integrin, Wnt/β-catenin, MAP kinase and NF-κB pathways. These results support the therapeutic potential of a combination strategy for aiding periodontal regeneration.

Comparison of transforaminal lumbar interbody fusion outcomes in patients receiving rhBMP-2 versus autograft.

PubMed

Khan, Taleef R; Pearce, Kalin R; McAnany, Steven J; Peters, Colleen M; Gupta, Munish C; Zebala, Lukas P

2018-03-01

Recombinant human bone morphogenetic protein 2 (rhBMP-2) plays a pivotal role in complex spine surgery. Despite its limited approval, the off-label use of rhBMP-2 is prevalent, particularly in transforaminal lumbar interbody fusions (TLIFs). To determine the effectiveness and safety of rhBMP-2 use in TLIF procedures versus autograft. Retrospective cohort study. Patients older than 18 years undergoing spine surgery for lumbar degenerative spine disease at a single academic institution. Clinical outcome was determined according to patient records. Radiographic outcome was determined according to plain X-rays and computed tomography (CT). A retrospective study from 1997 to 2014 was conducted on 191 adults undergoing anterior-posterior instrumented spinal fusion with TLIF at a single academic institution. Patient data were gathered from operative notes, follow-up clinic notes, and imaging studies to determine complications and fusion rates. One hundred eighty-seven patients fit the criteria, which included patients with a minimum of one TLIF, and had a minimum 2-year radiographic and clinical follow-up. Patients were further classified into a BMP group (n=83) or non-BMP group (n=104). Three logistic regression models were run using rhBMP-2 exposure as the independent variable. The respective outcome variables were TLIF-related complications (radiculitis, seroma, osteolysis, and ectopic bone), surgical complications, and all complications. Bone morphogenetic protein (n=83) and non-BMP (n=104) groups had similar baseline demographics (sex, diabetes, pre-existing cancer). On average, the BMP and non-BMP groups were similarly aged (51.9 vs. 47.9 years, p>.05), but the BMP group had a shorter follow-up time (3.03 vs. 4.06 years; p<.001) and fewer smokers (8 vs. 21 patients; p<.048). The fusion rate for the BMP and non-BMP groups was 92.7% and 92.3%, respectively. The pseudoarthrosis rate was 7.5% (14 of 187 patients). Radiculitis was observed in seven patients in the BMP group (8.4%) and two patients in the non-BMP group (1.9%). Seroma was observed in two patients in the BMP group (2.4%) and none in the non-BMP group. No deep infections were observed in the BMP group, and in one patient in the non-BMP group (0.96%). Although patients exposed to BMP were at a significantlygreater risk of developing radiculitis and seroma (odds ratio [OR]=4.53, confidence interval [CI]=1.42-14.5), BMP exposure was not a significant predictor of surgical complications (OR=0.32, CI=0.10-1.00) or overall complications (OR=1.11, CI=0.53-2.34). The outcome of TLIF-related complications was too rare and the confidence interval too wide for practical significance of the first model. Evidence supports the hypothesis that off-label use of rhBMP-2 in TLIF procedures is relatively effective for achieving bone fusion at rates similar to patients receiving autograft. Patients exhibited similar complication rates between the two groups, with the BMP group exhibiting slightly higher rates of radiculitis and seroma. Copyright © 2017 Elsevier Inc. All rights reserved.

BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells.

PubMed

Zhang, Han; Klausen, Christian; Zhu, Hua; Chang, Hsun-Ming; Leung, Peter C K

2015-11-01

Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

An Open-source Toolbox for Analysing and Processing PhysioNet Databases in MATLAB and Octave.

PubMed

Silva, Ikaro; Moody, George B

The WaveForm DataBase (WFDB) Toolbox for MATLAB/Octave enables integrated access to PhysioNet's software and databases. Using the WFDB Toolbox for MATLAB/Octave, users have access to over 50 physiological databases in PhysioNet. The toolbox provides access over 4 TB of biomedical signals including ECG, EEG, EMG, and PLETH. Additionally, most signals are accompanied by metadata such as medical annotations of clinical events: arrhythmias, sleep stages, seizures, hypotensive episodes, etc. Users of this toolbox should easily be able to reproduce, validate, and compare results published based on PhysioNet's software and databases.

A polymorphism in a conserved posttranscriptional regulatory motif alters bone morphogenetic protein 2 (BMP2) RNA:protein interactions.

PubMed

Fritz, David T; Jiang, Shan; Xu, Junwang; Rogers, Melissa B

2006-07-01

The bone morphogenetic protein (BMP)2 gene has been genetically linked to osteoporosis and osteoarthritis. We have shown that the 3'-untranslated regions (UTR) of BMP2 genes from mammals to fishes are extraordinarily conserved. This indicates that the BMP2 3'-UTR is under stringent selective pressure. We present evidence that the conserved region is a strong posttranscriptional regulator of BMP2 expression. Polymorphisms in cis-regulatory elements have been proven to influence susceptibility to a growing number of diseases. A common single nucleotide polymorphism (SNP) disrupts a putative posttranscriptional regulatory motif, an AU-rich element, within the BMP2 3'-UTR. The affinity of specific proteins for the rs15705 SNP sequence differs from their affinity for the normal human sequence. More importantly, the in vitro decay rate of RNAs with the SNP is higher than that of RNAs with the normal sequence. Such changes in mRNA:protein interactions may influence the posttranscriptional mechanisms that control BMP2 gene expression. The consequent alterations in BMP2 protein levels may influence the development or physiology of bone or other BMP2-influenced tissues.

Antagonistic BMP–cWNT signaling in the cnidarian Nematostella vectensis reveals insight into the evolution of mesoderm

PubMed Central

Wijesena, Naveen; Simmons, David K.

2017-01-01

Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta bone morphogenetic protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans, but there has been no direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (an anthozoan cnidarian). Using forward transcriptomics, we show that beta-catenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network (GRN) at the onset of gastrulation (24 h postfertilization) in N. vectensis. Surprisingly, beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oral–aboral axis in Nematostella. We also show functionally that the conserved “kernel” of the bilaterian heart mesoderm GRN is operational in N. vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesodermal tissue. PMID:28652368

Identification of key genes associated with the effect of estrogen on ovarian cancer using microarray analysis.

PubMed

Zhang, Shi-tao; Zuo, Chao; Li, Wan-nan; Fu, Xue-qi; Xing, Shu; Zhang, Xiao-ping

2016-02-01

To identify key genes related to the effect of estrogen on ovarian cancer. Microarray data (GSE22600) were downloaded from Gene Expression Omnibus. Eight estrogen and seven placebo treatment samples were obtained using a 2 × 2 factorial designs, which contained 2 cell lines (PEO4 and 2008) and 2 treatments (estrogen and placebo). Differentially expressed genes were identified by Bayesian methods, and the genes with P < 0.05 and |log2FC (fold change)| ≥0.5 were chosen as cut-off criterion. Differentially co-expressed genes (DCGs) and differentially regulated genes (DRGs) were, respectively, identified by DCe function and DRsort function in DCGL package. Topological structure analysis was performed on the important transcriptional factors (TFs) and genes in transcriptional regulatory network using tYNA. Functional enrichment analysis was, respectively, performed for DEGs and the important genes using Gene Ontology and KEGG databases. In total, 465 DEGs were identified. Functional enrichment analysis of DEGs indicated that ACVR2B, LTBP1, BMP7 and MYC involved in TGF-beta signaling pathway. The 2285 DCG pairs and 357 DRGs were identified. Topological structure analysis showed that 52 important TFs and 65 important genes were identified. Functional enrichment analysis of the important genes showed that TP53 and MLH1 participated in DNA damage response and the genes (ACVR2B, LTBP1, BMP7 and MYC) involved in TGF-beta signaling pathway. TP53, MLH1, ACVR2B, LTBP1 and BMP7 might participate in the pathogenesis of ovarian cancer.

BMP-2 up-regulates PTEN expression and induces apoptosis of pulmonary artery smooth muscle cells under hypoxia.

PubMed

Pi, Weifeng; Guo, Xuejun; Su, Liping; Xu, Weiguo

2012-01-01

To investigate the role of bone morphogenetic protein 2 (BMP-2) in regulation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and apoptosis of pulmonary artery smooth muscle cells (PASMCs) under hypoxia. Normal human PASMCs were cultured in growth medium (GM) and treated with BMP-2 from 5-80 ng/ml under hypoxia (5% CO(2)+94% N(2)+1% O(2)) for 72 hours. Gene expression of PTEN, AKT-1 and AKT-2 were determined by quantitative RT-PCR (QRT-PCR). Protein expression levels of PTEN, AKT and phosph-AKT (pAKT) were determined. Apoptosis of PASMCs were determined by measuring activities of caspases-3, -8 and -9. siRNA-smad-4, bpV(HOpic) (PTEN inhibitor) and GW9662 (PPARγ antagonist) were used to determine the signalling pathways. Proliferation of PASMCs showed dose dependence of BMP-2, the lowest proliferation rate was achieved at 60 ng/ml concentration under hypoxia (82.2±2.8%). BMP-2 increased PTEN gene expression level, while AKT-1 and AKT-2 did not change. Consistently, the PTEN protein expression also showed dose dependence of BMP-2. AKT activity significantly reduced in BMP-2 treated PASMCs. Increased activities of caspase-3, -8 and -9 of PASMCs were found after cultured with BMP-2. PTEN expression remained unchanged when Smad-4 expression was inhibited by siRNA-Smad-4. bpV(HOpic) and GW9662 (PPARγ inhibitor) inhibited PTEN protein expression and recovered PASMCs proliferation rate. BMP-2 increased PTEN expression under hypoxia in a dose dependent pattern. BMP-2 reduced AKT activity and increased caspase activity of PASMCs under hypoxia. The increased PTEN expression may be mediated through PPARγ signalling pathway, instead of BMP/Smad signalling pathway.

Bone morphogenetic protein 9 (BMP9) and BMP10 enhance tumor necrosis factor-α-induced monocyte recruitment to the vascular endothelium mainly via activin receptor-like kinase 2.

PubMed

Mitrofan, Claudia-Gabriela; Appleby, Sarah L; Nash, Gerard B; Mallat, Ziad; Chilvers, Edwin R; Upton, Paul D; Morrell, Nicholas W

2017-08-18

Bone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte-endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The role of the BMP signaling cascade in regulation of stem cell activity following massive small bowel resection in a rat.

PubMed

Sukhotnik, I; Berkowitz, D; Dorfman, T; Halabi, Salim; Pollak, Y; Bejar, J; Bitterman, A; Coran, A G

2016-02-01

Bone morphogenetic proteins (BMPs) are a group of growth factors that are implicated in intestinal growth, morphogenesis, differentiation, and homeostasis. The role of the BMP signaling cascade in stimulation of cell proliferation after massive small bowel resection is unknown. The purpose of this study was to evaluate the role of BMP signaling during intestinal adaptation in a rat model of short bowel syndrome (SBS). Male rats were divided into two groups: Sham rats underwent bowel transection and SBS rats underwent a 75 % bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's Digital Gene Expression analysis was used to determine the BMP signaling gene expression profiling. BMP-related genes and protein expression were determined using real-time PCR, Western blotting and immunohistochemistry. From the total number of 20,000 probes, 8 genes related to BMP signaling were investigated. From these genes, five genes were found to be up-regulated in jejunum (BMP1-10 %, BMP2-twofold increase, BMP3-10 %, BMP2R-12 % and STAT3-28 %) and four genes to be up-regulated in ileum (BMP1-16 %, BMP2-27 %, BMP3-10 %, and STAT3-20 %) in SBS vs sham animals with a relative change in gene expression level of 10 % or more. SBS rats also demonstrated a significant increase in BMP2 and STAT3 mRNA and protein levels (determined by real-time PCR and Western blot) compared to control animals. Two weeks following massive bowel resection in rats, the BMP signaling pathway is stimulated. BMP signaling may serve as an important mediator of reciprocal interactions between the epithelium and the underlying mesenchymal stroma during intestinal adaptation following massive bowel resection in a rat.

BMP9 Inhibits Proliferation and Metastasis of HER2-Positive SK-BR-3 Breast Cancer Cells through ERK1/2 and PI3K/AKT Pathways

PubMed Central

Ren, Wei; Liu, Yuehong; Wan, Shaoheng; Fei, Chang; Wang, Wei; Chen, Yingying; Zhang, Zhihui; Wang, Ting; Wang, Jinshu; Zhou, Lan; Weng, Yaguang; He, Tongchuan; Zhang, Yan

2014-01-01

Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer. PMID:24805814

BMP9 inhibits proliferation and metastasis of HER2-positive SK-BR-3 breast cancer cells through ERK1/2 and PI3K/AKT pathways.

PubMed

Ren, Wei; Liu, Yuehong; Wan, Shaoheng; Fei, Chang; Wang, Wei; Chen, Yingying; Zhang, Zhihui; Wang, Ting; Wang, Jinshu; Zhou, Lan; Weng, Yaguang; He, Tongchuan; Zhang, Yan

2014-01-01

Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.

Pharmacological activation of aldehyde dehydrogenase 2 promotes osteoblast differentiation via bone morphogenetic protein-2 and induces bone anabolic effect

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mittal, Monika; Pal, Subhashis; China, Shyamsundar

Aldehyde dehydrogenases (ALDHs) are a family of enzymes involved in detoxifying aldehydes. Previously, we reported that an ALDH inhibitor, disulfiram caused bone loss in rats and among ALDHs, osteoblast expressed only ALDH2. Loss-of-function mutation in ALDH2 gene is reported to cause bone loss in humans which suggested its importance in skeletal homeostasis. We thus studied whether activating ALDH2 by N-(1, 3-benzodioxol-5-ylmethyl)-2, 6-dichlorobenzamide (alda-1) had osteogenic effect. We found that alda-1 increased and acetaldehyde decreased the differentiation of rat primary osteoblasts and expressions of ALDH2 and bone morphogenetic protein-2 (BMP-2). Silencing ALDH2 in osteoblasts abolished the alda-1 effects. Further, alda-1 attenuatedmore » the acetaldehyde-induced lipid-peroxidation and oxidative stress. BMP-2 is essential for bone regeneration and alda-1 increased its expression in osteoblasts. We then showed that alda-1 (40 mg/kg dose) augmented bone regeneration at the fracture site with concomitant increase in BMP-2 protein compared with control. The osteogenic dose (40 mg/kg) of alda-1 attained a bone marrow concentration that was stimulatory for osteoblast differentiation, suggesting that the tissue concentration of alda-1 matched its pharmacologic effect. In addition, alda-1 promoted modeling-directed bone growth and peak bone mass achievement, and increased bone mass in adult rats which reiterated its osteogenic effect. In osteopenic ovariectomized (OVX) rats, alda-1 reversed trabecular osteopenia with attendant increase in serum osteogenic marker (procollagen type I N-terminal peptide) and decrease in oxidative stress. Alda-1 has no effect on liver and kidney function. We conclude that activating ALDH2 by alda-1 had an osteoanabolic effect involving increased osteoblastic BMP-2 production and decreased OVX-induced oxidative stress. - Highlights: • Alda-1 induced osteoblast differentiation that involved upregulation of ALDH2 and BMP-2 • Alda-1 attenuated acetaldehyde-induced inhibition of osteoblast differentiation • Alda-1 enhanced bone regeneration at the fracture site and peak bone mass achievement • Alda-1 reversed trabecular osteopenia in OVX rats via an osteoanabolic mechanism.« less

A Novel Role for the BMP Antagonist Noggin in Sensitizing Cells to Non-canonical Wnt-5a/Ror2/Disheveled Pathway Activation

PubMed Central

Bernatik, Ondrej; Radaszkiewicz, Tomasz; Behal, Martin; Dave, Zankruti; Witte, Florian; Mahl, Annika; Cernohorsky, Nicole H.; Krejci, Pavel; Stricker, Sigmar; Bryja, Vitezslav

2017-01-01

Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions. PMID:28523267

Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish.

PubMed

Lovely, C Ben; Swartz, Mary E; McCarthy, Neil; Norrie, Jacqueline L; Eberhart, Johann K

2016-06-01

The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face. © 2016. Published by The Company of Biologists Ltd.

Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish

PubMed Central

Swartz, Mary E.; McCarthy, Neil; Norrie, Jacqueline L.; Eberhart, Johann K.

2016-01-01

The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face. PMID:27122171

Role of BMP receptor traffic in synaptic growth defects in an ALS model.

PubMed

Deshpande, Mugdha; Feiger, Zachary; Shilton, Amanda K; Luo, Christina C; Silverman, Ethan; Rodal, Avital A

2016-10-01

TAR DNA-binding protein 43 (TDP-43) is genetically and functionally linked to amyotrophic lateral sclerosis (ALS) and regulates transcription, splicing, and transport of thousands of RNA targets that function in diverse cellular pathways. In ALS, pathologically altered TDP-43 is believed to lead to disease by toxic gain-of-function effects on RNA metabolism, as well as by sequestering endogenous TDP-43 and causing its loss of function. However, it is unclear which of the numerous cellular processes disrupted downstream of TDP-43 dysfunction lead to neurodegeneration. Here we found that both loss and gain of function of TDP-43 in Drosophila cause a reduction of synaptic growth-promoting bone morphogenic protein (BMP) signaling at the neuromuscular junction (NMJ). Further, we observed a shift of BMP receptors from early to recycling endosomes and increased mobility of BMP receptor-containing compartments at the NMJ. Inhibition of the recycling endosome GTPase Rab11 partially rescued TDP-43-induced defects in BMP receptor dynamics and distribution and suppressed BMP signaling, synaptic growth, and larval crawling defects. Our results indicate that defects in receptor traffic lead to neuronal dysfunction downstream of TDP-43 misregulation and that rerouting receptor traffic may be a viable strategy for rescuing neurological impairment. © 2016 Deshpande, Feiger, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Dragon enhances BMP signaling and increases transepithelial resistance in kidney epithelial cells.

PubMed

Xia, Yin; Babitt, Jodie L; Bouley, Richard; Zhang, Ying; Da Silva, Nicolas; Chen, Shanzhuo; Zhuang, Zhenjie; Samad, Tarek A; Brenner, Gary J; Anderson, Jennifer L; Hong, Charles C; Schneyer, Alan L; Brown, Dennis; Lin, Herbert Y

2010-04-01

The neuronal adhesion protein Dragon acts as a bone morphogenetic protein (BMP) coreceptor that enhances BMP signaling. Given the importance of BMP signaling in nephrogenesis and its putative role in the response to injury in the adult kidney, we studied the localization and function of Dragon in the kidney. We observed that Dragon localized predominantly to the apical surfaces of tubular epithelial cells in the thick ascending limbs, distal convoluted tubules, and collecting ducts of mice. Dragon expression was weak in the proximal tubules and glomeruli. In mouse inner medullary collecting duct (mIMCD3) cells, Dragon generated BMP signals in a ligand-dependent manner, and BMP4 is the predominant endogenous ligand for the Dragon coreceptor. In mIMCD3 cells, BMP4 normally signaled through BMPRII, but Dragon enhanced its signaling through the BMP type II receptor ActRIIA. Dragon and BMP4 increased transepithelial resistance (TER) through the Smad1/5/8 pathway. In epithelial cells isolated from the proximal tubule and intercalated cells of collecting ducts, we observed coexpression of ActRIIA, Dragon, and BMP4 but not BMPRII. Taken together, these results suggest that Dragon may enhance BMP signaling in renal tubular epithelial cells and maintain normal renal physiology.

Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries.

PubMed

Hernandez-Hurtado, Adelina A; Borrego-Soto, Gissela; Marino-Martinez, Ivan A; Lara-Arias, Jorge; Romero-Diaz, Viktor J; Abrego-Guerra, Adalberto; Vilchez-Cavazos, Jose F; Elizondo-Riojas, Guillermo; Martinez-Rodriguez, Herminia G; Espinoza-Juarez, Marcela A; Lopez-Romero, Gloria C; Robles-Zamora, Alejandro; Mendoza Lemus, Oscar F; Ortiz-Lopez, Rocio; Rojas-Martinez, Augusto

2016-01-01

Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

cAMP enhances BMP2-signaling through PKA and MKP1-dependent mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghayor, Chafik; Ehrbar, Martin; Miguel, Blanca San

2009-04-03

Recent studies suggest that the elevation of intracellular cyclic adenosine monophosphate (cAMP) and the activation of the protein kinase A regulate BMP-induced osteogenesis. However, the precise mechanisms underlying the enhancing effect of cAMP on BMP2 signaling were not completely revealed. In this study we investigated the effect of elevated cAMP level and PKA activation on the BMP2-induced osteoblastic differentiation in pluripotent C2C12 cells. Alkaline phosphatase activity and its mRNA were consistently induced by BMP2 treatment. The pretreatment of C2C12 cells with Forskolin, a cAMP generating agent, dbcAMP, an analogue of cAMP, or IBMX (3-isobutyl 1-methyl xanthine), and a nonspecific inhibitormore » of phosphodiesterases elicited further activation of alkaline phosphatase. Furthermore, elevated intracellular cAMP level increased BMP2-induced MKP1. On the other hand, BMP2-induced Erk phosphorylation (p44/p42) and cell proliferation were suppressed in the presence of cAMP. Thus, cAMP might enhance BMP2-induced osteoblastic differentiation by a MKP1-Erk-dependent mechanism.« less

Selective laser sintering fabrication of nano-hydroxyapatite/poly-ε-caprolactone scaffolds for bone tissue engineering applications

PubMed Central

Xia, Yan; Zhou, Panyu; Cheng, Xiaosong; Xie, Yang; Liang, Chong; Li, Chao; Xu, Shuogui

2013-01-01

The regeneration of functional tissue in osseous defects is a formidable challenge in orthopedic surgery. In the present study, a novel biomimetic composite scaffold, here called nano-hydroxyapatite (HA)/poly-ε-caprolactone (PCL) was fabricated using a selective laser sintering technique. The macrostructure, morphology, and mechanical strength of the scaffolds were characterized. Scanning electronic microscopy (SEM) showed that the nano-HA/PCL scaffolds exhibited predesigned, well-ordered macropores and interconnected micropores. The scaffolds have a range of porosity from 78.54% to 70.31%, and a corresponding compressive strength of 1.38 MPa to 3.17 MPa. Human bone marrow stromal cells were seeded onto the nano-HA/PCL or PCL scaffolds and cultured for 28 days in vitro. As indicated by the level of cell attachment and proliferation, the nano-HA/PCL showed excellent biocompatibility, comparable to that of PCL scaffolds. The hydrophilicity, mineralization, alkaline phosphatase activity, and Alizarin Red S staining indicated that the nano-HA/PCL scaffolds are more bioactive than the PCL scaffolds in vitro. Measurements of recombinant human bone morphogenetic protein-2 (rhBMP-2) release kinetics showed that after nano-HA was added, the material increased the rate of rhBMP-2 release. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both nano-HA/PCL scaffolds and PCL scaffolds were implanted in rabbit femur defects for 3, 6, and 9 weeks. The wounds were studied radiographically and histologically. The in vivo results showed that both nano-HA/PCL composite scaffolds and PCL scaffolds exhibited good biocompatibility. However, the nano-HA/PCL scaffolds enhanced the efficiency of new bone formation more than PCL scaffolds and fulfilled all the basic requirements of bone tissue engineering scaffolds. Thus, they show large potential for use in orthopedic and reconstructive surgery. PMID:24204147

A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

PubMed Central

Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

2012-01-01

In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

Ex vivo bone morphogenetic protein 2 gene delivery using periodontal ligament stem cells for enhanced re-osseointegration in the regenerative treatment of peri-implantitis.

PubMed

Park, Shin-Young; Kim, Kyoung-Hwa; Gwak, Eun-Hye; Rhee, Sang-Hoon; Lee, Jeong-Cheol; Shin, Seung-Yun; Koo, Ki-Tae; Lee, Yong-Moo; Seol, Yang-Jo

2015-01-01

Peri-implantitis is a chronic inflammatory process with advanced bone loss and impaired healing potential. For peri-implantitis treatment, tissue engineering can be applied to enhance bone regeneration of peri-implant defects. This study aimed to evaluate ex vivo bone morphogenetic protein 2 (BMP2) gene delivery using canine periodontal ligament stem cells (PDLSCs) for regeneration of peri-implantitis defects. Canine PDLSCs were transduced with adenoviral vectors containing BMP2 (BMP2/PDLSCs). After peri-implantitis was induced by ligature placement in six beagle dogs, regenerative procedures were performed; hydroxyapatite (HA) particles and collagen gel with autologous canine PDLSCs (PDLSC group) or BMP2/PDLSCs (BMP/PDLSC group) or without cells (control group) were grafted into the defects and covered by an absorbable membrane. Three months later, the animals were sacrificed. In vitro, BMP2/PDLSCs showed similar levels of stem cell properties to PDLSCs, such as colony-forming efficiency and expression of MSC markers STRO-1 and CD 146. BMP2/PDLSCs produced BMP-2 until day 21 at a concentration of 4-8 ng/mL. In vivo, the BMP2/PDLSC group showed significantly more new bone formation and re-osseointegration in peri-implantitis defects compared to the other groups. In conclusion, ex vivo BMP2 gene delivery using PDLSCs enhanced new bone formation and re-osseointegration in peri-implantitis defects. © 2014 Wiley Periodicals, Inc.

Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Si, Lina; Shi, Jin; Gao, Wenqun

2014-07-18

Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part bymore » increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.« less

Lack of Obvious Influence of PLLA Nanofibers on the Gene Expression of BMP-2 and VEGF during Growth and Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Schofer, Markus D.; Fuchs-Winkelmann, S.; Wack, C.; Rudisile, M.; Dersch, R.; Leifeld, I.; Wendorff, J.; Greiner, A.; Paletta, J. R. J.; Boudriot, U.

2009-01-01

Growth factors like bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) play an important role in bone remodeling and fracture repair. Therefore, with respect to tissue engineering, an artificial graft should have no negative impact on the expression of these factors. In this context, the aim of this study was to analyze the impact of poly(L-lactic acid) (PLLA) nanofibers on VEGF and BMP-2 gene expression during the time course of human mesenchymal stem cell (hMSC) differentiation towards osteoblasts. PLLA matrices were seeded with hMSCs and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of VEGF and BMP-2. Furthermore, BMP-2–enwoven PLLA nanofibers were used in order to elucidate whether initial down-regulation of growth factor expression could be compensated. Although there was a great interpatient variability with respect to the expression of VEGF and BMP-2, PLLA nanofibers tend to result in a down-regulation in BMP-2 expression during the early phase of cultivation. This effect was diminished in the case of VEGF gene expression. The initial down-regulation was overcome when BMP-2 was directly incorporated into the PLLA nanofibers by electrospinning. Furthermore, the incorporation of BMP-2 into the PLLA nanofibers resulted in an increase in VEGF gene expression. Summarized, the results indicate that the PLLA nanofibers have little effect on growth factor production. An enhancement in gene expression of BMP-2 and VEGF can be achieved by an incorporation of BMP-2 into the PLLA nanofibers. PMID:19412560

BMP-2/PLGA delayed-release microspheres composite graft, selection of bone particulate diameters, and prevention of aseptic inflammation for bone tissue engineering.

PubMed

Ji, Ye; Xu, Gong Ping; Zhang, Zhi Peng; Xia, Jing Jun; Yan, Jing Long; Pan, Shang Ha

2010-03-01

Autogenous bone grafts are widely used in the repair of bone defects. Growth factors such as bone morphogenetic protein 2 (BMP-2) can induce bone regeneration and enhance bone growth. The combination of an autogenous bone graft and BMP-2 may provide a better osteogenic effect than either treatment alone, but BMP-2 is easily inactivated in body fluid. The objective of this study was to develop a technique that can better preserve the in vivo activity of BMP-2 incorporated in bone grafts. In this study, we first prepared BMP-2/poly(lactic-co-glycolic acid) (PLGA) delayed-release microspheres, and then combined collagen, the delayed-release microspheres, and rat autologous bone particulates to form four groups of composite grafts with different combinations: collagen in group A; collagen combined with bone particulates in group B; collagen combined with BMP-2/PLGA delayed-release microspheres in group C; and collagen combined with both bone particulates and BMP-2/PLGA delayed-release microspheres in group D. The four groups of composite grafts were implanted into the gluteus maximus pockets in rats. The ectopic osteogenesis and ALP level in group D (experimental group) were compared with those in groups A, B, and C (control groups) to study whether it had higher osteogenic capability. Results showed that the composite graft design increased the utility of BMP-2 and reduced the required dose of BMP-2 and volume of autologous bone. The selection of bone particulate diameter had an impact on the osteogenetic potential of bone grafts. Collagen prevented the occurrence of aseptic inflammation and improved the osteoinductivity of BMP-2. These results showed that this composite graft design is effective and feasible for use in bone repair.

Creation of bony microenvironment with CaP and cell-derived ECM to enhance human bone-marrow MSC behavior and delivery of BMP-2

PubMed Central

Kang, Yunqing; Kim, Sungwoo; Khademhosseini, Ali; Yang, Yunzhi

2011-01-01

Extracellular matrix (ECM) comprises a rich meshwork of proteins and proteoglycans, which not only contains biological cues for cell behavior, but is also a reservoir for binding growth factors and controlling their release. Here we aimed to create a suitable bony microenvironment with cell-derived ECM and biodegradable β-tricalcium phosphate (β-TCP). More specifically, we investigated whether the ECM produced by bone marrow-derived mesenchymal stem cells (hBMSC) on a β-TCP scaffold can bind bone morphogenetic protein-2 (BMP-2) and control its release in a sustained manner, and further examined the effect of ECM and the BMP-2 released from ECM on cell behaviors. The ECM was obtained through culturing the hBMSC on a β-TCP porous scaffold and performing decellularization and sterilization. SEM, XPS, FTIR, and immunofluorescent staining results indicated the presence of ECM on the β-TCP and the amount of ECM increased with the incubation time. BMP-2 was loaded onto the β-TCP with and without ECM by immersing the scaffolds in the BMP-2 solution. The loading and release kinetics of the BMP-2 on the β-TCP/ECM were significantly slower than those on the β-TCP. The β-TCP/ECM exhibited a sustained release profile of the BMP-2, which was also affected by the amount of ECM. This is probably because the β-TCP/ECM has different binding mechanisms with BMP-2. The β-TCP/ECM promoted cell proliferation. Furthermore, the BMP-2-loaded β-TCP/ECM stimulated reorganization of the actin cytoskeleton, increased expression of alkaline phosphatase and calcium deposition by the cells compared to those without BMP-2 loading and the β-TCP with BMP-2 loading. PMID:21632105

Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response

PubMed Central

Lipchina, Inna; Elkabetz, Yechiel; Hafner, Markus; Sheridan, Robert; Mihailovic, Aleksandra; Tuschl, Thomas; Sander, Chris; Studer, Lorenz; Betel, Doron

2011-01-01

MicroRNAs are important regulators in many cellular processes, including stem cell self-renewal. Recent studies demonstrated their function as pluripotency factors with the capacity for somatic cell reprogramming. However, their role in human embryonic stem (ES) cells (hESCs) remains poorly understood, partially due to the lack of genome-wide strategies to identify their targets. Here, we performed comprehensive microRNA profiling in hESCs and in purified neural and mesenchymal derivatives. Using a combination of AGO cross-linking and microRNA perturbation experiments, together with computational prediction, we identified the targets of the miR-302/367 cluster, the most abundant microRNAs in hESCs. Functional studies identified novel roles of miR-302/367 in maintaining pluripotency and regulating hESC differentiation. We show that in addition to its role in TGF-β signaling, miR-302/367 promotes bone morphogenetic protein (BMP) signaling by targeting BMP inhibitors TOB2, DAZAP2, and SLAIN1. This study broadens our understanding of microRNA function in hESCs and is a valuable resource for future studies in this area. PMID:22012620

Cyclooxygenase-2 promotes pulmonary intravascular macrophage accumulation by exacerbating BMP signaling in rat experimental hepatopulmonary syndrome.

PubMed

Liu, Chang; Gao, Jing; Chen, Bing; Chen, Lin; Belguise, Karine; Yu, Weifeng; Lu, Kaizhi; Wang, Xiaobo; Yi, Bin

2017-08-15

One central factor in hepatopulmonary syndrome (HPS) pathogenesis is intravascular accumulation of activated macrophages in small pulmonary arteries. However, molecular mechanism underlying the macrophage accumulation in HPS is unknown. In this study, we aimed to explore whether elevated COX-2 induces the Bone morphogenic protein-2 (BMP-2)/Crossveinless-2 (CV-2) imbalance and then activation of BMP signaling pathway promotes the macrophage accumulation in Common Bile Duct Ligation (CBDL) rat lung. The COX-2/PGE2 signaling activation, the BMP-2/CV-2 imbalance and the activation of Smad1 were evaluated in CBDL rat lung and in cultured pulmonary microvascular endothelial cells (PMVECs) under the HPS serum stimulation. The effects of Parecoxib (COX-2 inhibitor), BMP-2 and CV-2 recombinant proteins on 4-week CBDL rat lung were determined, respectively. The COX-2/PGE2 signaling pathway was activated in CBDL rat lung in vivo and PMVECs in vitro, which was due to the activation of NF-κB P65. The inhibition of COX-2 by Parecoxib reduced macrophage accumulation, decreased lung angiogenesis and improved HPS. Meanwhile, the CBDL rat lung secreted more BMP-2 but less CV-2, and the imbalance between BMP-2 and CV-2 exacerbated the BMP signaling activation thus promoting the macrophage accumulation and lung angiogenesis. The BMP-2/CV-2 imbalance is dependent on the COX-2/PGE2 signaling pathway, and thus the effects of this imbalance can be reversed by adminstration of Parecoxib. Our findings indicate that inhibition of COX-2 by parecoxib can improve the HPS through the repression of BMP signaling and macrophage accumulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Immunohistological Localization of BMP-2, BMP-7, and Their Receptors in Knee Joints with Focal Cartilage Lesions

PubMed Central

Schmal, Hagen; Mehlhorn, Alexander T.; Pilz, Ingo H.; Dovi-Akue, David; Kirchhoff, Christina; Südkamp, Norbert P.; Gerlach, Ulrike; Lohrmann, Christian; Niemeyer, Philipp

2012-01-01

Introduction. Although it is well known that BMP-2 and BMP-7 play significant roles in cartilage metabolism, data about intra-articular expression and localization of these proteins and their receptors in humans are rare. Methods. Biopsies of synovia and debrided cartilage were taken in patients undergoing autologous chondrocyte implantation. Expression of BMP-2, BMP-7, and their receptors BMPR-1A, BMPR-1B and BMPR-2 were semiquantitatively evaluated by immunohistological staining. Results. BMP-7 was equally highly expressed in all cartilage and synovial biopsies. Increased levels of BMPR-1A, but not of BMPR-1B, and BMPR-2, were found in all synovial and 47% of all cartilage samples (P = 0.002). BMP-2 was positively scored in 47% of all cartilage and 40% of all synovial specimens. Defect size, KOSS, Henderson or Kellgren-Lawrence score did not statistically significant correlate with the expression of the analyzed proteins or Mankin and Pritzker scores. Duration of symptoms and localization of lesions were associated with KOSS (P < 0.02), but there was no influence of these parameters on protein expression. Conclusions. BMP-2, BMP-7, and BMPR-1A were expressed in cartilage and synovia of knees with focal cartilage lesions. Although defect localization and duration of symptoms decisively influence KOSS, there was no associated alteration of protein expression observed. PMID:22272175

The expression and crucial roles of BMP signaling in development of smooth muscle progenitor cells in the mouse embryonic gut.

PubMed

Torihashi, Shigeko; Hattori, Takako; Hasegawa, Hirotaka; Kurahashi, Masaaki; Ogaeri, Takunori; Fujimoto, Toyoshi

2009-03-01

Bone morphogenetic protein (BMP) signaling is essential for normal development of the gastrointestinal (GI) tract. BMPs also play multiple roles in vascular smooth muscle cells; however, the BMP signaling in the development of the GI musculature remains to be clarified. We investigated the expression of BMPs and their receptors in mouse embryonic GI tracts by immunohistochemistry and in situ hybridization. We demonstrated that BMP2, BMP receptor Ib and BMP receptor II were expressed in the smooth muscle progenitors from E12 to E13 for the first time. BMP signaling on smooth muscle differentiation was examined by implantation of agarose beads soaked with BMPs in the in vitro developmental model that is gut-like structures from mouse embryonic stem (ES) cells. BMP2 rather than BMP4 beads enhanced smooth muscle differentiation, and increased gut-like structures showing spontaneous contractions and expressing intensive alpha-smooth muscle actin immunoreactivity. This increase was confirmed by up-regulation of SM22 mRNA shown by real-time PCR. By addition of noggin beads or noggin to the medium at BMP2 bead implantation, the ratio of contractive gut-like structures decreased. Implantation of BMP2 beads at EB7 (EB--embryoid bodies) (corresponding to E12 or E13 of mouse embryo) showed the highest effects and up-regulation of transcription factors msx-1 after 24h. This increase was blocked by noggin, and msx-1 decreased to almost the control level after 60 h. BMP2 beads at EB7 increased platelet-derived growth factor-A (PDGF-A) in the differentiating smooth muscle cells. We have recently reported that PDGF-A is expressed in the developing inner circular smooth muscle and is crucial for the longitudinal smooth muscle differentiation. Taken together, BMP signaling was expressed for a short window in the smooth muscle progenitors and the signal, especially BMP2, plays an essential role in smooth muscle differentiation in cooperation with PDGF signaling.

TGF-β but not BMP signaling induces prechondrogenic condensation through ATP oscillations during chondrogenesis.

PubMed

Kwon, Hyuck Joon

2012-08-10

Although both TGF-β and BMP signaling enhance expression of adhesion molecules during chondrogenesis, TGF-β but not BMP signaling can initiate condensation of uncondensed mesenchymal cells. However, it remains unclear what causes the differential effects between TGF-β and BMP signaling on prechondrogenic condensation. Our previous report demonstrated that ATP oscillations play a critical role in prechondrogenic condensation. Thus, the current study examined whether ATP oscillations are associated with the differential actions of TGF-β and BMP signaling on prechondrogenic condensation. The result revealed that while both TGF-β1 and BMP2 stimulated chondrogenic differentiation, TGF-β1 but not BMP2 induced prechondrogenic condensation. It was also found that TGF-β1 but not BMP2 induced ATP oscillations and inhibition of TGF-β but not BMP signaling prevented insulin-induced ATP oscillations. Moreover, blockage of ATP oscillations inhibited TGF-β1-induced prechondrogenic condensation. In addition, TGF-β1-driven ATP oscillations and prechondrogenic condensation depended on Ca(2+) influx via voltage-dependent calcium channels. This study suggests that Ca(2+)-driven ATP oscillations mediate TGF-β-induced the initiation step of prechondrogenic condensation and determine the differential effects between TGF-β and BMP signaling on chondrogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

Endocytosis contributes to BMP2-induced Smad signalling and neuronal growth.

PubMed

Hegarty, Shane V; Sullivan, Aideen M; O'Keeffe, Gerard W

2017-03-16

Bone morphogenetic protein 2 (BMP2) is a neurotrophic factor which induces the growth of midbrain dopaminergic (DA) neurons in vitro and in vivo, and its neurotrophic effects have been shown to be dependent on activation of BMP receptors (BMPRs) and Smad 1/5/8 signalling. However, the precise intracellular cascades that regulate BMP2-BMPR-Smad-signalling-induced neurite growth remain unknown. Endocytosis has been shown to regulate Smad 1/5/8 signalling and differentiation induced by BMPs. However, these studies were carried out in non-neural cells. Indeed, there are scant reports regarding the role of endocytosis in BMP-Smad signalling in neurons. To address this, and to further characterise the mechanisms regulating the neurotrophic effects of BMP2, the present study examined the role of dynamin-dependent endocytosis in BMP2-induced Smad signalling and neurite growth in the SH-SY5Y neuronal cell line. The activation, temporal kinetics and magnitude of Smad 1/5/8 signalling induced by BMP2 were significantly attenuated by dynasore-mediated inhibition of endocytosis in SH-SY5Y cells. Furthermore, BMP2-induced increases in neurite length and neurite branching in SH-SY5Y cells were significantly reduced following inhibition of dynamin-dependent endocytosis using dynasore. This study demonstrates that BMP2-induced Smad signalling and neurite growth is regulated by dynamin-dependent endocytosis in a model of human midbrain dopaminergic neurons. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

PubMed

García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

2015-09-01

Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the blastocyst stage in the BMP5 group. Moreover, reverse transcription quantitative real-time polymerase chain reaction analysis showed a significant increase in the relative abundance of SOX2 in two-cell stage embryos, ID2 and OCT4 in eight-cell stage embryos, and NANOG and OCT4 in blastocysts derived from BMP5-treated embryos. In conclusion, our results report that early addition of BMP5 to the embryo culture medium had a positive effect on the blastocyst rate and affected the relative expression of BMP target and pluripotency genes, suggesting that BMP5 could play an important role in the preimplantation development of bovine embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

Immunohistochemical localization of bone morphogenetic proteins and the receptors in epiphyseal growth plate.

PubMed

Yazaki, Y; Matsunaga, S; Onishi, T; Nagamine, T; Origuchi, N; Yamamoto, T; Ishidou, Y; Imamura, T; Sakou, T

1998-01-01

The expression of bone morphogenetic proteins (BMPs) and BMP receptors (BMPRs) in the epiphyseal growth plate has not been clarified. In this study, we studied immunohistochemically the spatial and temporal localization of BMP-2/4, osteogenic protein-1 (OP-1, or BMP-7), and BMP receptors (BMPR-IA, BMPR-IB, and BMPR-II) in the epiphyseal plate of growing rats. The proximal parts of tibia in growing rats were observed. At 12 weeks after birth, BMP-2/4 and OP-1 were expressed markedly in proliferating and maturing chondrocytes. BMPR-IA, IB and II were clearly co-expressed in proliferating and maturing chondrocytes, and the expression was decreased in hypertrophic chondrocytes. At 24 weeks, the expression of BMP-2/4 and OP-1 was decreased, but BMPRs were still well-expressed in proliferating chondrocytes. The temporal and spatial expression of BMP and BMPR suggests that BMP and BMP receptors play roles in the multistep cascade of enchondral ossification in the epiphyseal growth plate.

Use of Bioresorbable Hydrogels and Genetic Engineering to Accomplish Rapid Stabilization and Healing in Segmental Long Bone Defects

DTIC Science & Technology

2013-04-29

transduction of human mesenchymal stem cells (MSCs), BMP2 was not detectable by Western blotting, whereas high levels of the protein were produced by A549 (human... mesenchymal stem cells , generating high levels of BMP2. When Ad5BMP2 or Ad5F35BMP2 were compared in vitro for their ability to induce BMP2 synthesis...in human mesenchymal stem cells and in vivo for their ability to stimulate formation of heterotopic bone, mineralized bone was radiologically

Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2.

PubMed

Xu, Emma-Ruoqi; Blythe, Emily E; Fischer, Gerhard; Hyvönen, Marko

2017-07-28

Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signaling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. Although the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC/BMP-binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2: collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analyzed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Alveolar ridge and maxillary sinus augmentation using rhBMP-2: a systematic review.

PubMed

Freitas, Rubens Moreno de; Spin-Neto, Rubens; Marcantonio Junior, Elcio; Pereira, Luís Antônio Violin Dias; Wikesjö, Ulf M E; Susin, Cristiano

2015-01-01

The aim of this systematic review was to evaluate clinical and safety data for recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge (ACS) carrier when used for alveolar ridge/maxillary sinus augmentation in humans. Clinical studies/case series published 1980 through June 2012 using rhBMP-2/ACS were searched. Studies meeting the following criteria were considered eligible for inclusion: >10 subjects at baseline and maxillary sinus or alveolar ridge augmentation not concomitant with implant placement. Seven of 69 publications were eligible for review. rhBMP-2/ACS yielded clinically meaningful bone formation for maxillary sinus augmentation that would allow placement of regular dental implants without consistent differences between rhBMP-2 concentrations. Nevertheless, the statistical analysis showed that sinus augmentation following autogenous bone graft was significantly greater (mean bone height: 1.6 mm, 95% CI: 0.5-2.7 mm) than for rhBMP-2/ACS (rhBMP-2 at 1.5 mg/mL). In extraction sockets, rhBMP-2/ACS maintained alveolar ridge height while enhancing alveolar ridge width. Safety reports did not represent concerns for the proposed indications. rhBMP-2/ACS appears a promising alternative to autogenous bone grafts for alveolar ridge/maxillary sinus augmentation; dose and carrier optimization may expand its efficacy, use, and clinical application. © 2013 Wiley Periodicals, Inc.

Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype

PubMed Central

Wu, Li-an; Feng, Junsheng; Wang, Lynn; Mu, Yan-dong; Baker, Andrew; Donly, Kevin J.; Harris, Stephen E.; MacDougall, Mary; Chen, Shuo

2011-01-01

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways. PMID:21271257

Phosphorous Application Improves Drought Tolerance of Phoebe zhennan.

PubMed

Tariq, Akash; Pan, Kaiwen; Olatunji, Olusanya A; Graciano, Corina; Li, Zilong; Sun, Feng; Sun, Xiaoming; Song, Dagang; Chen, Wenkai; Zhang, Aiping; Wu, Xiaogang; Zhang, Lin; Mingrui, Deng; Xiong, Qinli; Liu, Chenggang

2017-01-01

Phoebe zhennan (Gold Phoebe) is a threatened tree species in China and a valuable and important source of wood and bioactive compounds used in medicine. Apart from anthropogenic disturbances, several biotic constraints currently restrict its growth and development. However, little attention has been given to building adaptive strategies for its conservation by examining its morphological and physio-biochemical responses to drought stress, and the role of fertilizers on these responses. A randomized experimental design was used to investigate the effects of two levels of irrigation (well-watered and drought-stressed) and phosphorous (P) fertilization treatment (with and without P) to assess the morphological and physio-biochemical responses of P. zhennan seedlings to drought stress. In addition, we evaluated whether P application could mitigate the negative impacts of drought on plant growth and metabolism. Drought stress had a significant negative effect on the growth and metabolic processes of P. zhennan . Despite this, reduced leaf area, limited stomatal conductance, reduced transpiration rate, increased water use efficiency, enhanced antioxidant enzymes activities, and osmolytes accumulation suggested that the species has good adaptive strategies for tolerating drought stress. Application of P had a significant positive effect on root biomass, signifying its improved water extracting capacity from the soil. Moreover, P fertilization significantly increased leaf relative water content, net photosynthetic rate, and maximal quantum efficiency of PSII under drought stress conditions. This may be attributable to several factors, such as enhanced root biomass, decreased malondialdehyde content, and the up-regulation of chloroplast pigments, osmolytes, and nitrogenous compounds. However, P application had only a slight or negligible effect on the growth and metabolism of well-watered plants. In conclusion, P. zhennan has a strong capability for drought resistance, while P application facilitates and improves drought tolerance mostly through physio-biochemical adjustments, regardless of water availability. It is imperative to explore the underlying metabolic mechanisms and effects of different levels of P fertilization on P. zhennan under drought conditions in order to design appropriate conservation and management strategies for this species, which is at risk of extinction.

Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products.

PubMed

Bae, Hyun W; Zhao, Li; Kanim, Linda E A; Wong, Pamela; Delamarter, Rick B; Dawson, Edgar G

2006-05-20

Enzyme-linked immunosorbent assay was used to detect bone morphogenetic proteins (BMPs) 2, 4, and 7 in 9 commercially available ("off the shelf") demineralized bone matrix (DBM) product formulations using 3 different manufacturer's production lots of each DBM formulation. To evaluate and compare the quantity of BMPs among several different DBM formulations (inter-product variability), as well as examine the variability of these proteins in different production lots within the same DBM formulation (intra-product variability). DBMs are commonly used to augment available bone graft in spinal fusion procedures. Surgeons are presented with an ever-increasing variety of commercially available human DBMs from which to choose. Yet, there is limited information on a specific DBM product's osteoinductive efficacy, potency, and constancy. There were protein extracts from each DBM sample separately dialyzed 4 times against distilled water at 4 degrees C for 48 hours. The amount of BMP-2, BMP-4, and BMP-7 was determined using enzyme-linked immunosorbent assay. RESULTS.: The concentrations of detected BMP-2 and BMP-7 were low for all DBM formulations, only nanograms of BMP were extracted from each gram of DBM (20.2-120.6 ng BMP-2/g DBM product; 54.2-226.8 ng BMP-7/g DBM). The variability of BMP concentrations among different lots of the same DBM formulation, intra-product variability, was higher than the variability of concentrations among different DBM formulations, inter-product variability (coefficient of variation range BMP-2 [16.34% to 76.01%], P < 0.01; BMP-7 [3.71% to 82.08%], P < 0.001). BMP-4 was undetectable. The relative quantities of BMPs in DBMs are low, in the order of 1 x 10(-9) g of BMP/g of DBM. There is higher variability in concentration of BMPs among 3 different lots of the same DBM formulation than among different DBM formulations. This variability questions DBM products' reliability and, possibly, efficacy in providing consistent osteoinduction.

Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

PubMed

Rinker, Torri E; Philbrick, Brandon D; Temenoff, Johnna S

2017-07-01

Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation. Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic core-shell heparin-PEG microparticles presented here overcome this limitation by sequestering proteins through a PEG-based shell onto a protein-protective heparin core, temporarily isolating bound proteins from the cellular microenvironment, and re-delivering proteins only after degradation of the PEG-based shell. Thus, these core-shell microparticles have potential to be a novel tool to harness and isolate proteins produced in the cellular environment and then control when proteins are re-introduced for the most effective tissue regeneration and repair. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

BMP signaling modulates hepcidin expression in zebrafish embryos independent of hemojuvelin.

PubMed

Gibert, Yann; Lattanzi, Victoria J; Zhen, Aileen W; Vedder, Lea; Brunet, Frédéric; Faasse, Sarah A; Babitt, Jodie L; Lin, Herbert Y; Hammerschmidt, Matthias; Fraenkel, Paula G

2011-01-21

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

Local administration of bone morphogenetic protein-2 and bisphosphonate during non-weight-bearing treatment of ischemic osteonecrosis of the femoral head: an experimental investigation in immature pigs.

PubMed

Kim, Harry K W; Aruwajoye, Olumide; Du, Justin; Kamiya, Nobuhiro

2014-09-17

Non-weight-bearing decreases the femoral head deformity but increases bone resorption without increasing bone formation in an experimental animal model of Legg-Calvé-Perthes disease. We sought to determine if local administration of bone morphogenetic protein (BMP)-2 with or without bisphosphonate can increase the bone formation during the non-weight-bearing treatment in the large animal model of Legg-Calvé-Perthes disease. Eighteen piglets were surgically induced with femoral head ischemia. Immediately following the surgery, all animals received an above-the-knee amputation to enforce local non-weight-bearing (NWB). One to two weeks later, six animals received local BMP-2 to the necrotic head (BMP group), six received local BMP-2 and ibandronate (BMP+IB group), and the remaining six received no treatment (NWB group). All animals were killed at eight weeks after the induction of ischemia. Radiographic, microcomputed tomography (micro-CT), and histomorphometric assessments were performed. Radiographic assessment showed that the femoral heads in the NWB, BMP, and BMP+IB groups had a decrease of 20%, 14%, and 10%, respectively, in their mean epiphyseal quotient in comparison with the normal control group. Micro-CT analyses showed significantly higher femoral head bone volume in the BMP+IB group than in the BMP group (p = 0.02) and the NWB group (p < 0.001). BMP+IB and BMP groups had a significantly higher trabecular number (p < 0.01) and lower trabecular separation (p < 0.02) than the NWB group. In addition, the osteoclast number per bone surface was significantly lower in the BMP+IB group compared with the NWB group. Calcein labeling showed significantly higher bone formation in the BMP and BMP+IB groups than in the NWB group (p < 0.05). Heterotopic ossification was found in the capsule of four hips in the BMP+IB group but not in the BMP group. Administration of BMP-2 with bisphosphonate best decreased bone resorption and increased new bone formation during non-weight-bearing treatment of ischemic osteonecrosis in a pig model, but heterotopic ossification is a concern. This preclinical study provides new evidence that BMP-2 with bisphosphonate can effectively prevent the extreme bone loss associated with the non-weight-bearing treatment and increase new bone formation in the femoral head in this animal model of ischemic osteonecrosis. Copyright © 2014 by The Journal of Bone and Joint Surgery, Incorporated.

Complications Related to the Recombinant Human Bone Morphogenetic Protein 2 Use in Posterior Cervical Fusion.

PubMed

Takahashi, Shinji; Buser, Zorica; Cohen, Jeremiah R; Roe, Allison; Myhre, Sue L; Meisel, Hans-Joerg; Brodke, Darrel S; Yoon, S Tim; Park, Jong-Beom; Wang, Jeffrey C; Youssef, Jim A

2017-11-01

A retrospective cohort study. To compare the complications between posterior cervical fusions with and without recombinant human bone morphogenetic protein 2 (rhBMP2). Use of rhBMP2 in anterior cervical spinal fusion procedures can lead to potential complications such as neck edema, resulting in airway complications or neurological compression. However, there are no data on the complications associated with the "off-label" use of rhBMP2 in upper and lower posterior cervical fusion approaches. Patients from the PearlDiver database who had a posterior cervical fusion between 2005 and 2011 were identified. We evaluated complications within 90 days after fusion and data was divided in 2 groups: (1) posterior cervical fusion including upper cervical spine O-C2 (upper group) and (2) posterior cervical fusion including lower cervical spine C3-C7 (lower group). Complications were divided into: any complication, neck-related complications, wound-related complications, and other complications. Of the 352 patients in the upper group, 73 patients (20.7%) received rhBMP2, and 279 patients (79.3%) did not. Likewise, in the lower group of 2372 patients, 378 patients (15.9%) had surgery with rhBMP2 and 1994 patients (84.1%) without. In the upper group, complications were observed in 7 patients (9.6%) with and 34 patients (12%) without rhBMP2. In the lower group, complications were observed in 42 patients (11%) with and 276 patients (14%) without rhBMP2. Furthermore, in the lower group the wound-related complications were significantly higher in the rhBMP2 group (23 patients, 6.1%) compared with the non-rhBMP2 group (75 patients, 3.8%). Our data showed that the use of rhBMP2 does not increase the risk of complications in upper cervical spine fusion procedures. However, in the lower cervical spine, rhBMP2 may elevate the risk of wound-related complications. Overall, there were no major complications associated with the use of rhBMP2 for posterior cervical fusion approaches. Level III.

Bone morphogenetic protein-2 and bone therapy: successes and pitfalls.

PubMed

Poon, Bonnie; Kha, Tram; Tran, Sally; Dass, Crispin R

2016-02-01

Bone morphogenetic proteins (BMPs), more specifically BMP-2, are being increasingly used in orthopaedic surgery due to advanced research into osteoinductive factors that may enhance and improve bone therapy. There are many areas in therapy that BMP-2 is being applied to, including dental treatment, open tibial fractures, cancer and spinal surgery. Within these areas of treatment, there are many reports of successes and pitfalls. This review explores the use of BMP-2 and its successes, pitfalls and future prospects in bone therapy. The PubMed database was consulted to compile this review. With successes in therapy, there were descriptions of a more rapid healing time with no signs of rejection or infection attributed to BMP-2 treatment. Pitfalls included BMP-2 'off-label' use, which lead to various adverse effects. Our search highlighted that optimising treatment with BMP-2 is a direction that many researchers are exploring, with areas of current research interest including concentration and dose of BMP-2, carrier type and delivery. © 2015 Royal Pharmaceutical Society.

Bone regeneration by polyhedral microcrystals from silkworm virus

PubMed Central

Matsumoto, Goichi; Ueda, Takayo; Shimoyama, Junko; Ijiri, Hiroshi; Omi, Yasushi; Yube, Hisato; Sugita, Yoshihiko; Kubo, Katsutoshi; Maeda, Hatsuhiko; Kinoshita, Yukihiko; Arias, Duverney Gaviria; Shimabukuro, Junji; Kotani, Eiji; Kawamata, Shin; Mori, Hajime

2012-01-01

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing. PMID:23226833

Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Huiwu; Health and Science Center, SIBS CAS and SSMU, 225 South Chongqing Road, Shanghai 200025; Dai Kerong

2007-05-18

In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a {beta}-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified bymore » BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals.« less

Osteogenic potential of the human bone morphogenetic protein 2 gene activated nanobone putty.

PubMed

Tian, Xiao-bin; Sun, Li; Yang, Shu-hua; Zhang, Yu-kun; Hu, Ru-yin; Fu, De-hao

2008-04-20

Nanobone putty is an injectable and bioresorbable bone substitute. The neutral-pH putty resembles hard bone tissue, does not contain polymers or plasticizers, and is self-setting and nearly isothermic, properties which are helpful for the adhesion, proliferation, and function of bone cells. The aim of this study was to investigate the osteogenic potential of human bone morphogenetic protein 2 (hBMP2) gene activated nanobone putty in inducing ectopic bone formation, and the effects of the hBMP2 gene activated nanobone putty on repairing bone defects. Twenty four Kunming mice were randomly divided into two groups. The nanobone putty + hBMP2 plasmid was injected into the right thigh muscle pouches of the mice (experiment side). The nanobone putty + blank plasmid or nanobone putty was injected into the left thigh muscle pouches of the group 1 (control side 1) or group 2 (control side 2), respectively. The effects of ectopic bone formation were evaluated by radiography, histology, and molecular biology analysis at 2 and 4 weeks after operation. Bilateral 15 mm radial defects were made in forty-eight rabbits. These rabbits were randomly divided into three groups: Group A, nanobone putty + hBMP2 plasmid; Group B, putty + blank plasmid; Group C, nanobone putty only. Six rabbits with left radial defects served as blank controls. The effect of bone repairing was evaluated by radiography, histology, molecular biology, and biomechanical analysis at 4, 8, and 12 weeks after operation. The tissue from the experimental side of the mice expressed hBMP2. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed at 2 weeks after operation, and massive mature bone observed at 4 weeks. No bone formation was observed in the control side of the mice. The ALP activity in the experiment side of the mice was higher than that in the control side. The tissue of Group A rabbits expressed hBMP2 protein and higher ALP level. The new bone formation rate and antibending strength of group A was significantly higher than those of group B and C. The defects in blank control were not healed. The hBMP2 gene activated nanobone putty exhibited osteoinductive ability, and had a better bone defect repair capability than that of nanobone putty only.

Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries

PubMed Central

Hernandez-Hurtado, Adelina A.; Lara-Arias, Jorge; Romero-Diaz, Viktor J.; Abrego-Guerra, Adalberto; Vilchez-Cavazos, Jose F.; Elizondo-Riojas, Guillermo; Martinez-Rodriguez, Herminia G.; Espinoza-Juarez, Marcela A.; Mendoza Lemus, Oscar F.

2016-01-01

Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology. PMID:27818692

Negative regulation of BMP signaling by the ski oncoprotein.

PubMed

Luo, Kunxin

2003-01-01

The bone morphogenetic proteins (BMPs) play important roles in the regulation of multiple aspects of vertebrate development. BMPs signal through the cell surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. The activity of this signal pathway can be modulated both by extracellular factors that regulate the binding of BMPs to the receptor and by intracellular proteins that interact with the Smad proteins. We have shown that Ski is an important negative regulator of the Smad proteins. Ski can bind to the BMP-Smad protein complexes in response to BMP and repress their ability to activate BMP target genes through disruption of a functional Smad complex and through recruitment of transcriptional co-repressors. The antagonism of BMP signaling by Ski results in neural specification in Xenopus embryos and inhibition of osteoblast differentiation in mouse bone-marrow stromal progenitor cells. This ability to modulate BMP signaling by Ski may play an important role in the regulation of craniofacial, neuronal, and skeletal muscle development.

Evidence-Based Evaluation of Practice and Innovation in Physical Therapy Using the IDEAL-Physio Framework.

PubMed

Beard, David; Hamilton, David; Davies, Loretta; Cook, Jonathan; Hirst, Allison; McCulloch, Peter; Paez, Arsenio

2018-02-01

The IDEAL framework is an established method for initial and ongoing evaluations of innovation and practice for complex health care interventions. First derived for surgical sciences and embedded at a global level for evaluating surgery/surgical devices, the IDEAL framework is based on the principle that innovation and evaluation in clinical practice can, and should, evolve together in an ordered manner: from conception to development and then to validation by appropriate clinical studies and, finally, longer-term follow-up. This framework is highly suited to other complex, nonpharmacological interventions, such as physical therapist interventions. This perspective outlines the application of IDEAL to physical therapy in the new IDEAL-Physio framework. The IDEAL-Physio framework comprises 5 stages. In stage 1, the idea phase, formal data collection should begin. Stage 2a is the phase for iterative improvement and adjustment with thorough data recording. Stage 2b involves the onset of formal evaluation using systematically collected group or cohort data. Stage 3 is the phase for formal comparative assessment of treatment, usually involving randomized studies. Stage 4 involves long-term follow-up. The IDEAL-Physio framework is recommended as a method for guiding and evaluating both innovation and practice in physical therapy, with the overall goal of providing better evidence-based care. © 2017 American Physical Therapy Association.

Tbx20 Transcription Factor Is a Downstream Mediator for Bone Morphogenetic Protein-10 in Regulating Cardiac Ventricular Wall Development and Function*

PubMed Central

Zhang, Wenjun; Chen, Hanying; Wang, Yong; Yong, Weidong; Zhu, Wuqiang; Liu, Yunlong; Wagner, Gregory R.; Payne, R. Mark; Field, Loren J.; Xin, Hongbo; Cai, Chen-Leng; Shou, Weinian

2011-01-01

Bone morphogenetic protein 10 (BMP10) belongs to the TGFβ-superfamily. Previously, we had demonstrated that BMP10 is a key regulator for ventricular chamber formation, growth, and maturation. Ablation of BMP10 leads to hypoplastic ventricular wall formation, and elevated levels of BMP10 are associated with abnormal ventricular trabeculation/compaction and wall maturation. However, the molecular mechanism(s) by which BMP10 regulates ventricle wall growth and maturation is still largely unknown. In this study, we sought to identify the specific transcriptional network that is potentially mediated by BMP10. We analyzed and compared the gene expression profiles between α-myosin heavy chain (αMHC)-BMP10 transgenic hearts and nontransgenic littermate controls using Affymetrix mouse exon arrays. T-box 20 (Tbx20), a cardiac transcription factor, was significantly up-regulated in αMHC-BMP10 transgenic hearts, which was validated by quantitative RT-PCR and in situ hybridization. Ablation of BMP10 reduced Tbx20 expression specifically in the BMP10-expressing region of the developing ventricle. In vitro promoter analysis demonstrated that BMP10 was able to induce Tbx20 promoter activity through a conserved Smad binding site in the Tbx20 promoter proximal region. Furthermore, overexpression of Tbx20 in myocardium led to dilated cardiomyopathy that exhibited ventricular hypertrabeculation and an abnormal muscular septum, which phenocopied genetically modified mice with elevated BMP10 levels. Taken together, our findings demonstrate that the BMP10-Tbx20 signaling cascade is important for ventricular wall development and maturation. PMID:21890625

Genetic Variants of BMP2 and Their Association with the Risk of Non-Syndromic Tooth Agenesis

PubMed Central

Wang, Yuting; Gu, Ning; Ma, Lan; Xu, Min; Ma, Junqing; Zhang, Weibing; Pan, Yongchu; Wang, Lin

2016-01-01

Non-syndromic tooth agenesis (or non-syndromic congenitally missing tooth) is one of the most common congenital defects in humans affecting the craniofacial function and appearance. Single nucleotide polymorphisms (SNPs) have been associated with an individual’s susceptibility to these anomalies. The aim of the present study was therefore to investigate the roles of the potentially functional SNPs of BMP2 in the occurrence of tooth agenesis. Overall, four potentially functional SNPs of BMP2 (rs15705, rs235768, rs235769 and rs3178250) were selected, and their associations with the susceptibility of tooth agenesis were evaluated in a case-control study of 335 non-syndromic tooth agenesis cases and 444 healthy controls. The SNPs rs15705 and rs3178250 were found to be associated with an individual’s risk of tooth agenesis (P = 0.046 and P = 0.039, respectively). Both SNPs showed an increased risk of mandibular incisor agenesis (rs15705, AA/AC vs. CC = 1.58, 95% CI = [1.06–2.34], P = 0.024; rs3178250, TT/TC vs. CC = 1.60, 95% CI = [1.08–2.37], P = 0.020). Bioinformatics analysis indicated that these two SNPs located at the 3’-untranslated region (3’-UTR) of BMP2 might alter the binding ability of miR-1273d and miR-4639-5p, respectively, which was confirmed by luciferase activity assays in the 293A and COS7 cell lines (P < 0.001 in 293A and P < 0.01 in COS7 for miR-1273d; and P < 0.001 in both cells for miR-4639-5p). Furthermore, BMP2 mRNA expression decreased after transfecting either miR-1273d or miR-4639-5p into these two cell lines (P < 0.01 in 293A and P < 0.001 in COS7 for miR-1273d, and P < 0.01 in both cell lines for miR-4639-5p). Taken together, our findings indicate that rs15705 and rs317250 are associated with the susceptibility of non-syndromic tooth agenesis by possibly affecting miRNAs and mRNA interaction. PMID:27362534

PhysioDroid: Combining Wearable Health Sensors and Mobile Devices for a Ubiquitous, Continuous, and Personal Monitoring

PubMed Central

Villalonga, Claudia; Damas, Miguel

2014-01-01

Technological advances on the development of mobile devices, medical sensors, and wireless communication systems support a new generation of unobtrusive, portable, and ubiquitous health monitoring systems for continuous patient assessment and more personalized health care. There exist a growing number of mobile apps in the health domain; however, little contribution has been specifically provided, so far, to operate this kind of apps with wearable physiological sensors. The PhysioDroid, presented in this paper, provides a personalized means to remotely monitor and evaluate users' conditions. The PhysioDroid system provides ubiquitous and continuous vital signs analysis, such as electrocardiogram, heart rate, respiration rate, skin temperature, and body motion, intended to help empower patients and improve clinical understanding. The PhysioDroid is composed of a wearable monitoring device and an Android app providing gathering, storage, and processing features for the physiological sensor data. The versatility of the developed app allows its use for both average users and specialists, and the reduced cost of the PhysioDroid puts it at the reach of most people. Two exemplary use cases for health assessment and sports training are presented to illustrate the capabilities of the PhysioDroid. Next technical steps include generalization to other mobile platforms and health monitoring devices. PMID:25295301

PhysioDroid: combining wearable health sensors and mobile devices for a ubiquitous, continuous, and personal monitoring.

PubMed

Banos, Oresti; Villalonga, Claudia; Damas, Miguel; Gloesekoetter, Peter; Pomares, Hector; Rojas, Ignacio

2014-01-01

Technological advances on the development of mobile devices, medical sensors, and wireless communication systems support a new generation of unobtrusive, portable, and ubiquitous health monitoring systems for continuous patient assessment and more personalized health care. There exist a growing number of mobile apps in the health domain; however, little contribution has been specifically provided, so far, to operate this kind of apps with wearable physiological sensors. The PhysioDroid, presented in this paper, provides a personalized means to remotely monitor and evaluate users' conditions. The PhysioDroid system provides ubiquitous and continuous vital signs analysis, such as electrocardiogram, heart rate, respiration rate, skin temperature, and body motion, intended to help empower patients and improve clinical understanding. The PhysioDroid is composed of a wearable monitoring device and an Android app providing gathering, storage, and processing features for the physiological sensor data. The versatility of the developed app allows its use for both average users and specialists, and the reduced cost of the PhysioDroid puts it at the reach of most people. Two exemplary use cases for health assessment and sports training are presented to illustrate the capabilities of the PhysioDroid. Next technical steps include generalization to other mobile platforms and health monitoring devices.

PhysioDirect: Supporting physiotherapists to deliver telephone assessment and advice services within the context of a randomised trial

PubMed Central

Bishop, Annette; Gamlin, Jill; Hall, Jeanette; Hopper, Cherida; Foster, Nadine E.

2013-01-01

Physiotherapy-led telephone assessment and advice services for patients with musculoskeletal problems have been developed in many services in the UK, but high quality trial data on clinical and cost effectiveness has been lacking. In order to address this ‘The PhysioDirect trial’ (ISRCTN55666618), was a pragmatic randomised trial of a PhysioDirect telephone assessment and advice service. This paper describes the PhysioDirect system used in the trial and how physiotherapists were trained and supported to use the system and deliver the PhysioDirect service. The PhysioDirect system used in the trial was developed in Huntingdon and now serves a population of 350,000 people. When initiating or providing physiotherapy-led telephone assessment and advice services training and support for physiotherapists delivering care in this way is essential. An enhanced skill set is required for telephone assessment and advice particularly in listening and communication skills. In addition to an initial training programme, even experienced physiotherapists benefit from a period of skill consolidation to become proficient and confident in assessing patients and delivering care using the telephone. A computer-based system assists the delivery of a physiotherapy-led musculoskeletal assessment and advice service. Clinical Trials Registration Number (ISRCTN55666618). PMID:23219629

A Retrospective Analysis of Complications Associated With Bone Morphogenetic Protein 2 in Anterior Lumbar Interbody Fusion.

PubMed

Hindoyan, Kevork; Tilan, Justin; Buser, Zorica; Cohen, Jeremiah R; Brodke, Darrel S; Youssef, Jim A; Park, Jong-Beom; Yoon, S Tim; Meisel, Hans-Joerg; Wang, Jeffrey C

2017-04-01

Retrospective review. The aim of our study was to quantify the frequency of complications associated with recombinant human bone morphogenetic protein 2 (rhBMP-2) use in anterior lumbar interbody fusion (ALIF). The orthopedic subset of the Medicare database (PearlDiver) was queried for this retrospective cohort study using International Statistical Classification of Diseases 9 (ICD-9) and Current Procedure Terminology (CPT) codes for ALIF procedures with and without rhBMP-2 between 2005 and 2010. Frequencies of complications and reoperations were then identified within 1 year from the index procedure. Complications included reoperations, pulmonary embolus, deep vein thrombosis, myocardial infarction, nerve-related complications, incision and drainage procedures, wound, sepsis, pneumonia, urinary tract infections, respiratory, heterotopic ossification, retrograde ejaculation, radiculopathy, and other medical complications. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the statistical significance. We identified a total of 41 865 patients who had an ALIF procedure. A total of 14 384 patients received rhBMP-2 while 27 481 did not. Overall, 6016 (41.8%) complications within 1 year from surgery were noted within the group who received rhBMP-2 and 12 950 (47.1%) complications within 1 year from surgery were recorded in those who did not receive rhBMP-2 (OR = 0.81, CI = 0.77-0.84). Overall, exposure to rhBMP-2 was associated with significantly decreased odds of complications with exception to reoperation rates (0.9% rhBMP-2 vs 1.0% no rhBMP-2; OR = 0.88, CI = 0.71-1.09) and radiculopathy (4.4% rhBMP-2 vs 4.3% no rhBMP-2; OR = 1.02, CI = 0.93-1.13). The use of rhBMP-2 in patients undergoing ALIF procedure was associated with a significantly decreased rate of complications. Further studies are needed to elucidate a true incidence of complication.

A Retrospective Analysis of Complications Associated With Bone Morphogenetic Protein 2 in Anterior Lumbar Interbody Fusion

PubMed Central

Hindoyan, Kevork; Tilan, Justin; Cohen, Jeremiah R.; Brodke, Darrel S.; Youssef, Jim A.; Park, Jong-Beom; Yoon, S. Tim; Meisel, Hans-Joerg; Wang, Jeffrey C.

2017-01-01

Study Design: Retrospective review. Objective: The aim of our study was to quantify the frequency of complications associated with recombinant human bone morphogenetic protein 2 (rhBMP-2) use in anterior lumbar interbody fusion (ALIF). Methods: The orthopedic subset of the Medicare database (PearlDiver) was queried for this retrospective cohort study using International Statistical Classification of Diseases 9 (ICD-9) and Current Procedure Terminology (CPT) codes for ALIF procedures with and without rhBMP-2 between 2005 and 2010. Frequencies of complications and reoperations were then identified within 1 year from the index procedure. Complications included reoperations, pulmonary embolus, deep vein thrombosis, myocardial infarction, nerve-related complications, incision and drainage procedures, wound, sepsis, pneumonia, urinary tract infections, respiratory, heterotopic ossification, retrograde ejaculation, radiculopathy, and other medical complications. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the statistical significance. Results: We identified a total of 41 865 patients who had an ALIF procedure. A total of 14 384 patients received rhBMP-2 while 27 481 did not. Overall, 6016 (41.8%) complications within 1 year from surgery were noted within the group who received rhBMP-2 and 12 950 (47.1%) complications within 1 year from surgery were recorded in those who did not receive rhBMP-2 (OR = 0.81, CI = 0.77-0.84). Overall, exposure to rhBMP-2 was associated with significantly decreased odds of complications with exception to reoperation rates (0.9% rhBMP-2 vs 1.0% no rhBMP-2; OR = 0.88, CI = 0.71-1.09) and radiculopathy (4.4% rhBMP-2 vs 4.3% no rhBMP-2; OR = 1.02, CI = 0.93-1.13). Conclusions: The use of rhBMP-2 in patients undergoing ALIF procedure was associated with a significantly decreased rate of complications. Further studies are needed to elucidate a true incidence of complication. PMID:28507884

BMP2-Induced Inflammation Can Be Suppressed by the Osteoinductive Growth Factor NELL-1

PubMed Central

Shen, Jia; James, Aaron W.; Zara, Janette N.; Asatrian, Greg; Khadarian, Kevork; Zhang, James B.; Ho, Stephanie; Kim, Hyun Ju

2013-01-01

Bone-morphogenetic protein 2 (BMP2) is currently the only Food and Drug Administration-approved osteoinductive growth factor used in clinical settings for bone regeneration and repair. However, the use of BMP2 is encumbered by numerous clinical complications, including postoperative inflammation and life-threatening cervical swelling. Thus, methods to prevent BMP2-induced inflammation would have far-reaching clinical implications toward improving current BMP2-based methods for bone regeneration. For the first time, we investigate the potential role of the growth factor Nel-like molecule-1 (NELL-1) in inhibiting BMP2-induced inflammation. Adult rats underwent a femoral bone onlay procedure, treated with either BMP2 protein (4 mg/mL), NELL-1 protein (4 mg/mL), or both proteins combined. Animals were evaluated at 3, 7, and 14 days postoperatively by histology, histomorphometry, immunohistochemistry, and real-time PCR for markers of inflammation (TNFα, IL6). The relative levels of TNFα and IL6 in serum were also detected by ELISA. The mechanism for NELL-1's anti-inflammatory effect was further assessed through examining inflammatory markers and generation of reactive oxygen species (ROS) in the mouse embryonic fibroblast NIH3T3 cells. BMP2 significantly induced local inflammation, including an early and pronounced polymorphonuclear cell infiltration accompanied by increased expression of TNFα and IL6. Treatment with NELL-1 alone elicited no significant inflammatory response. However, NELL-1 significantly attenuated BMP2-induced inflammation by all markers and at all timepoints. These local findings were also confirmed using systemic serum inflammatory biomarkers (TNFα, IL6). In each case, NELL-1 fully reversed BMP2-induced systemic inflammation. Lastly, our findings were recapitulated in vitro, where NELL-1 suppressed BMP2 induced expression of inflammatory markers, as well as NF-κB transcriptional activity and generation of ROS. BMP2-induced inflammation is a serious public health concern with potentially life-threatening complications. In the present study, we observed that the growth factor, NELL-1, significantly attenuates or completely reverses BMP2-induced inflammation. The mechanisms of NELL-1's anti-inflammatory effect are only partially elucidated, and may include reduction of NF-κB transcriptional activity or ROS generation. PMID:23758588

Bone Formation in a Rat Tibial Defect Model Using Carboxymethyl Cellulose/BioC/Bone Morphogenic Protein-2 Hybrid Materials

PubMed Central

Kim, Hak-Jun; Park, Kyeongsoon; Kim, Sung Eun; Song, Hae-Ryong

2014-01-01

The objective of this study was to assess whether carboxymethyl cellulose- (CMC-) based hydrogel containing BioC (biphasic calcium phosphate (BCP); tricalcium phosphate (TCP) : hydroxyapatite (Hap) = 70 : 30) and bone morphogenic protein-2 (BMP-2) led to greater bone formation than CMC-based hydrogel containing BioC without BMP-2. In order to demonstrate bone formation at 4 and 8 weeks, plain radiographs, microcomputed tomography (micro-CT) evaluation, and histological studies were performed after implantation of all hybrid materials on an 8 mm defect of the right tibia in rats. The plain radiographs and micro-CT analyses revealed that CMC/BioC/BMP-2 (0.5 mg) led to much greater mineralization at 4 and 8 weeks than did CMC/BioC or CMC/Bio/BMP-2 (0.1 mg). Likewise, bone formation and bone remodeling studies revealed that CMC/BioC/BMP-2 (0.5 mg) led to a significantly greater amount of bone formation and bone remodeling at 4 and 8 weeks than did CMC/BioC or CMC/BioC/BMP-2 (0.1 mg). Histological studies revealed that mineralized bone tissue was present around the whole circumference of the defect site with CMC/BioC/BMP-2 (0.5 mg) but not with CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) at 4 and 8 weeks. These results suggest that CMC/BioC/BMP-2 hybrid materials induced greater bone formation than CMC/BioC hybrid materials. Thus, CMC/BioC/BMP-2 hybrid materials may be used as an injectable substrate to regenerate bone defects. PMID:24804202

Complications of Anterior Cervical Fusion using a Low-dose Recombinant Human Bone Morphogenetic Protein-2

PubMed Central

Kukreja, Sunil; Ahmed, Osama I; Haydel, Justin; Nanda, Anil

2015-01-01

Objective There are several reports, which documented a high incidence of complications following the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in anterior cervical fusions (ACFs). The objective of this study is to share our experience with low-dose rhBMP-2 in anterior cervical spine. Methods We performed a retrospective analysis of 197 patients who underwent anterior cervical fusion (ACF) with the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) during 2007-2012. A low-dose rhBMP-2 (0.7mg/level) sponge was placed exclusively within the cage. In 102 patients demineralized bone matrix (DBM) was filled around the BMP sponge. Incidence and severity of dysphagia was determined by 5 points SWAL-QOL scale. Results Two patients had prolonged hospitalization due to BMP unrelated causes. Following the discharge, 13.2%(n=26) patients developed dysphagia and 8.6%(n=17) patients complained of neck swelling. More than half of the patients (52.9%, n=9) with neck swelling also had associated dysphagia; however, only 2 of these patients necessitated readmission. Both of these patients responded well to the intravenous dexamethasone. The use of DBM did not affect the incidence and severity of complications (p>0.05). Clinico-radiological evidence of fusion was not observed in 2 patients. Conclusion A low-dose rhBMP-2 in ACFs is not without risk. However, the incidence and severity of complications seem to be lower with low-dose BMP placed exclusively inside the cage. Packing DBM putty around the BMP sponge does not affect the safety profile of rhBMP-2 in ACFs. PMID:26217385

Msx-1 is suppressed in bisphosphonate-exposed jaw bone analysis of bone turnover-related cell signalling after bisphosphonate treatment.

PubMed

Wehrhan, F; Hyckel, P; Amann, K; Ries, J; Stockmann, P; Schlegel, Ka; Neukam, Fw; Nkenke, E

2011-05-01

Bone-destructive disease treatments include bisphosphonates and antibodies against receptor activator for nuclear factor κB ligand (aRANKL). Osteonecrosis of the jaw (ONJ) is a side-effect. Aetiopathology models failed to explain their restriction to the jaw. The osteoproliferative transcription factor Msx-1 is expressed constitutively only in mature jaw bone. Msx-1 expression might be impaired in bisphosphonate-related ONJ. This study compared the expression of Msx-1, Bone Morphogenetic Protein (BMP)-2 and RANKL, in ONJ-affected and healthy jaw bone. An automated immunohistochemistry-based alkaline phosphatase-anti-alkaline phosphatase method was used on ONJ-affected and healthy jaw bone samples (n = 20 each): cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed to quantitatively compare Msx-1, BMP-2, RANKL and GAPDH mRNA levels. Labelling indices were significantly lower for Msx-1 (P < 0.03) and RANKL (P < 0.003) and significantly higher (P < 0.02) for BMP-2 in ONJ compared with healthy bone. Expression was sevenfold lower (P < 0.03) for Msx-1, 22-fold lower (P < 0.001) for RANKL and eightfold higher (P < 0.02) for BMP-2 in ONJ bone. Msx-1, RANKL suppression and BMP-2 induction were consistent with the bisphosphonate-associated osteopetrosis and impaired bone remodelling in BP- and aRANKL-induced ONJ. Msx-1 suppression suggested a possible explanation of the exclusivity of ONJ in jaw bone. Functional analyses of Msx-1- RANKL interaction during bone remodelling should be performed in the future. © 2011 John Wiley & Sons A/S.

RhBMP-2-induced radiculitis in patients undergoing transforaminal lumbar interbody fusion: relationship to dose.

PubMed

Villavicencio, Alan T; Burneikiene, Sigita

2016-10-01

Recombinant human bone morphogenetic protein-2 (rhBMP-2) remains the primary synthetic osteoinductive material used in spinal fusion surgery today. The early inflammation reaction to rhBMP-2 manifesting with radicular symptoms has been previously reported in patients undergoing transforaminal lumbar interbody fusion (TLIF). There is a disagreement with regard to the factors affecting its occurrence and whether such symptoms are dose dependent. The purpose of this analysis was to determine the incidence of rhBMP-2-induced radiculitis and its relationship to dose. A retrospective cohort analysis was performed of the prospectively collected data. All consecutive patients (n=204) who underwent one- or two-level TLIF and instrumented posterolateral fusion with an off-label rhBMP-2 use were included in this analysis. The patients who developed new radicular symptoms after initial improvement postoperatively and had sterile fluid collections indicative of inflammatory process, or in the absence of any structural abnormalities that would explain these symptoms on imaging studies, were deemed to have rhBMP-2-induced radiculitis. Magnetic resonance imaging (MRI) scans were obtained for all patients who developed postoperative radicular symptoms. Correlations between the total rhBMP-2 dose, dose per spinal level, and incidence of radiculitis were evaluated while controlling for age, sex, number of TLIF levels, and surgeon. The incidence of postoperative radiculitis was 11.3% (23 out of 204). The average total rhBMP-2 dose was 4.9 mg (range=2.1-12) and the average dose per spinal level was 3.8 mg (range=1.05-12). Logistic regression analysis did not identify any significant correlations between the rhBMP-2 doses and the incidence of radiculitis (p=.6). The incidence of rhBMP-2-induced radiculitis in patients undergoing TLIF is quite high, but there were no dose-related correlations found. The study, however, cannot rule out a possibility that a larger variation in bone morphogenetic protein (BMP) doses could still be a factor in the development of rhBMP-2-associated radiculitis. Copyright © 2016 Elsevier Inc. All rights reserved.

Cyst-Like Osteolytic Formations in Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Augmented Sheep Spinal Fusion.

PubMed

Pan, Hsin Chuan; Lee, Soonchul; Ting, Kang; Shen, Jia; Wang, Chenchao; Nguyen, Alan; Berthiaume, Emily A; Zara, Janette N; Turner, A Simon; Seim, Howard B; Kwak, Jin Hee; Zhang, Xinli; Soo, Chia

2017-07-01

Multiple case reports using recombinant human bone morphogenetic protein-2 (rhBMP-2) have reported complications. However, the local adverse effects of rhBMP-2 application are not well documented. In this report we show that, in addition to promoting lumbar spinal fusion through potent osteogenic effects, rhBMP-2 augmentation promotes local cyst-like osteolytic formations in sheep trabecular bones that have undergone anterior lumbar interbody fusion. Three months after operation, conventional computed tomography showed that the trabecular bones of the rhBMP-2 application groups could fuse, whereas no fusion was observed in the control group. Micro-computed tomography analysis revealed that the core implant area's bone volume fraction and bone mineral density increased proportionately with rhBMP-2 dose. Multiple cyst-like bone voids were observed in peri-implant areas when using rhBMP-2 applications, and these sites showed significant bone mineral density decreases in relation to the unaffected regions. Biomechanically, these areas decreased in strength by 32% in comparison with noncystic areas. Histologically, rhBMP-2-affected void sites had an increased amount of fatty marrow, thinner trabecular bones, and significantly more adiponectin- and cathepsin K-positive cells. Despite promoting successful fusion, rhBMP-2 use in clinical applications may result in local adverse structural alterations and compromised biomechanical changes to the bone. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Effects of Bone Morphogenic Proteins on Engineered Cartilage

NASA Technical Reports Server (NTRS)

Gooch, Keith, J.; Blunk, Torsten; Courter, Donald L.; Sieminski, Alisha; Vunjak-Novakovic, Gordana; Freed, Lisa E.

2007-01-01

A report describes experiments on the effects of bone morphogenic proteins (BMPs) on engineered cartilage grown in vitro. In the experiments, bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid scaffolds and cultured in, variously, a control medium or a medium supplemented with BMP-2, BMP-12, or BMP-13 in various concentrations. Under all conditions investigated, cell-polymer constructs cultivated for 4 weeks macroscopically and histologically resembled native cartilage. At a concentration of 100 ng/mL, BMP-2, BMP-12, or BMP-13 caused (1) total masses of the constructs to exceed those of the controls by 121, 80, or 62 percent, respectively; (2) weight percentages of glycosaminoglycans in the constructs to increase by 27, 18, or 15, respectively; and (3) total collagen contents of the constructs to decrease to 63, 89, or 83 percent of the control values, respectively. BMP-2, but not BMP-12 or BMP-13, promoted chondrocyte hypertrophy. These observations were interpreted as suggesting that the three BMPs increase the growth rates and modulate the compositions of engineered cartilage. It was also concluded that in vitro engineered cartilage is a suitable system for studying effects of BMPs on chondrogenesis in a well-defined environment.

BMP2 and BMP4 variations and risk of non-syndromic cleft lip and palate.

PubMed

Saket, Mitra; Saliminejad, Kioomars; Kamali, Koorosh; Moghadam, Fatemeh Aghakhani; Anvar, Nazanin Esmaeili; Khorram Khorshid, Hamid Reza

2016-12-01

Non-syndromic cleft lip with or without cleft palate (CL/P) is one of the most common congenital anomalies and arises from the interaction of environmental and genetic factors. The objective of this study was to investigate the association between the BMP2 (bone morphogenetic protein 2) and BMP4 (bone morphogenetic protein 4) polymorphisms with non-syndromic CL/P to clarify the potential role of these genes in the etiology of CL/P in Iranian population. The allelic and genotypic frequencies of BMP2 rs235768 A>T and BMP4 rs17563 T>C polymorphisms were determined in 107 unrelated Iranian subjects with non-syndromic CL/P and 186 control subjects using PCR and RFLP methods, and the results were compared with healthy controls. A p-value of <0.05 was considered statistically significant. The BMP2 rs235768 AT genotype was significantly higher (P=0.009, OR=3, 95% CI=1.3-7.0) in the CL/P (59.8%) than the control group (33.3%). Similarly, the BMP4 rs17563 TC genotype were significantly higher (P=0.008, OR=3.7, 95% CI=1.4-9.9) in the CL/P (70.0%) than the control group (44.6%). The BMP2 rs235768 A>T and BMP4 rs17563 T>C polymorphisms could be considered as the risk factor for non-syndromic CL/P in Iranian population. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

PubMed Central

Shen, Jia; James, Aaron W.; Zhang, Xinli; Pang, Shen; Zara, Janette N.; Asatrian, Greg; Chiang, Michael; Lee, Min; Khadarian, Kevork; Nguyen, Alan; Lee, Kevin S.; Siu, Ronald K.; Tetradis, Sotirios; Ting, Kang; Soo, Chia

2017-01-01

The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poor-quality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis. PMID:26772960

Trends Analysis of rhBMP Utilization in Single-Level Posterior Lumbar Interbody Fusion in the United States

PubMed Central

Lao, Lifeng; Cohen, Jeremiah R.; Brodke, Darrel S.; Youssef, Jim A.; Park, Jong-Beom; Yoon, S. Tim; Wang, Jeffrey C.; Meisel, Hans-Joerg

2017-01-01

Study Design: Retrospective study. Objectives: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been widely used in spinal fusion surgery, but there is little information on rhBMP-2 utilization in single-level posterior lumbar interbody fusion (PLIF). The purpose of our study was to evaluate the trends and demographics of rhBMP-2 utilization in single-level PLIF. Methods: Patients who underwent single-level PLIF from 2005 to 2011 were identified by searching ICD-9 diagnosis and procedure codes in the PearlDiver Patient Records Database, a national database of orthopedic insurance records. The year of procedure, age, gender, and region of the United States were recorded for each patient. Results were reported for each variable as the incidence of procedures identified per 100 000 patients searched in the database. Results: A total of 2735 patients had single-level PLIF. The average rate of single-level PLIF with rhBMP-2 maintained at a relatively stable level (28% to 31%) from 2005 to 2009, but decreased in 2010 (9.9%) and 2011 (11.8%). The overall incidence of single-level PLIF without rhBMP-2 (0.68 cases per 100 000 patients) was statistically higher (P < .01) compared to single-level PLIF with rhBMP-2 (0.21 cases per 100 000 patients). The average rate of single-level PLIF with rhBMP-2 utilization was the highest in West (30.1%), followed by Midwest (26.9%), South (20.5%), and Northeast (17.8%). The highest incidence of single-level PLIF with rhBMP-2 was observed in the age group <65 years (0.3 per 100 000 patients). Conclusions: To our knowledge, this is the first study to report on the demographics associated with rhBMP-2 use in single-level PLIF. There was a 3-fold increase in the rate of PLIF without rhBMP-2 compared to PLIF with rhBMP-2, with both procedures being mainly done in patients less than 65 years of age. PMID:28989840

Trends Analysis of rhBMP Utilization in Single-Level Posterior Lumbar Interbody Fusion in the United States.

PubMed

Lao, Lifeng; Cohen, Jeremiah R; Buser, Zorica; Brodke, Darrel S; Youssef, Jim A; Park, Jong-Beom; Yoon, S Tim; Wang, Jeffrey C; Meisel, Hans-Joerg

2017-10-01

Retrospective study. Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been widely used in spinal fusion surgery, but there is little information on rhBMP-2 utilization in single-level posterior lumbar interbody fusion (PLIF). The purpose of our study was to evaluate the trends and demographics of rhBMP-2 utilization in single-level PLIF. Patients who underwent single-level PLIF from 2005 to 2011 were identified by searching ICD-9 diagnosis and procedure codes in the PearlDiver Patient Records Database, a national database of orthopedic insurance records. The year of procedure, age, gender, and region of the United States were recorded for each patient. Results were reported for each variable as the incidence of procedures identified per 100 000 patients searched in the database. A total of 2735 patients had single-level PLIF. The average rate of single-level PLIF with rhBMP-2 maintained at a relatively stable level (28% to 31%) from 2005 to 2009, but decreased in 2010 (9.9%) and 2011 (11.8%). The overall incidence of single-level PLIF without rhBMP-2 (0.68 cases per 100 000 patients) was statistically higher ( P < .01) compared to single-level PLIF with rhBMP-2 (0.21 cases per 100 000 patients). The average rate of single-level PLIF with rhBMP-2 utilization was the highest in West (30.1%), followed by Midwest (26.9%), South (20.5%), and Northeast (17.8%). The highest incidence of single-level PLIF with rhBMP-2 was observed in the age group <65 years (0.3 per 100 000 patients). To our knowledge, this is the first study to report on the demographics associated with rhBMP-2 use in single-level PLIF. There was a 3-fold increase in the rate of PLIF without rhBMP-2 compared to PLIF with rhBMP-2, with both procedures being mainly done in patients less than 65 years of age.

Activation of Bmp2-Smad1 Signal and Its Regulation by Coordinated Alteration of H3K27 Trimethylation in Ras-Induced Senescence

PubMed Central

Kaneda, Atsushi; Fujita, Takanori; Anai, Motonobu; Yamamoto, Shogo; Nagae, Genta; Morikawa, Masato; Tsuji, Shingo; Oshima, Masanobu; Miyazono, Kohei; Aburatani, Hiroyuki

2011-01-01

Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF), we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP)-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence. PMID:22072987

Multifunctional phosphate-based inorganic-organic hybrid nanoparticles.

PubMed

Heck, Joachim G; Napp, Joanna; Simonato, Sara; Möllmer, Jens; Lange, Marcus; Reichardt, Holger M; Staudt, Reiner; Alves, Frauke; Feldmann, Claus

2015-06-17

Phosphate-based inorganic-organic hybrid nanoparticles (IOH-NPs) with the general composition [M](2+)[Rfunction(O)PO3](2-) (M = ZrO, Mg2O; R = functional organic group) show multipurpose and multifunctional properties. If [Rfunction(O)PO3](2-) is a fluorescent dye anion ([RdyeOPO3](2-)), the IOH-NPs show blue, green, red, and near-infrared fluorescence. This is shown for [ZrO](2+)[PUP](2-), [ZrO](2+)[MFP](2-), [ZrO](2+)[RRP](2-), and [ZrO](2+)[DUT](2-) (PUP = phenylumbelliferon phosphate, MFP = methylfluorescein phosphate, RRP = resorufin phosphate, DUT = Dyomics-647 uridine triphosphate). With pharmaceutical agents as functional anions ([RdrugOPO3](2-)), drug transport and release of anti-inflammatory ([ZrO](2+)[BMP](2-)) and antitumor agents ([ZrO](2+)[FdUMP](2-)) with an up to 80% load of active drug is possible (BMP = betamethason phosphate, FdUMP = 5'-fluoro-2'-deoxyuridine 5'-monophosphate). A combination of fluorescent dye and drug anions is possible as well and shown for [ZrO](2+)[BMP](2-)0.996[DUT](2-)0.004. Merging of functional anions, in general, results in [ZrO](2+)([RdrugOPO3]1-x[RdyeOPO3]x)(2-) nanoparticles and is highly relevant for theranostics. Amine-based functional anions in [MgO](2+)[RaminePO3](2-) IOH-NPs, finally, show CO2 sorption (up to 180 mg g(-1)) and can be used for CO2/N2 separation (selectivity up to α = 23). This includes aminomethyl phosphonate [AMP](2-), 1-aminoethyl phosphonate [1AEP](2-), 2-aminoethyl phosphonate [2AEP](2-), aminopropyl phosphonate [APP](2-), and aminobutyl phosphonate [ABP](2-). All [M](2+)[Rfunction(O)PO3](2-) IOH-NPs are prepared via noncomplex synthesis in water, which facilitates practical handling and which is optimal for biomedical application. In sum, all IOH-NPs have very similar chemical compositions but can address a variety of different functions, including fluorescence, drug delivery, and CO2 sorption.

Construction of doxycycline-mediated BMP-2 transgene combining with APA microcapsules for bone repair.

PubMed

Qian, Dongyang; Bai, Bo; Yan, Guangbin; Zhang, Shujiang; Liu, Qi; Chen, Yi; Tan, Xiaobo; Zeng, Yanjun

2016-01-01

The repairing of large segmental bone defects is difficult for clinical orthopedists at present. Gene therapy is regarded as a promising method for bone defects repair. The present study aimed to construct an effective and controllable Tet-On expression system for transferring hBMP-2 gene into bone marrow mesenchymal progenitor cells (BMSCs). Meanwhile, with combination of alginate-poly-L-lysine-alginate (APA) microencapsulation technology, we attempted to reduce the influence of immunologic rejection and examine the effect of the Tet-On expression system on osteogenesis of BMSCs. The adenovirus encoding hBMP-2 (ADV-hBMP2) was constructed using the means of molecular cloning. The ADV-hBMP2 and Adeno-X Tet-On virus was respectively transfected to the HEK293 for amplification and afterward BMSCs were co-infected with the virus of ADV-hBMP2 and the Adeno-X Tet-On. The expression of hBMP-2 was measured with induction by doxycycline (DOX) at different concentration by means of RT-PCR and ELISA. Combining Tet-On expression system and APA microcapsules with the use of the pulsed high-voltage electrostatic microcapsule instrument, we examined the expression level of hBMP-2 in APA microcapsules by ELISA as well as the osteogenesis by alizarin red S staining. An effective Tet-On expression system for transferring hBMP-2 gene into BMSCs was constructed successfully. Also, the expression of hBMP-2 could be regulated by concentration of DOX. The data exhibited that BMSCs in APA microcapsules maintained the capability of proliferation and differentiation. The combination of Tet-On expression system and APA microcapsules could promote the osteogenesis of BMSCs. According to the results, microencapsulated Tet-On expression system showed the effective characteristics of secreting hBMP-2 and enhancing osteogenesis, which would provide a promising way for bone repair.

Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

PubMed

Karyagina, A S; Boksha, I S; Grunina, T M; Demidenko, A V; Poponova, M S; Sergienko, O V; Lyashchuk, A M; Galushkina, Z M; Soboleva, L A; Osidak, E O; Bartov, M S; Gromov, A V; Lunin, V G

2017-05-01

Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models.

Obese rats supplemented with bitter melon display marked shifts in the expression of genes controlling inflammatory response and lipid metabolism by RNA-Seq analysis of colonic mucosa.

PubMed

Bai, Juan; Zhu, Ying; Dong, Ying

2018-06-01

Obesity is known to induce pathological changes in the gut and diets rich in complex carbohydrates that resist digestion in the small bowel can alter large bowel ecology. The purposes of this study were to identify the effects of bitter melon powder (BMP) on the global gene expression pattern in the colon mucosa of obese rats. Obese rats were fed a high-fat diet and treated without or with BMP for 8 weeks. Genome-wide expression profiles of the colon mucosa were determined by RNA sequencing (RNA-Seq) analysis at the end of experiment. A total of 87 genes were identified as differentially expressed (DE) between these two groups (fold change > 1.2). These results were further validated by quantitative RT-PCR, confirming the high reliability of the RNA-Seq. Interestingly, DE genes implicated in inflammation and lipid metabolism were found to be downregulated by BMP in the colon. Network between genes and the top 15 KEGG pathways showed that PRKCβ (protein kinase C beta) and Pla2g2a (phospholipase A2 group IIA) strongly interacted with surrounding pathways and genes. Results revealed that BMP supplement could remodel key colon functions by altering transcriptomic profile in obese rats.

Effect of rhBMP-2 on tibial plateau fractures in a canine model.

PubMed

Schaefer, Susan L; Lu, Yan; Seeherman, Howard; Li, X Jian; Lopez, Mandi J; Markel, Mark D

2009-04-01

This study was to determine the efficacy of recombinant human bone morphogenetic protien-2 (rhBMP-2)/calcium phosphate matrix (CPX) paste to accelerate healing in a canine articular fracture model with associated subchondral defect. rhBMP-2/CPX (BMP), CPX alone (CPX) or autogenous bone graft (ABG) was administered to a canine articular tibial plateau osteotomy with a subchondral defect in each of 21 female dogs. The unoperated contralateral limbs served as controls. Ground reaction forces, synovial fluid, radiographic changes, mechanical testing, bone density, and histology of bone and synovium were analyzed at 6 weeks after surgery. Radiographic analysis demonstrated that the BMP and CPX groups showed improved bony healing compared to the ABG group at week 6. Histomorphometric analysis demonstrated that the BMP group had significantly increased trabecular bone volume compared to the CPX and ABG groups. Mechanical testing revealed that the BMP group had significantly greater maximum failure loads than the ABG group. Histological analysis demonstrated that the BMP group had significantly less sub-synovial inflammation than CPX group. This study demonstrated that rhBMP-2/CPX accelerated healing of articular fractures with subchondral defect compared to ABG in most of the parameters evaluated, and had less subsynovial inflammation than the CPX alone in a canine model.

Dkk1 haploinsufficiency requires expression of Bmp2 for bone anabolic activity.

PubMed

Intini, Giuseppe; Nyman, Jeffry S

2015-06-01

Bone fractures remain a serious health burden and prevention and enhanced healing of fractures have been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1(+/-)) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2(c/c);Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1(+/-) mice were crossed with Bmp2(c/c);Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy. Copyright © 2015 Elsevier Inc. All rights reserved.

Dkk1 haploinsufficiency requires expression of Bmp2 for bone anabolic activity

PubMed Central

Intini, Giuseppe; Nyman, Jeffry S.

2015-01-01

Bone fractures remain a serious health burden and prevention and enhanced healing of fractures has been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1+/−) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2c/c;Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1+/− mice were crossed with Bmp2c/c;Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy. PMID:25603465

Polydopamine mediated assembly of hydroxyapatite nanoparticles and bone morphogenetic protein-2 on magnesium alloys for enhanced corrosion resistance and bone regeneration.

PubMed

Jiang, Yanan; Wang, Bi; Jia, Zhanrong; Lu, Xiong; Fang, Liming; Wang, Kefeng; Ren, Fuzeng

2017-10-01

Magnesium alloys have the great potential to be used as orthopedic implants due to their biodegradability and mechanical resemblance to human cortical bone. However, the rapid degradation in physiological environment with the evolution of hydrogen gas release hinders their clinical applications. In this study, we developed a novel functional and biocompatible coating strategy through polydopamine mediated assembly of hydroxyapatite nanoparticles and growth factor, bone morphogenetic protein-2 (BMP-2), onto the surface of AZ31 Mg alloys. Such functional coating has strong bonding with the substrate and can increase surface hydrophilicity of magnesium alloys. In vitro electrochemical corrosion and hydrogen evolution tests demonstrate that the coating can significantly enhance the corrosion resistance and therefore slow down the degradation of AZ31 Mg alloys. In vitro cell culture reveals that immobilization of HA nanoparticles and BMP-2 can obviously promote cell adhesion and proliferation. Furthermore, in vivo implantation tests indicate that with the synergistic effects of HA nanoparticles and BMP-2, the coating does not cause obvious inflammatory response and can significantly reduce the biodegradation rate of the magnesium alloys and induce the new bone formation adjacent to the implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2750-2761, 2017. © 2017 Wiley Periodicals, Inc.

Expression characteristics of BMP2, BMPR-IA and Noggin in different stages of hair follicle in yak skin.

PubMed

Song, Liang-Li; Cui, Yan; Yu, Si-Jiu; Liu, Peng-Gang; Liu, Jun; Yang, Xue; He, Jun-Feng; Zhang, Qian

2018-05-01

Bone morphogenetic protein 2 (BMP2), BMP receptor-IA (BMPR-IA), and the BMP2 antagonist Noggin are important proteins involved in regulating the hair follicle (HF) cycle in skin. In order to explore the expression profiles of BMP2, BMPR-IA, and Noggin in the HF cycle of yak skin, we collected adult yak skin in the telogen, proanagen, and midanagen phases of HFs and evaluated gene and protein expression by real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. qRT-PCR and western blotting results showed that BMP2 and BMPR-IA expression levels were highest in the telogen of HFs and higher than that of Noggin in the same phase. The expression of Noggin was significantly higher in proanagen and midanagen phases of HFs than in the telogen phase, with the highest expression observed in the proanagen phase. Moreover, the expression of Noggin in the proanagen phase was significantly higher than those of BMP2 and BMPR-IA during the same phase. Immunohistochemistry results showed that BMP2, BMPR-IA, and Noggin were expressed in the skin epidermis, sweat glands, sebaceous glands, HF outer root sheath, and hair matrix. In summary, the characteristic expression profiles of BMP2, BMPR-IA, and Noggin suggested that BMP2 and BMPR-IA had inhibitory effects on the growth of HFs in yaks, whereas Noggin promoted the growth of yak HFs, mainly by affecting skin epithelial cell activity. These results provide a basis for further studies of HF development and cycle transition in yak skin. Copyright © 2017. Published by Elsevier Inc.

Combination of Root Surface Modification with BMP-2 and Collagen Hydrogel Scaffold Implantation for Periodontal Healing in Beagle Dogs

PubMed Central

Kato, Akihito; Miyaji, Hirofumi; Ishizuka, Ryosuke; Tokunaga, Keisuke; Inoue, Kana; Kosen, Yuta; Yokoyama, Hiroyuki; Sugaya, Tsutomu; Tanaka, Saori; Sakagami, Ryuji; Kawanami, Masamitsu

2015-01-01

Objective : Biomodification of the root surface plays a major role in periodontal wound healing. Root surface modification with bone morphogenetic protein (BMP) stimulates bone and cementum-like tissue formation; however, severe ankylosis is simultaneously observed. Bio-safe collagen hydrogel scaffolds may therefore be useful for supplying periodontal ligament cells and preventing ankylosis. We examined the effects of BMP modification in conjunction with collagen hydrogel scaffold implantation on periodontal wound healing in dogs. Material and Methods: The collagen hydrogel scaffold was composed of type I collagen sponge and collagen hydrogel. One-wall infrabony defects (5 mm in depth, 3 mm in width) were surgically created in six beagle dogs. In the BMP/Col group, BMP-2 was applied to the root surface (loading dose; 1 µg/µl), and the defects were filled with collagen hydrogel scaffold. In the BMP or Col group, BMP-2 coating or scaffold implantation was performed. Histometric parameters were evaluated at 4 weeks after surgery. Results: Single use of BMP stimulated formation of alveolar bone and ankylosis. In contrast, the BMP/Col group frequently enhanced reconstruction of periodontal attachment including cementum-like tissue, periodontal ligament and alveolar bone. The amount of new periodontal ligament in the BMP/Col group was significantly greater when compared to all other groups. In addition, ankylosis was rarely observed in the BMP/Col group. Conclusion: The combination method using root surface modification with BMP and collagen hydrogel scaffold implantation facilitated the reestablishment of periodontal attachment. BMP-related ankylosis was suppressed by implantation of collagen hydrogel. PMID:25674172

Evaluation of a thiolated chitosan scaffold for local delivery of BMP-2 for osteogenic differentiation and ectopic bone formation.

PubMed

Bae, In-Ho; Jeong, Byung-Chul; Kook, Min-Suk; Kim, Sun-Hun; Koh, Jeong-Tae

2013-01-01

Thiolated chitosan (Thio-CS) is a well-established pharmaceutical excipient for drug delivery. However, its use as a scaffold for bone formation has not been investigated. The aim of this study was to evaluate the potential of Thio-CS in bone morphogenetic protein-2 (BMP-2) delivery and bone formation. In vitro study showed that BMP-2 interacted with the Thio-CS and did not affect the swelling behavior. The release kinetics of BMP-2 from the Thio-CS was slightly delayed (70%) within 7 days compared with that from collagen gel (Col-gel, 85%), which is widely used in BMP-2 delivery. The BMP-2 released from Thio-CS increased osteoblastic cell differentiation but did not show any cytotoxicity until 21 days. Analysis of the in vivo ectopic bone formation at 4 weeks of posttransplantation showed that use of Thio-CS for BMP-2 delivery induced more bone formation to a greater extent (1.8 fold) than that of Col-gel. However, bone mineral density in both bones was equivalent, regardless of Thio-CS or Col-gel carrier. Taken together, Thio-CS system might be useful for delivering osteogenic protein BMP-2 and present a promising bone regeneration strategy.

DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts.

PubMed

Kanomata, Kazuhiro; Kokabu, Shoichiro; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

2009-06-01

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

Retention of 125I-labeled recombinant human bone morphogenetic protein-2 by biphasic calcium phosphate or a composite sponge in a rabbit posterolateral spine arthrodesis model.

PubMed

Louis-Ugbo, John; Kim, Hak-Sun; Boden, Scott D; Mayr, Matthew T; Li, Ronald C; Seeherman, Howard; D'Augusta, Darren; Blake, Cara; Jiao, Aiping; Peckham, Steve

2002-09-01

The purpose of this study was to characterize the retention kinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2) applied to two calcium-based delivery matrices. Biphasic calcium phosphate (BCP) and a composite containing BCP in an absorbable collagen sponge (BCP/ACS) were evaluated using a spinal fusion model in rabbits. rhBMP-2 labeled with radioactive iodine (125I) was used as a tracer to assess in vivo retention of rhBMP-2 in the presence of these materials (nine animals per material studied). Over a 36 day study period, animals were assessed for the following: percent administered dose retained at the implant site as measured by scintigraphic imaging (counting) with a gamma camera (all animals), radiography of the implant site (all animals), radioactivity in blood and plasma (all animals), and radioactivity in the urine and feces (three animals for each material). Radioactivity data were corrected for the decay of 125I and the attenuation between the implant in vivo and the gamma camera. Differences observed between the two materials for the area under the retention vs. time profile (AUC; 988%*day for BCP vs. 1070%*day for BCP/ACS, p = 0.57) and the mean residence time (MRT; 10.2 days for BCP vs. 7.6 days for BCP/ACS, p = 0.06) were not statistically significant. Initial retention/incorporation of rhBMP-2 was slightly higher for rhBMP-2/BCP/ACS than for rhBMP-2/BCP (96.8% vs. 86.0%, p < 0.05). Animals receiving rhBMP-2/BCP showed a longer terminal retention half-life (t1/2) than did those receiving rhBMP-2/BCP/ACS (7.5 vs. 4.5 days, p < 0.05). The urinary radioactivity recovery data supported the data obtained by scintigraphy. Over the 36 day collection period, essentially complete recovery of radioactivity (dose) in urine was observed for rhBMP-2/BCP and rhBMP-2/BCP/ACS and the majority of the radioactivity (approximately 95%) was soluble in trichloroacetic acid, suggesting extensive catabolism of rhBMP-2 before renal excretion. Fecal recovery of radioactivity was low, approximately 2-3%. In conclusion, rhBMP-2 was retained at the implant site when delivered with either BCP or BCP/ACS based on mean residence time and area under the retention curve vs. time profile. Use of these matrices resulted in detectable rhBMP-2 levels at the surgical site for over a week in contrast to data reported with several other matrices that lasted less time. Systemic catabolism and elimination of the rhBMP-2 was extensive and systemic presence of the protein was negligible.

Signaling by Bone Morphogenetic Proteins directs formation of an ectodermal signaling center that regulates craniofacial development

PubMed Central

Foppiano, Silvia; Hu, Diane; Marcucio, Ralph S.

2008-01-01

We previously described a signaling center, the Frontonasal Ectodermal Zone (FEZ) that regulates growth and patterning of the frontonasal process (FNP). The FEZ is comprised of FNP ectoderm flanking a boundary between Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. Our objective was to examine BMP signaling during formation of the FEZ. We blocked BMP signaling throughout the FNP prior to FEZ formation by infecting chick embryos at stage 10 (HH10) with a replication competent avian retrovirus encoding the BMP antagonist Noggin. We assessed gene expression patterns in the FNP 72 hours after infection (~HH22) and observed that Shh expression was reduced or absent. In the mesenchyme we observed that Bmp2 transcripts were absent while the Bmp4 expression domain was expanded proximally. In addition to the molecular changes, infected embryos also exhibited facial malformations at 72 and 96 hours after infection suggesting that the FEZ did not form. Our data indicate that reduced cell proliferation, but not apoptosis, in the mesenchyme contributed to the phenotype that we observed. Additionally, adding exogenous SHH into the mesenchyme of RCAS-Noggin infected embryos did not restore Bmp2 and Bmp4 to a normal pattern of expression. These data indicate that BMP signaling mediates interactions between tissues in the FNP that regulate FEZ formation; and that the correct pattern of Bmp2 and Bmp4, but not Bmp7, expression in the FNP mesenchyme requires signaling by the BMP pathway. PMID:18028903

Microcontact printing of BMP-2 and its effect on human chondrocytes behavior

NASA Astrophysics Data System (ADS)

Pan, Chang-Jiang; Nie, Yu-Dong

2010-01-01

The present study is to investigate human chondrocytes behavior on microcontact printed bone morphogenetic protein-2 (BMP-2) lines on polystyrene (PS) surface. It was found that the cells aligned with BMP lines and expressed type II and VI collagen. The chondrocytes in vitro cultured on BMP lines were elongated, which resulted in altered cell morphology. Taking all these results into consideration, BMP-2 lines enhance cell adhesion, restrict spreading, and increase type II and VI collagen expression. The results represented in this study may be an approach to the problem of engineering reparative cartilage in vitro.

BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis

PubMed Central

Jiang, Ming; Ku, Wei-Yao; Zhou, Zhongren; Dellon, Evan S.; Falk, Gary W.; Nakagawa, Hiroshi; Wang, Mei-Lun; Liu, Kuancan; Wang, Jun; Katzka, David A.; Peters, Jeffrey H.; Lan, Xiaopeng; Que, Jianwen

2015-01-01

Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barrett’s intestinal differentiation; however, in mice, basal progenitor cell–specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE. PMID:25774506

Biphasic effects of FGF2 on odontoblast differentiation involve changes in the BMP and Wnt signaling pathways.

PubMed

Sagomonyants, Karen; Mina, Mina

2014-08-01

Odontoblast differentiation during physiological and reparative dentinogenesis is dependent upon multiple signaling molecules, including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs) and Wingless/Integrated (Wnt) ligands. Recent studies in our laboratory showed that continuous exposure of primary dental pulp cultures to FGF2 exerted biphasic effects on the expression of markers of dentinogenesis. In the present study, we examined the possible involvement of the BMP and Wnt signaling pathways in mediating the effects of FGF2 on dental pulp cells. Our results showed that stimulatory effects of FGF2 on dentinogenesis during the proliferation phase of growth were associated with increased expression of the components of the BMP (Bmp2, Dlx5, Msx2, Osx) and Wnt (Wnt10a, Wisp2) pathways, and decreased expression of an inhibitor of the Wnt signaling, Nkd2. Further addition of FGF2 during the differentiation/mineralization phase of growth resulted in decreased expression of components of the BMP signaling (Bmp2, Runx2, Osx) and increased expression of inhibitors of the Wnt signaling (Nkd2, Dkk3). This suggests that both BMP and Wnt pathways may be involved in mediating the effects of FGF2 on dental pulp cells.

Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro

PubMed Central

Garrett, I.R.; Chen, D.; Gutierrez, G.; Zhao, M.; Escobedo, A.; Rossini, G.; Harris, S.E.; Gallwitz, W.; Kim, K.B.; Hu, S.; Crews, C.M.; Mundy, G.R.

2003-01-01

We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic β subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor–1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss. PMID:12782679

A living thick nanofibrous implant bifunctionalized with active growth factor and stem cells for bone regeneration.

PubMed

Eap, Sandy; Keller, Laetitia; Schiavi, Jessica; Huck, Olivier; Jacomine, Leandro; Fioretti, Florence; Gauthier, Christian; Sebastian, Victor; Schwinté, Pascale; Benkirane-Jessel, Nadia

2015-01-01

New-generation implants focus on robust, durable, and rapid tissue regeneration to shorten recovery times and decrease risks of postoperative complications for patients. Herein, we describe a new-generation thick nanofibrous implant functionalized with active containers of growth factors and stem cells for regenerative nanomedicine. A thick electrospun poly(ε-caprolactone) nanofibrous implant (from 700 μm to 1 cm thick) was functionalized with chitosan and bone morphogenetic protein BMP-7 as growth factor using layer-by-layer technology, producing fish scale-like chitosan/BMP-7 nanoreservoirs. This extracellular matrix-mimicking scaffold enabled in vitro colonization and bone regeneration by human primary osteoblasts, as shown by expression of osteocalcin, osteopontin, and bone sialoprotein (BSPII), 21 days after seeding. In vivo implantation in mouse calvaria defects showed significantly more newly mineralized extracellular matrix in the functionalized implant compared to a bare scaffold after 30 days' implantation, as shown by histological scanning electron microscopy/energy dispersive X-ray microscopy study and calcein injection. We have as well bifunctionalized our BMP-7 therapeutic implant by adding human mesenchymal stem cells (hMSCs). The activity of this BMP-7-functionalized implant was again further enhanced by the addition of hMSCs to the implant (living materials), in vivo, as demonstrated by the analysis of new bone formation and calcification after 30 days' implantation in mice with calvaria defects. Therefore, implants functionalized with BMP-7 nanocontainers associated with hMSCs can act as an accelerator of in vivo bone mineralization and regeneration.

Osteoblastic differentiation of human and equine adult bone marrow-derived mesenchymal stem cells when BMP-2 or BMP-7 homodimer genetic modification is compared to BMP-2/7 heterodimer genetic modification in the presence and absence of dexamethasone.

PubMed

Carpenter, Ryan S; Goodrich, Laurie R; Frisbie, David D; Kisiday, John D; Carbone, Beth; McIlwraith, C Wayne; Centeno, Christopher J; Hidaka, Chisa

2010-10-01

Bone marrow-derived mesenchymal stem cells (BMDMSCs) have been targeted for use in enhancement of bone healing; and their osteogenic potential may be further augmented by genes encoding bone morphogenetic proteins (BMP's). The purpose of this study was to compare the effect of genetic modification of human and equine BMDMSCs with BMP-2 or -7 or BMP-2 and -7 on their osteoblastogenic differentiation in the presence or absence of dexamethasone. The BMDMSCs were harvested from the iliac crest of three human donors and tuber coxae of three equine donors. Monolayer cells were genetically modified using adenovirus vectors encoding BMP-2, -7 or both and cultured in the presence or absence of dexamethasone. Expression of BMPs was confirmed by enzyme linked immunosorbent assay (ELISA). To evaluate osteoblastic differentiation, cellular morphology was assessed every other day and expression and secretion of alkaline phosphatase (ALP), as well as expression levels of osteonectin (OSTN), osteocalcin (OCN), and runt-related transcription factor-2 (Runx2) were measured for up to 14 days. Human and equine BMDMSCs showed a capacity for osteogenic differentiation regardless of genetic modification or dexamethasone supplementation. Dexamethasone supplementation was more important for osteoblastogenic differentiation of equine BMDMSCs than human BMDMSCs. Genetic modification of BMDMSCs increased ALP secretion with AdBMP-2 homodimer having the greatest effect in both human and equine cells compared to AdBMP 7 or AdBMP 2/7. BMP protein elution rates reached their maximal concentration between day 4 and 8 and remained relatively stable thereafter, suggesting that genetically modified BMDMSCs could be useful for cell-based delivery of BMPs to a site of bone formation. Published by Wiley Periodicals, Inc. J Orthop Res 28:1330-1337, 2010.

A peptide that blocks the interaction of NF-κB p65 subunit with Smad4 enhances BMP2-induced osteogenesis.

PubMed

Urata, Mariko; Kokabu, Shoichiro; Matsubara, Takuma; Sugiyama, Goro; Nakatomi, Chihiro; Takeuchi, Hiroshi; Hirata-Tsuchiya, Shizu; Aoki, Kazuhiro; Tamura, Yukihiko; Moriyama, Yasuko; Ayukawa, Yasunori; Matsuda, Miho; Zhang, Min; Koyano, Kiyoshi; Kitamura, Chiaki; Jimi, Eijiro

2018-09-01

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity. © 2018 Wiley Periodicals, Inc.

Enhancement of Tendon–Bone Healing for Anterior Cruciate Ligament (ACL) Reconstruction Using Bone Marrow-Derived Mesenchymal Stem Cells Infected with BMP-2

PubMed Central

Dong, Yu; Zhang, Qingguo; Li, Yunxia; Jiang, Jia; Chen, Shiyi

2012-01-01

At present, due to the growing attention focused on the issue of tendon–bone healing, we carried out an animal study of the use of genetic intervention combined with cell transplantation for the promotion of this process. Here, the efficacy of bone marrow stromal cells infected with bone morphogenetic protein-2 (BMP-2) on tendon–bone healing was determined. A eukaryotic expression vector containing the BMP-2 gene was constructed and bone marrow-derived mesenchymal stem cells (bMSCs) were infected with a lentivirus. Next, we examined the viability of the infected cells and the mRNA and protein levels of BMP-2-infected bMSCs. Gastrocnemius tendons, gastrocnemius tendons wrapped by bMSCs infected with the control virus (bMSCs+Lv-Control), and gastrocnemius tendons wrapped by bMSCs infected with the recombinant BMP-2 virus (bMSCs+Lv-BMP-2) were used to reconstruct the anterior cruciate ligament (ACL) in New Zealand white rabbits. Specimens from each group were harvested four and eight weeks postoperatively and evaluated using biomechanical and histological methods. The bMSCs were infected with the lentivirus at an efficiency close to 100%. The BMP-2 mRNA and protein levels in bMSCs were significantly increased after lentiviral infection. The bMSCs and BMP-2-infected bMSCs on the gastrocnemius tendon improved the biomechanical properties of the graft in the bone tunnel; specifically, bMSCs infected with BMP-2 had a positive effect on tendon–bone healing. In the four-week and eight-week groups, bMSCs+Lv-BMP-2 group exhibited significantly higher maximum loads of 29.3 ± 7.4 N and 45.5 ± 11.9 N, respectively, compared with the control group (19.9 ± 6.4 N and 21.9 ± 4.9 N) (P = 0.041 and P = 0.001, respectively). In the eight-week groups, the stiffness of the bMSCs+Lv-BMP-2 group (32.5 ± 7.3) was significantly higher than that of the bMSCs+Lv-Control group (22.8 ± 7.4) or control groups (12.4 ± 6.0) (p = 0.036 and 0.001, respectively). Based on the histological findings, there was an increased amount of perpendicular collagen fibers formed between the tendon and bone in the bMSCs+Lv-Control and bMSCs+Lv-BMP-2 group, compared with the gastrocnemius tendons. The proliferation of cartilage-like cells and the formation of fibrocartilage-like tissue were highest within the bone tunnels in the bMSCs+Lv-BMP-2 group. These results suggest that this lentivirus can be used to efficiently infect bMSCs with BMP-2. Furthermore, tendons wrapped by bMSCs+Lv-BMP-2 improved tendon–bone healing. PMID:23202970

Bone Regeneration Using N-Methyl-2-pyrrolidone as an Enhancer for Recombinant Human Bone Morphogenetic Protein-2 in a Rabbit Sinus Augmentation Model

PubMed Central

Thoma, Daniel S.

2017-01-01

The aim of this study was to determine whether N-methyl-2-pyrrolidone (NMP) can decrease the dose of recombinant human bone morphogenetic protein-2 (rhBMP-2) in sinus augmentation of rabbits. In each of 15 rabbits, 2 sinuses were randomly grafted using 1 of 3 treatment modalities: (i) biphasic calcium phosphate (BCP; control), (ii) rhBMP-2-coated BCP (BMP), or (iii) rhBMP-2-coated BCP soaked in NMP solution (BMP/NMP). The rabbits were sacrificed 2 weeks postoperatively. Histologic and histomorphometric analyses were performed. Bone formation in all groups was predominantly located close to the access window and the lateral walls. Newly formed bone within the total augmented area (NBTA) was greatest in BMP/NMP (1.94 ± 0.69 mm2), followed by BMP (1.50 ± 0.72 mm2) and BCP (1.28 ± 0.52 mm2) (P > 0.05). In the center of the augmentation (NBROI_C) and the area close to the sinus membrane (NBROI_M), BMP/NMP produced the largest area of NB (NBROI_C: 0.10 ± 0.11 mm2; NBROI_M: 0.17 ± 0.08 mm2); the corresponding NB values for BCP were 0.05 ± 0.05 mm2 and 0.08 ± 0.09 mm2, respectively (P > 0.05 for all comparisons). The effect of NMP on bone regeneration was inconsistent between the specimens. Adding NMP as an adjunct to rhBMP-2-coated BCP produced inconsistent effects on bone regeneration, resulting in no significant benefit compared to controls. PMID:28680881

Bone Regeneration Using N-Methyl-2-pyrrolidone as an Enhancer for Recombinant Human Bone Morphogenetic Protein-2 in a Rabbit Sinus Augmentation Model.

PubMed

Lim, Hyun-Chang; Thoma, Daniel S; Yoon, So-Ra; Cha, Jae-Kook; Lee, Jung-Seok; Jung, Ui-Won

2017-01-01

The aim of this study was to determine whether N-methyl-2-pyrrolidone (NMP) can decrease the dose of recombinant human bone morphogenetic protein-2 (rhBMP-2) in sinus augmentation of rabbits. In each of 15 rabbits, 2 sinuses were randomly grafted using 1 of 3 treatment modalities: (i) biphasic calcium phosphate (BCP; control), (ii) rhBMP-2-coated BCP (BMP), or (iii) rhBMP-2-coated BCP soaked in NMP solution (BMP/NMP). The rabbits were sacrificed 2 weeks postoperatively. Histologic and histomorphometric analyses were performed. Bone formation in all groups was predominantly located close to the access window and the lateral walls. Newly formed bone within the total augmented area (NB TA ) was greatest in BMP/NMP (1.94 ± 0.69 mm 2 ), followed by BMP (1.50 ± 0.72 mm 2 ) and BCP (1.28 ± 0.52 mm 2 ) ( P > 0.05). In the center of the augmentation (NB ROI_C ) and the area close to the sinus membrane (NB ROI_M ), BMP/NMP produced the largest area of NB (NB ROI_C : 0.10 ± 0.11 mm 2 ; NB ROI_M : 0.17 ± 0.08 mm 2 ); the corresponding NB values for BCP were 0.05 ± 0.05 mm 2 and 0.08 ± 0.09 mm 2 , respectively ( P > 0.05 for all comparisons). The effect of NMP on bone regeneration was inconsistent between the specimens. Adding NMP as an adjunct to rhBMP-2-coated BCP produced inconsistent effects on bone regeneration, resulting in no significant benefit compared to controls.

Differing impact of the deletion of hemochromatosis-associated molecules HFE and transferrin receptor-2 on the iron phenotype of mice lacking bone morphogenetic protein 6 or hemojuvelin.

PubMed

Latour, Chloé; Besson-Fournier, Céline; Meynard, Delphine; Silvestri, Laura; Gourbeyre, Ophélie; Aguilar-Martinez, Patricia; Schmidt, Paul J; Fleming, Mark D; Roth, Marie-Paule; Coppin, Hélène

2016-01-01

Hereditary hemochromatosis, which is characterized by inappropriately low levels of hepcidin, increased dietary iron uptake, and systemic iron accumulation, has been associated with mutations in the HFE, transferrin receptor-2 (TfR2), and hemojuvelin (HJV) genes. However, it is still not clear whether these molecules intersect in vivo with bone morphogenetic protein 6 (BMP6)/mothers against decapentaplegic (SMAD) homolog signaling, the main pathway up-regulating hepcidin expression in response to elevated hepatic iron. To answer this question, we produced double knockout mice for Bmp6 and β2-microglobulin (a surrogate for the loss of Hfe) and for Bmp6 and Tfr2, and we compared their phenotype (hepcidin expression, Bmp/Smad signaling, hepatic and extrahepatic tissue iron accumulation) with that of single Bmp6-deficient mice and that of mice deficient for Hjv, alone or in combination with Hfe or Tfr2. Whereas the phenotype of Hjv-deficient females was not affected by loss of Hfe or Tfr2, that of Bmp6-deficient females was considerably worsened, with decreased Smad5 phosphorylation, compared with single Bmp6-deficient mice, further repression of hepcidin gene expression, undetectable serum hepcidin, and massive iron accumulation not only in the liver but also in the pancreas, the heart, and the kidneys. These results show that (1) BMP6 does not require HJV to transduce signal to hepcidin in response to intracellular iron, even if the loss of HJV partly reduces this signal, (2) another BMP ligand can replace BMP6 and significantly induce hepcidin expression in response to extracellular iron, and (3) BMP6 alone is as efficient at inducing hepcidin as the other BMPs in association with the HJV/HFE/TfR2 complex; they provide an explanation for the compensatory effect of BMP6 treatment on the molecular defect underlying Hfe hemochromatosis in mice. © 2015 by the American Association for the Study of Liver Diseases.

Leiomyoma-derived transforming growth factor-β impairs bone morphogenetic protein-2-mediated endometrial receptivity.

PubMed

Doherty, Leo F; Taylor, Hugh S

2015-03-01

To determine whether transforming growth factor (TGF)-β3 is a paracrine signal secreted by leiomyoma that inhibits bone morphogenetic protein (BMP)-mediated endometrial receptivity and decidualization. Experimental. Laboratory. Women with symptomatic leiomyomas. Endometrial stromal cells (ESCs) and leiomyoma cells were isolated from surgical specimens. Leiomyoma-conditioned media (LCM) was applied to cultured ESC. The TGF-β was blocked by two approaches: TGF-β pan-specific antibody or transfection with a mutant TGF-β receptor type II. Cells were then treated with recombinant human BMP-2 to assess BMP responsiveness. Expression of BMP receptor types 1A, 1B, 2, as well as endometrial receptivity mediators HOXA10 and leukemia inhibitory factor (LIF). Enzyme-linked immunosorbent assay showed elevated TGF-β levels in LCM. LCM treatment of ESC reduced expression of BMP receptor types 1B and 2 to approximately 60% of pretreatment levels. Preincubation of LCM with TGF-β neutralizing antibody or mutant TGF receptor, but not respective controls, prevented repression of BMP receptors. HOXA10 and LIF expression was repressed in recombinant human BMP-2 treated, LCM exposed ESC. Pretreatment of LCM with TGF-β antibody or transfection with mutant TGF receptor prevented HOXA10 and LIF repression. Leiomyoma-derived TGF-β was necessary and sufficient to alter endometrial BMP-2 responsiveness. Blockade of TGF-β prevents repression of BMP-2 receptors and restores BMP-2-stimulated expression of HOXA10 and LIF. Blockade of TGF signaling is a potential strategy to improve infertility and pregnancy loss associated with uterine leiomyoma. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effect of Emdogain enamel matrix derivative and BMP-2 on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells.

PubMed

Fawzy El-Sayed, Karim M; Dörfer, Christof; Ungefroren, Hendrick; Kassem, Neemat; Wiltfang, Jörg; Paris, Sebastian

2014-07-01

The objective of this study was to evaluate the effect of Emdogain (Enamel Matrix Derivative, EMD) and Bone Morphogenetic Protein-2 (BMP-2), either solely or in combination, on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells. Stem/progenitor cells were isolated from human alveolar bone proper, magnetically sorted using STRO-1 antibodies, characterized flowcytometrically for their surface markers' expression, and examined for colony formation and multilineage differentiation potential. Subsequently, cells were treated over three weeks with 100 μg/ml Emdogain (EMD-Group), or 100 ng/ml BMP-2 (BMP-Group), or a combination of 100 ng/ml BMP-2 and 100 μg/ml Emdogain (BMP/EMD-Group). Unstimulated stem/progenitor cells (MACS(+)-Group) and osteoblasts (OB-Group) served as controls. Osteogenic gene expression was analyzed using RTq-PCR after 1, 2 and 3 weeks (N = 3/group). Mineralized nodule formation was evaluated by Alizarin-Red staining. BMP and EMD up-regulated the osteogenic gene expression. The BMP Group showed significantly higher expression of Collagen-I, III, and V, Alkaline phosphatase and Osteonectin compared to MACS(+)- and OB-Group (p < 0.05; Two-way ANOVA/Bonferroni) with no mineralized nodule formation. Under in-vitro conditions, Emdogain and BMP-2 up-regulate the osteogenic gene expression of stem/progenitor cells. The combination of BMP-2 and Emdogain showed no additive effect and would not be recommended for a combined clinical stimulation. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects.

PubMed

Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

2015-01-01

The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m(2)/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration.

Bone morphogenetic protein 2 and decorin expression in old fracture fragments and surrounding tissues.

PubMed

Han, X G; Wang, D K; Gao, F; Liu, R H; Bi, Z G

2015-09-21

Bone morphogenetic protein 2 (BMP-2) can promote fracture healing. Although the complex role BMP-2 in bone formation is increasingly understood, the role of endogenous BMP-2 in nonunion remains unclear. Decorin (DCN) can promote the formation of bone matrix and calcium deposition to control bone morphogenesis. In this study, tissue composition and expression of BMP-2 and DCN were detected in different parts of old fracture zones to explore inherent anti-fibrotic ability and osteogenesis. Twenty-three patients were selected, including eight cases of delayed union and 15 cases of nonunion. Average duration of delayed union or nonunion was 15 months. Fracture fragments and surrounding tissues, including bone grafts, marrow cavity contents, and sticking scars, were categorically sampled during surgery. Through observation and histological testing, component comparisons were made between fracture fragments and surrounding tissue. The expression levels of DCN and BMP-2 in different tissues were detected by immunohistochemical staining and real-time polymerase chain reaction. The expression of DCN and BMP- 2 in different parts of the nonunion area showed that, compared with bone graft and marrow cavity contents, sticking scars had the highest expression of BMP-2. Compared with the marrow cavity contents and sticking scars, bone grafts had the highest expression of DCN. The low antifibrotic and osteogenic activity of the nonunion area was associated with non-co-expression of BMP-2 and DCN. Therefore, the co-injection of osteogenic factor BMP and DCN into the nonunion area can improve the induction of bone formation and enhance the conversion of the old scar, thereby achieving better nonunion treatment.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Li-An; Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an; Yuan, Guohua

Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show thatmore » transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.« less

Evaluation of rhBMP-2/collagen/TCP-HA bone graft with and without bone marrow cells in the canine femoral multi defect model.

PubMed

Luangphakdy, V; Shinohara, K; Pan, H; Boehm, C; Samaranska, A; Muschler, G F

2015-01-12

Recombinant human bone morphogenetic protein-2, when applied to an absorbable type 1 bovine collagen sponge (rhBMP-2/ACS) is an effective therapy in many bone grafting settings. Bone marrow aspirate (BMA) has also been used as a source of transplantable osteogenic connective tissue progenitors. This study was designed to characterize the performance of a scaffold comprising rhBMP-2/ACS in which the sponge wraps around tri-calcium phosphate hydroxyapatite granules (rhBMP-2/ACS/TCP-HA) and to test the hypothesis that addition of BMA will improve the performance of this construct in the Canine Femoral Multi Defect Model. In each subject, two sites were grafted with rhBMP-2/ACS/TCP-HA scaffold loaded with BMA clot and two other sites with rhBMP-2/ACS/TCP-HA scaffold loaded with wound blood (WB). After correction for unresorbed TCP-HA granules, sites grafted with rhBMP-2/ACS/TCP-HA+BMA and rhBMP-2/ACS/TCP-HA+WB were similar, with mean percent bone volumes of 10.9 %±1.2 and 11.2 %±1.2, respectively. No differences were seen in quantitative histomorphometry. While bone formation using both constructs was robust, this study did not support the hypothesis that the addition of unprocessed bone marrow aspirate clot improved bone regeneration in a site engrafted with rhBMP-2/ACS/TCP-HA+BMA. In contrast to prior studies using this model, new bone formation was greater at the center of the defect where TCP-HA was distributed. This finding suggests a potential synergy between rhBMP-2 and the centrally placed ceramic and cellular components of the graft construct. Further optimization may also require more uniform distribution of TCP-HA, alternative cell delivery strategies, and a more rigorous large animal segmental defect model.

Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis

PubMed Central

Xiao, Changchun; Shim, Jae-hyuck; Klüppel, Michael; Zhang, Samuel Shao-Min; Dong, Chen; Flavell, Richard A.; Fu, Xin-Yuan; Wrana, Jeffrey L.; Hogan, Brigid L.M.; Ghosh, Sankar

2003-01-01

Bone morphogenetic proteins (Bmps) are members of the transforming growth factor β (TGFβ) superfamily that play critical roles during mouse embryogenesis. Signaling by Bmp receptors is mediated mainly by Smad proteins. In this study, we show that a targeted null mutation of Ecsit, encoding a signaling intermediate of the Toll pathway, leads to reduced cell proliferation, altered epiblast patterning, impairment of mesoderm formation, and embryonic lethality at embryonic day 7.5 (E7.5), phenotypes that mimic the Bmp receptor type1a (Bmpr1a) null mutant. In addition, specific Bmp target gene expression is abolished in the absence of Ecsit. Biochemical analysis demonstrates that Ecsit associates constitutively with Smad4 and associates with Smad1 in a Bmp-inducible manner. Together with Smad1 and Smad4, Ecsit binds to the promoter of specific Bmp target genes. Finally, knock-down of Ecsit with Ecsit-specific short hairpin RNA inhibits both Bmp and Toll signaling. Therefore, these results show that Ecsit functions as an essential component in two important signal transduction pathways and establishes a novel role for Ecsit as a cofactor for Smad proteins in the Bmp signaling pathway. PMID:14633973

[A novel tissue-engineered bone constructed by using human adipose-derived stem cells and biomimetic calcium phosphate scaffold coprecipitated with bone morphogenetic protein-2].

PubMed

Jiang, W R; Zhang, X; Liu, Y S; Wu, G; Ge, Y J; Zhou, Y S

2017-02-18

To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2), and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo. The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study. Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM), respectively. The supernatants were collected and used to culture hASCs in vitro. Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 14 and 21 days, the calcification deposition was determined by alizarin red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study, 6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups: (1) BioCaP scaffold only, (2) BioCaP scaffold+hASCs, (3) BMP-2-BioCaP scaffold, (4) BMP-2-BioCaP scaffold+hASCs (test group). After 4 weeks of implantation, hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs. SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight, plate-like and sharp-edged crystal units, and the length of the crystal units varied between 5 and 10 μm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days, and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2-BioCaP. ALP activity was higher by the induction of OM+BMP-2-BioCaP than of the other groups (P<0.01). More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (OPN) and osteocalcin (OC) were determined in the OM+BMP-2-BioCaP group at different time points (P<0.01). HE staining showed that, in the test group and BMP-2-BioCaP scaffold group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the BMP-2-BioCaP scaffold group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups. No obvious positive results were found in the other groups. BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo. The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

The nursing management of diarrhoea and constipation before and after the implementation of a bowel management protocol.

PubMed

McKenna, S; Wallis, M; Brannelly, A; Cawood, J

2001-02-01

Intensive care unit (ICU) patients frequently suffer problems associated with both diarrhoea and constipation. Strategies to optimise the management of these conditions need to focus on improving the communication between staff and ensuring effective treatment is implemented. The team involved in this study developed a Bowel Management Protocol (BMP). The effect of this BMP on the documentation of assessment and management of diarrhoea and constipation was evaluated using a quasi-experimental research design. Data were collected via a retrospective audit of medical records. Two groups of patient records were randomly sampled. The records of 60 patients who were admitted to ICU in the 6 months before the introduction of the BMP were accessed together with the records of 60 patients admitted in the 6 months following the introduction of the BMP. Data were collected regarding patient demographics and the assessment and management of bowel function before and after BMP introduction. The results indicated that a BMP improved documentation of the assessment of bowel function. In addition, there was an improvement in the documentation of nursing intervention in the presence of constipation and diarrhoea. These results have to be interpreted with caution because, despite random sampling over two 6 month periods, there were statistically significant differences in age, length of stay, method of feeding and medical diagnosis between the two groups. Further research into the effectiveness of using a BMP is recommended.

Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3.

PubMed

Dwivedi, Prem P; Grose, Randall H; Filmus, Jorge; Hii, Charles S T; Xian, Cory J; Anderson, Peter J; Powell, Barry C

2013-08-01

From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis. Copyright © 2013 Elsevier Inc. All rights reserved.

Suppression of BMP-7 by histone deacetylase 2 promoted apoptosis of renal tubular epithelial cells in acute kidney injury

PubMed Central

Ma, Taotao; Huang, Cheng; Xu, Qingqing; Yang, Yang; Liu, Yaru; Meng, Xiaoming; Li, Jun; Ye, Min; Liang, Hong

2017-01-01

Cisplatin, a highly effective and widely used chemotherapeutic agent, has a major limitation for its nephrotoxicity. Currently, there are no therapies available to treat or prevent cisplatin nephrotoxicity. We recently identified a novel strategy for attenuating its nephrotoxicity in chemotherapy by histone deacetylase (HDAC) inhibitors via epigenetic modification to enhance bone morphogenetic protein 7 (BMP-7) expression. Cisplatin upregulated the activity of HDAC2 in the kidney. Inhibition of HDAC with clinically used trichostatin A (TSA) or valproic acid (VPA) suppressed cisplatin-induced kidney injury and epithelial cell apoptosis. Overexpression of HDAC2 promotes CP-treated tubular epithelium cells apoptosis. Chromatin immunoprecipitation assay clearly detected HDAC2 assosiation with BMP-7 promoter. Western blot and immunofluorescence results demonstrated that the expression of BMP-7 was clearly induced by TSA or VPA in vivo and in vitro. Interestingly, administration of recombinant BMP-7 (rhBMP-7) reduced cisplatin-induced kidney dysfunction. Moreover, BMP-7 treatment suppressed epithelial cell apoptosis and small interfering RNA-based knockdown of BMP-7 expression abolished HDAC inhibitors suppression of epithelial cell apoptosis in vitro. Results of current study indicated that TSA or VPA inhibited apoptosis of renal tubular epithelial cells via promoting the level of BMP-7 epigenetically through targeting HDAC2. Hence, HDAC inhibitors could be useful therapeutic agents for the prevention of cisplatin nephrotoxicity. PMID:29072686

Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

PubMed Central

Yuasa, Masato; Yamada, Tsuyoshi; Taniyama, Takashi; Masaoka, Tomokazu; Xuetao, Wei; Yoshii, Toshitaka; Horie, Masaki; Yasuda, Hiroaki; Uemura, Toshimasa; Okawa, Atsushi; Sotome, Shinichi

2015-01-01

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2. PMID:25659106

Tridax procumbens flavonoids promote osteoblast differentiation and bone formation.

PubMed

Al Mamun, Md Abdullah; Hosen, Mohammad Jakir; Islam, Kamrul; Khatun, Amina; Alam, M Masihul; Al-Bari, Md Abdul Alim

2015-11-18

Tridax procumbens flavonoids (TPFs) are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts. TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs) including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2. Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Posterolateral lumbar fusion using Escherichia coli-derived rhBMP-2/hydroxyapatite in the mini pig.

PubMed

Kong, Chang-Bae; Lee, Jae Hyup; Baek, Hae-Ri; Lee, Choon-Ki; Chang, Bong-Soon

2014-12-01

Hydroxyapatite (HA) is used as a bone graft extender for posterolateral spinal fusion in human. It is also useful as a recombinant human bone morphogenetic protein (rhBMP)-2 carrier because of its high affinity for rhBMP-2. To assess the osteoinductivity of Escherichia coli-derived rhBMP-2 (E-BMP-2) using HA granules as a carrier and to evaluate the bone-forming ability depending on the different dosages of E-BMP-2. A mini-pig lumbar posterolateral fusion model using microcomputed tomography (μCT) scanning. Thirty-one adult male mini pigs were randomized into a single control group (n=8) without E-BMP-2 and two experimental groups with two different doses of E-BMP-2 (1 mg per side, n=8 and 3 mg per side, n=15). Outcome was measured by plain radiography, manual palpation, CT, three-dimensional μCT, and histologic examinations. Bilateral intertransverse process arthrodesis was performed, and E-BMP-2 (0, 1.0, 3.0 mg per side) was implanted into the intertransverse space using HA granules as a carrier. Three mini pigs were removed because of death. Among 28 experimental subjects, 19 animals achieved solid bony union. The fusion rates were 37.5% for control group, 71.4% for 1 mg group, and 84.6% for 3 mg group. Fusion rates were significantly different among groups (p=.031). However, there was no statistically significant difference in fusion rates between 1 and 3 mg groups (p=.587). Thirty-eight intertransverse fusion masses of 19 subjects underwent μCT scanning. The bone volumes determined by μCT were 12,603±3,240 mm(3) for control group, 18,718±3,000 mm(3) for 1 mg group, and 26,768±7,256 mm(3) for 3 mg group, and the difference between groups was statistically significant (p<.001). This study shows that E-BMP-2 has osteoinductive activity in dose-dependent fashion, and porous HA granule is suitable for E-BMP-2 carrier in a porcine posterolateral fusion model. These preliminary findings suggest that E-BMP-2-adsorbed porous HA granules could be a novel effective bone graft substitute. Copyright © 2014 Elsevier Inc. All rights reserved.

Trends Analysis of rhBMP2 Utilization in Single-Level Anterior Lumbar Interbody Fusion in the United States

PubMed Central

Lao, Lifeng; Cohen, Jeremiah R.; Buser, Zorica; Brodke, Darrel S.; Yoon, S. Tim; Youssef, Jim A.; Park, Jong-Beom; Meisel, Hans-Joerg; Wang, Jeffrey C.

2017-01-01

Study Design: Retrospective case study. Objective: To evaluate the trends and demographics of recombinant human bone morphogenetic protein 2 (rhBMP2) utilization in single-level anterior lumbar interbody fusion (ALIF) in the United States. Methods: Patients who underwent single-level ALIF from 2005 to 2011 were identified by searching ICD-9 diagnosis and procedure codes in the PearlDiver Patient Records Database (PearlDiver Technologies, Fort Wayne, IN), a national database of orthopedic insurance records. The year of procedure, age, gender, and region of the United States were analyzed for each patient. Results: A total of 921 patients were identified who underwent a single-level ALIF in this study. The average rate of single-level ALIF with rhBMP2 utilization increased (35%-48%) from 2005 to 2009, but sharply decreased to 16.7% in 2010 and 15.0% in 2011. The overall incidence of single-level ALIF without rhBMP2 (0.20 cases per 100 000 patients) was more than twice of the incidence of single-level ALIF with rhBMP2 (0.09 cases per 100 000 patients). The average rate of single-level ALIF with rhBMP2 utilization is highest in West (41.4%), followed by Midwest (33.3%), South (26.5%) and Northeast (22.2%). The highest incidence of single-level ALIF with rhBMP2 was observed in the group aged less than 65 years (compared with any other age groups, P < .001), with an incidence of 0.21 per 100 000 patients. Conclusions: The incidence of rhBMP2 utilization in single-level ALIF increased from 2006 to 2009, but decreased in 2010 and 2011. The Northeast region had the lowest incidence of rhBMP2 utilization. The group aged less than 65 years trended to have the higher incidence of single-level ALIF with rhBMP2 utilization. PMID:29662743

Trends Analysis of rhBMP2 Utilization in Single-Level Anterior Lumbar Interbody Fusion in the United States.

PubMed

Lao, Lifeng; Cohen, Jeremiah R; Buser, Zorica; Brodke, Darrel S; Yoon, S Tim; Youssef, Jim A; Park, Jong-Beom; Meisel, Hans-Joerg; Wang, Jeffrey C

2018-04-01

Retrospective case study. To evaluate the trends and demographics of recombinant human bone morphogenetic protein 2 (rhBMP2) utilization in single-level anterior lumbar interbody fusion (ALIF) in the United States. Patients who underwent single-level ALIF from 2005 to 2011 were identified by searching ICD-9 diagnosis and procedure codes in the PearlDiver Patient Records Database (PearlDiver Technologies, Fort Wayne, IN), a national database of orthopedic insurance records. The year of procedure, age, gender, and region of the United States were analyzed for each patient. A total of 921 patients were identified who underwent a single-level ALIF in this study. The average rate of single-level ALIF with rhBMP2 utilization increased (35%-48%) from 2005 to 2009, but sharply decreased to 16.7% in 2010 and 15.0% in 2011. The overall incidence of single-level ALIF without rhBMP2 (0.20 cases per 100 000 patients) was more than twice of the incidence of single-level ALIF with rhBMP2 (0.09 cases per 100 000 patients). The average rate of single-level ALIF with rhBMP2 utilization is highest in West (41.4%), followed by Midwest (33.3%), South (26.5%) and Northeast (22.2%). The highest incidence of single-level ALIF with rhBMP2 was observed in the group aged less than 65 years (compared with any other age groups, P < .001), with an incidence of 0.21 per 100 000 patients. The incidence of rhBMP2 utilization in single-level ALIF increased from 2006 to 2009, but decreased in 2010 and 2011. The Northeast region had the lowest incidence of rhBMP2 utilization. The group aged less than 65 years trended to have the higher incidence of single-level ALIF with rhBMP2 utilization.

Fabrication of nano-silver particles using Cymodocea serrulata and its cytotoxicity effect against human lung cancer A549 cells line

NASA Astrophysics Data System (ADS)

Palaniappan, P.; Sathishkumar, G.; Sankar, R.

2015-03-01

The present study reports, green synthesis of bioactive silver nanoparticles (AgNPs) under different temperature (60 °C, room temperature and 4° refrigerator) using the aqueous extract of sea grass Cymodocea serrulata as a potential bioreductant. Increased temperature fabricates more AgNPs compare to room temperature and refrigerator condition. At first the reduction of Ag+ ions were confirmed through color change which produces an absorbance spectra at 420 nm in UV-Visible spectrophotometer. Additionally various exclusive instrumentations such as X-ray diffraction (XRD), Dynamic light scattering (DLS), scanning electron microscope (SEM) analysis and Transmission electron microscope (TEM) were authorizes the biosynthesis and physio-chemical characterization of AgNPs. From Fourier transform infrared spectroscopy (FTIR) analysis, it was identified that the water soluble fractions of the sea grass mainly responsible for reduction of ionic silver (Ag+) into (Ag0) nano-ranged particles and also they act as stabilizing agent to sustain the durability of NPs for long period of time. Further, synthesized AgNPs shows potential cytotoxicity against human lung cancer A549 cells (LD50-100 μg/ml). The overall results suggest that C. serrulata is a valuable bioresource to generate rapid and eco-friendly bioactive AgNPs towards cancer therapy.

Physio-Biochemical Composition and Untargeted Metabolomics of Cumin (Cuminum cyminum L.) Make It Promising Functional Food and Help in Mitigating Salinity Stress

PubMed Central

Pandey, Sonika; Patel, Manish Kumar; Mishra, Avinash; Jha, Bhavanath

2015-01-01

Cumin is an annual, aromatic, herbaceous, medicinal, spice plant, most widely used as a food additive and flavoring agent in different cuisines. The study is intended to comprehensively analyse physiological parameters, biochemical composition and metabolites under salinity stress. Seed germination index, rate of seed emergence, rate of seed germination, mean germination time, plant biomass, total chlorophyll and carotenoid contents decreased concomitantly with salinity. In contrast, total antioxidant activity, H2O2, proline and MDA contents increased concurrently with stress treatments. Total phenolic and flavonoid contents were decreased initially about 1.4-fold at 50 mM, and thereafter increased about 1.2-fold at 100 mM NaCl stress. Relative water content remained unchanged up to 50 mM NaCl stress, and thereafter decreased significantly. About 2.8-fold electrolyte leakage was found in 50 mM, which increases further 4-fold at 100 mM NaCl stress. Saturated fatty acids (FAs) increased gradually with salinity, whereas unsaturation index and degree of unsaturation change arbitrarily along with the percent quantity of unsaturated FAs. Total lipid and fatty acid composition were significantly influenced by salinity stress. A total of 45 differentially expressed metabolites were identified, including luteolin, salvianolic acid, kaempferol and quercetin, which are phenolic, flavonoid or alkaloids in nature and contain antioxidant activities. Additionally, metabolites with bioactivity such as anticancerous (docetaxel) and antimicrobial (megalomicin) properties were also identified. The study evidenced that plant shoots are a rich source of metabolites, essential amino acids, phenolic compounds and fatty acids, which unveil the medicinal potential of this plant, and also provide useful insight about metabolic responses under salinity stress. PMID:26641494

Physio-Biochemical Composition and Untargeted Metabolomics of Cumin (Cuminum cyminum L.) Make It Promising Functional Food and Help in Mitigating Salinity Stress.

PubMed

Pandey, Sonika; Patel, Manish Kumar; Mishra, Avinash; Jha, Bhavanath

2015-01-01

Cumin is an annual, aromatic, herbaceous, medicinal, spice plant, most widely used as a food additive and flavoring agent in different cuisines. The study is intended to comprehensively analyse physiological parameters, biochemical composition and metabolites under salinity stress. Seed germination index, rate of seed emergence, rate of seed germination, mean germination time, plant biomass, total chlorophyll and carotenoid contents decreased concomitantly with salinity. In contrast, total antioxidant activity, H2O2, proline and MDA contents increased concurrently with stress treatments. Total phenolic and flavonoid contents were decreased initially about 1.4-fold at 50 mM, and thereafter increased about 1.2-fold at 100 mM NaCl stress. Relative water content remained unchanged up to 50 mM NaCl stress, and thereafter decreased significantly. About 2.8-fold electrolyte leakage was found in 50 mM, which increases further 4-fold at 100 mM NaCl stress. Saturated fatty acids (FAs) increased gradually with salinity, whereas unsaturation index and degree of unsaturation change arbitrarily along with the percent quantity of unsaturated FAs. Total lipid and fatty acid composition were significantly influenced by salinity stress. A total of 45 differentially expressed metabolites were identified, including luteolin, salvianolic acid, kaempferol and quercetin, which are phenolic, flavonoid or alkaloids in nature and contain antioxidant activities. Additionally, metabolites with bioactivity such as anticancerous (docetaxel) and antimicrobial (megalomicin) properties were also identified. The study evidenced that plant shoots are a rich source of metabolites, essential amino acids, phenolic compounds and fatty acids, which unveil the medicinal potential of this plant, and also provide useful insight about metabolic responses under salinity stress.

Molecular characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of genes encoding membrane proteins of Borrelia burgdorferi.

PubMed Central

Ramamoorthy, R; Povinelli, L; Philipp M, T

1996-01-01

An expression library made with Borrelia burgdorferi DNA in the vector lambda ZapII was screened with serum from a monkey infected with the Lyme disease agent. This serum killed B. burgdorferi in vitro by an antibody-dependent, complement-mediated mechanism and contained antibodies to at least seven spirochetal antigens, none of which were the major outer surface proteins OspA or OspB. Among several positive clones, a clone containing the B. burgdorferi bmpA gene encoding the immunodominant antigen P39 was obtained. Chromosome walking and DNA sequence analysis permitted the identification of two additional upstream genes homologous to the bmpA gene and its related companion, bmpB. The first of these was the recently characterized bmpC gene, and adjacent to it was the fourth and new member of this class, which has been designated bmpD. The gene product encoded by bmpD is 34l residues long, contains a signal sequence with a potential signal peptidase II cleavage site, and has 26% identity with TmpC of Treponema pallidum. Southern blotting confirmed the tandem arrangement of all four bmp genes in the chromosome of B. burgdorferi JD1. However, Northern (RNA) blotting revealed that bmpD is expressed as a monocistronic transcript, which indicates that it is not part of an operon at the bmp locus. The bmpD gene was found to be conserved in representative members of the three species of the B. burgdorferi sensu lato complex, suggesting that it serves an important biological function in the spirochete. PMID:8606088

Controllable mineral coatings on scaffolds as carriers for growth factor release for bone tissue engineering

NASA Astrophysics Data System (ADS)

Saurez-Gonzalez, Darilis

The work presented in this document, focused on the development and characterization of mineral coatings on scaffold materials to serve as templates for growth factor binding and release. Mineral coatings were formed using a biomimetic approach that consisted in the incubation of scaffolds in modified simulated body fluids (mSBF). To modulate the properties of the mineral coating, which we hypothesized would dictate growth factor release, we used carbonate (HCO3) concentration in mSBF of 4.2 mM, 25mM, and 100mM. Analysis of the mineral coatings formed using scanning electron microscopy indicated growth of a continuous layer of mineral with different morphologies. X-ray diffraction analysis showed peaks associated with hydroxyapatite. FTIR data confirmed the substitution of HCO3 in the mineral. As the extent of HCO3 substitution increased, the coating exhibited more rapid dissolution kinetics in an environment deficient in calcium and phosphate. The mineral coatings provided an effective mechanism for bioactive growth factor binding and release. Peptide versions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2) were bound with efficiencies up to 90% to mineral-coated PCL scaffolds. Recombinant human vascular endothelial growth factor (rhVEGF) also bound to mineral coated scaffolds with lower efficiency (20%) and released with faster release kinetics compared to peptides growth factor. Released rhVEGF induced human umbilical vein endothelial cell (HUVEC) proliferation in vitro and enhanced blood vessel formation in vivo in an intramuscular sheep model. In addition to the use the mineral coatings for single growth factor release, we expanded the concept and bound both an angiogenic (rhVEGF) and osteogenic (mBMP2) growth factor by a simple double dipping process. Sustained release of both growth factors was demonstrated for over 60 days. Released rhVEGF enhanced blood vessel formation in vivo in sheep and its biological activity was not affected by the presence of mBMP2. The approach for growth factor binding and release from mineral coatings can be adapted to different materials and medical devices and provide a simple and adaptable mechanism for sustained release of single or dual growth factors.

A Long-Acting BMP-2 Release System Based on Poly(3-hydroxybutyrate) Nanoparticles Modified by Amphiphilic Phospholipid for Osteogenic Differentiation

PubMed Central

Peng, Xiaochun; Chen, Yunsu; Li, Yamin; Wang, Yiming

2016-01-01

We explored a novel poly(3-hydroxybutyrate) (PHB) nanoparticle loaded with hydrophilic recombinant human BMP-2 with amphiphilic phospholipid (BPC-PHB NP) for a rapid-acting and long-acting delivery system of BMP-2 for osteogenic differentiation. The BPC-PHB NPs were prepared by a solvent evaporation method and showed a spherical particle with a mean particle size of 253.4 nm, mean zeta potential of −22.42 mV, and high entrapment efficiency of 77.18%, respectively. For BPC-PHB NPs, a short initial burst release of BMP-2 from NPs in 24 h was found and it has steadily risen to reach about 80% in 20 days for in vitro test. BPC-PHB NPs significantly reduced the burst release of BMP-2, as compared to that of PHB NPs loading BMP-2 without PL (B-PHB NPs). BPC-PHB NPs maintained the content of BMP-2 for a long-term osteogenic differentiation. The OCT-1 cells with BPC-PHB NPs have high ALP activity in comparison with others. The gene markers for osteogenic differentiation were significantly upregulated for sample with BPC-PHB NPs, implying that BPC-PHB NPs can be used as a rapid-acting and long-acting BMP-2 delivery system for osteogenic differentiation. PMID:27379249

Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling

PubMed Central

Qin, Song; Niu, Wenze; Iqbal, Nida; Smith, Derek K.; Zhang, Chun-Li

2014-01-01

Neural stem cells (NSCs) are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates bone morphogenetic protein (BMP)-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP) and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity. PMID:24782704

Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling.

PubMed

Qin, Song; Niu, Wenze; Iqbal, Nida; Smith, Derek K; Zhang, Chun-Li

2014-01-01

Neural stem cells (NSCs) are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates bone morphogenetic protein (BMP)-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP) and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

PubMed

Luo, Jiang-Yun; Zhang, Yang; Wang, Li; Huang, Yu

2015-07-15

Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Increased bone morphogenetic protein signaling contributes to age-related declines in neurogenesis and cognition.

PubMed

Meyers, Emily A; Gobeske, Kevin T; Bond, Allison M; Jarrett, Jennifer C; Peng, Chian-Yu; Kessler, John A

2016-02-01

Aging is associated with decreased neurogenesis in the hippocampus and diminished hippocampus-dependent cognitive functions. Expression of bone morphogenetic protein 4 (BMP4) increases with age by more than 10-fold in the mouse dentate gyrus while levels of the BMP inhibitor, noggin, decrease. This results in a profound 30-fold increase in phosphorylated-SMAD1/5/8, the effector of canonical BMP signaling. Just as observed in mice, a profound increase in expression of BMP4 is observed in the dentate gyrus of humans with no known cognitive abnormalities. Inhibition of BMP signaling either by overexpression of noggin or transgenic manipulation not only increases neurogenesis in aging mice, but remarkably, is associated with a rescue of cognitive deficits to levels comparable to young mice. Additive benefits are observed when combining inhibition of BMP signaling and environmental enrichment. These findings indicate that increased BMP signaling contributes significantly to impairments in neurogenesis and to cognitive decline associated with aging, and identify this pathway as a potential druggable target for reversing age-related changes in cognition. Copyright © 2016 Elsevier Inc. All rights reserved.

Inkjet-based biopatterning of SDF-1β augments BMP-2-induced repair of critical size calvarial bone defects in mice

PubMed Central

Herberg, Samuel; Kondrikova, Galina; Periyasamy-Thandavan, Sudharsan; Howie, R. Nicole; Elsalanty, Mohammed E.; Weiss, Lee; Campbell, Phil; Hill, William D.; Cray, James J.

2014-01-01

Background A major problem in craniofacial surgery is non-healing bone defects. Autologous reconstruction remains the standard of care for these cases. Bone morphogenetic protein-2 (BMP-2) therapy has proven its clinical utility, although non-targeted adverse events occur due to the high milligram-level doses used. Ongoing efforts explore the use of different growth factors, cytokines, or chemokines, as well as co-therapy to augment healing. Methods Here we utilize inkjet-based biopatterning to load acellular DermaMatrix delivery matrices with nanogram-level doses of BMP-2, stromal cell-derived factor-1β (SDF-1β), transforming growth factor-β1 (TGF-β1), or co-therapies thereof. We tested the hypothesis that bioprinted SDF-1β co-delivery enhances BMP-2 and TGF-β1-driven osteogenesis both in-vitro and in-vivo using a mouse calvarial critical size defect (CSD) model. Results Our data showed that BMP-2 bioprinted in low-doses induced significant new bone formation by four weeks post-operation. TGF-β1 was less effective compared to BMP-2, and SDF-1β therapy did not enhance osteogenesis above control levels. However, co-delivery of BMP-2 + SDF-1β was shown to augment BMP-2-induced bone formation compared to BMP-2 alone. In contrast, co-delivery of TGF-β1 + SDF-1β decreased bone healing compared to TGF-β1 alone. This was further confirmed in vitro by osteogenic differentiation studies using MC3T3-E1 pre-osteoblasts. Conclusions Our data indicates that sustained release delivery of a low-dose growth factor therapy using biopatterning technology can aid in healing CSD injuries. SDF-1β augments the ability for BMP-2 to drive healing, a result confirmed in vivo and in vitro; however, because SDF-1β is detrimental to TGF-β1-driven osteogenesis, its’ effect on osteogenesis is not universal. PMID:25016095

Prediction of Biological Motion Perception Performance from Intrinsic Brain Network Regional Efficiency

PubMed Central

Wang, Zengjian; Zhang, Delong; Liang, Bishan; Chang, Song; Pan, Jinghua; Huang, Ruiwang; Liu, Ming

2016-01-01

Biological motion perception (BMP) refers to the ability to perceive the moving form of a human figure from a limited amount of stimuli, such as from a few point lights located on the joints of a moving body. BMP is commonplace and important, but there is great inter-individual variability in this ability. This study used multiple regression model analysis to explore the association between BMP performance and intrinsic brain activity, in order to investigate the neural substrates underlying inter-individual variability of BMP performance. The resting-state functional magnetic resonance imaging (rs-fMRI) and BMP performance data were collected from 24 healthy participants, for whom intrinsic brain networks were constructed, and a graph-based network efficiency metric was measured. Then, a multiple linear regression model was used to explore the association between network regional efficiency and BMP performance. We found that the local and global network efficiency of many regions was significantly correlated with BMP performance. Further analysis showed that the local efficiency rather than global efficiency could be used to explain most of the BMP inter-individual variability, and the regions involved were predominately located in the Default Mode Network (DMN). Additionally, discrimination analysis showed that the local efficiency of certain regions such as the thalamus could be used to classify BMP performance across participants. Notably, the association pattern between network nodal efficiency and BMP was different from the association pattern of static directional/gender information perception. Overall, these findings show that intrinsic brain network efficiency may be considered a neural factor that explains BMP inter-individual variability. PMID:27853427

BMP-2-regenerated calvarial bone: a biomechanical appraisal in a large animal model.

PubMed

Cray, James; Henderson, Sarah E; Smith, Darren M; Kinsella, Christopher R; Bykowski, Michael; Cooper, Gregory M; Almarza, Alejandro J; Losee, Joseph E

2014-11-01

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is gaining popularity in craniofacial applications. Calvarial defects are, under normal circumstances, subjected to only minimal levels of the biomechanical stresses known to play an important role in osteogenesis, yet regenerated calvarial bone must be capable of withstanding traumatic forces such that the underlying neurocapsule is protected. The aim of this study is to, for the first time, assess the biomechanical properties of calvarial bone regenerated with derivations of a commercially available rhBMP-2-based system. Standardized calvarial defects were created in 23 adult male canines. These defects were treated with rhBMP-2 on one of several carriers. After 24 weeks, the biomechanical properties of the rhBMP-2-generated bone were compared to those of controls with a modified punch-out test (Bluehill 2; Instron, Norwood, Mass) and compared using a paired nonparametric analyses (SPSS, 17.0, Chicago, Ill). In a previously published report, defects across all the rhBMP-2 therapy groups were observed to have a mean rate of 99.5% radio-opacity at 24 weeks indicating nearly full bony coverage of the calvarial defect (compared to 32.7% in surgical controls). For ultimate load, ultimate energy, and first peak energy, there were significant differences (P<0.05) with the control native bone having more robust biomechanical properties than the rhBMP-2-generated bone. We conclude from these findings that rhBMP-2-generated calvarial bone is significantly less protective against trauma than native bone at 6 months. Further investigation is required to assess the efficacy of rhBMP-2 in healing calvarial defects in the longer term.

The Efficacy of Cyclic Injection of Bone Morphogenetic Protein-2 in Large-Scale Calvarial Bone Defects.

PubMed

Choi, Jin Mi; Jeong, Woo Shik; Park, Eun Jung; Choi, Jong Woo

2017-03-01

Bone morphogenetic protein-2 (BMP-2) appears to be one of the most potent growth factors thus far studied. However, recent publications on the clinical application of BMP-2 revealed that its correct control is the paramount issue in clinical practice. For improving BMP-2 delivery, the cyclic administration might be an alternative. Accordingly, the authors cyclically injected BMP-2 in a cyclic injection model of large cranial defects to maintain the proper dosage during the bone healing process. A 10-mm diameter calvarial bone defect was produced using a round drill in 8-week-old Sprague-Dawley rats. Silk-hydroxyapatite scaffolds soaked in the appropriate concentration of BMP-2 were implanted into the defect. The animals were split into 4 single-injection groups and 3 multiple-injection groups; the latter groups received weekly subcutaneous injections of BMP-2 solution (1, 5, and 10 μg/mL) for 4 weeks, whereas the former groups received a single injection of BMP-2 at these concentrations. Each rat underwent computed tomography at 8 weeks. In terms of total volumes of the new bone, the 5 μg/mL multiple-injection BMP-2 group had significantly greater increases in bone volume than the single-injection groups. In terms of bone thickness, the multiple-injection groups had better outcomes than the single-injection groups. Thus, the cyclic injection protocol restored the original thickness without overgrowth. Cyclic injection of BMP-2 permits more accurate dosage control than single injection and improves thickness and dense bone regeneration. Therefore, it may represent a promising approach for future clinical trials. Further investigation using a greater number of animals is required.

* Calvarial Bone Regeneration Is Enhanced by Sequential Delivery of FGF-2 and BMP-2 from Layer-by-Layer Coatings with a Biomimetic Calcium Phosphate Barrier Layer.

PubMed

Gronowicz, Gloria; Jacobs, Emily; Peng, Tao; Zhu, Li; Hurley, Marja; Kuhn, Liisa T

2017-12-01

A drug delivery coating for synthetic bone grafts has been developed to provide sequential delivery of multiple osteoinductive factors to better mimic aspects of the natural regenerative process. The coating is composed of a biomimetic calcium phosphate (bCaP) layer that is applied to a synthetic bone graft and then covered with a poly-l-Lysine/poly-l-Glutamic acid polyelectrolyte multilayer (PEM) film. Bone morphogenetic protein-2 (BMP-2) was applied before the coating process directly on the synthetic bone graft and then, bCaP-PEM was deposited followed by adsorption of fibroblast growth factor-2 (FGF-2) into the PEM layer. Cells access the FGF-2 immediately, while the bCaP-PEM temporally delays the cell access to BMP-2. In vitro studies with cells derived from mouse calvarial bones demonstrated that Sca-1 and CD-166 positive osteoblast progenitor cells proliferated in response to media dosing with FGF-2. Coated scaffolds with BMP-2 and FGF-2 were implanted in mouse calvarial bone defects and harvested at 1 and 3 weeks. After 1 week in vivo, proliferation of cells, including Sca-1+ progenitors, was observed with low dose FGF-2 and BMP-2 compared to BMP-2 alone, indicating that in vivo delivery of FGF-2 activated a similar population of cells as shown by in vitro testing. At 3 weeks, FGF-2 and BMP-2 delivery increased bone formation more than BMP-2 alone, particularly in the center of the defect, confirming that the proliferation of the Sca-1 positive osteoprogenitors by FGF-2 was associated with increased bone healing. Areas of bone mineralization were positive for double fluorochrome labeling of calcium and alkaline phosphatase staining of osteoblasts, along with increased TRAP+ osteoclasts, demonstrating active bone formation distinct from the bone-like collagen/hydroxyapatite scaffold. In conclusion, the addition of a bCaP layer to PEM delayed access to BMP-2 and allowed the FGF-2 stimulated progenitors to populate the scaffold before differentiating in response to BMP-2, leading to improved bone defect healing.

Maxillary sinus floor elevation using BMP-2 and Nell-1 gene-modified bone marrow stromal cells and TCP in rabbits.

PubMed

Xia, Lunguo; Xu, Yuanjin; Chang, Qing; Sun, Xiaojuan; Zeng, Deliang; Zhang, Wenjie; Zhang, Xiuli; Zhang, Zhiyuan; Jiang, Xinquan

2011-07-01

This study evaluated the synergistic osteogenic effect of bone morphogenetic protein-2 (BMP-2) and Nel-like molecule-1 (Nell-1) genes in a rabbit maxillary sinus floor elevation model. Bone marrow stromal cells (bMSCs) were cultured and transduced with AdEGFP, AdNell-1, AdBMP-2, or AdNell-1 + AdBMP-2 overexpression virus. These gene-modified autologous bMSCs were then combined with a β-tricalcium phosphate (β-TCP) granule scaffold and used to elevate the maxillary sinus floor in rabbits. bMSCs cotransduced with AdNell-1 + AdBMP-2 demonstrated a synergistic effect on osteogenic differentiation as detected by real-time PCR analysis on markers of runt-related transcription factor-2, osteocalcin, collagen type 1, alkaline phosphatase activity, and calcium deposits in vitro. As for maxillary sinus floor elevation in a rabbit model in vivo, AdNell-1 + AdBMP-2 gene-transduced autologeous bMSCs/β-TCP complex had the largest bone area and most mature bone structure among the groups, as detected by HE staining and immunohistochemistry at weeks 2 and 8 after implantation. Our data suggested that the BMP-2 and Nell-1 genes possessed a synergistic effect on osteogenic differentiation of bMSCs, while bMSCs modified with the BMP-2 and Nell-1 genes could promote new bone formation and maturation in the rabbit maxillary sinus model.

Bone generation in the reconstruction of a critical size calvarial defect in an experimental model.

PubMed

Por, Yong-Chen; Barceló, C Raul; Salyer, Kenneth E; Genecov, David G; Troxel, Karen; Gendler, El; Elsalanty, Mohammed E; Opperman, Lynne A

2008-03-01

This study was designed to investigate the optimal combination of known osteogenic biomaterials with shape conforming struts to achieve calvarial vault reconstruction, using a canine model. Eighteen adolescent beagles were divided equally into 6 groups. A critical-size defect of 6 x 2 cm traversed the sagittal suture. The biomaterials used for calvarial reconstruction were demineralized perforated bone matrix (DBM), recombinant human bone morphogenetic protein 2 (rhBMP2), and autogenous platelet-rich plasma (PRP). The struts used were cobalt chrome (metal) or resorbable plate. The groupings were as follows: 1) DBM + metal, 2) DBM + PRP + metal, 3) DBM + PRP + resorbable plate, 4) DBM + rhBMP2 + metal, 5) DBM + rhBMP2 + PRP + metal, and 6) DBM + rhBMP2 + resorbable plate. Animals were killed at 3 months after surgery. There was no mortality or major complications. Analysis was performed macroscopically and histologically and with computed tomography. There was complete bony regeneration in the rhBMP2 groups only. Non-rhBMP2 groups had minimal bony ingrowth from the defect edges and on the dural surface, a finding confirmed by computed tomographic scan and histology. Platelet-rich plasma did not enhance bone regeneration. Shape conformation was good with both metal and resorbable plate. rhBMP2, but not PRP, accelerated calvarial regeneration in 3 months. The DBMs in the rhBMP2 groups were substituted by new trabecular bone. Shape molding was good with both metal and resorbable plate.

BMP2 induces PANC-1 cell invasion by MMP-2 overexpression through ROS and ERK.

PubMed

Liu, Jun; Ben, Qi-Wen; Yao, Wei-Yan; Zhang, Jian-Jun; Chen, Da-Fan; He, Xiang-Yi; Li, Lei; Yuan, Yao-Zong

2012-06-01

The emerging roles of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers have drawn great attention in cancer research. We hypothesized that BMP2 promotes cancer metastasis by modulating MMP-2 secretion and activity through intracellular ROS regulation and ERK activation in human pancreatic cancer. Our data show that stimulation of PANC-1 cells with BMP2 induced MMP-2 secretion and activation, associated with decreased E-cadherin expression, resulting in epithelial-to-mesenchymal transformation (EMT) and cell invasion. Blockade of ROS by the ROS scavenger, 2-MPG, abolished cell invasion, inhibited the EMT process and decreased MMP-2 expression, suggesting ROS accumulation caused an increase in MMP-2 expression in BMP2-stimulated PANC-1 cell invasion. Furthermore, treatment of PANC-1 cells with 2-MPG or ERK inhibitor PD98059 reduced the phosphorylation of ERK, resulting in attenuation of BMP2-induced cell invasion and MMP-2 activation. Taken together, these results suggest that BMP2 induces the cell invasion of PANC-1 cells by enhancing MMP-2 secretion and acting through ROS accumulation and ERK activation.

Enhanced Healing of Rat Calvarial Defects with MSCs Loaded on BMP-2 Releasing Chitosan/Alginate/Hydroxyapatite Scaffolds

PubMed Central

He, Xiaoning; Liu, Yang; Yuan, Xue; Lu, Li

2014-01-01

In this study, we designed a chitosan/alginate/hydroxyapatite scaffold as a carrier for recombinant BMP-2 (CAH/B2), and evaluated the release kinetics of BMP-2. We evaluated the effect of the CAH/B2 scaffold on the viability and differentiation of bone marrow mesenchymal stem cells (MSCs) by scanning electron microscopy, MTS, ALP assay, alizarin-red staining and qRT-PCR. Moreover, MSCs were seeded on scaffolds and used in a 8 mm rat calvarial defect model. New bone formation was assessed by radiology, hematoxylin and eosin staining 12 weeks postoperatively. We found the release kinetics of BMP-2 from the CAH/B2 scaffold were delayed compared with those from collagen gel, which is widely used for BMP-2 delivery. The BMP-2 released from the scaffold increased MSC differentiation and did not show any cytotoxicity. MSCs exhibited greater ALP activity as well as stronger calcium mineral deposition, and the bone-related markers Col1α, osteopontin, and osteocalcin were upregulated. Analysis of in vivo bone formation showed that the CAH/B2 scaffold induced more bone formation than other groups. This study demonstrates that CAH/B2 scaffolds might be useful for delivering osteogenic BMP-2 protein and present a promising bone regeneration strategy. PMID:25084008

Periodontal Tissues, Maxillary Jaw Bone, and Tooth Regeneration Approaches: From Animal Models Analyses to Clinical Applications

PubMed Central

Batool, Fareeha; Strub, Marion; Petit, Catherine; Bugueno, Isaac Maximiliano; Bornert, Fabien; Clauss, François; Kuchler-Bopp, Sabine; Benkirane-Jessel, Nadia

2018-01-01

This review encompasses different pre-clinical bioengineering approaches for periodontal tissues, maxillary jaw bone, and the entire tooth. Moreover, it sheds light on their potential clinical therapeutic applications in the field of regenerative medicine. Herein, the electrospinning method for the synthesis of polycaprolactone (PCL) membranes, that are capable of mimicking the extracellular matrix (ECM), has been described. Furthermore, their functionalization with cyclosporine A (CsA), bone morphogenetic protein-2 (BMP-2), or anti-inflammatory drugs’ nanoreservoirs has been demonstrated to induce a localized and targeted action of these molecules after implantation in the maxillary jaw bone. Firstly, periodontal wound healing has been studied in an induced periodontal lesion in mice using an ibuprofen-functionalized PCL membrane. Thereafter, the kinetics of maxillary bone regeneration in a pre-clinical mouse model of surgical bone lesion treated with BMP-2 or BMP-2/Ibuprofen functionalized PCL membranes have been analyzed by histology, immunology, and micro-computed tomography (micro-CT). Furthermore, the achievement of innervation in bioengineered teeth has also been demonstrated after the co-implantation of cultured dental cell reassociations with a trigeminal ganglia (TG) and the cyclosporine A (CsA)-loaded poly(lactic-co-glycolic acid) (PLGA) scaffold in the jaw bone. The prospective clinical applications of these different tissue engineering approaches could be instrumental in the treatment of various periodontal diseases, congenital dental or cranio-facial bone anomalies, and post-surgical complications. PMID:29772691

Localized expression of a dpp/BMP2/4 ortholog in a coral embryo

PubMed Central

Hayward, David C.; Samuel, Gabrielle; Pontynen, Patricia C.; Catmull, Julian; Saint, Robert; Miller, David J.; Ball, Eldon E.

2002-01-01

As the closest outgroup to the Bilateria, the Phylum Cnidaria is likely to be critical to understanding the origins and evolution of body axes. Proteins of the decapentaplegic (DPP)/bone morphogenetic protein (BMP) 2/4 subfamily are central to the specification of the dorsoventral (D/V) axis in bilateral animals, albeit with an axis inversion between arthropods and chordates. We show that a dpp/BMP2/4 ortholog (bmp2/4-Am) is present in the reef-building scleractinian coral, Acropora millepora (Class Anthozoa) and that it is capable of causing phenotypic effects in Drosophila that mimic those of the endogenous dpp gene. We also show that, during coral embryonic development, bmp2/4-Am expression is localized in an ectodermal region adjacent to the blastopore. Thus, a representative of the DPP/BMP2/4 subfamily of ligands was present in the common ancestor of diploblastic and triploblastic animals where it was probably expressed in a localized fashion during development. A localized source of DPP/BMP2/4 may have already been used in axis formation in this ancestor, or it may have provided a means by which an axis could evolve in triploblastic animals. PMID:12048233

rhBMP-2 (ACS and CRM formulations) overcomes pseudarthrosis in a New Zealand white rabbit posterolateral fusion model.

PubMed

Lawrence, James P; Waked, Walid; Gillon, Thomas J; White, Andrew P; Spock, Christopher R; Biswas, Debdut; Rosenberger, Patricia; Troiano, Nancy; Albert, Todd J; Grauer, Jonathan N

2007-05-15

The study design consisted of a New Zealand white rabbit model of pseudarthrosis repair. Study groups consisting of no graft, autograft, or recombinant human bone morphogenetic protein-2 (rhBMP-2) with absorbable collagen sponge (ACS) or compression resistant matrix (CRM) were evaluated. To evaluate the relative efficacy of bone graft materials (autograft, ACS, and CRM). rhBMP-2 has been shown to have a 100% fusion rate in a primary rabbit fusion model, even in the presence of nicotine, which is known to inhibit fusion. Seventy-two New Zealand white rabbits underwent posterolateral lumbar fusion with iliac crest autograft. To establish pseudarthroses, nicotine was administered to all animals. At 5 weeks, the spines were explored and all pseudarthroses were redecorticated and implanted with no graft, autograft, rhBMP-2/ACS, or rhBMP-2/CRM. At 10 weeks, fusions were assessed by manual palpation and histology. Eight rabbits (11%) were lost to complications. At 5 weeks, 66 (97%) had pseudarthroses. At 10 weeks, attempted pseudarthrosis repairs were fused in 1 of 16 of no graft rabbits (6%), 5 of 17 autograft rabbits (29%), and 31 of 31 rhBMP-2 rabbits (with ACS or CRM) (100%). Histologic analysis demonstrated more mature bone formation in the rhBMP-2 groups. The 2 rhBMP-2 formulations led to significantly higher fusion rates and histologic bone formation than no graft and autograft controls in this pseudarthrosis repair model.

BMP2-loaded hollow hydroxyapatite microspheres exhibit enhanced osteoinduction and osteogenicity in large bone defects

PubMed Central

Xiong, Long; Zeng, Jianhua; Yao, Aihua; Tu, Qiquan; Li, Jingtang; Yan, Liang; Tang, Zhiming

2015-01-01

The regeneration of large bone defects is an osteoinductive, osteoconductive, and osteogenic process that often requires a bone graft for support. Limitations associated with naturally autogenic or allogenic bone grafts have demonstrated the need for synthetic substitutes. The present study investigates the feasibility of using novel hollow hydroxyapatite microspheres as an osteoconductive matrix and a carrier for controlled local delivery of bone morphogenetic protein 2 (BMP2), a potent osteogenic inducer of bone regeneration. Hollow hydroxyapatite microspheres (100±25 μm) with a core (60±18 μm) and a mesoporous shell (180±42 m2/g surface area) were prepared by a glass conversion technique and loaded with recombinant human BMP2 (1 μg/mg). There was a gentle burst release of BMP2 from microspheres into the surrounding phosphate-buffered saline in vitro within the initial 48 hours, and continued at a low rate for over 40 days. In comparison with hollow hydroxyapatite microspheres without BMP2 or soluble BMP2 without a carrier, BMP2-loaded hollow hydroxyapatite microspheres had a significantly enhanced capacity to reconstitute radial bone defects in rabbit, as shown by increased serum alkaline phosphatase; quick and complete new bone formation within 12 weeks; and great biomechanical flexural strength. These results indicate that BMP2-loaded hollow hydroxyapatite microspheres could be a potential new option for bone graft substitutes in bone regeneration. PMID:25609957

Sox2 in the dermal papilla niche controls hair growth by fine-tuning Bmp signaling in differentiating hair shaft progenitors

PubMed Central

Clavel, Carlos; Grisanti, Laura; Zemla, Roland; Rezza, Amelie; Barros, Rita; Sennett, Rachel; Mazloom, Amin; Chung, Chi-Yeh; Cai, Xiaoqiang; Cai, Chen-Leng; Pevny, Larysa; Nicolis, Silvia; Ma’ayan, Avi; Rendl, Michael

2012-01-01

SUMMARY How dermal papilla (DP) niche cells regulate hair follicle progenitors to control hair growth remains unclear. Using Tbx18Cre to target embryonic DP precursors, we ablate the transcription factor Sox2 early and efficiently, resulting in diminished hair shaft outgrowth. We find that DP niche expression of Sox2 controls the migration rate of differentiating hair shaft progenitors. Transcriptional profiling of Sox2 null DPs reveals increased Bmp6 and decreased Bmp inhibitor Sostdc1, a direct Sox2 transcriptional target. Subsequently, we identify upregulated Bmp signaling in knockout hair shaft progenitors and demonstrate that Bmps inhibit cell migration, an effect that can be attenuated by Sostdc1. A shorter and Sox2-negative hair type lacks Sostdc1 in the DP and shows reduced migration and increased Bmp activity of hair shaft progenitors. Collectively, our data identify Sox2 as a key regulator of hair growth that controls progenitor migration by fine-tuning Bmp-mediated mesenchymal-epithelial crosstalk. PMID:23153495

Initial Experience With rhBMP-2 Delivered in a Compressive Resistant Matrix for Mandibular Reconstruction in 5 Dogs.

PubMed

Boudrieau, Randy J

2015-05-01

To document cumulative initial experience and long-term follow-up of the use of rhBMP-2/CRM for reconstruction of large mandibular defects (≥5 cm) in dogs. Retrospective case series. Dogs (n = 5). Medical records (October 1999-April 2011) of dogs that had mandibular reconstruction for defects/resections of ≥5 cm using rhBMP-2/CRM were reviewed. Signalment, preoperative assessment/rationale for mandibular reconstruction, surgical methods, postoperative assessment of the reconstruction (evaluation of occlusion), and complications were recorded. A definitive histologic diagnosis was obtained in dogs that had mandibular resection for mass removal. Long-term complications were determined. A minimum time frame of 2-year in-hospital follow-up was required for case inclusion. Mandibular reconstruction was successfully performed in all dogs' defects where gaps of 5-9 cm were bridged. Surgical reconstruction rapidly restored cosmetic appearance and function. All dogs healed with new bone formation across the gap. New bone formation was present within the defects as early as 2 weeks after surgery based on palpation, and new bone formation bridging the gap was documented radiographically by 16 weeks. Minor complications occurred in all dogs in the early postoperative period, and included early firm swelling and gingival dehiscence in 1 dog; late plate exposure in 3 dogs; and exuberant/cystic bone formation in 2 dogs (related to concentration/formulation of rhBMP-2/CRM). Two dogs had minor long-term complications of late plate exposure and a non-vital canine tooth; the plates and the affected canine tooth were removed. Long-term in-hospital follow-up was 5.3 years (range, 2-12.5 years); further long-term telephone follow-up was 6.3 years (range, 2-12.5 years). All owners were pleased with the outcome and would repeat the surgery again under similar circumstances. The efficacy and success of this mandibular reconstruction technique, using rhBMP-2/CRM with plate fixation, was demonstrated with bridging of large mandibular defects regardless of the underlying cause, and with excellent cosmetic and functional results. Complications were common, but considered minor and easily treated. The complications encountered revealed the importance of tailoring the use of BMPs and fixation methods to this specific anatomic location and indication. © Copyright 2014 by The American College of Veterinary Surgeons.

Enhanced Healing of Segmental Bone Defects by Modulation of the Mechanical Environment

DTIC Science & Technology

2012-10-01

5.5 µg BMP-2, it was largely disorganized, woven bone with non-osseous soft tissue interspersed. The highest 4 dose (11 µg) of BMP-2, in contrast...various doses of BMP-2. Top row: 16x magnification Bottom row: 100x magnification N= new cortex M= marrow T=trabecular bone F= fibrous tissue ...areas of cartilagenous tissue (figure 5) it was clear that substantial areas of cartilage remained in the defects treated with 5.5 µg BMP-2. These may

Enhanced Bone Formation in Segmental Defects with BMP2 in a Biologically Relevant Molecular Context

DTIC Science & Technology

2016-10-16

gun shots . These do not heal on their own once a ‘critical size’ segment of bone is missing. One strategy to induce healing is to use bone-inducing...accelerate BMP2-induced bone formation by presenting the growth factor in a more biologically relevant context. This is based on our observation...that manganese increases the binding of BMP2 to COMP. The next steps are to validate these observations using BMP2:COMP on HA/PLG scaffolds in-vitro

Systems biology derived source-sink mechanism of BMP gradient formation

PubMed Central

Zinski, Joseph; Bu, Ye; Wang, Xu; Dou, Wei

2017-01-01

A morphogen gradient of Bone Morphogenetic Protein (BMP) signaling patterns the dorsoventral embryonic axis of vertebrates and invertebrates. The prevailing view in vertebrates for BMP gradient formation is through a counter-gradient of BMP antagonists, often along with ligand shuttling to generate peak signaling levels. To delineate the mechanism in zebrafish, we precisely quantified the BMP activity gradient in wild-type and mutant embryos and combined these data with a mathematical model-based computational screen to test hypotheses for gradient formation. Our analysis ruled out a BMP shuttling mechanism and a bmp transcriptionally-informed gradient mechanism. Surprisingly, rather than supporting a counter-gradient mechanism, our analyses support a fourth model, a source-sink mechanism, which relies on a restricted BMP antagonist distribution acting as a sink that drives BMP flux dorsally and gradient formation. We measured Bmp2 diffusion and found that it supports the source-sink model, suggesting a new mechanism to shape BMP gradients during development. PMID:28826472

Systems biology derived source-sink mechanism of BMP gradient formation.

PubMed

Zinski, Joseph; Bu, Ye; Wang, Xu; Dou, Wei; Umulis, David; Mullins, Mary C

2017-08-09

A morphogen gradient of Bone Morphogenetic Protein (BMP) signaling patterns the dorsoventral embryonic axis of vertebrates and invertebrates. The prevailing view in vertebrates for BMP gradient formation is through a counter-gradient of BMP antagonists, often along with ligand shuttling to generate peak signaling levels. To delineate the mechanism in zebrafish, we precisely quantified the BMP activity gradient in wild-type and mutant embryos and combined these data with a mathematical model-based computational screen to test hypotheses for gradient formation. Our analysis ruled out a BMP shuttling mechanism and a bmp transcriptionally-informed gradient mechanism. Surprisingly, rather than supporting a counter-gradient mechanism, our analyses support a fourth model, a source-sink mechanism, which relies on a restricted BMP antagonist distribution acting as a sink that drives BMP flux dorsally and gradient formation. We measured Bmp2 diffusion and found that it supports the source-sink model, suggesting a new mechanism to shape BMP gradients during development.

[Hand Therapy in the Treatment of Patients with CRPS].

PubMed

Körbler, C; Pfau, M; Becker, F; Koester, U; Werdin, F

2015-06-01

In the modern treatment of CRPS a multidisciplinary concept is firmly established (MMPT, multimodal pain therapy). Besides medical therapy and psychotherapy, physio- and occupational therapy count as basic treatment options. Although physio- and occupational therapy (in the following called hand therapy) are the most important basic treatments, the therapy is hardly standardised and there are few scientific investigations concerning their application. Therefore the purpose of this paper is to present the applied hand therapeutic techniques with regard to function/performance, application and effectiveness, and to derive a suitable treatment algorithm. The techniques used in hand therapy are presented and reviewed in regard to their effectiveness by means of a literature search. It turns out that exercise therapy, manual therapy, graded motor imaging, CO2 baths and occupational therapy have a proven benefit for the patients. Although for many of the treatments reliable evidence-based data are lacking a treatment algorithm was established but there is a strong need for further investigations concerning the therapeutic effectiveness in the treatment of CRPS. © Georg Thieme Verlag KG Stuttgart · New York.

A living thick nanofibrous implant bifunctionalized with active growth factor and stem cells for bone regeneration

PubMed Central

Eap, Sandy; Keller, Laetitia; Schiavi, Jessica; Huck, Olivier; Jacomine, Leandro; Fioretti, Florence; Gauthier, Christian; Sebastian, Victor; Schwinté, Pascale; Benkirane-Jessel, Nadia

2015-01-01

New-generation implants focus on robust, durable, and rapid tissue regeneration to shorten recovery times and decrease risks of postoperative complications for patients. Herein, we describe a new-generation thick nanofibrous implant functionalized with active containers of growth factors and stem cells for regenerative nanomedicine. A thick electrospun poly(ε-caprolactone) nanofibrous implant (from 700 μm to 1 cm thick) was functionalized with chitosan and bone morphogenetic protein BMP-7 as growth factor using layer-by-layer technology, producing fish scale-like chitosan/BMP-7 nanoreservoirs. This extracellular matrix-mimicking scaffold enabled in vitro colonization and bone regeneration by human primary osteoblasts, as shown by expression of osteocalcin, osteopontin, and bone sialoprotein (BSPII), 21 days after seeding. In vivo implantation in mouse calvaria defects showed significantly more newly mineralized extracellular matrix in the functionalized implant compared to a bare scaffold after 30 days’ implantation, as shown by histological scanning electron microscopy/energy dispersive X-ray microscopy study and calcein injection. We have as well bifunctionalized our BMP-7 therapeutic implant by adding human mesenchymal stem cells (hMSCs). The activity of this BMP-7-functionalized implant was again further enhanced by the addition of hMSCs to the implant (living materials), in vivo, as demonstrated by the analysis of new bone formation and calcification after 30 days’ implantation in mice with calvaria defects. Therefore, implants functionalized with BMP-7 nanocontainers associated with hMSCs can act as an accelerator of in vivo bone mineralization and regeneration. PMID:25709432

Comparison of the osteogenesis and fusion rates between activin A/BMP-2 chimera (AB204) and rhBMP-2 in a beagle's posterolateral lumbar spine model.

PubMed

Zheng, Guang Bin; Yoon, Byung-Hak; Lee, Jae Hyup

2017-10-01

Activin A/BMP-2 chimera (AB204) could promote bone healing more effectively than recombinant bone morphogenetic protein 2 (rhBMP-2) with much lower dose in a rodent model, but there is no report about the effectiveness of AB204 in a large animal model. The purpose of this study was to compare the osteogenesis and fusion rate between AB204 and rhBMP-2 using biphasic calcium phosphate (BCP) as a carrier in a beagle's posterolateral lumbar fusion model. This is a randomized control animal study. Seventeen male beagle dogs were included. Bilateral posterolateral fusion was performed at the L1-L2 and L4-L5 levels. Biphasic calcium phosphate (2 cc), rhBMP-2 (50 µg)+BCP (2 cc), or AB204 (50 µg)+BCP (2 cc) were implanted into the intertransverse space randomly. X-ray was performed at 4 and 8 weeks. After 8 weeks, the animals were sacrificed, and new bone formation and fusion rate were evaluated by manual palpation, computed tomography (CT), and undecalcified histology. The AB204 group showed significantly higher fusion rate (90%) than the rhBMP-2 group (15%) or the Osteon group (6.3%) by manual palpation. On x-ray and CT assessment, fusion rate and the volume of newly formed bone were also significantly higher in AB204 group than other groups. In contrast, more osteolysis was found in rhBMP-2 group (40%) than in AB204 group (10%) on CT study. In histologic results, new bone formation was sufficient between transverse processes in AB204 group, and obvious trabeculation and bone remodeling were observed. But in rhBMP-2 group, new bone formation was less than AB204 group and osteolysis was observed between the intertransverse spaces. A low dose of AB204 with BCP as a carrier significantly promotes the fusion rate in a large animal model when compared with the rhBMP-2. These findings demonstrate that AB204 could be an alternative to rhBMP-2 to improve fusion rate. Copyright © 2017 Elsevier Inc. All rights reserved.

Bone morphogenetic protein use in spine surgery-complications and outcomes: a systematic review.

PubMed

Faundez, Antonio; Tournier, Clément; Garcia, Matthieu; Aunoble, Stéphane; Le Huec, Jean-Charles

2016-06-01

Because of significant complications related to the use of autologous bone grafts in spinal fusion surgery, bone substitutes and growth factors such as bone morphogenetic protein (BMP) have been developed. One of them, recombinant human (rh) BMP-2, has been approved by the Food and Drug Administration (FDA) for use under precise conditions. However, rhBMP-2-related side effects have been reported, used in FDA-approved procedures, but also in off-label use.A systematic review of clinical data was conducted to analyse the rhBMP-2-related adverse events (AEs), in order to assess their prevalence and the associated surgery practices. Medline search with keywords "bone morphogenetic protein 2", "lumbar spine", "anterolateral interbody fusion" (ALIF) and the filter "clinical trial". FDA published reports were also included. Study assessment was made by authors (experienced spine surgeons), based on quality of study designs and level of evidence. Extensive review of randomised controlled trials (RCTs) and controlled series published up to the present point, reveal no evidence of a significant increase of AEs related to rhBMP-2 use during ALIF surgeries, provided that it is used following FDA guidelines. Two additional RCTs performed with rhBMP-2 in combination with allogenic bone dowels reported increased bone remodelling in BMP-treated patients. This AE was transient and had no consequence on the clinical outcome of the patients. No other BMP-related AEs were reported in these studies. This literature review confirms that the use of rhBMP-2 following FDA-approved recommendations (i.e. one-level ALIF surgery with an LT-cage) is safe. The rate of complications is low and the AEs had been identified by the FDA during the pre-marketing clinical trials. The clinical efficiency of rhBMP-2 is equal or superior to that of allogenic or autologous bone graft in respect to fusion rate, low back pain disability, patient satisfaction and rate of re-operations. For all other off-label use, the safety and effectiveness of rhBMP-2 have not been established, and further RCTs with high level of evidence are required.

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway.

PubMed

Yang, Hongpeng; Guo, Yue; Wang, Dawei; Yang, Xiaofei; Ha, Chengzhi

2018-01-02

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.

Peptide-laden mesoporous silica nanoparticles with promoted bioactivity and osteo-differentiation ability for bone tissue engineering.

PubMed

Luo, Zuyuan; Deng, Yi; Zhang, Ranran; Wang, Mengke; Bai, Yanjie; Zhao, Qiang; Lyu, Yalin; Wei, Jie; Wei, Shicheng

2015-07-01

Combination of mesoporous silica materials and bioactive factors is a promising niche-mimetic solution as a hybrid bone substitution for bone tissue engineering. In this work, we have synthesized biocompatible silica-based nanoparticles with abundant mesoporous structure, and incorporated bone-forming peptide (BFP) derived from bone morphogenetic protein-7 (BMP-7) into the mesoporous silica nanoparticles (MSNs) to obtain a slow-release system for osteogenic factor delivery. The chemical characterization demonstrates that the small osteogenic peptide is encapsulated in the mesoporous successfully, and the nitrogen adsorption-desorption isotherms suggest that the peptide encapsulation has no influence on mesoporous structure of MSNs. In the cell experiment, the peptide-laden MSNs (p-MSNs) show higher MG-63 cell proliferation, spreading and alkaline phosphatase (ALP) activity than the bare MSNs, indicating good in vitro cytocompatibility. Simultaneously, the osteogenesis-related proteins expression and calcium mineral deposition disclose enhanced osteo-differentiation of human mesenchymal stem cells (hMSCs) under the stimulation of the p-MSNs, confirming that BFP released from MSNs could significantly promote the osteogenic differentiation of hMSCs, especially at 500μg/mL of p-MSNs concentration. The peptide-modified MSNs with better bioactivity and osteogenic differentiation make it a potential candidate as bioactive material for bone repairing, bone regeneration, and bio-implant coating applications. Copyright © 2015 Elsevier B.V. All rights reserved.

BmP02 Atypically Delays Kv4.2 Inactivation: Implication for a Unique Interaction between Scorpion Toxin and Potassium Channel

PubMed Central

Wu, Bin; Zhu, Yan; Shi, Jian; Tao, Jie; Ji, Yonghua

2016-01-01

BmP02, a short-chain peptide with 28 residues from the venom of Chinese scorpion Buthus martensi Karsch, has been reported to inhibit the transient outward potassium currents (Ito) in rat ventricular muscle cells. However, it remains unclear whether BmP02 modulates the Kv4.2 channel, one of the main contributors to Ito. The present study investigated the effects of BmP02 on Kv4.2 kinetics and its underlying molecular mechanism. The electrophysiological recordings showed that the inactivation of Kv4.2 expressed in HEK293T cells was significantly delayed by BmP02 in a dose-response manner with EC50 of ~850 nM while the peak current, activation and voltage-dependent inactivation of Kv4.2 were not affected. Meanwhile, the recovery from inactivation of Kv4.2 was accelerated and the deactivation was slowed after the application of BmP02. The site-directed mutagenesis combined with computational modelling identified that K347 and K353, located in the turret motif of the Kv4.2, and E4/E5, D20/D21 in BmP02 are key residues to interact with BmP02 through electrostatic force. These findings not only reveal a novel interaction between Kv4.2 channel and its peptidyl modulator, but also provide valuable information for design of highly-selective Kv4.2 modulators. PMID:27690098

Genetic analyses of bone morphogenetic protein 2, 4 and 7 in congenital combined pituitary hormone deficiency.

PubMed

Breitfeld, Jana; Martens, Susanne; Klammt, Jürgen; Schlicke, Marina; Pfäffle, Roland; Krause, Kerstin; Weidle, Kerstin; Schleinitz, Dorit; Stumvoll, Michael; Führer, Dagmar; Kovacs, Peter; Tönjes, Anke

2013-12-01

The complex process of development of the pituitary gland is regulated by a number of signalling molecules and transcription factors. Mutations in these factors have been identified in rare cases of congenital hypopituitarism but for most subjects with combined pituitary hormone deficiency (CPHD) genetic causes are unknown. Bone morphogenetic proteins (BMPs) affect induction and growth of the pituitary primordium and thus represent plausible candidates for mutational screening of patients with CPHD. We sequenced BMP2, 4 and 7 in 19 subjects with CPHD. For validation purposes, novel genetic variants were genotyped in 1046 healthy subjects. Additionally, potential functional relevance for most promising variants has been assessed by phylogenetic analyses and prediction of effects on protein structure. Sequencing revealed two novel variants and confirmed 30 previously known polymorphisms and mutations in BMP2, 4 and 7. Although phylogenetic analyses indicated that these variants map within strongly conserved gene regions, there was no direct support for their impact on protein structure when applying predictive bioinformatics tools. A mutation in the BMP4 coding region resulting in an amino acid exchange (p.Arg300Pro) appeared most interesting among the identified variants. Further functional analyses are required to ultimately map the relevance of these novel variants in CPHD.

Genetic analyses of bone morphogenetic protein 2, 4 and 7 in congenital combined pituitary hormone deficiency

PubMed Central

2013-01-01

Background The complex process of development of the pituitary gland is regulated by a number of signalling molecules and transcription factors. Mutations in these factors have been identified in rare cases of congenital hypopituitarism but for most subjects with combined pituitary hormone deficiency (CPHD) genetic causes are unknown. Bone morphogenetic proteins (BMPs) affect induction and growth of the pituitary primordium and thus represent plausible candidates for mutational screening of patients with CPHD. Methods We sequenced BMP2, 4 and 7 in 19 subjects with CPHD. For validation purposes, novel genetic variants were genotyped in 1046 healthy subjects. Additionally, potential functional relevance for most promising variants has been assessed by phylogenetic analyses and prediction of effects on protein structure. Results Sequencing revealed two novel variants and confirmed 30 previously known polymorphisms and mutations in BMP2, 4 and 7. Although phylogenetic analyses indicated that these variants map within strongly conserved gene regions, there was no direct support for their impact on protein structure when applying predictive bioinformatics tools. Conclusions A mutation in the BMP4 coding region resulting in an amino acid exchange (p.Arg300Pro) appeared most interesting among the identified variants. Further functional analyses are required to ultimately map the relevance of these novel variants in CPHD. PMID:24289245

Epigenetic effects of green tea polyphenols in cancer

PubMed Central

Henning, Susanne M; Wang, Piwen; Carpenter, Catherine L; Heber, David

2014-01-01

Epigenetics describes heritable alterations of gene expression and chromatin organization without changes in DNA sequence. Both hypermethylation and hypomethylation of DNA can affect gene expression and the multistep process of carcinogenesis. Epigenetic changes are reversible and may be targeted by dietary interventions. Bioactive compounds from green tea (GT) such as (–)-epigallocatechin gallate have been shown to alter DNA methyltransferase activity in studies of esophageal, oral, skin, Tregs, lung, breast and prostate cancer cells, which may contribute to the chemopreventive effect of GT. Three out of four mouse model studies have confirmed the inhibitory effect of (–)-epigallocatechin gallate on DNA methylation. A human study demonstrated that decreased methylation of CDX2 and BMP-2 in gastric carcinoma was associated with higher GT consumption. It is the goal of this review to summarize our current knowledge of the potential of GT to alter epigenetic processes, which may be useful in chemoprevention. PMID:24283885

Evaluation of implants coated with rhBMP-2 using two different coating strategies: a critical-size supraalveolar peri-implant defect study in dogs.

PubMed

Lee, Jaebum; Decker, John F; Polimeni, Giuseppe; Cortella, Carlo Alberto; Rohrer, Michael D; Wozney, John M; Hall, Jan; Susin, Cristiano; Wikesjö, Ulf M E

2010-06-01

Implants coated with recombinant human bone morphogenetic protein-2 (rhBMP-2) induce relevant bone formation but also resident bone remodelling. To compare the effect of implants fully or partially coated with rhBMP-2 on new bone formation and resident bone remodelling. Twelve, male, adult, Hound Labrador mongrel dogs were used. Critical-size, supraalveolar, peri-implant defects received titanium porous oxide surface implants coated in their most coronal aspect with rhBMP-2 (coronal-load/six animals) or by immersion of the entire implant in an rhBMP-2 solution (soak-load/six animals) for a total of 30 mug rhBMP-2/implant. All implants were air-dried. The animals were euthanized at 8 weeks for histometric evaluation. Clinical healing was uneventful. Supraalveolar bone formation was not significantly affected by the rhBMP-2 application protocol. New bone height and area averaged (+/- SE) 3.4 +/- 0.2 versus 3.5 +/- 0.4 mm and 2.6 +/- 0.4 versus 2.5 +/- 0.7 mm(2) for coronal-load and soak-load implants, respectively (p>0.05). The corresponding bone density and bone-implant contact (BIC) recordings averaged 38.0 +/- 3.8%versus 34.4 +/- 5.6% and 25.0 +/- 3.8%versus 31.2 +/- 3.3% (p>0.05). In contrast, resident bone remodelling was significantly influenced by the rhBMP-2 application protocol. Bone density outside the implants threads averaged 74.7 +/- 3.8% and 50.8 +/- 4.1% for coronal-load and soak-load implants, respectively (p<0.05); bone density within the thread area averaged 51.8 +/- 1.2% and 37.8 +/- 2.9%, and BIC 70.1 +/- 6.7% and 43.3 +/- 3.9% (p<0.05). Local application of rhBMP-2 appears to be a viable technology to support local bone formation and osseointegration. Coronal-load implants obviate resident bone remodelling without compromising new bone formation.

The Penultimate Arginine of the Carboxyl Terminus Determines Slow Desensitization in a P2X Receptor from the Cattle Tick Boophilus microplus

PubMed Central

Bavan, Selvan; Farmer, Louise; Singh, Shire K.; Straub, Volko A.; Guerrero, Felix D.

2011-01-01

P2X ion channels have been functionally characterized from a range of eukaryotes. Although these receptors can be broadly classified into fast and slow desensitizing, the molecular mechanisms underlying current desensitization are not fully understood. Here, we describe the characterization of a P2X receptor from the cattle tick Boophilus microplus (BmP2X) displaying extremely slow current kinetics, little desensitization during ATP application, and marked rundown in current amplitude between sequential responses. ATP (EC50, 67.1 μM) evoked concentration-dependent currents at BmP2X that were antagonized by suramin (IC50, 4.8 μM) and potentiated by the antiparasitic drug amitraz. Ivermectin did not potentiate BmP2X currents, but the mutation M362L conferred ivermectin sensitivity. To investigate the mechanisms underlying slow desensitization we generated intracellular domain chimeras between BmP2X and the rapidly desensitizing P2X receptor from Hypsibius dujardini. Exchange of N or C termini between these fast- and slow-desensitizing receptors altered the rate of current desensitization toward that of the donor channel. Truncation of the BmP2X C terminus identified the penultimate residue (Arg413) as important for slow desensitization. Removal of positive charge at this position in the mutant R413A resulted in significantly faster desensitization, which was further accentuated by the negatively charged substitution R413D. R413A and R413D, however, still displayed current rundown to sequential ATP application. Mutation to a positive charge (R413K) reconstituted the wild-type phenotype. This study identifies a new determinant of P2X desensitization where positive charge at the end of the C terminal regulates current flow and further demonstrates that rundown and desensitization are governed by distinct mechanisms. PMID:21212138

Expression of bone morphogenetic proteins 4, 6 and 7 is downregulated in kidney allografts with interstitial fibrosis and tubular atrophy.

PubMed

Furic-Cunko, Vesna; Kes, Petar; Coric, Marijana; Hudolin, Tvrtko; Kastelan, Zeljko; Basic-Jukic, Nikolina

2015-07-01

Bone morphogenetic proteins (BMPs) are pleiotropic growth factors. This paper investigates the connection between the expression pattern of BMPs in kidney allograft tissue versus the cause of allograft dysfunction. The expression pattern of BMP2, BMP4, BMP6 and BMP7 in 50 kidney allografts obtained by transplant nephrectomy is investigated. Immunohistochemical staining is semiquantitatively evaluated for intensity to identify the expression pattern of BMPs in normal and allograft kidney tissues. The expression of BMP4 is unique between different tubular cell types in grafts without signs of fibrosis. This effect is not found in specimens with high grades of interstitial fibrosis and tubular atrophy (IFTA). In samples with IFTA grades II and III, the BMP7 expression is reduced in a significant fraction of specimens relative to those without signs of IFTA. The expression pattern of BMP6 indicates that its activation may be triggered by the act of transplantation and subsequent reperfusion injury. The expression of BMP2 is strong in all types of tubular epithelial cells and does not differ between the compared allografts and control kidney specimens. The intensity and expression pattern of BMP4, BMP6 and BMP7 in transplanted kidney tissue are found to be dependent upon the length of the transplanted period, the clinical indication for transplant nephrectomy and signs of IFTA in kidney tissue.

A new molecular logic for BMP-mediated dorsoventral patterning in the leech Helobdella.

PubMed

Kuo, Dian-Han; Weisblat, David A

2011-08-09

Bone morphogenetic protein (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Neurologic impairment from ectopic bone in the lumbar canal: a potential complication of off-label PLIF/TLIF use of bone morphogenetic protein-2 (BMP-2).

PubMed

Wong, David A; Kumar, Anant; Jatana, Sanjay; Ghiselli, Gary; Wong, Katherine

2008-01-01

Bone morphogenetic protein-2 (BMP-2) (Infuse) has been approved for use in anterior lumbar fusion in conjunction with an LT cage. However, off-label use is seen with anterior cervical fusion, posterior lumbar interbody fusion (PLIF), and transforaminal lumbar interbody fusion (TLIF). The Federal Food and Drug Administration trial of BMP-2 in a PLIF application was halted because of a high incidence of ectopic bone forming in the neural canal (75%). The authors did not find a correlation between ectopic bone and increased leg pain. They concluded that the ectopic bone was a radiographic phenomenon and not associated with clinical findings. Complications using BMP in the cervical spine have been reported. Heretofore, there has not been a similar warning voiced for use of BMP in a lumbar PLIF or TLIF. The purpose was to report five cases of ectopic bone in the canal associated with PLIF/TLIF off-label use of BMP-2 potentially contributing to abnormal neurologic findings. This is an observational cohort study of patients referred to a tertiary care private medical center. This was a retrospective chart review of patients referred to a tertiary spine institute with complications after surgery where BMP-2 had been used in an off-label PLIF or TLIF application. Patient demographics, operating room (OR) notes from the index BMP surgery, imaging studies, and current clinical status were reviewed. Five cases of ectopic bone in the spinal canal with potential neurologic compromise were identified. It does appear that ectopic bone in the spinal canal associated with BMP-2 use in PLIF or TLIF may contribute to symptomatic neurologic findings in rare cases. Revision surgeries are difficult. This article challenges a previous publication, which concluded that the high incidence of ectopic bone was of no clinical significance. Isolating BMP anteriorly in the disc space using layered barriers of bone graft between the BMP and the annular defect may reduce the incidence of ectopic bone in the spinal canal. Surgeons need to weigh the benefits versus risks of any technology used off label when making treatment decisions with their patients.

Downregulated bone morphogenetic protein signaling in nitrofen-induced congenital diaphragmatic hernia.

PubMed

Makanga, Martine; Dewachter, Céline; Maruyama, Hidekazu; Vuckovic, Aline; Rondelet, Benoit; Naeije, Robert; Dewachter, Laurence

2013-08-01

Bone morphogenetic proteins (BMP) have been shown to play crucial roles in not only lung and heart development, but also in the pathogenesis of pulmonary vascular remodeling in pulmonary hypertension (PH). We therefore hypothesized that BMP signaling could be altered in nitrofen-induced congenital diaphragmatic hernia (CDH) and associated PH. Pregnant rats were exposed to either 100 mg nitrofen or vehicle on embryonic day (E) 9.5. On E17 and E21, fetuses were delivered by cesarean section, killed and checked for left-sided CDH. The tissue was then harvested for pathobiological evaluation. In nitrofen-induced CDH, pulmonary expressions of BMP4, BMP receptor (BMPR) type 2 and Id1 decreased on E17 and E21. On E17, pulmonary gremlin-1 expression increased, while BMP7 decreased. In the lungs, Id1 expression was correlated to BMP4 and BMPR2 and inversely correlated to gremlin-1 expression. Myocardial expressions of BMPR2, BMPR1A, BMP7 and SERCA-2A decreased, while gremlin-1 and noggin expressions increased on E17. On E21, myocardial expressions of Id1 and SERCA-2A decreased, while gremlin-1 expression increased. Moreover, BMPR2 and BMPR1A expressions were correlated to SERCA-2A expression and inversely correlated to pro-apoptotic Bax/Bcl2 ratio within the myocardium. Downregulation of BMP signaling seems to contribute to pulmonary and myocardial anomalies observed in nitrofen-induced CDH.

Bone morphogenetic protein type I receptor inhibition induces cleft palate associated with micrognathia and cleft lower lip in mice.

PubMed

Lai, Yongzhen; Xie, Changfu; Zhang, Shixian; Gan, Guowu; Wu, Di; Chen, Weihui

2016-07-01

Gain-of- and loss-of-function studies have demonstrated that changes in bone morphogenetic protein (BMP) signaling during embryo development cause craniofacial malformations, including cleft palate. It remains uncertain whether BMP signaling could be targeted pharmacologically to affect craniofacial morphogenesis. Pregnant C57Bl/6J mice were treated with the BMP type I receptor inhibitor LDN-193189 at the dose of 3, 6, or 9 mg/kg twice a day by intraperitoneal injection from embryonic day 10.5 (E10.5) to E15.5. At E16.5, embryos were investigated by facial measurement analysis and histology to determine the optimal concentration for malformation. Subsequent embryonic phenotypes were analyzed in detail by histology, whole-mount skeletal staining, micro-computed tomography, and palatal organic culture. We further used immunohistochemistry to analyze protein expression of the BMP-mediated canonical and noncanonical signaling components. The optimal concentration of LDN-193189 was determined to be 6 mg/kg. In utero, LDN-193189 exposures induced partial clefting of the anterior palate or complete cleft palate, which was attributed to a reduced cell proliferation rate in the secondary palate, and delayed palatal elevation caused by micrognathia. Analysis of signal transduction in palatal shelves at E12.5 and E13.5 identified a significant reduction of BMP/Smad signaling (p-Smad1/5/8) and unchanged BMP noncanonical signaling (p-p38, p-Erk1/2) after treatment with LDN-193189. The results of this study indicate that LDN-193189 can be used to manipulate BMP signaling by selectively targeting the BMP/Smad signaling pathway to affect palatal morphogenesis and produce phenotypes mimicking those caused by genetic mutations. This work established a novel mouse model for teratogen-induced cleft palate. Birth Defects Research (Part A) 106:612-623, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): cellular localization, developmental profiles, and response to unilateral ovariectomy.

PubMed

García-López, Ángel; Sánchez-Amaya, María Isabel; Halm, Silke; Astola, Antonio; Prat, Francisco

2011-12-01

Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass. Copyright © 2011 Elsevier Inc. All rights reserved.

Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment.

PubMed

Todeschi, Maria R; El Backly, Rania M; Varghese, Oommen P; Hilborn, Jöns; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-07-01

This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation. Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation. Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.

THE RGM/DRAGON FAMILY OF BMP CO-RECEPTORS

PubMed Central

Corradini, Elena; Babitt, Jodie L.; Lin, Herbert Y.

2013-01-01

The BMP signaling pathway controls a number of cell processes during development and in adult tissues. At the cellular level, ligands of the BMP family act by binding a hetero-tetrameric signaling complex, composed of two type I and two type II receptors. BMP ligands make use of a limited number of receptors, which in turn activate a common signal transduction cascade at the intracellular level. A complex regulatory network is required in order to activate the signaling cascade at proper times and locations, and to generate specific downstream effects in the appropriate cellular context. One such regulatory mechanism is the repulsive guidance molecule (RGM) family of BMP co-receptors. This article reviews the current knowledge regarding the structure, regulation, and function of RGMs, focusing on known and potential roles of RGMs in physiology and pathophysiology. PMID:19897400

A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis.

PubMed

Kerr, Georgina; Sheldon, Helen; Chaikuad, Apirat; Alfano, Ivan; von Delft, Frank; Bullock, Alex N; Harris, Adrian L

2015-04-01

Activin receptor-like kinase 1 (ALK1, encoded by the gene ACVRL1) is a type I BMP/TGF-β receptor that mediates signalling in endothelial cells via phosphorylation of SMAD1/5/8. During angiogenesis, sprouting endothelial cells specialise into tip cells and stalk cells. ALK1 synergises with Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2 and thereby represses tip cell formation and angiogenic sprouting. The ALK1-Fc soluble protein fusion has entered clinic trials as a therapeutic strategy to sequester the high-affinity extracellular ligand BMP9. Here, we determined the crystal structure of the ALK1 intracellular kinase domain and explored the effects of a small molecule kinase inhibitor K02288 on angiogenesis. K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses. In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition. Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis. Such activity may be advantageous for small molecule inhibitors currently in preclinical development for specific BMP gain of function conditions, including diffuse intrinsic pontine glioma and fibrodysplasia ossificans progressiva, as well as more generally for other applications in tumour biology.

Generation of microgrooved silica nanotube membranes with sustained drug delivery and cell contact guidance ability by using a Teflon microfluidic chip

NASA Astrophysics Data System (ADS)

Chen, Song; Shi, Xuetao; Chinnathambi, Shanmugavel; Wu, Hongkai; Hanagata, Nobutaka

2013-02-01

Silica nanotubes have been extensively applied in the biomedical field. However, very little attention has been paid to the fabrication and application of micropatterned silica nanotubes. In the present study, microgrooved silica nanotube membranes were fabricated in situ by microgrooving silica-coated collagen hybrid fibril hydrogels in a Teflon microfluidic chip followed by calcination for removal of collagen fibrils. Scanning electron microscopy images showed that the resulting silica nanotube membranes displayed a typical microgroove/ridge surface topography with ˜50 μm microgroove width and ˜120 μm ridge width. They supported adsorption of bone morphogenetic protein 2 (BMP-2) and exhibited a sustained release behavior for BMP-2. After culturing with osteoblast MC3T3-E1 cells, they induced an enhanced osteoblast differentiation due to the release of biologically active BMP-2 and a strong contact guidance ability to directly align and elongate osteoblasts due to the presence of microgrooved surface topography, indicating their potential application as a multi-functional cell-supporting matrix for tissue generation.

Osteoinductive activity of insulin-functionalized cell culture surfaces obtained using diazonium chemistry

NASA Astrophysics Data System (ADS)

Mikulska, Anna; Filipowska, Joanna; Osyczka, Anna; Nowakowska, Maria; Szczubiałka, Krzysztof

2014-12-01

Polymeric surfaces suitable for cell culture (DR/Pec) were constructed from diazoresin (DR) and pectin (Pec) in a form of ultrathin films using the layer-by-layer (LbL) technique. The surfaces were functionalized with insulin using diazonium chemistry. Such functionalized surfaces were used to culture human mesenchymal stem cells (hMSCs) to assess their suitability for bone tissue engineering and regeneration. The activity of insulin immobilized on the surfaces (DR/Pec/Ins) was compared to that of insulin dissolved in the culture medium. Human MSC grown on insulin-immobilized DR/Pec surfaces displayed increased proliferation and higher osteogenic activity. The latter was determined by means of alkaline phosphatase (ALP) activity, which increases at early stages of osteoblasts differentiation. Insulin dissolved in the culture medium did not stimulate cell proliferation and its osteogenic activity was significantly lower. Addition of recombinant human bone morphogenetic protein 2 (rhBMP-2) to the culture medium further increased ALP activity in hMSCs indicating additive osteogenic action of immobilized insulin and rhBMP-2

Combined sustained release of BMP2 and MMP10 accelerates bone formation and mineralization of calvaria critical size defect in mice.

PubMed

Reyes, Ricardo; Rodríguez, Jose Antonio; Orbe, Josune; Arnau, María Rosa; Évora, Carmen; Delgado, Araceli

2018-11-01

The effect of dual delivery of bone morphogenetic protein-2 (BMP-2) and matrix metalloproteinase 10 (MMP10) on bone regeneration was investigated in a murine model of calvarial critical-size defect, hypothesizing that it would result in an enhanced bone formation. Critical-size calvarial defects (4 mm diameter) were created in mice and PLGA microspheres preloaded with either BMP-2, MMP10 or a microsphere combination of both were transplanted into defect sites at different doses. Empty microspheres were used as the negative control. Encapsulation efficiency was assessed and in vivo release kinetics of BMP-2 and MMP10 were examined over 14 days. Histological analyses were used to analyze bone formation after four and eight weeks. Combination with MMP10 (30 ng) significantly enhanced BMP-2 (600 ng)-mediated osteogenesis, as confirmed by the increase in percentage of bone fill (p < .05) at four weeks. Moreover, it also increased mineral apposition rate (p < .05), measured by double labeling with tetracycline and calceine. MMP10 accelerates bone repair by enhancing BMP-2-promoted bone healing and improving the mineralization rate. In conclusion combination of MMP10 and BMP-2 may become a promising strategy for repair and regeneration of bone defects.

Binding Interactions of Keratin-Based Hair Fiber Extract to Gold, Keratin, and BMP-2

PubMed Central

de Guzman, Roche C.; Tsuda, Shanel M.; Ton, Minh-Thi N.; Zhang, Xiao; Esker, Alan R.; Van Dyke, Mark E.

2015-01-01

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD) of 1.8 × 10−4 M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10−7 M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10−5 M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration. PMID:26317522

Improving anti-felting characteristics of Merino wool fiber by 2.5 MHz atmosphere pressure air plasma

NASA Astrophysics Data System (ADS)

Chandwani, Nisha; Dave, Purvi; Jain, Vishal; Nema, Sudhir; Mukherjee, Subroto

2017-04-01

The present work investigates the effect of high frequency (2.5 MHz) Dielectric Barrier Discharge (DBD) in air on surface characteristics of Merino wool as a function of plasma exposure time (5s to 15s). The FE-SEM (Field Emission Scanning Electron Microscopy), EDS (Energy Dispersive X-ray spectrum) and Derivative ATR-FTIR (Attenuated Total Reflection- Fourier Transform Infrared) Spectroscopy are used to study physio-chemical changes induced by plasma. These physio-chemical properties of fibers can be co-related with the felting behaviour of the wool fiber, which leads to shrinkage and pilling of garments while laundering. Felting occurs mainly because of presence of outermost hydrophobic cuticle layer having sharp scales. The FE-SEM analysis of wool fiber surface reveals that cuticle scales on wool fiber become blunt after plasma processing. The ATR-FTIR analysis along with second order derivative spectroscopy demonstrates the cleavage of di-sulphide bonds of cuticle and formation of sulphur-oxygen groups such as Cystine Sulphonate (-S-SO3-), cysteic acid (-SO3-), cystine monoxide(-SO-S-), cysteine di-oxide (-SO2-S-). A possible explanation about how the combined effect of morphological and chemical changes induced by plasma results in minimizing the felting of wool fibers is discussed.

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity‡

PubMed Central

Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B.; Titus, Louisa; Boden, Scott D.

2010-01-01

The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1ΔSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell-based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post-translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression-based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP-1. Among them, we selected SVAK-3, a compound that showed a dose-dependent potentiation of BMP-2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP-1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP-2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored. PMID:19862690

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

PubMed

Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

2009-12-01

The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1DeltaSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell-based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post-translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression-based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP-1. Among them, we selected SVAK-3, a compound that showed a dose-dependent potentiation of BMP-2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP-1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP-2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored.

Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response

NASA Technical Reports Server (NTRS)

Sorescu, George P.; Sykes, Michelle; Weiss, Daiana; Platt, Manu O.; Saha, Aniket; Hwang, Jinah; Boyd, Nolan; Boo, Yong C.; Vega, J. David; Taylor, W. Robert;

Effects of recombinant dentin sialoprotein in dental pulp cells.

PubMed

Lee, S-Y; Kim, S-Y; Park, S-H; Kim, J-J; Jang, J-H; Kim, E-C

2012-04-01

Dentin sialophosphoprotein (DSPP) is critical for dentin mineralization. However, the function of dentin sialoprotein (DSP), the cleaved product of DSPP, remains unclear. This study aimed to investigate the signal transduction pathways and effects of recombinant human DSP (rh-DSP) on proliferation, migration, and odontoblastic differentiation in human dental pulp cells (HDPCs). The exogenous addition of rh-DSP enhanced the proliferation and migration of HDPCs in dose- and time-dependent manners. rh-DSP markedly increased ALP activity, calcium nodule formation, and levels of odontoblastic marker mRNA. rh-DSP increased BMP-2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist, noggin. Furthermore, rh-DSP phosphorylated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), Akt, and IκB-α, and induced the nuclear translocation of the NF-κB p65 subunit. Analysis of these data demonstrates a novel signaling function of rh-DSP for the promotion of growth, migration, and differentiation in HDPCS via the BMP/Smad, JNK, ERK, MAPK, and NF-κB signaling pathways, suggesting that rh-DSP may have therapeutic utility in dentin regeneration or dental pulp tissue engineering.

Dura Mater Stimulates Human Adipose-Derived Stromal Cells to Undergo Bone Formation in Mouse Calvarial Defects

PubMed Central

Levi, Benjamin; Nelson, Emily R.; Li, Shuli; James, Aaron W.; Hyun, Jeong S.; Montoro, Daniel T.; Lee, Min; Glotzbach, Jason P.; Commons, George W.; Longaker, Michael T.

2015-01-01

Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2–Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair. PMID:21656608

Borrelia burgdorferi basic membrane protein A could induce chemokine production in murine microglia cell line BV2.

PubMed

Zhao, Hua; Liu, Aihua; Cui, Yuhui; Liang, Zhang; Li, Bingxue; Bao, Fukai

2017-10-01

Lyme neuroborreliosis is a nervous system infectious disease caused by Borrelia burgdorferi (B. burgdorferi). It has been demonstrated that cytokines induced by B. burgdorferi are related to Lyme neuroborreliosis. Microglia is known as a key player in the immune responses that occur within the central nervous system. In response to inflammation, it will be activated and generate cytokines and chemokines. Experiments in vitro cells have showed that B. Burgdorferi membrane protein A (BmpA), a major immunogen of B. Burgdorferi, could induce Lyme arthritis and stimulate human and murine lymphocytes to produce inflammatory cytokines. In our study, the murine microglia BV2 cell line was used as a cell model to explore the stimulating effects of recombinant BmpA (rBmpA); Chemokine chip, ELISA and QPCR technology were used to measure the production of chemokines from microglial cells stimulated by rBmpA. Compared with the negative control group, CXCL2, CCL22, and CCL5 concentrations in the cell supernatant increased significantly after the rBmpA stimulation; the concentration of these chemokines increased with rBmpA concentration increasing; the mRNA expression levels of chemokines (CXCL2, CCL22, and CCL5) in murine BV2 cells increased significantly with 10 μg/mL and 20 μg/mL rBmpA stimulation; CXCL13 was not change after the rBmpA stimulation. Our study shows that chemokines, such as CXCL2, CCL22, and CCL5 were up-regulated by the rBmpA in the BV2 cells. The production of chemokines in Lyme neuroborreliosis may be mainly from microglia cells and the rBmpA may be closely related with the development of Lyme neuroborreliosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo.

PubMed

Haillot, Emmanuel; Molina, Maria Dolores; Lapraz, François; Lepage, Thierry

2015-01-01

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo

PubMed Central

Haillot, Emmanuel; Molina, Maria Dolores; Lapraz, François; Lepage, Thierry

2015-01-01

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis. PMID:26352141

Lack of Association of Bone Morphogenetic Protein 2 Gene Haplotypes with Bone Mineral Density, Bone Loss, or Risk of Fractures in Men

PubMed Central

Varanasi, Satya S.; Tuck, Stephen P.; Mastana, Sarabjit S.; Dennison, Elaine; Cooper, Cyrus; Vila, Josephine; Francis, Roger M.; Datta, Harish K.

2011-01-01

Introduction. The association of bone morphogenetic protein 2 (BMP2) with BMD and risk of fracture was suggested by a recent linkage study, but subsequent studies have been contradictory. We report the results of a study of the relationship between BMP2 genotypes and BMD, annual change in BMD, and risk of fracture in male subjects. Materials and Methods. We tested three single-nucleotide polymorphisms (SNPs) across the BMP2 gene, including Ser37Ala SNP, in 342 Caucasian Englishmen, comprising 224 control and 118 osteoporotic subjects. Results. BMP2 SNP1 (Ser37Ala) genotypes were found to have similar low frequency in control subjects and men with osteoporosis. The major informative polymorphism, BMP2 SNP3 (Arg190Ser), showed no statistically significant association with weight, height, BMD, change in BMD at hip or lumbar spine, and risk of fracture. Conclusion. There were no genotypic or haplotypic effects of the BMP2 candidate gene on BMD, change in BMD, or fracture risk identified in this cohort. PMID:22013543

Biochemical methane potential (BMP) tests: Reducing test time by early parameter estimation.

PubMed

Da Silva, C; Astals, S; Peces, M; Campos, J L; Guerrero, L

2018-01-01

Biochemical methane potential (BMP) test is a key analytical technique to assess the implementation and optimisation of anaerobic biotechnologies. However, this technique is characterised by long testing times (from 20 to >100days), which is not suitable for waste utilities, consulting companies or plants operators whose decision-making processes cannot be held for such a long time. This study develops a statistically robust mathematical strategy using sensitivity functions for early prediction of BMP first-order model parameters, i.e. methane yield (B 0 ) and kinetic constant rate (k). The minimum testing time for early parameter estimation showed a potential correlation with the k value, where (i) slowly biodegradable substrates (k≤0.1d -1 ) have a minimum testing times of ≥15days, (ii) moderately biodegradable substrates (0.1

Lack of effect of bone morphogenetic protein 2 and 4 gene polymorphisms on bone density in postmenopausal Turkish women.

PubMed

Ozkan, Z S; Deveci, D; Onalan Etem, E; Yüce, H

2010-11-30

We investigated the effect of bone morphogenetic protein 2 and 4 (BMP-2 and -4) gene polymorphisms on bone density in postmenopausal Turkish women with osteoporosis. The frequency of single-nucleotide polymorphisms (SNPs) of BMP-2 and -4 genes was analyzed in 101 osteoporotic-postmenopausal women and 52 postmenopausal women with positive bone mineral density scores. We evaluated the frequency of the thymine→cytosine nucleotide variation at position 538 for BMP-4 and the transposition of adenine→thymine at codon 190 for BMP-2, with PCR. The proportions of genotypes observed for the BMP-2 SNP in the osteoporotic group were AA (47.5%), AT (39.6%), TT (12.9%), and in the non-osteoporotic group they were AA (48.1%), AT (40.4%), TT (11.5%). The corresponding frequencies for the BMP-4 SNP in the osteoporotic group were TT (30.7%), TC (45.5%), CC (23.8%), and in the non-osteoporotic group they were TT (26.9%), TC (40.4%), CC (32.7%). There were no significant differences in the frequencies of these genotypes between the patient and control groups. We conclude that genetic variations in BMP-2 and -4 do not substantially contribute to lumbar spine bone mineral density in postmenopausal Turkish women.

Microstructured Titanium Regulates Interleukin Production by Osteoblasts, an Effect Modulated by Exogenous BMP-2

PubMed Central

Hyzy, Sharon; Olivares-Navarrete, Rene; Hutton, Daphne L.; Tan, Christian; Boyan, Barbara D.; Schwartz, Zvi

2013-01-01

Microtextured implant surfaces increase osteoblast differentiation in vitro and enhance bone-to-implant contact in vivo and clinically. These implants may be used in combination with recombinant human bone morphogenetic protein 2 (rhBMP-2) to enhance peri-implant bone formation. However, the effect of surface modifications alone or in combination with rhBMP-2 on osteoblast-produced inflammatory microenvironment is unknown. MG63 cells were cultured on tissue culture polystyrene or titanium substrates: smooth pretreated (PT, Ra=0.2μm), sandblasted/acid-etched (SLA, Ra=3.2μm), or hydrophilic-SLA (modSLA). Expression and protein production of pro-inflammatory interleukins (IL1b, IL6, IL8, IL17) and anti-inflammatory interleukins (IL10) were measured in cells with or without rhBMP-2. To determine which BMP signaling pathways were involved, cultures were incubated with BMP pathway inhibitors to blocking Smad (dorsomorphin), TAB/TAK1 ((5Z)-7-oxozeaenol), or PKA (H-8) signaling. Culture on rough SLA and modSLA surfaces decreased pro-inflammatory interleukins and increased anti-inflammatory IL10. This effect was negated in cells treated with rhBMP-2, which caused an increase in pro-inflammatory interleukins and a decrease in anti-inflammatory interleukins through TAB/TAK signaling. The results suggest that surface microtexture modulates the inflammatory process during osseointegration, an effect that may enhance healing. However, rhBMP-2 in combination with microtextured titanium implants can influence the effect of cells on these surfaces, and may adversely affect cells involved in osseointegration. PMID:23123301

BMP type II receptors have redundant roles in the regulation of hepatic hepcidin gene expression and iron metabolism.

PubMed

Mayeur, Claire; Leyton, Patricio A; Kolodziej, Starsha A; Yu, Binglan; Bloch, Kenneth D

2014-09-25

Expression of hepcidin, the hepatic hormone controlling iron homeostasis, is regulated by bone morphogenetic protein (BMP) signaling. We sought to identify which BMP type II receptor expressed in hepatocytes, ActR2a or BMPR2, is responsible for regulating hepcidin gene expression. We studied Bmpr2 heterozygous mice (Bmpr2(+/-)), mice with hepatocyte-specific deficiency of BMPR2, mice with global deficiency of ActR2a, and mice in which hepatocytes lacked both BMPR2 and ActR2a. Hepatic hepcidin messenger RNA (mRNA) levels, serum hepcidin and iron levels, and tissue iron levels did not differ in wild-type mice, Bmpr2(+/-) mice, and mice in which either BMPR2 or ActR2a was deficient. Deficiency of both BMP type II receptors markedly reduced hepatic hepcidin gene expression and serum hepcidin levels leading to severe iron overload. Iron injection increased hepatic hepcidin mRNA levels in mice deficient in either BMPR2 or ActR2a, but not in mice deficient in both BMP type II receptors. In addition, in mouse and human primary hepatocytes, deficiency of both BMPR2 and ActR2a profoundly decreased basal and BMP6-induced hepcidin gene expression. These results suggest that BMP type II receptors, BMPR2 and ActR2a, have redundant roles in the regulation of hepatic hepcidin gene expression and iron metabolism. © 2014 by The American Society of Hematology.

Bone formation at recombinant human bone morphogenetic protein-2-coated titanium implants in the posterior mandible (Type II bone) in dogs.

PubMed

Wikesjö, Ulf M E; Xiropaidis, Andreas V; Qahash, Mohammed; Lim, Won Hee; Sorensen, Rachel G; Rohrer, Michael D; Wozney, John M; Hall, Jan

2008-11-01

Conventional oral/maxillofacial implants reach osseointegration over several months during which the titanium fixtures interact with alveolar bone. The objective of this study was to determine if adsorbing recombinant human bone morphogenetic protein-2 (rhBMP-2) onto a titanium porous oxide (TPO) implant surface might enhance or accelerate local bone formation and support osseointegration in a large animal oral/maxillofacial orthotopic model. Endosseous implants with a TPO surface were installed into the edentulated posterior mandible in eight adult Hound Labrador mongrel dogs. The implant surface had been adsorbed with rhBMP-2 at 0.2 or 4.0 mg/ml. TPO implants without rhBMP-2 served as control. Treatments were randomized between jaw quadrants. Mucosal flaps were advanced and sutured leaving the implants submerged. Clinical and radiographic evaluations were made immediately post-surgery, at day 10 (suture removal), and week 4 and 8 post-surgery. The animals received fluorescent bone markers at week 3, 4, and at week 8 post-surgery, when they were euthanized for histologic analysis. TPO implants coated with rhBMP-2 exhibited dose-dependent bone remodelling including immediate resorption and formation of implant adjacent bone, and early establishment of clinically relevant osseointegration. The resulting bone-implant contact, although clinically respectable, appeared significantly lower for rhBMP-2-coated implants compared with the control [rhBMP-2 (0.2 mg/ml) 43.3+/-10.8%versus 71.7+/-7.8%, p<0.02; rhBMP-2 (4.0 mg/ml) 35.4+/-10.6%versus 68.2+/-11.0%, p<0.03]. rhBMP-2 adsorbed onto TPO implant surfaces initiates dose-dependent peri-implant bone re-modelling resulting in the formation of normal, physiologic bone and clinically relevant osseointegration within 8 weeks.

Expression and Function of Xmsx-2B in Dorso-Ventral Axis Formation in Gastrula Embryos.

PubMed

Onitsuka, I; Takeda, M; Maéno, M

2000-11-01

Msx is a homeodomain-containing transcriptional factor that plays an essential role in pattern formation in vertebrata and invertebrata embryos. In Xenopus laevis, two msx genes have been identified (Xmsx-1 and Xmsx-2). In the present study, we attempted to elucidate the expression and function of Xmsx-2B (formerly designated as Xhox7.1') in early embryogenesis. Whole mount in situ hybridization analyses showed that the expression pattern of Xmsx-2B at gastrula and neurula stages was very similar to that of Xmsx-1: the transcript of Xmsx-2B was observed in ventral and lateral sides of the embryo. At the tailbud stage, however, the expression pattern of Xmsx-2B in neural tissues was distinct from that of Xmsx-1. An RNA injection experiment revealed that, like BMP-4, Xmsx-2B has a strong ventralizing activity. In the Xmsx-2B -injected embryos, differentiation of axial structures such as the notochord, muscle, and neural tissue was completely suppressed, whereas alpha-globin mRNA, a blood cell marker, was highly expressed. Simultaneous injection of Xmsx-1 and Xmsx-2B RNAs showed that they function in an additive manner. It was also shown that coinjection of Xmsx-2B with a dominant-negative BMP-4 receptor (tBR), which can induce formation of secondary axis when injected alone in ventral blastomeres, suppressed secondary axis formation. Furthermore, Xmsx-2B also suppressed secondary axis formation, which was induced by a dominant-negative form of Xmsx-1 (VP16/msx-1). Therefore, like Xmsx-1, Xmsx-2B is a downstream nuclear factor of the BMP-4-derived ventralizing signal, and these two factors probably share the same target molecules. In conclusion, Xmsx-1 and Xmsx-2B function in dorso-ventral axis formation in early Xenopus laevis development.

Spinal neuromodulation as a novel surgical option for failed back surgery syndrome following rhBMP exuberant bony growth in instrumented lumbar fusion: A case report and literature review

PubMed Central

Ghaly, Ramsis F.; Lissounov, Alexei; Tverdohleb, Tatiana; Kohanchi, David; Candido, Kenneth D.; Knezevic, Nebojsa Nick

2016-01-01

Background: Bone morphogenic protein (BMP) for instrumented lumbar fusion was approved in 2002, and since then has led to an increasing incidence of BMP-related neuropathic pain. These patients are usually resistant to conventional medical therapy and frequently undergo multiple surgical revisions without any pain relief. Case Description: A 58-year-old male was referred to the author's outpatient clinic after four lumbar surgeries did not provide satisfactory pain relief. During his 10 years of suffering from low back pain after an injury, the patient was resistant to conventional and interventional treatment options. He was experiencing severe back pain rated 10/10, as well as right lower extremity pain, numbness, tingling, and motor deficits. Outside spine specialists had performed revision surgeries for BMP-related exuberant bone formation at L5–S1, which included the removal of the ipsilateral hardware and debridement of intradiscal and intraforamina heterotrophic exuberant bony formation. The author implanted the patient with a permanent continuous spinal cord stimulator, after which he achieved complete pain relief (0/10) and restoration of motor, sensory, autonomic, and sphincter functions. Conclusion: This is the first reported case of restorative function with neuromodulation therapy in a BMP-induced postoperative complication, which is considered as a primarily inflammatory process, rather than nerve root compression due to exuberant bony formation. We hypothesize that neuromodulation may enhance blood flow and interfere with inflammatory processes, in addition to functioning by the accepted gate control theory mechanism. The neuromodulation therapy should be strongly considered as a therapeutic approach, even with confirmed BMP-induced postoperative radiculitis, rather than proposing multiple surgical revisions. PMID:27843683

Topical bisphosphonate augments fixation of bone-grafted hydroxyapatite coated implants, BMP-2 causes resorption-based decrease in bone.

PubMed

Baas, Jorgen; Vestermark, Marianne; Jensen, Thomas; Bechtold, Joan; Soballe, Kjeld; Jakobsen, Thomas

2017-04-01

Bone allograft is used in total joint arthroplasties in order to enhance implant fixation. BMPs are known to stimulate new bone formation within allograft, but also known to accelerate graft resorption. Bisphosphonates are strong inhibitor of bone resorption. The aim of this study was to investigate whether the bisphosphonate zoledronate was able to counteract the accelerated graft resorption without interfering with the BMP induced bone formation. In the present study the two drugs alone and in combination were studied in our canine model of impaction bone grafting. We included 10 dogs in this study. Cancellous allograft bone grafts were soaked in either saline or zoledronate solution (0.005mg/mL) and then vehicle or BMP2 (0.15mg rhBMP2) was added. This produced four treatment groups: A) control, B) BMP2, C) zoledronate and D) BMP2+zoledronate. The allograft treated with A, B, C or D was impacted into a circumferential defect of 2.5mm around HA-coated porous Ti implants. Each dog received all four treatment groups with two implants in the distal part of each femur. The group with allograft soaked in zoledronate (C) showed better biomechanical fixation than all other groups (p<0.05). It had less allograft resorption compared to all other groups (p<0.005) without any statistically significant change in new bone formation. The addition of BMP2 to the allograft did not increase new bone formation significantly, but did accelerate allograft resorption. This was also the case where the allograft was treated with BMP2 and zoledronate in combination (D). This caused a decrease in mechanical implant fixation in both these groups compared to the control group, however only statistically significant for the BMP2 group compared to control. The study shows that topical zoledronate can be a valuable tool for augmenting bone grafts when administered optimally. The use of BMP2 in bone grafting procedures seems associated with a high risk of bone resorption and mechanical weakening. Copyright © 2017 Elsevier Inc. All rights reserved.

Topical Bisphosphonate Augments Fixation of Bone-grafted Hydroxyapatite coated Implants, BMP-2 causes Resorption-based decrease in Bone

PubMed Central

Baas, Jorgen; Vestermark, Marianne; Jensen, Thomas; Bechtold, Joan; Soballe, Kjeld; Jakobsen, Thomas

2017-01-01

Bone allograft is used in total joint artroplasties in order to enhance implant fixation. BMPs are known to stimulate new bone formation within allograft, but also known to accelerate graft resorption. Bisphosphonates are strong inhibitor of bone resorption. The aim of this study was to investigate whether the bisphosphonate zoledronate was able to counteract the accelerated graft resorption without interfering with the BMP induced bone formation. In the present study the two drugs alone and in combination were studied in our canine model of impaction bone grafting. We included 10 dogs in this study. Cancellous allograft bone grafts were soaked in either saline or zoledronate solution (0.005 mg/mL) and then vehicle or BMP2 (0.15 mg rhBMP2) was added. This produced four treatment groups: A) control B) BMP2 C) zoledronate and D) BMP2+ zoledronate. The allograft treated with A,B,C or D was impacted into a circumferential defect of 2.5 mm around HA-coated porous Ti implants. Each dog received all four treatment groups with two implants in the distal part of each femur. The group with allograft soaked in zoledronate (C) showed better biomechanical fixation than all other groups (p<0.05). It had less allograft resorption compared to all other groups (p<0.005) without any statistically significant change in new bone formation. The addition of BMP2 to the allograft did not increase new bone formation significantly, but did accelerate allograft resorption. This was also the case where the allograft was treated with BMP2 and zoledronate in combination (D). This caused a decrease in mechanical implant fixation in both these groups compared to the control group, however only statistically significant for the BMP2 group compared to control. The study shows that topical zoledronate can be a valuable tool for augmenting bone grafts when administered optimally. The use of BMP2 in bone grafting procedures seems associated with a high risk of bone resorption and mechanical weakening. PMID:28082076

Differential growth factor control of bone formation through osteoprogenitor differentiation.

PubMed

Chaudhary, L R; Hofmeister, A M; Hruska, K A

2004-03-01

The osteogenic factors bone morphogenetic protein (BMP-7), platelet-derived growth factor (PDGF)-BB, and fibroblast growth factor (FGF-2) regulate the recruitment of osteoprogenitor cells and their proliferation and differentiation into mature osteoblasts. However, their mechanisms of action on osteoprogenitor cell growth, differentiation, and bone mineralization remain unclear. Here, we tested the hypothesis that these osteogenic agents were capable of regulating osteoblast differentiation and bone formation in vitro. Normal human bone marrow stromal (HBMS) cells were treated with BMP-7 (40 ng ml(-1)), PDGF-BB (20 ng ml(-1)), FGF-2 (20 ng ml(-1)), or FGF-2 plus BMP-7 for 28 days in a serum-containing medium with 10 mM beta-glycerophosphate and 50 microg ml(-1) ascorbic acid. BMP-7 stimulated a morphological change to cuboidal-shaped cells, increased alkaline phosphatase (ALKP) activity, bone sialoprotein (BSP) gene expression, and alizarin red S positive nodule formation. Hydroxyapatite (HA) crystal deposition in the nodules was demonstrated by Fourier transform infrared (FTIR) spectroscopy only in BMP-7- and dexamethasone (DEX)-treated cells. DEX-treated cells appeared elongated and fibroblast-like compared to BMP-7-treated cells. FGF-2 did not stimulate ALKP, and cell morphology was dystrophic. PDGF-BB had little or no effect on ALKP activity and biomineralization. Alizarin Red S staining of cells and calcium assay indicated that BMP-7, DEX, and FGF-2 enhanced calcium mineral deposition, but FTIR spectroscopic analysis demonstrated no formation of HA similar to human bone in control, PDGF-BB-, and FGF-2-treated samples. Thus, FGF-2 stimulated amorphous octacalcium phosphate mineral deposition that failed to mature into HA. Interestingly, FGF-2 abrogated BMP-7-induced ALKP activity and HA formation. Results demonstrate that BMP-7 was competent as a sole factor in the differentiation of human bone marrow stromal cells to bone-forming osteoblasts confirmed by FTIR examination of mineralized matrix. Other growth factors, PDGF, and FGF-2 were incompetent as sole factors, and FGF-2 inhibited BMP-7-stimulated osteoblast differentiation.

Effect of recombinant human bone morphogenetic protein-2 on bone regeneration and osseointegration of dental implants.

PubMed

Sykaras, N; Triplett, R G; Nunn, M E; Iacopino, A M; Opperman, L A

2001-08-01

Recombinant human bone morphogenetic protein-2 (rhBMP-2) induced bone regeneration and osseointegration was evaluated in bony defects created within the hollow chamber of endosseous dental implants in 14 foxhound dogs. Bilateral extractions of mandibular premolars were performed and surgical implantation of 104 hollow cylinder implants followed after 8 weeks of healing. Experimental implants had their hollow chamber filled with 20 microg of rhBMP-2 delivered with a bovine collagen carrier, whereas the control implants had their apical chamber left empty. Dogs were followed for 2, 4, 8 and 12 weeks. Histomorphometric evaluation and immunohistochemical analysis were performed. Minimal bone was regenerated at 2 weeks for both groups. At 4 weeks, bone fill averaged 23.48% for the rhBMP-2 and 5.98% for the control group (P<0.05). At 8 weeks, mean bone fill was 20.94% and 7.75% for the rhBMP-2 and the controls, respectively (P<0.05). At 12 weeks, mean bone fill was 31.39% and 24.31% for the rhBMP-2 and control implants, respectively (P>0.05). Bone-implant contact (BIC) increased for both groups over time and at 8 weeks the rhBMP-2 BIC value was 18.65% and for the control 7.22% (P<0.05). At 12 weeks, the BIC was 43.78% and 21.05% for the rhBMP-2 and the control group, respectively (P<0.05). Immunohistochemical staining for type II collagen was positive only for parts of the collagen carrier and formation of cartilaginous intermediate was not observed in any of the specimens. The results suggest that, in confined defects adjacent to dental implants, rhBMP-2 can induce bone regeneration in close apposition to the implant surface.

Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity

PubMed Central

Alsamarah, Abdelaziz; LaCuran, Alecander E.; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

2015-01-01

Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

Repulsive Guidance Molecules (RGMs) and Neogenin in Bone Morphogenetic Protein (BMP) signaling

PubMed Central

Tian, Chenxi; Liu, Jun

2015-01-01

Summary Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGFβ) superfamily. BMPs mediate a highly conserved signal transduction cascade through the type I and type II serine/threonine kinase receptors and intracellular Smad proteins. The BMP pathway regulates multiple developmental and homeostatic processes. Mutations in this pathway can cause various diseases in humans, such as skeletal disorders, cardiovascular diseases and various cancers. Multiple levels of regulation, including extracellular regulation, help to ensure proper spatiotemporal control of BMP signaling in the right cellular context. The family of repulsive guidance molecules (RGMs) and the type I trans-membrane protein neogenin, a paralog of DCC (Deleted in Colorectal Cancer), have been implicated in modulating the BMP pathway. In this review, we discuss the properties and functions of RGM proteins and neogenin, focusing on their roles in the modulation of BMP signal transduction. PMID:23740870

Analysis of Recombinant Human Bone Morphogenetic Protein-2 Use in the Treatment of Lumbar Degenerative Spondylolisthesis.

PubMed

Yao, Qingqiang; Cohen, Jeremiah R; Buser, Zorica; Park, Jong-Beom; Brodke, Darrel S; Meisel, Hans-Joerg; Youssef, Jim A; Wang, Jeffrey C; Yoon, S Tim

2016-12-01

Study Design  Retrospective database review. Objective  To identify trends of the recombinant human bone morphogenetic protein-2 (rhBMP-2) use in the treatment of lumbar degenerative spondylolisthesis (LDS). Methods  PearlDiver Patient Record Database was used to identify patients who underwent lumbar fusion for LDS between 2005 and 2011. The distribution of bone morphogenetic protein use rate (BR) in various surgical procedures was recorded. Patient numbers, reoperation numbers, BR, and per year BR (PYBR) were stratified by geographic region, gender, and age. Results  There were 11,335 fusion surgeries, with 3,461 cases using rhBMP-2. Even though PYRB increased between 2005 and 2008, there was a significant decrease in 2010 for each procedure: 404 (34.5%) for posterior interbody fusion, 1,282 (34.3%) for posterolateral plus posterior interbody fusion (PLPIF), 1,477 (29.2%) for posterolateral fusion, and 335 (22.4%) for anterior lumbar interbody fusion. In patients using rhBMP-2, the reoperation rate was significantly lower than in patients not using rhBMP-2 (0.69% versus 1.07%, p  < 0.0001). Male patients had higher PYBR compared with female patients in 2008 and 2009 ( p  < 0.05). The West region and PLPIF had the highest BR and PYBR. Conclusions Our data shows that the revision rates were significantly lower in patients treated with rhBMP-2 compared with patients not treated with rhBMP-2. Furthermore, rhBMP-2 use in LDS varied by year, region, gender, and type of fusion technique. In the West region, the posterior approach and patients 65 to 69 years of age had the highest rate of rhBMP-2 use.

Analysis of Recombinant Human Bone Morphogenetic Protein-2 Use in the Treatment of Lumbar Degenerative Spondylolisthesis

PubMed Central

Yao, Qingqiang; Cohen, Jeremiah R.; Buser, Zorica; Park, Jong-Beom; Brodke, Darrel S.; Meisel, Hans-Joerg; Youssef, Jim A.; Wang, Jeffrey C.; Yoon, S. Tim

2016-01-01

Study Design Retrospective database review. Objective To identify trends of the recombinant human bone morphogenetic protein-2 (rhBMP-2) use in the treatment of lumbar degenerative spondylolisthesis (LDS). Methods PearlDiver Patient Record Database was used to identify patients who underwent lumbar fusion for LDS between 2005 and 2011. The distribution of bone morphogenetic protein use rate (BR) in various surgical procedures was recorded. Patient numbers, reoperation numbers, BR, and per year BR (PYBR) were stratified by geographic region, gender, and age. Results There were 11,335 fusion surgeries, with 3,461 cases using rhBMP-2. Even though PYRB increased between 2005 and 2008, there was a significant decrease in 2010 for each procedure: 404 (34.5%) for posterior interbody fusion, 1,282 (34.3%) for posterolateral plus posterior interbody fusion (PLPIF), 1,477 (29.2%) for posterolateral fusion, and 335 (22.4%) for anterior lumbar interbody fusion. In patients using rhBMP-2, the reoperation rate was significantly lower than in patients not using rhBMP-2 (0.69% versus 1.07%, p < 0.0001). Male patients had higher PYBR compared with female patients in 2008 and 2009 (p < 0.05). The West region and PLPIF had the highest BR and PYBR. Conclusions Our data shows that the revision rates were significantly lower in patients treated with rhBMP-2 compared with patients not treated with rhBMP-2. Furthermore, rhBMP-2 use in LDS varied by year, region, gender, and type of fusion technique. In the West region, the posterior approach and patients 65 to 69 years of age had the highest rate of rhBMP-2 use. PMID:27853658

Delayed Expression of Circulating TGF-β1 and BMP-2 Levels in Human Nonunion Long Bone Fracture Healing.

PubMed

Hara, Yoshiaki; Ghazizadeh, Mohammad; Shimizu, Hajime; Matsumoto, Hisashi; Saito, Nobuyuki; Yagi, Takanori; Mashiko, Kazuki; Mashiko, Kunihiro; Kawai, Makoto; Yokota, Hiroyuki

2017-01-01

The healing process of bone fracture requires a well-controlled multistage and sequential order beginning immediately after the injury. However, complications leading to nonunion exist, creating serious problems and costs for patients. Transforming growth factor-beta 1 (TGF-β1) and bone morphogenic protein 2 (BMP-2) are two major growth factors involved in human bone fracture healing by promoting various stages of bone ossification. In this study, we aimed to determine the role of these factors during the fracture healing of human long bones and assess their impacts on nonunion condition. We performed a comprehensive analysis of plasma TGF-β1 and BMP-2 levels in blood samples from 10 patients with proved nonunion and 10 matched patients with normal union following a predetermined time schedule. The concentrations of TGF-β1 and BMP-2 were measured at each time point using a solid-phase ELISA. TGF-β1 and BMP-2 levels were detectable in all patients. For all patients, a maximal peak for TGF-β1 was found at 3-week. In normal union group, TGF-β1 showed a maximal peak at 2-week while nonunion group had a delayed maximal peak at 3-week. Plasma levels of BMP-2 for all patients and for normal union group reached a maximal peak at 1-week, but nonunion group showed a delayed maximal peak at 2-week. In general, plasma TGF-β1 or BMP-2 level was not significantly different between normal union and nonunion groups. The expression levels of TGF-β1 and BMP-2 appeared to be delayed in nonunion patients which could play an important role in developing an early marker of fracture union condition and facilitate improved patient's management.

Arsenite suppression of BMP signaling in human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillips, Marjorie A.; Qin, Qin; Hu, Qin

2013-06-15

Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction,more » BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression. - Highlights: • BMP induces FOXN1 transcription. • BMP induces DUSP2 and DUSP14, suppressing ERK activation. • Arsenite suppresses levels of phosphorylated Smad1/5 and FOXN1 and DUSP mRNA. • These actions rationalize arsenite suppression of keratinocyte differentiation.« less

Autologous implantation of BMP2-expressing dermal fibroblasts to improve bone mineral density and architecture in rabbit long bones.

PubMed

Ishihara, Akikazu; Weisbrode, Steve E; Bertone, Alicia L

2015-10-01

Cell-mediated gene therapy may treat bone fragility disorders. Dermal fibroblasts (DFb) may be an alternative cell source to stem cells for orthopedic gene therapy because of their rapid cell yield and excellent plasticity with bone morphogenetic protein-2 (BMP2) gene transduction. Autologous DFb or BMP2-expressing autologous DFb were administered in twelve rabbits by two delivery routes; a transcortical intra-medullar infusion into tibiae and delayed intra-osseous injection into femoral drill defects. Both delivery methods of DFb-BMP2 resulted in a successful cell engraftment, increased bone volume, bone mineral density, improved trabecular bone microarchitecture, greater bone defect filling, external callus formation, and trabecular surface area, compared to non-transduced DFb or no cells. Cell engraftment within trabecular bone and bone marrow tissue was most efficiently achieved by intra-osseous injection of DFb-BMP2. Our results suggested that BMP2-expressing autologous DFb have enhanced efficiency of engraftment in target bones resulting in a measurable biologic response by the bone of improved bone mineral density and bone microarchitecture. These results support that autologous implantation of DFb-BMP2 warrants further study on animal models of bone fragility disorders, such as osteogenesis imperfecta and osteoporosis to potentially enhance bone quality, particularly along with other gene modification of these diseases. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Adipose-derived stem cells and BMP-2 delivery in chitosan-based 3D constructs to enhance bone regeneration in a rat mandibular defect model.

PubMed

Fan, Jiabing; Park, Hyejin; Lee, Matthew K; Bezouglaia, Olga; Fartash, Armita; Kim, Jinku; Aghaloo, Tara; Lee, Min

2014-08-01

Reconstructing segmental mandiblular defects remains a challenge in the clinic. Tissue engineering strategies provide an alternative option to resolve this problem. The objective of the present study was to determine the effects of adipose-derived stem cells (ASCs) and bone morphogenetic proteins-2 (BMP-2) in three-dimensional (3D) scaffolds on mandibular repair in a small animal model. Noggin expression levels in ASCs were downregulated by a lentiviral short hairpin RNA strategy to enhance ASC osteogenesis (ASCs(Nog-)). Chitosan (CH) and chondroitin sulfate (CS), natural polysaccharides, were fabricated into 3D porous scaffolds, which were further modified with apatite coatings for enhanced cellular responses and efficient delivery of BMP-2. The efficacy of 3D apatite-coated CH/CS scaffolds supplemented with ASCs(Nog-) and BMP-2 were evaluated in a rat critical-sized mandibular defect model. After 8 weeks postimplantation, the scaffolds treated with ASCs(Nog-) and BMP-2 significantly promoted rat mandibular regeneration as demonstrated by micro-computerized tomography, histology, and immunohistochemistry, compared with the groups treated with ASCs(Nog-) or BMP-2 alone. These results suggest that our combinatorial strategy of ASCs(Nog-)+BMP-2 in 3D apatite microenvironments can significantly promote mandibular regeneration, and these may provide a potential tissue engineering approach to repair large bony defects.

Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression.

PubMed

Xie, Xiaohua; Liu, Chao; Zhang, Hua; Jani, Priyam H; Lu, Yongbo; Wang, Xiaofang; Zhang, Bin; Qin, Chunlin

2016-05-05

Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2(f/f);Bmp4(f/f)ameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling.

Abrogation of epithelial BMP2 and BMP4 causes Amelogenesis Imperfecta by reducing MMP20 and KLK4 expression

PubMed Central

Xie, Xiaohua; Liu, Chao; Zhang, Hua; Jani, Priyam H.; Lu, Yongbo; Wang, Xiaofang; Zhang, Bin; Qin, Chunlin

2016-01-01

Amelogenesis Imperfecta (AI) can be caused by the deficiencies of enamel matrix proteins, molecules responsible for the transportation and secretion of enamel matrix components, and proteases processing enamel matrix proteins. In the present study, we discovered the double deletion of bone morphogenetic protein 2 (Bmp2) and bone morphogenetic protein 4 (Bmp4) in the dental epithelium by K14-cre resulted in hypoplastic enamel and reduced density in X-ray radiography as well as shortened enamel rods under scanning electron microscopy. Such enamel phenotype was consistent with the diagnosis of hypoplastic amelogenesis imperfecta. Histological and molecular analyses revealed that the removal of matrix proteins in the mutant enamel was drastically delayed, which was coincided with the greatly reduced expression of matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4). Although the expression of multiple enamel matrix proteins was down-regulated in the mutant ameloblasts, the cleavage of ameloblastin was drastically impaired. Therefore, we attributed the AI primarily to the reduction of MMP20 and KLK4. Further investigation found that BMP/Smad4 signaling pathway was down-regulated in the K14-cre;Bmp2f/f;Bmp4f/fameloblasts, suggesting that the reduced MMP20 and KLK4 expression may be due to the attenuated epithelial BMP/Smad4 signaling. PMID:27146352

BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia

PubMed Central

Feng, Yanmin; Feng, Xue; Wang, Xiuxia; Gan, Haiyun; Wang, Lixian; Lin, Xiwen

2016-01-01

Spermatogenesis is sustained by the proliferation and differentiation of spermatogonial stem cells (SSCs). However, the molecules controlling these processes remain largely unknown. Here, we developed a simplified high concentration serum-containing system for the culture of mouse SSCs. Analysis of SSCs markers and transplantation results revealed that the cultured spermatogonia retained stem cell characteristics after long-term in vitro propagation. Using this culture system, the expression and function of bone morphogenetic protein 4 (BMP4) were explored. Immunostaining showed that BMP4 was predominantly expressed in germ cells and that its level increased as spermatogenesis progresses. BMP4 receptors BMPR1A and BMPRII were present in spermatogonia, spermatocytes, and round spermatids. Moreover, despite the mRNAs of these two genes being present in mouse Sertoli cells, only BMPRII was detected by using Western blotting assays. While exogenous BMP4 by itself did not induce the expression of Stra8 and c-Kit, two marker genes of differentiating spermatogonia, a significant cooperative effect of BMP4 and retinoic acid (RA) was observed. Moreover, pretreatment of cultured spermatogonia with the BMP4 antagonist Noggin could inhibit RA-induced expression of these two marker genes. In conclusion, BMP4 may exert autocrine effects and act cooperatively with RA to induce the differentiation of spermatogonia in vivo. PMID:27795714

Continuous delivery of rhBMP2 and rhVEGF165 at a certain ratio enhances bone formation in mandibular defects over the delivery of rhBMP2 alone--An experimental study in rats.

PubMed

Lohse, N; Moser, N; Backhaus, S; Annen, T; Epple, M; Schliephake, H

2015-12-28

The aim of the present study was to test the hypothesis that different amounts of vascular endothelial growth factor and bone morphogenic protein differentially affect bone formation when applied for repair of non-healing defects in the rat mandible. Porous composite PDLLA/CaCO3 carriers were fabricated as slow release carriers and loaded with rhBMP2 and rhVEGF165 in 10 different dosage combinations using gas foaming with supercritical carbon dioxide. They were implanted in non-healing defects of the mandibles of 132 adult Wistar rats with additional lateral augmentation. Bone formation was assessed both radiographically (bone volume) and by histomorphometry (bone density). The use of carriers with a ratio of delivery of VEGF/BMP between 0.7 and 1.2 was significantly related to the occurrence of significant increases in radiographic bone volume and/or histologic bone density compared to the use of carriers with a ratio of delivery of ≤ 0.5 when all intervals and all outcome parameters were considered. Moreover, simultaneous delivery at this ratio helped to "save" rhBMP2 as both bone volume and bone density after 13 weeks were reached/surpassed using half the dosage required for rhBMP2 alone. It is concluded, that the combined delivery of rhVEGF165 and rhBMP2 for repair of critical size mandibular defects can significantly enhance volume and density of bone formation over delivery of rhBMP2 alone. It appears from the present results that continuous simultaneous delivery of rhVEGF165 and rhBMP2 at a ratio of approximately 1 is favourable for the enhancement of bone formation. Copyright © 2015. Published by Elsevier B.V.

The human chondrosarcoma HCS-2/8 cell line is responsive to BMP-7, but not to IL-1beta.

PubMed

Saas, Joachim; Gebauer, Matthias; Jacobi, Carsten; Haag, Jochen; Takigawa, Masaharu; Aigner, Thomas

2005-05-01

Cultures of primary chondrocytes as in vitro model systems for studying the cellular behavior of chondrocytes are notoriously difficult to cultivate and propagate. One way to circumvent these problems appears to be the use of immortalized/immortal chondrocytic cell lines. In the present study, we were interested whether the chondrosarcoma derived HCS-2/8 cells are suitable for studying major cellular reaction pattern in response to key anabolic (BMP-7) and catabolic (IL-1beta) factors. Therefore, we used cDNA array and real-time PCR technology in order to evaluate gene expression triggered by stimulation with IL-1beta (0,1-100 ng/ml) and BMP-7 in confluent monolayer cultures. HCS-2/8 cells hardly responded to IL-1beta, but showed good responsiveness to BMP-7. We found 12 genes up- and 17 significantly down-regulated by BMP-7 (out of 340 investigated genes). Besides the expected activation of anabolic genes chondrocytic cells after BMP-stimulation try to neutralize activation of the BMP-signalling cascade by expressing intra- and extracellular BMP-antagonists. Chondrosarcoma derived cell lines are a potential substitute for primary articular chondrocytes promising consistent expression of a differentiated chondrocyte phenotype with sufficient proliferative capacity. However, as shown by this study one needs to carefully select the cell line depending on the effects which one intends to study. In this respect, HCS-2/8 cells are a validated tool for studying BMP-effects on chondrocytes, but not e.g. effects of interleukin-1.

Anterior Cruciate Ligament-Derived Stem Cells Transduced With BMP2 Accelerate Graft-Bone Integration After ACL Reconstruction.

PubMed

Kawakami, Yohei; Takayama, Koji; Matsumoto, Tomoyuki; Tang, Ying; Wang, Bing; Mifune, Yutaka; Cummins, James H; Warth, Ryan J; Kuroda, Ryosuke; Kurosaka, Masahiro; Fu, Freddie H; Huard, Johnny

2017-03-01

Strong graft-bone integration is a prerequisite for successful graft remodeling after reconstruction of the anterior cruciate ligament (ACL) using soft tissue grafts. Novel strategies to accelerate soft tissue graft-bone integration are needed to reduce the need for bone-tendon-bone graft harvest, reduce patient convalescence, facilitate rehabilitation, and reduce total recovery time after ACL reconstruction. The application of ACL-derived stem cells with enhanced expression of bone morphogenetic protein 2 (BMP2) onto soft tissue grafts in the form of cell sheets will both accelerate and improve the quality of graft-bone integration after ACL reconstruction in a rat model. Controlled laboratory study. ACL-derived CD34+ cells were isolated from remnant human ACL tissues, virally transduced to express BMP2, and embedded within cell sheets. In a rat model of ACL injury, bilateral single-bundle ACL reconstructions were performed, in which cell sheets were wrapped around tendon autografts before reconstruction. Four groups containing a total of 48 rats (96 knees) were established (n = 12 rats; 24 knees per group): CD34+BMP2 (100%), CD34+BMP2 (25%), CD34+ (untransduced), and a control group containing no cells. Six rats from each group were euthanized 2 and 4 weeks after surgery, and each graft was harvested for immunohistochemical and histological analyses. The remaining 6 rats in each group were euthanized at 4 and 8 weeks to evaluate in situ tensile load to failure in each femur-graft-tibia complex. In vitro, BMP2 transduction promoted the osteogenic differentiation of ACL-derived CD34+ cells while retaining their intrinsic multipotent capabilities. Osteoblast densities were greatest in the BMP2 (100%) and BMP2 (25%) groups. Bone tunnels in the CD34+BMP2 (100%) and CD34+BMP2 (25%) groups had the smallest cross-sectional areas according to micro-computed tomography analyses. Graft-bone integration occurred most rapidly in the CD34+BMP2 (25%) group. Tensile load to failure was significantly greater in the groups containing stem cells at 4 and 8 weeks after surgery. Tensile strength was greatest in the CD34+BMP2 (100%) group at 4 weeks, and in the CD34+BMP2 (25%) group at 8 weeks. ACL-derived CD34+ cells transduced with BMP2 accelerated graft-bone integration after ACL reconstruction using soft tissue autografts in a rat model, as evidenced by improved histological appearance and graft-bone interface biology along with tensile load to failure at each time point up to 8 weeks after surgery. A primary disadvantage of using soft tissue grafts for ACL reconstruction is the prolonged time required for bony ingrowth, which delays the initiation of midsubstance graft remodeling. The lack of consistent correlation between the appearance of a "healed" ACL on postoperative magnetic resonance imaging and readiness to return to sport results in athletes being released to sport before the graft is ready to handle high-intensity loading. Therefore, it is desirable to identify strategies that accelerate graft-bone integration, which would reduce the time to biologic fixation, improve the reliability of biologic fixation, allow for accelerated rehabilitation, and potentially reduce the incidence of early graft pullout and late midsubstance failure.

3-Dimensional functionalized polycaprolactone-hyaluronic acid hydrogel constructs for bone tissue engineering.

PubMed

Hamlet, Stephen M; Vaquette, Cedryck; Shah, Amit; Hutmacher, Dietmar W; Ivanovski, Saso

2017-04-01

Alveolar bone regeneration remains a significant clinical challenge in periodontology and dental implantology. This study assessed the mineralized tissue forming potential of 3-D printed medical grade polycaprolactone (mPCL) constructs containing osteoblasts (OB) encapsulated in a hyaluronic acid (HA)-hydrogel incorporating bone morphogenetic protein-7 (BMP-7). HA-hydrogels containing human OB ± BMP-7 were prepared. Cell viability, osteogenic gene expression, mineralized tissue formation and BMP-7 release in vitro, were assessed by fluorescence staining, RT-PCR, histological/μ-CT examination and ELISA respectively. In an athymic rat model, subcutaneous ectopic mineralized tissue formation in mPCL-hydrogel constructs was assessed by μ-CT and histology. Osteoblast encapsulation in HA-hydrogels did not detrimentally effect cell viability, and 3-D culture in osteogenic media showed mineralized collagenous matrix formation after 6 weeks. BMP-7 release from the hydrogel was biphasic, sustained and increased osteogenic gene expression in vitro. After 4 weeks in vivo, mPCL-hydrogel constructs containing BMP-7 formed significantly more volume (mm 3 ) of vascularized bone-like tissue. Functionalized mPCL-HA hydrogel constructs provide a favourable environment for bone tissue engineering. Although encapsulated cells contributed to mineralized tissue formation within the hydrogel in vitro and in vivo, their addition did not result in an improved outcome compared to BMP-7 alone. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

BMP regulates regional gene expression in the dorsal otocyst through canonical and non-canonical intracellular pathways

PubMed Central

2016-01-01

The inner ear consists of two otocyst-derived, structurally and functionally distinct components: the dorsal vestibular and ventral auditory compartments. BMP signaling is required to form the vestibular compartment, but how it complements other required signaling molecules and acts intracellularly is unknown. Using spatially and temporally controlled delivery of signaling pathway regulators to developing chick otocysts, we show that BMP signaling regulates the expression of Dlx5 and Hmx3, both of which encode transcription factors essential for vestibular formation. However, although BMP regulates Dlx5 through the canonical SMAD pathway, surprisingly, it regulates Hmx3 through a non-canonical pathway involving both an increase in cAMP-dependent protein kinase A activity and the GLI3R to GLI3A ratio. Thus, both canonical and non-canonical BMP signaling establish the precise spatiotemporal expression of Dlx5 and Hmx3 during dorsal vestibular development. The identification of the non-canonical pathway suggests an intersection point between BMP and SHH signaling, which is required for ventral auditory development. PMID:27151948

Essential Roles of Epithelial Bone Morphogenetic Protein Signaling During Prostatic Development

PubMed Central

Omori, Akiko; Miyagawa, Shinichi; Ogino, Yukiko; Harada, Masayo; Ishii, Kenichiro; Sugimura, Yoshiki; Ogino, Hajime; Nakagata, Naomi

2014-01-01

Prostate is a male sex-accessory organ. The prostatic epithelia consist primarily of basal and luminal cells that differentiate from embryonic urogenital sinus epithelia. Prostate tumors are believed to originate in the basal and luminal cells. However, factors that promote normal epithelial differentiation have not been well elucidated, particularly for bone morphogenetic protein (Bmp) signaling. This study shows that Bmp signaling prominently increases during prostatic differentiation in the luminal epithelia, which is monitored by the expression of phosphorylated Smad1/5/8. To elucidate the mechanism of epithelial differentiation and the function of Bmp signaling during prostatic development, conditional male mutant mouse analysis for the epithelial-specific Bmp receptor 1a (Bmpr1a) was performed. We demonstrate that Bmp signaling is indispensable for luminal cell maturation, which regulates basal cell proliferation. Expression of the prostatic epithelial regulatory gene Nkx3.1 was significantly reduced in the Bmpr1a mutants. These results indicate that Bmp signaling is a key factor for prostatic epithelial differentiation, possibly by controlling the prostatic regulatory gene Nkx3.1. PMID:24731097

A poly (lactide-co-glycolide) film loaded with abundant bone morphogenetic protein-2: A substrate promoting osteoblast attachment, proliferation and differentiation in bone tissue engineering.

PubMed

Qu, Xiangyang; Cao, Yujiang; Chen, Cong; Die, Xiaohong; Kang, Quan

2014-12-10

We explored a novel biodegradable poly (lactide-co-glycolide) (PLGA) film loaded with over 80 wt% bone morphogenetic protein (BMP-2), which was regarded as a substrate promoting osteoblast attachment, proliferation and differentiation for application of bone tissue engineering. Using phospholipid as a surfactant, BMP-2 was modified as a complex (PBC) for dispersing in PLGA/dichloromethane solution. The PLGA film loaded with BMP-2 and phospholipid complex (PBC-PF) showed rough and draped morphology with high entrapment efficiency exceeding 80% and good hydrophilicity respectively. The in-vitro release study of BMP-2 showed that about 50% BMP-2 was slowly and continuously released from PBC-PF within 5 weeks and had a short initial burst release only in the last 1.5 days, which was better than serious burst release of PLGA film loaded with pure BMP-2 without phospholipid (BMP-PF) controlling. By comparison with other PLGA films and tissue culture plates, it was confirmed that PBC-PF significantly promoted the attachment, proliferation and differentiation of osteoblasts with higher entrapment efficiency and better sustained release. These advantages illustrated that PBC-PF could be a potential substrate providing long-term requisite growth factors for osteoblasts, which might be applied in bone tissue engineering. This article is protected by copyright. All rights reserved. Copyright © 2014 Wiley Periodicals, Inc., A Wiley Company.

A poly(lactide-co-glycolide) film loaded with abundant bone morphogenetic protein-2: A substrate-promoting osteoblast attachment, proliferation, and differentiation in bone tissue engineering.

PubMed

Qu, Xiangyang; Cao, Yujiang; Chen, Cong; Die, Xiaohong; Kang, Quan

2015-08-01

We explored a novel biodegradable poly(lactide-co-glycolide) (PLGA) film loaded with over 80 wt % bone morphogenetic protein (BMP)-2, which was regarded as a substrate-promoting osteoblast attachment, proliferation, and differentiation for application of bone tissue engineering. Using phospholipid as a surfactant, BMP-2 was modified as a complex (PBC) for dispersing in PLGA/dichloromethane solution. The PLGA film loaded with BMP-2 and phospholipid complex (PBC-PF) showed rough and draped morphology with high entrapment efficiency exceeding 80% and good hydrophilicity, respectively. The in vitro release study of BMP-2 showed that about 50% BMP-2 was slowly and continuously released from PBC-PF within 5 weeks and had a short initial burst release only in the last 1.5 days, which was better than serious burst release of PLGA film loaded with pure BMP-2 without phospholipid (BMP-PF) as control. By comparison with other PLGA films and tissue culture plates, it was confirmed that PBC-PF significantly promoted the attachment, proliferation, and differentiation of osteoblasts with higher entrapment efficiency and better sustained release. These advantages illustrated that PBC-PF could be a potential substrate providing long-term requisite growth factors for osteoblasts, which might be applied in bone tissue engineering. © 2015 Wiley Periodicals, Inc.

Influences of Reduced Expression of Maternal Bone Morphogenetic Protein 2 on Embryonic Development

PubMed Central

Singh, Ajeet P.; Castranio, Trisha; Scott, Greg; Guo, Dayong; Harris, Marie A.; Ray, Manas; Harris, Stephan E.; Mishina, Yuji

2009-01-01

Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. During a course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeting events in embryonic stem cells, in the 3’ untranslated region of exon 3 of Bmp2, reduced the expression levels of Bmp2 both in embryonic and maternal tissues. Some of the embryos that were genotyped as transheterozygous for the floxed allele with the neo cassette over the conventional null allele (fn/−) showed a lethal phenotype including defects in cephalic neural tube closure and ventral abdominal wall closure. Embryos exhibiting these abnormalities were increased when genotypes of the pregnant females were different; when expression levels of Bmp2 in maternal tissues were lower, a larger proportion of fn/− embryos exhibit these abnormalities. These results suggest that the expression levels of Bmp2 together in both in embryonic and maternal tissues influence the normal neural tube closure and body wall closure with different thresholds. PMID:18769073

[In vitro differentiation of synovial-derived mesenchymal stem cells infected by adenovirus vector mediated by bone morphogenetic protein 2/7 genes into fibrocartilage cells in rabbits].

PubMed

Fu, Peiliang; Zhang, Lei; Wu, Haishan; Cong, Ruijun; Chen, Song; Ding, Zheru; Hu, Kaimen

2013-03-01

To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 +/- 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

Influence of bone morphogenetic protein and proportion of hydroxyapatite on new bone formation in biphasic calcium phosphate graft: two pilot studies in animal bony defect model.

PubMed

Yun, Pil-Young; Kim, Young-Kyun; Jeong, Kyung-In; Park, Ju-Cheol; Choi, Yeon-Jo

2014-12-01

The purpose of these two pilot studies using animal bony defect models was to evaluate the influence of bone morphogenetic protein (BMP) and proportion of hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) in biphasic calcium phosphate (BCP) graft on new bone formation. In this study, four kinds of synthetic osteoconductive bone materials known for bone growth scaffold, OSTEON™II(HA:β-TCP 30:70), OSTEON™III (HA:β-TCP 20:80), OSTEON™II Collagen, and OSTEON™III Collagen, were prepared as BCP graft materials. In pilot study 1, three BCP materials (OSTEON™II, OSTEON™III, and OSTEON™II Collagen) were grafted in rabbit calvarial defects after impregnating in rhBMP-2. OSTEON™II without the rhBMP-2 impregnation was included in the study as the control. The amount of new bone was examined and measured histologically at 2, 4, and 8 weeks. In pilot study 2, four BCP materials (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) were grafted in beagle dog mandibular defects after soaking in the rhBMP-2. The amount of total bone and new bone were measured three-dimensionally using microCT and healing process was examined histologically at 2, 4, and 8 weeks. In pilot study 1, rhBMP-2 impregnated groups showed more new bone formation than the rhBMP-2 free group. In pilot study 2, increased new bone formation was observed in time-dependent manner after graft of BCP and BCP-collagen (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) impregnated with rhBMP-2. Also, BCP with a higher proportion of HA (30% HA) showed more favorable result in new bone formation and space maintenance, especially at the 8 weeks. From the results of the pilot studies, rhBMP-2 played positive roles in new bone formation and BCP could become a scaffold candidate for rhBMP-2 impregnation to induce new bone formation. Moreover, BCP with a higher proportion of HA (30% HA) could be considered more appropriate for rhBMP-2 carrier. Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: loss of chondrocyte stability.

PubMed

Krawczak, David A; Westendorf, Jennifer J; Carlson, Cathy S; Lewis, Jack L

2009-06-01

The aim of this study was to determine the effects of bone morphogenetic protein-2 (BMP-2) on articular chondrocyte tissues grown as monolayers in vitro for up to 8 weeks. Articular chondrocytes were isolated from New Zealand White rabbits and plated in monolayer cultures. The cultures were supplemented with 100 ng/mL of BMP-2 for up to 8 weeks and the extracellular matrix (ECM) composition, material properties, and messenger RNA (mRNA) expression were analyzed. mRNA expression of cartilage-specific genes, type II collagen, and aggrecan showed that BMP-2 enhanced chondrocyte stability for up to 3 weeks. After 3 weeks in culture, there was substantially more type I collagen expression and more osteopontin and runt-related transcription factor 2 expression in 5- and 8-week cultures treated with BMP-2 than in controls. Additionally, matrix metalloproteinase-13 and ADAMTS-5 (A disintegrin-like and metalloproteinase with thrombospondin 5) were upregulated in 5- and 8-week cultures treated with BMP-2, coinciding with a loss of ECM density, collagen, and proteoglycan. Eight-week tissue stimulated with BMP-2 was more fragile and tore more easily when removed from the culture dish as compared to controls, suggesting temporal limitations to the effectiveness of BMP-2 in monolayer systems and perhaps other models to enhance the generation of a cartilage-like tissue for tissue engineering purposes.

Effectiveness and harms of recombinant human bone morphogenetic protein-2 in spine fusion: a systematic review and meta-analysis.

PubMed

Fu, Rongwei; Selph, Shelley; McDonagh, Marian; Peterson, Kimberly; Tiwari, Arpita; Chou, Roger; Helfand, Mark

2013-06-18

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is used as a bone graft substitute in spinal fusion, which unites (fuses) bones in the spine. The accuracy and completeness of journal publications of industry-sponsored trials on the effectiveness and harms of rhBMP-2 has been called into question. To independently assess the effectiveness and harms of rhBMP-2 in spinal fusion and reporting bias in industry-sponsored journal publications. Individual-patient data (IPD) from 17 industry-sponsored studies; related internal documents; and searches of MEDLINE (1996 to August 2012), other databases, and reference lists. Randomized, controlled trials (RCTs) and cohort studies of rhBMP-2 versus any control and uncontrolled studies of harms. Effectiveness outcomes in IPD were recalculated using consistent definitions. Study characteristics and results were abstracted by 1 investigator and confirmed by another. Two investigators independently assessed quality using predefined criteria. Thirteen RCTs and 31 cohort studies were included. For lumbar spine fusion, rhBMP-2 and iliac crest bone graft were similar in overall success, fusion, and other effectiveness measures and in risk for any adverse event, although rates were high across interventions (77% to 93% at 24 months from surgery). For anterior lumbar interbody fusion, rhBMP-2 was associated with nonsignificantly increased risk for retrograde ejaculation and urogenital problems. For anterior cervical spine fusion, rhBMP-2 was associated with increased risk for wound complications and dysphagia. At 24 months, the cancer risk was increased with rhBMP-2 (risk ratio, 3.45 [95% CI, 1.98 to 6.00]), but event rates were low and cancer was heterogeneous. Early journal publications misrepresented the effectiveness and harms through selective reporting, duplicate publication, and underreporting. Outcome assessment was not blinded, and ascertainment of harms in trials was poor. No trials were truly independent of industry sponsorship. In spinal fusion, rhBMP-2 has no proven clinical advantage over bone graft and may be associated with important harms, making it difficult to identify clear indications for rhBMP-2. Earlier disclosure of all relevant data would have better informed clinicians and the public than the initial published trial reports did. Yale University and Medtronic.

Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration.

PubMed

González-Sastre, Alejandro; Molina, Ma Dolores; Saló, Emili

2012-01-01

Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain further insight into the role of this signalling pathway in planarians, we have isolated and functionally characte-rized the inhibitory Smads (I-Smads) in Schmidtea mediterranea. Two I-Smad homologues have been identified: Smed-smad6/7-1 and Smed-smad6/7-2. Expression of smad6/7-1 was detected in the parenchyma, while smad6/7-2 was found to be ex-pressed in the central nervous system and the eyes. Neither single smad6/7-1 and smad6/7-2 nor double smad6/7-1,-2 silencing gave rise to any apparent disruption of the DV axis. However, both regenerating and intact smad6/7-2 (RNAi) planarians showed defects in eye morphogenesis and displayed small, rounded eyes that lacked the anterior subpopulation of photoreceptor cells. The number of pigment cells was also reduced in these animals at later stages of regeneration. In contrast, after low doses of Smed-bmp(RNAi), planarians regenerated larger eyes in which the anterior subpopulation of photoreceptor cells was expanded. Our results suggest that Smed-smad6/7-2 and Smed-bmp control the re-specification and maintenance of anterior photoreceptor cell number in S. mediterranea.

Development of a Model System to Evaluate Local Recurrence in Osteosarcoma and Assessment of the Effects of Bone Morphogenetic Protein-2.

PubMed

Geller, David S; Singh, Michael Y; Zhang, Wendong; Gill, Jonathan; Roth, Michael E; Kim, Mimi Y; Xie, Xianhong; Singh, Christopher K; Dorfman, Howard D; Villanueva-Siles, Esperanza; Park, Amy; Piperdi, Sajida; Gorlick, Richard

2015-07-01

It is increasingly relevant to better define what constitutes an adequate surgical margin in an effort to improve reconstructive longevity and functional outcomes following osteosarcoma surgery. In addition, nonunion remains a challenging problem in some patients following allograft reconstruction. Bone morphogenetic protein-2 (BMP-2) could enhance osseous union, but has been historically avoided due to concerns that it may promote tumor recurrence. An orthotopic xenograft murine model was utilized to describe the natural temporal course of osteosarcoma growth. Tumors were treated either with surgery alone, surgery and single-agent chemotherapy, or surgery and dual-agent chemotherapy to assess the relationship between surgical margin and local recurrence. The effect of BMP-2 on local recurrence was similarly assessed. Osteosarcoma tumor growth was categorized into reproducible phases. Margins greater than 997 μm resulted in local control following surgery alone. Margins greater than 36 μm resulted in local control following surgery and single-agent chemotherapy. Margins greater than 12 μm resulted in local control following surgery and dual-agent chemotherapy. The application of exogenous BMP-2 does not confer an increased risk of local recurrence. This model reliably reproduces the clinical, radiographic, and surgical conditions encountered in human osteosarcoma. It successfully incorporates relevant chemotherapy, further paralleling the human experience. Surgical margins required to achieve local control in osteosarcoma can be reduced using single-agent chemotherapy and further decreased using dual-agent chemotherapy. The application of BMP-2 does not increase local recurrence in this model. ©2014 American Association for Cancer Research.

Thyroid Hormone-Induced Hypertrophy in Mesenchymal Stem Cell Chondrogenesis Is Mediated by Bone Morphogenetic Protein-4

PubMed Central

Karl, Alexandra; Olbrich, Norman; Pfeifer, Christian; Berner, Arne; Zellner, Johannes; Kujat, Richard; Angele, Peter; Nerlich, Michael

2014-01-01

Chondrogenic differentiating mesenchymal stem cells (MSCs) express markers of hypertrophic growth plate chondrocytes. As hypertrophic cartilage undergoes ossification, this is a concern for the application of MSCs in articular cartilage tissue engineering. To identify mechanisms that elicit this phenomenon, we used an in vitro hypertrophy model of chondrifying MSCs for differential gene expression analysis and functional experiments with the focus on bone morphogenetic protein (BMP) signaling. Hypertrophy was induced in chondrogenic MSC pellet cultures by transforming growth factor β (TGFβ) and dexamethasone withdrawal and addition of triiodothyronine. Differential gene expression analysis of BMPs and their receptors was performed. Based on these results, the in vitro hypertrophy model was used to investigate the effect of recombinant BMP4 and the BMP inhibitor Noggin. The enhancement of hypertrophy could be shown clearly by an increased cell size, alkaline phosphatase activity, and collagen type X deposition. Upon induction of hypertrophy, BMP4 and the BMP receptor 1B were upregulated. Addition of BMP4 further enhanced hypertrophy in the absence, but not in the presence of TGFβ and dexamethasone. Thyroid hormone induced hypertrophy by upregulation of BMP4 and this induced enhancement of hypertrophy could be blocked by the BMP antagonist Noggin. BMP signaling is an important modulator of the late differentiation stages in MSC chondrogenesis and the thyroid hormone induces this pathway. As cartilage tissue engineering constructs will be exposed to this factor in vivo, this study provides important insight into the biology of MSC-based cartilage. Furthermore, the possibility to engineer hypertrophic cartilage may be helpful for critical bone defect repair. PMID:23937304

Secreted phosphoprotein 24 kD (Spp24) inhibits growth of human pancreatic cancer cells caused by BMP-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Chen-Shuang; Tian, Haijun, E-mail: haijuntianmd@gmail.com; Department of Orthopaedic Surgery, University of California, Los Angeles, Los Angeles, CA

The emerging role of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers has drawn great attention in cancer research. In this study, we report that BMP-2 can promote the proliferation of the pancreatic tumor cell line, PANC-1. Secreted phosphoprotein 24 kD (Spp24), a BMP binding protein, did not affect the proliferation of the cells but promoted the apoptosis of the cells in vitro. In a xeneograft tumor model using PANC-1 cells, BMP-2 dramatically promoted tumor growth, while Spp24 not only abolished the effect of BMP-2, but also dramatically induced tumor shrinking when used alone. Activation of Smad1/5/8 participatedmore » in this process as demonstrated by immunohistochemical staining of phosphorylated Smad 1/5/8. We conclude that Spp24 can be developed into a therapeutic agent that could be employed in clinical situations where the inhibition of BMPs and related proteins is advantageous. - Highlights: • Spp24 effectively inhibited the in vivo tumor growth of PANC-1. • BMP-2 dramatically promoted tumor growth by promoting PANC-1 proliferation. • Spp24 abolished the tumor growth effect of BMP-2 by promoting PANC-1 apoptosis. • Spp24 may be a candidate as a therapeutic agent of pancreatic cancer.« less

The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians.

PubMed

Molina, M Dolores; Saló, Emili; Cebrià, Francesc

2007-11-01

The bone morphogenetic protein (BMP) pathway has been shown to play an important role in the establishment of the dorsoventral axis during development in both vertebrate and invertebrate species. In an attempt to unravel the role of BMPs in pattern formation during planarian regeneration, we studied this signaling pathway in Schmidtea mediterranea. Here, we functionally characterize planarian homologues of two key elements of the pathway: Smed-BMP and Smed-Smad1. Whole-mount in situ hybridization showed that Smed-BMP is expressed at the planarian dorsal midline, suggesting a role in dorsoventral patterning, while Smed-Smad1 is widely expressed throughout the mesenchyme and in the central nervous system. RNA interference (RNAi) knockdowns of Smed-BMP or Smed-Smad1 led to the disappearance of dorsal markers along with the ectopic expression of ventral markers on the dorsal side of the treated animals. In almost all cases, a duplicated central nervous system differentiated dorsally after Smed-BMP or Smed-Smad1 RNAi. These defects were observed not only during regeneration but also in intact non-regenerating animals. Our results suggest that the BMP signaling pathway is conserved in planarians and that it plays a key role in the regeneration and maintenance of the dorsoventral axis.

SNW1 Is a Critical Regulator of Spatial BMP Activity, Neural Plate Border Formation, and Neural Crest Specification in Vertebrate Embryos

PubMed Central

Wu, Mary Y.; Ramel, Marie-Christine; Howell, Michael; Hill, Caroline S.

2011-01-01

Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and thus neural crest specification. PMID:21358802

Exogenous γ-aminobutyric Acid (GABA) Application Improved Early Growth, Net Photosynthesis, and Associated Physio-Biochemical Events in Maize.

PubMed

Li, Wu; Liu, Jianhua; Ashraf, Umair; Li, Gaoke; Li, Yuliang; Lu, Wenjia; Gao, Lei; Han, Fuguang; Hu, Jianguang

2016-01-01

γ-aminobutyric acid (GABA) is an endogenous signaling molecule and involved in growth regulations and plant development, however, a little information is available on the consequences of exogenous GABA application on growth, development, and associated physio-biochemical processes in maize. The present study examined the GABA-induced regulations in early growth, net photosynthetic rate, gas exchange, osmoregulation, and enzymatic activities in three maize cultivars, i.e., Yuecainuo 6, Zhengtian 68, and Yuecainuo 2. Two levels of GABA, i.e., 0 mg L(-1) and 50 mg L(-1), in solution form, with total application volume of 100 ml per pot containing 15 maize seedlings were exogenously applied. Results revealed that exogenous GABA application improved seedling growth in terms of seedling length and biomass accumulation in all maize cultivars at both 3 and 7 days after treatment (DAT). It also promoted net photosynthesis and variably affected gas exchange attributes, i.e., stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr), as well as leaves SPAD value. Furthermore, lipid peroxidation [in terms of malondialdehyde (MDA)] under GABA treated maize seedlings were also remained variable; however, osmolyte accumulation (protein and proline) and activities of anti-oxidants enzymes, i.e., super-oxide dismutase and peroxidase were also affected differently at both 3 and 7 DAT in all maize cultivars. Furthermore, enzymes involved in nitrogen metabolism, e.g., nitrate reductase and glutamine synthetase were improved. These results suggest the involvement of GABA in various physio-metablical mechanisms which might lead to improvement in morphological growth of maize. In future, research is still needed at molecular and genetic levels to unravel the involvement of GABA-mediated regulations in growth and its associated physio-biochemical mechanisms.

Cholesteryl group- and acryloyl group-bearing pullulan nanogel to deliver BMP2 and FGF18 for bone tissue engineering.

PubMed

Fujioka-Kobayashi, Masako; Ota, Masato S; Shimoda, Asako; Nakahama, Ken-ichi; Akiyoshi, Kazunari; Miyamoto, Youji; Iseki, Sachiko

2012-10-01

To create a drug delivery system that allows the controlled release of proteins, such as growth factors, over a long-term period, cholesteryl group- and acryloyl group-bearing pullulan (CHPOA) nanogels were aggregated to form fast-degradable hydrogels (CHPOA/hydrogels) by cross-linking with thiol-bearing polyethylene glycol. The gold standard of clinical bone reconstruction therapy with a physiologically active material is treatment with recombinant human bone morphogenetic protein 2 (BMP2); however, this approach has limitations, such as inflammation, poor cost-efficiency, and varying interindividual susceptibility. In this study, two distinct growth factors, BMP2 and recombinant human fibroblast growth factor 18 (FGF18), were applied to a critical-size skull bone defect for bone repair by the CHPOA/hydrogel system. The CHPOA-FGF18/hydrogel displayed identical results to the control CHPOA-PBS/hydrogel, and the CHPOA-BMP2/hydrogel treatment imperfectly induced bone repair. By contrast, the CHPOA-FGF18 + BMP2/hydrogel treatment strongly enhanced and stabilized the BMP2-dependent bone repair, inducing osteoprogenitor cell infiltration inside and around the hydrogel. This report indicates that the CHPOA/hydrogel system can successfully deliver two different proteins to the bone defect to induce effective bone repair. The combination of the CHPOA/hydrogel system with the growth factors FGF18 and BMP2 might be a step towards efficient bone tissue engineering. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthetic design of growth factor sequestering extracellular matrix mimetic hydrogel for promoting in vivo bone formation.

PubMed

Yan, Hong Ji; Casalini, Tommaso; Hulsart-Billström, Gry; Wang, Shujiang; Oommen, Oommen P; Salvalaglio, Matteo; Larsson, Sune; Hilborn, Jöns; Varghese, Oommen P

2018-04-01

Synthetic scaffolds that possess an intrinsic capability to protect and sequester sensitive growth factors is a primary requisite for developing successful tissue engineering strategies. Growth factors such as recombinant human bone morphogenetic protein-2 (rhBMP-2) is highly susceptible to premature degradation and to provide a meaningful clinical outcome require high doses that can cause serious side effects. We discovered a unique strategy to stabilize and sequester rhBMP-2 by enhancing its molecular interactions with hyaluronic acid (HA), an extracellular matrix (ECM) component. We found that by tuning the initial protonation state of carboxylic acid residues of HA in a covalently crosslinked hydrogel modulate BMP-2 release at physiological pH by minimizing the electrostatic repulsion and maximizing the Van der Waals interactions. At neutral pH, BMP-2 release is primarily governed by Fickian diffusion, whereas at acidic pH both diffusion and electrostatic interactions between HA and BMP-2 become important as confirmed by molecular dynamics simulations. Our results were also validated in an in vivo rat ectopic model with rhBMP-2 loaded hydrogels, which demonstrated superior bone formation with acidic hydrogel as compared to the neutral counterpart. We believe this study provides new insight on growth factor stabilization and highlights the therapeutic potential of engineered matrices for rhBMP-2 delivery and may help to curtail the adverse side effects associated with the high dose of the growth factor. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration.

PubMed

Xu, Kang; Sun, Yanjun; Kh Al-Ani, Mohanad; Wang, Chunli; Sha, Yongqiang; Sung, Kl Paul; Dong, Nianguo; Qiu, Xuefeng; Yang, Li

2018-01-03

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80-90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young's were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect regeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Stepwise verification of bone regeneration using recombinant human bone morphogenetic protein-2 in rat fibula model

PubMed Central

2017-01-01

Objectives The purpose of this study was to introduce our three experiments on bone morphogenetic protein (BMP) and its carriers performed using the critical sized segmental defect (CSD) model in rat fibula and to investigate development of animal models and carriers for more effective bone regeneration. Materials and Methods For the experiments, 14, 16, and 24 rats with CSDs on both fibulae were used in Experiments 1, 2, and 3, respectively. BMP-2 with absorbable collagen sponge (ACS) (Experiments 1 and 2), autoclaved autogenous bone (AAB) and fibrin glue (FG) (Experiment 3), and xenogenic bone (Experiment 2) were used in the experimental groups. Radiographic and histomorphological evaluations were performed during the follow-up period of each experiment. Results Significant new bone formation was commonly observed in all experimental groups using BMP-2 compared to control and xenograft (porcine bone) groups. Although there was some difference based on BMP carrier, regenerated bone volume was typically reduced by remodeling after initially forming excessive bone. Conclusion BMP-2 demonstrates excellent ability for bone regeneration because of its osteoinductivity, but efficacy can be significantly different depending on its delivery system. ACS and FG showed relatively good bone regeneration capacity, satisfying the essential conditions of localization and release-control when used as BMP carriers. AAB could not provide release-control as a BMP carrier, but its space-maintenance role was remarkable. Carriers and scaffolds that can provide sufficient support to the BMP/carrier complex are necessary for large bone defects, and AAB is thought to be able to act as an effective scaffold. The CSD model of rat fibula is simple and useful for initial estimate of bone regeneration by agents including BMPs. PMID:29333367

Polyglutamate directed coupling of bioactive peptides for the delivery of osteoinductive signals on allograft bone

PubMed Central

Culpepper, Bonnie K.; Bonvallet, Paul P.; Reddy, Michael S.; Ponnazhagan, Selvarangan; Bellis, Susan L.

2012-01-01

Allograft bone is commonly used as an alternative to autograft, however allograft lacks many osteoinductive factors present in autologous bone due to processing. In this study, we investigated a method to reconstitute allograft with osteoregenerative factors. Specifically, an osteoinductive peptide from collagen I, DGEA, was engineered to express a heptaglutamate (E7) domain, which binds the hydroxyapatite within bone mineral. Addition of E7 to DGEA resulted in 9× greater peptide loading on allograft, and significantly greater retention after a 5-day interval with extensive washing. When factoring together greater initial loading and retention, the E7 domain directed a 45-fold enhancement of peptide density on the allograft surface. Peptide-coated allograft was also implanted subcutaneously into rats and it was found that E7DGEA was retained in vivo for at least 3 months. Interestingly, E7DGEA peptides injected intravenously accumulated within bone tissue, implicating a potential role for E7 domains in drug delivery to bone. Finally, we determined that, as with DGEA, the E7 modification enhanced coupling of a bioactive BMP2-derived peptide on allograft. These results suggest that E7 domains are useful for coupling many types of bone-regenerative molecules to the surface of allograft to reintroduce osteoinductive signals and potentially advance allograft treatments. PMID:23182349

Continuity resection of the mandible after ameloblastoma - feasibility of oral rehabilitation with rhBMP-2 associated to bovine xenograft followed by implant installation.

PubMed

Lustosa, Romulo Maciel; Macedo, Diogo de Vasconcelos; Iwaki, Lilian Cristina Vessoni; Tolentino, Elen de Souza; Hasse, Paulo Norberto; Marson, Giordano Bruno de Oliveira; Iwaki Filho, Liogi

2015-10-01

Recombinant human morphogenetic protein (rhBMP) is a graft alternative for extensive mandibular reconstruction after tumor resections. However, the feasibility of rhBMP-2 to receive osseointegrated implants and prosthetic rehabilitation has been rarely reported. This study reports on a case of an extensive solid ameloblastoma along the mandibular body. The treatment consisted of resection followed by off-label use of rhBMP type 2 associated with bovine bone xenograft. Eleven months postoperatively, the patient was prosthetically rehabilitated with dental implants, without evidence of resorption or complications. The literature on mandibular reconstructions using rhBMP and their feasibility for future osseointegrated implant placement was also reviewed. Based on the presented case, the association between rhBMP-2 and a bovine bone xenograft could be considered a feasible option for the reconstruction and rehabilitation of large mandibular defects after tumor resection. According to the literature, the use of rhBMP as a graft material is encouraging, with good clinical outcome. However, there are no long-term studies demonstrating success and survival rates of implants placed in these grafts. Future investigations will be required to ascertain the long-term survival of implants in areas grafted with rhBMP. Also, there is a lack of information regarding the prosthetic rehabilitation of these patients. Copyright © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.