Sample records for bone forming cell
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Evidence that Resorption of Bone by Rat Peritoneal Macrophages Occurs in an Acidic Environment
NASA Technical Reports Server (NTRS)
Blair, H. C.
1985-01-01
Skeletal loss in space, like any form of osteoporosis, reflects a relative imbalance of the activities of cells resorbing (degrading) or forming bone. Consequently, prevention of weightlessness induced bone loss may theoretically be accomplished by (1) stimulating bone formation or (2) inhibiting bone resorption. This approach, however, requires fundamental understanding of the mechanisms by which cells form or degrade bone, information not yet at hand. An issue central to bone resorption is the pH at which resorption takes place. The pH dependent spectral shift of a fluorescent dye (fluorescein isothiocyanate) conjugated to bone matrix was used to determine the pH at the resorptive cell bone matrix interface. Devitalized rat bone was used as the substrate, and rat peritoneal macrophages were used as the bone resorbing cells. The results suggest that bone resorption is the result of generation of an acidic microenvironment at the cell matrix junction.
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Komatsu, Koichiro; Shimada, Akemi; Shibata, Tatsuya; Wada, Satoshi; Ideno, Hisashi; Nakashima, Kazuhisa; Amizuka, Norio; Noda, Masaki; Nifuji, Akira
2013-11-01
Bisphosphonates (BPs) are a major class of antiresorptive drug, and their molecular mechanisms of antiresorptive action have been extensively studied. Recent studies have suggested that BPs target bone-forming cells as well as bone-resorbing cells. We previously demonstrated that local application of a nitrogen-containing BP (N-BP), alendronate (ALN), for a short period of time increased bone tissue in a rat tooth replantation model. Here, we investigated cellular mechanisms of bone formation by ALN. Bone histomorphometry confirmed that bone formation was increased by local application of ALN. ALN increased proliferation of bone-forming cells residing on the bone surface, whereas it suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment. In vitro studies revealed that pulse treatment with ALN promoted osteocalcin expression. To track the target cells of N-BPs, we applied fluorescence-labeled ALN (F-ALN) in vivo and in vitro. F-ALN was taken into bone-forming cells both in vivo and in vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiation in vitro and it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formation in vivo. DC also blocked the inhibition of Rap1A prenylation by ALN in the osteoblastic cells. These data suggest that local application of ALN promotes bone formation by stimulating proliferation and differentiation of bone-forming cells as well as inhibiting osteoclast function. These effects may occur through endocytic incorporation of ALN and subsequent inhibition of protein prenylation.
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Supplying osteogenesis to dead bone using an osteogenic matrix cell sheet.
Uchihara, Yoshinobu; Akahane, Manabu; Okuda, Akinori; Shimizu, Takamasa; Masuda, Keisuke; Kira, Tsutomu; Kawate, Kenji; Tanaka, Yasuhito
2018-02-22
To evaluate whether osteogenic matrix cell sheets can supply osteogenesis to dead bone. Femur bone fragments (5 mm in length) were obtained from Fisher 344 rats and irradiated by a single exposure of 60 Gy to produce bones that were no longer viable. Osteogenic matrix cell sheets were created from rat bone marrow-derived stromal cells (BMSCs). After wrapping the dead bone with an osteogenic matrix cell sheet, it was subcutaneously transplanted into the back of a rat and harvested after 4 weeks. Bone formation around the dead bone was evaluated by X-ray imaging and histology. Alkaline phosphatase (ALP) and osteocalcin (OC) mRNA expression levels were measured to confirm osteogenesis of the transplanted bone. The contribution of donor cells to bone formation was assessed using the Sry gene and PKH26. After the cell sheet was transplanted together with dead bone, X-ray images showed abundant calcification around the dead bone. In contrast, no newly formed bone was seen in samples that were transplanted without the cell sheet. Histological sections also showed newly formed bone around dead bone in samples transplanted with the cell sheet, whereas many empty lacunae and no newly formed bone were observed in samples transplanted without the cell sheet. ALP and OC mRNA expression levels were significantly higher in dead bones transplanted with cell sheets than in those without a cell sheet (P < 0.01). Sry gene expression and cells derived from cell sheets labeled with PKH26 were detected in samples transplanted with a cell sheet, indicating survival of donor cells after transplantation. Our study indicates that osteogenic matrix cell sheet transplantation can supply osteogenesis to dead bone. Copyright © 2018. Published by Elsevier B.V.
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Bone regeneration performance of surface-treated porous titanium.
Amin Yavari, Saber; van der Stok, Johan; Chai, Yoke Chin; Wauthle, Ruben; Tahmasebi Birgani, Zeinab; Habibovic, Pamela; Mulier, Michiel; Schrooten, Jan; Weinans, Harrie; Zadpoor, Amir Abbas
2014-08-01
The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid-alkali (AcAl), alkali-acid-heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Andersen, Thomas L; Søe, Kent; Sondergaard, Teis E; Plesner, Torben; Delaisse, Jean-Marie
2010-02-01
Osteolytic lesions are a hallmark of multiple myeloma. They are due to the hyperactivity of bone resorbing osteoclasts and hypoactivity of bone forming osteoblasts, in response to neighbouring myeloma cells. This study identified a structure that deeply affects this response, because of its impact on the physical organisation of the myeloma cell microenvironment. The proximity between myeloma cells and osteoclasts or osteoblasts was shown to be conditioned by the recently discovered layer of flat cells that separates the osteoclasts and osteoblasts from the bone marrow, by forming a canopy over bone remodelling compartment (BRC). These canopies are frequently disrupted in myeloma, and this disruption correlates with increased proximity and density of myeloma cells. In vitro evidence indicates that this disruption may be due to direct contact between myeloma and BRC canopy cells. Importantly, this disruption and increased proximity and density of myeloma cells coincides with key myeloma-induced bone events, such as osteolytic lesions, impaired bone formation despite increased bone resorption, and fusion of myeloma cells with osteoclasts thereby forming myeloma-osteoclast hybrid cells. These findings strongly support a critical role of BRC canopies in myeloma-induced bone disease. BRC canopies could therefore be considered as a new therapeutic target.
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Zhang, Zhifang; Shively, John E
2010-11-15
Bone generation and maintenance involve osteoblasts, osteoclasts, and osteocytes which originate from unique precursors and rely on key growth factors for differentiation. However, an incomplete understanding of bone forming cells during wound healing has led to an unfilled clinical need such as nonunion of bone fractures. Since circulating monocytes are often recruited to sites of injury and may differentiate into various cell types including osteoclasts, we investigated the possibility that circulating monocytes in the context of tissue injury may also contribute to bone repair. In particular, we hypothesized that LL-37 (produced from hCAP-18, cathelicidin), which recruits circulating monocytes during injury, may play a role in bone repair. Treatment of monocytes from blood with LL-37 for 6 days resulted in their differentiation to large adherent cells. Growth of LL-37-differentiated monocytes on osteologic discs reveals bone-like nodule formation by scanning electron microscopy (SEM). In vivo transplantation studies in NOD/SCID mice show that LL-37-differentiated monocytes form bone-like structures similar to endochondral bone formation. Importantly, LL-37-differentiated monocytes are distinct from conventional monocyte-derived osteoclasts, macrophages, and dendritic cells and do not express markers of the mesenchymal stem cells (MSC) lineage, distinguishing them from the conventional precursors of osteoblasts. Furthermore, LL-37 differentiated monocytes express intracellular proteins of both the osteoblast and osteoclast lineage including osteocalcin (OC), osteonectin (ON), bone sialoprotein II (BSP II), osteopontin (OP), RANK, RANKL, MMP-9, tartrate resistant acid phosphatase (TRAP), and cathepsin K (CK). Blood derived monocytes treated with LL-37 can be differentiated into a novel bone forming cell that functions both in vitro and in vivo. We propose the name monoosteophil to indicate their monocyte derived lineage and their bone forming phenotype. These cells may have wide ranging implications in the clinic including repair of broken bones and treatment of osteoporosis.
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Lorenz, Jonas; Kubesch, Alica; Korzinskas, Tadas; Barbeck, Mike; Landes, Constantin; Sader, Robert A; Kirkpatrick, Charles J; Ghanaati, Shahram
2015-12-01
This study compared the material-specific tissue response to the synthetic, hydroxyapatite-based bone substitute material NanoBone (NB) with that of the xenogeneic, bovine-based bone substitute material Bio-Oss (BO). The sinus cavities of 14 human patients were augmented with NB and BO in a split-mouth design. Six months after augmentation, bone biopsies were extracted for histological and histomorphometric investigation prior to dental implant insertion. The following were evaluated: the cellular inflammatory pattern, the induction of multinucleated giant cells, vascularization, the relative amounts of newly formed bone, connective tissue, and the remaining bone substitute material. NB granules were well integrated in the peri-implant tissue and were surrounded by newly formed bone tissue. Multinucleated giant cells were visible on the surfaces of the remaining granules. BO granules were integrated into the newly formed bone tissue, which originated from active osteoblasts on their surface. Histomorphometric analysis showed a significantly higher number of multinucleated giant cells and blood vessels in the NB group compared to the BO group. No statistical differences were observed in regard to connective tissue, remaining bone substitute, and newly formed bone. The results of this study highlight the different cellular reactions to synthetic and xenogeneic bone substitute materials. The significantly higher number of multinucleated giant cells within the NB implantation bed seems to have no effect on its biodegradation. Accordingly, the multinucleated giant cells observed within the NB implantation bed have characteristics more similar to those of foreign body giant cells than to those of osteoclasts.
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[Bone remodeling and modeling/mini-modeling.
Hasegawa, Tomoka; Amizuka, Norio
Modeling, adapting structures to loading by changing bone size and shapes, often takes place in bone of the fetal and developmental stages, while bone remodeling-replacement of old bone into new bone-is predominant in the adult stage. Modeling can be divided into macro-modeling(macroscopic modeling)and mini-modeling(microscopic modeling). In the cellular process of mini-modeling, unlike bone remodeling, bone lining cells, i.e., resting flattened osteoblasts covering bone surfaces will become active form of osteoblasts, and then, deposit new bone onto the old bone without mediating osteoclastic bone resorption. Among the drugs for osteoporotic treatment, eldecalcitol(a vitamin D3 analog)and teriparatide(human PTH[1-34])could show mini-modeling based bone formation. Histologically, mature, active form of osteoblasts are localized on the new bone induced by mini-modeling, however, only a few cell layer of preosteoblasts are formed over the newly-formed bone, and accordingly, few osteoclasts are present in the region of mini-modeling. In this review, histological characteristics of bone remodeling and modeling including mini-modeling will be introduced.
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Blood-Forming Stem Cell Transplants
... to Ask about Your Treatment Research Blood-Forming Stem Cell Transplants On This Page What are bone marrow ... Considering becoming a bone marrow or a blood stem cell donor? View this video on YouTube. Follow a ...
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Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism
Chen, Julia C.; Hoey, David A.; Chua, Mardonn; Bellon, Raymond; Jacobs, Christopher R.
2016-01-01
It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitment in vitro. In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cells in vivo. Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitment in vivo and that the primary cilium contributes to this process.—Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism. PMID:26675708
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Lu, Helen H; Kofron, Michelle D; El-Amin, Saadiq F; Attawia, Mohammed A; Laurencin, Cato T
2003-06-13
Over 800,000 bone grafting procedures are performed in the United States annually, creating a demand for viable alternatives to autogenous bone, the grafting standard in osseous repair. The objective of this study was to examine the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype and in vitro bone formation by muscle-derived cells. Specifically, we evaluated the ability of bone morphogenetic protein-7 (BMP-7), delivered from a poly(lactide-co-glycolide) (PLAGA) matrix, to induce the differentiation of cells derived from rabbit skeletal muscle into osteoblast-like cells and subsequently form mineralized tissue. Results confirmed that muscle-derived cells attached and proliferated on the PLAGA substrates. BMP-7 released from PLAGA induced the muscle-derived cells to increase bone marker expression and form mineralized cultures. These results demonstrate the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype by muscle-derived cells and present a new paradigm for bone tissue engineering.
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James, Aaron W.; Zara, Janette N.; Zhang, Xinli; Askarinam, Asal; Goyal, Raghav; Chiang, Michael; Yuan, Wei; Chang, Le; Corselli, Mirko; Shen, Jia; Pang, Shen; Stoker, David; Wu, Ben
2012-01-01
Adipose tissue is an ideal source of mesenchymal stem cells for bone tissue engineering: it is largely dispensable and readily accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which leads to unreliable bone formation. In the present study, we prospectively purified human perivascular stem cells (PSCs) from adipose tissue and compared their bone-forming capacity with that of traditionally derived SVF. PSCs are a population (sorted by fluorescence-activated cell sorting) of pericytes (CD146+CD34−CD45−) and adventitial cells (CD146−CD34+CD45−), each of which we have previously reported to have properties of mesenchymal stem cells. Here, we found that PSCs underwent osteogenic differentiation in vitro and formed bone after intramuscular implantation without the need for predifferentiation. We next sought to optimize PSCs for in vivo bone formation, adopting a demineralized bone matrix for osteoinduction and tricalcium phosphate particle formulation for protein release. Patient-matched, purified PSCs formed significantly more bone in comparison with traditionally derived SVF by all parameters. Recombinant bone morphogenetic protein 2 increased in vivo bone formation but with a massive adipogenic response. In contrast, recombinant Nel-like molecule 1 (NELL-1; a novel osteoinductive growth factor) selectively enhanced bone formation. These studies suggest that adipose-derived human PSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, PSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy. Finally, NELL-1 is a candidate growth factor able to induce human PSC osteogenesis. PMID:23197855
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Bone formation in vitro and in nude mice by human osteosarcoma cells.
Ogose, A; Motoyama, T; Hotta, T; Watanabe, H; Takahashi, H E
1995-01-01
Osteosarcomas contain variable amounts of bony tissue, but the mechanism of bone formation by osteosarcoma is not well understood. While a number of cultured human osteosarcoma cell lines have been established, they are maintained by different media and differ qualitatively with regard to bone formation. We examined different media for their ability to support bone formation in vitro and found the alpha-modification of Eagle's minimal essential medium supplemented with beta glycerophosphate was best for this purpose, because it contained the proper calcium and phosphate concentrations. Subsequently, we compared seven human osteosarcoma cell lines under the same experimental conditions to clarify their ability to induce bone formation. NOS-1 cells most frequently exhibited features of bone formation in vitro and in nude mice. Collagen synthesis by tumour cells themselves seemed to be the most important factor for bone volume. However, even HuO9 cells, which lacked collagen synthesis and failed to form bone in vitro, successfully formed tumours containing bone in nude mice. Histological analysis of HuO9 cells in diffusion chambers implanted in nude mice and the findings of polymerase chain reaction indicated that the phenomenon was probably due to bone morphogenetic protein.
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Bone remodelling: its local regulation and the emergence of bone fragility.
Martin, T John; Seeman, Ego
2008-10-01
Bone modelling prevents the occurrence of damage by adapting bone structure - and hence bone strength - to its loading circumstances. Bone remodelling removes damage, when it inevitably occurs, in order to maintain bone strength. This cellular machinery is successful during growth, but fails during advancing age because of the development of a negative balance between the volumes of bone resorbed and formed during remodelling by the basic multicellular unit (BMU), high rates of remodelling during midlife in women and late in life in both sexes, and a decline in periosteal bone formation. together resulting in bone loss and structural decay each time a remodelling event occurs. The two steps in remodelling - resorption of a volume of bone by osteoclasts and formation of a comparable volume by osteoblasts - are sequential, but the regulatory events leading to these two fully differentiated functions are not. Reparative remodelling is initiated by damage producing osteocyte apoptosis, which signals the location of damage via the osteocyte canalicular system to endosteal lining cells which forms the canopy of a bone-remodelling compartment (BRC). Within the BRC, local recruitment of osteoblast precursors from the lining cells, the marrow and circulation, direct contact with osteoclast precursors, osteoclastogenesis and molecular cross-talk between precursors, mature cells, cells of the immune system, and products of the resorbed matrix, titrate the birth, work and lifespan of the cells of this multicellular remodelling machinery to either remove or form a net volume of bone appropriate to the mechanical requirements.
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Tirkkonen, Laura; Halonen, Heidi; Hyttinen, Jari; Kuokkanen, Hannu; Sievänen, Harri; Koivisto, Anna-Maija; Mannerström, Bettina; Sándor, George K. B.; Suuronen, Riitta; Miettinen, Susanna; Haimi, Suvi
2011-01-01
Mechanical stimulation is an essential factor affecting the metabolism of bone cells and their precursors. We hypothesized that vibration loading would stimulate differentiation of human adipose stem cells (hASCs) towards bone-forming cells and simultaneously inhibit differentiation towards fat tissue. We developed a vibration-loading device that produces 3g peak acceleration at frequencies of 50 and 100 Hz to cells cultured on well plates. hASCs were cultured using either basal medium (BM), osteogenic medium (OM) or adipogenic medium (AM), and subjected to vibration loading for 3 h d–1 for 1, 7 and 14 day. Osteogenesis, i.e. differentiation of hASCs towards bone-forming cells, was analysed using markers such as alkaline phosphatase (ALP) activity, collagen production and mineralization. Both 50 and 100 Hz vibration frequencies induced significantly increased ALP activity and collagen production of hASCs compared with the static control at 14 day in OM. A similar trend was detected for mineralization, but the increase was not statistically significant. Furthermore, vibration loading inhibited adipocyte differentiation of hASCs. Vibration did not affect cell number or viability. These findings suggest that osteogenic culture conditions amplify the stimulatory effect of vibration loading on differentiation of hASCs towards bone-forming cells. PMID:21613288
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Yuanzheng, Chen; Yan, Gao; Ting, Li; Yanjie, Fu; Peng, Wu; Nan, Bai
2015-05-01
Autologous bone graft has been regarded as the criterion standard for the repair of alveolar cleft. However, the most prominent issue in alveolar cleft treatment is the high absorption rate of the bone graft. The authors' objective was to investigate the effects of an autologous iliac bone, bone marrow-derived mesenchymal stem cell, and platelet-rich fibrin mixture on the repair of dog alveolar cleft. Twenty beagle dogs with unilateral alveolar clefts created by surgery were divided randomly into four groups: group A underwent repair with an autologous iliac bone, bone marrow-derived mesenchymal stem cell, and platelet-rich fibrin mixture; group B underwent repair with autologous iliac bone and bone marrow-derived mesenchymal stem cells; group C underwent repair with autologous iliac bone and platelet-rich fibrin; and group D underwent repair with autologous iliac bone as the control. One day and 6 months after transplantation, the transplant volumes and bone mineral density were assessed by quantitative computed tomography. All of the transplants were harvested for hematoxylin and eosin staining 6 months later. Bone marrow-derived mesenchymal stem cells and platelet-rich fibrin transplants formed the greatest amounts of new bone among the four groups. The new bone formed an extensive union with the underlying maxilla in groups A, B, and C. Transplants with the bone marrow-derived mesenchymal stem cells, platelet-rich fibrin, and their mixture retained the majority of their initial volume, whereas the transplants in the control group showed the highest absorption rate. Bone mineral density of transplants with the bone marrow-derived mesenchymal stem cells, platelet-rich fibrin, and their mixture 6 months later was significantly higher than in the control group (p < 0.05), and was the highest in bone marrow-derived mesenchymal stem cells and platelet-rich fibrin mixed transplants. Hematoxylin and eosin staining showed that the structure of new bones formed the best in group A. Both bone marrow-derived mesenchymal stem cells and platelet-rich fibrin are capable of improving the repair of dog alveolar cleft, and the mixture of them is more potent than each one of them used singly for enhancing new bone regeneration.
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Leukemia is cancer of the white blood cells. White blood cells help your body fight infection. Your blood cells form in your bone marrow. In leukemia, the bone marrow produces abnormal white blood cells. ...
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Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells
Chan, Charles K. F.; Lindau, Paul; Jiang, Wen; Chen, James Y.; Zhang, Lillian F.; Chen, Ching-Cheng; Seita, Jun; Sahoo, Debashis; Kim, Jae-Beom; Lee, Andrew; Park, Sujin; Nag, Divya; Gong, Yongquan; Kulkarni, Subhash; Luppen, Cynthia A.; Theologis, Alexander A.; Wan, Derrick C.; DeBoer, Anthony; Seo, Eun Young; Vincent-Tompkins, Justin D.; Loh, Kyle; Walmsley, Graham G.; Kraft, Daniel L.; Wu, Joseph C.; Longaker, Michael T.; Weissman, Irving L.
2013-01-01
Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells. PMID:23858471
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Honda, Yoshitomo; Takeda, Yoshihiro; Li, Peiqi; Huang, Anqi; Sasayama, Satoshi; Hara, Eiki; Uemura, Naoya; Ueda, Mamoru; Hashimoto, Masanori; Arita, Kenji; Matsumoto, Naoyuki; Hashimoto, Yoshiya; Baba, Shunsuke; Tanaka, Tomonari
2018-04-11
Chemical modification of gelatin using epigallocatechin gallate (EGCG) promotes bone formation in vivo. However, further improvements are required to increase the mechanical strength and bone-forming ability of fabricated EGCG-modified gelatin sponges (EGCG-GS) for practical applications in regenerative therapy. In the present study, we investigated whether vacuum heating-induced dehydrothermal cross-linking of EGCG-GS enhances bone formation in critical-sized rat calvarial defects. The bone-forming ability of vacuum-heated EGCG-GS (vhEGCG-GS) and other sponges was evaluated by micro-computed tomography and histological staining. The degradation of sponges was assessed using protein assays, and cell morphology and proliferation were verified by scanning electron microscopy and immunostaining using osteoblastic UMR106 cells in vitro. Four weeks after the implantation of sponges, greater bone formation was detected for vhEGCG-GS than for EGCG-GS or vacuum-heated gelatin sponges (dehydrothermal cross-linked sponges without EGCG). In vitro experiments revealed that the relatively low degradability of vhEGCG-GS supports cell attachment, proliferation, and cell-cell communication on the matrix. These findings suggest that vacuum heating enhanced the bone forming ability of EGCG-GS, possibly via the dehydrothermal cross-linking of EGCG-GS, which provides a scaffold for cells, and by maintaining the pharmacological effect of EGCG.
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Leukemia is cancer of the white blood cells. White blood cells help your body fight infection. Your blood cells form in your bone marrow. In leukemia, the bone marrow produces abnormal white blood cells. ...
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Leukemia is cancer of the white blood cells. White blood cells help your body fight infection. Your blood cells form in your bone marrow. In leukemia, the bone marrow produces abnormal white blood cells. ...
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Pessa-Morikawa, Tiina; Niku, Mikael; Iivanainen, Antti
2012-03-01
The CD34 glycoprotein is an important marker of hematopoietic stem cells. We used a polyclonal rabbit anti-bovine CD34 antibody to stain fetal and adult bovine bone marrow cells. Flow cytometry revealed a low side scatter (SSC(low)) population of cells that were CD34(+) but negative for leukocyte lineage markers CD11b, CD14 or CD2. Hematopoietic colony assays with CD34(+) and CD34(-) bone marrow cells suggested that the colony-forming potential in SSC(low) bone marrow cells was confined to the CD34(+) fraction. In contrast, this population was not enriched for cells expressing high aldehyde dehydrogenase activity, a metabolic marker that has been used to characterize hematopoietic stem cells. Thus, the CD34 antigen can be used to identify and isolate bovine bone marrow cells exhibiting clonogenic potential in vitro. Moreover, the proportion of CD34(+) cells is very high in fetal bovine bone marrow, indicating it as a rich source of hematopoietic progenitors. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Study of tissue engineered bone nodules by Fourier transform infrared spectroscopy.
Aydin, Halil Murat; Hu, Bin; Suso, Josep Sulé; El Haj, Alicia; Yang, Ying
2011-02-21
The key criteria for assessing the success of bone tissue engineering are the quality and quantity of the produced minerals within the cultured constructs. The accumulation of calcium ions and inorganic phosphates in culture medium serves as nucleating agents for the formation of hydroxyapatite, which is the main inorganic component of bone. Bone nodule formation is one of the hallmarks of mineralization in such cell cultures. In this study, we developed a new two-step procedure to accelerate bone formation in which mouse bone cell aggregates were produced first on various chemically treated non-adhesive substrates. After this step, the bone cells' growth and mineralization were followed in conventional culture plates. The number and size of cell aggregates were studied with light microscopy. The minerals' formation in the form of nodules produced by the cell aggregates and the bone crystal quality were studied with Fourier Transform Infrared (FTIR) spectroscopy. The FTIR spectra of the ash specimens (mineral phase only) from thermal gravimetric analysis (TGA) provided valuable information of the quality of the minerals. The υ(4) PO(4) region (550-650 cm(-1)), which reveals apatitic and non-apatitic HPO(4) or PO(4) environments, and phosphate region (910-1180 cm(-1)) were examined for the minerals produced in the form of nodules. The peak position and intensity of the spectra demonstrate that the quality of the bone produced by cell aggregates, especially from the bigger ones, which were formed on Plunoric treated substrates, exhibit a composition more similar to that of native bone. This work establishes a new protocol for high quality bone formation and characterization, with the potential to be applied to bone tissue engineering.
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Induced Pluripotent Stem Cell Derived Mesenchymal Stem Cells for Attenuating Age-Related Bone Loss
2012-07-01
Mesenchymal stem cell (MSC) differentiation towards the bone forming osteoblastic lineage decreases as a function of age and may contribute to age-related...problem of age-related reduced availability of MSC we propose to examine the bone anabolic potential of induced pluripotent stem cell (iPS) derived MSC
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Kojima, Hiroko; Uemura, Toshimasa
2005-01-28
Core binding factor alpha-1 (Cbfa1), known as an essential transcription factor for osteogenic lineage, has two major N-terminal isoforms: Pebp2alphaA and Til-1. To study the roles of these isoforms in bone regeneration, we applied an adenoviral vector carrying their genes to transduce primary osteoprogenitor cells in vitro and in vivo. Overexpression of the two isoforms induced rapid and marked osteoblast differentiation, with Til-1 being more effective in vitro, by examination of the alkaline phosphatase activity, calcium content, and Alizarin red staining. Til-1 overexpressing cells/porous ceramic composites were transplanted into subcutaneous and bone defect sites in Fischer rats (cultured bone transplantation model) and markedly affected in vivo bone formation and osteoblast markers. The results demonstrated that the reconstitution of bone tissues, such as cortical bone and trabecular bone was accelerated by implantation of Til-1 overexpressing cells/porous ceramic composites. Moreover, the new bone formation by Til-1 overexpression appeared to reflect replacement of new bone within the implant boundaries. To ascertain whether implanted Cbfa1 overexpressing cells could differentiate into osteogenic cells to create bone or whether it stimulated the surrounding recipient tissue to regenerate bone, implanted male donor cells were visualized by fluorescent in situ hybridization analysis. The proportion of implanted cells in the presumptive bone forming region was over 80% and did not change throughout from 3 days to 8 weeks after implantation. These findings suggested that the newly formed bone in the porous area of the scaffold is mostly produced by the implanted donor cells or their derived cells, effectively by Til-1 overexpression.
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USDA-ARS?s Scientific Manuscript database
Ovariectomy (OVX)-induced bone loss has been linked to increased bone turnover and higher bone matrix collagen degradation as the result of osteoclast activation. However, the role of degraded collagen matrix in the fate of resident bone-forming cells is unclear. In this report, we show that OVX-i...
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Leukemia is cancer of the white blood cells. White blood cells help your body fight infection. Your blood cells form in your bone marrow. In leukemia, however, the bone marrow produces abnormal white blood ...
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Leukemia is cancer of the white blood cells. White blood cells help your body fight infection. Your blood cells form in your bone marrow. In leukemia, however, the bone marrow produces abnormal white blood ...
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Yukita, Akira; Yoshiba, Kunihiko; Yoshiba, Nagako; Takahashi, Masafumi; Nakamura, Hiroaki
2012-01-01
Dental pulp is involved in the formation of bone-like tissue in response to external stimuli. However, the origin of osteoblast-like cells constructing this tissue and the mechanism of their induction remain unknown. We therefore evaluated pulp mineralization induced by transplantation of a green fluorescent protein (GFP)–labeled tooth into a GFP-negative hypodermis of host rats. Five days after the transplantation, the upper pulp cavity became necrotic; however, cell-rich hard tissue was observed adjacent to dentin at the root apex. At 10 days, woven bone-like tissue was formed apart from the dentin in the upper pulp. After 20 days, these hard tissues expanded and became histologically similar to bone. GFP immunoreactivity was detected in the hard tissue-forming cells within the root apex as well as in the upper pulp. Furthermore, immunohistochemical observation of α–smooth muscle actin, a marker for undifferentiated cells, showed a positive reaction in cells surrounding this bone-like tissue within the upper pulp but not in those within the root apex. Immunoreactivities of Smad4, Runx2, and Osterix were detected in the hard tissue-forming cells within both areas. These results collectively suggest that the dental pulp contains various types of osteoblast progenitors and that these cells might thus induce bone-like tissue in severely injured pulp. PMID:22899860
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Isern, Joan; García-García, Andrés; Martín, Ana M; Arranz, Lorena; Martín-Pérez, Daniel; Torroja, Carlos; Sánchez-Cabo, Fátima; Méndez-Ferrer, Simón
2014-01-01
Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin− MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin+ cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP+ Pdgfrα− cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation. DOI: http://dx.doi.org/10.7554/eLife.03696.001 PMID:25255216
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Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow.
Falla, N; Van Vlasselaer; Bierkens, J; Borremans, B; Schoeters, G; Van Gorp, U
1993-12-15
In the presence of beta-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 x 10(5) cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 x 10(4) cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 x 10(4) or 1 of 5 x 10(3) plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.
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Engineering tubular bone using mesenchymal stem cell sheets and coral particles
DOE Office of Scientific and Technical Information (OSTI.GOV)
Geng, Wenxin; Ma, Dongyang; Yan, Xingrong
Highlights: • We developed a novel engineering strategy to solve the limitations of bone grafts. • We fabricated tubular constructs using cell sheets and coral particles. • The composite constructs showed high radiological density and compressive strength. • These characteristics were similar to those of native bone. -- Abstract: The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheetsmore » and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.« less
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The osteocyte: key player in regulating bone turnover
Goldring, Steven R
2015-01-01
Osteocytes are the most abundant cell type in bone and are distributed throughout the mineralised bone matrix forming an interconnected network that ideally positions them to sense and to respond to local biomechanical and systemic stimuli to regulate bone remodelling and adaptation. The adaptive process is dependent on the coordinated activity of osteoclasts and osteoblasts that form a so called bone multicellular unit that remodels cortical and trabecular bone through a process of osteoclast-mediated bone resorption, followed by a phase of bone formation mediated by osteoblasts. Osteocytes mediate their effects on bone remodelling via both cell–cell interactions with osteoclasts and osteoblasts, but also via signaling through the release of soluble mediators. The remodelling process provides a mechanism for adapting the skeleton to local biomechanical factors and systemic hormonal influences and for replacing bone that has undergone damage from repetitive mechanical loading. PMID:26557372
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Scheven, B A; Wassenaar, A M; Kawilarang-de Haas, E W; Nijweide, P J
1987-07-01
Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.
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CD146/MCAM defines functionality of human bone marrow stromal stem cell populations.
Harkness, Linda; Zaher, Walid; Ditzel, Nicholas; Isa, Adiba; Kassem, Moustapha
2016-01-11
Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous hMSC population. Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146(+) and hMSC-CD146(-) cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high-content analysis and additionally for their ability to differentiate toward osteogenesis in vitro and form bone in vivo, and their migrational ability in vivo and in vitro was investigated. In vitro, the two cell populations exhibited similar growth rate and differentiation capacity to osteoblasts and adipocytes on the basis of gene expression and protein production of lineage-specific markers. In vivo, hMSC-CD146(+) and hMSC-CD146(-) cells formed bone and bone marrow organ when implanted subcutaneously in immune-deficient mice. Bone was enriched in hMSC-CD146(-) cells (12.6 % versus 8.1 %) and bone marrow elements enriched in implants containing hMSC-CD146(+) cells (0.5 % versus 0.05 %). hMSC-CD146(+) cells exhibited greater chemotactic attraction in a transwell migration assay and, when injected intravenously into immune-deficient mice following closed femoral fracture, exhibited wider tissue distribution and significantly increased migration ability as demonstrated by bioluminescence imaging. Our studies demonstrate that CD146 defines a subpopulation of hMSCs capable of bone formation and in vivo trans-endothelial migration and thus represents a population of hMSCs suitable for use in clinical protocols of bone tissue regeneration.
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NOS2 deficiency has no influence on the radiosensitivity of the hematopoietic system.
Li, Chengcheng; Luo, Yi; Shao, Lijian; Meng, Aimin; Zhou, Daohong
2018-01-01
Previous studies have shown that inhibition of inducible NO synthase (NOS2 or iNOS) with an inhibitor can selectively protect several normal tissues against radiation during radiotherapy. However, the role of NOS2 in ionizing radiation (IR)-induced bone marrow (BM) suppression is unknown and thus was investigated in the present study using NOS2 - / - and wild-type mice 14 days after they were exposed to a sublethal dose of total body irradiation (TBI). The effects of different doses of IR (1, 2 and 4 Gy) on the apoptosis and colony-forming ability of bone marrow cells from wild-type (WT) and NOS2 - / - mice were investigated in vitro. In addition, we exposed NOS2 - / - mice and WT mice to 6-Gy TBI or sham irradiation. They were euthanized 14 days after TBI for analysis of peripheral blood cell counts and bone marrow cellularity. Colony-forming unit-granulocyte and macrophage, burst-forming unit-erythroid and CFU-granulocyte, erythroid, macrophage in bone marrow cells from the mice were determined to evaluate the function of hematopoietic progenitor cells (HPCs), and the ability of hematopoietic stem cells (HSCs) to self-renew was analysed by the cobblestone area forming cell assay. The cell cycling of HPCs and HSCs were measured by flow cytometry. Exposure to 2 and 4 Gy IR induced bone marrow cell apoptosis and inhibited the proliferation of HPCs in vitro. However, there was no difference between the cells from WT mice and NOS2 - / - mice in response to IR exposure in vitro. Exposure of WT mice and NOS2 - / - mice to 6 Gy TBI decreased the white blood cell, red blood cell, and platelet counts in the peripheral blood and bone marrow mononuclear cells, and reduced the colony-forming ability of HPCs (P < 0.05), damaged the clonogenic function of HSCs. However, these changes were not significantly different in WT and NOS2 - / - mice. These data suggest that IR induces BM suppression in a NOS2-independent manner.
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The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts.
ten Harkel, Bas; Schoenmaker, Ton; Picavet, Daisy I; Davison, Noel L; de Vries, Teun J; Everts, Vincent
2015-01-01
Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones--cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K.
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The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts
ten Harkel, Bas; Schoenmaker, Ton; Picavet, Daisy I.; Davison, Noel L.; de Vries, Teun J.; Everts, Vincent
2015-01-01
Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones—cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K. PMID:26426806
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Geurtzen, Karina; Knopf, Franziska; Wehner, Daniel; Huitema, Leonie F A; Schulte-Merker, Stefan; Weidinger, Gilbert
2014-06-01
Zebrafish have an unlimited capacity to regenerate bone after fin amputation. In this process, mature osteoblasts dedifferentiate to osteogenic precursor cells and thus represent an important source of newly forming bone. By contrast, differentiated osteoblasts do not appear to contribute to repair of bone injuries in mammals; rather, osteoblasts form anew from mesenchymal stem cells. This raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, a special feature of the lepidotrichia bone of the fish fin, or a process found more generally in fish bone. Here, we show that dedifferentiation of mature osteoblasts is not restricted to fin regeneration after amputation, but also occurs during repair of zebrafish fin fractures and skull injuries. In both models, mature osteoblasts surrounding the injury downregulate the expression of differentiation markers, upregulate markers of the pre-osteoblast state and become proliferative. Making use of photoconvertible Kaede protein as well as Cre-driven genetic fate mapping, we show that osteoblasts migrate to the site of injury to replace damaged tissue. Our findings suggest a fundamental role for osteoblast dedifferentiation in reparative bone formation in fish and indicate that adult fish osteoblasts display elevated cellular plasticity compared with mammalian bone-forming cells. © 2014. Published by The Company of Biologists Ltd.
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Anemia - hemolytic ... bones that helps form all blood cells. Hemolytic anemia occurs when the bone marrow isn't making ... destroyed. There are several possible causes of hemolytic anemia. Red blood cells may be destroyed due to: ...
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Sphene ceramics for orthopedic coating applications: an in vitro and in vivo study.
Ramaswamy, Yogambha; Wu, Chengtie; Dunstan, Colin R; Hewson, Benjamin; Eindorf, Tanja; Anderson, Gail I; Zreiqat, Hala
2009-10-01
The host response to titanium alloy (Ti-6Al-4V) is not always favorable as a fibrous layer may form at the skeletal tissue-device interface, causing aseptic loosening. Recently, sphene (CaTiSiO(5)) ceramics were developed by incorporating Ti in the Ca-Si system, and found to exhibit improved chemical stability. The aim of this study is to evaluate the in vitro response of human osteoblast-like cells, human osteoclasts and human microvascular endothelial cells to sphene ceramics and determine whether coating Ti-6Al-4V implants with sphene enhances anchorage to surrounding bone. The study showed that sphene ceramics support human osteoblast-like cell attachment with organized cytoskeleton structure and express increased mRNA levels of osteoblast-related genes. Sphene ceramics were able to induce the differentiation of monocytes to form functional osteoclasts with the characteristic features of f-actin and alpha(v)beta(3) integrin, and express osteoclast-related genes. Human endothelial cells were also able to attach and express the endothelial cell markers ZO-1 and VE-Cadherin when cultured on sphene ceramics. Histological staining, enzyme histochemistry and immunolabelling were used for identification of mineralized bone and bone remodelling around the coated implants. Ti-6Al-4V implants coated with sphene showed new bone formation and filled the gap between the implants and existing bone in a manner comparable to that of the hydroxyapatite coatings used as control. The new bone was in direct contact with the implants, whereas fibrous tissue formed between the bone and implant with uncoated Ti-6Al-4V. The in vivo assessment of sphene-coated implants supports our in vitro observation and suggests that they have the ability to recruit osteogenic cells, and thus support bone formation around the implants and enhance osseointegration.
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Intrasinusoidal pattern of bone marrow infiltration by hepatosplenic T-cell lymphoma.
Butler, Liesl Ann; Juneja, Surender
2018-04-01
Hepatosplenic T-cell lymphoma is a rare, aggressive form of extranodal lymphoma, which frequently involves the bone marrow. An intrasinusoidal pattern of infiltration is characteristic of the disease and is often best appreciated on immunohistochemical staining. Bone marrow biopsy can be a useful diagnostic tool.
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Dong, Jinlei; Thu, Hnin Ei; Abourehab, Mohammed A S; Hussain, Zahid
2018-05-18
Osteoporosis is a medical condition of fragile bones with an increased susceptibility to bone fracture. Despite having availability of a wide range of pharmacological agents, prevalence of this metabolic disorder is continuously escalating. Owing to excellent biomedical achievements of nanomedicines in the last few decades, we aimed combo delivery of bone anti-resorptive agent, alendronate (ALN), and bone density enhancing drug, curcumin (CUR), in the form of polymeric nanoparticles. To further optimize the therapeutic efficacy, the prepared ALN/CUR nanoparticles were decorated with hyaluronic acid which is a well-documented biomacromolecule having exceptional bone regenerating potential. The optimized nanoformulation was evaluated for bone regeneration efficacy by assessing the time-mannered modulation in the proliferation, differentiation, and mineralization of MC3T3-E1 cells, a pre-osteoblast model. Moreover, the time-mannered expressions of various bone-forming protein biomarkers including bone morphogenetic protein, runt related transcription factor 2, and osteocalcin were assessed in the cell lysates. Results revealed that HA-ALN/CUR NPs provoke remarkable increase in proliferation, differentiation, and mineralization in the ECM of MC3T3-E1 cells which ultimately leads to enhanced bone formation. This new strategy employing simultaneous delivery of anti-resorptive and bone forming agents would open new horizons for the scientists as an efficient alternative pharmacotherapy for the management of osteoporosis. Copyright © 2017. Published by Elsevier B.V.
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NASA Astrophysics Data System (ADS)
Lau, P.; Hellweg, C. E.; Kirchner, S.; Baumstark-Khan, C.
2005-08-01
During long-term space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. Bone loss during space flight is about 1-2% per month. Bone is continually being remodelled under the influence of three types of highly specialized cells. Osteoblasts, the bone forming cells, osteoclasts, the bone resorbing cells and finally osteocytes preserve the homeostasis of bone formation and resorption. In vitro 3- dimensional cell culture of osteoblastic cell lines on microcarrier beads might be a better model to evaluate changes in bone cell morphology, function and differentiation under influence of spaceflight related factors than the conventional 2-D monolayer culture technique. Furthermore, it allows production of a greater amount of cells compared to the monolayer culture. Aim of this study is to examine the effects of culturing the immortalized murine osteoblastic cell line OCT-1 in a 3- dimensional environment on cell morphology and proliferation rate.
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A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone
Thibaudeau, Laure; Taubenberger, Anna V.; Holzapfel, Boris M.; Quent, Verena M.; Fuehrmann, Tobias; Hesami, Parisa; Brown, Toby D.; Dalton, Paul D.; Power, Carl A.; Hollier, Brett G.; Hutmacher, Dietmar W.
2014-01-01
ABSTRACT The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact ‘organ’ bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo. PMID:24713276
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Ye, Xinhai; Yin, Xiaofan; Yang, Dawei; Tan, Jian; Liu, Guangpeng
2012-07-01
Tissue engineering approaches using the combination of porous ceramics and bone marrow mesenchymal stem cells (BMSCs) represent a promising bone substitute for repairing large bone defects. Nevertheless, optimal conditions for constructing tissue-engineered bone have yet to be determined. It remains unclear if transplantation of predifferentiated BMSCs is superior to undifferentiated BMSCs or freshly isolated bone marrow mononucleated cells (BMNCs) in terms of new bone formation in vivo. The aim of this study was to investigate the effect of in vitro osteogenic differentiation (β-glycerophosphate, dexamethasone, and l-ascorbic acid) of human BMSCs on the capability to form tissue-engineered bone in unloaded conditions after subcutaneous implantation in nude mice. After isolation from human bone marrow aspirates, BMNCs were divided into three parts: one part was seeded onto porous beta-tricalcium phosphate ceramics immediately and transplanted in a heterotopic nude mice model; two parts were expanded in vitro to passage 2 before cell seeding and in vivo transplantation, either under osteogenic conditions or not. Animals were sacrificed for micro-CT and histological evaluation at 4, 8, 12, 16, and 20 weeks postimplantation. The results showed that BMSCs differentiated into osteo-progenitor cells after induction, as evidenced by the altered cell morphology and elevated alkaline phosphatase activity and calcium deposition, but their clonogenicity, proliferating rate, and seeding efficacy were not significantly affected by osteogenic differentiation, compared with undifferentiated cells. Extensive new bone formed in the pores of all the scaffolds seeded with predifferentiated BMSCs at 4 weeks after implantation, and maintained for 20 weeks. On the contrary, scaffolds containing undifferentiated BMSCs revealed limited bone formation only in 1 out of 6 cases at 8 weeks, and maintained for 4 weeks. For scaffolds with BMNCs, woven bone was observed sporadically only in one case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration.
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Tooth-bone morphogenesis during postnatal stages of mouse first molar development
Lungová, Vlasta; Radlanski, Ralf J; Tucker, Abigail S; Renz, Herbert; Míšek, Ivan; Matalová, Eva
2011-01-01
The first mouse molar (M1) is the most common model for odontogenesis, with research particularly focused on prenatal development. However, the functional dentition forms postnatally, when the histogenesis and morphogenesis of the tooth is completed, the roots form and the tooth physically anchors into the jaw. In this work, M1 was studied from birth to eruption, assessing morphogenesis, proliferation and apoptosis, and correlating these with remodeling of the surrounding bony tissue. The M1 completed crown formation between postnatal (P) days 0–2, and the development of the tooth root was initiated at P4. From P2 until P12, cell proliferation in the dental epithelium reduced and shifted downward to the apical region of the forming root. In contrast, proliferation was maintained or increased in the mesenchymal cells of the dental follicle. At later stages, before tooth eruption (P20), cell proliferation suddenly ceased. This withdrawal from the cell cycle correlated with tooth mineralization and mesenchymal differentiation. Apoptosis was observed during all stages of M1 postnatal morphogenesis, playing a role in the removal of cells such as osteoblasts in the mandibular region and working together with osteoclasts to remodel the bone around the developing tooth. At more advanced developmental stages, apoptotic cells and bodies accumulated in the cell layers above the tooth cusps, in the path of eruption. Three-dimensional reconstruction of the developing postnatal tooth and bone indicates that the alveolar crypts form by resorption underneath the primordia, whereas the ridges form by active bone growth between the teeth and roots to form a functional complex. PMID:21418206
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Tan, Shuang; Zhang, Binbin; Zhu, Xiaomei; Ao, Ping; Guo, Huajie; Yi, Weihong; Zhou, Guang-Qian
2014-01-01
Age-related bone loss and osteoporosis are associated with bone remodeling changes that are featured with decreased trabecular and periosteal bone formation relative to bone resorption. Current anticatabolic therapies focusing on the inhibition of bone resorption may not be sufficient in the prevention or reversal of age-related bone deterioration and there is a big need in promoting osteoblastogenesis and bone formation. Enhanced understanding of the network formed by key signaling pathways and molecules regulating bone forming cells in health and diseases has therefore become highly significant. The successful development of agonist/antagonist of the PTH and Wnt signaling pathways are profits of the understanding of these key pathways. As the core component of an approved antiosteoporosis agent, strontium takes its effect on osteoblasts at multilevel through multiple pathways, representing a good example in revealing and exploring anabolic mechanisms. The recognition of strontium effects on bone has led to its expected application in a variety of biomaterial scaffolds used in tissue engineering strategies aiming at bone repairing and regeneration. While summarizing the recent progress in these respects, this review also proposes the new approaches such as systems biology in order to reveal new insights in the pathology of osteoporosis as well as possible discovery of new therapies. PMID:24800251
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Kagami, Hideaki; Agata, Hideki; Inoue, Minoru; Asahina, Izumi; Tojo, Arinobu; Yamashita, Naohide; Imai, Kohzoh
2014-06-01
Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. Human bone marrow stromal cells (BMSCs) are the most commonly used cell source for bone tissue engineering. Although it is known that cell culture and induction protocols significantly affect the in vivo bone forming ability of BMSCs, the responsible factors of clinical outcome are poorly understood. The results from recent studies using human BMSCs have shown that factors such as passage number and length of osteogenic induction significantly affect ectopic bone formation, although such differences hardly affected the alkaline phosphatase activity or gene expression of osteogenic markers. Application of basic fibroblast growth factor helped to maintain the in vivo osteogenic ability of BMSCs. Importantly, responsiveness of those factors should be tested under clinical circumstances to improve the bone tissue engineering further. In this review, clinical application of bone tissue engineering was reviewed with putative underlying mechanisms.
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Wegman, F; Poldervaart, M T; van der Helm, Y J; Oner, F C; Dhert, W J; Alblas, J
2015-07-27
Bone morphogenetic protein-2 (BMP-2) gene delivery has shown to induce bone formation in vivo in cell-based tissue engineering. In addition, the chemoattractant stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is known to recruit multipotent stromal cells towards its release site where it enhances vascularisation and possibly contributes to osteogenic differentiation. To investigate potential cooperative behaviour for bone formation, we investigated combined release of BMP-2 and SDF-1α on ectopic bone formation in mice. Multipotent stromal cell-seeded and cell-free constructs with BMP-2 plasmid DNA and /or SDF-1α loaded onto gelatin microparticles, were implanted subcutaneously in mice for a period of 6 weeks. Histological analysis and histomorphometry revealed that the onset of bone formation and the formed bone volume were both enhanced by the combination of BMP-2 and SDF-1α compared to controls in cell-seeded constructs. Samples without seeded multipotent stromal cells failed to induce any bone formation. We conclude that the addition of stromal cell-derived factor-1α to a cell-seeded alginate based bone morphogenetic protein-2 plasmid DNA construct has an additive effect on bone formation and can be considered a promising combination for bone regeneration.
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Hiraga, Toru; Ninomiya, Tadashi; Hosoya, Akihiro; Takahashi, Masafumi; Nakamura, Hiroaki
2009-01-01
Periodontal ligament (PDL) is a unique connective tissue that not only connects cementum and alveolar bone to support teeth, but also plays an important role in reconstructing periodontal tissues. Previous studies have suggested that PDL cells have osteogenic potential; however, they lack precise histological examinations. Here, we studied bone-like matrix formation by PDL cells in rats using morphological techniques. Rat and human PDL cells exhibited substantial alkaline phosphatase activity and induced mineralization in vitro. RT-PCR analyses showed that PDL cells expressed the osteoblast markers, Runx2, osterix, and osteocalcin. These results suggest that PDL cells share similar phenotypes with osteoblasts. To examine the bone-like matrix formation in vivo, PDL cells isolated from green fluorescent protein (GFP)-transgenic rats were inoculated with hydroxyapatite (HA) disks into wild-type rats. Five weeks after the implantation, the pores in HA disks were occupied by GFP-positive cells. Mineralized matrix formation was also found on the surface of HA pores. At 12 weeks, some of the pores were filled with bone-like mineralized matrices (BLMM), which were positive for the bone matrix proteins, osteopontin, bone sialoprotein, and osteocalcin. Immunohistochemical examination revealed that most of the osteoblast- and osteocyte-like cells on or in the BLMM were GFP-positive, suggesting that the BLMM were directly formed by the inoculated PDL cells. On the pore surfaces, Sharpey's fiber-like structures embedded in cementum-like mineralized layers were also observed. These results collectively suggest that PDL cells have the ability to form periodontal tissues and could be a useful source for regenerative therapies of periodontal diseases.
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Mesenchymal Stem Cell Spheroids Retain Osteogenic Phenotype Through α2β1 Signaling
Murphy, Kaitlin C.; Hoch, Allison I.; Harvestine, Jenna N.; Zhou, Dejie
2016-01-01
The induction of mesenchymal stem cells (MSCs) toward the osteoblastic lineage using osteogenic supplements prior to implantation is one approach under examination to enhance their bone-forming potential. MSCs rapidly lose their induced phenotype upon removal of the soluble stimuli; however, their bone-forming potential can be sustained when provided with continued instruction via extracellular matrix (ECM) cues. In comparison with dissociated cells, MSC spheroids exhibit improved survival and secretion of trophic factors while maintaining their osteogenic potential. We hypothesized that entrapment of MSC spheroids formed from osteogenically induced cells would exhibit better preservation of their bone-forming potential than would dissociated cells from monolayer culture. Spheroids exhibited comparable osteogenic potential and increased proangiogenic potential with or without osteogenic preconditioning versus monolayer-cultured MSCs. Spheroids were then entrapped in collagen hydrogels, and the osteogenic stimulus was removed. In comparison with entrapped dissociated MSCs, spheroids exhibited significantly increased markers of osteogenic differentiation. The capacity of MSC spheroids to retain their osteogenic phenotype upon withdrawal of inductive cues was mediated by α2β1 integrin binding to cell-secreted ECM. These results demonstrate the capacity of spheroidal culture to sustain the mineral-producing phenotype of MSCs, thus enhancing their contribution toward bone formation and repair. Significance Despite the promise of mesenchymal stem cells (MSCs) for cell-based therapies for tissue repair and regeneration, there is little evidence that transplanted MSCs directly contribute to new bone formation, suggesting that induced cells rapidly lose their osteogenic phenotype or undergo apoptosis. In comparison with dissociated cells, MSC spheroids exhibit increased trophic factor secretion and improved cell survival. The loss of phenotype represents a significant clinical challenge for cell therapies, yet there is no evidence for whether MSC spheroids retain their osteogenic phenotype upon entrapment in a clinically relevant biomaterial. These findings demonstrate that MSC spheroids retain their osteogenic phenotype better than do dissociated MSCs, and this is due to integrin engagement with the cell-secreted extracellular matrix. These data provide evidence for a novel approach for potentiating the use of MSCs in bone repair. PMID:27365484
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Cai, Yun-Feng; Zhen, Zuo-Jun; Min, Jun; Fang, Tian-Ling; Chu, Zhong-Hua; Chen, Ji-Sheng
2004-11-15
To explore the feasibility of direct separation, selective proliferation and differentiation of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. Whole bone marrow cells of rats cultured in routine medium were replaced with conditioning selection media containing 20 mL/L, 50 mL/L, 70 mL/L, and 100 mL/L cholestatic sera, respectively, after they attached to the plates. The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures. Then the selected BDLSC were induced to proliferate and differentiate with the addition of hepatocyte growth factor (HGF). The morphology and phenotypic markers of BDLSC were characterized using immunohistochemistry, RT-PCR and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. Bone marrow cells formed fibroblast-like but not hepatocyte-like colonies in the presence of 20 mL/L cholestatic serum. In 70 mL/L cholestatic serum, BDLSC colonies could be selected but could not maintain good growth status. In 100 mL/L cholestatic serum, all of the bone marrow cells were unable to survive. A 50 mL/L cholestatic serum was the optimal concentration for the selection of BDLSC at which BDLSC could survive while the other populations of the bone marrow cells could not. The selected BDLSC proliferated and differentiated after HGF was added. Hepatocyte-like colony-forming units (H-CFU) then were formed. H-CFU expressed markers of embryonic hepatocytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors (HNF-1alpha and HNF-3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. The selected medium containing cholestatic serum can select BDLSC from whole bone marrow cells. It will be a new way to provide a readily available alternate source of cells for clinical hepatocyte therapy.
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Utilization of microgravity bioreactors for differentiation of mammalian skeletal tissue
NASA Technical Reports Server (NTRS)
Klement, B. J.; Spooner, B. S.
1993-01-01
Bioreactor cell and tissue culture vessels can be used to study bone development in a simulated microgravity environment. These vessels will also provide an advantageous, low maintenance culture system on space station Freedom. Although many types of cells and tissues can potentially utilize this system, our particular interest is in developing bone tissue. We have characterized an organ culture system utilizing embryonic mouse pre-metatarsal mesenchyme, documenting morphogenesis and differentiation as cartilage rods are formed, with subsequent terminal chondrocyte differentiation to hypertrophied cells. Further development to form bone tissue is achieved by supplementation of the culture medium. Research using pre-metatarsal tissue, combined with the bioreactor culture hardware, could give insight into the advantages and/or disadvantages of conditions experienced in microgravity. Studies such as these have the potential to enhance understanding of bone development and adult bone physiology, and may help define the processes of bone demineralization experienced in space and in pathological conditions here on earth.
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Origins of endothelial and osteogenic cells in the subcutaneous collagen gel implant.
Bilic-Curcic, I; Kalajzic, Z; Wang, L; Rowe, D W
2005-11-01
The interdependent relationship between vascular endothelial cells and osteoblasts during bone formation and fracture healing has been long appreciated. This paper reports a heterotopic implant model using FGF-2-expanded bone marrow stromal cells (BMSC) derived from Tie2eGFP (endothelial marker) and pOBCol3.6GFPcyan or topaz (early osteoblast marker) transgenic mice to appreciate the host/donor relationships of cells participating in the process of heterotopic bone formation. The study included various combinations of Tie2eGFP and pOBCol3.6GFPcyan and topaz transgenics as BMSC or whole bone marrow (WBM) donors and also as recipients. Rat tail collagen was used as a carrier of donor cells and implantation was done in lethally irradiated mice rescued with WBM injection. Development of ossicles in the implants was followed weekly during the 4- to 5-week long post-implantation period. By 4-5 weeks after total body irradiation (TBI) and implantation, a well-formed bone spicule had developed that was invested with bone marrow. Experiments showed absolute dominance of donor-derived cells in the formation of endothelial-lined vessels inside the implants as well as the marrow stromal-derived osteogenic cells. Host-derived fibroblasts and osteogenic cells were confined to the fibrous capsule surrounding the implant. In addition, cells lining the endosteal surface of newly formed marrow space carrying a pOBCol3.6GFP marker were observed that were contributed by WBM donor cells and the host. Thus, FGF-2-expanded BMSC appear to be a source of endothelial and osteogenic progenitor cells capable of eliciting heterotopic bone formation independent of cells from the host. This model should be useful for understanding the interactions between these two cell types that control osteogenic differentiation in vivo.
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Osteopetroses, emphasizing potential approaches to treatment.
Teti, Anna; Econs, Michael J
2017-09-01
Osteopetroses are a heterogeneous group of rare genetic bone diseases sharing the common hallmarks of reduced osteoclast activity, increased bone mass and high bone fragility. Osteoclasts are bone resorbing cells that contribute to bone growth and renewal through the erosion of the mineralized matrix. Alongside the bone forming activity by osteoblasts, osteoclasts allow the skeleton to grow harmonically and maintain a healthy balance between bone resorption and formation. Osteoclast impairment in osteopetroses prevents bone renewal and deteriorates bone quality, causing atraumatic fractures. Osteopetroses vary in severity and are caused by mutations in a variety of genes involved in bone resorption or in osteoclastogenesis. Frequent signs and symptoms include osteosclerosis, deformity, dwarfism and narrowing of the bony canals, including the nerve foramina, leading to hematological and neural failures. The disease is autosomal, with only one extremely rare form associated so far to the X-chromosome, and can have either recessive or dominant inheritance. Recessive ostepetroses are generally lethal in infancy or childhood, with a few milder forms clinically denominated intermediate osteopetroses. Dominant osteopetrosis is so far associated only with mutations in the CLCN7 gene and, although described as a benign form, it can be severely debilitating, although not at the same level as recessive forms, and can rarely result in reduced life expectancy. Severe osteopetroses due to osteoclast autonomous defects can be treated by Hematopoietic Stem Cell Transplant (HSCT), but those due to deficiency of the pro-osteoclastogenic cytokine, RANKL, are not suitable for this procedure. Likewise, it is unclear as to whether HSCT, which has high intrinsic risks, results in clinical improvement in autosomal dominant osteopetrosis. Therefore, there is an unmet medical need to identify new therapies and studies are currently in progress to test gene and cell therapies, small interfering RNA approach and novel pharmacologic treatments. Copyright © 2017 Elsevier Inc. All rights reserved.
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Expression of CD44v6 as matrix-associated ectodomain in the bone development.
Nakajima, Kosei; Taniguchi, Kazumi; Mutoh, Ken-ichiro
2010-08-01
This study describes the expression of CD44v6 in the bone development and is the first study of its kind to the authors' best knowledge. The CD44 family is a family of transmembrane glycoproteins that acts as cell adhesion molecules binding cells to other cells as well as cells to the extracellular matrix. It has been suggested that the CD44v6, a family member of CD44, is closely related to the osteosarcoma metastasis. In general, when cancer cells metastasize, they revert to their immature forms. In the present study, therefore, we have investigated CD44v6 and the standard form of CD44 (CD44st) in two types of immature forms of bone tissues: developmentally immature stages from fetuses to adults as well as experimentally immature stages using fracture models. CD44st expression was identified in osteoblasts, osteocytes, and in the peripheral portion of the bone matrix from the fetal to young ages of rats. Many more intense reactions for CD44v6 were observed in the bone matrix than CD44st in fetal stages. In experimental fracture models, positive immunoreactions to CD44st were clearly observed in the osteoblasts and osteocytes. CD44v6-positive immunoreactivity, however, was not detected in either osteoblasts or the bone matrix. In conclusion, CD44v6 is expressed in the embryonic stages and may be involved in the bone matrix formation as a matrix-associated ectodomain during normal ontogenetic development but not involved in the process of fracture healing.
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Engineering Vascularized Bone Grafts by Integrating a Biomimetic Periosteum and β-TCP Scaffold
2015-01-01
Treatment of large bone defects using synthetic scaffolds remain a challenge mainly due to insufficient vascularization. This study is to engineer a vascularized bone graft by integrating a vascularized biomimetic cell-sheet-engineered periosteum (CSEP) and a biodegradable macroporous beta-tricalcium phosphate (β-TCP) scaffold. We first cultured human mesenchymal stem cells (hMSCs) to form cell sheet and human umbilical vascular endothelial cells (HUVECs) were then seeded on the undifferentiated hMSCs sheet to form vascularized cell sheet for mimicking the fibrous layer of native periosteum. A mineralized hMSCs sheet was cultured to mimic the cambium layer of native periosteum. This mineralized hMSCs sheet was first wrapped onto a cylindrical β-TCP scaffold followed by wrapping the vascularized HUVEC/hMSC sheet, thus generating a biomimetic CSEP on the β-TCP scaffold. A nonperiosteum structural cell sheets-covered β-TCP and plain β-TCP were used as controls. In vitro studies indicate that the undifferentiated hMSCs sheet facilitated HUVECs to form rich capillary-like networks. In vivo studies indicate that the biomimetic CSEP enhanced angiogenesis and functional anastomosis between the in vitro preformed human capillary networks and the mouse host vasculature. MicroCT analysis and osteocalcin staining show that the biomimetic CSEP/β-TCP graft formed more bone matrix compared to the other groups. These results suggest that the CSEP that mimics the cellular components and spatial configuration of periosteum plays a critical role in vascularization and osteogenesis. Our studies suggest that a biomimetic periosteum-covered β-TCP graft is a promising approach for bone regeneration. PMID:24858072
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Mousa, Aisha; Cui, Cui; Song, Aimei; Myneni, Vamsee D; Sun, Huifang; Li, Jin Jin; Murshed, Monzur; Melino, Gerry; Kaartinen, Mari T
2017-01-01
Appropriate bone mass is maintained by bone-forming osteoblast and bone-resorbing osteoclasts. Mesenchymal stem cell (MSC) lineage cells control osteoclastogenesis via expression of RANKL and OPG (receptor activator of nuclear factor κB ligand and osteoprotegerin), which promote and inhibit bone resorption, respectively. Protein crosslinking enzymes transglutaminase 2 (TG2) and Factor XIII-A (FXIII-A) have been linked to activity of myeloid and MSC lineage cells; however, in vivo evidence has been lacking to support their function. In this study, we show in mice that TG2 and FXIII-A control monocyte-macrophage cell differentiation into osteoclasts as well as RANKL production in MSCs and in adipocytes. Long bones of mice lacking TG2 and FXIII-A transglutaminases, show compromised biomechanical properties and trabecular bone loss in axial and appendicular skeleton. This was caused by increased osteoclastogenesis, a cellular phenotype that persists in vitro. The increased potential of TG2 and FXIII-A deficient monocytes to form osteoclasts was reversed by chemical inhibition of TG activity, which revealed the presence of TG1 in osteoclasts and assigned different roles for the TGs as regulators of osteoclastogenesis. TG2- and FXIII-A-deficient mice had normal osteoblast activity, but increased bone marrow adipogenesis, MSCs lacking TG2 and FXIII-A showed high adipogenic potential and significantly increased RANKL expression as well as upregulated TG1 expression. Chemical inhibition of TG activity in the null cells further increased adipogenic potential and RANKL production. Altered differentiation of TG2 and FXIII-A null MSCs was associated with plasma fibronectin (FN) assembly defect in cultures and FN retention in serum and marrow in vivo instead of assembly into bone. Our findings provide new functions for TG2, FXIII-A and TG1 in bone cells and identify them as novel regulators of bone mass, plasma FN homeostasis, RANKL production and myeloid and MSC cell differentiation. PMID:28387755
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Weigand, Annika; Beier, Justus P; Schmid, Rafael; Knorr, Tobias; Kilian, David; Götzl, Rebekka; Gerber, Thomas; Horch, Raymund E; Boos, Anja M
2017-03-01
For decades, researchers have been developing a range of promising strategies in bone tissue engineering with the aim of producing a significant clinical benefit over existing therapies. However, a major problem concerns the traditional use of xenogeneic substances for the expansion of cells, which complicates direct clinical transfer. The study's aim was to establish a totally autologous sheep model as a basis for further preclinical studies and future clinical application. Ovine mesenchymal stromal cells (MSC) were cultivated in different concentrations (0%, 2%, 5%, 10%, and 25%) of either autologous serum (AS) or fetal calf serum (FCS). With an increase of serum concentration, enhanced metabolic activity and proliferation could be observed. There were minor differences between MSC cultivated in AS or FCS, comparing gene and protein expression of osteogenic and stem cell markers, morphology, and osteogenic differentiation. MSC implanted subcutaneously in the sheep model, together with a nanostructured bone substitute, either in stable block or moldable putty form, induced similar vascularization and remodeling of the bone substitute irrespective of cultivation of MSC in AS or FCS and osteogenic differentiation. The bone substitute in block form together with MSC proved particularly advantageous in the induction of ectopic bone formation compared to the cell-free control and putty form. It could be demonstrated that AS is suitable for replacement of FCS for cultivation of ovine MSC for bone tissue engineering purposes. Substantial progress has been made in the development of a strictly xenogeneic-free preclinical animal model to bring future clinical application of bone tissue engineering strategies within reach.
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Zhao, Nan; Zhu, Donghui
2014-01-01
Magnesium (Mg) biomaterials are a new generation of biodegradable materials and have promising potential for orthopedic applications. After implantation in bone tissues, these materials will directly interact with extracellular matrix (ECM) biomolecules and bone cells. Type I collagen, the major component of bone ECM, forms the architecture scaffold that provides physical support for bone cell attachment. However, it is still unknown how Mg substrate affects collagen assembly on top of it as well as subsequent cell attachment and growth. Here, we studied the effects of collagen monomer concentration, pH, assembly time, and surface roughness of two Mg materials (pure Mg and AZ31) on collagen fibril formation. Results showed that formation of fibrils would not initiate until the monomer concentration reached a certain level depending on the type of Mg material. The thickness of collagen fibril increased with the increase of assembly time. The structures of collagen fibrils formed on semi-rough surfaces of Mg materials have a high similarity to that of native bone collagen. Next, cell attachment and growth after collagen assembly were examined. Materials with rough surface showed higher collagen adsorption but compromised bone cell attachment. Interestingly, surface roughness and collagen structure did not affect cell growth on AZ31 for up to a week. Findings from this work provide some insightful information on Mg-tissue interaction at the interface and guidance for future surface modifications of Mg biomaterials. PMID:25303459
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Yoshioka, Tomokazu; Mishima, Hajime; Sakai, Shinsuke; Uemura, Toshimasa
2013-10-01
The purpose of this study was to evaluate the long-term results of cartilage repair after allogeneic transplantation of cartilaginous aggregates formed from bone marrow-derived cells. Bone marrow cells were harvested from 12-day-old rabbits. The cells were subjected to a monolayer culture, and the spindle-shaped cells attached to the flask surface were defined as bone marrow-derived mesenchymal cells. After the monolayer culture, a 3-dimensional cartilaginous aggregate was formed using a bioreactor with chondrogenesis. We created osteochondral defects, measuring 5 mm in diameter and 4 mm in depth, at the femoral trochlea of 10-week-old rabbits. Two groups were established, the transplanted group in which the cartilaginous aggregate was transplanted into the defect, and the control group in which the defect was left untreated. Twenty-six and 52 weeks after surgery, the rabbits were sacrificed and their tissue repair status was evaluated macroscopically (International Cartilage Repair Society [ICRS] score) and histologically (O'Driscoll score). The ICRS scores were as follows: at week 26, 7.2 ± 0.5 and 7.6 ± 0.8; at week 52, 7.6 ± 1.1 and 9.7 ± 0.7, for the transplanted and control groups, respectively. O'Driscoll scores were as follows: at week 26, 12.6 ± 1.9 and 10.1 ± 1.9; at week 52, 9.6 ± 3.0 and 14.0 ± 1.4, each for transplanted and control groups, respectively. No significant differences were observed between the groups. This study demonstrates that allogeneic transplantation of cartilaginous aggregates formed from bone marrow-derived cells produces comparable long-term results based on macroscopic and histological outcome measures when compared with osteochondral defects that are left untreated.
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Douglas, Timothy E L; Łapa, Agata; Samal, Sangram Keshari; Declercq, Heidi A; Schaubroeck, David; Mendes, Ana C; der Voort, Pascal Van; Dokupil, Agnieszka; Plis, Agnieszka; De Schamphelaere, Karel; Chronakis, Ioannis S; Pamuła, Elżbieta; Skirtach, Andre G
2017-12-01
Mineralization of hydrogel biomaterials is considered desirable to improve their suitability as materials for bone regeneration. Calcium carbonate (CaCO 3 ) has been successfully applied as a bone regeneration material, but hydrogel-CaCO 3 composites have received less attention. Magnesium (Mg) has been used as a component of calcium phosphate biomaterials to stimulate bone-forming cell adhesion and proliferation and bone regeneration in vivo, but its effect as a component of carbonate-based biomaterials remains uninvestigated. In the present study, gellan gum (GG) hydrogels were mineralized enzymatically with CaCO 3 , Mg-enriched CaCO 3 and magnesium carbonate to generate composite biomaterials for bone regeneration. Hydrogels loaded with the enzyme urease were mineralized by incubation in mineralization media containing urea and different ratios of calcium and magnesium ions. Increasing the magnesium concentration decreased mineral crystallinity. At low magnesium concentrations calcite was formed, while at higher concentrations magnesian calcite was formed. Hydromagnesite (Mg 5 (CO 3 ) 4 (OH) 2 .4H 2 O) formed at high magnesium concentration in the absence of calcium. The amount of mineral formed and compressive strength decreased with increasing magnesium concentration in the mineralization medium. The calcium:magnesium elemental ratio in the mineral formed was higher than in the respective mineralization media. Mineralization of hydrogels with calcite or magnesian calcite promoted adhesion and growth of osteoblast-like cells. Hydrogels mineralized with hydromagnesite displayed higher cytotoxicity. In conclusion, enzymatic mineralization of GG hydrogels with CaCO 3 in the form of calcite successfully reinforced hydrogels and promoted osteoblast-like cell adhesion and growth, but magnesium enrichment had no definitive positive effect. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.
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Wang, Zhong-Shan; Feng, Zhi-Hong; Wu, Guo-Feng; Bai, Shi-Zhu; Dong, Yan; Chen, Fa-Ming; Zhao, Yi-Min
2016-06-21
Periodontal regeneration involves the restoration of at least three unique tissues: cementum, periodontal ligament tissue (PDL) and alveolar bone tissue. Here, we first isolated human PDL stem cells (PDLSCs) and jaw bone mesenchymal stem cells (JBMSCs). These cells were then induced to form cell sheets using an ascorbic acid-rich approach, and the cell sheet properties, including morphology, thickness and gene expression profile, were compared. Platelet-rich fibrin (PRF) derived from human venous blood was then fabricated into bioabsorbable fibrin scaffolds containing various growth factors. Finally, the in vivo potential of a cell-material construct based on PDLSC sheets, PRF scaffolds and JBMSC sheets to form periodontal tissue was assessed in a nude mouse model. In this model, PDLSC sheet/PRF/JBMSC sheet composites were placed in a simulated periodontal space comprising human treated dentin matrix (TDM) and hydroxyapatite (HA)/tricalcium phosphate (TCP) frameworks. Eight weeks after implantation, the PDLSC sheets tended to develop into PDL-like tissues, while the JBMSC sheets tended to produce predominantly bone-like tissues. In addition, the PDLSC sheet/PRF/JBMSC sheet composites generated periodontal tissue-like structures containing PDL- and bone-like tissues. Further improvements in this cell transplantation design may have the potential to provide an effective approach for future periodontal tissue regeneration.
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Translational Control in Bone Marrow Failure
2015-05-01
HCLS1 associated protein X-1 (HAX1), cause hereditary forms of neutropenia . Previously, competing hypotheses have posited that mutant forms of...derived induced pluripotent stem cell (iPSC) model of ELANE-associated neutropenia . During the second year of this project, in order to facilitate...pathology. 3 2. KEY WORDS neutropenia bone marrow failure neutrophil elastase ELANE HAX1 alternate translation induced pluripotent stem cells (iPSC
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Normal Collagen and Bone Production by Gene-targeted Human Osteogenesis Imperfecta iPSCs
Deyle, David R; Khan, Iram F; Ren, Gaoying; Wang, Pei-Rong; Kho, Jordan; Schwarze, Ulrike; Russell, David W
2012-01-01
Osteogenesis imperfecta (OI) is caused by dominant mutations in the type I collagen genes. In principle, the skeletal abnormalities of OI could be treated by transplantation of patient-specific, bone-forming cells that no longer express the mutant gene. Here, we develop this approach by isolating mesenchymal cells from OI patients, inactivating their mutant collagen genes by adeno-associated virus (AAV)-mediated gene targeting, and deriving induced pluripotent stem cells (iPSCs) that were expanded and differentiated into mesenchymal stem cells (iMSCs). Gene-targeted iMSCs produced normal collagen and formed bone in vivo, but were less senescent and proliferated more than bone-derived MSCs. To generate iPSCs that would be more appropriate for clinical use, the reprogramming and selectable marker transgenes were removed by Cre recombinase. These results demonstrate that the combination of gene targeting and iPSC derivation can be used to produce potentially therapeutic cells from patients with genetic disease. PMID:22031238
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Aging of marrow stromal (skeletal) stem cells and their contribution to age-related bone loss.
Bellantuono, Ilaria; Aldahmash, Abdullah; Kassem, Moustapha
2009-04-01
Marrow stromal cells (MSC) are thought to be stem cells with osteogenic potential and therefore responsible for the repair and maintenance of the skeleton. Age related bone loss is one of the most prevalent diseases in the elder population. It is controversial whether MSC undergo a process of aging in vivo, leading to decreased ability to form and maintain bone homeostasis with age. In this review we summarize evidence of MSC involvement in age related bone loss and suggest new emerging targets for intervention.
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[Optimizing histological image data for 3-D reconstruction using an image equalizer].
Roth, A; Melzer, K; Annacker, K; Lipinski, H G; Wiemann, M; Bingmann, D
2002-01-01
Bone cells form a wired network within the extracellular bone matrix. To analyse this complex 3D structure, we employed a confocal fluorescence imaging procedure to visualize live bone cells within their native surrounding. By means of newly developed image processing software, the "Image-Equalizer", we aimed to enhanced the contrast and eliminize artefacts in such a way that cell bodies as well as fine interconnecting processes were visible.
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Di Maggio, Nunzia; Martella, Elisa; Frismantiene, Agne; Resink, Therese J.; Schreiner, Simone; Lucarelli, Enrico; Jaquiery, Claude; Schaefer, Dirk J.; Martin, Ivan; Scherberich, Arnaud
2017-01-01
Stromal vascular fraction (SVF) cells of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, adipose-derived stromal/stem cells (ASC), even after minimal monolayer expansion, display poor osteogenic capacity in vivo. We investigated whether ASC bone-forming capacity may be maintained by culture within a self-produced extracellular matrix (ECM) that recapitulates the native environment. SVF cells expanded without passaging up to 28 days (Unpass-ASC) deposited a fibronectin-rich extracellular matrix and displayed greater clonogenicity and differentiation potential in vitro compared to ASC expanded only for 6 days (P0-ASC) or for 28 days with regular passaging (Pass-ASC). When implanted subcutaneously, Unpass-ASC produced bone tissue similarly to SVF cells, in contrast to P0- and Pass-ASC, which mainly formed fibrous tissue. Interestingly, clonogenic progenitors from native SVF and Unpass-ASC expressed low levels of the fibronectin receptor α5 integrin (CD49e), which was instead upregulated in P0- and Pass-ASC. Mechanistically, induced activation of α5β1 integrin in Unpass-ASC led to a significant loss of bone formation in vivo. This study shows that ECM and regulation of α5β1-integrin signaling preserve ASC progenitor properties, including bone tissue-forming capacity, during in vitro expansion. PMID:28290502
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Osteogenically differentiated mesenchymal stem cells and ceramics for bone tissue engineering.
Ohgushi, Hajime
2014-02-01
In the human body, cells having self-renewal and multi-differentiation capabilities reside in many tissues and are called adult stem cells. In bone marrow tissue, two types of stem cells are well known: hematopoietic stem cells and mesenchymal stem cells (MSCs). Though the number of MSCs in bone marrow tissue is very low, it can be increased by in vitro culture of the marrow, and culture-expanded MSCs are available for various tissue regeneration. The culture-expanded MSCs can further differentiate into osteogenic cells such as bone forming osteoblasts by culturing the MSCs in an osteogenic medium. This paper discusses osteogenically differentiated MSCs derived from the bone marrow of patients. Importantly, the differentiation can be achieved on ceramic surfaces which demonstrate mineralized bone matrix formation as well as appearance of osteogenic cells. The cell/matrix/ceramic constructs could show immediate in vivo bone formation and are available for bone reconstruction surgery. Currently, MSCs are clinically available for the regeneration of various tissues due to their high proliferation/differentiation capabilities. However, the capabilities are still limited and thus technologies to improve or recover the inherent capabilities of MSCs are needed.
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Cordyceps sinensis health supplement enhances recovery from taxol-induced leukopenia.
Liu, Wei-Chung; Chuang, Wei-Ling; Tsai, Min-Lung; Hong, Ji-Hong; McBride, William H; Chiang, Chi-Shiun
2008-04-01
This study aimed to evaluate the ability of the health food supplement Cordyceps sinensis (CS) to ameliorate suppressive effects of chemotherapy on bone marrow function as a model for cancer treatment. Mice were treated with Taxol (17 mg/kg body wt) one day before oral administration of a hot-water extract of CS (50 mg/kg daily) that was given daily for 3 weeks. White blood cell counts in peripheral blood of mice receiving Taxol were at 50% of normal levels on day 28 but had recovered completely in mice treated with CS. In vitro assays showed that CS enhanced the colony-forming ability of both granulocyte macrophage colony forming unit (GM-CFU) and osteogenic cells from bone marrow preparations and promoted the differentiation of bone marrow mesenchymal stromal cells into adipocytes, alkaline phosphatase-positive osteoblasts, and bone tissue. This result could be attributed to enhanced expression of Cbfa1 (core binding factor a) and BMP-2 (bone morphogenetic protein) with concurrent suppression of ODF (osteoclast differentiation factor/RANK [receptor activator of NF-kappaB]) ligand. In summary, CS enhances recovery of mice from leukopenia caused by Taxol treatment. It appears to do so by protecting both hematopoietic progenitor cells directly and the bone marrow stem cell niche through its effects on osteoblast differentiation.
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Tormin, Ariane; Li, Ou; Brune, Jan Claas; Walsh, Stuart; Schütz, Birgit; Ehinger, Mats; Ditzel, Nicholas; Kassem, Moustapha
2011-01-01
Nonhematopoietic bone marrow mesenchymal stem cells (BM-MSCs) are of central importance for bone marrow stroma and the hematopoietic environment. However, the exact phenotype and anatomical distribution of specified MSC populations in the marrow are unknown. We characterized the phenotype of primary human BM-MSCs and found that all assayable colony-forming units-fibroblast (CFU-Fs) were highly and exclusively enriched not only in the lin−/CD271+/CD45−/CD146+ stem-cell fraction, but also in lin−/CD271+/CD45−/CD146−/low cells. Both populations, regardless of CD146 expression, shared a similar phenotype and genotype, gave rise to typical cultured stromal cells, and formed bone and hematopoietic stroma in vivo. Interestingly, CD146 was up-regulated in normoxia and down-regulated in hypoxia. This was correlated with in situ localization differences, with CD146 coexpressing reticular cells located in perivascular regions, whereas bone-lining MSCs expressed CD271 alone. In both regions, CD34+ hematopoietic stem/progenitor cells were located in close proximity to MSCs. These novel findings show that the expression of CD146 differentiates between perivascular versus endosteal localization of non-hematopoietic BM-MSC populations, which may be useful for the study of the hematopoietic environment. PMID:21415267
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Maiborodin, I V; Yakimova, N V; Matveyeva, V A; Pekarev, O G; Maiborodina, E I; Pekareva, E O
2011-04-01
Results of injection of autologous bone marrow mesenchymal stem cells with transfected GFP gene into the rat uterine horn cicatrix were studied by light microscopy. Large groups of blood vessels with blood cells inside were seen after injection of autologous bone marrow cells into the cicatrix on the right horn, formed 2 months after its ligation; no groups of vessels of this kind were found in the cicatrix in the contralateral horn. Examination of unstained sections in reflected UV light showed sufficiently bright fluorescence in the endothelium and outer vascular membrane in the uterine horn cicatrix only on the side of injection. Hence, autologous mesenchymal stem cells injected into the cicatrix formed the blood vessels due to differentiation into endotheliocytes and pericytes. The expression of GFP gene not only in the vascular endothelium, but also in vascular outer membranes indicated that autologous mesenchymal stem cells differentiated in the endothelial and pericytic directions.
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Matrix Elasticity of Void-Forming Hydrogels Controls Transplanted Stem Cell-Mediated Bone Formation
Huebsch, Nathaniel; Lippens, Evi; Lee, Kangwon; Mehta, Manav; Koshy, Sandeep T; Darnell, Max C; Desai, Rajiv; Madl, Christopher M.; Xu, Maria; Zhao, Xuanhe; Chaudhuri, Ovijit; Verbeke, Catia; Kim, Woo Seob; Alim, Karen; Mammoto, Akiko; Ingber, Donald E.; Duda, Georg N; Mooney, David J.
2015-01-01
The effectiveness of stem-cell therapies has been hampered by cell death and limited control over fate1. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype2–4. Stem cell behavior can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials5–7, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem-cell behaviors in situ. PMID:26366848
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Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation
NASA Astrophysics Data System (ADS)
Huebsch, Nathaniel; Lippens, Evi; Lee, Kangwon; Mehta, Manav; Koshy, Sandeep T.; Darnell, Max C.; Desai, Rajiv M.; Madl, Christopher M.; Xu, Maria; Zhao, Xuanhe; Chaudhuri, Ovijit; Verbeke, Catia; Kim, Woo Seob; Alim, Karen; Mammoto, Akiko; Ingber, Donald E.; Duda, Georg N.; Mooney, David J.
2015-12-01
The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel’s elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel’s elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.
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Gao, Xueqin; Usas, Arvydas; Lu, Aiping; Kozemchak, Adam; Tang, Ying; Poddar, Minakshi; Sun, Xuying; Cummins, James H; Huard, Johnny
2016-08-01
This study investigated the role of cyclooxygenase-2 (COX-2) expression by donor and host cells in muscle-derived stem cell (MDSC)-mediated bone regeneration utilizing a critical size calvarial defect model. We found that BMP4/green fluorescent protein (GFP)-transduced MDSCs formed significantly less bone in COX-2 knock-out (Cox-2KO) than in COX-2 wild-type (WT) mice. BMP4/GFP-transduced Cox-2KO MDSCs also formed significantly less bone than transduced WT MDSCs when transplanted into calvarial defects created in CD-1 nude mice. The impaired bone regeneration in the Cox-2KO MDSCBMP4/GFP group is associated with downregulation of BMP4-pSMAD1/5 signaling, decreased osteogenic differentiation and lowered proliferation capacity after transplantation, compared with WT MDSCBMP4/GFP cells. The Cox-2KO MDSCBMP4/GFP group demonstrated a reduction in cell survival and direct osteogenic differentiation in vitro These effects were mediated in part by the downregulation of Igf1 and Igf2. In addition, the Cox-2KO MDSCBMP4/GFP cells recruited fewer macrophages than the WT MDSC/BMP4/GFP cells in the early phase after injury. We concluded that the bone regeneration capacity of Cox-2KO MDSCs was impaired because of a reduction in cell proliferation and survival capacities, reduction in osteogenic differentiation and a decrease in the ability of the cells to recruit host cells to the injury site. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Terranova, Lisa; Dragusin, Diana Maria; Mallet, Romain; Vasile, Eugeniu; Stancu, Izabela-Cristina; Behets, Catherine; Chappard, Daniel
2017-02-01
Non-biodegradable porous polystyrene (PS) scaffolds, composed of microfibers, have been prepared by electrospinning for the reconstruction of large bone defects. PS microfibers were prepared by incorporating β-TCP grains inside the polymer or grafting gold nanoparticles surface functionalized with mercaptosuccinic acid. Cytocompatibility of the three types of scaffolds (PS, β-TCP-PS and Au-PS) was studied by seeding human mesenchymal stem cells. Biocompatibility was evaluated by implanting β-TCP-PS and Au-PS scaffolds into a critical size (4mm) calvarial defect in mice. Calvaria were taken 6, 9, and 12 weeks after implantation; newly formed bone and cellular response was analyzed by microcomputed tomography (microCT) and histology. β-TCP-PS scaffolds showed a significantly higher cell proliferation in vitro than on PS or Au-PS alone; clearly, the presence of β-TCP grains improved cytocompatibility. Biocompatibility study in the mouse calvaria model showed that β-TCP-PS scaffolds were significantly associated with more newly-formed bone than Au-PS. Bone developed by osteoconduction from the defect margins to the center. A dense fibrous connective tissue containing blood vessels was identified histologically in both types of scaffolds. There was no inflammatory foci nor giant cell in these areas. AuNPs aggregates were identified histologically in the fibrosis and also incorporated in the newly-formed bone matrix. Although the different types of PS microfibers appeared cytocompatible during the in vitro experiment, they appeared biotolerated in vivo since they induced a fibrotic reaction associated with newly formed bone. Copyright © 2016 Elsevier Ltd. All rights reserved.
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HORSE SPECIES SYMPOSIUM: Use of mesenchymal stem cells in fracture repair in horses.
Govoni, K E
2015-03-01
Equine bone fractures are often catastrophic, potentially fatal, and costly to repair. Traditional methods of healing fractures have limited success, long recovery periods, and a high rate of reinjury. Current research in the equine industry has demonstrated that stem cell therapy is a promising novel therapy to improve fracture healing and reduce the incidence of reinjury; however, reports of success in horses have been variable and limited. Stem cells can be derived from embryonic, fetal, and adult tissue. Based on the ease of collection, opportunity for autologous cells, and proven success in other models, adipose- or bone marrow-derived mesenchymal stem cells (MSC) are often used in equine therapies. Methods for isolation, proliferation, and differentiation of MSC are well established in rodent and human models but are not well characterized in horses. There is recent evidence that equine bone marrow MSC are able to proliferate in culture for several passages in the presence of autologous and fetal bovine serum, which is important for expansion of cells. Mesenchymal stem cells have the capacity to differentiate into osteoblasts, the bone forming cells, and this complex process is regulated by a number of transcription factors including runt-related transcription factor 2 (Runx2) and osterix (Osx). However, it has not been well established if equine MSC are regulated in a similar manner. The data presented in this review support the view that equine bone marrow MSC are regulated by the same transcription factors that control the differentiation of rodent and human MSC into osteoblasts. Although stem cell therapy is promising in equine bone repair, additional research is needed to identify optimal methods for reintroduction and potential manipulations to improve their ability to form new bone.
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Solid Free-form Fabrication Technology and Its Application to Bone Tissue Engineering
Lee, Jin Woo; Kim, Jong Young; Cho, Dong-Woo
2010-01-01
The development of scaffolds for use in cell-based therapies to repair damaged bone tissue has become a critical component in the field of bone tissue engineering. However, design of scaffolds using conventional fabrication techniques has limited further advancement, due to a lack of the required precision and reproducibility. To overcome these constraints, bone tissue engineers have focused on solid free-form fabrication (SFF) techniques to generate porous, fully interconnected scaffolds for bone tissue engineering applications. This paper reviews the potential application of SFF fabrication technologies for bone tissue engineering with respect to scaffold fabrication. In the near future, bone scaffolds made using SFF apparatus should become effective therapies for bone defects. PMID:24855546
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β3-Adrenergic Regulation of EPC Features Through Manipulation of the Bone Marrow MSC Niche.
Vafaei, Rana; Nassiri, Seyed Mahdi; Siavashi, Vahid
2017-12-01
Mesenchymal stem cells (MSCs) reside in a specific niche in the bone marrow, however, biological features of this niche are still not fully understood. Given the interactions of MSCs with endothelial cells in different tissues, bone marrow MSC niche may influence the biological features of endothelial progenitor cells (EPCs). To understand the role of the sympathetic nervous system in regulation of the MSC niche, we examined whether the manipulation of the MSC niche via β3-adrenergic signals will affect EPC features. A selective β3 agonist (BRL37344) or a β3 antagonist (SR59230A) was administered in mice for 2 weeks to determine the potential effects of these regimens on the population of CD133 + stem cells in the bone marrow. Then, bone marrow-derived MSCs and EPCs were harvested and expanded from the mice to examine the effect of changes in the MSC niche on EPC features. Improved MSC colony forming potency with increased bone marrow stromal cell-derived factor 1 (SDF-1) (also known as C-X-C motif chemokine 12 [CXCL12]) expression was shown as a result of intensification of the bone marrow adrenergic signals through BRL37344 injection. On the other hand, the blockage of these signals limited the expression level of SDF-1 and resulted in bone marrow enrichment of CD133 + cells. Manipulation of the MSC niche and decreased SDF-1 expression via SR59230A injection also prompted EPCs to form more colonies with augmented proliferation and differentiation capacity. Overall, our results indicate that the β3-adrenergic signals regulate the MSC niche, thereby resulting in modulation of EPC biological features. J. Cell. Biochem. 118: 4753-4761, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
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Hou, Tianyong; Li, Zhiqiang; Luo, Fei; Xie, Zhao; Wu, Xuehui; Xing, Junchao; Dong, Shiwu; Xu, Jianzhong
2014-07-01
The need for suitable bone grafts is high; however, there are limitations to all current graft sources, such as limited availability, the invasive harvest procedure, insufficient osteoinductive properties, poor biocompatibility, ethical problems, and degradation properties. The lack of osteoinductive properties is a common problem. As an allogenic bone graft, demineralized bone matrix (DBM) can overcome issues such as limited sources and comorbidities caused by invasive harvest; however, DBM is not sufficiently osteoinductive. Bone marrow has been known to magnify osteoinductive components for bone reconstruction because it contains osteogenic cells and factors. Mesenchymal stem cells (MSCs) derived from bone marrow are the gold standard for cell seeding in tissue-engineered biomaterials for bone repair, and these cells have demonstrated beneficial effects. However, the associated high cost and the complicated procedures limit the use of tissue-engineered bone constructs. To easily enrich more osteogenic cells and factors to DBM by selective cell retention technology, DBM is modified by a nanoscale self-assembling peptide (SAP) to form a composite DBM/SAP scaffold. By decreasing the pore size and increasing the charge interaction, DBM/SAP scaffolds possess a much higher enriching yield for osteogenic cells and factors compared with DBM alone scaffolds. At the same time, SAP can build a cellular microenvironment for cell adhesion, proliferation, and differentiation that promotes bone reconstruction. As a result, a suitable bone graft fabricated by DBM/SAP scaffolds and bone marrow represents a new strategy and product for bone transplantation in the clinic. Copyright © 2014 Elsevier Ltd. All rights reserved.
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The Bony Side of Endothelial Cells in Prostate Cancer.
Peng, Jia; Kang, Yibin
2017-06-05
Prostate cancer bone metastases are primarily osteoblastic, but the source of bone-forming cells in these lesions remains poorly defined. In this issue of Developmental Cell, Lin et al. (2017) demonstrate that tumor-associated endothelial cells can give rise to osteoblasts in prostate cancer through endothelial-to-osteoblast (EC-to-OSB) conversion. Copyright © 2017 Elsevier Inc. All rights reserved.
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A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions
NASA Astrophysics Data System (ADS)
Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace
2016-08-01
Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.
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Grardel, B; Hardouin, P
1992-01-01
Thirty two cases of the association of mastocytosis and bone lesions were collected in a multicentre study. Five cases involved osteocondensation forms. However, most often (27 cases), there was osteoporosis (OP). The diagnosis was made in the absence of obvious risk factors, and thus often in men (2/3 of patients), when there was the association of pigmented urticaria and an excess of mast cells in bone biopsies. Laboratory, radiological and isotope scan findings are often non-specific, being identical to those encountered in common OP. The histomorphometric profile involves an association of decreased cancellous bone volume, increased area of resorption and decreased bone formation parameters. Progression to malignant mastocytosis occurs essentially in diffuse osteocondensation forms and is rare in OP types. Emphasis must be placed on the importance of qualitative study of bone marrow, using specific stains, since the diagnosis may be missed in the absence of typical skin lesions. Conversely, since a simple increase in mast cell count is possible during common OP, a search for mast cell nodules is important in order to establish the diagnosis with certitude.
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Dos Santos Neves, Juliana; Franchin, Marcelo; Rosalen, Pedro Luiz; Omar, Nadia Fayez; Dos Santos, Mariana Albuquerque; Paschoal, Jonas Augusto Rizzato; Novaes, Pedro Duarte
2016-05-13
Hancornia speciosa Gomes, commonly known as Mangabeira, is a Brazilian native fruit tree belonging to the Apocynaceae family. In folk medicine, the latex obtained from Mangabeira's trunk has been used as an adjunct therapy for bone fractures. Few pharmacological studies on the Hancornia speciosa latex have been developed and despite its popular use for bone healing there is no data about its biological effect on bone. The present study aimed to investigate the osteogenic potential of Hancornia speciosa latex in rat calvaria, as well as its phytochemical profile. A neutral gel composition containing 5% latex was topical applied to a critical size bone defect and over intact calvaria of rats. Areas of newly formed bone on the borders of the defect and of calvaria periosteum were quantified, as well as the percentage of BrdU-positive cells and total cells in the periosteum at different periods of time after latex application. The cytotoxicity of the latex aqueous phase was evaluated in rat calvarial cells in vitro by MTT assay and its phytochemical profile was investigated by ESI-MS/MS. The area of newly formed bone on the borders of the calvaria defect was larger in rats that received latex at 15 and 30 days of healing. After 3 days of latex application over the intact calvaria, the periosteum area was increased and newly formed bone was observed after 5 and 11 days. There was also an increase in periosteum cell proliferation and population followed latex application on calvaria (p<0.05). The latex aqueous phase limited rat calvarial cell viability in vitro in concentrations larger than 0.6mg/mL. Chlorogenic acid and naringenin-7-O-glucoside were identified in the latex aqueous phase, along with catechin and procyanidin compounds. There was a stimulus for periosteum cell proliferation and bone formation when Hancornia speciosa latex was topically applied on rat calvaria. In addition, chlorogenic acid and naringenin-7-O-glucoside present in Hancornia speciosa latex may contribute to its effects on bone formation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
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microRNAs as regulators of adipogenic differentiation of mesenchymal stem cells.
Hamam, Dana; Ali, Dalia; Kassem, Moustapha; Aldahmash, Abdullah; Alajez, Nehad M
2015-02-15
microRNAs (miRNAs) constitute complex regulatory network, fine tuning the expression of a myriad of genes involved in different biological and physiological processes, including stem cell differentiation. Mesenchymal stem cells (MSCs) are multipotent stem cells present in the bone marrow stroma, and the stroma of many other tissues, and can give rise to a number of mesoderm-type cells including adipocytes and osteoblasts, which form medullary fat and bone tissues, respectively. The role of bone marrow fat in bone mass homeostasis is an area of intensive investigation with the aim of developing novel approaches for enhancing osteoblastic bone formation through inhibition of bone marrow fat formation. A number of recent studies have reported several miRNAs that enhance or inhibit adipogenic differentiation of MSCs and with potential use in microRNA-based therapy to regulate adipogenesis in the context of treating bone diseases and metabolic disorders. The current review focuses on miRNAs and their role in regulating adipogenic differentiation of MSCs.
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Zhang, Bing; Zhang, Pei-biao; Wang, Zong-liang; Lyu, Zhong-wen; Wu, Han
2017-01-01
Objective: A new therapeutic strategy using nanocomposite scaffolds of grafted hydroxyapatite (g-HA)/poly(lactide-co-glycolide) (PLGA) carried with autologous mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) was assessed for the therapy of critical bone defects. At the same time, tissue response and in vivo mineralization of tissue-engineered implants were investigated. Methods: A composite scaffold of PLGA and g-HA was fabricated by the solvent casting and particulate-leaching method. The tissue-engineered implants were prepared by seeding the scaffolds with autologous bone marrow MSCs in vitro. Then, mineralization and osteogenesis were observed by intramuscular implantation, as well as the repair of the critical radius defects in rabbits. Results: After eight weeks post-surgery, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) revealed that g-HA/PLGA had a better interface of tissue response and higher mineralization than PLGA. Apatite particles were formed and varied both in macropores and micropores of g-HA/PLGA. Computer radiographs and histological analysis revealed that there were more and more quickly formed new bone formations and better fusion in the bone defect areas of g-HA/PLGA at 2–8 weeks post-surgery. Typical bone synostosis between the implant and bone tissue was found in g-HA/PLGA, while only fibrous tissues formed in PLGA. Conclusions: The incorporation of g-HA mainly improved mineralization and bone formation compared with PLGA. The application of MSCs can enhance bone formation and mineralization in PLGA scaffolds compared with cell-free scaffolds. Furthermore, it can accelerate the absorption of scaffolds compared with composite scaffolds. PMID:29119734
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Zhang, Bing; Zhang, Pei-Biao; Wang, Zong-Liang; Lyu, Zhong-Wen; Wu, Han
A new therapeutic strategy using nanocomposite scaffolds of grafted hydroxyapatite (g-HA)/ poly(lactide-co-glycolide) (PLGA) carried with autologous mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) was assessed for the therapy of critical bone defects. At the same time, tissue response and in vivo mineralization of tissue-engineered implants were investigated. A composite scaffold of PLGA and g-HA was fabricated by the solvent casting and particulate-leaching method. The tissue-engineered implants were prepared by seeding the scaffolds with autologous bone marrow MSCs in vitro. Then, mineralization and osteogenesis were observed by intramuscular implantation, as well as the repair of the critical radius defects in rabbits. After eight weeks post-surgery, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) revealed that g-HA/PLGA had a better interface of tissue response and higher mineralization than PLGA. Apatite particles were formed and varied both in macropores and micropores of g-HA/PLGA. Computer radiographs and histological analysis revealed that there were more and more quickly formed new bone formations and better fusion in the bone defect areas of g-HA/PLGA at 2-8 weeks post-surgery. Typical bone synostosis between the implant and bone tissue was found in g-HA/PLGA, while only fibrous tissues formed in PLGA. The incorporation of g-HA mainly improved mineralization and bone formation compared with PLGA. The application of MSCs can enhance bone formation and mineralization in PLGA scaffolds compared with cell-free scaffolds. Furthermore, it can accelerate the absorption of scaffolds compared with composite scaffolds.
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Wang, Zhong-Shan; Feng, Zhi-Hong; Wu, Guo-Feng; Bai, Shi-Zhu; Dong, Yan; Chen, Fa-Ming; Zhao, Yi-Min
2016-01-01
Periodontal regeneration involves the restoration of at least three unique tissues: cementum, periodontal ligament tissue (PDL) and alveolar bone tissue. Here, we first isolated human PDL stem cells (PDLSCs) and jaw bone mesenchymal stem cells (JBMSCs). These cells were then induced to form cell sheets using an ascorbic acid-rich approach, and the cell sheet properties, including morphology, thickness and gene expression profile, were compared. Platelet-rich fibrin (PRF) derived from human venous blood was then fabricated into bioabsorbable fibrin scaffolds containing various growth factors. Finally, the in vivo potential of a cell-material construct based on PDLSC sheets, PRF scaffolds and JBMSC sheets to form periodontal tissue was assessed in a nude mouse model. In this model, PDLSC sheet/PRF/JBMSC sheet composites were placed in a simulated periodontal space comprising human treated dentin matrix (TDM) and hydroxyapatite (HA)/tricalcium phosphate (TCP) frameworks. Eight weeks after implantation, the PDLSC sheets tended to develop into PDL-like tissues, while the JBMSC sheets tended to produce predominantly bone-like tissues. In addition, the PDLSC sheet/PRF/JBMSC sheet composites generated periodontal tissue-like structures containing PDL- and bone-like tissues. Further improvements in this cell transplantation design may have the potential to provide an effective approach for future periodontal tissue regeneration. PMID:27324079
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Osteogenic differentiation capacity of human skeletal muscle-derived progenitor cells.
Oishi, Teruyo; Uezumi, Akiyoshi; Kanaji, Arihiko; Yamamoto, Naoki; Yamaguchi, Asami; Yamada, Harumoto; Tsuchida, Kunihiro
2013-01-01
Heterotopic ossification (HO) is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56(+) and PDGFRα(+) cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56(+) cells and PDGFRα(+) cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα(+) cells formed bone-like tissue and showed successful engraftment, while CD56(+) cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα(+) cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs) are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα(+) cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα(+) cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα(+) cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα(+) cells. Our results suggest that PDGFRα(+) cells may be the major source of HO and that the newly identified miRNAs may regulate osteogenic differentiation process of PDGFRα(+) cells.
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Kokorev, O V; Khodorenko, V N; Radkevich, A A; Dambaev, G Ts; Gunter, V E
2016-08-01
We studied the structure of porous permeable titanium nickelide used as the scaffold. In vitro population of the porous scaffold with multipotent mesenchymal stem bone marrow cells on days 7, 14, 21, and 28 was analyzed by scanning electron microscopy. Stage-by-stage histogenesis of the tissues formed from the bone marrow cells in the titanium nickelide scaffold in vivo is described in detail. Using mesenchymal stem cells, we demonstrated that porous permeable titanium nickelide scaffolds are unique incubators for cell cultures applicable for tissue engineering.
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Miyamoto, Takeshi
2013-07-01
Macrophages play a pivotal role in host defense against multiple foreign materials such as bacteria, parasites and artificial devices. Some macrophage lineage cells, namely osteoclasts and foreign body giant cells (FBGCs), form multi-nuclear giant cells by the cell-cell fusion of mono-nuclear cells. Osteoclasts are bone-resorbing cells, and are formed in the presence of RANKL on the surface of bones, while FBGCs are formed in the presence of IL-4 or IL-13 on foreign materials such as artificial joints, catheters and parasites. Recently, fusiogenic mechanisms and the molecules required for the cell-cell fusion of these macrophage lineage cells were, at least in part, clarified. Dendritic cell specific transmembrane protein (DC-STAMP) and osteoclast stimulatory transmembrane protein (OC-STAMP), both of which comprise seven transmembrane domains, are required for both osteoclast and FBGC cell-cell fusion. STAT6 was demonstrated to be required for the cell-cell fusion of FBGCs but not osteoclasts. In this review, advances in macrophage cell-cell fusion are discussed.
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Götz, Werner; Gerber, Thomas; Michel, Barbara; Lossdörfer, Stefan; Henkel, Kai-Olaf; Heinemann, Friedhelm
2008-10-01
Bone substitute biomaterials may be osteogenic, osteoconductive or osteoinductive. To test for these probable characteristics in a new nanoporous grafting material consisting of nanocrystalline hydroxyapatite embedded in a porous silica gel matrix (NanoBone(s)), applied in humans, we studied biopsies from 12 patients before dental implantation following various orofacial augmentation techniques with healing times of between 3.5 and 12 months. Sections from decalcified specimens were investigated using histology, histochemistry [periodic acid Schiff, alcian blue staining and tartrate-resistant acid phosphatase (TRAP)] and immunohistochemistry, with markers for osteogenesis, bone remodelling, resorption and vessel walls (alkaline phosphatase, bone morphogenetic protein-2, collagen type I, ED1, osteocalcin, osteopontin, runx2 and Von-Willebrand factor). Histologically, four specific stages of graft transformation into lamellar bone could be characterized. During early stages of healing, bone matrix proteins were absorbed by NanoBone(s) granules, forming a proteinaceous matrix, which was invaded by small vessels and cells. We assume that the deposition of these molecules promotes early osteogenesis in and around NanoBone(s) and supports the concomitant degradation probably by osteoclast-like cells. TRAP-positive osteoclast-like cells were localized directly on the granular surfaces. Runx2-immunoreactive pre-osteoblasts, which are probably involved in direct osteogenesis forming woven bone that is later transformed into lamellar bone, were attracted. Graft resorption and bone apposition around the graft granules appear concomitantly. We postulate that NanoBone(s) has osteoconductive and biomimetic properties and is integrated into the host's physiological bone turnover at a very early stage.
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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
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Kay, Alasdair Gawain; Dale, Tina Patricia; Akram, Khondoker Mehedi; Mohan, Param; Hampson, Karen; Maffulli, Nicola; Spiteri, Monica A; El Haj, Alicia Jennifer; Forsyth, Nicholas Robert
2015-01-01
Human mesenchymal stem cells (hMSC) are multipotent progenitor cells. We propose the optimization of hMSC isolation and recovery using the application of a controlled hypoxic environment. We evaluated oxygen, glucose and serum in the recovery of hMSC from bone marrow (BMhMSC). Colony forming units-fibroblastic, cell numbers, tri-lineage differentiation, immunofluorescence and microarray were used to confirm and characterize BMhMSC. In an optimized (2% O(2), 4.5 g/l glucose and 5% serum) environment both colony forming units-fibroblastic (p = 0.01) and cell numbers (p = 0.0001) were enhanced over standard conditions. Transcriptional analysis identified differential expression of bone morphogenetic protein 2 (BMP2) and, putatively, chemokine (C-X-C motif) receptor 2 (CXCR2) signaling pathways. We have detailed a potential milestone in the process of refinement of the BMhMSC isolation process.
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Rodriguez-Menocal, Luis; Shareef, Shahjahan; Salgado, Marcela; Shabbir, Arsalan; Van Badiavas, Evangelos
2015-03-13
Recent evidence has shown that bone marrow cells play critical roles during the inflammatory, proliferative and remodeling phases of cutaneous wound healing. Among the bone marrow cells delivered to wounds are stem cells, which can differentiate into multiple tissue-forming cell lineages to effect, healing. Gaining insight into which lineages are most important in accelerating wound healing would be quite valuable in designing therapeutic approaches for difficult to heal wounds. In this report we compared the effect of different bone marrow preparations on established in vitro wound healing assays. The preparations examined were whole bone marrow (WBM), whole bone marrow (long term initiating/hematopoietic based) cultured cells (BMC), and bone marrow derived mesenchymal stem cells (BM-MSC). We also applied these bone marrow preparations in two murine models of radiation induced delayed wound healing to determine which had a greater effect on healing. Angiogenesis assays demonstrated that tube formation was stimulated by both WBM and BMC, with WBM having the greatest effect. Scratch wound assays showed higher fibroblast migration at 24, 48, and 72 hours in presence of WBM as compared to BM-MSC. WBM also appeared to stimulate a greater healing response than BMC and BM-MSC in a radiation induced delayed wound healing animal model. These studies promise to help elucidate the role of stem cells during repair of chronic wounds and reveal which cells present in bone marrow might contribute most to the wound healing process.
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Li, Deqiang; Li, Ming; Liu, Peilai; Zhang, Yuankai; Lu, Jianxi; Li, Jianmin
2014-11-01
Repair of bone defects, particularly critical-sized bone defects, is a considerable challenge in orthopaedics. Tissue-engineered bones provide an effective approach. However, previous studies mainly focused on the repair of bone defects in small animals. For better clinical application, repairing critical-sized bone defects in large animals must be studied. This study investigated the effect of a tissue-engineered bone for repairing critical-sized bone defect in sheep. A tissue-engineered bone was constructed by culturing bone marrow mesenchymal-stem-cell-derived osteoblast cells seeded in a porous β-tricalcium phosphate ceramic (β-TCP) scaffold in a perfusion bioreactor. A critical-sized bone defect in sheep was repaired with the tissue-engineered bone. At the eighth and 16th week after the implantation of the tissue-engineered bone, X-ray examination and histological analysis were performed to evaluate the defect. The bone defect with only the β-TCP scaffold served as the control. X-ray showed that the bone defect was successfully repaired 16 weeks after implantation of the tissue-engineered bone; histological sections showed that a sufficient volume of new bones formed in β-TCP 16 weeks after implantation. Eight and 16 weeks after implantation, the volume of new bones that formed in the tissue-engineered bone group was more than that in the β-TCP scaffold group (P < 0.05). Tissue-engineered bone improved osteogenesis in vivo and enhanced the ability to repair critical-sized bone defects in large animals.
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Yue, Rui; Shen, Bo; Morrison, Sean J
2016-01-01
Bone marrow stromal cells maintain the adult skeleton by forming osteoblasts throughout life that regenerate bone and repair fractures. We discovered that subsets of these stromal cells, osteoblasts, osteocytes, and hypertrophic chondrocytes secrete a C-type lectin domain protein, Clec11a, which promotes osteogenesis. Clec11a-deficient mice appeared developmentally normal and had normal hematopoiesis but reduced limb and vertebral bone. Clec11a-deficient mice exhibited accelerated bone loss during aging, reduced bone strength, and delayed fracture healing. Bone marrow stromal cells from Clec11a-deficient mice showed impaired osteogenic differentiation, but normal adipogenic and chondrogenic differentiation. Recombinant Clec11a promoted osteogenesis by stromal cells in culture and increased bone mass in osteoporotic mice in vivo. Recombinant human Clec11a promoted osteogenesis by human bone marrow stromal cells in culture and in vivo. Clec11a thus maintains the adult skeleton by promoting the differentiation of mesenchymal progenitors into mature osteoblasts. In light of this, we propose to call this factor Osteolectin. DOI: http://dx.doi.org/10.7554/eLife.18782.001 PMID:27976999
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Mesenchymal Stem Cells: Time to Change the Name!
Caplan, Arnold I
2017-06-01
Mesenchymal stem cells (MSCs) were officially named more than 25 years ago to represent a class of cells from human and mammalian bone marrow and periosteum that could be isolated and expanded in culture while maintaining their in vitro capacity to be induced to form a variety of mesodermal phenotypes and tissues. The in vitro capacity to form bone, cartilage, fat, etc., became an assay for identifying this class of multipotent cells and around which several companies were formed in the 1990s to medically exploit the regenerative capabilities of MSCs. Today, there are hundreds of clinics and hundreds of clinical trials using human MSCs with very few, if any, focusing on the in vitro multipotential capacities of these cells. Unfortunately, the fact that MSCs are called "stem cells" is being used to infer that patients will receive direct medical benefit, because they imagine that these cells will differentiate into regenerating tissue-producing cells. Such a stem cell treatment will presumably cure the patient of their medically relevant difficulties ranging from osteoarthritic (bone-on-bone) knees to various neurological maladies including dementia. I now urge that we change the name of MSCs to Medicinal Signaling Cells to more accurately reflect the fact that these cells home in on sites of injury or disease and secrete bioactive factors that are immunomodulatory and trophic (regenerative) meaning that these cells make therapeutic drugs in situ that are medicinal. It is, indeed, the patient's own site-specific and tissue-specific resident stem cells that construct the new tissue as stimulated by the bioactive factors secreted by the exogenously supplied MSCs. Stem Cells Translational Medicine 2017;6:1445-1451. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kimura, Hiroaki; Akiyama, Haruhiko; Nakamura, Takashi
We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterix-null embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblasticmore » cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TN-W suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.« less
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Surface microtopography modulates sealing zone development in osteoclasts cultured on bone
Addadi, Lia; Geiger, Benjamin
2017-01-01
Bone homeostasis is continuously regulated by the coordinated action of bone-resorbing osteoclasts and bone-forming osteoblasts. Imbalance between these two cell populations leads to pathological bone diseases such as osteoporosis and osteopetrosis. Osteoclast functionality relies on the formation of sealing zone (SZ) rings that define the resorption lacuna. It is commonly assumed that the structure and dynamic properties of the SZ depend on the physical and chemical properties of the substrate. Considering the unique complex structure of native bone, elucidation of the relevant parameters affecting SZ formation and stability is challenging. In this study, we examined in detail the dynamic response of the SZ to the microtopography of devitalized bone surfaces, taken from the same area in cattle femur. We show that there is a significant enrichment in large and stable SZs (diameter larger than 14 µm; lifespan of hours) in cells cultured on rough bone surfaces, compared with small and fast turning over SZ rings (diameter below 7 µm; lifespan approx. 7 min) formed on smooth bone surfaces. Based on these results, we propose that the surface roughness of the physiologically relevant substrate of osteoclasts, namely bone, affects primarily the local stability of growing SZs. PMID:28202594
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Papadimou, Evangelia; Morigi, Marina; Iatropoulos, Paraskevas; Xinaris, Christodoulos; Tomasoni, Susanna; Benedetti, Valentina; Longaretti, Lorena; Rota, Cinzia; Todeschini, Marta; Rizzo, Paola; Introna, Martino; Grazia de Simoni, Maria; Remuzzi, Giuseppe; Goligorsky, Michael S; Benigni, Ariela
2015-04-14
The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs), also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes-formation of "domes" and tubule-like structures-and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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Browne, Christopher; Bishop, Julius; Yang, Yunzhi
2014-01-01
The induced membrane has been widely used in the treatment of large bone defects but continues to be limited by a relatively lengthy healing process and a requisite two stage surgical procedure. Here we report the development and characterization of a synthetic biomimetic induced membrane (BIM) consisting of an inner highly pre-vascularized cell sheet and an outer osteogenic layer using cell sheet engineering. The pre-vascularized inner layer was formed by seeding human umbilical vein endothelial cells (HUVECs) on a cell sheet comprised of a layer of undifferentiated human bone marrow-derived mesenchymal stem cells (hMSCs). The outer osteogenic layer was formed by inducing osteogenic differentiation of hMSCs. In vitro results indicated the undifferentiated hMSCs cell sheet facilitated the alignment of HUVECs and significantly promoted the formation of vascular-like networks. Furthermore, seeded HUVECs rearranged the extracellular matrix produced by hMSCs sheet. After subcutaneously implantation, the composite constructs showed rapid vascularization and anastomosis with the host vascular system, forming functional blood vessels in vivo. Osteogenic potential of the BIM was evidenced by immunohistochemistry staining of osteocalcin, tartrate-resistant acid phosphatase (TRAP) staining, and alizarin red staining. In summary, the synthetic BIM showed rapid vascularization, significant anastomoses, and osteogenic potential in vivo. This synthetic BIM has the potential for treatment of large bone defects in the absence of infection. PMID:24747351
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Zhang, Dan; Gao, Peng; Li, Qin; Li, Jinda; Li, Xiaojuan; Liu, Xiaoning; Kang, Yunqing; Ren, Liling
2017-06-05
There is a critical need for the management of large bone defects. The purpose of this study was to engineer a biomimetic periosteum and to combine this with a macroporous β-tricalcium phosphate (β-TCP) scaffold for bone tissue regeneration. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) were harvested and cultured in different culture media to form undifferentiated rBMSC sheets (undifferentiated medium (UM)) and osteogenic cell sheets (osteogenic medium (OM)). Simultaneously, rBMSCs were differentiated to induced endothelial-like cells (iECs), and the iECs were further cultured on a UM to form a vascularized cell sheet. At the same time, flow cytometry was used to detect the conversion rates of rBMSCs to iECs. The pre-vascularized cell sheet (iECs/UM) and the osteogenic cell sheet (OM) were stacked together to form a biomimetic periosteum with two distinct layers, which mimicked the fibrous layer and cambium layer of native periosteum. The biomimetic periostea were wrapped onto porous β-TCP scaffolds (BP/β-TCP) and implanted in the calvarial bone defects of rats. As controls, autologous periostea with β-TCP (AP/β-TCP) and β-TCP alone were implanted in the calvarial defects of rats, with a no implantation group as another control. At 2, 4, and 8 weeks post-surgery, implants were retrieved and X-ray, microcomputed tomography (micro-CT), histology, and immunohistochemistry staining analyses were performed. Flow cytometry results showed that rBMSCs were partially differentiated into iECs with a 35.1% conversion rate in terms of CD31. There were still 20.97% rBMSCs expressing CD90. Scanning electron microscopy (SEM) results indicated that cells from the wrapped cell sheet on the β-TCP scaffold apparently migrated into the pores of the β-TCP scaffold. The histology and immunohistochemistry staining results from in vivo implantation indicated that the BP/β-TCP and AP/β-TCP groups promoted the formation of blood vessels and new bone tissues in the bone defects more than the other two control groups. In addition, micro-CT showed that more new bone tissue formed in the BP/β-TCP and AP/β-TCP groups than the other groups. Inducing rBMSCs to iECs could be a good strategy to obtain an endothelial cell source for prevascularization. Our findings indicate that the biomimetic periosteum with porous β-TCP scaffold has a similar ability to promote osteogenesis and angiogenesis in vivo compared to the autologous periosteum. This function could result from the double layers of biomimetic periosteum. The prevascularized cell sheet served a mimetic fibrous layer and the osteogenic cell sheet served a cambium layer of native periosteum. The biomimetic periosteum with a porous ceramic scaffold provides a new promising method for bone healing.
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Multi-scale osteointegration and neovascularization of biphasic calcium phosphate bone scaffolds
NASA Astrophysics Data System (ADS)
Lan, Sheeny K.
Bone grafts are utilized clinically to guide tissue regeneration. Autologous bone and allogeneic bone are the current clinical standards. However, there are significant limitations to their use. To address the need for alternatives to autograft and allograft, researchers have worked to develop synthetic grafts, also referred to as scaffolds. Despite extensive efforts in this area, a gap persists between basic research and clinical application. In particular, solutions for repairing critical size and/or load-bearing defects are lacking. The aim of this thesis work was to address two critical barriers preventing design of successful tissue engineering constructs for bone regeneration within critical size and/or load-bearing defects. Those barriers are insufficient osteointegration and slow neovascularization. In this work, the effects of scaffold microporosity, recombinant human bone morphogenetic protein-2 delivery and endothelial colony forming cell vasculogenesis were evaluated in the context of bone formation in vivo. This was accomplished to better understand the role of these factors in bone regeneration, which may translate to improvements in tissue engineering construct design. Biphasic calcium phosphate (BCP) scaffolds with controlled macro- and microporosity were implanted in porcine mandibular defects. Evaluation of the BCP scaffolds after in vivo implantation showed, for the first time, osteocytes embedded in bone within scaffold micropores (< 10 microm) as well as the most extensive bone growth into micropores to date with bone penetration throughout rods 394 microm in diameter. The result is the first truly osteointegrated bone scaffolds with integration occurring at both the macro and micro length scales, leaving no "dead space" or discontinuities of bone in the defect site. The scaffold forms a living composite upon integration with regenerating bone and this has significant implications with regard to improved scaffold mechanical properties. The presence of osteocytes within scaffold micropores is an indication of scaffold osteoinductivity because a chemotactic factor must be present to induce cell migration into pores on the order of the cell diameter. It is likely that the scaffold undergoes in vivo modifications involving formation of a biological apatite layer within scaffold micropores and possibly co-precipitation of endogenous osteoinductive proteins. To further investigate the effects of scaffold osteoinductivity, BCP scaffolds were implanted in porcine mandibular defects with rhBMP-2, which was partially sequestered in the micropores. Cell migration into osteoinductive scaffold micropores can be enhanced through the delivery of exogenous rhBMP-2 further promoting multi-scale osteointegration. Finally, endothelial colony forming cells (ECFCs) isolated from human umbilical cord blood (UCB) were evaluated in terms of their in vivo vasculogenic potential in the context of bone formation. This work was completed to determine if ECFCs could be utilized in a bone tissue engineering construct to promote neovascularization. ECFCs were combined with a BCP scaffold and rhBMP-2 and implanted subcutaneously on the abdominal wall of NOD/SCID mice. The result was formation of perfused human vessels within BCP scaffold macropores that were present at 4 weeks. The high density and persistence of human vessels at four weeks indicates that human UCB ECFCs exceed their reported in vivo vasculogenic potential when combined with rhBMP-2 and a BCP scaffold. This shows a dual role for BMP-2 in the context of bone regeneration. Collectively, the thesis demonstrates that (1) the design of synthetic bone scaffolds should include controlled multi-scale porosity to promote multi-scale osteointegration, which may significantly improve scaffold mechanical properties and (2) human umbilical cord blood-derived endothelial colony forming cells have potential for promoting neovascularization in a bone defect when combined with rhBMP-2.
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Stem cell- and scaffold-based tissue engineering approaches to osteochondral regenerative medicine
Sundelacruz, Sarah; Kaplan, David L.
2009-01-01
In osteochondral tissue engineering, cell recruitment, proliferation, differentiation, and patterning are critical for forming biologically and structurally viable constructs for repair of damaged or diseased tissue. However, since constructs prepared ex vivo lack the multitude of cues present in the in vivo microenvironment, cells often need to be supplied with external biological and physical stimuli to coax them towards targeted tissue functions. To determine which stimuli to present to cells, bioengineering strategies can benefit significantly from endogenous examples of skeletogenesis. As an example of developmental skeletogenesis, the developing limb bud serves as an excellent model system in which to study how an osteochondral structures form from undifferentiated precursor cells. Alongside skeletal formation during embryogenesis, bone also possesses innate regenerative capacity, displaying remarkable ability to heal after damage. Bone fracture healing shares many features with bone development, driving the hypothesis that the regenerative process generally recapitulates development. Similarities and differences between the two modes of bone formation may offer insight into the special requirements for healing damaged or diseased bone. Thus, endogenous fracture healing, as an example of regenerative skeletogenesis, may also inform bioengineering strategies. In this review, we summarize the key cellular events involving stem and progenitor cells in developmental and regenerative skeletogenesis, and discuss in parallel the corresponding cell- and scaffold-based strategies that tissue engineers employ to recapitulate these events in vitro. PMID:19508851
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Matthews, Brya G; Roeder, Emilie; Wang, Xi; Aguila, Hector Leonardo; Lee, Sun-Kyeong; Grcevic, Danka; Kalajzic, Ivo
2017-10-01
Osteogenesis imperfecta (OI) is a disease caused by defects in type I collagen production that results in brittle bones. While the pathology is mainly caused by defects in the osteoblast lineage, there is also elevated bone resorption by osteoclasts resulting in high bone turnover in severe forms of the disease. Osteoclasts originate from hematopoietic myeloid cells, however changes in hematopoiesis have not been previously documented in OI. In this study, we evaluated hematopoietic lineage distribution and osteoclast progenitor cell frequency in bone marrow, spleen and peripheral blood of osteogenesis imperfecta murine (OIM) mice, a model of severe OI. We found splenomegaly in all ages examined, and expansion of myeloid lineage cells (CD11b + ) in bone marrow and spleen of 7-9week old male OIM animals. OIM spleens also showed an increased frequency of purified osteoclast progenitors. This phenotype is suggestive of chronic inflammation. Isolated osteoclast precursors from both spleen and bone marrow formed osteoclasts more rapidly than wild-type controls. We found that serum TNFα levels were increased in OIM, as was IL1α in OIM females. We targeted inflammation therapeutically by treating growing animals with murine TNFR2:Fc, a compound that blocks TNFα activity. Anti-TNFα treatment marginally decreased spleen mass in OIM females, but failed to reduce bone resorption, or improve bone parameters or fracture rate in OIM animals. We have demonstrated that OIM mice have changes in their hematopoietic system, and form osteoclasts more rapidly even in the absence of OI osteoblast signals, however therapy targeting TNFα did not improve disease parameters. Copyright © 2017 Elsevier Inc. All rights reserved.
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Endochondral ossification is required for haematopoietic stem-cell niche formation.
Chan, Charles K F; Chen, Ching-Cheng; Luppen, Cynthia A; Kim, Jae-Beom; DeBoer, Anthony T; Wei, Kevin; Helms, Jill A; Kuo, Calvin J; Kraft, Daniel L; Weissman, Irving L
2009-01-22
Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.
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NASA Astrophysics Data System (ADS)
D'Ambrogio, A.
Skeletal system has two main functions, to provide mechanical integrity for both locomotion and protection and to play an important role in mineral homeostasis. There is extensive evidence showing loss of bone mass during long-term Space-Flights. The loss is due to a break in the equilibrium between the activity of osteoblasts (the cells that forms bone) and the activity of osteoclasts (the cells that resorbs bone). Surprisingly, there is scanty information about the possible altered gene expression occurring in cells that form bone in microgravity.(Just 69 articles result from a "gene expression in microgravity" MedLine query.) Gene-chip or microarray technology allows to screen thousands of genes at the same time: the use of this technology on samples coming from cells exposed to microgravity could provide us with many important informations. For example, the identification of the molecules or structures which are the first sensors of the mechanical stress derived from lack of gravity, could help in understanding which is the first event leading to bone loss due to long-term exposure to microgravity. Consequently, this structure could become a target for a custom-designed drug. It is evident that bone mass loss, observed during long-time stay in Space, represents an accelerated model of what happens in aging osteoporosis. Therefore, the discovery and design of drugs able to interfere with the bone-loss process, could help also in preventing negative physiological processes normally observed on Earth. Considering the aims stated above, my research is designed to:
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Hydroxyapatite/collagen bone-like nanocomposite.
Kikuchi, Masanori
2013-01-01
Our group has succeeded to synthesize material with bone-like nanostructure and bone-like inorganic and organic composition via self-organization mechanism between them using simultaneous titration method under controlled pH and temperature. The hydroxyapatite/collagen (HAp/Col) bone-like nanocomposite completely incorporated into bone remodeling process to be substituted by new bone. Cells cultured on the HAp/Col revealed very interesting reactions. Osteoblast-like MG63 cells showed upregulation of alkaline phosphatase >3 times greater than MG63 cells cultured on tissue culture polystyrene (TCPS). MG63 cells 3-dimensionally cultured in a "HAp/Col sponge," a porous HAp/Col having sponge-like viscoelasticity, accumulated calcium phosphate nodules on extracellular matrices they secreted. Bone marrow cells co-cultured with osteoblasts on HAp/Col differentiated to osteoclasts without differentiation supplements. This phenomenon is not found in cells cultured on hydroxyapatite ceramics and TCPS, and rarely in cells cultured on dentin. These results suggest that HAp/Col is a good candidate for tissue engineering of bone as well as bone filler. In a clinical test as a bone filler, the HAp/Col sponge was significantly better than porous β-tricalcium phosphate. The HAp/Col sponge has been approved by the Japanese government and will be used as greatly needed bone filler in patients. In addition to the above, HAp/Col coating on titanium revealed higher osteo-conductivity than HAp-coated titanium and bare titanium and improved direct bonding between titanium and newly formed bone. The HAp/Col coating may be used for metal devices requiring osseointegration.
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Jacome-Galarza, Christian E.; Lee, Sun-Kyeong; Lorenzo, Joseph A.; LeonardoAguila, Hector
2012-01-01
Osteoclasts are specialized bone resorbing cells that derive from monocyte precursors. We have identified three populations of cells with high osteoclastogenic potential in murine bone marrow, which expressed the phenotype: B220−CD3−CD11b−/low CD115+ and either CD117hi, CD117intermediate or CD117low. We have evaluated these populations for their ability to also generate macrophages and dendritic cells. At a single cell level, the population expressing higher CD117 levels was able to generate bone-resorbing osteoclasts, phagocytic macrophages and antigen-presenting dendritic cells in vitro with efficiencies of over 90 percent, indicating that there exists a common developmental pathway for these cell types. Cells with osteoclastogenic potential also exist in blood and peripheral hematopoietic organs. Their functional meaning and/or their relationship with bone marrow progenitors is not well established. Hence, we characterized murine peripheral cell populations for their ability to form osteoclasts, macrophages and dendritic cells in vitro. The spleen and peripheral blood monocyte progenitors share phenotypic markers with bone marrow progenitors, but differ in their expression of CD11b, which was low in bone marrow but high in periphery. We propose that circulating monocyte progenitors are derived from a common bone marrow osteoclasts/macrophage/dendritic cell progenitor (OcMDC), which we have now characterized at a clonal level. However, the lineage relationship between the bone marrow and peripheral monocyte progenitors has yet to be defined. PMID:23165930
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Mishima, Hajime; Sakai, Shinsuke; Uemura, Toshimasa
2013-01-01
Objective: The purpose of this study was to evaluate the long-term results of cartilage repair after allogeneic transplantation of cartilaginous aggregates formed from bone marrow–derived cells. Methods: Bone marrow cells were harvested from 12-day-old rabbits. The cells were subjected to a monolayer culture, and the spindle-shaped cells attached to the flask surface were defined as bone marrow–derived mesenchymal cells. After the monolayer culture, a 3-dimensional cartilaginous aggregate was formed using a bioreactor with chondrogenesis. We created osteochondral defects, measuring 5 mm in diameter and 4 mm in depth, at the femoral trochlea of 10-week-old rabbits. Two groups were established, the transplanted group in which the cartilaginous aggregate was transplanted into the defect, and the control group in which the defect was left untreated. Twenty-six and 52 weeks after surgery, the rabbits were sacrificed and their tissue repair status was evaluated macroscopically (International Cartilage Repair Society [ICRS] score) and histologically (O’Driscoll score). Results: The ICRS scores were as follows: at week 26, 7.2 ± 0.5 and 7.6 ± 0.8; at week 52, 7.6 ± 1.1 and 9.7 ± 0.7, for the transplanted and control groups, respectively. O’Driscoll scores were as follows: at week 26, 12.6 ± 1.9 and 10.1 ± 1.9; at week 52, 9.6 ± 3.0 and 14.0 ± 1.4, each for transplanted and control groups, respectively. No significant differences were observed between the groups. Conclusions: This study demonstrates that allogeneic transplantation of cartilaginous aggregates formed from bone marrow–derived cells produces comparable long-term results based on macroscopic and histological outcome measures when compared with osteochondral defects that are left untreated. PMID:26069678
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kawata, Shigehisa; Suzuki, Jun; Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871
2006-11-10
Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types ofmore » cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.« less
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Schmidt, Linnea; Taiyab, Aftab; Melvin, Vida Senkus; Jones, Kenneth L; Williams, Trevor
2018-05-10
The bones of the cranial vault are formed directly from mesenchymal cells through intramembranous ossification rather than via a cartilage intermediate. Formation and growth of the skull bones involves the interaction of multiple cell:cell signaling pathways, with Fibroblast Growth Factors (FGFs) and their receptors exerting prominent influence. Mutations within this pathway are the most frequent cause of craniosynostosis, which is a common human craniofacial developmental abnormality characterized by the premature fusion of the cranial sutures. Here, we have developed new mouse models to investigate how different levels of increased Fgf signaling can impact the formation of the calvarial bones and associated sutures. While moderate Fgf8 overexpression resulted in delayed ossification followed by craniosynostosis of the coronal suture, higher Fgf8 levels promoted a loss of ossification and favored cartilage over bone formation across the skull. In contrast, endochondral bones were still able to form and ossify in the presence of increased Fgf8 , though the growth and mineralization of these bones were impacted to varying extents. Expression analysis demonstrated that abnormal skull chondrogenesis was accompanied by changes in genes required for Wnt signaling. Moreover, further analysis indicated that the pathology was associated with decreased Wnt signaling since the reduction in ossification could be partially rescued by halving Axin2 gene dosage. Taken together, these findings indicate that mesenchymal cells of the skull are not fated to form bone but can be forced into a chondrogenic fate via manipulation of FGF8 signaling. These results have implications for evolution of the different methods of ossification as well as for therapeutic intervention in craniosynostosis. © 2018. Published by The Company of Biologists Ltd.
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[Malignant T-cell lymphoma with osteomyelitis-like bone infiltration].
Mittelmeier, H; Schmitt, O
1980-01-01
After a short review on the late literature, existing about various forms of acute lymphoblastic leucemias, it is reported on a rare case of malignant T-cell-Lymphoma with ostemyelitis-like, painfull bone infiltration. The clinical symptoms, as well as differential-diagnostic criterias to other leucemias are described.
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Wang, Wen; Strecker, Sara; Liu, Yaling; Wang, Liping; Assanah, Fayekah; Smith, Spenser; Maye, Peter
2015-02-01
Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously had been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into osteoblast, adipocyte, and stromal cell lineages after transplantation into a parietal bone defect. In vivo examination of CTGF reporter expression in bone tissue sections revealed that it marked cells highly localized to the trabecular bone region and was not expressed in the perichondrium or periosteum. Mesenchymal cells retaining high CTGF reporter expression were adjacent to, but distinct from mature osteoblasts lining bone surfaces and endothelial cells forming the vascular sinuses. Comparison of CTGF and Osterix reporter expression in bone tissue sections indicated an inverse correlation between the strength of CTGF expression and osteoblast maturation. Down-regulation of CTGF reporter expression also occurred during in vitro osteogenic differentiation. Collectively, our studies indicate that CTGF reporter mice selectively identify a subpopulation of bone marrow mesenchymal progenitor cells that reside in the trabecular bone region. Copyright © 2014 Elsevier Inc. All rights reserved.
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Wang, Wen; Strecker, Sara; Liu, Yaling; Wang, Liping; Assanah, Fayekah; Smith, Spenser; Maye, Peter
2014-01-01
Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously has been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into osteoblast, adipocyte, and stromal cell lineages after transplantation into a parietal bone defect. In vivo examination of CTGF reporter expression in bone tissue sections revealed it marked cells highly localized to the trabecular bone region and was not expressed in the perichondrium or periosteum. Mesenchymal cells retaining high CTGF reporter expression were adjacent to, but distinct from mature osteoblasts lining bone surfaces and endothelial cells forming the vascular sinuses. Comparison of CTGF and Osterix reporter expression in bone tissue sections indicated an inverse correlation between the strength of CTGF expression and osteoblast maturation. Down-regulation of CTGF reporter expression also occurred during in vitro osteogenic differentiation. Collectively, our studies indicate that CTGF reporter mice selectively identify a subpopulation of bone marrow mesenchymal progenitor cells that reside in the trabecular bone region. PMID:25464947
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Engineering a humanized bone organ model in mice to study bone metastases.
Martine, Laure C; Holzapfel, Boris M; McGovern, Jacqui A; Wagner, Ferdinand; Quent, Verena M; Hesami, Parisa; Wunner, Felix M; Vaquette, Cedryck; De-Juan-Pardo, Elena M; Brown, Toby D; Nowlan, Bianca; Wu, Dan Jing; Hutmacher, Cosmo Orlando; Moi, Davide; Oussenko, Tatiana; Piccinini, Elia; Zandstra, Peter W; Mazzieri, Roberta; Lévesque, Jean-Pierre; Dalton, Paul D; Taubenberger, Anna V; Hutmacher, Dietmar W
2017-04-01
Current in vivo models for investigating human primary bone tumors and cancer metastasis to the bone rely on the injection of human cancer cells into the mouse skeleton. This approach does not mimic species-specific mechanisms occurring in human diseases and may preclude successful clinical translation. We have developed a protocol to engineer humanized bone within immunodeficient hosts, which can be adapted to study the interactions between human cancer cells and a humanized bone microenvironment in vivo. A researcher trained in the principles of tissue engineering will be able to execute the protocol and yield study results within 4-6 months. Additive biomanufactured scaffolds seeded and cultured with human bone-forming cells are implanted ectopically in combination with osteogenic factors into mice to generate a physiological bone 'organ', which is partially humanized. The model comprises human bone cells and secreted extracellular matrix (ECM); however, other components of the engineered tissue, such as the vasculature, are of murine origin. The model can be further humanized through the engraftment of human hematopoietic stem cells (HSCs) that can lead to human hematopoiesis within the murine host. The humanized organ bone model has been well characterized and validated and allows dissection of some of the mechanisms of the bone metastatic processes in prostate and breast cancer.
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Singh, Anju; Lester, Chantel; Drapp, Rebecca; Hu, Dorothy Z; Glimcher, Laurie H; Jones, Dallas
2015-02-01
Cell-based bone regeneration strategies offer promise for traumatic bone injuries, congenital defects, non-union fractures and other skeletal pathologies. Postnatal bone remodeling and fracture healing provide evidence that an osteochondroprogenitor cell is present in adult life that can differentiate to remodel or repair the fractured bone. However, cell-based skeletal repair in the clinic is still in its infancy, mostly due to poor characterization of progenitor cells and lack of knowledge about their in vivo behavior. Here, we took a combined approach of high-throughput screening, flow-based cell sorting and in vivo transplantation to isolate markers that identify osteochondroprogenitor cells. We show that the presence of tetraspanin CD9 enriches for osteochondroprogenitors within CD105(+) mesenchymal cells and that these cells readily form bone upon transplantation. In addition, we have used Thy1.2 and the ectonucleotidase CD73 to identify subsets within the CD9(+) population that lead to endochondral or intramembranous-like bone formation. Utilization of this unique cell surface phenotype to enrich for osteochondroprogenitor cells will allow for further characterization of the molecular mechanisms that regulate their osteogenic properties. © 2015. Published by The Company of Biologists Ltd.
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Brucella and Osteoarticular Cell Activation: Partners in Crime
Giambartolomei, Guillermo H.; Arriola Benitez, Paula C.; Delpino, M. Victoria
2017-01-01
Osteoarticular brucellosis is the most common presentation of human active disease although its prevalence varies widely. The three most common forms of osteoarticular involvement are sacroiliitis, spondylitis, and peripheral arthritis. The molecular mechanisms implicated in bone damage have been recently elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and the receptor activator of nuclear factor kappa-B ligand (RANKL)-the natural modulator of bone homeostasis are involved. These processes are driven by inflammatory cells, like monocytes/macrophages, neutrophils, Th17 CD4+ T, and B cells. In addition, Brucella abortus has a direct effect on osteoarticular cells and tilts homeostatic bone remodeling. These bacteria inhibit bone matrix deposition by osteoblasts (the only bone cells involved in bone deposition), and modify the phenotype of these cells to produce matrix metalloproteinases (MMPs) and cytokine secretion, contributing to bone matrix degradation. B. abortus also affects osteoclasts (cells naturally involved in bone resorption) by inducing an increase in osteoclastogenesis and osteoclast activation; thus, increasing mineral and organic bone matrix resorption, contributing to bone damage. Given that the pathology induced by Brucella species involved joint tissue, experiments conducted on synoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits synoviocyte apoptosis. Brucella is an intracellular bacterium that replicates preferentially in the endoplasmic reticulum of macrophages. The analysis of B. abortus-infected synoviocytes indicated that bacteria also replicate in their reticulum suggesting that they could use this cell type for intracellular replication during the osteoarticular localization of the disease. Finally, the molecular mechanisms of osteoarticular brucellosis discovered recently shed light on how the interaction between B. abortus and immune and osteoarticular cells may play an important role in producing damage in joint and bone. PMID:28265268
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Distinct bone marrow blood vessels differentially regulate haematopoiesis.
Itkin, Tomer; Gur-Cohen, Shiri; Spencer, Joel A; Schajnovitz, Amir; Ramasamy, Saravana K; Kusumbe, Anjali P; Ledergor, Guy; Jung, Yookyung; Milo, Idan; Poulos, Michael G; Kalinkovich, Alexander; Ludin, Aya; Kollet, Orit; Shakhar, Guy; Butler, Jason M; Rafii, Shahin; Adams, Ralf H; Scadden, David T; Lin, Charles P; Lapidot, Tsvee
2016-04-21
Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.
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A Spontaneous 3D Bone-On-a-Chip for Bone Metastasis Study of Breast Cancer Cells.
Hao, Sijie; Ha, Laura; Cheng, Gong; Wan, Yuan; Xia, Yiqiu; Sosnoski, Donna M; Mastro, Andrea M; Zheng, Si-Yang
2018-03-01
Bone metastasis occurs at ≈70% frequency in metastatic breast cancer. The mechanisms used by tumors to hijack the skeleton, promote bone metastases, and confer therapeutic resistance are poorly understood. This has led to the development of various bone models to investigate the interactions between cancer cells and host bone marrow cells and related physiological changes. However, it is challenging to perform bone studies due to the difficulty in periodic sampling. Herein, a bone-on-a-chip (BC) is reported for spontaneous growth of a 3D, mineralized, collagenous bone tissue. Mature osteoblastic tissue of up to 85 µm thickness containing heavily mineralized collagen fibers naturally formed in 720 h without the aid of differentiation agents. Moreover, co-culture of metastatic breast cancer cells is examined with osteoblastic tissues. The new bone-on-a-chip design not only increases experimental throughput by miniaturization, but also maximizes the chances of cancer cell interaction with bone matrix of a concentrated surface area and facilitates easy, frequent observation. As a result, unique hallmarks of breast cancer bone colonization, previously confirmed only in vivo, are observed. The spontaneous 3D BC keeps the promise as a physiologically relevant model for the in vitro study of breast cancer bone metastasis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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[Osteostimulating effect of bone xenograft on bone tissue regeneration].
Balin, V N; Balin, D V; Iordanishvili, A K; Musikin, M I
2015-01-01
The aim of experimental case-control study performed in 28 dogs divided in 2 groups was to assess local tissue reactions on bone xenograft transplantation; dynamics of bone remodeling and formation at the site of bone defect wall contacting with bone xenograft; dynamics and mechanisms of xenograft remodeling. Transplantation of xenograft in conventional bone defects did not cause inflammatory of destructive reactions because of high biocompatibility of the material. At transplantation site active fibrous bone trabeculae formation filling the spaces between xenograft participles was observed. On the 90th day newly formed bone showed lammelar structure. Simultaneously from the 42d day the invasion of cell elements from recipient bed into the material was seen leading to xenograft resorption. The observed dynamics may be assessed as gradual substitution of xenograft with newly formed host bone structures.
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NASA Astrophysics Data System (ADS)
Ohnishi, Hiroe; Oda, Yasuaki; Ohgushi, Hajime
2010-02-01
Mesenchymal stem cells (MSCs) are adult stem cells which show differentiation capabilities toward various cell lineages. We have already used MSCs for treatments of osteoarthritis, bone necrosis and bone tumor. For this purpose, culture expanded MSCs were combined with various ceramics and then implanted. Because of rejection response to allogeneic MSC implantation, we have utilized patients' own MSCs for the treatment. Bone marrow is a good cell source of MSCs, although the MSCs also exist in adipose tissue. When comparing osteogenic differentiation of these MSCs, bone marrow MSCs show more extensive bone forming capability than adipose MSCs. Thus, the bone marrow MSCs are useful for bone tissue regeneration. However, the MSCs show limited proliferation and differentiation capabilities that hindered clinical applications in some cases. Recent advances reveal that transduction of plural transcription factors into human adult cells results in generation of new type of stem cells called induced pluripotent stem cells (iPS cells). A drawback of the iPS cells for clinical applications is tumor formation after their in vivo implantation; therefore it is difficult to use iPS cells for the treatment. To circumvent the problem, we transduced a single factor of either SOX2 or NANOG into the MSCs and found high proliferation as well as osteogenic differentiation capabilities of the MSCs. The stem cells could be combined with bioceramics for clinical applications. Here, we summarize our recent technologies using adult stem cells in viewpoints of bone tissue regeneration.
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Endothelial-to-Osteoblast Conversion Generates Osteoblastic Metastasis of Prostate Cancer.
Lin, Song-Chang; Lee, Yu-Chen; Yu, Guoyu; Cheng, Chien-Jui; Zhou, Xin; Chu, Khoi; Murshed, Monzur; Le, Nhat-Tu; Baseler, Laura; Abe, Jun-Ichi; Fujiwara, Keigi; deCrombrugghe, Benoit; Logothetis, Christopher J; Gallick, Gary E; Yu-Lee, Li-Yuan; Maity, Sankar N; Lin, Sue-Hwa
2017-06-05
Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2 cre /Osx f/f /SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osx f/f /SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa. Copyright © 2017 Elsevier Inc. All rights reserved.
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Razawy, Wida; van Driel, Marjolein
2018-01-01
Abstract The IL‐23/Th17 axis has been implicated in the development of autoimmune diseases, such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA). RA and PsA are heterogeneous diseases with substantial burden on patients. Increasing evidence suggests that the IL‐23 signaling pathway may be involved in the development of autoimmunity and erosive joint damage. IL‐23 can act either directly or indirectly on bone forming osteoblasts as well as on bone resorbing osteoclasts. As IL‐23 regulates the activity of cells of the bone, it is conceivable that in addition to inflammation‐mediated joint erosion, IL‐23 may play a role in physiological bone remodeling. In this review, we focus on the role of IL‐23 in autoimmune arthritis in patients and murine models, and provide an overview of IL‐23 producing and responding cells in autoimmune arthritic joints. In addition, we discuss the role of IL‐23 on bone forming osteoblasts and bone resorbing osteoclasts regarding inflammation‐mediated joint damage and bone remodeling. At last, we briefly discuss the clinical implications of targeting this pathway for joint damage and systemic bone loss in autoimmune arthritis. PMID:29148561
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Dumont, Bruno; Castronovo, Vincent; Peulen, Olivier; Blétard, Noëlla; Clézardin, Philippe; Delvenne, Philippe; De Pauw, Edwin A; Turtoi, Andrei; Bellahcène, Akeila
2012-04-06
The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface and in the extracellular space, establishing the first contacts with the target tissue. In this study, we had the rare opportunity to analyze a bone metastatic lesion and its corresponding breast primary tumor obtained simultaneously from the same patient. Using mass spectrometry, we undertook a proteomic study on cell surface and extracellular protein-enriched material. We provide a repertoire of significantly modulated proteins, some with yet unknown roles in the bone metastatic process as well as proteins notably involved in cancer cell invasiveness and in bone metabolism. The comparison of these clinical data with those previously obtained using a human osteotropic breast cancer cell line highlighted an overlapping group of proteins. Certain differentially expressed proteins are validated in the present study using immunohistochemistry on a retrospective collection of breast tumors and matched bone metastases. Our exclusive set of selected proteins supports the setup of further investigations on both clinical samples and experimental bone metastasis models that will help to reveal the finely coordinated expression of proteins that favor the development of metastases in the bone microenvironment.
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Electron Micrographs of Quail Limb Bones formed in microgravity
NASA Technical Reports Server (NTRS)
2003-01-01
Electron micrographs of quail limb bones that formed under the influence of microgravity show decreased mineralization compared to bones formed in normal gravity. The letters B and C indicate bone and cartilage sides of the sample, respectively, with the arrows marking the junction between bone and cartilage cells. The asterisks indicate where mineralization begins. The bone that developed during spaceflight (top) shows less mineral compared to the control sample (bottom); the control sample clearly shows mineral deposits (dark spots) that are absent in the flight sample. Quail eggs are small and develop quickly, making them ideal for space experiments. In late 2001, the Avian Development Facility (ADF) made its first flight and carried eggs used in two investigations, development and function of the irner-ear balance system in normal and altered gravity environments, and skeletal development in embryonic quail.
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Microfibrous β-TCP/collagen scaffolds mimic woven bone in structure and composition.
Zhang, Shen; Zhang, Xin; Cai, Qing; Wang, Bo; Deng, Xuliang; Yang, Xiaoping
2010-12-01
Woven bone, as the initial form of bone tissue, is always found in developing and repairing bone. It is thought of as a temporary scaffold for the deposition of osteogenic cells and the laying down of lamellar bone. Thus, we hypothesize that a matrix which resembles the architecture and components of woven bone can provide an osteoblastic microenvironment for bone cell growth and new bone formation. In this study, woven-bone-like beta-tricalcium phosphate (β-TCP)/collagen scaffolds were fabricated by sol-gel electrospinning and impregnating methods. Optimization studies on sol-gel synthesis and electrospinning process were conducted respectively to prepare pure β-TCP fibers with dimensions close to mineralized collagen fibrils in woven bone. The collagen-coating layer prepared by impregnation had an adhesive role that held the β-TCP fibers together, and resulted in rapid degradation and matrix mineralization in in vitro tests. MG63 osteoblast-like cells seeded on the resultant scaffolds showed three-dimensional (3D) morphologies, and merged into multicellular layers after 7 days culture. Cytotoxicity test further revealed that extracts from the resultant scaffolds could promote the proliferation of MG63 cells. Therefore, the woven-bone-like matrix that we constructed favored the attachment and proliferation of MG63 cells in three dimensions. It has great potential ability to shorten the time of formation of new bone.
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Cohn Yakubovich, Doron; Tawackoli, Wafa; Sheyn, Dmitriy; Kallai, Ilan; Da, Xiaoyu; Pelled, Gadi; Gazit, Dan; Gazit, Zulma
2015-12-22
A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro-computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis. The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7(th) day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7(th) and 10(th) postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.
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Analysis of bone marrow plasma cells in patients with solitary bone plasmacytoma.
Bhaskar, Archana; Gupta, Ritu; Sharma, Atul; Kumar, Lalit; Jain, Paresh
Local radiotherapy is the treatment of choice for solitary bone plasmacytoma (SBP) and the role of adjuvant systemic chemotherapy in preventing progression to multiple myeloma (MM) is controversial. The purpose of this study was to examine the presence of systemic disease in the form of neoplastic plasma cells (PC) in bone marrow of patients with SBP. Flow cytometric immunophenotyping of PC was carried out on bone marrow aspirate of 7 patients using monoclonal antibodies: CD19 FITC, CD45 FITC, CD20 FITC, CD52 PE, CD117 PE, CD56 PE, CD38 PerCP-Cy5.5, CD138 APC, anti-kappa (κ) FITC and anti-lambda (λ) PE. The neoplastic as well as normal PC were identified in bone marrow aspirate of all the patients at the time of diagnosis; the neoplastic PC ranged from 0.1%to 0.7% of all BM cells and 33.5% to 89.7% of total BMPC. The κ:λ ratio was normal in all the samples ranging from 0.5% to 1.6%. The present work shows the presence of systemic disease in the form of neoplastic PC in bone marrow of patients with SBP. Prospective studies would be required to study if the levels of neoplastic PC in the bone marrow may help us identify patients who are likely to progress to overt MM and benefit from systemic chemotherapy.
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Chitosan-Based Bilayer Hydroxyapatite Nanorod Composite Scaffolds for Osteochondral Regeneration
NASA Astrophysics Data System (ADS)
Swanson, Shawn
Osteochondral defects involve injury to bone and cartilage. As articular cartilage is worn down, bone in the joint begins to rub together, causing bone spurs. This is known as osteoarthritis, and is a common issue among the aging population. This problem presents an interesting opportunity for tissue engineering. Tissue engineering is an approach to treatment of tissue defects where synthetic, three dimensional (3-D) scaffolds are implanted in a defect to facilitate healing. The osteochondral scaffold consists of two regions in the form of a bilayer scaffold- one to mimic bone with osteoconductive properties, and one to mimic cartilage with biomimetic properties. One approach to improving the osteoconductivity of tissue engineering scaffolds is the addition of hydroxyapatite (HAp), the main mineral phase in bone. HAp with nanorod morphology is desirable because it is biomimetic for the calcium phosphate found in bone. Incorporating HAp nanorods in bone tissue engineering scaffolds to form a composite material may increase scaffold osteoconductivity. The cartilage scaffold is fabricated from chitosan and hyaluronic acid (HA). HA is a known component of cartilage and thus is biomimetic. The bilayer scaffolds were seeded with osteoblast-like MG-63 cells to investigate cell migration and were evaluated with Alamar Blue proliferation assay. The cells successfully migrated to the bone region of the scaffold, indicating that the bilayer scaffold provides a promising osteochondral scaffold.
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Cao, Lei; Li, Xiaokang; Zhou, Xiaoshu; Li, Yong; Vecchio, Kenneth S; Yang, Lina; Cui, Wei; Yang, Rui; Zhu, Yue; Guo, Zheng; Zhang, Xing
2017-03-22
Sea urchin spines (Heterocentrotus mammillatus), with a hierarchical open-cell structure similar to that of human trabecular bone and superior mechanical property (compressive strength ∼43.4 MPa) suitable for machining to shape, were explored for potential applications of bone defect repair. Finite element analyses reveal that the compressive stress concentrates along the dense growth rings and dissipates through strut structures of the stereoms, indicating that the exquisite mesostructures play an important role in high strength-to-weight ratios. The fracture strength of magnesium-substituted tricalcium phosphate (β-TCMP) scaffolds produced by hydrothermal conversion of urchin spines is about 9.3 MPa, comparable to that of human trabecular bone. New bone forms along outer surfaces of β-TCMP scaffolds after implantation in rabbit femoral defects for one month and grows into the majority of the inner open-cell spaces postoperation in three months, showing tight interface between the scaffold and regenerative bone tissue. Fusion of beagle lumbar facet joints using a Ti-6Al-4V cage and β-TCMP scaffold can be completed within seven months with obvious biodegradation of the β-TCMP scaffold, which is nearly completely degraded and replaced by newly formed bone ten months after implantation. Thus, sea urchin spines suitable for machining to shape have advantages for production of biodegradable artificial grafts for bone defect repair.
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Wang, Zhifa; Weng, Yanming; Lu, Shengjun; Zong, Chunlin; Qiu, Jianyong; Liu, Yanpu; Liu, Bin
2015-08-01
To analyze the effects of platelet-rich fibrin (PRF) on mesenchymal stem cells (MSCs) in vitro and investigate in vivo bone formation by MSC sheets with PRF. Cell proliferation and expression of osteogenesis-related genes within MSC sheets were assessed upon exposure to PRF from the same donors. We then injected MSC sheet fragments with or without PRF subcutaneously in nude mice and assessed bone formation by micro-computed tomography and histological analyses. PRF significantly stimulated MSC proliferation and osteogenesis in vitro. MSC sheets injected with or without PRF formed new bone, but those with PRF produced significantly more and denser bone. MSC sheets can be used to generate tissue engineered bone upon injection, and PRF increases the osteogenic capacity of MSC sheets in vitro and in vivo. © 2014 Wiley Periodicals, Inc.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhu Lingling; Zaidi, Samir; Peng Yuanzhen
Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized (von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obsmore » at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate.« less
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Use of NASA Bioreactor in Engineering Tissue for Bone Repair
NASA Technical Reports Server (NTRS)
Duke, Pauline
1998-01-01
This study was proposed in search for a new alternative for bone replacement or repair. Because the systems commonly used in repair of bony defects form bone by going through a cartilaginous phase, implantation of a piece of cartilage could enhance the healing process by having a more advanced starting point. However, cartilage has seldom been used to replace bone due, in part, to the limitations in conventional culture systems that did not allow production of enough tissue for implants. The NASA-developed bioreactors known as STLV (Slow Turning Lateral Vessel) provide homogeneous distribution of cells, nutrients, and waste products, with less damaging turbulence and shear forces than conventional systems. Cultures under these conditions have higher growth rates, viability, and longevity, allowing larger "tissue-like" aggregates to form, thus opening the possibilities of producing enough tissue for implantation, along with the inherent advantages of in vitro manipulations. To assure large numbers of cells and to eliminate the use of timed embryos, we proposed to use an immortalized mouse limb bud cell line as the source of cells.
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NF-κB RelB Negatively Regulates Osteoblast Differentiation and Bone Formation
Yao, Zhenqiang; Li, Yanyun; Yin, Xiaoxiang; Dong, Yufeng; Xing, Lianping; Boyce, Brendan F.
2013-01-01
RelA-mediated NF-κB canonical signaling promotes mesenchymal progenitor cell (MPC) proliferation, but inhibits differentiation of mature osteoblasts (OBs) and thus negatively regulates bone formation. Previous studies suggest that NF-κB RelB may also negatively regulate bone formation through non-canonical signaling, but they involved a complex knockout mouse model and the molecular mechanisms involved were not investigated. Here, we report that RelB−/− mice develop age-related increased trabecular bone mass associated with increased bone formation. RelB−/− bone marrow stromal cells expanded faster in vitro and have enhanced OB differentiation associated with increased expression of the osteoblastogenic transcription factor, Runx2. In addition, RelB directly targeted the Runx2 promoter to inhibit its activation. Importantly, RelB−/− bone-derived MPCs formed bone more rapidly than wild-type cells after they were injected into a murine tibial bone defect model. Our findings indicate that RelB negatively regulates bone mass as mice age and limits bone formation in healing bone defects, suggesting that inhibition of RelB could reduce age-related bone loss and enhance bone repair. PMID:24115294
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Kayacan, Mehmet C; Baykal, Yakup B; Karaaslan, Tamer; Özsoy, Koray; Alaca, İlker; Duman, Burhan; Delikanlı, Yunus E
2018-04-01
This study investigated the design and osseointegration process of transitive porous implants that can be used in humans and all trabecular and compact bone structure animals. The aim was to find a way of forming a strong and durable tissue bond on the bone-implant interface. Massive and transitive porous implants were produced on a direct metal laser sintering machine, surgically implanted into the skulls of sheep and kept in place for 12 weeks. At the end of the 12-week period, the Massive and porous implants removed from the sheep were investigated by scanning electron microscopy (SEM) to monitor the osseointegration process. In the literature, each study has selected standard sizes for pore diameter in the structures they use. However, none of these involved transitional porous structures. In this study, as opposed to standard pores, there were spherical or elliptical pores at the micro level, development channels and an inner region. Bone cells developed in the inner region. Transitive pores grown gradually in accordance with the natural structure of the bone were modeled in the inner region for cells to develop. Due to this structure, a strong and durable tissue bond could be formed at the bone-implant interface. Osseointegration processes of Massive vs. porous implants were compared. It was observed that cells were concentrated on the surface of Massive implants. Therefore, osseointegration between implant and bone was less than that of porous implants. In transitive porous implants, as opposed to Massive implants, an outer region was formed in the bone-implant interface that allowed tissue development.
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2016-01-01
Recovery of still-soft tissue structures, including blood vessels and osteocytes, from dinosaur bone after demineralization was reported in 2005 and in subsequent publications. Despite multiple lines of evidence supporting an endogenous source, it was proposed that these structures arose from contamination from biofilm-forming organisms. To test the hypothesis that soft tissue structures result from microbial invasion of the fossil bone, we used two different biofilm-forming microorganisms to inoculate modern bone fragments from which organic components had been removed. We show fundamental morphological, chemical and textural differences between the resultant biofilm structures and those derived from dinosaur bone. The data do not support the hypothesis that biofilm-forming microorganisms are the source of these structures. PMID:26926069
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Schweitzer, Mary Higby; Moyer, Alison E; Zheng, Wenxia
2016-01-01
Recovery of still-soft tissue structures, including blood vessels and osteocytes, from dinosaur bone after demineralization was reported in 2005 and in subsequent publications. Despite multiple lines of evidence supporting an endogenous source, it was proposed that these structures arose from contamination from biofilm-forming organisms. To test the hypothesis that soft tissue structures result from microbial invasion of the fossil bone, we used two different biofilm-forming microorganisms to inoculate modern bone fragments from which organic components had been removed. We show fundamental morphological, chemical and textural differences between the resultant biofilm structures and those derived from dinosaur bone. The data do not support the hypothesis that biofilm-forming microorganisms are the source of these structures.
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Osteoblasts are target cells for transformation in c-fos transgenic mice
1993-01-01
We have generated transgenic mice expressing the proto-oncogene c-fos from an H-2Kb class I MHC promoter as a tool to identify and isolate cell populations which are sensitive to altered levels of Fos protein. All homozygous H2-c-fosLTR mice develop osteosarcomas with a short latency period. This phenotype is specific for c-fos as transgenic mice expressing the fos- and jun-related genes, fosB and c-jun, from the same regulatory elements do not develop any pathology despite high expression in bone tissues. The c-fos transgene is not expressed during embryogenesis but is expressed after birth in bone tissues before the onset of tumor formation, specifically in putative preosteoblasts, bone- forming osteoblasts, osteocytes, as well as in osteoblastic cells present within the tumors. Primary and clonal cell lines established from c-fos-induced tumors expressed high levels of exogenous c-fos as well as the bone cell marker genes, type I collagen, alkaline phosphatase, and osteopontin/2ar. In contrast, osteocalcin/BGP expression was either low or absent. All cell lines were tumorigenic in vivo, some of which gave rise to osteosarcomas, expressing exogenous c- fos mRNA, and Fos protein in osteoblastic cells. Detailed analysis of one osteogenic cell line, P1, and several P1-derived clonal cell lines indicated that bone-forming osteoblastic cells were transformed by Fos. The regulation of osteocalcin/BGP and alkaline phosphatase gene expression by 1,25-dihydroxyvitamin D3 was abrogated in P1-derived clonal cells, whereas glucocorticoid responsiveness was unaltered. These results suggest that high levels of Fos perturb the normal growth control of osteoblastic cells and exert specific effects on the expression of the osteoblast phenotype. PMID:8335693
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Biology of bone and how it orchestrates the form and function of the skeleton
NASA Technical Reports Server (NTRS)
Sommerfeldt, D. W.; Rubin, C. T.
2001-01-01
The principal role of the skeleton is to provide structural support for the body. While the skeleton also serves as the body's mineral reservoir, the mineralized structure is the very basis of posture, opposes muscular contraction resulting in motion, withstands functional load bearing, and protects internal organs. Although the mass and morphology of the skeleton is defined, to some extent, by genetic determinants, it is the tissue's ability to remodel--the local resorption and formation of bone--which is responsible for achieving this intricate balance between competing responsibilities. The aim of this review is to address bone's form-function relationship, beginning with extensive research in the musculoskeletal disciplines, and focusing on several recent cellular and molecular discoveries which help understand the complex interdependence of bone cells, growth factors, physical stimuli, metabolic demands, and structural responsibilities. With a clinical and spine-oriented audience in mind, the principles of bone cell and molecular biology and physiology are presented, and an attempt has been made to incorporate epidemiologic data and therapeutic implications. Bone research remains interdisciplinary by nature, and a deeper understanding of bone biology will ultimately lead to advances in the treatment of diseases and injuries to bone itself.
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Phase field approaches of bone remodeling based on TIP
NASA Astrophysics Data System (ADS)
Ganghoffer, Jean-François; Rahouadj, Rachid; Boisse, Julien; Forest, Samuel
2016-01-01
The process of bone remodeling includes a cycle of repair, renewal, and optimization. This adaptation process, in response to variations in external loads and chemical driving factors, involves three main types of bone cells: osteoclasts, which remove the old pre-existing bone; osteoblasts, which form the new bone in a second phase; osteocytes, which are sensing cells embedded into the bone matrix, trigger the aforementioned sequence of events. The remodeling process involves mineralization of the bone in the diffuse interface separating the marrow, which contains all specialized cells, from the newly formed bone. The main objective advocated in this contribution is the setting up of a modeling and simulation framework relying on the phase field method to capture the evolution of the diffuse interface between the new bone and the marrow at the scale of individual trabeculae. The phase field describes the degree of mineralization of this diffuse interface; it varies continuously between the lower value (no mineral) and unity (fully mineralized phase, e.g. new bone), allowing the consideration of a diffuse moving interface. The modeling framework is the theory of continuous media, for which field equations for the mechanical, chemical, and interfacial phenomena are written, based on the thermodynamics of irreversible processes. Additional models for the cellular activity are formulated to describe the coupling of the cell activity responsible for bone production/resorption to the kinetics of the internal variables. Kinetic equations for the internal variables are obtained from a pseudo-potential of dissipation. The combination of the balance equations for the microforce associated to the phase field and the kinetic equations lead to the Ginzburg-Landau equation satisfied by the phase field with a source term accounting for the dissipative microforce. Simulations illustrating the proposed framework are performed in a one-dimensional situation showing the evolution of the diffuse interface separating new bone from marrow.
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Markina, Elena; Andreeva, Elena; Andrianova, Irina; Sotnezova, Elena; Buravkova, Ludmila
2018-05-02
Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45 + MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.
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Ismail, Tarek; Osinga, Rik; Todorov, Atanas; Haumer, Alexander; Tchang, Laurent A; Epple, Christian; Allafi, Nima; Menzi, Nadia; Largo, René D; Kaempfen, Alexandre; Martin, Ivan; Schaefer, Dirk J; Scherberich, Arnaud
2017-11-01
Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. The standard of care, vascularized bone grafts, is limited by donor site morbidity and restricted availability. The aim of this study was to generate and test engineered, axially vascularized SVF cells-based bone substitutes in a rat model of AVN. SVF cells were isolated from lipoaspirates and cultured onto porous hydroxyapatite scaffolds within a perfusion-based bioreactor system for 5days. The resulting constructs were inserted into devitalized bone cylinders mimicking AVN-affected bone. A ligated vascular bundle was inserted upon subcutaneous implantation of constructs in nude rats. After 1 and 8weeks in vivo, bone formation and vascularization were analyzed. Newly-formed bone was found in 80% of SVF-seeded scaffolds after 8weeks but not in unseeded controls. Human ALU+cells in the bone structures evidenced a direct contribution of SVF cells to bone formation. A higher density of regenerative, M2 macrophages was observed in SVF-seeded constructs. In both experimental groups, devitalized bone was revitalized by vascularized tissue after 8 weeks. SVF cells-based osteogenic constructs revitalized fully necrotic bone in a challenging AVN rat model of clinically-relevant size. SVF cells contributed to accelerated initial vascularization, to bone formation and to recruitment of pro-regenerative endogenous cells. Avascular necrosis (AVN) of bone often requires surgical treatment with autologous bone grafts, which is surgically demanding and restricted by significant donor site morbidity and limited availability. This paper describes a de novo engineered axially-vascularized bone graft substitute and tests the potential to revitalize dead bone and provide efficient new bone formation in a rat model. The engineering of an osteogenic/vasculogenic construct of clinically-relevant size with stromal vascular fraction of human adipose, combined to an arteriovenous bundle is described. This construct revitalized and generated new bone tissue. This successful approach proposes a novel paradigm in the treatment of AVN, in which an engineered, vascularized osteogenic graft would be used as a germ to revitalize large volumes of necrotic bone. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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IFN-γ stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation
Gao, Yuhao; Grassi, Francesco; Ryan, Michaela Robbie; Terauchi, Masakazu; Page, Karen; Yang, Xiaoying; Weitzmann, M. Neale; Pacifici, Roberto
2006-01-01
T cell–produced cytokines play a pivotal role in the bone loss caused by inflammation, infection, and estrogen deficiency. IFN-γ is a major product of activated T helper cells that can function as a pro- or antiresorptive cytokine, but the reason why IFN-γ has variable effects in bone is unknown. Here we show that IFN-γ blunts osteoclast formation through direct targeting of osteoclast precursors but indirectly stimulates osteoclast formation and promotes bone resorption by stimulating antigen-dependent T cell activation and T cell secretion of the osteoclastogenic factors RANKL and TNF-α. Analysis of the in vivo effects of IFN-γ in 3 mouse models of bone loss — ovariectomy, LPS injection, and inflammation via silencing of TGF-β signaling in T cells — reveals that the net effect of IFN-γ in these conditions is that of stimulating bone resorption and bone loss. In summary, IFN-γ has both direct anti-osteoclastogenic and indirect pro-osteoclastogenic properties in vivo. Under conditions of estrogen deficiency, infection, and inflammation, the net balance of these 2 opposing forces is biased toward bone resorption. Inhibition of IFN-γ signaling may thus represent a novel strategy to simultaneously reduce inflammation and bone loss in common forms of osteoporosis. PMID:17173138
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The Differentiation Balance of Bone Marrow Mesenchymal Stem Cells Is Crucial to Hematopoiesis
Zhang, Weiwei; Ran, Qian; Xiang, Yang; Zhong, Jiang F.; Li, Shengwen Calvin
2018-01-01
Bone marrow mesenchymal stem cells (BMSCs), the important component and regulator of bone marrow microenvironment, give rise to hematopoietic-supporting stromal cells and form hematopoietic niches for hematopoietic stem cells (HSCs). However, how BMSC differentiation affects hematopoiesis is poorly understood. In this review, we focus on the role of BMSC differentiation in hematopoiesis. We discussed the role of BMSCs and their progeny in hematopoiesis. We also examine the mechanisms that cause differentiation bias of BMSCs in stress conditions including aging, irradiation, and chemotherapy. Moreover, the differentiation balance of BMSCs is crucial to hematopoiesis. We highlight the negative effects of differentiation bias of BMSCs on hematopoietic recovery after bone marrow transplantation. Keeping the differentiation balance of BMSCs is critical for hematopoietic recovery. This review summarises current understanding about how BMSC differentiation affects hematopoiesis and its potential application in improving hematopoietic recovery after bone marrow transplantation. PMID:29765406
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Nitschke, Lars; Floyd, Helen; Ferguson, David J.P.; Crocker, Paul R.
1999-01-01
CD22 is a B cell–specific transmembrane protein known to function as a negative regulator of B cell signaling. It has also been implicated in cell adhesion through recognition of α2,6-linked sialic acids on glycans of target cells. Previous studies showed that CD22-deficient mice had a strongly reduced population of mature recirculating B cells in the bone marrow despite normal B cell development. Using a soluble recombinant form of the receptor (CD22-Fc), we demonstrate here that sialylated ligands for CD22 are expressed on sinusoidal endothelial cells of murine bone marrow but not on endothelial cells in other tissues examined. Injection of CD22-Fc revealed that the CD22 ligands in the bone marrow were accessible to the circulation. Treatment of mice with either CD22-Fc or affinity-purified anti-CD22 antibody led to an ∼50% reduction in mature recirculating B cells in the bone marrow without affecting numbers in the spleen. Finally, consistent with the notion that CD22 is a homing receptor, we show that compared with wild-type mice, CD22-deficient animals have a lower number of immunoglobulin M–secreting plasma cells in the bone marrow. PMID:10224292
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Sims, Natalie A
2016-10-01
Bone renews itself and changes shape throughout life to account for the changing needs of the body; this requires co-ordinated activities of bone resorbing cells (osteoclasts), bone forming cells (osteoblasts) and bone's internal cellular network (osteocytes). This review focuses on paracrine signaling by the IL-6 family of cytokines between bone cells, bone marrow, and skeletal muscle in normal physiology and in pathological states where their levels may be locally or systemically elevated. These functions include the support of osteoclast formation by osteoblast lineage cells in response to interleukin 6 (IL-6), interleukin 11 (IL-11), oncostatin M (OSM) and cardiotrophin 1 (CT-1). In addition it will discuss how bone-resorbing osteoclasts promote osteoblast activity by secreting CT-1, which acts as a "coupling factor" on osteocytes, osteoblasts, and their precursors to promote bone formation. OSM, produced by osteoblast lineage cells and macrophages, stimulates bone formation via osteocytes. IL-6 family cytokines also mediate actions of other bone formation stimuli like parathyroid hormone (PTH) and mechanical loading. CT-1, OSM and LIF suppress marrow adipogenesis by shifting commitment of pluripotent precursors towards osteoblast differentiation. Ciliary neurotrophic factor (CNTF) is released as a myokine from skeletal muscle and suppresses osteoblast differentiation and bone formation on the periosteum (outer bone surface in apposition to muscle). Finally, IL-6 acts directly on marrow-derived osteoclasts to stimulate release of "osteotransmitters" that act through the cortical osteocyte network to stimulate bone formation on the periosteum. Each will be discussed as illustrations of how the extended family of IL-6 cytokines acts within the skeleton in physiology and may be altered in pathological conditions or by targeted therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Xiao, Juan; Wu, Yong-Ji; Han, Bing; Dong, Hong-Yan; Chen, Shi-Ping
2013-08-01
To investigate the isolation, purification and ex vivo expansion of CD34(+)CD59(+) cells from the bone marrow of children with paroxysmal nocturnal hemoglobinuria (PNH), to evaluate the capability of long-term hematopoietic reconstruction of the expanded CD34(+)CD59(+) cells, and to provide a laboratory basis for novel treatment of PNH. CD34(+)CD59(+) cells were isolated from the bone marrow mononuclear cells of children with PNH using immunomagnetic beads and flow cytometer in sequence. The isolated cells were subjected to ex vivo expansion in the presence of different combinations of hematopoietic growth factors for two weeks. The colony-forming cells and long-term culture-initiating cells (LTC-ICs) were cultured and counted. The optimal combination of hematopoietic growth factors for ex vivo expansion was stem cell factor+interleukin (IL)-3+IL-6+FLT3 ligand+thrombopoietin+ery-thropoietin, and maximum expansion (30.4 ± 6.7 folds) was seen on day 7 of days 4 to 14 of ex vivo expansion. After ex vivo expansion, CD34(+)CD59(+) cells remained CD59-positive, retained strong capability of forming colony-forming units, and could still form LTC-ICs. There was no significant difference in capability of forming LTC-ICs between CD34(+)CD59(+) cells before and after expansion. The expansion capability of CD34(+)CD59(+) cells from children with PNH was significantly lower than that of CD34(+) cells from normal controls (P<0.01). The CD34(+)CD59(+) cells from children with PNH can be expanded in vitro. Post-expansion CD34(+)CD59(+) cells retain capability of long-term hematopoietic reconstruction. CD34(+)CD59(+) cells showed no trend towards PNH clone during culture. Ex vivo expansion of CD34(+)CD59(+) cells from children with PNH might be practical in performing autologous transplantation clinically for these children.
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Allogeneic adipose-derived stem cells regenerate bone in a critical-sized ulna segmental defect
Wen, Congji; Yan, Hai; Fu, Shibo; Qian, Yunliang
2016-01-01
Adipose-derived stem cells (ASCs) with multilineage potential can be induced into osteoblasts, adipocytes and chondrocytes. ASCs as seed cell are widely used in the field of tissue engineering, but most studies either use autologous cells as the source or an immunodeficient animal as the host. In our present study, we explored the feasibility of applying allogeneic ASCs and demineralized bone matrix (DBM) scaffolds for repairing tubular bone defects without using immunosuppressive therapy. Allogeneic ASCs were expanded and seeded on DBM scaffolds and induced to differentiate along the osteogenic lineage. Eight Sprague–Dawley (SD) rats were used in this study and bilateral critical-sized defects (8 mm) of the ulna were created and divided into two groups: with ASC-DBM constructs or DBM alone. The systemic immune response and the extent of bone healing were evaluated post-operatively. Twenty-four weeks after implantation, digital radiography (DR) testing showed that new bones had formed in the experimental group. By contrast, no bone tissue formation was observed in the control group. This study demonstrated that allogeneic ASCs could promote bone regeneration and repair tubular bone defects combined with DBM by histologically typical bone without systemic immune response PMID:25819682
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Davis, Hannah M; Pacheco-Costa, Rafael; Atkinson, Emily G; Brun, Lucas R; Gortazar, Arancha R; Harris, Julia; Hiasa, Masahiro; Bolarinwa, Surajudeen A; Yoneda, Toshiyuki; Ivan, Mircea; Bruzzaniti, Angela; Bellido, Teresita; Plotkin, Lilian I
2017-06-01
Skeletal aging results in apoptosis of osteocytes, cells embedded in bone that control the generation/function of bone forming and resorbing cells. Aging also decreases connexin43 (Cx43) expression in bone; and osteocytic Cx43 deletion partially mimics the skeletal phenotype of old mice. Particularly, aging and Cx43 deletion increase osteocyte apoptosis, and osteoclast number and bone resorption on endocortical bone surfaces. We examined herein the molecular signaling events responsible for osteocyte apoptosis and osteoclast recruitment triggered by aging and Cx43 deficiency. Cx43-silenced MLO-Y4 osteocytic (Cx43 def ) cells undergo spontaneous cell death in culture through caspase-3 activation and exhibit increased levels of apoptosis-related genes, and only transfection of Cx43 constructs able to form gap junction channels reverses Cx43 def cell death. Cx43 def cells and bones from old mice exhibit reduced levels of the pro-survival microRNA miR21 and, consistently, increased levels of the miR21 target phosphatase and tensin homolog (PTEN) and reduced phosphorylated Akt, whereas PTEN inhibition reduces Cx43 def cell apoptosis. miR21 reduction is sufficient to induce apoptosis of Cx43-expressing cells and miR21 deletion in miR21 fl/fl bones increases apoptosis-related gene expression, whereas a miR21 mimic prevents Cx43 def cell apoptosis, demonstrating that miR21 lies downstream of Cx43. Cx43 def cells release more osteoclastogenic cytokines [receptor activator of NFκB ligand (RANKL)/high-mobility group box-1 (HMGB1)], and caspase-3 inhibition prevents RANKL/HMGB1 release and the increased osteoclastogenesis induced by conditioned media from Cx43 def cells, which is blocked by antagonizing HMGB1-RAGE interaction. These findings identify a novel Cx43/miR21/HMGB1/RANKL pathway involved in preventing osteocyte apoptosis that also controls osteoclast formation/recruitment and is impaired with aging. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
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CELLS INVOLVED IN THE IMMUNE RESPONSE
Abdou, Nabih I.; Richter, Maxwell
1969-01-01
Rabbits were made immunologically tolerant to either human serum albumin or bovine gamma globulin by the neonatal administration of antigen. At 10 wk of age, they were challenged with the tolerogenic antigen and found to be non-responsive. However, these tolerant rabbits could respond with humoral antibody formation directed toward the tolerogenic antigen if they were treated with normal, allogeneic bone marrow or bone marrow obtained from a rabbit made tolerant toward a different antigen. They were incapable of responding if they were given bone marrow obtained from a rabbit previously made tolerant to the tolerogenic antigen. Irradiated rabbits were unable to respond if treated with tolerant bone marrow, but could respond well if given normal bone marrow. Since it has previously been demonstrated that the antibody-forming cell, in an irradiated recipient of allogeneic bone marrow, is of recipient and not donor origin, the data presented strongly indicate that the unresponsive cell in the immunologically tolerant rabbit is the antigen-reactive cell. PMID:4183777
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Wu, Wei; Le, Andrew V.; Mendez, Julio J.; Chang, Julie; Niklason, Laura E.
2015-01-01
Adipose-derived mesenchymal cells (ACs) and bone marrow-derived mesenchymal cells (BMCs) have been widely used for bone regeneration and can be seeded on a variety of rigid scaffolds. However, to date, a direct comparison of mesenchymal cells (MC) harvested from different tissues from the same donor and cultured in identical osteogenic conditions has not been investigated. Indeed, it is unclear whether marrow-derived or fat-derived MC possess intrinsic differences in bone-forming capabilities, since within-patient comparisons have not been previously done. This study aims at comparing ACs and BMCs from three donors ranging in age from neonatal to adult. Matched cells from each donor were studied in three distinct bioreactor settings, to determine the best method to create a viable osseous engineered construct. Human ACs and BMCs were isolated from each donor, cultured, and seeded on decellularized porcine bone (DCB) constructs. The constructs were then subjected to either static or dynamic (stirring or perfusion) bioreactor culture conditions for 7–21 days. Afterward, the constructs were analyzed for cell adhesion and distribution and osteogenic differentiation. ACs demonstrated higher seeding efficiency than BMCs. However, static and dynamic culture significantly increased BMCs proliferation more than ACs. In all conditions, BMCs demonstrated stronger osteogenic activity as compared with ACs, through higher alkaline phosphatase activity and gene expression for various bony markers. Conversely, ACs expressed more collagen I, which is a nonspecific matrix molecule in most connective tissues. Overall, dynamic bioreactor culture conditions enhanced osteogenic gene expression in both ACs and BMCs. Scaffolds seeded with BMCs in dynamic stirring culture conditions exhibit the greatest osteogenic proliferation and function in vitro, proving that marrow-derived MC have superior bone-forming potential as compared with adipose-derived cells. PMID:25668104
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Guerrero, Julien; Oliveira, Hugo; Catros, Sylvain; Siadous, Robin; Derkaoui, Sidi-Mohammed; Bareille, Reine; Letourneur, Didier; Amédée, Joëlle
2015-03-01
Current approaches in bone tissue engineering have shown limited success, mostly owing to insufficient vascularization of the construct. A common approach consists of co-culture of endothelial cells and osteoblastic cells. This strategy uses cells from different sources and differentiation states, thus increasing the complexity upstream of a clinical application. The source of reparative cells is paramount for the success of bone tissue engineering applications. In this context, stem cells obtained from human bone marrow hold much promise. Here, we analyzed the potential of human whole bone marrow cells directly expanded in a three-dimensional (3D) polymer matrix and focused on the further characterization of this heterogeneous population and on their ability to promote angiogenesis and osteogenesis, both in vitro and in vivo, in a subcutaneous model. Cellular aggregates were formed within 24 h and over the 12-day culture period expressed endothelial and bone-specific markers and a specific junctional protein. Ectopic implantation of the tissue-engineered constructs revealed osteoid tissue and vessel formation both at the periphery and within the implant. This work sheds light on the potential clinical use of human whole bone marrow for bone regeneration strategies, focusing on a simplified approach to develop a direct 3D culture without two-dimensional isolation or expansion.
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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.
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Zhang, Luo; Han, De-min; Ge, Wen-tong; Zhou, Bing; Xian, Jun-fang; Liu, Zhong-yan; Wang, Kui-ji; He, Fei
2005-12-01
To investigate the anatomical interaction between uncinate process and agger nasi cell to better understand the anatomy of the frontal sinus drainage pathway by endoscopy, spiral computed tomography (CT) and sectioning. Twenty-one skeletal skulls (forty-two sides) and one cadaver head (two sides) were studied by spiral CT together with endoscopy and collodion embedded thin sectioning at coronal plane. The sections with the thickness of 100 microm were stained with hemotoxylin and eosin. Under endoscopy, a leaflet of bone to the middle turbinate, which is given off by uncinate process, forms the anterior insertion of the middle turbinate onto the lateral nasal wall. The middle portion of the uncinate process attached to the frontal process of the maxilla in all of the skeletal nasal cavities, as well as the lacrimal bone in 78.6% of the skeletal nasal cavities. On CT scans, the agger nasi cell is present in 90.5% of the skeletal nasal cavities. While the lateral wall of the agger nasi cell is formed by lacrimal bone, the medial wall of the agger nasi cell is formed by uncinate process. And the anterior wall is formed by the frontal process of the maxilla. The superior portion of the uncinate process forms the medial, posterior and top wall of the agger nasi cells. The superior portion of the uncinate extends into the frontal recess and may insert into lamina papyracea (33.3%), skull base (9.5%), middle turbinate, combination of these (57.2%). The agger nasi cell is the key that unlocks the frontal recess.
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The cell biology and role of resorptive cells in diseases: A review.
Babaji, Prashant; Devanna, Raghu; Jagtap, Kiran; Chaurasia, Vishwajit Rampratap; Jerry, Jeethu John; Choudhury, Basanta Kumar; Duhan, Dinesh
2017-01-01
Resorptive cells are responsible for the resorption of mineralized matrix of hard tissues. Bone-resorbing cells are called osteoclasts; however, they can resorb mineralized dental tissues or calcified cartilage and then they are called odontoclasts and chondroclasts, respectively. Resorptive cells form when mononuclear precursors derived from a monocyte-macrophage cell lineage are attracted to certain mineralized surfaces and subsequently fuse and adhere onto them for exerting their resorbing activity. These cells are responsible for degradation of calcified extracellular matrix composed of organic molecules and hydroxyapatite. The activity of these cells can be observed in both physiological and pathological processes throughout life and their activity is mainly required in bone turnover and growth, spontaneous and induced (orthodontic) tooth movement, tooth eruption, and bone fracture healing, as well as in pathological conditions such as osteoporosis, osteoarthritis, and bone metastasis. In addition, they are responsible for daily control of calcium homeostasis. Clastic cells also resorb the primary teeth for shedding before the permanent teeth erupt into the oral cavity.
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Erythroid colony induction without erythropoietin by Friend leukemia virus in vitro.
Clarke, B J; Axelrad, A A; Shreeve, M M; McLeod, D L
1975-09-01
Erythroid colonies could be produced without the addition of erythropeietin in plasma cultures seeded with bone marrow cells from normal C3Hf/Bi mice by exposure of the cells in vitro to medium from a cell line (IS) that continuously produces Friend leukemia virus in culture. The activity in the culture medium was viral rather than erythropoietin-like, since it was sedimentable by high-speed centrifugation and heat labile. Erythroid colonies did not develop when the bone marrow cells exposed to virus-containing medium were from mice genetically resistant to Friend virus. IS culture medium contained both Friend spleen focus-forming and XC-plaque-forming activities. No erythroid colonies were induced when genetically sensitive cells were exposed to a preparation from which the spleen focus-forming activity had been removed, but which contained XC plaque-forming activity in high concentration. Thus the spleen focus-forming component of Friend virus appeared to be responsible for inducing erythroid colony formation without erythropoietin in vitro. Some erythroid colonies were also found in control cultures to which neither virus nor erythropoietin had been added. Reduction in the concentration of fetal calf serum in the culture medium substantially decreased the number of these colonies but had only a minor effect on the number of virus-induced colonies. The number of erythroid colonies produced after 2 days of culture without erythropoietin or fetal calf serum was approximately proportional to the titer of Friend spleen focus-forming virus to whcih the bone marrow cells had been exposed. This system should prove useful for investigation in vitro of Friend virus--host cell interactions which lead to erythropoietin-independent erythropoiesis.
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Lipid Osteoclastokines Regulate Breast Cancer Bone Metastasis
Krzeszinski, Jing Y.; Schwaid, Adam G.; Cheng, Wing Yin; Jin, Zixue; Gallegos, Zachary R.; Saghatelian, Alan
2017-01-01
Bone metastasis is a deadly consequence of cancers, in which osteoclast forms a vicious cycle with tumor cells. Bone metastasis attenuation by clinical usage of osteoclast inhibitors and in our osteopetrotic mouse genetic models with β-catenin constitutive activation or peroxisome proliferator-activated receptor γ deficiency fully support the important role of osteoclast in driving the bone metastatic niche. However, the mechanisms for this “partnership in crime” are underexplored. Here we show that osteoclasts reprogram their lipid secretion to support cancer cells. Metabolomic profiling reveals elevated prometastatic arachidonic acid (AA) but reduced antimetastatic lysophosphatidylcholines (LPCs). This shift in lipid osteoclastokines synergistically stimulates tumor cell proliferation, migration, survival, and expression of prometastatic genes. Pharmacologically, combined treatment with LPCs and BW-755C, an inhibitor of AA signaling via blocking lipoxygenase and cyclooxygenase, impedes breast cancer bone metastasis. Our findings elucidate key paracrine mechanisms for the osteoclast-cancer vicious cycle and uncover important therapeutic targets for bone metastasis. PMID:27967239
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Guided bone augmentation using ceramic space-maintaining devices: the impact of chemistry
Anderud, Jonas; Abrahamsson, Peter; Jimbo, Ryo; Isaksson, Sten; Adolfsson, Erik; Malmström, Johan; Naito, Yoshihito; Wennerberg, Ann
2015-01-01
The purpose of the study was to evaluate histologically, whether vertical bone augmentation can be achieved using a hollow ceramic space maintaining device in a rabbit calvaria model. Furthermore, the chemistry of microporous hydroxyapatite and zirconia were tested to determine which of these two ceramics are most suitable for guided bone generation. 24 hollow domes in two different ceramic materials were placed subperiosteal on rabbit skull bone. The rabbits were sacrificed after 12 weeks and the histology results were analyzed regarding bone-to-material contact and volume of newly formed bone. The results suggest that the effect of the microporous structure of hydroxyapatite seems to facilitate for the bone cells to adhere to the material and that zirconia enhance a slightly larger volume of newly formed bone. In conclusion, the results of the current study demonstrated that ceramic space maintaining devices permits new bone formation and osteoconduction within the dome. PMID:25792855
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Handschel, Jörg; Naujoks, Christian; Depprich, Rita; Lammers, Lydia; Kübler, Norbert; Meyer, Ulrich; Wiesmann, Hans-Peter
2011-07-14
Extracorporeal formation of mineralized bone-like tissue is still an unsolved challenge in tissue engineering. Embryonic stem cells may open up new therapeutic options for the future and should be an interesting model for the analysis of fetal organogenesis. Here we describe a technique for culturing embryonic stem cells (ESCs) in the absence of artificial scaffolds which generated mineralized miromasses. Embryonic stem cells were harvested and osteogenic differentiation was stimulated by the addition of dexamethasone, ascorbic acid, and ß-glycerolphosphate (DAG). After three days of cultivation microspheres were formed. These spherical three-dimensional cell units showed a peripheral zone consisting of densely packed cell layers surrounded by minerals that were embedded in the extracellular matrix. Alizarine red staining confirmed evidence of mineralization after 10 days of DAG stimulation in the stimulated but not in the control group. Transmission electron microscopy demonstrated scorching crystallites and collagenous fibrils as early indication of bone formation. These extracellular structures resembled hydroxyl apatite-like crystals as demonstrated by distinct diffraction patterns using electron diffraction analysis. The micromass culture technique is an appropriate model to form three-dimensional bone-like micro-units without the need for an underlying scaffold. Further studies will have to show whether the technique is applicable also to pluripotent stem cells of different origin. © 2011 Handschel et al; licensee BioMed Central Ltd.
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Rodriguez, Rudy U; Kemper, Nathan; Breathwaite, Erick; Dutta, Sucharita M; Hsu, Erin L; Hsu, Wellington K; Francis, Michael P
2016-07-26
Bone repair frequently requires time-consuming implant construction, particularly when using un-formed implants with poor handling properties. We therefore developed osteoinductive, micro-fibrous surface patterned demineralized bone matrix (DBM) fibers for engineering both defect-matched and general three-dimensional implants. Implant molds were filled with demineralized human cortical bone fibers there were compressed and lyophilized, forming mechanically strong shaped DBM scaffolds. Enzyme linked immunosorbent assays and mass spectrometry confirmed that DBM fibers contained abundant osteogenic growth factors (bone morphogenetic proteins, insulin-like growth factor-I) and extracellular matrix proteins. Mercury porosimetry and mechanical testing showed interconnected pores within the mechanically stable, custom DBM fiber scaffolds. Mesenchymal stem cells readily attached to the DBM and showed increasing metabolic activity over time. DBM fibers further increased alkaline phosphatase activity in C2C12 cells. In vivo, DBM implants elicited osteoinductive potential in a mouse muscle pouch, and also promoted spine fusion in a rat arthrodesis model. DBM fibers can be engineered into custom-shaped, osteoinductive and osteoconductive implants with potential for repairing osseous defects with precise fitment, potentially reducing operating time. By providing pre-formed and custom implants, this regenerative allograft may improve patient outcomes following surgical bone repair, while further advancing personalized orthopedic and craniomaxillofacial medicine using three-dimensional-printed tissue molds.
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Tissue-Engineered Autologous Grafts for Facial Bone Reconstruction
Bhumiratana, Sarindr; Bernhard, Jonathan C.; Alfi, David M.; Yeager, Keith; Eton, Ryan E.; Bova, Jonathan; Shah, Forum; Gimble, Jeffrey M.; Lopez, Mandi J.; Eisig, Sidney B.; Vunjak-Novakovic, Gordana
2016-01-01
Facial deformities require precise reconstruction of the appearance and function of the original tissue. The current standard of care—the use of bone harvested from another region in the body—has major limitations, including pain and comorbidities associated with surgery. We have engineered one of the most geometrically complex facial bones by using autologous stromal/stem cells, without bone morphogenic proteins, using native bovine bone matrix and a perfusion bioreactor for the growth and transport of living grafts. The ramus-condyle unit (RCU), the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalized approach in skeletally mature Yucatan minipigs (human-scale preclinical model). We used clinically approved decellularized bovine trabecular bone as a scaffolding material, and crafted it into an anatomically correct shape using image-guided micromilling, to fit the defect. Autologous adipose-derived stromal/stem cells were seeded into the scaffold and cultured in perfusion for 3 weeks in a specialized bioreactor to form immature bone tissue. Six months after implantation, the engineered grafts maintained their anatomical structure, integrated with native tissues, and generated greater volume of new bone and greater vascular infiltration than either non-seeded anatomical scaffolds or untreated defects. This translational study demonstrates feasibility of facial bone reconstruction using autologous, anatomically shaped, living grafts formed in vitro, and presents a platform for personalized bone tissue engineering. PMID:27306665
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Razawy, Wida; van Driel, Marjolein; Lubberts, Erik
2018-02-01
The IL-23/Th17 axis has been implicated in the development of autoimmune diseases, such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA). RA and PsA are heterogeneous diseases with substantial burden on patients. Increasing evidence suggests that the IL-23 signaling pathway may be involved in the development of autoimmunity and erosive joint damage. IL-23 can act either directly or indirectly on bone forming osteoblasts as well as on bone resorbing osteoclasts. As IL-23 regulates the activity of cells of the bone, it is conceivable that in addition to inflammation-mediated joint erosion, IL-23 may play a role in physiological bone remodeling. In this review, we focus on the role of IL-23 in autoimmune arthritis in patients and murine models, and provide an overview of IL-23 producing and responding cells in autoimmune arthritic joints. In addition, we discuss the role of IL-23 on bone forming osteoblasts and bone resorbing osteoclasts regarding inflammation-mediated joint damage and bone remodeling. At last, we briefly discuss the clinical implications of targeting this pathway for joint damage and systemic bone loss in autoimmune arthritis. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Wu, Chengtie; Chang, Jiang
2012-01-01
The impact of bone diseases and trauma in the whole world has increased significantly in the past decades. Bioactive glasses are regarded as an important bone regeneration material owing to their generally excellent osteoconductivity and osteostimulativity. A new class of bioactive glass, referred to as mesoporous bioglass (MBG), was developed 7 years ago, which possess a highly ordered mesoporous channel structure and a highly specific surface area. The study of MBG for drug/growth factor delivery and bone tissue engineering has grown significantly in the past several years. In this article, we review the recent advances of MBG materials, including the preparation of different forms of MBG, composition–structure relationship, efficient drug/growth factor delivery and bone tissue engineering application. By summarizing our recent research, the interaction of MBG scaffolds with bone-forming cells, the effect of drug/growth factor delivery on proliferation and differentiation of tissue cells and the in vivo osteogenesis of MBG scaffolds are highlighted. The advantages and limitations of MBG for drug delivery and bone tissue engineering have been compared with microsize bioactive glasses and nanosize bioactive glasses. The future perspective of MBG is discussed for bone regeneration application by combining drug delivery with bone tissue engineering and investigating the in vivo osteogenesis mechanism in large animal models. PMID:23741607
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Misdorp, W.; Van Der Heul, R. O.
1976-01-01
Tumours of bones and joints are not infrequent in dogs but are rare in other domestic animals. In the dog, most bone tumours are malignant; osteosarcomas are by far the most frequently encountered tumours, especially in giant breeds and boxers. The following main categories of bone tumour are described: bone-forming, cartilage-forming, giant cell, marrow, vascular, miscellaneous, metastatic, unclassified, and tumour-like lesions. The tumours of joints and related structures are classified as synovial sarcomas, fibroxanthomas, and malignant giant cell tumour of soft tissues. ImagesFig. 21Fig. 22Fig. 23Fig. 24Fig. 17Fig. 18Fig. 19Fig. 20Fig. 29Fig. 30Fig. 31Fig. 32Fig. 33Fig. 34Fig. 35Fig. 36Fig. 25Fig. 26Fig. 27Fig. 28Fig. 1Fig. 2Fig. 3Fig. 4Fig. 37Fig. 38Fig. 39Fig. 40Fig. 5Fig. 6Fig. 7Fig. 8Fig. 13Fig. 14Fig. 15Fig. 16Fig. 9Fig. 10Fig. 11Fig. 12 PMID:1086157
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2017-05-02
iss051e034105 (May 2, 2017) --- Commander Peggy Whitson is working on the OsteoOmics bone cell study that utilizes the Microgravity Science Glovebox inside the U.S. Destiny laboratory. OsteoOmics investigates the molecular mechanisms that dictate bone loss in microgravity by examining osteoblasts, which form bone, and osteoclasts, which dissolves bone. This leads to better preventative care or therapeutic treatments for people suffering bone loss as a result of bone diseases like osteopenia and osteoporosis, or for patients on prolonged bed rest.
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Endothelial Notch activity promotes angiogenesis and osteogenesis in bone
NASA Astrophysics Data System (ADS)
Ramasamy, Saravana K.; Kusumbe, Anjali P.; Wang, Lin; Adams, Ralf H.
2014-03-01
Blood vessel growth in the skeletal system and osteogenesis seem to be coupled, suggesting the existence of molecular crosstalk between endothelial and osteoblastic cells. Understanding the nature of the mechanisms linking angiogenesis and bone formation should be of great relevance for improved fracture healing or prevention of bone mass loss. Here we show that vascular growth in bone involves a specialized, tissue-specific form of angiogenesis. Notch signalling promotes endothelial cell proliferation and vessel growth in postnatal long bone, which is the opposite of the well-established function of Notch and its ligand Dll4 in the endothelium of other organs and tumours. Endothelial-cell-specific and inducible genetic disruption of Notch signalling in mice not only impaired bone vessel morphology and growth, but also led to reduced osteogenesis, shortening of long bones, chondrocyte defects, loss of trabeculae and decreased bone mass. On the basis of a series of genetic experiments, we conclude that skeletal defects in these mutants involved defective angiocrine release of Noggin from endothelial cells, which is positively regulated by Notch. Administration of recombinant Noggin, a secreted antagonist of bone morphogenetic proteins, restored bone growth and mineralization, chondrocyte maturation, the formation of trabeculae and osteoprogenitor numbers in endothelial-cell-specific Notch pathway mutants. These findings establish a molecular framework coupling angiogenesis, angiocrine signals and osteogenesis, which may prove significant for the development of future therapeutic applications.
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Inoue, Yuji; Iriyama, Aya; Ueno, Shuji; Takahashi, Hidenori; Kondo, Mineo; Tamaki, Yasuhiro; Araie, Makoto; Yanagi, Yasuo
2007-08-01
Because there is no effective treatment for this retinal degeneration, potential application of cell-based therapy has attracted considerable attention. Several investigations support that bone marrow mesenchymal stem cells (MSCs) can be used for a broad spectrum of indications. Bone marrow MSCs exert their therapeutic effect in part by secreting trophic factors to promote cell survival. The current study investigates whether bone marrow MSCs secrete factor(s) to promote photoreceptor cell survival and whether subretinal transplantation of bone marrow MSCs promotes photoreceptor survival in a retinal degeneration model using Royal College of Surgeons (RCS) rats. In vitro, using mouse retinal cell culture, it was demonstrated that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that the secreted factor(s) from the MSCs promote photoreceptor cell survival. In vivo, the MSCs were injected into the subretinal space of the RCS rats and histological analysis, real-time RT-PCR and electrophysiological analysis demonstrated that the subretinal transplantation of MSCs delays retinal degeneration and preserves retinal function in the RCS rats. These results suggest that MSC is a useful cell source for cell-replacement therapy for some forms of retinal degeneration.
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A review of fibrin and fibrin composites for bone tissue engineering
Noori, Alireza; Ashrafi, Seyed Jamal; Vaez-Ghaemi, Roza; Hatamian-Zaremi, Ashraf; Webster, Thomas J
2017-01-01
Tissue engineering has emerged as a new treatment approach for bone repair and regeneration seeking to address limitations associated with current therapies, such as autologous bone grafting. While many bone tissue engineering approaches have traditionally focused on synthetic materials (such as polymers or hydrogels), there has been a lot of excitement surrounding the use of natural materials due to their biologically inspired properties. Fibrin is a natural scaffold formed following tissue injury that initiates hemostasis and provides the initial matrix useful for cell adhesion, migration, proliferation, and differentiation. Fibrin has captured the interest of bone tissue engineers due to its excellent biocompatibility, controllable biodegradability, and ability to deliver cells and biomolecules. Fibrin is particularly appealing because its precursors, fibrinogen, and thrombin, which can be derived from the patient’s own blood, enable the fabrication of completely autologous scaffolds. In this article, we highlight the unique properties of fibrin as a scaffolding material to treat bone defects. Moreover, we emphasize its role in bone tissue engineering nanocomposites where approaches further emulate the natural nanostructured features of bone when using fibrin and other nanomaterials. We also review the preparation methods of fibrin glue and then discuss a wide range of fibrin applications in bone tissue engineering. These include the delivery of cells and/or biomolecules to a defect site, distributing cells, and/or growth factors throughout other pre-formed scaffolds and enhancing the physical as well as biological properties of other biomaterials. Thoughts on the future direction of fibrin research for bone tissue engineering are also presented. In the future, the development of fibrin precursors as recombinant proteins will solve problems associated with using multiple or single-donor fibrin glue, and the combination of nanomaterials that allow for the incorporation of biomolecules with fibrin will significantly improve the efficacy of fibrin for numerous bone tissue engineering applications. PMID:28761338
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A review of fibrin and fibrin composites for bone tissue engineering.
Noori, Alireza; Ashrafi, Seyed Jamal; Vaez-Ghaemi, Roza; Hatamian-Zaremi, Ashraf; Webster, Thomas J
2017-01-01
Tissue engineering has emerged as a new treatment approach for bone repair and regeneration seeking to address limitations associated with current therapies, such as autologous bone grafting. While many bone tissue engineering approaches have traditionally focused on synthetic materials (such as polymers or hydrogels), there has been a lot of excitement surrounding the use of natural materials due to their biologically inspired properties. Fibrin is a natural scaffold formed following tissue injury that initiates hemostasis and provides the initial matrix useful for cell adhesion, migration, proliferation, and differentiation. Fibrin has captured the interest of bone tissue engineers due to its excellent biocompatibility, controllable biodegradability, and ability to deliver cells and biomolecules. Fibrin is particularly appealing because its precursors, fibrinogen, and thrombin, which can be derived from the patient's own blood, enable the fabrication of completely autologous scaffolds. In this article, we highlight the unique properties of fibrin as a scaffolding material to treat bone defects. Moreover, we emphasize its role in bone tissue engineering nanocomposites where approaches further emulate the natural nanostructured features of bone when using fibrin and other nanomaterials. We also review the preparation methods of fibrin glue and then discuss a wide range of fibrin applications in bone tissue engineering. These include the delivery of cells and/or biomolecules to a defect site, distributing cells, and/or growth factors throughout other pre-formed scaffolds and enhancing the physical as well as biological properties of other biomaterials. Thoughts on the future direction of fibrin research for bone tissue engineering are also presented. In the future, the development of fibrin precursors as recombinant proteins will solve problems associated with using multiple or single-donor fibrin glue, and the combination of nanomaterials that allow for the incorporation of biomolecules with fibrin will significantly improve the efficacy of fibrin for numerous bone tissue engineering applications.
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A novel three-dimensional bone chip organ culture.
Kuttenberger, Johannes; Polska, Elzbieta; Schaefer, Birgit M
2013-07-01
The objective of this study was to develop a 3D bone chip organ culture model. We aimed to collect in vitro evidence of the ability of vital bone chips to promote new bone formation. We developed a 3D in vitro hypoxic bone chip organ culture model. Histology of the bone chips was performed before and after culture and immunohistochemistry after 3-week culture. The 3D culture supernatants were tested for the presence of pro-angiogenic growth factors, TGFβ1, GADPH, bone alkaline phosphatase, osteocalcin, osteonectin, osteopontin, bone sialoprotein and collagen type I. Histology after culture revealed bone chips in a matrix of fibrin remnants and a fibrous-appearing matter. Collagen type I- and IV-positive structures were also identified. Cells could be seen on the surface of the bone chips, with spindle-shaped cells bridging the bone chip particles. Pro-angiogenic growth factors and TGFβ1were detected in the 3D cell culture supernatants. The transcripts for osteocalcin, bone sialoprotein and collagen type I were revealed only via PCR. Our results indicate that bone chips in our 3D organ culture remain vital and may stimulate the growth of a bone-forming matrix. The use of autogenous bone chips for oral and maxillofacial bone augmentation procedures is widespread in clinical practice. The rationale for this is that if bone chips remain vital in vivo, they could provide an environment promoting new bone formation through growth factors and cells. This 3D culture method is an essential tool for investigating the behaviour of bone chips.
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Murphy, Kaitlin C.; Hughbanks, Marissa L.; Binder, Bernard Y.K.; Vissers, Caroline B.; Leach, J. Kent
2014-01-01
Mesenchymal stem/stromal cells (MSCs) are under examination for use in cell therapies to repair bone defects resulting from trauma or disease. MSCs secrete proangiogenic cues and can be induced to differentiate into bone-forming osteoblasts, yet there is limited evidence that these events can be achieved in parallel. Manipulation of the cell delivery vehicle properties represents a candidate approach for directing MSC function in bone healing. We hypothesized that the biophysical properties of a fibrin gel could simultaneously regulate the proangiogenic and osteogenic potential of entrapped MSCs. Fibrin gels were formed by supplementation with NaCl (1.2, 2.3, and 3.9% w/v) to modulate gel biophysical properties without altering protein concentrations. MSCs entrapped in 1.2% w/v NaCl gels were the most proangiogenic in vitro, yet cells in 3.9% w/v gels exhibited the greatest osteogenic response. Compared to the other groups, MSCs entrapped in 2.3% w/v gels provided the best balance between proangiogenic potential, osteogenic potential, and gel contractility. The contribution of MSCs to bone repair was then examined when deployed in 2.3% w/v NaCl gels and implanted into an irradiated orthotopic bone defect. Compared to acellular gels after 3 weeks of implantation, defects treated with MSC-loaded fibrin gels exhibited significant increases in vessel density, early osteogenesis, superior morphology, and increased cellularity of repair tissue. Defects treated with MSC-loaded gels exhibited increased bone formation after 12 weeks compared to blank gels. These results confirm that fibrin gel properties can be modulated to simultaneously promote both the proangiogenic and osteogenic potential of MSCs, and fibrin gels modified by supplementation with NaCl are promising carriers for MSCs to stimulate bone repair in vivo. PMID:25527322
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The expression of ADAM12 (meltrin alpha) in human giant cell tumours of bone.
Tian, B L; Wen, J M; Zhang, M; Xie, D; Xu, R B; Luo, C J
2002-12-01
To examine the expression of ADAM12 (meltrin alpha), a member of the disintegrin and metalloprotease (ADAM) family, in human giant cell tumours of the bone, skeletal muscle tissue from human embryos, and human adult skeletal muscle tissue. ADAM12 mRNA was detected by reverse transcription polymerase chain reaction and in situ hybridisation. ADAM12 mRNA was detected in 14 of the 20 giant cell tumours of bone and in three of the six tumour cell cultures. The expression of ADAM12 in cells cultured from the tumour was linked to the presence of multinucleated giant cells. ADAM12 mRNA could not be detected in the five adult skeletal muscle tissue samples, although it was found in the two embryonic skeletal muscle tissue samples. ADAM12 mRNA was localised to the cytoplasm of multinucleated giant cells and some mononuclear stromal cells. These results indicate that multinucleated giant cells are formed by the cell fusion of mononuclear stromal cells in giant cell tumours of bone and that ADAM12 is involved in the cell fusion process.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nagata, T.; Todescan, R.; Goldberg, H.A.
Secreted phosphoprotein I (SPPI) is a prominent structural protein in mineralized connective tissues. Rat bone cells in culture produce several forms of SPPI that differ in post-translational modifications such as phosphorylation and sulphation. To determine the significance of protein sulphation in bone formation, the synthesis of SPPI was studied in vitro using rat bone marrow cells (RBMC) which form bone-like tissue when grown in the presence of dexamethasone (Dex) and beta-glycerophosphate (beta-GP). In the presence of 10(-7) M Dex SPPI expression was stimulated 4-5-fold. Radiolabelling multilayered RBMCs for 48 h with (35S)-methionine, Na2(35SO4), or Na3(32PO4) revealed that two major phosphorylatedmore » forms of SPPI were secreted into the culture medium: a highly phosphorylated form migrating at 44 kDa on 15% SDS-PAGE and a less phosphorylated 55 kDa form. In the mineralized tissue formed in the presence of Dex and beta-GP, both forms of SPPI, in addition to proteoglycans and a 67 kDa protein, incorporated significant amounts of (35SO4). Sulphation of SPPI was not observed in the absence of mineral formation, indicating that the sulphation of SPPI is closely associated with mineralization and that it can be used as a sensitive and specific marker for the osteoblastic phenotype.« less
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[Neuro-skeletal biology and its importance for clinical osteology].
Zofková, I
2012-01-01
Bone remodeling is determined by function of two basic cell forms--bone resorbing osteoclasts and bone formation activating osteoblasts. Both cells are under control of a variety of endogenic and environmental factors, which ensure balance between bone resorption and bone formation. This article reviews the neuro-hormonal factors with osteoanabolic (central isoform of serotonin, melatonin, cannabinoids, beta 1 adrenergic system, oxytocin, ACTH and TSH) or osteocatabolic effects (neuropeptide Y, neuromedin U, beta2 adrenergic system). The dual effects of the beta-adrenergic system, serotonin and leptin are also discussed. The goal of studies focused on neuro-skeletal interaction is to synthesize new molecules, which can modify osteo-anabolic or osteo-catabolic pathways.
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Schröder, Heinz C; Müller, Werner E G
2014-01-01
Summary Calcium carbonate is the material that builds up the spicules of the calcareous sponges. Recent results revealed that the calcium carbonate/biocalcite-based spicular skeleton of these animals is formed through an enzymatic mechanism, such as the skeleton of the siliceous sponges, evolutionarily the oldest animals that consist of biosilica. The enzyme that mediates the calcium carbonate deposition has been identified as a carbonic anhydrase (CA) and has been cloned from the calcareous sponge species Sycon raphanus. Calcium carbonate deposits are also found in vertebrate bones besides the main constituent, calcium phosphate/hydroxyapatite (HA). Evidence has been presented that during the initial phase of HA synthesis poorly crystalline carbonated apatite is deposited. Recent data summarized here indicate that during early bone formation calcium carbonate deposits enzymatically formed by CA, act as potential bioseeds for the precipitation of calcium phosphate mineral onto bone-forming osteoblasts. Two different calcium carbonate phases have been found during CA-driven enzymatic calcium carbonate deposition in in vitro assays: calcite crystals and round-shaped vaterite deposits. The CA provides a new target of potential anabolic agents for treatment of bone diseases; a first CA activator stimulating the CA-driven calcium carbonate deposition has been identified. In addition, the CA-driven calcium carbonate crystal formation can be frozen at the vaterite state in the presence of silintaphin-2, an aspartic acid/glutamic acid-rich sponge-specific protein. The discovery that calcium carbonate crystals act as bioseeds in human bone formation may allow the development of novel biomimetic scaffolds for bone tissue engineering. Na-alginate hydrogels, enriched with biosilica, have recently been demonstrated as a suitable matrix to embed bone forming cells for rapid prototyping bioprinting/3D cell printing applications. PMID:24991497
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Wang, Xiaohong; Schröder, Heinz C; Müller, Werner E G
2014-01-01
Calcium carbonate is the material that builds up the spicules of the calcareous sponges. Recent results revealed that the calcium carbonate/biocalcite-based spicular skeleton of these animals is formed through an enzymatic mechanism, such as the skeleton of the siliceous sponges, evolutionarily the oldest animals that consist of biosilica. The enzyme that mediates the calcium carbonate deposition has been identified as a carbonic anhydrase (CA) and has been cloned from the calcareous sponge species Sycon raphanus. Calcium carbonate deposits are also found in vertebrate bones besides the main constituent, calcium phosphate/hydroxyapatite (HA). Evidence has been presented that during the initial phase of HA synthesis poorly crystalline carbonated apatite is deposited. Recent data summarized here indicate that during early bone formation calcium carbonate deposits enzymatically formed by CA, act as potential bioseeds for the precipitation of calcium phosphate mineral onto bone-forming osteoblasts. Two different calcium carbonate phases have been found during CA-driven enzymatic calcium carbonate deposition in in vitro assays: calcite crystals and round-shaped vaterite deposits. The CA provides a new target of potential anabolic agents for treatment of bone diseases; a first CA activator stimulating the CA-driven calcium carbonate deposition has been identified. In addition, the CA-driven calcium carbonate crystal formation can be frozen at the vaterite state in the presence of silintaphin-2, an aspartic acid/glutamic acid-rich sponge-specific protein. The discovery that calcium carbonate crystals act as bioseeds in human bone formation may allow the development of novel biomimetic scaffolds for bone tissue engineering. Na-alginate hydrogels, enriched with biosilica, have recently been demonstrated as a suitable matrix to embed bone forming cells for rapid prototyping bioprinting/3D cell printing applications.
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Chen, Shuang-Tao; Zhang, Wei-Ping; Liu, Chang-An; Wang, Jun-Jiang; Song, Heng-Yi; Chai, Zhi-wen
2013-03-01
To discuss the feasibility of vascular bundle implantation combined with allogeneic bone marrow stromal cells (BMSCs) transplantation in treating rabbit femoral head osteonecrosis and bone defect, in order to explore a new method for the treatment of femoral head necrosis. Thirty-six New Zealand rabbits were randomly divided into three groups,with 12 rabbits in each group. Bilateral femoral heads of the rabbits were studied in the experiment. The models were made by liquid nitrogen frozen, and the femoral heads were drilled to cause bone defect. Group A was the control group,group B was stem cells transplantaion group of allograft marrow stromal,and group C was stem cells transplantation group of allograft marrow stromal combined with vascular bundle implantation. Three rabbits of each group were sacrificed respectively at 2, 4, 8, 12 weeks after operation. All specimens of the femoral heads were sliced for HE staining. Furthermore ,vascular density and the percentage of new bone trabecula of femoral head coronary section in defect area were measured and analyzed statistically. In group C,new bone trabecula and original micrangium formed at the 2nd week after operation; new bone trabecula was lamellar and interlaced with abundant micrangium at the 8th week;at the 12th week,the broadened,coarsened bone trabecula lined up regularly,and the mature bone trabecula and new marrow were visible. At the 2nd week after operation,there was no statistical significance in the percentage of new bone trabecula of femoral head coronary section in defect area between group B and C. While at 4, 8, 12 week after operation, vascular density and the percentage of new bone trabecula of femoral head coronary section in defect area of group C was higher than that of group B. Allogeneic bone marrow stromal cells cultured in vivo can form new bone trabecula, and can be applied to allotransplant. Vascular bundle implanted into the bone defect area of femoral head necrosis could improve blood supply, and promote the formation of bone trabecula.
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Advances in Bone Marrow Stem Cell Therapy for Retinal Dysfunction
Park, Susanna S.; Moisseiev, Elad; Bauer, Gerhard; Anderson, Johnathon D.; Grant, Maria B.; Zam, Azhar; Zawadzki, Robert J.; Werner, John S.; Nolta, Jan A.
2016-01-01
The most common cause of untreatable vision loss is dysfunction of the retina. Conditions, such as age-related macular degeneration, diabetic retinopathy and glaucoma remain leading causes of untreatable blindness worldwide. Various stem cell approaches are being explored for treatment of retinal regeneration. The rationale for using bone marrow stem cells to treat retinal dysfunction is based on preclinical evidence showing that bone marrow stem cells can rescue degenerating and ischemic retina. These stem cells have primarily paracrine trophic effects although some cells can directly incorporate into damaged tissue. Since the paracrine trophic effects can have regenerative effects on multiple cells in the retina, the use of this cell therapy is not limited to a particular retinal condition. Autologous bone marrow-derived stem cells are being explored in early clinical trials as therapy for various retinal conditions. These bone marrow stem cells include mesenchymal stem cells, mononuclear cells and CD34+ cells. Autologous therapy requires no systemic immunosuppression or donor matching. Intravitreal delivery of CD34+ cells and mononuclear cells appears to be tolerated and is being explored since some of these cells can home into the damaged retina after intravitreal administration. The safety of intravitreal delivery of mesenchymal stem cells has not been well established. This review provides an update of the current evidence in support of the use of bone marrow stem cells as treatment for retinal dysfunction. The potential limitations and complications of using certain forms of bone marrow stem cells as therapy are discussed. Future directions of research include methods to optimize the therapeutic potential of these stem cells, non-cellular alternatives using extracellular vesicles, and in vivo high-resolution retinal imaging to detect cellular changes in the retina following cell therapy. PMID:27784628
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Protective effects of deferasirox and N-acetyl-L-cysteine on iron overload-injured bone marrow.
Shen, J C; Zhang, Y C; Zhao, M F
2017-10-19
Using an iron overload mouse model, we explored the protective effect of deferasirox (DFX) and N-acetyl-L-cysteine (NAC) on injured bone marrow hematopoietic stem/progenitor cells (HSPC) induced by iron overload. Mice were intraperitoneally injected with 25 mg iron dextran every 3 days for 4 weeks to establish an iron overload (Fe) model. DFX or NAC were co-administered with iron dextran in two groups of mice (Fe+DFX and Fe+NAC), and the function of HSPCs was then examined. Iron overload markedly decreased the number of murine HSPCs in bone marrow. Subsequent colony-forming cell assays showed that iron overload also decreased the colony forming capacity of HSPCs, the effect of which could be reversed by DFX and NAC. The bone marrow hematopoiesis damage caused by iron overload could be alleviated by DFX and NAC.
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Journey into Bone Models: A Review
Scheinpflug, Julia; Pfeiffenberger, Moritz; Damerau, Alexandra; Schwarz, Franziska; Textor, Martin; Lang, Annemarie
2018-01-01
Bone is a complex tissue with a variety of functions, such as providing mechanical stability for locomotion, protection of the inner organs, mineral homeostasis and haematopoiesis. To fulfil these diverse roles in the human body, bone consists of a multitude of different cells and an extracellular matrix that is mechanically stable, yet flexible at the same time. Unlike most tissues, bone is under constant renewal facilitated by a coordinated interaction of bone-forming and bone-resorbing cells. It is thus challenging to recreate bone in its complexity in vitro and most current models rather focus on certain aspects of bone biology that are of relevance for the research question addressed. In addition, animal models are still regarded as the gold-standard in the context of bone biology and pathology, especially for the development of novel treatment strategies. However, species-specific differences impede the translation of findings from animal models to humans. The current review summarizes and discusses the latest developments in bone tissue engineering and organoid culture including suitable cell sources, extracellular matrices and microfluidic bioreactor systems. With available technology in mind, a best possible bone model will be hypothesized. Furthermore, the future need and application of such a complex model will be discussed. PMID:29748516
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Journey into Bone Models: A Review.
Scheinpflug, Julia; Pfeiffenberger, Moritz; Damerau, Alexandra; Schwarz, Franziska; Textor, Martin; Lang, Annemarie; Schulze, Frank
2018-05-10
Bone is a complex tissue with a variety of functions, such as providing mechanical stability for locomotion, protection of the inner organs, mineral homeostasis and haematopoiesis. To fulfil these diverse roles in the human body, bone consists of a multitude of different cells and an extracellular matrix that is mechanically stable, yet flexible at the same time. Unlike most tissues, bone is under constant renewal facilitated by a coordinated interaction of bone-forming and bone-resorbing cells. It is thus challenging to recreate bone in its complexity in vitro and most current models rather focus on certain aspects of bone biology that are of relevance for the research question addressed. In addition, animal models are still regarded as the gold-standard in the context of bone biology and pathology, especially for the development of novel treatment strategies. However, species-specific differences impede the translation of findings from animal models to humans. The current review summarizes and discusses the latest developments in bone tissue engineering and organoid culture including suitable cell sources, extracellular matrices and microfluidic bioreactor systems. With available technology in mind, a best possible bone model will be hypothesized. Furthermore, the future need and application of such a complex model will be discussed.
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2015-01-01
Bone remodeling relies on the coordinated functioning of osteoblasts, bone-forming cells, and osteoclasts, bone-resorbing cells. The effects of specific chemical and physical bone features on the osteoclast adhesive apparatus, the sealing zone ring, and their relation to resorption functionality are still not well-understood. We designed and implemented a correlative imaging method that enables monitoring of the same area of bone surface by time-lapse light microscopy, electron microscopy, and atomic force microscopy before, during, and after exposure to osteoclasts. We show that sealing zone rings preferentially develop around surface protrusions, with lateral dimensions of several micrometers, and ∼1 μm height. Direct overlay of sealing zone rings onto resorption pits on the bone surface shows that the rings adapt to pit morphology. The correlative procedure presented here is noninvasive and performed under ambient conditions, without the need for sample labeling. It can potentially be applied to study various aspects of cell-matrix interactions. PMID:26682493
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Ogawa, Munehiro; Tohma, Yasuaki; Ohgushi, Hajime; Takakura, Yoshinori; Tanaka, Yasuhito
2012-01-01
To establish the methods of demonstrating early fixation of metal implants to bone, one side of a Cobalt-Chromium (CoCr) based alloy implant surface was seeded with rabbit marrow mesenchymal cells and the other side was left unseeded. The mesenchymal cells were further cultured in the presence of ascorbic acid, β-glycerophosphate and dexamethasone, resulting in the appearance of osteoblasts and bone matrix on the implant surface. Thus, we succeeded in generating tissue-engineered bone on one side of the CoCr implant. The CoCr implants were then implanted in rabbit bone defects. Three weeks after the implantation, evaluations of mechanical test, undecalcified histological section and electron microscope analysis were performed. Histological and electron microscope images of the tissue engineered surface exhibited abundant new bone formation. However, newly formed bone tissue was difficult to detect on the side without cell seeding. In the mechanical test, the mean values of pull-out forces were 77.15 N and 44.94 N for the tissue-engineered and non-cell-seeded surfaces, respectively. These findings indicate early bone fixation of the tissue-engineered CoCr surface just three weeks after implantation.
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Ogawa, Munehiro; Tohma, Yasuaki; Ohgushi, Hajime; Takakura, Yoshinori; Tanaka, Yasuhito
2012-01-01
To establish the methods of demonstrating early fixation of metal implants to bone, one side of a Cobalt-Chromium (CoCr) based alloy implant surface was seeded with rabbit marrow mesenchymal cells and the other side was left unseeded. The mesenchymal cells were further cultured in the presence of ascorbic acid, β-glycerophosphate and dexamethasone, resulting in the appearance of osteoblasts and bone matrix on the implant surface. Thus, we succeeded in generating tissue-engineered bone on one side of the CoCr implant. The CoCr implants were then implanted in rabbit bone defects. Three weeks after the implantation, evaluations of mechanical test, undecalcified histological section and electron microscope analysis were performed. Histological and electron microscope images of the tissue engineered surface exhibited abundant new bone formation. However, newly formed bone tissue was difficult to detect on the side without cell seeding. In the mechanical test, the mean values of pull-out forces were 77.15 N and 44.94 N for the tissue-engineered and non-cell-seeded surfaces, respectively. These findings indicate early bone fixation of the tissue-engineered CoCr surface just three weeks after implantation. PMID:22754313
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Stem cells and bone: a historical perspective.
Bianco, Paolo
2015-01-01
Bone physiology and stem cells were tightly intertwined with one another, both conceptually and experimentally, long before the current explosion of interest in stem cells and so-called regenerative medicine. Bone is home to the two best known and best characterized systems of postnatal stem cells, and it is the only organ in which two stem cells and their dependent lineages coordinate the overall adaptive responses of two major physiological systems. All along, the nature and the evolutionary significance of the interplay of bone and hematopoiesis have remained a major scientific challenge, but also allowed for some of the most spectacular developments in cell biology-based medicine, such as hematopoietic stem cell transplantation. This question recurs in novel forms at multiple turning points over time: today, it finds in the biology of the "niche" its popular phrasing. Entirely new avenues of investigation emerge as a new view of bone in physiology and medicine is progressively established. Looking at bone and stem cells in a historical perspective provides a unique case study to highlight the general evolution of science in biomedicine since the end of World War II to the present day. A paradigm shift in science and in its relation to society and policies occurred in the second half of the XXth century, with major implications thereof for health, industry, drug development, market and society. Current interest in stem cells in bone as in other fields is intertwined with that shift. New opportunities and also new challenges arise. This article is part of a Special Issue entitled "Stem cells and bone". Copyright © 2014. Published by Elsevier Inc.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Krieg, A.M.; Gourley, M.F.; Steinberg, A.D.
1991-05-01
Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymicmore » epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.« less
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Martin-Millan, Marta; Almeida, Maria; Ambrogini, Elena; Han, Li; Zhao, Haibo; Weinstein, Robert S.; Jilka, Robert L.; O'Brien, Charles A.; Manolagas, Stavros C.
2010-01-01
Estrogens attenuate osteoclastogenesis and stimulate osteoclast apoptosis, but the molecular mechanism and contribution of these effects to the overall antiosteoporotic efficacy of estrogens remain controversial. We selectively deleted the estrogen receptor (ER)α from the monocyte/macrophage cell lineage in mice (ERαLysM−/−) and found a 2-fold increase in osteoclast progenitors in the marrow and the number of osteoclasts in cancellous bone, along with a decrease in cancellous bone mass. After loss of estrogens these mice failed to exhibit the expected increase in osteoclast progenitors, the number of osteoclasts in bone, and further loss of cancellous bone. However, they lost cortical bone indistinguishably from their littermate controls. Mature osteoclasts from ERαLysM−/− were resistant to the proapoptotic effect of 17β-estradiol. Nonetheless, the effects of estrogens on osteoclasts were unhindered in mice bearing an ERα knock-in mutation that prevented binding to DNA. Moreover, a polymeric form of estrogen that is not capable of stimulating the nuclear-initiated actions of ERα was as effective as 17β-estradiol in inducing osteoclast apoptosis in cells with the wild-type ERα. We conclude that estrogens attenuate osteoclast generation and life span via cell autonomous effects mediated by DNA-binding-independent actions of ERα. Elimination of these effects is sufficient for loss of bone in the cancellous compartment in which complete perforation of trabeculae by osteoclastic resorption precludes subsequent refilling of the cavities by the bone-forming osteoblasts. However, additional effects of estrogens on osteoblasts, osteocytes, and perhaps other cell types are required for their protective effects on the cortical compartment, which constitutes 80% of the skeleton. PMID:20053716
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Sinclair, Sarina S Kay; Horton, C Olsen; Jeray, Kyle J; Tanner, Stephanie L; Burgl, Karen J L
2015-01-01
Mesenchymal stem cells (MSCs) are of therapeutic interest to clinicians and researchers, as they have been shown to augment the osteogenic properties of bone grafts. MSCs are known to be prevalent in bone marrow, but are still limited in numbers. Hence, additional sources of MSCs are beneficial to increasing grafting potential. Aspirate material collected using the Reamer/Irrigator/Aspirator (RIA) device (Synthes; Paoli, PA) during reaming of the femoral shaft consists of three main components: bone fragments, liquid flow-through, and a fat layer. Currently, only the bone and liquid layers have been examined for osteoinductive elements, and the bone fragments are exclusively used as autologous bone graft. In the present study, a method to promote cellular outgrowth, tapping proliferative capacity from the previously discarded fatty layer of RIA aspirate, is described. Proliferating cells were successfully isolated from the bone and fatty layers of a consenting patient and found to be viable after liquid nitrogen storage. The osteogenic differentiation potential of the cells isolated from the fat and bone layers was assessed. Cells from both layers of the aspirate expressed statistically significant levels (p < 0.05) of the bone cell marker alkaline phosphatase compared to the control cells, suggesting differentiation along the osteoblastic pathway. Results from this pilot study indicate that the traditionally discarded fatty element of RIA aspirate may be a source of MSCs with bone-forming capabilities and the described isolation technique is effective. Combining the aspirate fatty and bony elements may enhance the clinical success of the RIA autograft.
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On Orbit Osteobiology Experiments: from "STROMA" to "MDS" -from in vitro to in vivo
NASA Astrophysics Data System (ADS)
Liu, Yi; Cancedda, Ranieri
Spaceflight causes profound changes in the skeleton, in particular, in the weight-loading bones. Uncoupling of bone remodeling equilibrium between bone formation and resorption is con-sidered responsible for the microgravity-induced bone loss. These changes result in weak-ened and brittle bones prone to fracture on re-entry and in accelerated osteoporosis, making bone deterioration a major problem obstructing the prospects of long-duration manned space flight. Osteoblasts (bone forming cells) and osteocytes (bone resorption cells) are known to be mechano-sensors. Short-exposure of osteoblasts to simulated microgravity ensnarled cell adhe-sion and cytoskeleton. Also osteoblast precursors such as bone marrow stroma cells (BMSC) were shown to be sensitive to mechanical loading. We performed a series of STROMA space-flight experiments by culturing BMSC or co-culturing osteoblasts and osteoclast precursors in automated bioreactors on orbit. Genechip analysis revealed an inhibition of cell proliferation and an unexpected activation of nervous system development genes by spaceflight. To unravel effects of microgravity on genes governing bone mass, transgenic mice with a higher bone mass were flown to orbit inside the Mice Drawer System (MDS) payload. The MDS experiment was launched inside Shuttle Discovery in STS-128 on August 28 2009 at 23:58 EST, and returned to earth by Shuttle Atlantis in STS129 on November 27 2009 at 9:47 EST, marking it as the first long duration animal experiment on the International Space Station (ISS).
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A monoclonal antibody that recognizes B cells and B cell precursors in mice
DOE Office of Scientific and Technical Information (OSTI.GOV)
Coffman, R.L.; Weissman, I.L.
1981-02-01
The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.
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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.
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Improved autologous cortical bone harvest and viability with 2Flute otologic burs.
Roth, Adam A; Tang, Pei-Ciao; Ye, Michael J; Mohammad, Khalid S; Nelson, Rick F
2018-01-01
To determine if 2Flute (Stryker Corporation, Kalamazoo, MI) otologic burs improve the size, cellular content, and bone healing of autologous cortical bone grafts harvested during canal wall reconstruction (CWR) tympanomastoidectomy with mastoid obliteration. Institutional review board-approved prospective cohort study. Human autologous cortical bone chips were harvested using various burs (4 and 6 mm diameter; multiflute, and 2Flute [Stryker Corporation]) from patients undergoing CWR tympanomastoidectomy for the treatment of chronic otitis media with cholesteatoma. Bone chip size, cell counts, cellular gene expression, and new bone formation were quantified. Bone chips were significantly larger when harvested with 2Flute (Stryker Corporation) bur compared to multiflute burs at both 6 mm diameter (113 ± 14 μm 2 vs. 66 ± 8 μm 2 ; P < 0.05) and 4 mm diameter (70 ± 8 μm 2 vs. 50 ± 3 μm 2 ; P < 0.05). After 2 weeks in culture, cell numbers were significantly higher when harvested with 2Flute (Stryker Corporation) bur compared to multiflute burs at both 6 mm diameter (48.7 ± 3 vs. 31.8 ± 3 cells/μg bone; P < 0.05) and 4 mm diameter (27.6 ± 1.2 vs. 8.8 ± 1.2 cells/μg bone; P < 0.05). Bone-derived cells express osteoblast markers (alkaline phosphatase, osteocalcin). Cultured cells are able to form new bone in culture, and bone formation is facilitated by the presence of bone chips. Use of 2Flute (Stryker Corporation) otologic burs for human autologous cortical bone harvest results in more viable bone fragments, with larger bone chips and more osteoblasts. Future studies are needed to determine if this leads to improved bone healing. NA. Laryngoscope, 128:E41-E46, 2018. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.
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In vivo response to starch-based scaffolds designed for bone tissue engineering applications.
Salgado, A J; Coutinho, O P; Reis, R L; Davies, J E
2007-03-15
Our purpose was to evaluate the in vivo endosseous response to three starch-based scaffolds implanted in rats (n = 54). We implanted the three scaffold groups; a 50/50 (wt %) blend of corn starch and ethylene-vinyl alcohol (SEVA-C), the same composition coated with a biomimetic calcium phosphate (Ca-P) layer (SEVA-C/CaP), and a 50/50 (wt %) blend of corn starch and cellulose acetate (SCA), all produced by extrusion with blowing agents, into distal femurs proximal to the epiphyseal plate, for 1, 3, or 6 weeks. Our results showed that at 1 week considerable reparative bone formed around all scaffold groups, although the bone was separated from the scaffold by an intervening soft tissue interfacial zone that comprised two distinct compartments: the surface of the scaffold was occupied by multinucleate giant cells and the compartment between these cells and the surrounding bone was occupied by a streaming fibrous-like tissue. The extracellular matrix of the latter was continuous with the extracellular bone matrix itself, labeled positively for osteocalcin and appeared mineralized by back-scattered electron imaging. All three scaffolds showed a similar tissue response, with the soft tissue interface diminishing with time. No bone contact was observed with SEVA-C at any time point, only transitory bone contact was observed with SEVA-C/CaP at 3 weeks, but SCA exhibited direct bone contact at 6 weeks where 56.23 +/- 6.46% of the scaffold surface was occupied by bone. We conclude that all materials exhibited a favorable bony response and that the rapidly forming initial "connective tissue" seen around all scaffolds was a very early form of bone formation.
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Kiviranta, Riku; Yamana, Kei; Saito, Hiroaki; Ho, Daniel K.; Laine, Julius; Tarkkonen, Kati; Nieminen-Pihala, Vappu; Hesse, Eric; Correa, Diego; Määttä, Jorma; Tessarollo, Lino; Rosen, Evan D.; Horne, William C.; Jenkins, Nancy A.; Copeland, Neal G.; Warming, Soren
2013-01-01
Bone homeostasis is maintained by the coupled actions of hematopoietic bone-resorbing osteoclasts (OCs) and mesenchymal bone-forming osteoblasts (OBs). Here we identify early B cell factor 1 (Ebf1) and the transcriptional coregulator Zfp521 as components of the machinery that regulates bone homeostasis through coordinated effects in both lineages. Deletion of Zfp521 in OBs led to impaired bone formation and increased OB-dependent osteoclastogenesis (OC-genesis), and deletion in hematopoietic cells revealed a strong cell-autonomous role for Zfp521 in OC progenitors. In adult mice, the effects of Zfp521 were largely caused by repression of Ebf1, and the bone phenotype of Zfp521+/− mice was rescued in Zfp521+/−:Ebf1+/− mice. Zfp521 interacted with Ebf1 and repressed its transcriptional activity. Accordingly, deletion of Zfp521 led to increased Ebf1 activity in OBs and OCs. In vivo, Ebf1 overexpression in OBs resulted in suppressed bone formation, similar to the phenotype seen after OB-targeted deletion of Zfp521. Conversely, Ebf1 deletion led to cell-autonomous defects in both OB-dependent and cell-intrinsic OC-genesis, a phenotype opposite to that of the Zfp521 knockout. Thus, we have identified the interplay between Zfp521 and Ebf1 as a novel rheostat for bone homeostasis. PMID:23569325
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Nguyen, Minh K; Jeon, Oju; Dang, Phuong N; Huynh, Cong T; Varghai, Davood; Riazi, Hooman; McMillan, Alexandra; Herberg, Samuel; Alsberg, Eben
2018-06-06
RNA interference (RNAi) may be an effective and valuable tool for promoting the growth of functional tissue, as short interfering RNA (siRNA) and microRNA (miRNA) can block the expression of genes that have negative effects on tissue regeneration. Our group has recently reported that the localized and sustained presentation of siRNA against noggin (siNoggin) and miRNA-20a from in situ forming poly(ethylene glycol) (PEG) hydrogels enhanced osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hMSCs). Here, the capacity of the hydrogel system to accelerate bone formation in a rat calvarial bone defect model is presented. After 12 weeks post-implantation, the hydrogels containing encapsulated hMSCs and miRNA-20a resulted in more bone formation in the defects than the hydrogels containing hMSCs without siRNA or with negative control siRNA. This localized and sustained RNA interfering molecule delivery system may provide an excellent platform for healing bony defects and other tissues. Delivery of RNAi molecules may be a valuable strategy to guide cell behavior for tissue engineering applications, but to date there have been no reports of a biomaterial system capable of both encapsulation of cells and controlled delivery of incorporated RNA. Here, we present PEG hydrogels that form in situ via Michael type reaction, and that permit encapsulation of hMSCs and the concomitant controlled delivery of siNoggin and/or miRNA-20a. These RNAs were chosen to suppress noggin, a BMP-2 antagonist, and/or PPAR-γ, a negative regulator of BMP-2-mediated osteogenesis, and therefore promote osteogenic differentiation of hMSCs and subsequent bone repair in critical-sized rat calvarial defects. Simultaneous delivery of hMSCs and miRNA-20a enhanced repair of these defects compared to hydrogels containing hMSCs without siRNA or with negative control siRNA. This in situ forming PEG hydrogel system offers an exciting platform for healing critical-sized bone defects by localized, controlled delivery of RNAi molecules to encapsulated hMSCs and surrounding cells. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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Lopez-Heredia, Marco A; Łapa, Agata; Reczyńska, Katarzyna; Pietryga, Krzysztof; Balcaen, Lieve; Mendes, Ana C; Schaubroeck, David; Van Der Voort, Pascal; Dokupil, Agnieszka; Plis, Agnieszka; Stevens, Chris V; Parakhonskiy, Bogdan V; Samal, Sangram Keshari; Vanhaecke, Frank; Chai, Feng; Chronakis, Ioannis S; Blanchemain, Nicolas; Pamuła, Elżbieta; Skirtach, Andre G; Douglas, Timothy E L
2018-04-27
Mineralization of hydrogels is desirable prior to applications in bone regeneration. CaCO 3 is a widely used bone regeneration material and Mg, when used as a component of calcium phosphate biomaterials, has promoted bone-forming cell adhesion and proliferation and bone regeneration. In this study, gellan gum (GG) hydrogels were mineralized with carbonates containing different amounts of calcium (Ca) and magnesium (Mg) by alternate soaking in, firstly, a calcium and/or magnesium ion solution and, secondly, a carbonate ion solution. This alternate soaking cycle was repeated five times. Five different calcium and/or magnesium ion solutions, containing different molar ratios of Ca to Mg ranging from Mg-free to Ca-free were compared. Carbonate mineral formed in all sample groups subjected to the Ca:Mg elemental ratio in the carbonate mineral formed was higher than in the respective mineralizing solution. Mineral formed in the absence of Mg was predominantly CaCO 3 in the form of a mixture of calcite and vaterite. Increasing the Mg content in the mineral formed led to the formation of magnesian calcite, decreased the total amount of the mineral formed and its crystallinity. Hydrogel mineralization and increasing Mg content in mineral formed did not obviously improve proliferation of MC3T3-E1 osteoblast-like cells or differentiation after 7 days. This article is protected by copyright. All rights reserved.
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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.
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Stimulation of Mucosal Mast Cell Growth in Normal and Nude Rat Bone Marrow Cultures
NASA Astrophysics Data System (ADS)
Haig, David M.; McMenamin, Christine; Gunneberg, Christian; Woodbury, Richard; Jarrett, Ellen E. E.
1983-07-01
Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.
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Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model
NASA Astrophysics Data System (ADS)
Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther
During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX-immunostaining. Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (von-Kossa staining, Alizarin Red staining). During the process of osteoblastic cell differentiation, the expression of the bone specific marker genes osteocalcin (OCN) and osteopontin (OPN) were recorded by quantitative real time reverse transcription PCR (qRT-PCR). Compared with standard culture conditions, the osteogenic marker genes OCN and OPN were highly expressed during the differentiation process induced either by osteo-inductive media additives (50 µg/ml ascorbic acid, 10 mmol/l β-glycero phosphate) or by sparsely ionizing radiation (X-rays). After 21 days of postirradiation incubation sparsely ionizing radiation could be shown to induce the formation of bone-like nodules (von-Kossa staining) for OCT-1 and MC3T3-E1 S4 cells but nor for MC3T3- E1 S24 cells. Ionizing radiation leads to a cell cycle arrest which is resolved in a dose and time dependent way. This was accompanied by a dose dependent regulation of the cyclin kinase inhibitor CDKN1A (p21/WAF) and transforming growth factor beta 1 (TGF-β1). TGF-β1 is known to affect osteoblast differentiation, matrix formation and mineralization. Modulation of its expression could influence the expression of main osteogenic transcription factors. For exposure with high LET radiation a pronounced cell cycle block was evident. The expression of the osteogenic marker genes OCN and Osterix (OSX) was increased in the OCT-1 cells with differentiation potential for exposure to α particles and accelerated carbon and argon ions. The results on the expression of differentiation markers during radiation-induced premature differentiation of bone cells of the osteoblast lineage show that densely ionizing radiation results in expression of proteins essential for bone formation and consequently in an increase in bone volume. Such an effect has been observed in in-vivo carbon ion irradiated rats. As radiation dependent permanent cell cycle blocks lead to a depletion of proliferation-competent cells from the osteoblastic precursor pool in the body, a gradual decrease of bone mass in weightlessness may be attributed to synergistic effects of radiation and weightlessness.
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Joint diseases: from connexins to gap junctions.
Donahue, Henry J; Qu, Roy W; Genetos, Damian C
2017-12-19
Connexons form the basis of hemichannels and gap junctions. They are composed of six tetraspan proteins called connexins. Connexons can function as individual hemichannels, releasing cytosolic factors (such as ATP) into the pericellular environment. Alternatively, two hemichannel connexons from neighbouring cells can come together to form gap junctions, membrane-spanning channels that facilitate cell-cell communication by enabling signalling molecules of approximately 1 kDa to pass from one cell to an adjacent cell. Connexins are expressed in joint tissues including bone, cartilage, skeletal muscle and the synovium. Indicative of their importance as gap junction components, connexins are also known as gap junction proteins, but individual connexin proteins are gaining recognition for their channel-independent roles, which include scaffolding and signalling functions. Considerable evidence indicates that connexons contribute to the function of bone and muscle, but less is known about the function of connexons in other joint tissues. However, the implication that connexins and gap junctional channels might be involved in joint disease, including age-related bone loss, osteoarthritis and rheumatoid arthritis, emphasizes the need for further research into these areas and highlights the therapeutic potential of connexins.
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The Osteogenic Niche Promotes Early-Stage Bone Colonization of Disseminated Breast Cancer Cells
Wang, Hai; Yu, Cuijuan; Gao, Xia; Welte, Thomas; Muscarella, Aaron M.; Tian, Lin; Zhao, Hong; Zhao, Zhen; Du, Shiyu; Tao, Jianning; Lee, Brendan; Westbrook, Thomas F.; Wong, Stephen T. C.; Jin, Xin; Rosen, Jeffrey M.; Osborne, C. Kent; Zhang, Xiang H.-F.
2014-01-01
Summary Breast cancer bone micrometastases can remain asymptomatic for years before progressing into overt lesions. The biology of this process, including the microenvironment niche and supporting pathways, is unclear. We find that bone micrometastases predominantly reside in a niche that exhibits features of osteogenesis. Niche interactions are mediated by heterotypic adherens junctions (hAJs) involving cancer-derived E-cadherin and osteogenic N-cadherin, the disruption of which abolishes niche-conferred advantages. We further elucidate that hAJ activates the mTOR pathway in cancer cells, which drives the progression from single cells to micrometastases. Human datasets analyses support the roles of AJ and the mTOR pathway in bone colonization. Our study illuminates the initiation of bone colonization, and provides potential therapeutic targets to block progression toward osteolytic metastases. Significance In advanced stages, breast cancer bone metastases are driven by paracrine crosstalk among cancer cells, osteoblasts, and osteoclasts, which constitute a vicious osteolytic cycle. Current therapies targeting this process limit tumor progression, but do not improve patient survival. On the other hand, bone micrometastases may remain indolent for years before activating the vicious cycle, providing a therapeutic opportunity to prevent macrometastases. Here, we show that bone colonization is initiated in a microenvironment niche exhibiting active osteogenesis. Cancer and osteogenic cells form heterotypic adherens junctions, which enhance mTOR activity and drive early-stage bone colonization prior to osteolysis. These results reveal a strong connection between osteogenesis and micrometastasis and suggest potential therapeutic targets to prevent bone macrometastases. PMID:25600338
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Dental pulp stem cells for in vivo bone regeneration: a systematic review of literature.
Morad, Golnaz; Kheiri, Lida; Khojasteh, Arash
2013-12-01
This review of literature was aimed to assess in vivo experiments which have evaluated the efficacy of dental pulp stem cells (DPSCs) for bone regeneration. An electronic search of English-language papers was conducted on PubMed database. Studies that assessed the use of DPSCs in bone regeneration in vivo were included and experiments evaluating regeneration of hard tissues other than bone were excluded. The retrieved articles were thoroughly reviewed according to the source of stem cell, cell carrier, the in vivo experimental model, defect type, method of evaluating bone regeneration, and the obtained results. Further assessment of the results was conducted by classifying the studies based on the defect type. Seventeen papers formed the basis of this systematic review. Sixteen out of 17 experiments were performed on animal models with mouse and rat being the most frequently used animal models. Seven out of 17 animal studies, contained subcutaneous pockets on back of the animal for stem cell implantation. In only one study hard tissue formation was not observed. Other types of defects used in the retrieved studies, included cranial defects and mandibular bone defects, in all of which bone formation was reported. When applied in actual bone defects, DPSCs were capable of regenerating bone. Nevertheless, a precise conclusion regarding the efficiency of DPSCs for bone regeneration is yet to be made, considering the limited number of the in vivo experiments and the heterogeneity within their methods. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Biofabricated Structures Reconstruct Functional Urinary Bladders in Radiation-injured Rat Bladders.
Imamura, Tetsuya; Shimamura, Mitsuru; Ogawa, Teruyuki; Minagawa, Tomonori; Nagai, Takashi; Silwal Gautam, Sudha; Ishizuka, Osamu
2018-05-08
The ability to repair damaged urinary bladders through the application of bone marrow-derived cells is in the earliest stages of development. We investigated the application of bone marrow-derived cells to repair radiation-injured bladders. We used a three-dimensional (3D) bioprinting robot system to biofabricate bone marrow-derived cell structures. We then determined if the biofabricated structures could restore the tissues and functions of radiation-injured bladders. The bladders of female 10-week-old Sprague-Dawley (SD) rats were irradiated with 2-Gy once a week for 5 weeks. Adherent and proliferating bone marrow-derived cells harvested from the femurs of male 17-week-old green fluorescence protein-transfected Tg-SD rats were cultured in collagen-coated flasks. Bone marrow-derived cell spheroids were formed in 96-well plates. Three layers of spheroids were assembled by the bioprinter onto a 9x9 microneedle array. The assembled spheroids were perfusion cultured for 7 days, and then the microneedle array was removed. Two weeks after the last radiation treatment, the biofabricated structures were transplanted into an incision on the anterior wall of the bladders (n=10). Control rats received the same surgery but without the biofabricated structures (sham-structure, n=12). At 2 and 4 weeks after surgery, the sham-structure control bladder tissues exhibited disorganized smooth muscle layers, decreased nerve cells, and significant fibrosis with increased presence of fibrosis-marker P4HB-positive cells and hypoxia-marker HIF1α-positive cells. The transplanted structures survived within the recipient tissues, and blood vessels extended within them from the recipient tissues. The bone marrow-derived cells in the structures differentiated into smooth muscle cells and formed smooth muscle clusters. The recipient tissues near the transplanted structures had distinct smooth muscle layers and reconstructed nerve cells, and only minimal fibrosis with decreased presence of P4HB- and HIF1α-positive cells. At 4 weeks after surgery, the sham-structure control rats exhibited significant urinary frequency symptoms with irregular and short voiding intervals, and low micturition volumes. In contrast, the structure-transplanted rats had regular micturition with longer voiding intervals and higher micturition volumes compared to the control rats. Further, the residual volume of the structure-transplanted rats was lower than for the controls. Therefore, transplantation of biofabricated bone marrow-derived cell structures reconstructed functional bladders.
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Jokanović, Vukoman; Čolović, Božana; Marković, Dejan; Petrović, Milan; Soldatović, Ivan; Antonijević, Djordje; Milosavljević, Petar; Sjerobabin, Nikola; Sopta, Jelena
2017-05-24
This study examined the potential of a new porous calcium hydroxyapatite scaffold covered with poly (lactide-co-glycolide) (PLGA) as a bone substitute, identifying its advantages over Geistlich Bio-Oss®, considered the gold standard, in in vivo biofunctionality investigations. Structural and morphological properties of the new scaffold were analyzed by scanning electron and atomic force microscopy. The biofunctionality assays were performed on New Zealand white rabbits using new scaffold for filling full-thickness defects of critical size. The evaluated parameters were: the presence of macrophages, giant cells, monoocytes, plasma cells, granulocytes, neoangiogenesis, fibroplasia, and the percentage of mineralization. Parallel biofunctionality assays were performed using Geistlich Bio-Oss®. The appearance of bone defects 12 weeks after the new scaffold implantation showed the presence of a small number of typical immune response cells. Furthermore, significantly reduced number of capillary buds, low intensity of fibroplasia and high degree of mineralization in a lamellar pattern indicated that the inflammation process has been almost completely overcome and that the new bone formed was in the final phase of remodeling. All biofunctionality assays proved the new scaffold's suitability as a bone substitute for applications in maxillofacial surgery. It showed numerous biological advantages over Geistlich Bio-Oss® which was reflected mainly as a lower number of giant cells surrounding implanted material and higher degree of mineralization in new formed bone.
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Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.
Ando, Kazunori; Shibata, Eri; Hans, Stefan; Brand, Michael; Kawakami, Atsushi
2017-12-04
Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.
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Westerweel, Peter E; Teraa, Martin; Rafii, Shahin; Jaspers, Janneke E; White, Ian A; Hooper, Andrea T; Doevendans, Pieter A; Verhaar, Marianne C
2013-01-01
Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired -at least partly- due to dysfunction of the bone marrow stromal compartment. Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1(+)Flk-1(+) EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34(+) hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell-endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients.
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Schumacher, M; Lode, A; Helth, A; Gelinsky, M
2013-12-01
In the present study, the in vitro effects of novel strontium-modified calcium phosphate bone cements (SrCPCs), prepared using two different approaches on human-bone-marrow-derived mesenchymal stem cells (hMSCs), were evaluated. Strontium ions, known to stimulate bone formation and therefore already used in systemic osteoporosis therapy, were incorporated into a hydroxyapatite-forming calcium phosphate bone cement via two simple approaches: incorporation of strontium carbonate crystals and substitution of Ca(2+) by Sr(2+) ions during cement setting. All modified cements released 0.03-0.07 mM Sr(2+) under in vitro conditions, concentrations that were shown not to impair the proliferation or osteogenic differentiation of hMSCs. Furthermore, strontium modification led to a reduced medium acidification and Ca(2+) depletion in comparison to the standard calcium phosphate cement. In indirect and direct cell culture experiments with the novel SrCPCs significantly enhanced cell proliferation and differentiation were observed. In conclusion, the SrCPCs described here could be beneficial for the local treatment of defects, especially in the osteoporotic bone. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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Nanoceramics on osteoblast proliferation and differentiation in bone tissue engineering.
Sethu, Sai Nievethitha; Namashivayam, Subhapradha; Devendran, Saravanan; Nagarajan, Selvamurugan; Tsai, Wei-Bor; Narashiman, Srinivasan; Ramachandran, Murugesan; Ambigapathi, Moorthi
2017-05-01
Bone, a highly dynamic connective tissue, consist of a bioorganic phase comprising osteogenic cells and proteins which lies over an inorganic phase predominantly made of CaPO 4 (biological apatite). Injury to bone can be due to mechanical, metabolic or inflammatory agents also owing pathological conditions like fractures, osteomyelitis, osteolysis or cysts may arise in enameloid, chondroid, cementum, or chondroid bone which forms the intermediate tissues of the body. Bone tissue engineering (BTE) applies bioactive scaffolds, host cells and osteogenic signals for restoring damaged or diseased tissues. Various bioceramics used in BTE can be bioactive (like glass ceramics and hydroxyapatite bioactive glass), bioresorbable (like tricalcium phosphates) or bioinert (like zirconia and alumina). Limiting the size of these materials to nano-scale has resulted in a higher surface area to volume ratio thereby improving multi-functionality, solubility, surface catalytic activity, high heat and electrical conductivity. Nanoceramics have been found to induce osteoconduction, osteointegration, osteogenesis and osteoinduction. The present review aims at summarizing the interactions of nanoceramics and osteoblast/stem cells for promoting the proliferation and differentiation of the osteoblast cells by nanoceramics as superior bone substitutes in bone tissue engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.
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Gleitz, Hélène Fe; Kramann, Rafael; Schneider, Rebekka K
2018-06-01
Bone marrow fibrosis is the continuous replacement of blood-forming cells in the bone marrow with excessive scar tissue, leading to failure of the body to produce blood cells and ultimately to death. Myofibroblasts are fibrosis-driving cells and are well characterized in solid organ fibrosis, but their role and cellular origin in bone marrow fibrosis have remained obscure. Recent work has demonstrated that Gli1 + and leptin receptor + mesenchymal stromal cells are progenitors of fibrosis-causing myofibroblasts in the bone marrow. Genetic ablation or pharmacological inhibition of Gli1 + mesenchymal stromal cells ameliorated fibrosis in mouse models of myelofibrosis. Conditional deletion of the platelet-derived growth factor (PDGF) receptor-α (PDGFRA) gene (Pdgfra) and inhibition of PDGFRA by imatinib in leptin receptor + stromal cells suppressed their expansion and ameliorated bone marrow fibrosis. Understanding the cellular and molecular mechanisms in the haematopoietic stem cell niche that govern the mesenchymal stromal cell-to-myofibroblast transition and myofibroblast expansion will be critical to understand the pathogenesis of bone marrow fibrosis in both malignant and non-malignant conditions, and will guide the development of novel therapeutics. In this review, we summarize recent discoveries of mesenchymal stromal cells as part of the haematopoietic niche and as myofibroblast precursors, and discuss potential therapeutic strategies in the specific targeting of fibrotic transformation in bone marrow fibrosis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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Lynch, Maureen E; Chiou, Aaron E; Lee, Min Joon; Marcott, Stephen C; Polamraju, Praveen V; Lee, Yeonkyung; Fischbach, Claudia
2016-08-01
Dynamic mechanical loading is a strong anabolic signal in the skeleton, increasing osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) and increasing the bone-forming activity of osteoblasts, but its role in bone metastatic cancer is relatively unknown. In this study, we integrated a hydroxyapatite-containing three-dimensional (3D) scaffold platform with controlled mechanical stimulation to investigate the effects of cyclic compression on the interplay between breast cancer cells and BM-MSCs as it pertains to bone metastasis. BM-MSCs cultured within mineral-containing 3D poly(lactide-co-glycolide) (PLG) scaffolds differentiated into mature osteoblasts, and exposure to tumor-derived soluble factors promoted this process. When BM-MSCs undergoing osteogenic differentiation were exposed to conditioned media collected from mechanically loaded breast cancer cells, their gene expression of osteopontin was increased. This was further enhanced when mechanical compression was simultaneously applied to BM-MSCs, leading to more uniformly deposited osteopontin within scaffold pores. These results suggest that mechanical loading of 3D scaffold-based culture models may be utilized to evaluate the role of physiologically relevant physical cues on bone metastatic breast cancer. Furthermore, our data imply that cyclic mechanical stimuli within the bone microenvironment modulate interactions between tumor cells and BM-MSCs that are relevant to bone metastasis.
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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
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Deng, Yuan; Jiang, Chuan; Li, Cuidi; Li, Tao; Peng, Mingzheng; Wang, Jinwu; Dai, Kerong
2017-07-17
Synthetic bone scaffolds have potential application in repairing large bone defects, however, inefficient vascularization after implantation remains the major issue of graft failure. Herein, porous β-tricalcium phosphate (β-TCP) scaffolds with calcium silicate (CS) were 3D printed, and pre-seeded with co-cultured human umbilical cord vein endothelial cells (HUVECs) and human bone marrow stromal cells (hBMSCs) to construct tissue engineering scaffolds with accelerated vascularization and better bone formation. Results showed that in vitro β-TCP scaffolds doped with 5% CS (5%CS/β-TCP) were biocompatible, and stimulated angiogenesis and osteogenesis. The results also showed that 5%CS/β-TCP scaffolds not only stimulated co-cultured cells angiogenesis on Matrigel, but also stimulated co-cultured cells to form microcapillary-like structures on scaffolds, and promoted migration of BMSCs by stimulating co-cultured cells to secrete PDGF-BB and CXCL12 into the surrounding environment. Moreover, 5%CS/β-TCP scaffolds enhanced vascularization and osteoinduction in comparison with β-TCP, and synergized with co-cultured cells to further increase early vessel formation, which was accompanied by earlier and better ectopic bone formation when implanted subcutaneously in nude mice. Thus, our findings suggest that porous 5%CS/β-TCP scaffolds seeded with co-cultured cells provide new strategy for accelerating tissue engineering scaffolds vascularization and osteogenesis, and show potential as treatment for large bone defects.
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Ozasa, Ryosuke; Matsugaki, Aira; Isobe, Yoshihiro; Saku, Taro; Yun, Hui-Suk; Nakano, Takayoshi
2018-02-01
Bone tissue has anisotropic microstructure based on collagen/biological apatite orientation, which plays essential roles in the mechanical and biological functions of bone. However, obtaining an appropriate anisotropic microstructure during the bone regeneration process remains a great challenging. A powerful strategy for the control of both differentiation and structural development of newly-formed bone is required in bone tissue engineering, in order to realize functional bone tissue regeneration. In this study, we developed a novel anisotropic culture model by combining human induced pluripotent stem cells (hiPSCs) and artificially-controlled oriented collagen scaffold. The oriented collagen scaffold allowed hiPSCs-derived osteoblast alignment and further construction of anisotropic bone matrix which mimics the bone tissue microstructure. To the best of our knowledge, this is the first report showing the construction of bone mimetic anisotropic bone matrix microstructure from hiPSCs. Moreover, we demonstrated for the first time that the hiPSCs-derived osteoblasts possess a high level of intact functionality to regulate cell alignment. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 360-369, 2018. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc.
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Bone Marrow Aspirate Concentrate-Enhanced Marrow Stimulation of Chondral Defects
Eichler, Hermann; Orth, Patrick
2017-01-01
Mesenchymal stem cells (MSCs) from bone marrow play a critical role in osteochondral repair. A bone marrow clot forms within the cartilage defect either as a result of marrow stimulation or during the course of the spontaneous repair of osteochondral defects. Mobilized pluripotent MSCs from the subchondral bone migrate into the defect filled with the clot, differentiate into chondrocytes and osteoblasts, and form a repair tissue over time. The additional application of a bone marrow aspirate (BMA) to the procedure of marrow stimulation is thought to enhance cartilage repair as it may provide both an additional cell population capable of chondrogenesis and a source of growth factors stimulating cartilage repair. Moreover, the BMA clot provides a three-dimensional environment, possibly further supporting chondrogenesis and protecting the subchondral bone from structural alterations. The purpose of this review is to bridge the gap in our understanding between the basic science knowledge on MSCs and BMA and the clinical and technical aspects of marrow stimulation-based cartilage repair by examining available data on the role and mechanisms of MSCs and BMA in osteochondral repair. Implications of findings from both translational and clinical studies using BMA concentrate-enhanced marrow stimulation are discussed. PMID:28607559
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3D Printing of Lotus Root-Like Biomimetic Materials for Cell Delivery and Tissue Regeneration.
Feng, Chun; Zhang, Wenjie; Deng, Cuijun; Li, Guanglong; Chang, Jiang; Zhang, Zhiyuan; Jiang, Xinquan; Wu, Chengtie
2017-12-01
Biomimetic materials have drawn more and more attention in recent years. Regeneration of large bone defects is still a major clinical challenge. In addition, vascularization plays an important role in the process of large bone regeneration and microchannel structure can induce endothelial cells to form rudimentary vasculature. In recent years, 3D printing scaffolds are major materials for large bone defect repair. However, these traditional 3D scaffolds have low porosity and nonchannel structure, which impede angiogenesis and osteogenesis. In this study, inspired by the microstructure of natural plant lotus root, biomimetic materials with lotus root-like structures are successfully prepared via a modified 3D printing strategy. Compared with traditional 3D materials, these biomimetic materials can significantly improve in vitro cell attachment and proliferation as well as promote in vivo osteogenesis, indicating potential application for cell delivery and bone regeneration.
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3D Printing of Lotus Root‐Like Biomimetic Materials for Cell Delivery and Tissue Regeneration
Feng, Chun; Zhang, Wenjie; Deng, Cuijun; Li, Guanglong; Chang, Jiang; Zhang, Zhiyuan
2017-01-01
Abstract Biomimetic materials have drawn more and more attention in recent years. Regeneration of large bone defects is still a major clinical challenge. In addition, vascularization plays an important role in the process of large bone regeneration and microchannel structure can induce endothelial cells to form rudimentary vasculature. In recent years, 3D printing scaffolds are major materials for large bone defect repair. However, these traditional 3D scaffolds have low porosity and nonchannel structure, which impede angiogenesis and osteogenesis. In this study, inspired by the microstructure of natural plant lotus root, biomimetic materials with lotus root‐like structures are successfully prepared via a modified 3D printing strategy. Compared with traditional 3D materials, these biomimetic materials can significantly improve in vitro cell attachment and proliferation as well as promote in vivo osteogenesis, indicating potential application for cell delivery and bone regeneration. PMID:29270348
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Activation of NF-kappa B Signaling Promotes Growth of Prostate Cancer Cells in Bone
Jin, Renjie; Sterling, Julie A.; Edwards, James R.; DeGraff, David J.; Lee, Changki; Park, Serk In; Matusik, Robert J.
2013-01-01
Patients with advanced prostate cancer almost invariably develop osseous metastasis. Although many studies indicate that the activation of NF-κB signaling appears to be correlated with advanced cancer and promotes tumor metastasis by influencing tumor cell migration and angiogenesis, the influence of altered NF-κB signaling in prostate cancer cells within boney metastatic lesions is not clearly understood. While C4-2B and PC3 prostate cancer cells grow well in the bone, LNCaP cells are difficult to grow in murine bone following intraskeletal injection. Our studies show that when compared to LNCaP, NF-κB activity is significantly higher in C4-2B and PC3, and that the activation of NF-κB signaling in prostate cancer cells resulted in the increased expression of the osteoclast inducing genes PTHrP and RANKL. Further, conditioned medium derived from NF-κB activated LNCaP cells induce osteoclast differentiation. In addition, inactivation of NF-κB signaling in prostate cancer cells inhibited tumor formation in the bone, both in the osteolytic PC3 and osteoblastic/osteoclastic mixed C4-2B cells; while the activation of NF-κB signaling in LNCaP cells promoted tumor establishment and proliferation in the bone. The activation of NF-κB in LNCaP cells resulted in the formation of an osteoblastic/osteoclastic mixed tumor with increased osteoclasts surrounding the new formed bone, similar to metastases commonly seen in patients with prostate cancer. These results indicate that osteoclastic reaction is required even in the osteoblastic cancer cells and the activation of NF-κB signaling in prostate cancer cells increases osteoclastogenesis by up-regulating osteoclastogenic genes, thereby contributing to bone metastatic formation. PMID:23577181
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Cardoso, A A; Li, M L; Batard, P; Hatzfeld, A; Brown, E L; Levesque, J P; Sookdeo, H; Panterne, B; Sansilvestri, P; Clark, S C
1993-01-01
Using optimal culture conditions in which the transforming growth factor beta 1 (TGF-beta 1) inhibitory loop has been interrupted by antisense TGF-beta 1 oligonucleotides or anti-TGF-beta serum, we have compared the proliferative capacities and the abilities of the CD34+ CD38- cell populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38- fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of colony-forming units (CFU)-granulocyte/erythrocyte/macrophage/megakaryocyte, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult transplantation. In addition, the colonies resulting from the umbilical cord blood samples were significantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparations, as estimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is partially compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbilical cord blood is a suitable source of cells for adult transplantation. PMID:7690969
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Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation
Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo
2015-01-01
Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696
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Mukhopadhyay, Keya De; Bandyopadhyay, Abhik; Chang, Ting-Tung A; Elkahloun, Abdel G; Cornell, John E; Yang, Junhua; Goins, Beth A; Yeh, I-Tien; Sun, Lu-Zhe
2011-01-01
The origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other's metastatic behavior. ER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC. The presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-β1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines. Spontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity.
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NASA Astrophysics Data System (ADS)
Huang, Zhi; Chen, Yan; Feng, Qing-Ling; Zhao, Wei; Yu, Bo; Tian, Jing; Li, Song-Jian; Lin, Bo-Miao
2011-09-01
For reconstruction of irregular bone defects, injectable biomaterials are more appropriate than the preformed biomaterials. We herein develop a biomimetic in situ-forming composite consisting of chitosan (CS) and mineralized collagen fibrils (nHAC), which has a complex hierarchical structure similar to natural bone. The CS/nHAC composites with or without mesenchymal stem cells (MSCs) are injected into cancellous bone defects at the distal end of rabbit femurs. Defects are assessed by radiographic, histological diagnosis and Raman microscopy until 12 weeks. The results show that MSCs improve the biocompatibility of CS/nHAC composites and enhance new bone formation in vivo at 12 weeks. It can be concluded that the injectable CS/nHAC composites combined with MSCs may be a novel method for reconstruction of irregular bone defects.
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NASA Technical Reports Server (NTRS)
Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)
1998-01-01
The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.
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Large-scale progenitor cell expansion for multiple donors in a monitored hollow fibre bioreactor.
Lambrechts, Toon; Papantoniou, Ioannis; Rice, Brent; Schrooten, Jan; Luyten, Frank P; Aerts, Jean-Marie
2016-09-01
With the increasing scale in stem cell production, a robust and controlled cell expansion process becomes essential for the clinical application of cell-based therapies. The objective of this work was the assessment of a hollow fiber bioreactor (Quantum Cell Expansion System from Terumo BCT) as a cell production unit for the clinical-scale production of human periosteum derived stem cells (hPDCs). We aimed to demonstrate comparability of bioreactor production to standard culture flask production based on a product characterization in line with the International Society of Cell Therapy in vitro benchmarks and supplemented with a compelling quantitative in vivo bone-forming potency assay. Multiple process read-outs were implemented to track process performance and deal with donor-to-donor-related variation in nutrient needs and harvest timing. The data show that the hollow fiber bioreactor is capable of robustly expanding autologous hPDCs on a clinical scale (yield between 316 million and 444 million cells starting from 20 million after ± 8 days of culture) while maintaining their in vitro quality attributes compared with the standard flask-based culture. The in vivo bone-forming assay on average resulted in 10.3 ± 3.7% and 11.0 ± 3.8% newly formed bone for the bioreactor and standard culture flask respectively. The analysis showed that the Quantum system provides a reproducible cell expansion process in terms of yields and culture conditions for multiple donors. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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Identification of full-length dentin matrix protein 1 in dentin and bone.
Huang, Bingzhen; Maciejewska, Izabela; Sun, Yao; Peng, Tao; Qin, Disheng; Lu, Yongbo; Bonewald, Lynda; Butler, William T; Feng, Jian; Qin, Chunlin
2008-05-01
Dentin matrix protein 1 (DMP1) has been identified in the extracellular matrix (ECM) of dentin and bone as the processed NH(2)-terminal and COOH-terminal fragment. However, the full-length form of DMP1 has not been identified in these tissues. The focus of this investigation was to search for the intact full-length DMP1 in dentin and bone. We used two types of anti-DMP1 antibodies to identify DMP1: one type specifically recognizes the NH(2)-terminal region and the other type is only reactive to the COOH-terminal region of the DMP1 amino acid sequence. An approximately 105-kDa protein, extracted from the ECM of rat dentin and bone, was recognized by both types of antibodies; and the migration rate of this protein was identical to the recombinant mouse full-length DMP1 made in eukaryotic cells. We concluded that this approximately 105-kDa protein is the full-length form of DMP1, which is considerably less abundant than its processed fragments in the ECM of dentin and bone. We also detected the full-length form of DMP1 and its processed fragments in the extract of dental pulp/odontoblast complex dissected from rat teeth. In addition, immunofluorescence analysis showed that in MC3T3-E1 cells the NH(2)-terminal and COOH-terminal fragments of DMP1 are distributed differently. Our findings indicate that the majority of DMP1 must be cleaved within the cells that synthesize it and that minor amounts of uncleaved DMP1 molecules are secreted into the ECM of dentin and bone.
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Panaroni, Cristina; Gioia, Roberta; Lupi, Anna; Besio, Roberta; Goldstein, Steven A.; Kreider, Jaclynn; Leikin, Sergey; Vera, Juan Carlos; Mertz, Edward L.; Perilli, Egon; Baruffaldi, Fabio; Villa, Isabella; Farina, Aurora; Casasco, Marco; Cetta, Giuseppe; Rossi, Antonio; Frattini, Annalisa; Marini, Joan C.; Vezzoni, Paolo
2009-01-01
Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [α1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases. PMID:19414862
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Cementoblast Delivery for Periodontal Tissue Engineering
Zhao, Ming; Jin, Qiming; Berry, Janice E.; Nociti, Francisco H.; Giannobile, William V.; Somerman, Martha J.
2008-01-01
Background Predictable periodontal regeneration following periodontal disease is a major goal of therapy. The objective of this proof of concept investigation was to evaluate the ability of cementoblasts and dental follicle cells to promote periodontal regeneration in a rodent periodontal fenestration model. Methods The buccal aspect of the distal root of the first mandibular molar was denuded of its periodontal ligament (PDL), cementum, and superficial dentin through a bony window created bilaterally in 12 athymic rats. Treated defects were divided into three groups: 1) carrier alone (PLGA polymer sponges), 2) carrier + follicle cells, and 3) carrier + cementoblasts. Cultured murine primary follicle cells and immortalized cementoblasts were delivered to the defects via biodegradable PLGA polymer sponges, and mandibulae were retrieved 3 weeks and 6 weeks post-surgery for histological evaluation. In situ hybridization, for gene expression of bone sialoprotein (BSP) and osteocalcin (OCN), and histomorphometric analysis were further done on 3-week specimens. Results Three weeks after surgery, histology of defects treated with carrier alone indicated PLGA particles, fibrous tissue, and newly formed bone scattered within the defect area. Defects treated with carrier + follicle cells had a similar appearance, but with less formation of bone. In contrast, in defects treated with carrier + cementoblasts, mineralized tissues were noted at the healing site with extension toward the root surface, PDL region, and laterally beyond the buccal plate envelope of bone. No PDL-bone fibrous attachment was observed in any of the groups at this point. In situ hybridization showed that the mineralized tissue formed by cementoblasts gave strong signals for both BSP and OCN genes, confirming its nature as cementum or bone. The changes noted at 3 weeks were also observed at 6 weeks. Cementoblast-treated and carrier alone-treated defects exhibited complete bone bridging and PDL formation, whereas follicle cell-treated defects showed minimal evidence of osteogenesis. No new cementum was formed along the root surface in the above two groups. Cementoblast-treated defects were filled with trabeculated mineralized tissue similar to, but more mature, than that seen at 3 weeks. Furthermore, the PDL region was maintained with well-organized collagen fibers connecting the adjacent bone to a thin layer of cementum-like tissue observed on the root surface. Neoplastic changes were observed at the superficial portions of the implants in two of the 6-week cementoblast-treated specimens, possibly due in part to the SV40-transformed nature of the implanted cell line. Conclusions This pilot study demonstrates that cementoblasts have a marked ability to induce mineralization in periodontal wounds when delivered via polymer sponges, while implanted dental follicle cells seem to inhibit periodontal healing. These results confirm the selective behaviors of different cell types in vivo and support the role of cementoblasts as a tool to better understand periodontal regeneration and cementogenesis. PMID:15025227
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Monocytes Induce STAT3 Activation in Human Mesenchymal Stem Cells to Promote Osteoblast Formation
Nicolaidou, Vicky; Wong, Mei Mei; Redpath, Andia N.; Ersek, Adel; Baban, Dilair F.; Williams, Lynn M.; Cope, Andrew P.; Horwood, Nicole J.
2012-01-01
A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair. PMID:22802946
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Thiolloy, Sophie; Edwards, James R; Fingleton, Barbara; Rifkin, Daniel B; Matrisian, Lynn M; Lynch, Conor C
2012-01-01
Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases.
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The impact of simulated and real microgravity on bone cells and mesenchymal stem cells.
Ulbrich, Claudia; Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Magnusson, Nils; Infanger, Manfred; Grosse, Jirka; Eilles, Christoph; Sundaresan, Alamelu; Grimm, Daniela
2014-01-01
How microgravity affects the biology of human cells and the formation of 3D cell cultures in real and simulated microgravity (r- and s-µg) is currently a hot topic in biomedicine. In r- and s-µg, various cell types were found to form 3D structures. This review will focus on the current knowledge of tissue engineering in space and on Earth using systems such as the random positioning machine (RPM), the 2D-clinostat, or the NASA-developed rotating wall vessel bioreactor (RWV) to create tissue from bone, tumor, and mesenchymal stem cells. To understand the development of 3D structures, in vitro experiments using s-µg devices can provide valuable information about modulations in signal-transduction, cell adhesion, or extracellular matrix induced by altered gravity conditions. These systems also facilitate the analysis of the impact of growth factors, hormones, or drugs on these tissue-like constructs. Progress has been made in bone tissue engineering using the RWV, and multicellular tumor spheroids (MCTS), formed in both r- and s-µg, have been reported and were analyzed in depth. Currently, these MCTS are available for drug testing and proteomic investigations. This review provides an overview of the influence of µg on the aforementioned cells and an outlook for future perspectives in tissue engineering.
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Sununliganon, Laddawun; Singhatanadgit, Weerachai
2012-01-01
Cells derived from the periodontal ligament (PDL) have previously been reported to have stem cell-like characteristics (PDL stem cells; PDLSCs) and play an important part in bone engineering, including that of alveolar bone. However, these populations have been heterogeneous, and thus far no specific marker has yet been established from adult human stem cells derived from PDL tissue. We have previously isolated highly purified single cell-derived PDLSC clones and delineated their phenotypic and functional characteristics. In this report, we further obtained three homogeneous and distinct PDLSC clones demonstrating low, moderate and high mineralized matrix forming ability-namely PC12, PC4 and PC3, respectively, and the expression of mesenchymal stem cell pathway-specific genes in these clones was investigated. PCR array revealed that the expression of intercellular adhesion molecule 1 (ICAM1), integrin beta 1 (ITGB1) and telomerase reverse transcriptase (TERT) was associated with highly osteogenic PDLSC clones, as determined by the expression of key osteoblastic markers and their ability to form alizarin red S positive mineralized matrix in vitro. The present results suggest that these three mesenchymal stem cell-associated markers could potentially be used to isolate PDLSCs with high osteogenic capability for engineering new bone.
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The Impact of Simulated and Real Microgravity on Bone Cells and Mesenchymal Stem Cells
Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Magnusson, Nils; Infanger, Manfred; Grosse, Jirka; Eilles, Christoph
2014-01-01
How microgravity affects the biology of human cells and the formation of 3D cell cultures in real and simulated microgravity (r- and s-µg) is currently a hot topic in biomedicine. In r- and s-µg, various cell types were found to form 3D structures. This review will focus on the current knowledge of tissue engineering in space and on Earth using systems such as the random positioning machine (RPM), the 2D-clinostat, or the NASA-developed rotating wall vessel bioreactor (RWV) to create tissue from bone, tumor, and mesenchymal stem cells. To understand the development of 3D structures, in vitro experiments using s-µg devices can provide valuable information about modulations in signal-transduction, cell adhesion, or extracellular matrix induced by altered gravity conditions. These systems also facilitate the analysis of the impact of growth factors, hormones, or drugs on these tissue-like constructs. Progress has been made in bone tissue engineering using the RWV, and multicellular tumor spheroids (MCTS), formed in both r- and s-µg, have been reported and were analyzed in depth. Currently, these MCTS are available for drug testing and proteomic investigations. This review provides an overview of the influence of µg on the aforementioned cells and an outlook for future perspectives in tissue engineering. PMID:25110709
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Rafii, Shahin; Jaspers, Janneke E.; White, Ian A.; Hooper, Andrea T.; Doevendans, Pieter A.; Verhaar, Marianne C.
2013-01-01
Background Circulating Endothelial Progenitor Cell (EPC) levels are reduced in diabetes mellitus. This may be a consequence of impaired mobilization of EPC from the bone marrow. We hypothesized that under diabetic conditions, mobilization of EPC from the bone marrow to the circulation is impaired –at least partly– due to dysfunction of the bone marrow stromal compartment. Methods Diabetes was induced in mice by streptozotocin injection. Circulating Sca-1+Flk-1+ EPC were characterized and quantified by flow cytometry at baseline and after mobilization with G-CSF/SCF injections. In vivo hemangiogenic recovery was tested by 5-FU challenge. Interaction within the bone marrow environment between CD34+ hematopoietic progenitor cells (HPC) and supporting stroma was assessed by co-cultures. To study progenitor cell–endothelial cell interaction under normoglycemic and hyperglycemic conditions, a co-culture model using E4Orf1-transfected human endothelial cells was employed. Results In diabetic mice, bone marrow EPC levels were unaffected. However, circulating EPC levels in blood were lower at baseline and mobilization was attenuated. Diabetic mice failed to recover and repopulate from 5-FU injection. In vitro, primary cultured bone marrow stroma from diabetic mice was impaired in its capacity to support human CFU-forming HPC. Finally, hyperglycemia hampered the HPC supportive function of endothelial cells in vitro. Conclusion EPC mobilization is impaired under experimental diabetic conditions and our data suggest that diabetes induces alterations in the progenitor cell supportive capacity of the bone marrow stroma, which could be partially responsible for the attenuated EPC mobilization and reduced EPC levels observed in diabetic patients. PMID:23555959
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Mesenchymal Stem Cells: Time to Change the Name!
2017-01-01
Summary Mesenchymal stem cells (MSCs) were officially named more than 25 years ago to represent a class of cells from human and mammalian bone marrow and periosteum that could be isolated and expanded in culture while maintaining their in vitro capacity to be induced to form a variety of mesodermal phenotypes and tissues. The in vitro capacity to form bone, cartilage, fat, etc., became an assay for identifying this class of multipotent cells and around which several companies were formed in the 1990s to medically exploit the regenerative capabilities of MSCs. Today, there are hundreds of clinics and hundreds of clinical trials using human MSCs with very few, if any, focusing on the in vitro multipotential capacities of these cells. Unfortunately, the fact that MSCs are called “stem cells” is being used to infer that patients will receive direct medical benefit, because they imagine that these cells will differentiate into regenerating tissue‐producing cells. Such a stem cell treatment will presumably cure the patient of their medically relevant difficulties ranging from osteoarthritic (bone‐on‐bone) knees to various neurological maladies including dementia. I now urge that we change the name of MSCs to Medicinal Signaling Cells to more accurately reflect the fact that these cells home in on sites of injury or disease and secrete bioactive factors that are immunomodulatory and trophic (regenerative) meaning that these cells make therapeutic drugs in situ that are medicinal. It is, indeed, the patient's own site‐specific and tissue‐specific resident stem cells that construct the new tissue as stimulated by the bioactive factors secreted by the exogenously supplied MSCs. Stem Cells Translational Medicine 2017;6:1445–1451 PMID:28452204
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Dong, Liang; Wang, Shao-Jie; Zhao, Xin-Rong; Zhu, Yu-Fang; Yu, Jia-Kuo
2017-10-17
Synthetic polymeric scaffolds are commonly used in bone tissue engineering (BTE) due to their biocompatibility and adequate mechanical properties. However, their hydrophobicity and the lack of specific cell recognition sites confined their practical application. In this study, to improve the cell seeding efficiency and osteoinductivity, an injectable thermo-sensitive chitosan hydrogel (CSG) was incorporated into a 3D-printed poly(ε-caprolactone) (PCL) scaffold to form a hybrid scaffold. To demonstrate the feasibility of this hybrid system for BTE application, rabbit bone marrow mesenchymal stem cells (BMMSCs) and bone morphogenetic protein-2 (BMP-2) were encapsulated in CSG. Pure PCL scaffolds were used as controls. Cell proliferation and viability were investigated. Osteogenic gene expressions of BMMSCs in various scaffolds were determined with reverse transcription polymerase chain reaction (RT-PCR). Growth factor releasing profile and mechanical tests were performed. CCK-8 assay confirmed greater cell retention and proliferation in chitosan and hybrid groups. Confocal microscopy showed even distribution of cells in the hybrid system. After 2-week osteogenic culture in vitro, BMMSCs in hybrid and chitosan scaffolds showed stronger osteogenesis and bone-matrix formation. To conclude, chitosan/PCL hybrid scaffolds are a favorable platform for BTE due to its capacity to carry cells and drugs, and excellent mechanical strength.
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Schopper, C; Moser, D; Wanschitz, F; Watzinger, F; Lagogiannis, G; Spassova, E; Ewers, R
1999-01-01
Sinus grafting, a popular and standard treatment for maxillary atrophy, uses a variety of grafting materials. In this study, specimens obtained 6 months after sinus grafting with Algipore were evaluated under light microscopy and showed osseoformation, xenograft degradation, and bone ingrowth into particles. Osteoblastic cells were embedded in the intracorpuscular bone matrix, which indicated that xenograft particles are an osseoconductive scaffold and stimulate matrix deposition. Acute inflammatory responses after insertion of Algipore did not occur. Particles were degraded during physiologic bone remodeling, and newly formed bone gradually replaced resorbed biomaterial.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yasumizu, R.; Hiai, H.; Sugiura, K.
1988-09-15
The transplantation of bone marrow cells from BALB/c (but not C57BL/6 and C3H/HeN) mice was observed to lead to the development of thymic lymphomas (leukemias) in AKR/J mice. Two leukemic cell lines, CAK1.3 and CAK4.4, were established from the primary culture of two thymic lymphoma, and surface phenotypes of these cell lines found to be H-2d and Thy-1.2+, indicating that these lymphoma cells are derived from BALB/c donor bone marrow cells. Further analyses of surface markers revealed that CAK1.3 is L3T4+ Lyt2+ IL2R-, whereas CAK4.4 is L3T4- Lyt2- IL2R+. Both CAK1.3 and CAK4.4 were transplantable into BALB/c but not AKR/Jmore » mice, further indicating that these cells are of BALB/c bone marrow donor origin. The cells were found to produce XC+-ecotropic viruses, but xenotropic and mink cell focus-forming viruses were undetectable. Inasmuch as thymic lymphomas are derived from bone marrow cells of leukemia-resistant BALB/c strain of mice under the allogeneic environment of leukemia-prone AKR/J mice, this animal model may serve as a useful tool not only for the analysis of leukemic relapse after bone marrow transplantation but also for elucidation of the mechanism of leukemogenesis.« less
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PATHOLOGICAL SPROUTING OF ADULT NOCICEPTORS IN CHRONIC PROSTATE CANCER-INDUCED BONE PAIN
Jimenez-Andrade, Juan M.; Bloom, Aaron P.; Stake, James I.; Mantyh, William G.; Taylor, Reid N.; Freeman, Katie T.; Ghilardi, Joseph R.; Kuskowski, Michael A.; Mantyh, Patrick W.
2012-01-01
Pain frequently accompanies cancer. What remains unclear is why this pain frequently becomes more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression, sensory nerve fibers that innervate the tumor-bearing tissue undergo a pathological sprouting and reorganization, which in other non-malignant pathologies has been shown to generate and maintain chronic pain. Injection of canine prostate cancer cells into mouse bone induces a remarkable sprouting of calcitonin gene related peptide (CGRP+) and neurofilament 200 kDa (NF200+) sensory nerve fibers. Nearly all sensory nerve fibers that undergo sprouting also co-express tropomyosin receptor kinase A (TrkA+). This ectopic sprouting occurs in sensory nerve fibers that are in close proximity to colonies of prostate cancer cells, tumor-associated stromal cells and newly formed woven bone, which together form sclerotic lesions that closely mirror the osteoblastic bone lesions induced by metastatic prostate tumors in humans. Preventive treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. Interestingly, RT-PCR analysis indicated that the prostate cancer cells themselves do not express detectable levels of mRNA coding for NGF. This suggests that the tumor-associated stromal cells express and release NGF, which drives the pathological reorganization of nearby TrkA+ sensory nerve fibers. Therapies that prevent this reorganization of sensory nerve fibers may provide insight into the evolving mechanisms that drive cancer pain and lead to more effective control of this chronic pain state. PMID:21048122
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Current progress in bioactive ceramic scaffolds for bone repair and regeneration.
Gao, Chengde; Deng, Youwen; Feng, Pei; Mao, Zhongzheng; Li, Pengjian; Yang, Bo; Deng, Junjie; Cao, Yiyuan; Shuai, Cijun; Peng, Shuping
2014-03-18
Bioactive ceramics have received great attention in the past decades owing to their success in stimulating cell proliferation, differentiation and bone tissue regeneration. They can react and form chemical bonds with cells and tissues in human body. This paper provides a comprehensive review of the application of bioactive ceramics for bone repair and regeneration. The review systematically summarizes the types and characters of bioactive ceramics, the fabrication methods for nanostructure and hierarchically porous structure, typical toughness methods for ceramic scaffold and corresponding mechanisms such as fiber toughness, whisker toughness and particle toughness. Moreover, greater insights into the mechanisms of interaction between ceramics and cells are provided, as well as the development of ceramic-based composite materials. The development and challenges of bioactive ceramics are also discussed from the perspective of bone repair and regeneration.
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Current Progress in Bioactive Ceramic Scaffolds for Bone Repair and Regeneration
Gao, Chengde; Deng, Youwen; Feng, Pei; Mao, Zhongzheng; Li, Pengjian; Yang, Bo; Deng, Junjie; Cao, Yiyuan; Shuai, Cijun; Peng, Shuping
2014-01-01
Bioactive ceramics have received great attention in the past decades owing to their success in stimulating cell proliferation, differentiation and bone tissue regeneration. They can react and form chemical bonds with cells and tissues in human body. This paper provides a comprehensive review of the application of bioactive ceramics for bone repair and regeneration. The review systematically summarizes the types and characters of bioactive ceramics, the fabrication methods for nanostructure and hierarchically porous structure, typical toughness methods for ceramic scaffold and corresponding mechanisms such as fiber toughness, whisker toughness and particle toughness. Moreover, greater insights into the mechanisms of interaction between ceramics and cells are provided, as well as the development of ceramic-based composite materials. The development and challenges of bioactive ceramics are also discussed from the perspective of bone repair and regeneration. PMID:24646912
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Huang, Lu; Tian, Mengkun; Wu, Dong; ...
2017-11-24
In this paper, a new materials platform, utilizing the amorphous microstructure of bulk metallic glasses (BMGs) and the versatility of ion implantation, was developed for the fundamental investigation of cell responses to substrate-rigidity variations in the gigapascal modulus range, which was previously unattainable with polymeric materials. The surface rigidity of a Zr-Al- Ni-Cu-Y BMG was modulated with low-energy Ar-ion implantation owing to the impartment of Ar nanobubbles into the amorphous matrix. Surface softening was achieved due to the formation of nanobubble-doped transitional zones in the Zrbased BMG substrate. Bone-forming cell studies on this newly designed platform demonstrated that mechanical cues,more » accompanied with the potential effects of other surface properties (i.e. roughness, morphology, and chemistry), contributed to modulating cell behaviors. Cell adhesion and actin filaments were found to be less established on less stiff surfaces, especially on the surface with an elastic modulus of 51 GPa. Cell growth appeared to be affected by surface mechanical properties. A lower stiffness was generally related to a higher growth rate. Findings in this study broadened our fundamental understanding concerning the mechanosensing of bone cells on stiff substrates. It also suggests that surface mechano-engineering of metallic materials could be a potential strategy to promote osseointegration of such materials for bone-implant applications. Further investigations are proposed to fine tune the ion implantation variables in order to further distinguish the surface-mechanical effect on bone-forming cell activities from the contributions of other surface properties.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Huang, Lu; Tian, Mengkun; Wu, Dong
In this paper, a new materials platform, utilizing the amorphous microstructure of bulk metallic glasses (BMGs) and the versatility of ion implantation, was developed for the fundamental investigation of cell responses to substrate-rigidity variations in the gigapascal modulus range, which was previously unattainable with polymeric materials. The surface rigidity of a Zr-Al- Ni-Cu-Y BMG was modulated with low-energy Ar-ion implantation owing to the impartment of Ar nanobubbles into the amorphous matrix. Surface softening was achieved due to the formation of nanobubble-doped transitional zones in the Zrbased BMG substrate. Bone-forming cell studies on this newly designed platform demonstrated that mechanical cues,more » accompanied with the potential effects of other surface properties (i.e. roughness, morphology, and chemistry), contributed to modulating cell behaviors. Cell adhesion and actin filaments were found to be less established on less stiff surfaces, especially on the surface with an elastic modulus of 51 GPa. Cell growth appeared to be affected by surface mechanical properties. A lower stiffness was generally related to a higher growth rate. Findings in this study broadened our fundamental understanding concerning the mechanosensing of bone cells on stiff substrates. It also suggests that surface mechano-engineering of metallic materials could be a potential strategy to promote osseointegration of such materials for bone-implant applications. Further investigations are proposed to fine tune the ion implantation variables in order to further distinguish the surface-mechanical effect on bone-forming cell activities from the contributions of other surface properties.« less
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Nair, Manitha B; Varma, H K; Menon, K V; Shenoy, Sachin J; John, Annie
2009-06-01
Segmental bone defects resulting from trauma or pathology represent a common and significant clinical problem. In this study, a triphasic ceramic (calcium silicate, hydroxyapatite and tricalcium phosphate)-coated hydroxyapatite (HASi) having the benefits of both HA (osteointegration, osteoconduction) and silica (degradation) was used as a bone substitute for the repair of segmental defect (2 cm) created in a goat femur model. Three experimental goat femur implant groups--(a) bare HASi, (b) osteogenic-induced goat bone marrow-derived mesenchymal stem cells cultured HASi (HASi+C) and (c) osteogenic-induced goat bone marrow-derived mesenchymal stem cells cultured HASi+platelet-rich plasma (HASi+CP)--were designed and efficacy performance in the healing of the defect was evaluated. In all the groups, the material united with host bone without any inflammation and an osseous callus formed around the implant. This reflects the osteoconductivity of HASi where the cells have migrated from the cut ends of host bone. The most observable difference between the groups appeared in the mid region of the defect. In bare HASi groups, numerous osteoblast-like cells could be seen together with a portion of material. However, in HASi+C and HASi+CP, about 60-70% of that area was occupied by woven bone, in line with material degradation. The interconnected porous nature (50-500 microm), together with the chemical composition of the HASi, facilitated the degradation of HASi, thereby opening up void spaces for cellular ingrowth and bone regeneration. The combination of HASi with cells and PRP was an added advantage that could promote the expression of many osteoinductive proteins, leading to faster bone regeneration and material degradation. Based on these results, we conclude that bare HASi can aid in bone regeneration but, with the combination of cells and PRP, the sequence of healing events are much faster in large segmental bone defects in weight-bearing areas in goats.
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Building strong bones: molecular regulation of the osteoblast lineage.
Long, Fanxin
2011-12-22
The past 15 years have witnessed tremendous progress in the molecular understanding of osteoblasts, the main bone-forming cells in the vertebrate skeleton. In particular, all of the major developmental signals (including WNT and Notch signalling), along with an increasing number of transcription factors (such as RUNX2 and osterix), have been shown to regulate the differentiation and/or function of osteoblasts. As evidence indicates that osteoblasts may also regulate the behaviour of other cell types, a clear understanding of the molecular identity and regulation of osteoblasts is important beyond the field of bone biology.
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Immature myeloid cells are critical for enhancing bone fracture healing through angiogenic cascade
Levy, Seth; Feduska, Joseph M.; Sawant, Anandi; Gilbert, Shawn; Hensel, Jonathan A.; Ponnazhagan, Selvarangan
2016-01-01
Bone fractures heal with overlapping phases of inflammation, cell proliferation, and bone remodeling. Osteogenesis and angiogenesis work in concert to control many stages of this process, and when one is impaired it leads to failure of bone healing, termed a nonunion. During fracture repair, there is an infiltration of immune cells at the fracture site that not only mediate the inflammatory responses, but we hypothesize they also exert influence on neovasculature. Thus, further understanding the effects of immune cell participation throughout fracture healing will reveal additional knowledge as to why some fractures heal while others form nonunions, and lead to development of novel therapeutics modulating immune cells, to increase fracture healing and prevent nonunions. Using novel femoral segmental and critical-size defect models in mice, we identified a systemic and significant increase in immature myeloid cell (IMC) infiltration during the initial phase of fracture healing until boney union is complete. Using gemcitabine to specifically ablate the IMC population, we confirmed delayed bone healing. Further, adoptive transfer of IMC increased bone growth in a nonunion model, signifying the role of this unique cell population in fracture healing. We also identified IMC post-fracture have the ability to increase endothelial cell migration, and tube formation, signaling the essential communication between the immune system and angiogenesis as a requirement for proper bone healing. Based on this data we propose that IMC may play a significant role in fracture healing and therapeutic targeting of IMC after fracture would minimize the chances of eventual nonunion pathology. PMID:27664567
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Immature myeloid cells are critical for enhancing bone fracture healing through angiogenic cascade.
Levy, Seth; Feduska, Joseph M; Sawant, Anandi; Gilbert, Shawn R; Hensel, Jonathan A; Ponnazhagan, Selvarangan
2016-12-01
Bone fractures heal with overlapping phases of inflammation, cell proliferation, and bone remodeling. Osteogenesis and angiogenesis work in concert to control many stages of this process, and when one is impaired it leads to failure of bone healing, termed a nonunion. During fracture repair, there is an infiltration of immune cells at the fracture site that not only mediate the inflammatory responses, but we hypothesize they also exert influence on neovasculature. Thus, further understanding the effects of immune cell participation throughout fracture healing will reveal additional knowledge as to why some fractures heal while others form nonunions, and lead to development of novel therapeutics modulating immune cells, to increase fracture healing and prevent nonunions. Using novel femoral segmental and critical-size defect models in mice, we identified a systemic and significant increase in immature myeloid cell (IMC) infiltration during the initial phase of fracture healing until boney union is complete. Using gemcitabine to specifically ablate the IMC population, we confirmed delayed bone healing. Further, adoptive transfer of IMC increased bone growth in a nonunion model, signifying the role of this unique cell population in fracture healing. We also identified IMC post-fracture have the ability to increase endothelial cell migration, and tube formation, signaling the essential communication between the immune system and angiogenesis as a requirement for proper bone healing. Based on this data we propose that IMC may play a significant role in fracture healing and therapeutic targeting of IMC after fracture would minimize the chances of eventual nonunion pathology. Copyright © 2016 Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Hirata, Eri; Ménard-Moyon, Cécilia; Venturelli, Enrica; Takita, Hiroko; Watari, Fumio; Bianco, Alberto; Yokoyama, Atsuro
2013-11-01
Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF-CNT) showed the same effect as FGF alone. In addition, FGF-CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF-CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF-CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications.
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Spaceflight and the skeleton: lessons for the earthbound
NASA Technical Reports Server (NTRS)
Bikle, D. D.; Halloran, B. P.; Morey-Holton, E.
1997-01-01
Loss of bone during extended space flight has long been a concern that could limit the ability of humans to explore the universe. Surprisingly the available data do not support the concept that weightlessness leads inexorably to a depleted skeleton unable to withstand the stress of a return to a 1g environment. Nevertheless, some bone loss does occur especially in those bones most stressed by gravity prior to flight, providing confirmation of the proposal formulated over a century ago by Julius Wolff that mechanical stress determines the form and function of bone. Although the phenomenon of bone loss with skeletal unloading, whether by space flight or immobilization or just taking a load off your feet (literally) is well established, the mechanisms by which bone senses load and adjusts to it are not so clear. What actually is the stimulus and what are the sensors? What are the target cells? How do the sensors communicate the message into the cells, and by what pathways do the cells respond? What is the role of endocrine factors versus paracrine or autocrine factors in mediating or modulating the response? None of these questions has been answered with certainty, but as will become apparent in this review, we have some clues directing us to the answers. Although the focus of this review concerns space flight, it seems highly likely that the mechanisms mediating the transmission of mechanical load to changes in bone formation and resorption apply equally well to all forms of disuse osteoporosis, and are likely to be the same mechanisms affected by other etiologies of osteoporosis.
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Space flight and the skeleton: lessons for the earthbound
NASA Technical Reports Server (NTRS)
Bikle, D. D.; Halloran, B. P.; Morey-Holton, E.
1997-01-01
Loss of bone during extended space flight has long been a concern that could limit the ability of humans to explore the universe. Surprisingly, the available data do not support the concept that weightlessness leads inexorably to a depleted skeleton unable to withstand the stress of a return to a 1-g environment. Nevertheless, some bone loss does occur, especially in those bones most stressed by gravity prior to flight, which provides confirmation of the proposal formulated over a century ago by Julius Wolff that mechanical stress determines the form and function of bone. Although the phenomenon of bone loss with skeletal unloading, whether by space flight or immobilization or just taking a load off your feet (literally) is well established, the mechanisms by which bone senses load and adjusts to it are not so clear. What actually is the stimulus, and what are the sensors? What are the target cells? How do the sensors communicate the message into the cells, and by what pathways do the cells respond? What is the role of endocrine, factors vs. paracrine or autocrine factors in mediating or modulating the response? None of these questions has been answered with certainty, but, as will become apparent in this review, we have some clues directing us to the answers. Although the focus of this review concerns space flight, it seems highly likely that the mechanisms mediating the transmission of mechanical load to changes in bone formation and resorption apply equally well to all forms of disuse osteoporosis and are likely to be the same mechanisms affected by other etiologies of osteoporosis.
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Characterization of bone marrow derived mesenchymal stem cells in suspension
2012-01-01
Introduction Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs). Methods To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice. Results S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production. Conclusions These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs. PMID:23083975
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Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration.
Kim, Beom-Su; Sung, Hark-Mo; You, Hyung-Keun; Lee, Jun
2014-10-01
Fibrin polymers are widely used in the tissue engineering field as biomaterials. Although numerous researchers have studied the fabrication of scaffolds using fibrin glue (FG) and bone powder, the effects of varied fibrinogen content during the fabrication of scaffolds on human mesenchymal stem cells (hMSCs) and bone regeneration remain poorly understood. In this study, we formulated scaffolds using demineralized bone powder and various fibrinogen concentrations and analyzed the microstructure and mechanical properties. Cell proliferation, cell viability, and osteoblast differentiation assays were performed. The ability of the scaffold to enhance bone regeneration was evaluated using a rabbit calvarial defect model. Micro-computed tomography (micro-CT) showed that bone powders were uniformly distributed on the scaffolds, and scanning electron microscopy (SEM) showed that the fibrin networks and flattened fibrin layers connected adjacent bone powder particles. When an 80 mg/mL fibrinogen solution was used to formulate scaffolds, the porosity decreased 41.6 ± 3.6%, while the compressive strength increased 1.16 ± 0.02 Mpa, when compared with the values for the 10 mg/mL fibrinogen solution. Proliferation assays and SEM showed that the scaffolds prepared using higher fibrinogen concentrations supported and enhanced cell adhesion and proliferation. In addition, mRNA expression of alkaline phosphatase and osteocalcin in cells grown on the scaffolds increased with increasing fibrinogen concentration. Micro-CT and histological analysis revealed that newly formed bone was stimulated in the scaffold implantation group. Our results demonstrate that optimization of the fibrinogen content of fibrin glue/bone powder scaffolds will be beneficial for bone tissue engineering. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Liu, Jin; Nam, Hwa Kyung; Campbell, Cassie; Gasque, Kellen Cristina da Silva; Millán, José Luis; Hatch, Nan E.
2014-01-01
Tissue-nonspecific alkaline phosphatase (TNAP) is an enzyme present on the surface of mineralizing cells and their derived matrix vesicles that promotes hydroxyapatite crystal growth. Hypophosphatasia (HPP) is an inborn-error-of-metabolism that, dependent upon age of onset, features rickets or osteomalacia due to loss-of function mutations in the gene (Alpl) encoding TNAP. Craniosynostosis is prevalent in infants with HPP and other forms of rachitic disease but how craniosynostosis develops in these disorders is unknown. Objectives: Because craniosynostosis carries high morbidity, we are investigating craniofacial skeletal abnormalities in Alpl−/− mice to establish these mice as a model of HPP-associated craniosynostosis and determine mechanisms by which TNAP influences craniofacial skeletal development. Methods: Cranial bone, cranial suture and cranial base abnormalities were analyzed by micro-CT and histology. Craniofacial shape abnormalities were quantified using digital calipers. TNAP expression was suppressed in MC3T3E1(C4) calvarial cells by TNAP-specific shRNA. Cells were analyzed for changes in mineralization, gene expression, proliferation, apoptosis, matrix deposition and cell adhesion. Results: Alpl−/− mice feature craniofacial shape abnormalities suggestive of limited anterior-posterior growth. Craniosynostosis in the form of bony coronal suture fusion is present by three weeks after birth. Alpl−/− mice also exhibit marked histologic abnormalities of calvarial bones and the cranial base involving growth plates, cortical and trabecular bone within two weeks of birth. Analysis of calvarial cells in which TNAP expression was suppressed by shRNA indicates that TNAP deficiency promotes aberrant osteoblastic gene expression, diminished matrix deposition, diminished proliferation, increased apoptosis and increased cell adhesion. Conclusions: These findings demonstrate that Alpl−/− mice exhibit a craniofacial skeletal phenotype similar to that seen in infants with HPP, including true bony craniosynostosis in the context of severely diminished bone mineralization. Future studies will be required to determine if TNAP deficiency and other forms of rickets promote craniosynostosis directly through abnormal calvarial cell behavior, or indirectly due to deficient growth of the cranial base. PMID:25014884
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See, Eugene Yong-Shun; Toh, Siew Lok; Goh, James Cho-Hong
2011-10-01
The aim of this study was to develop a tissue engineering approach in regenerating the annulus fibrosus (AF) as part of an overall strategy to produce a tissue-engineered intervertebral disc (IVD) replacement. To determine whether a rehabilitative simulation regime on bone marrow–derived mesenchymal stem cell cell-sheet is able to aid the regeneration of the AF. No previous study has used bone marrow–derived mesenchymal stem cell cell-sheets simulated by a rehabilitative regime to regenerate the AF. The approach was to use bone marrow–derived stem cells to form cell-sheets and incorporating them onto silk scaffolds to simulate the native lamellae of the AF. The in vitro experimental model used to study the efficacy of such a system was made up of the tissue engineering AF construct wrapped around a silicone disc to form a simulated IVD-like assembly. The assembly was cultured within a custom-designed bioreactor that provided a compressive mechanical stimulation onto the silicone disc. The silicone nucleus pulposus would bulge radially and compress the simulated AF to mimic the physiological conditions. The simulated IVD-like assembly was compressed using a rehabilitative regime that lasted for 4 weeks at 0.25 Hz, for 15 minutes each day. With the rehabilitative regime, the cell-sheets remained viable but showed a decrease in cell numbers and viability. Gene expression analysis showed significant upregulation of IVD-related genes and there was an increased ratio of collagen type II to collagen type I found within the extracellular matrix. The results suggested that a rehabilitative regime caused extensive remodeling to take place within the simulated IVD-like assembly, producing extracellular matrix similar to that found in the inner AF.
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Feng, Yuping; Wang, Jiao; Ling, Shixin; Li, Zhuo; Li, Mingsheng; Li, Qiongyi; Ma, Zongren; Yu, Sijiu
2014-01-01
The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells following induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined specific neuronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuronal-specific proteins, including βIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differentiation medium differentiated into a multilayered neural network-like structure with long nerve fibers that was composed of several parallel microfibers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sectioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve. PMID:25598779
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Periodontal regeneration using engineered bone marrow mesenchymal stromal cells.
Yang, Yi; Rossi, Fabio M V; Putnins, Edward E
2010-11-01
Regeneration of lost periodontium is a challenge in that both hard (alveolar bone, cementum) and soft (periodontal ligament) connective tissues need to be restored to their original architecture. Bone marrow mesenchymal stromal cells (BM-MSCs) appear to be an attractive candidate for connective tissue regeneration. We hypothesized that BM-MSCs are able to sense biological cues from the local microenvironment and organize appropriately to contribute to the regeneration of both soft and hard periodontal connective tissues. To test this hypothesis, we transplanted GFP(+) rat BM-MSCs expanded ex vivo on microcarrier gelatin beads into a surgically created rat periodontal defect. After three weeks, evidence of regeneration of bone, cementum and periodontal ligament was observed in both transplanted and control animals. However, the animals that received BM-MSCs regenerated significantly greater new bone. In addition, the animals that had received the cells and beads transplant had significantly more appropriately orientated periodontal ligament fibers, indicative of functional restoration. Finally, donor-derived BM-MSCs were found integrated in newly formed bone, cementum and periodontal ligament, suggesting that they can directly contribute to the regeneration of cells of these tissues. Copyright © 2010 Elsevier Ltd. All rights reserved.
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The Nell-1 Growth Factor Stimulates Bone Formation by Purified Human Perivascular Cells
Zhang, Xinli; Péault, Bruno; Chen, Weiwei; Li, Weiming; Corselli, Mirko; James, Aaron W.; Lee, Min; Siu, Ronald K.; Shen, Pang; Zheng, Zhong; Shen, Jia; Kwak, Jinny; Zara, Janette N.; Chen, Feng; Zhang, Hong; Yin, Zack; Wu, Ben; Ting, Kang
2011-01-01
The search for novel sources of stem cells other than bone marrow mesenchymal stem cells (MSCs) for bone regeneration and repair has been a critical endeavor. We previously established an effective protocol to homogeneously purify human pericytes from multiple fetal and adult tissues, including adipose, bone marrow, skeletal muscle, and pancreas, and identified pericytes as a primitive origin of human MSCs. In the present study, we further characterized the osteogenic potential of purified human pericytes combined with a novel osteoinductive growth factor, Nell-1. Purified pericytes grown on either standard culture ware or human cancellous bone chip (hCBC) scaffolds exhibited robust osteogenic differentiation in vitro. Using a nude mouse muscle pouch model, pericytes formed significant new bone in vivo as compared to scaffold alone (hCBC). Moreover, Nell-1 significantly increased pericyte osteogenic differentiation, both in vitro and in vivo. Interestingly, Nell-1 significantly induced pericyte proliferation and was observed to have pro-angiogenic effects, both in vitro and in vivo. These studies suggest that pericytes are a potential new cell source for future efforts in skeletal regenerative medicine, and that Nell-1 is a candidate growth factor able to induce pericyte osteogenic differentiation. PMID:21615216
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Minashima, Takeshi; Quirno, Martin; Lee, You Jin; Kirsch, Thorsten
2017-01-01
The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. In this study we show increased fatty degeneration of the bone marrow of adult ank/ank mice, which lack a functional ANK protein. In addition, isolated bone marrow stromal cells (BMSCs) isolated from ank/ank mice showed a decreased proliferation rate and osteogenic differentiation potential, and an increased adipogenic differentiation potential compared to BMSCs isolated from wild type (WT) littermates. Wnt signaling pathway PCR array analysis revealed that Wnt ligands, Wnt receptors and Wnt signaling proteins that stimulate osteoblast differentiation were expressed at markedly lower levels in ank/ank BMSCs than in WT BMSCs. Lack of ANK function also resulted in impaired bone fracture healing, as indicated by a smaller callus formed and delayed bone formation in the callus site. Whereas 5 weeks after fracture, the fractured bone in WT mice was further remodeled and restored to original shape, the fractured bone in ank/ank mice was not fully restored and remodeled to original shape. In conclusion, our study provides evidence that ANK plays a critical role in the adipogenic/osteogenic fate decision of adult mesenchymal precursor cells. ANK functions in precursor cells are required for osteogenic differentiation of these cells during adult bone homeostasis and repair, whereas lack of ANK functions favors adipogenic differentiation. PMID:28286238
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Lynch, Maureen E.; Chiou, Aaron E.; Lee, Min Joon; Marcott, Stephen C.; Polamraju, Praveen V.; Lee, Yeonkyung
2016-01-01
Dynamic mechanical loading is a strong anabolic signal in the skeleton, increasing osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) and increasing the bone-forming activity of osteoblasts, but its role in bone metastatic cancer is relatively unknown. In this study, we integrated a hydroxyapatite-containing three-dimensional (3D) scaffold platform with controlled mechanical stimulation to investigate the effects of cyclic compression on the interplay between breast cancer cells and BM-MSCs as it pertains to bone metastasis. BM-MSCs cultured within mineral-containing 3D poly(lactide-co-glycolide) (PLG) scaffolds differentiated into mature osteoblasts, and exposure to tumor-derived soluble factors promoted this process. When BM-MSCs undergoing osteogenic differentiation were exposed to conditioned media collected from mechanically loaded breast cancer cells, their gene expression of osteopontin was increased. This was further enhanced when mechanical compression was simultaneously applied to BM-MSCs, leading to more uniformly deposited osteopontin within scaffold pores. These results suggest that mechanical loading of 3D scaffold-based culture models may be utilized to evaluate the role of physiologically relevant physical cues on bone metastatic breast cancer. Furthermore, our data imply that cyclic mechanical stimuli within the bone microenvironment modulate interactions between tumor cells and BM-MSCs that are relevant to bone metastasis. PMID:27401765
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Mai, Ronald; Hagedorn, Manolo Gunnar; Gelinsky, Michael; Werner, Carsten; Turhani, Dritan; Späth, Heike; Gedrange, Tomas; Lauer, Günter
2006-09-01
The aim of this study was to evaluate the ectopic bone formation using tissue engineered cell-seeded constructs with two different scaffolds and primary human maxillary osteoblasts in nude rats over an implantation period of up to 96 days. Collagen I-coated Poly(3)hydroxybutyrate (PHB) embroidery and hydroxyapatite (HAP) collagen tapes were seeded with primary human maxillary osteoblasts (hOB) and implanted into athymic rnu/run rats. A total of 72 implants were placed into the back muscles of 18 rats. 24, 48 and 96 days after implantation, histological and histomorphometric analyses were made. The osteoblastic character of the cells was confirmed by immunocytochemistry and RT-PCR for osteocalcin. Histological analysis demonstrated that all cell-seeded constructs induced ectopic bone formation after 24, 48 and 96 days of implantation. There was more mineralized tissue in PHB constructs than in HAP-collagen tapes (at day 24; p < 0.05). Bone formation decreased with the increasing length of the implantation period. Osteocalcin expression verified the osteoblastic character of the cell-seeded constructs after implantation time. No bone formation and no osteocalcin expression were found in the control groups. Cell-seeded constructs either with PHB embroidery or HAP-collagen tapes can induce ectopic bone formation. However, the amount of bone formed decreased with increasing length of implantation.
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Mesenchymal Stem Cells for Osteochondral Tissue Engineering
Ng, Johnathan; Bernhard, Jonathan; Vunjak-Novakovic, Gordana
2017-01-01
Summary Mesenchymal stem cells (MSC) are of major interest to regenerative medicine, because of the ease of harvesting from a variety of sources (including bone marrow and fat aspirates) and ability to form a range of mesenchymal tissues, in vitro and in vivo. We focus here on the use of MSCs for engineering of cartilage, bone, and complex osteochondral tissue constructs, using protocols that replicate some aspects of the natural mesodermal development. For engineering of human bone, we discuss some of the current advances, and highlight the use of perfusion bioreactors for supporting anatomically exact human bone grafts. For engineering of human cartilage, we discuss limitations of current approaches, and highlight engineering of stratified, mechanically functional human cartilage interfaced with bone by mesenchymal condensation of MSCs. Taken together, the current advances enable engineering physiologically relevant bone, cartilage and osteochondral composites, and physiologically relevant studies of osteochondral development and disease. PMID:27236665
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Genetics Home Reference: Diamond-Blackfan anemia
... developing certain cancers, including a cancer of blood-forming tissue known as acute myeloid leukemia (AML) and ... proteins may increase the self-destruction of blood-forming cells in the bone marrow, resulting in anemia. ...
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Ptychographic X-ray nanotomography quantifies mineral distributions in human dentine
NASA Astrophysics Data System (ADS)
Zanette, I.; Enders, B.; Dierolf, M.; Thibault, P.; Gradl, R.; Diaz, A.; Guizar-Sicairos, M.; Menzel, A.; Pfeiffer, F.; Zaslansky, P.
2015-03-01
Bones are bio-composites with biologically tunable mechanical properties, where a polymer matrix of nanofibrillar collagen is reinforced by apatite mineral crystals. Some bones, such as antler, form and change rapidly, while other bone tissues, such as human tooth dentine, develop slowly and maintain constant composition and architecture for entire lifetimes. When studying apatite mineral microarchitecture, mineral distributions or mineralization activity of bone-forming cells, representative samples of tissue are best studied at submicrometre resolution while minimizing sample-preparation damage. Here, we demonstrate the power of ptychographic X-ray tomography to map variations in the mineral content distribution in three dimensions and at the nanometre scale. Using this non-destructive method, we observe nanostructures surrounding hollow tracts that exist in human dentine forming dentinal tubules. We reveal unprecedented quantitative details of the ultrastructure clearly revealing the spatially varying mineralization density. Such information is essential for understanding a variety of natural and therapeutic effects for example in bone tissue healing and ageing.
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Developing bioactive composite scaffolds for bone tissue engineering
NASA Astrophysics Data System (ADS)
Chen, Yun
Poly(L-lactic acid) (PLLA) films were fabricated using the method of dissolving and evaporation. PLLA scaffold was prepared by solid-liquid phase separation of polymer solutions and subsequent sublimation of solvent. Bonelike apatite coating was formed on PLLA films, PLLA scaffolds and poly(glycolic acid) (PGA) scaffolds in 24 hours through an accelerated biomimetic process. The ion concentrations in the simulated body fluid (SBF) were nearly 5 times of those in human blood plasma. The apatite formed was characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The apatite formed in 5SBF was similar in morphology and composition to that formed in the classical biomimetic process employing SBF or 1.5SBF, and similar to that of natural bone. This indicated that the biomimetic apatite coating process could be accelerated by using concentrated simulated body fluid at 37°C. Besides saving time, the accelerated biomimetic process is particularly significant to biodegradable polymers. Some polymers which degrade too fast to be coated with apatite by a classical biomimetic process, for example PGA, could be coated with bone-like apatite in an accelerated biomimetic process. Collagen and apatite were co-precipitated as a composite coating on poly(L-lactic acid) (PLLA) in an accelerated biomimetic process. The incubation solution contained collagen (1g/L) and simulated body fluid (SBF) with 5 times inorganic ionic concentrations as human blood plasma. The coating formed on PLLA films and scaffolds after 24 hours incubation was characterized using EDX, XRD, FTIR, and SEM. It was shown that the coating contained carbonated bone-like apatite and collagen, the primary constituents of natural bone. SEM showed a complex composite coating of submicron bone-like apatite particulates combined with collagen fibrils. This work provided an efficient process to obtain bone-like apatite/collagen composite coating. Saos-2 osteoblast-like cells were used to evaluate the cellular behaviors on these biomimetic coatings. Cell morphologies on the surfaces of PLLA films and scaffolds, PLLA films and scaffolds with apatite coating, and PLLA films and scaffolds with apatite/collagen composite coating were studied by SEM. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium bromide (MTT) assay. In addition, differentiated cell function was assessed by measuring alkaline phosphatase activity. These results suggested that the apatite coating and apatite/collagen composite coating fabricated through the accelerated biomimetic processes could improve the interactions between osteoblasts and PLLA. The composite coating was more effective than apatite coating in improving such interactions. PLLA scaffolds coated with submicron collagen fibrils and submicron apatite paticulates are expected to be one of the promising 3D substrates for bone tissue engineering. To facilitate coating into scaffolds, the flowing condition was introduced into the accelerated biomimetic process. The apatite formed in the different sites in the scaffold was characterized using SEM. It was found that the accelerated biomimetic process performed in the flowing condition yielded more uniform spatial distribution of apatite particles than that in the regular shaking condition. This work provides a novel condition for obtaining uniform spatial distribution of bone-like apatite within the scaffolds in a timely manner, which is expected to facilitate uniform distribution of attached cells within the scaffoldsin vitro and in vivo.
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Endogenous Bone Regeneration Is Dependent Upon a Dynamic Oxygen Event
2014-01-01
endosteum and nail epithelium (Fig. 2G ). By DPA 21 hypoxyprobe signaling is still apparent in osteoblasts in the newly formed trabecular bone, but to a...affiliated with cells that surround the vascular lumen (Fig. 2G ’). This signal within the distal tip of P3 expands at DPA 21, associated...vivo (Fig. 3G -I). Digit slices were cultured at 21% oxygen and 5% CO2. Bone formation was assessed using Alizarin red staining. Bone morphology is
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NASA Astrophysics Data System (ADS)
Tamin, R. Y.; Soeroso, Y.; Amir, L.; Idrus, E.
2017-08-01
Chronic periodontitis is an oral disease in which the destruction of periodontal tissue leads to tooth loss. Regenerative therapy for attachment cannot be applied to one wall bone defects owing to the minimal existing healthy bone. Tissue engineering in the form of cell sheets has been developed to overcome this limitation. In a previous study, cell sheet application to a one wall bone defect in Macaca nemestrina showed good clinical results. To evaluate the effectiveness of cell sheet application histologically, the level of periostin expression in the gingival crevicular fluid (GCF) of M. nemestrina was determined. Periostin is a 90-kDa protein that regulates coordination and interaction for regeneration and tissue repair. A laboratory observation study was performed to see the differences in periostin levels in samples collected from M. nemestrina’s GCF, where a cell sheet was applied to the bone defect. Gel electrophoresis with SDS-PAGE was performed to detect periostin expression based on its molecular weight and to compare the expression band between the cell sheet and the control at 1, 2, and 3 weeks after treatment. The gel electrophoresis result shows different thicknesses of the protein band around the molecular weight of periostin between the cell sheet groups.
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Zoledronate induces apoptosis in cells from fibro-cellular membrane of unicameral bone cyst (UBC).
Yu, John; Chang, Seong-Sil; Suratwala, Sanjeev; Chung, Woo-Sik; Abdelmessieh, Peter; Lee, Hahn-Jun; Yang, Jay; Lee, Francis Young-In
2005-09-01
Unicameral bone cyst (UBC) is a benign cystic lesion in children which is prone to fracture. Various treatments are available, but recurrence after different types of percutaneous injection therapy can cause bone destruction and pathologic fracture. The potential therapeutic effects of anti-resorptive agents, such as bisphosphonates, have not been investigated for UBC. The objective of this study was to characterize the cells from the fibro-cellular membrane of unicameral bone cyst (UBC cells) and to determine whether zoledronate, a nitrogen-containing bisphosphonate, could induce apoptosis in UBC cells. Flow cytometry and immunoblotting were performed in order to determine whether zoledronate induced apoptosis. Cells derived from normal human trabecular bones were used as controls against UBC cells to compare the effect of zoledronate in inducing apoptosis. Immunohisto/cytochemistry (IHC/ICC) and mini-array analyses were performed on tissues and cultured cells. Isolated peripheral blood mononuclear cells were incubated with conditioned media from the UBC cells to determine whether they are capable of inducing osteoclastogenesis. UBC membrane is composed of cells staining positively with CD68, SDF-1, STRO-1 and RANKL, but in vitro cells showed no staining with antibodies to CD68 and STRO-1, suggesting that there was a clonal selection of stromal cells during cell culture. UBC cells also express RUNX2 (runt-related transcription factor-2, core binding factor-1), a key transcription factor for osteoblastic differentiation. In addition, media collected from UBC cells induced a generation of multi-nucleated osteoclast-like cells of peripheral blood mononuclear cells. Zoledronate induced apoptosis of UBC cells in a dose-dependent manner. Apoptosis was evidenced by induction of the active cleaved form of caspase-3. The baseline apoptotic fractions were similar in UBC cells and trabecular bone cells. However, in the overall apoptotic fractions in this study, trabecular bone cells showed 17.2% of apoptosis, significantly lower than 24.2% of UBC cells (p-value=0.007). With the various zoledronate concentrations, mean apoptotic fractions of trabecular bone cells was 19.2%, significantly lower than 27.8% of UBC cells (p-value=0.040). With GGOH co-treatment in various zoledronate concentrations, 15.1% apoptosis was shown in trabecular bone cells, which was not significantly lower than 20.6% of UBC cells (p-value=0.076). This data suggests that zoledronate causes apoptosis in both UBC and trabecular bone cells by inhibition of the mevalonate pathway. In addition to the known anti-osteoclastogenic effect of bisphosphonates, the GGOH inhibitory effects of zoledronate were more prominent in UBC cells than trabecular bone cells, indicating their potential therapeutic role in UBC.
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Deletion of Adseverin in Osteoclasts Affects Cell Structure But Not Bone Metabolism.
Cao, Yixuan; Wang, Yongqiang; Sprangers, Sara; Picavet, Daisy I; Glogauer, Michael; McCulloch, Christopher A; Everts, Vincent
2017-08-01
Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and adseverin null mice, as measured by dual-energy X-ray absorptiometry, there were no differences of bone mineral content or bone mineral density, indicating no change of bone metabolism. In tibiae, TRAcP + osteoclasts were formed in comparable numbers in adseverin null and wild-type mice. Ultrastructural analysis showed normal and similar abundance of ruffled borders, sealing zones, and mitochondria, and with no difference of osteoclast nuclear numbers. In contrast, analyses of long bone showed that in the absence of adseverin osteoclasts were smaller (120 ± 13 vs. 274 ± 19 µm 2 ; p < 0.05), as were nuclear size and the surface area of cytoplasm. The nuclei of adseverin null osteoclasts exhibited more heterochromatin (31 ± 3%) than wild-type cells (8 ± 1%), suggesting that adseverin affects cell differentiation. The data indicate that in healthy, developing tissues, adseverin contributes to the regulation of osteoclast structure but not to bone metabolism in vivo.
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CELLS INVOLVED IN THE IMMUNE RESPONSE
Singhal, Sharwan K.; Richter, Maxwell
1968-01-01
Cell suspensions of immune rabbit lymph nodes and spleen were capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine when maintained in culture with the specific antigen in vitro. They did not respond to other, non-cross-reacting antigens. The blastogenic response obtained with immune lymph node cells could be correlated with the antibody synthesizing capacity of fragment cultures prepared from the same lymph nodes. Cell suspensions of immune bone marrow responded to non-cross-reacting antigens only whereas cell suspensions of immune thymus, sacculus rotundus, and appendix did not respond when exposed to any of the antigens tested. On the other hand, neither fragments nor cell suspensions prepared from lymph nodes, spleen, and thymus of normal, unimmunized rabbits responded with antibody formation and blastogenesis when exposed to any of the antigens. However, normal bone marrow cells responded with marked blastogenesis and tritiated thymidine uptake. The specificity of this in vitro bone marrow response was demonstrated by the fact that the injection of a protein antigen in vivo resulted in the loss of reactivity by the marrow cell to that particular antigen but not to the other, non-cross-reacting antigens. Furthermore, bone marrow cells of tolerant rabbits failed to respond to the specific antigen in vitro. It was also demonstrated that normal bone marrow cells incubated with antigen are capable of forming antibody which could be detected by the fluorescent antibody technique. This response of the bone marrow cells has been localized to the lymphocyte-rich fraction of the bone marrow. It is concluded that the bone marrow lymphocyte, by virtue of its capacity to react with blastogenesis and mitosis and with antibody formation upon initial exposure to the antigen, a capacity not possessed by lymphocytes of the other lymphoid organs, has a preeminent role in the sequence of cellular events culminating in antibody formation. PMID:4176224
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NASA Astrophysics Data System (ADS)
Katkova, Olena; Rodionova, Natalia; Shevel, Ivan
2016-07-01
Microgravity and long-term hypokinesia induce reduction both in bone mass and mineral saturation, which can lead to the development of osteoporosis and osteopenia. (Oganov, 2003). Reorganizations and adaptive remodeling processes in the skeleton bones occur in the topographical interconnection with blood capillaries and perivascular cells. Radioautographic studies with 3H- thymidine (Kimmel, Fee, 1980; Rodionova, 1989, 2006) have shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic. Hence the study of populations of perivascular stromal cells in areas of destructive changes is actual. Perivascular cells from metaphysis of the rat femoral bones under conditions of modeling microgravity were studied using electron microscopy and cytochemistry (hindlimb unloading, 28 days duration) and biosatellite «Bion-M1» (duration of flight from April 19 till May 19, 2013 on C57, black mice). It was revealed that both control and test groups populations of the perivascular cells are not homogeneous in remodeling adaptive zones. These populations comprise of adjacent to endothelium poorly differentiated forms and isolated cells with signs of differentiation (specific increased volume of rough endoplasmic reticulum in cytoplasm). Majority of the perivascular cells in the control group (modeling microgravity) reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In poorly differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of experimental animals reaction to the alkaline phosphatase is registered not in all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. Under microgravity some poorly differentiated perivascular cells reveal signs of destruction. Thus it was found that number of the alkaline phosphatase containing cells (i.e. osteogenic cells) declines in perivascular cells population. It is one of the mechanisms of the osteogenic process decrease of intensity in bones because of lessening support loading on the bone skeleton. In the adaptive remodeling zones of bone tissue (near the vascular canals) in experiments fibroblasts and fibrosis zones were found - areas filled with non-mineralized collagen fibrils on the bones surfaces. Hence it should be considered that decrease (removal) of support loading slows down osteogenic differentiation of the part of perivascular cells and stimulates differentiation of the fibroblast cells. Obtained data is considered as one of the cellular mechanisms of the adaptive reactions development in spongy bone under microgravity which could lead to the bone mass loss.
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[Progress of Masquelet technique to repair bone defect].
Yin, Qudong; Sun, Zhenzhong; Gu, Sanjun
2013-10-01
To summarize the progress of Masquelet technique to repair bone defect. The recent literature concerning the application of Masquelet technique to repair bone defect was extensively reviewed and summarized. Masquelet technique involves a two-step procedure. First, bone cement is used to fill the bone defect after a thorough debridement, and an induced membrane structure surrounding the spacer formed; then the bone cement is removed after 6-8 weeks, and rich cancellous bone is implanted into the induced membrane. Massive cortical bone defect is repaired by new bone forming and consolidation. Experiments show that the induced membrane has vascular system and is also rich in vascular endothelial growth factor, transforming growth factor beta1, bone morphogenetic protein 2, and bone progenitor cells, so it has osteoinductive property; satisfactory results have been achieved in clinical application of almost all parts of defects, various types of bone defect and massive defect up to 25 cm long. Compared with other repair methods, Masquelet technique has the advantages of reliable effect, easy to operate, few complications, low requirements for recipient site, and wide application. Masquelet technique is an effective method to repair bone defect and is suitable for various types of bone defect, especially for bone defects caused by infection and tumor resection.
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Antika, Lucia Dwi; Lee, Eun-Jung; Kim, Yun-Ho; Kang, Min-Kyung; Park, Sin-Hye; Kim, Dong Yeon; Oh, Hyeongjoo; Choi, Yean-Jung; Kang, Young-Hee
2017-11-01
Osteoporosis is one of the most prevalent forms of age-related bone diseases. Increased bone loss with advancing age has become a grave public health concern. This study examined whether phlorizin and phloretin, dihydrochalcones in apple peels, inhibited senile osteoporosis through enhancing osteoblastogenic bone formation in cell-based and aged mouse models. Submicromolar phloretin and phlorizin markedly stimulated osteoblast differentiation of MC3T3-E1 cells with increased transcription of Runx2 and osteocalcin. Senescence-accelerated resistant mouse strain prone-6 (SAMP6) mice were orally supplemented with 10 mg/kg phlorizin and phloretin daily for 12 weeks. Male senescence-accelerated resistant mouse strain R1 mice were employed as a nonosteoporotic age-matched control. Oral administration of ploretin and phorizin boosted bone mineralization in all the bones of femur, tibia and vertebra of SAMP6. In particular, phlorizin reduced serum RANKL/OPG ratio and diminished TRAP-positive osteoclasts in trabecular bones of SAMP6. Additionally, treating phlorizin to SAMP6 inhibited the osteoporotic resorption in distal femoral bones through up-regulating expression of BMP-2 and collagen-1 and decreasing production of matrix-degrading cathepsin K and MMP-9. Finally, phlorizin and phloretin antagonized GSK-3β induction and β-catenin phosphorylation in osteoblasts and aged mouse bones. Therefore, phlorizin and phloretin were potential therapeutic agents encumbering senile osteoporosis through promoting bone-forming osteoblastogenesis via modulation of GSK-3β/β-catenin-dependent signaling. Copyright © 2017 Elsevier Inc. All rights reserved.
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Analysis of Mouse Growth Plate Development
Mangiavini, Laura; Merceron, Christophe; Schipani, Ernestina
2016-01-01
To investigate skeletal development, pathophysiological mechanisms of cartilage and bone disease, and eventually assess innovative treatments, the mouse is a very important resource. During embryonic development, mesenchymal condensations are formed, and cells within these mesenchymal condensations either directly differentiate into osteoblasts and give origin to intramembranous bone, or differentiate into chondrocytes and form a cartilaginous anlage. The cartilaginous anlage or fetal growth plate is then replaced with bone. This process is also called endochondral bone development, and it is responsible for the generation of most of our skeleton. In this Review, we will discuss in detail the most common in vivo and in vitro techniques our laboratory is currently using for the analysis of the mouse fetal growth plate during development. PMID:26928664
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Demineralized dentin matrix composite collagen material for bone tissue regeneration.
Li, Jianan; Yang, Juan; Zhong, Xiaozhong; He, Fengrong; Wu, Xiongwen; Shen, Guanxin
2013-01-01
Demineralized dentin matrix (DDM) had been successfully used in clinics as bone repair biomaterial for many years. However, particle morphology of DDM limited it further applications. In this study, DDM and collagen were prepared to DDM composite collagen material. The surface morphology of the material was studied by scanning electron microscope (SEM). MC3T3-E1 cells responses in vitro and tissue responses in vivo by implantation of DDM composite collagen material in bone defect of rabbits were also investigated. SEM analysis showed that DDM composite collagen material evenly distributed and formed a porous scaffold. Cell culture and animal models results indicated that DDM composite collagen material was biocompatible and could support cell proliferation and differentiation. Histological evaluation showed that DDM composite collagen material exhibited good biocompatibility, biodegradability and osteoconductivity with host bone in vivo. The results suggested that DDM composite collagen material might have a significant clinical advantage and potential to be applied in bone and orthopedic surgery.
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Harris, Stephen E; Rediske, Michael; Neitzke, Rebecca; Rakian, Audrey
2017-01-01
The periodontium is a complex tissue with epithelial components and a complex set of mesodermal derived alveolar bone, cellular and a cellular cementum, and tendon like ligaments (PDL). The current evidence demonstrates that the major pool of periodontal stem cells is derived from a population of micro vascular associated aSMA-positive stem/progenitor (PSC) cells that by lineage tracing form all three major mesodermal derived components of the periodontium. With in vitro aSMA+ stem cells, transcriptome and chip- seq experiments, the gene network and enhancer maps were determined at several differentiation states of the PSC. Current work on the role of the Bmp2 gene in the periodontal stem cell differentiation demonstrated that this Wnt regulated gene, Bmp2, is necessary for differentiation to all three major mesodermal derived component of the periodontium. The mechanism and use of Sclerostin antibody as an activator of Wnt signaling and Bmp2 gene as a potential route to treat craniofacial bone loss is discussed. As well, the mechanism and use of Pth in the treatment of periodontal bone loss or other craniofacial bone loss is presented in this review. PMID:29457146
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Central Nervous System Fibrosis Is Associated with Fibrocyte-Like Infiltrates
Aldrich, Amy; Kielian, Tammy
2011-01-01
Fibrotic wall formation is essential for limiting pathogen dissemination during brain abscess development. However, little is known about the regulation of fibrotic processes in the central nervous system (CNS). Most CNS injury responses are associated with hypertrophy of resident astrocytes, a process termed reactive gliosis. Studies of fibrosis outside the CNS have identified two bone marrow–derived cell types, fibrocytes and alternatively activated M2 macrophages, as key mediators of fibrosis. The current study used bone marrow chimeras generated from green fluorescent protein transgenic mice to evaluate the appearance of these cell types and whether bone marrow–derived cells were capable of acquiring fibrotic characteristics during brain abscess development. Immunofluorescence staining revealed partial overlap between green fluorescent protein, α-smooth muscle actin, and procollagen, suggesting that a population of cells forming the brain abscess capsule originate from a bone marrow precursor. In addition, the influx of fibrocyte-like cells into brain abscesses immediately preceded the onset of fibrotic encapsulation. Fibrotic wall formation was also associated with increased numbers of alternatively activated M2 microglia and macrophages. To our knowledge, this is the first study demonstrating that bone marrow–derived infiltrates are capable of expressing fibrotic molecules during CNS inflammation. PMID:22015460
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Liu, Xian; Chen, Wenchuan; Zhang, Chi; Thein-Han, Wahwah; Hu, Kevin; Reynolds, Mark A; Bao, Chongyun; Wang, Ping; Zhao, Liang; Xu, Hockin H K
2017-06-01
A major challenge in repairing large bone defects with tissue-engineered constructs is the poor vascularization in the defect. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of seeded cells. To achieve favorable regenerative effects, prevascularization of tissue-engineered constructs by co-culturing of endothelial cells and bone cells is a promising strategy. The aim of this study was to investigate the effects of human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) co-cultured with human umbilical vein endothelial cells (HUVECs) for prevascularization of calcium phosphate cement (CPC) scaffold on bone regeneration in vivo for the first time. HUVECs co-cultured with hiPSC-MSCs formed microcapillary-like structures in vitro. HUVECs promoted mineralization of hiPSC-MSCs on CPC scaffolds. Four groups were tested in a cranial bone defect model in nude rats: (1) CPC scaffold alone (CPC control); (2) HUVEC-seeded CPC (CPC-HUVEC); (3) hiPSC-MSC-seeded CPC (CPC-hiPSC-MSC); and (4) HUVECs co-cultured with hiPSC-MSCs on CPC scaffolds (co-culture group). After 12 weeks, the co-culture group achieved the greatest new bone area percentage of 46.38% ± 3.8% among all groups (p < 0.05), which was more than four folds of the 10.61% ± 1.43% of CPC control. In conclusion, HUVECs co-cultured with hiPSC-MSCs substantially promoted bone regeneration. The novel construct of HUVECs co-cultured with hiPSC-MSCs delivered via CPC scaffolds is promising to enhance bone and vascular regeneration in orthopedic applications.
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Chen, Ching-Yun; Tseng, Kuo-Yun; Lai, Yen-Liang; Chen, Yo-Shen; Lin, Feng-Huei; Lin, Shankung
2017-01-01
Many studies have indicated that loss of the osteoblastogenic potential in bone marrow mesenchymal stem cells (bmMSCs) is the major component in the etiology of the aging-related bone deficit. But how the bmMSCs lose osteogenic capability in aging is unclear. Using 2-dimentional cultures, we examined the dose response of human bmMSCs, isolated from adult and aged donors, to exogenous insulin-like growth factor 1 (IGF-1), a growth factor regulating bone formation. The data showed that the mitogenic activity and the osteoblastogenic potential of bmMSCs in response to IGF-1 were impaired with aging, whereas higher doses of IGF-1 increased the proliferation rate and osteogenic potential of aging bmMSCs. Subsequently, we seeded IGF-1-overexpressing aging bmMSCs into calcium-alginate scaffolds and incubated in a bioreactor with constant perfusion for varying time periods to examine the effect of IGF-1 overexpression to the bone-forming capability of aging bmMSCs. We found that IGF-1 overexpression in aging bmMSCs facilitated the formation of cell clusters in scaffolds, increased the cell survival inside the cell clusters, induced the expression of osteoblast markers, and enhanced the biomineralization of cell clusters. These results indicated that IGF-1 overexpression enhanced cells' osteogenic capability. Thus, our data suggest that the aging-related loss of osteogenic potential in bmMSCs can be attributed in part to the impairment in bmMSCs' IGF-1 signaling, and support possible application of IGF-1-overexpressing autologous bmMSCs in repairing bone defect of the elderly and in producing bone graft materials for repairing large scale bone injury in the elderly.
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Cellular Mechanisms Underlying Bone-Forming Cell Proliferative Response to Hypergravity
NASA Technical Reports Server (NTRS)
Vercoutere, W.; Parra, M.; DaCosta, M.; Wing, A.; Roden, C.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.
2004-01-01
Life on Earth has evolved under the continuous influence of gravity (1-g). As humans explore and develop space, however, we must learn to adapt to an environment with little or no gravity. Studies indicate that lack of weightbearing for vertebrates occurring with immobilization, paralysis, or in a microgravity environment may cause muscle and bone atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) (consisting of molecules such as collagen, fibronectin, and laminin) in mechanosensitive tissues. We test for the presence of gravity-sensitive pathways in bone-forming cells (osteoblasts) using hypergravity applied by a cell culture centrifuge. Stimulation of 50 times gravity (50-g) increased proliferation in primary rat osteoblasts for cells grown on collagen Type I and fibronectin, but not on laminin or uncoated surfaces. Survival was also enhanced during hypergravity stimulation by the presence of ECM. Bromodeoxyuridine incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. Reverse transcription-polymerase chain reaction was used to test for all possible integrins. Our combined results indicate that beta1 and/or beta3 integrin subunits may be involved. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signalling pathways which are sensitive to g-level. Further research to define the mechanisms involved will provide direction so that we may better adapt and counteract bone atrophy caused by the lack of weightbearing.
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Yang, Mo; Li, Karen; Ng, Pak Cheung; Chuen, Carmen Ka Yee; Lau, Tze Kin; Cheng, Yuan Shan; Liu, Yuan Sheng; Li, Chi Kong; Yuen, Patrick Man Pan; James, Anthony Edward; Lee, Shuk Man; Fok, Tai Fai
2007-07-01
Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5-hydroxytryptamine (5-HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony-forming unit-fibroblast (CFU-F) formation, and antiapoptosis of megakaryoblastic M-07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony-forming unit-mixed [CFU-GEMM]) and multilineage committed progenitors (burst-forming unit/colony-forming unit-erythroid [BFU/CFU-E], colony-forming unit-granulocyte macrophage, colony-forming unit-megakaryocyte, CD61+ CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU-E, and CFU-GEMM engraftment in BM of animals 6 weeks post-transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor stimulated BM CFU-F formation. In M-07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase-3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5-HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation. Disclosure of potential conflicts of interest is found at the end of this article.
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Shimizu, Kazunori; Ito, Akira; Yoshida, Tatsuro; Yamada, Yoichi; Ueda, Minoru; Honda, Hiroyuki
2007-08-01
An in vitro reconstruction of three-dimensional (3D) tissues without the use of scaffolds may be an alternative strategy for tissue engineering. We have developed a novel tissue engineering strategy, termed magnetic force-based tissue engineering (Mag-TE), in which magnetite cationic liposomes (MCLs) with a positive charge at the liposomal surface, and magnetic force were used to construct 3D tissue without scaffolds. In this study, human mesenchymal stem cells (MSCs) magnetically labeled with MCLs were seeded onto an ultra-low attachment culture surface, and a magnet (4000 G) was placed on the reverse side. The MSCs formed multilayered sheet-like structures after a 24-h culture period. MSCs in the sheets constructed by Mag-TE maintained an in vitro ability to differentiate into osteoblasts, adipocytes, or chondrocytes after a 21-day culture period using each induction medium. Using an electromagnet, MSC sheets constructed by Mag-TE were harvested and transplanted into the bone defect in the crania of nude rats. Histological observation revealed that new bone surrounded by osteoblast-like cells was formed in the defect area 14 days after transplantation with MSC sheets, whereas no bone formation was observed in control rats without the transplant. These results indicated that Mag-TE could be used for the transplantation of MSC sheets using magnetite nanoparticles and magnetic force, providing novel methodology for bone tissue engineering.
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Cell Mechanisms of Bone Tissue Loss Under Space Flight Conditions
NASA Astrophysics Data System (ADS)
Rodionova, Natalia
Investigations on the space biosatellites has shown that the bone skeleton is one of the most im-portant targets of the effect space flight factors on the organism. Bone tissue cells were studied by electron microscopy in biosamples of rats' long bones flown on the board american station "SLS-2" and in experiments with modelling of microgravity ("tail suspension" method) with using autoradiography. The analysis of data permits to suppose that the processes of remod-eling in bone tissue at microgravity include the following succession of cell-to-cell interactions. Osteocytes as mechanosensory cells are first who respond to a changing "mechanical field". The next stage is intensification of osteolytic processes in osteocytes, leading to a volume en-largement of the osteocytic lacunae and removal of the "excess bone". Then mechanical signals have been transmitted through a system of canals and processes of the osteocytic syncitium to certain superficial bone zones and are perceived by osteoblasts and bone-lining cells (superficial osteocytes), as well as by the bone-marrow stromal cells. The sensitivity of stromal cells, pre-osteoblasts and osteoblasts, under microgravity was shown in a number of works. As a response to microgravity, the system of stromal cells -preosteoblasts -osteoblasts displays retardation of proliferation, differentiation and specific functions of osteogenetic cells. This is supported by the 3H-thymidine studies of the dynamics of differentiation of osteogenetic cells in remodeling zones. But unloading is not adequate and in part of the osteocytes are apoptotic changes as shown by our electron microscopic investigations. An osteocytic apoptosis can play the role in attraction the osteoclasts and in regulation of bone remodeling. The apoptotic bodies with a liquid flow through a system of canals are transferred to the bone surface, where they fulfil the role of haemoattractants for monocytes come here and form osteoclasts. The osteoclasts destroy bone tissue. The macrophages are incorporated into resorption lacunaes and utilize the organic matrix and cellular detritus. The products are secreted to remodeling zones and act as haemoattractants for recruiting and subsequent differentiation here of the osteogenic precursor cells. However, as shown by our results with 3H-glycine, in absence of mechanical stimulus the activization of osteoblastogenesis either doesn't occur, or takes place on a smaller scale. According to our electron-microscopic data a load deficit leads to an adaptive differentiation of fibroblasts and adipocytes in this remodeling zones. This sequence of events is considered as a mechanism of bone tissue loss which underlies the development of osteopenia and osteoporosis under space flight condition.
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Hoch, Allison I; Duhr, Ralph; Di Maggio, Nunzia; Mehrkens, Arne; Jakob, Marcel; Wendt, David
2017-12-01
Bone marrow-derived mesenchymal stromal cells (BMSC), when expanded directly within 3D ceramic scaffolds in perfusion bioreactors, more reproducibly form bone when implanted in vivo as compared to conventional expansion on 2D polystyrene dishes/flasks. Since the bioreactor-based expansion on 3D ceramic scaffolds encompasses multiple aspects that are inherently different from expansion on 2D polystyrene, we aimed to decouple the effects of specific parameters among these two model systems. We assessed the effects of the: 1) 3D scaffold vs. 2D surface; 2) ceramic vs. polystyrene materials; and 3) BMSC niche established within the ceramic pores during in vitro culture, on subsequent in vivo bone formation. While BMSC expanded on 3D polystyrene scaffolds in the bioreactor could maintain their in vivo osteogenic potential, results were similar as BMSC expanded in monolayer on 2D polystyrene, suggesting little influence of the scaffold 3D environment. Bone formation was most reproducible when BMSC are expanded on 3D ceramic, highlighting the influence of the ceramic substrate. The presence of a pre-formed niche within the scaffold pores had negligible effects on the in vivo bone formation. The results of this study allow a greater understanding of the parameters required for perfusion bioreactor-based manufacturing of osteogenic grafts for clinical applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Abarrategi, Ander; Moreno-Vicente, Carolina; Martínez-Vázquez, Francisco Javier; Civantos, Ana; Ramos, Viviana; Sanz-Casado, José Vicente; Martínez-Corriá, Ramón; Perera, Fidel Hugo; Mulero, Francisca; Miranda, Pedro; López-Lacomba, José Luís
2012-01-01
Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.
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Abarrategi, Ander; Moreno-Vicente, Carolina; Martínez-Vázquez, Francisco Javier; Civantos, Ana; Ramos, Viviana; Sanz-Casado, José Vicente; Martínez-Corriá, Ramón; Perera, Fidel Hugo; Mulero, Francisca; Miranda, Pedro; López-Lacomba, José Luís
2012-01-01
Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures. PMID:22470527
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Mesenchymal progenitor cells for the osteogenic lineage.
Ono, Noriaki; Kronenberg, Henry M
2015-09-01
Mesenchymal progenitors of the osteogenic lineage provide the flexibility for bone to grow, maintain its function and homeostasis. Traditionally, colony-forming-unit fibroblasts (CFU-Fs) have been regarded as surrogates for mesenchymal progenitors; however, this definition cannot address the function of these progenitors in their native setting. Transgenic murine models including lineage-tracing technologies based on the cre-lox system have proven to be useful in delineating mesenchymal progenitors in their native environment. Although heterogeneity of cell populations of interest marked by a promoter-based approach complicates overall interpretation, an emerging complexity of mesenchymal progenitors has been revealed. Current literatures suggest two distinct types of bone progenitor cells; growth-associated mesenchymal progenitors contribute to explosive growth of bone in early life, whereas bone marrow mesenchymal progenitors contribute to the much slower remodeling process and response to injury that occurs mainly in adulthood. More detailed relationships of these progenitors need to be studied through further experimentation.
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Piemontese, Marilina; Onal, Melda; Xiong, Jinhu; Han, Li; Thostenson, Jeff D; Almeida, Maria; O'Brien, Charles A
2016-04-11
Autophagy maintains cell function and homeostasis by recycling intracellular components. This process is also required for morphological changes associated with maturation of some cell types. Osteoblasts are bone forming cells some of which become embedded in bone and differentiate into osteocytes. This transformation includes development of long cellular projections and a reduction in endoplasmic reticulum and mitochondria. We examined the role of autophagy in osteoblasts by deleting Atg7 using an Osterix1-Cre transgene, which causes recombination in osteoblast progenitors and their descendants. Mice lacking Atg7 in the entire osteoblast lineage had low bone mass and fractures associated with reduced numbers of osteoclasts and osteoblasts. Suppression of autophagy also reduced the amount of osteocyte cellular projections and led to retention of endoplasmic reticulum and mitochondria in osteocytes. These results demonstrate that autophagy in osteoblasts contributes to skeletal homeostasis and to the morphological changes associated with osteocyte formation.
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Collin-Osdoby, P; Rothe, L; Bekker, S; Anderson, F; Osdoby, P
2000-03-01
High nitric oxide (NO) levels inhibit osteoclast (OC)-mediated bone resorption in vivo and in vitro, and nitrate donors protect against estrogen-deficient bone loss in postmenopausal women. Conversely, decreased NO production potentiates OC bone resorption in vitro and is associated with in vivo bone loss in rats and humans. Previously, we reported that bone sections from rats administered aminoguanidine (AG), a selective inhibitor of NO production via inducible NO synthase, exhibited both increased OC resorptive activity as well as greater numbers of OC. Here, we investigated further whether AG promoted osteoclastogenesis, in addition to stimulating mature OC function, using a modified in vivo chick chorioallantoic membrane (CAM) system and an in vitro chick bone marrow OC-like cell developmental model. AG, focally administered in small agarose plugs placed directly adjacent to a bone chip implanted on the CAM, dose-dependently elicited neoangiogenesis while stimulating the number, size, and bone pit resorptive activity of individual OC ectopically formed in vivo. In addition to enhancing OC precursor recruitment via neoangiogenesis, AG also exerted other vascular-independent effects on osteoclastogenesis. Thus, AG promoted the in vitro fusion and formation from bone marrow precursor cells of larger OC-like cells that contained more nuclei per cell and exhibited multiple OC differentiation markers. AG stimulated development was inversely correlated with declining medium nitrite levels. In contrast, three different NO donors each dose-dependently inhibited in vitro OC-like cell development while raising medium nitrite levels. Therefore, NO sensitively regulates OC-mediated bone resorption through affecting OC recruitment (angiogenesis), formation (fusion and differentiation), and bone resorptive activity in vitro and in vivo. Possibly, the stimulation of neoangiogenesis and OC-mediated bone remodeling via AG or other pro-angiogenic agents may find clinical applications in reconstructive surgery, fracture repair, or the treatment of avascular necrosis.
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Generation of a Bone Organ by Human Adipose-Derived Stromal Cells Through Endochondral Ossification.
Osinga, Rik; Di Maggio, Nunzia; Todorov, Atanas; Allafi, Nima; Barbero, Andrea; Laurent, Frédéric; Schaefer, Dirk Johannes; Martin, Ivan; Scherberich, Arnaud
2016-08-01
: Recapitulation of endochondral ossification (ECO) (i.e., generation of marrow-containing ossicles through a cartilage intermediate) has relevance to develop human organotypic models for bone or hematopoietic cells and to engineer grafts for bone regeneration. Unlike bone marrow-derived stromal cells (also known as bone marrow-derived mesenchymal stromal/stem cells), adipose-derived stromal cells (ASC) have so far failed to form a bone organ by ECO. The goal of the present study was to assess whether priming human ASC to a defined stage of chondrogenesis in vitro allows their autonomous ECO upon ectopic implantation. ASC were cultured either as micromass pellets or into collagen sponges in chondrogenic medium containing transforming growth factor-β3 and bone morphogenetic protein-6 for 4 weeks (early hypertrophic templates) or for two additional weeks in medium supplemented with β-glycerophosphate, l-thyroxin, and interleukin1-β to induce hypertrophic maturation (late hypertrophic templates). Constructs were implanted in vivo and analyzed after 8 weeks. In vitro, ASC deposited cartilaginous matrix positive for glycosaminoglycans, type II collagen, and Indian hedgehog. Hypertrophic maturation induced upregulation of type X collagen, bone sialoprotein, and matrix metalloproteinase13 (MMP13). In vivo, both early and late hypertrophic templates underwent cartilage remodeling, as assessed by MMP13- and tartrate-resistant acid phosphatase-positive staining, and developed bone ossicles, including bone marrow elements, although to variable degrees of efficiency. In situ hybridization for human-specific sequences and staining with a human specific anti-CD146 antibody demonstrated the direct contribution of ASC to bone and stromal tissue formation. In conclusion, despite their debated skeletal progenitor nature, human ASC can generate bone organs through ECO when suitably primed in vitro. Recapitulation of endochondral ossification (ECO) (i.e., generation of marrow-containing ossicles through a cartilage intermediate) has relevance to develop human organotypic models for bone or hematopoietic cells and to engineer grafts for bone regeneration. This study demonstrated that expanded, human adult adipose-derived stromal cells can generate ectopic bone through ECO, as previously reported for bone marrow stromal cells. This system can be used as a model in a variety of settings for mimicking ECO during development, physiology, or pathology (e.g., to investigate the role of BMPs, their receptors, and signaling pathways). The findings have also translational relevance in the field of bone regeneration, which, despite several advances in the domains of materials and surgical techniques, still faces various limitations before being introduced in the routine clinical practice. ©AlphaMed Press.
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Ma, Hongshi; Luo, Jian; Sun, Zhe; Xia, Lunguo; Shi, Mengchao; Liu, Mingyao; Chang, Jiang; Wu, Chengtie
2016-12-01
Primary bone cancer brings patients great sufferings. To deal with the bone defects resulted from cancer surgery, biomaterials with good bone-forming ability are necessary to repair bone defects. Meanwhile, in order to prevent possible tumor recurrence, it is essential that the remaining tumor cells around bone defects are completely killed. However, there are few biomaterials with the ability of both cancer therapy and bone regeneration until now. Here, we fabricated a 3D-printed bioceramic scaffold with a uniformly self-assembled Ca-P/polydopamine nanolayer surface. Taking advantage of biocompatibility, biodegradability and the excellent photothermal effect of polydopamine, the bifunctional scaffolds with mussel-inspired nanostructures could be used as a satisfactory and controllable photothermal agent, which effectively induced tumor cell death in vitro, and significantly inhibited tumor growth in mice. In addition, owing to the nanostructured surface, the prepared polydopamine-modified bioceramic scaffolds could support the attachment and proliferation of rabbit bone mesenchymal stem cells (rBMSCs), and significantly promoted the formation of new bone tissues in rabbit bone defects even under photothermal treatment. Therefore, the mussel-inspired nanostructures in 3D-printed bioceramic exhibited a remarkable capability for both cancer therapy and bone regeneration, offering a promising strategy to construct bifunctional biomaterials which could be widely used for therapy of tumor-induced tissue defects. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells
DOE Office of Scientific and Technical Information (OSTI.GOV)
Bhargava, U.; Bar-Lev, M.; Bellows, C.G.
When cells enzymatically digested from 21 d fetal rat calvaria are grown in ascorbic acid and Na beta-glycerophosphate, they form discrete three-dimensional nodular structures with the histological and immunohistochemical appearance of woven bone. The present investigation was undertaken to verify that bone-like features were identifiable at the ultrastructural level. The nodules formed on top of a fibroblast-like multilayer of cells. The upper surface of the nodules was lined by a continuous layer of cuboidal osteoblastic cells often seen to be joined by adherens junctions. Numerous microvilli, membrane protrusions, and coated pits could be seen on the upper surface of thesemore » cells, their cytoplasm contained prominent RER and Golgi membranes, and processes extended from their lower surfaces into a dense, highly organized collagenous matrix. Some osteocyte-like cells were completely embedded within this matrix; they also displayed RER and prominent processes which extended through the matrix and often made both adherens and gap junctional contacts with the processes of other cells. The fibroblastic cells not participating in nodule formation were surrounded by a less dense collagenous matrix and, in contrast to the matrix of the nodules, it did not mineralize. An unmineralized osteoid-like layer was seen directly below the cuboidal top layer of cells. A mineralization front was detectable below this in which small, discrete structures resembling matrix vesicles and feathery mineral crystals were evident and frequently associated with the collagen fibrils. More heavily mineralized areas were seen further into the nodule. Electron microprobe and electron and X-ray diffraction analysis confirmed the mineral to be hydroxyapatite.« less
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Composites structures for bone tissue reconstruction
NASA Astrophysics Data System (ADS)
Neto, W.; Santos, João.; Avérous, L.; Schlatter, G.; Bretas, Rosario.
2015-05-01
The search for new biomaterials in the bone reconstitution field is growing continuously as humane life expectation and bone fractures increase. For this purpose, composite materials with biodegradable polymers and hydroxyapatite (HA) have been used. A composite material formed by a film, nanofibers and HA has been made. Both, the films and the non-woven mats of nanofibers were formed by nanocomposites made of butylene adipate-co-terephthalate (PBAT) and HA. The techniques used to produce the films and nanofibers were spin coating and electrospinning, respectively. The composite production and morphology were evaluated. The composite showed an adequate morphology and fibers size to be used as scaffold for cell growth.
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Solitary plasmacytoma of the mandible - a rare entity.
Baad, Rajendra; Kapse, Sonam C; Rathod, Nanita; Sonawane, Kishor; Thete, Sanjay Gangadhar; Kumar, M Naveen
2013-06-01
Plasma cell dyscrasias (multiple myeloma, solitary plasmocytoma of bone and extra medullary plasmocytoma) are cha¬racterized by a monoclonal neoplastic proliferation of plasma cells of which Solitary plasmocytoma of bone (SPB) is a localized form. SPB is most frequently seen in vertebrae and secondarily in long bones. Its presence in jaws is extremely rare. The malignant plasma cells express monotypic cytoplasmic immunoglobulins and plasma cell-associated antigens, with an absence of immature B-cell antigens. Here we report a unique case of plasmacytoma in the right side of mandible, a chronology for diagnosis of the lesion is also reviewed along with clinical, radiographic, histopathological and immunohistochemical evidence. How to cite this article: Baad R, Kapse S C, Rathod N, Sonawane K, Thete S G, Naveen M K. Solitary Plasmacytoma of the Mandible - A Rare Entity. J Int Oral Health 2013; 5(3):97-101.
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NASA Technical Reports Server (NTRS)
Makuch, Lauren A.
2004-01-01
Humans reach peak bone mass at age 30. After this point, we lose 1 to 2 percent of bone mass each decade. In the microgravity environment of space, astronauts lose bone mass at an accelerated rate of 1 to 2 percent each month. When astronauts travel to Mars, they may be in space for as long as 3 years. During this time, they may lose about half of their bone mass from weight-bearing bones. This loss may be irreversible. The drastic loss in bone that astronauts experience in space makes them much more vulnerable to fractures. In addition, the corresponding removal of calcium from bone results in higher levels of calcium in the blood, which increases the risk of developing kidney stones. Currently, studies are being conducted which investigate factors governing bone adaptation and mechanotransduction. Bone is constantly adapting in response to mechanical stimuli. Increased mechanical loading stimulates bone formation and suppresses bone resorption. Reduction in mechanical loading caused by bedrest, disuse, or microgravity results in decreased bone formation and possibly increased bone resorption. Osteoblasts and osteoclasts are the two main cell types that participate in bone remodeling. Osteoblasts are anabolic (bone-forming) cells and osteoclasts are catabolic (bone-resorbing) cells. In microgravity, the activity of osteoblasts slows down and the activity of osteoclasts may speed up, causing a loss of bone density. Mechanotransduction, the molecular mechanism by which mechanical stimuli are converted to biochemical signals, is not yet understood. Exposure of cells to fluid flow imposes a shear stress on the cells. Several studies have shown that the shear stress that results from fluid flow induces a cellular response similar to that induced by mechanical loading. Thus, fluid flow can be used as an in vitro model to simulate the mechanical stress that bone cells experience in vivo. Previous in vitro studies have shown that fluid flow induces several responses in osteoblasts, including increased proliferation, osteoblastic differentiation, alkaline phosphatase activity, and production of nitric oxide, prostaglandins, and osteopontin. Several proteins have been implicated in osteoblastic mechanotransduction including Bone Morphogenetic Protein-2 (BMP-2), parathyroid hormone, 1,25-dihydroxyvitamin D3 receptor, osteopontin (OPN), osteoprotegerin (OPG), and alkaline phosphatase (AP). We will characterize relative levels of each protein in mineralizing or non-mineralizing MC3T3 osteoblastic cells that have been exposed to fluid flow compared to non-fluid flow using immunofluorescent staining and two- photon laser microscopy as well as western blotting. Because calcium-mediated pathways are important in osteoblastic signaling, we will transfect MC3T3 cells with cameleon probes for Ca2+ containing YFP and CFP. Results will be analyzed using FRET/FLIM to study differential release of intracellular Ca(2+) in response to fluid flow and conditions inducing matrix mineralization. In addition, we plan to conduct several microarray experiments to determine differential gene expression in MC3T3 cells in response to fluid flow and conditions inducing mineralization.
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Henning, Robert J
2011-01-01
Myocardial infarction is the leading cause of death among people in industrialized nations. Although the heart has some ability to regenerate after infarction, myocardial restoration is inadequate. Consequently, investigators are currently exploring the use of human embryonic stem cells (hESCs), skeletal myoblasts and adult bone marrow stem cells to limit infarct size. hESCs are pluripotent cells that can regenerate myocardium in infarcted hearts, attenuate heart remodeling and contribute to left ventricle (LV) systolic force development. Since hESCs can form heart teratomas, investigators are differentiating hESCs toward cardiac progenitor cells prior to transplantation into hearts. Large quantities of hESCs cardiac progenitor cells, however, must be generated, immune rejection must be prevented and grafts must survive over the long term to significantly improve myocardial performance. Transplanted autologous skeletal myoblasts can survive in infarcted myocardium in small numbers, proliferate, differentiate into skeletal myofibers and increase the LV ejection fraction. These cells, however, do not form electromechanical connections with host cardiomyocytes. Consequently, electrical re-entry can occur and cause cardiac arrhythmias. Autologous bone marrow mononuclear cells contain hematopoietic and mesenchymal stem cells. In several meta-analyses, patients with coronary disease who received autologous bone marrow cells by intracoronary injection show significant 3.7% (range: 1.9-5.4%) increases in LV ejection fraction, decreases in LV end-systolic volume of -4.8 ml (range: -1.4 to -8.2 ml) and reductions in infarct size of 5.5% (-1.9 to -9.1%), without experiencing arrhythmias. Bone marrow cells appear to release biologically active factors that limit myocardial damage. Unfortunately, bone marrow cells from patients with chronic diseases propagate poorly and can die prematurely. Substantial challenges must be addressed and resolved to advance the use of stem cells in cardiac repair including identifying the optimal stem cell(s) that permit transplantation without requirements for host immune suppression; timing of stem cell transplantation that maximizes chemoattraction of stem cells to infarcts; and determining the optimal technique for injecting stem cells for cardiac repair. Techniques must be developed to enhance survival and propagation of stem cells in the myocardium. These studies will require close cooperation and interaction of scientists and clinicians. Cell-based cardiac repair in the 21st century will offer new hope for millions of patients worldwide with myocardial infarctions who, otherwise, would suffer from the relentless progression of heart disease to heart failure and death.
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NASA Astrophysics Data System (ADS)
Rodionova, Natalia; Katkova, Olena
Adaptive remodeling processes in the skeleton bones occur in the close topographical interconnection with blood capillaries followed by perivascular cells. Radioautographic studies with 3H- thymidine (Kimmel D.B., Fee W.S., 1980; Rodionova N.V., 1989, 2006) has shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic ones. Using electron microscopy and cytochemistry we studied perivsacular cells in metaphysis of the rats femoral bones under conditions of modeling microgravity (28 days duration) and in femoral bones metaphyses of rats flown on board of the space laboratory (Spacelab - 2) It was revealed that population of the perivascular cells is not homogeneous in adaptive zones of the remodeling in both control and test groups (lowering support loading). This population comprises adjacent to endothelium little differentiated forms and isolated cells with differentiation features (specific volume of rough endoplasmic reticulum in cytoplasm is increased). Majority of the perivascular cells in the control group reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In little differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of animals under microgravitaty reaction to the alkaline phosphatase is registered not for all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. There is also visible trend of individual alkaline phosphatase containing perivascular cells amounts decrease (i.e. osteogenic cells-precursors). Under microgravity some little differentiated perivascular cells reveal destruction signs. Found decrease trend of the alkaline phosphatase containing cells (i.e. osteogenic cells) number in perivascular cells population. It is one of the mechanisms of the osteogenic process intensity decrease in bones due to lowering support loading on the bone skeleton. In particular this is confirmed by the fact that in the zones of adaptive remodeling we found fibroblasts and fibrosis zones - areas filled with non mineralized collagen fibrils on the bones surfaces. Hence it should be considered that lowering (removal) support loading slows down (or blocks) osteogenic differentiation of the perivascular cells part and stimulates differentiation of the fibroblast cells. Obtained data considered as one of the cellular mechanisms of the adaptive reactions development in spongy bone under microgravity which could lead to the bone mass loss.
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Gu, Yifei; Huang, Wenhai; Rahaman, Mohamed N; Day, Delbert E
2013-11-01
Previous studies have evaluated the capacity of porous scaffolds composed of a single bioactive glass to regenerate bone. In the present study, scaffolds composed of a mixture of two different bioactive glasses (silicate 13-93 and borate 13-93B3) were created and evaluated for their response to osteogenic MLO-A5 cells in vitro and their capacity to regenerate bone in rat calvarial defects in vivo. The scaffolds, which have similar microstructures (porosity=58-67%) and contain 0, 25, 50 and 100 wt.% 13-93B3 glass, were fabricated by thermally bonding randomly oriented short fibers. The silicate 13-93 scaffolds showed a better capacity to support cell proliferation and alkaline phosphatase activity than the scaffolds containing borate 13-93B3 fibers. The amount of new bone formed in the defects implanted with the 13-93 scaffolds at 12 weeks was 31%, compared to values of 25, 17 and 20%, respectively, for the scaffolds containing 25, 50 and 100% 13-93B3 glass. The amount of new bone formed in the 13-93 scaffolds was significantly higher than in the scaffolds containing 50 and 100% 13-93B3 glass. While the 13-93 fibers were only partially converted to hydroxyapatite at 12 weeks, the 13-93B3 fibers were fully converted and formed a tubular morphology. Scaffolds composed of an optimized mixture of silicate and borate bioactive glasses could provide the requisite architecture to guide bone regeneration combined with a controllable degradation rate that could be beneficial for bone and tissue healing. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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Schaeren, Stefan; Jaquiéry, Claude; Wolf, Francine; Papadimitropoulos, Adam; Barbero, Andrea; Schultz-Thater, Elke; Heberer, Michael; Martin, Ivan
2010-03-15
In this study, we addressed whether Bone Sialoprotein (BSP) coating of various substrates could enhance the in vitro osteogenic differentiation and in vivo bone formation capacity of human Bone Marrow Stromal Cells (BMSC). Moreover, we tested whether synthetic polymer-based porous scaffolds, despite the absence of a mineral component, could support ectopic bone formation by human BMSC if coated with BSP. Adsorption of recombinant human BSP on tissue culture-treated polystyrene (TCTP), beta-tricalcium phosphate (Osteologic) or synthetic polymer (Polyactive) substrates was dose dependent, but did not consistently accelerate or enhance in vitro BMSC osteogenic differentiation, as assessed by the mRNA expression of osteoblast-related genes. Similarly, BSP coating of porous beta-tricalcium phosphate scaffolds (Skelite) did not improve the efficiency of bone tissue formation following loading with BMSC and ectopic implantation in nude mice. Finally, Polyactive foams seeded with BMSC did not form bone tissue in the same ectopic assay, even if coated with BSP. We conclude that BSP coating of a variety of substrates is not directly associated with an enhancement of osteoprogenitor cell differentiation in vitro or in vivo, and that presentation of BSP on polymeric materials is not sufficient to prime BMSC functional osteoblastic differentiation in vivo. (c) 2009 Wiley Periodicals, Inc.
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Short-term implantation effects of a DCPD-based calcium phosphate cement.
Frayssinet, P; Gineste, L; Conte, P; Fages, J; Rouquet, N
1998-06-01
Calcium phosphate cements can be handled in paste form and set in a wet medium after precipitation of calcium phosphate crystals in the implantation site. Depending on the products entering into the chemical reaction leading to the precipitation of calcium phosphates, different phases can be obtained with different mechanical properties, setting times and injectability. We tested a cement composed of a powder, containing beta-tricalcium phosphate (beta-TCP) and sodium pyrophosphate mixed with a solution of phosphoric and sulphuric acids. The cement set under a dicalcium phosphate dihydrate (DCPD)-based matrix containing beta-TCP particles. This was injected with a syringe into a defect drilled in rabbit condyles, the control being an identical defect left empty in the opposite condyle. The condyles were analysed histologically 2, 6 and 18 weeks after implantation. After injection into the bone defect the cement set and formed a porous calcium phosphate structure. Two different calcium phosphate phases with different solubility rates could be identified by scanning electron microscopy (SEM) observation. The less-soluble fragments could be degraded by cell phagocytosis in cell compartments of low pH or integrated in the newly formed bone matrix. The degradation rate of the material was relatively high but compatible with the ingrowth of bone trabeculae within the resorbing material. The ossification process was different from the creeping substitution occurring at the ceramic contact. Bone did not form directly at the cement surface following the differentiation of osteoblasts at the material surface. The trabeculae came to the material surface from the edges of the implantation site. Bone formation in the implantation site was significantly higher than in the control region during the first week of implantation. In conclusion, this material set in situ was well tolerated, inducing a mild foreign-body reaction, which did not impair its replacement by newly formed bone within a few weeks.
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Coronado-Cerda, Erika Evangelina; Franco-Molina, Moisés Armides; Mendoza-Gamboa, Edgar; Prado-García, Heriberto; Rivera-Morales, Lydia Guadalupe; Zapata-Benavides, Pablo; Rodríguez-Salazar, María del Carmen; Caballero-Hernandez, Diana; Tamez-Guerra, Reyes Silvestre; Rodríguez-Padilla, Cristina
2016-01-01
Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU) is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE) or IMMUNEPOTENT CRP® (ICRP) is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM) cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM), cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients. PMID:27191003
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Gopalakrishnapillai, Anilkumar; Kolb, E Anders; Dhanan, Priyanka; Mason, Robert W; Napper, Andrew; Barwe, Sonali P
2015-01-01
The bone marrow microenvironment plays an important role in acute lymphoblastic leukemia (ALL) cell proliferation, maintenance, and resistance to chemotherapy. Annexin II (ANX2) is abundantly expressed on bone marrow cells and complexes with p11 to form ANX2/p11-hetero-tetramer (ANX2T). We present evidence that p11 is upregulated in refractory ALL cell lines and patient samples. A small molecule inhibitor that disrupts ANX2/p11 interaction (ANX2T inhibitor), an anti-ANX2 antibody, and knockdown of p11, abrogated ALL cell adhesion to osteoblasts, indicating that ANX2/p11 interaction facilitates binding and retention of ALL cells in the bone marrow. Furthermore, ANX2T inhibitor increased the sensitivity of primary ALL cells co-cultured with osteoblasts to dexamethasone and vincristine induced cell death. Finally, in an orthotopic leukemia xenograft mouse model, the number of ALL cells homing to the bone marrow was reduced by 40-50% in mice injected with anti-ANX2 antibody, anti-p11 antibody or ANX2T inhibitor compared to respective controls. In a long-term engraftment assay, the percentage of ALL cells in mouse blood, bone marrow and spleen was reduced in mice treated with agents that disrupt ANX2/p11 interaction. These data show that disruption of ANX2/p11 interaction results in reduced ALL cell adhesion to osteoblasts, increased ALL cell sensitization to chemotherapy, and suppression of ALL cell homing and engraftment.
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MedlinePlus Videos and Cool Tools
... body's tissues in exchange for carbon dioxide, which is carried to and eliminated by the lungs. Red blood cells are formed in the red bone marrow ... 2 days. The body makes about two million red blood cells every second. Blood is made up of both cellular and liquid components. ...
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Nga, Nguyen Kim; Hoai, Tran Thanh; Viet, Pham Hung
2015-04-01
This study presents a facile synthesis of biomimetic hydroxyapatite nanorod/poly(D,L) lactic acid (HAp/PDLLA) scaffolds with the use of solvent casting combined with a salt-leaching technique for bone-tissue engineering. Field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive X-ray spectroscopy were used to observe the morphologies, pore structures of synthesized scaffolds, interactions between hydroxyapatite nanorods and poly(D,L) lactic acid, as well as the compositions of the scaffolds, respectively. Porosity of the scaffolds was determined using the liquid substitution method. Moreover, the apatite-forming capability of the scaffolds was evaluated through simulated body fluid (SBF) incubation tests, whereas the viability, attachment, and distribution of human osteoblast cells (MG 63 cell line) on the scaffolds were determined through alamarBlue assay and confocal laser microscopy after nuclear staining with 4',6-diamidino-2-phenylindole and actin filaments of a cytoskeleton with Oregon Green 488 phalloidin. Results showed that hydroxyapatite nanorod/poly(D,L) lactic acid scaffolds that mimic the structure of natural bone were successfully produced. These scaffolds possessed macropore networks with high porosity (80-84%) and mean pore sizes ranging 117-183 μm. These scaffolds demonstrated excellent apatite-forming capabilities. The rapid formation of bone-like apatites with flower-like morphology was observed after 7 days of incubation in SBFs. The scaffolds that had a high percentage (30 wt.%) of hydroxyapatite demonstrated better cell adhesion, proliferation, and distribution than those with low percentages of hydroxyapatite as the days of culture increased. This work presented an efficient route for developing biomimetic composite scaffolds, which have potential applications in bone-tissue engineering. Copyright © 2015 Elsevier B.V. All rights reserved.
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Ng, Angela M H; Tan, K K; Phang, M Y; Aziyati, O; Tan, G H; Isa, M R; Aminuddin, B S; Naseem, M; Fauziah, O; Ruszymah, B H I
2008-05-01
Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering. Copyright 2007 Wiley Periodicals, Inc.
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Wilson, C E; Dhert, W J A; Van Blitterswijk, C A; Verbout, A J; De Bruijn, J D
2002-12-01
Bone tissue engineering using patient derived cells seeded onto porous scaffolds has gained much attention in recent years. Evaluating the viability of these 3D constructs is an essential step in optimizing the process. The alamarBlue (aB) assay was evaluated for its potential to follow in vitro cell proliferation on architecturally standardized hydroxyapatite scaffolds. The impact of the aB assayed and seeding density on subsequent in vivo bone formation was investigated. Twelve scaffolds were seeded with various densities from 250 to 2.5x10(6) cells/scaffold and assay by aB at 5 time points during the 7-day culture period. Twelve additional scaffolds were seeded with 2.5x10(5) cells/scaffold. Two control and 2 aB treated scaffolds were subcutaneously implanted into each of 6 nude mice for 6 weeks. Four observers ranked bone formation using a pair wise comparison of histological sections form each mouse. The aB assay successfully followed cell proliferation, however, the diffusion kinetics of the 3D constructs must be considered. The influence of in vitro aB treatment on subsequent in vivo bone formation cannot be ruled out but was not shown to be significant in the current study. The aB assay appears to be quite promising for evaluating a maximum or end-point viability of 3D tissue engineered constructs. Finally, higher seeding densities resulted in more observed bone formation.
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NASA Technical Reports Server (NTRS)
Ishaug-Riley, S. L.; Crane, G. M.; Gurlek, A.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)
1997-01-01
Porous biodegradable poly(DL-lactic-co-glycolic acid) foams were seeded with rat marrow stromal cells and implanted into the rat mesentery to investigate in vivo bone formation at an ectopic site. Cells were seeded at a density of 6.83 x 10(5) cells/cm2 onto polymer foams having pore sizes ranging from either 150 to 300 to 710 microns and cultured for 7 days in vitro prior to implantation. The polymer/cell constructs were harvested after 1, 7, 28, or 49 days in vivo and processed for histology and gel permeation chromatography. Visual observation of hematoxylin and eosin-stained sections and von Kossa-stained sections revealed the formation of mineralized bonelike tissue in the constructs within 7 days postimplantation. Ingrowth of vascular tissue was also found adjacent to the islands of bone, supplying the necessary metabolic requirements to the newly formed tissue. Mineralization and bone tissue formation were investigated by histomorphometry. The average penetration depth of mineralized tissue in the construct ranged from 190 +/- 50 microns for foams with 500-710-microns pores to 370 +/- 160 microns for foams with 150-300-microns pores after 49 days in vivo. The mineralized bone volume per surface area and total bone volume per surface area had maximal values of 0.28 +/- 0.21 mm (500-710-microns pore size, day 28) and 0.038 +/- 0.024 mm (150-300-microns, day 28), respectively. As much as 11% of the foam volume penetrated by bone tissue was filled with mineralized tissue. No significant trends over time were observed for any of the measured values (penetration depth, bone volume/surface area, or percent mineralized bone volume). These results suggest the feasibility of bone formation by osteoblast transplantation in an orthotopic site where not only bone formation from transplanted cells but also ingrowth from adjacent bone may occur.
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2013-01-01
Background In this study we evaluated a novel approach to guide the bone marrow-driven articular cartilage repair response in skeletally aged rabbits. We hypothesized that dispersed chitosan particles implanted close to the bone marrow degrade in situ in a molecular mass-dependent manner, and attract more stromal cells to the site in aged rabbits compared to the blood clot in untreated controls. Methods Three microdrill hole defects, 1.4 mm diameter and 2 mm deep, were created in both knee trochlea of 30 month-old New Zealand White rabbits. Each of 3 isotonic chitosan solutions (150, 40, 10 kDa, 80% degree of deaceylation, with fluorescent chitosan tracer) was mixed with autologous rabbit whole blood, clotted with Tissue Factor to form cylindrical implants, and press-fit in drill holes in the left knee while contralateral holes received Tissue Factor or no treatment. At day 1 or day 21 post-operative, defects were analyzed by micro-computed tomography, histomorphometry and stereology for bone and soft tissue repair. Results All 3 implants filled the top of defects at day 1 and were partly degraded in situ at 21 days post-operative. All implants attracted neutrophils, osteoclasts and abundant bone marrow-derived stromal cells, stimulated bone resorption followed by new woven bone repair (bone remodeling) and promoted repair tissue-bone integration. 150 kDa chitosan implant was less degraded, and elicited more apoptotic neutrophils and bone resorption than 10 kDa chitosan implant. Drilled controls elicited a poorly integrated fibrous or fibrocartilaginous tissue. Conclusions Pre-solidified implants elicit stromal cells and vigorous bone plate remodeling through a phase involving neutrophil chemotaxis. Pre-solidified chitosan implants are tunable by molecular mass, and could be beneficial for augmented marrow stimulation therapy if the recruited stromal cells can progress to bone and cartilage repair. PMID:23324433
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Thiolloy, Sophie; Edwards, James R.; Fingleton, Barbara; Rifkin, Daniel B.; Matrisian, Lynn M.; Lynch, Conor C.
2012-01-01
Background Breast to bone metastases frequently induce a “vicious cycle” in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. Methodology/Principal Findings To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). Conclusion/Significance Collectively, these studies identify a novel “mini-vicious cycle” between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases. PMID:22238668
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USDA-ARS?s Scientific Manuscript database
Chronic consumption by experimental animals of a typical Western diet high in saturated fats and cholesterol during postnatal life has been demonstrated to impair skeletal development. However, the underlying mechanism by which high fat, energy dense diets affect bone-forming cell phenotypes is poor...
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USDA-ARS?s Scientific Manuscript database
Chronic consumption by experimental animals of a typical Western diet high in saturated fats and cholesterol during postnatal life has been demonstrated to impair skeletal development. However, underlying mechanism by which high fat, energy dense diets affect bone forming cell phenotypes is poorly u...
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Murphy, Kaitlin C.; Fang, Sophia Y.; Leach, J. Kent
2014-01-01
Mesenchymal stem cells (MSC) have great therapeutic potential for the repair of nonhealing bone defects due to their proliferative capacity, multilineage potential, trophic factor secretion, and lack of immunogenicity. However, a major barrier to the translation of cell-based therapies into clinical practice is ensuring their survival and function upon implantation into the defect site. We hypothesized that forming MSC into more physiologic 3-dimensional spheroids, rather than employing dissociated cells from 2-dimensional monolayer culture, would enhance their survival when exposed to a harsh microenvironment while maintaining their osteogenic potential. MSC spheroids were formed using the hanging drop method with increasing cell numbers. Compared to larger spheroids, the smallest spheroids which contained 15,000 cells exhibited increased metabolic activity, reduced apoptosis, and the most uniform distribution of proliferating cells. Spheroids were then entrapped in fibrin gels and cultured in serum-free media and 1% oxygen. Compared to identical numbers of dissociated MSC in fibrin gels, spheroids exhibited significantly reduced apoptosis and secreted up to 100-fold more VEGF. We also observed that fibrin gels containing spheroids and those containing an equivalent number of dissociated cells exhibited similar expression levels of early and late markers of osteogenic differentiation. These data demonstrate that MSC spheroids exhibit greater resistance to apoptosis and enhanced proangiogenic potential, while maintaining similar osteogenic potential to dissociated MSC entrapped in a clinically relevant biomaterial, supporting the use of MSC spheroids in cell-based approaches to bone repair. PMID:24781147
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Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.
Nah, H D; Pacifici, M; Gerstenfeld, L C; Adams, S L; Kirsch, T
2000-03-01
Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.
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Mizukawa, Yuri; Iwasaka, Masakazu
2013-01-01
In the present study, a cellular level response of Cyto-aa3 oxidation was investigated in real time under both time-varying and strong static magnetic fields of 5 T. Two kinds of cells, a slime mold, Physarum polycephalum, and bone forming cells, MC-3T3-E1, were used for the experiments. The oxidation level of the Cyto-aa3 was calculated by optical absorptions at 690 nm, 780 nm and 830 nm. The sample, fiber-optics and an additional optical fiber for light stimulation were set in a solenoidal coil or the bore of a 5-T superconducting magnet. The solenoidal coil for time-varying magnetic fields produced sinusoidal magnetic fields of 6 mT. The slime mold showed a periodic change in Cyto-aa3 oxidation, and the oxidation-reduction cycle of Cyto-aa3 was apparently changed when visible-light irradiated the slime mold. Similarly to the case with light, time-varying magnetic stimulations changed the oxidation-reduction cycle during and after the stimulation for 10 minutes. The same phenomena were observed in the MC-3T3-E1 cell assembly, although their cycle rhythm was comparatively random. Finally, magnetic field exposure of up to 5 T exhibited a distinct suppression of Cyto-aa3 oscillation in the bone forming cells. Exposure up to 5 T was repeated five times, and the change in Cyto-aa3 oxidation reproducibly occurred.
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Multiple vertebral fractures in young man as first manifestation of systemic mastocytosis.
Carrasco Cubero, Carmen; Chamizo Carmona, Eugenio
Systemic mastocytosis (SM) is a clonal disease of mast cell progenitors from the bone marrow. The clinical picture varies from asymptomatic forms (indolent) to a highly aggressive form with a very short (mast cell leukemia) survival. Between 28-34% of patients with SM are related to bone condition at the time of diagnosis and 16% have symptomatic fractures. The presentation of SM as clinical vertebral fractures in young men is rare. Here, we describe a case of established osteoporosis as the only manifestation of SM. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.
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Circular RNAs and hereditary bone diseases.
Zhai, Naixiang; Lu, Yanqin; Wang, Yanzhou; Ren, Xiuzhi; Han, Jinxiang
2018-02-01
Circular RNA (circRNA) is a non-linear form of RNA derived from exonic, intronic, and exon-intron gene regions. circRNAs are characterized by covalent closed loops, highly stable nuclease resistance, and specific expression in species and developmental stages. CircRNA molecules have been identified as playing roles in the regulation of cell transcription, transcriptional expression after translation, interactions with microRNAs, and protein coding. A high stability and tissue- and disease-specific expression allow circRNAs to serve as potential biomarkers both for diseases and prognosis. CircRNAs function in bone remodeling by directly participating in bone-related signaling pathways and by forming the circRNA-miRNA-mRNA axis. Studies have seldom reported on the low incidence of circRNAs in genetic bone disorders. The current study reviews the characteristics of circRNAs and recent research on their role in rare hereditary bone diseases.
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Cheng, Shaohong; Xing, Weirong; Pourteymoor, Sheila; Mohan, Subburaman
2014-10-01
We have previously shown that the increase in osterix (Osx) expression during osteoblast maturation is dependent on the activity of the prolyl hydroxylase domain-containing protein 2 (Phd2), a key regulator of protein levels of the hypoxia-inducible factor family proteins in many tissues. In this study, we generated conditional Phd2 knockout mice (cKO) in osteoblast lineage cells by crossing floxed Phd2 mice with a Col1α2-iCre line to investigate the function of Phd2 in vivo. The cKO mice developed short stature and premature death at 12 to 14 weeks of age. Bone mineral content, bone area, and bone mineral density were decreased in femurs and tibias, but not vertebrae of the cKO mice compared to WT mice. The total volume (TV), bone volume (BV), and bone volume fraction (BV/TV) in the femoral trabecular bones of cKO mice were significantly decreased. Cross-sectional area of the femoral mid-diaphysis was also reduced in the cKO mice. The reduced bone size and trabecular bone volume in the cKO mice were a result of impaired bone formation but not bone resorption as revealed by dynamic histomorphometric analyses. Bone marrow stromal cells derived from cKO mice formed fewer and smaller nodules when cultured with mineralization medium. Quantitative RT-PCR and immunohistochemistry detected reduced expression of Osx, osteocalcin, and bone sialoprotein in cKO bone cells. These data indicate that Phd2 plays an important role in regulating bone formation in part by modulating expression of Osx and bone formation marker genes. © 2014 American Society for Bone and Mineral Research.
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Wang, Yan; Zhou, Lili; Li, Chen; Xie, Han; Lu, Yuwang; Wu, Ying; Liu, Hongwei
2015-01-01
Periodontitis, a disease leads to the formation of periodontal defect, can result in tooth loss if left untreated. The therapies to repair/regenerate periodontal tissues have attracted lots of attention these years. Bone marrow-derived cells (BMDCs), a group of cells containing heterogeneous stem/progenitor cells, are capable of homing to injured tissues and participating in tissue repair/regeneration. The amplification of autologous BMDCs’ potential in homing for self-repair/regeneration, therefore, might be considered as an alternative therapy except for traditional cell transplantation. However, the knowledge of the BMDCs’ homing and participation in periodontal repair/regeneration is still known little. For the purpose of directly observing BMDCs’ involvement in periodontal repair, chimeric mouse models were established to make their bone marrow cells reconstituted with cells expressing green enhanced fluorescence protein (EGFP) in this study. One month after bone marrow transplantation, periodontal defects were made on the mesial side of bilateral maxillary first molars in chimeric mice. The green fluorescence protein-positive (GFP+) BMDCS in periodontal defect regions were examined by bioluminescent imaging and immunofluorescence staining. GFP+ BMDCs were found to aggregate in the periodontal defect regions and emerge in newly-formed bones or fibers. Some of them also co-expressed markers of fibroblasts, osteoblasts or vascular endothelial cells. These results indicated that BMDCs might contribute to the formation of new fibers, bones and blood vessels during periodontal repair. In conclusion, we speculated that autologous BMDCs were capable of negotiating into the surgical sites created by periodontal operation and participating in tissue repair. PMID:26722424
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An Animal Model of Chronic Aplastic Bone Marrow Failure Following Pesticide Exposure in Mice
Chatterjee, Sumanta; Chaklader, Malay; Basak, Pratima; Das, Prosun; Das, Madhurima; Pereira, Jacintha Archana; Dutta, Ranjan Kumar; Chaudhuri, Samaresh; Law, Sujata
2010-01-01
The wide use of pesticides for agriculture, domestic and industrial purposes and evaluation of their subsequent effect is of major concern for public health. Human exposure to these contaminants especially bone marrow with its rapidly renewing cell population is one of the most sensitive tissues to these toxic agents represents a risk for the immune system leading to the onset of different pathologies. In this experimental protocol we have developed a mouse model of pesticide(s) induced hypoplastic/aplastic marrow failure to study quantitative changes in the bone marrow hematopoietic stem cell (BMHSC) population through flowcytometric analysis, defects in the stromal microenvironment through short term adherent cell colony (STACC) forming assay and immune functional capacity of the bone marrow derived cells through cell mediated immune (CMI) parameter study. A time course dependent analysis for consecutive 90 days were performed to monitor the associated changes in the marrow’s physiology after 30th, 60th and 90th days of chronic pesticide exposure. The peripheral blood showed maximum lowering of the blood cell count after 90 days which actually reflected the bone marrow scenario. Severe depression of BMHSC population, immune profile of the bone marrow derived cells and reduction of adherent cell colonies pointed towards an essentially empty and hypoplastic marrow condition that resembled the disease aplastic anemia. The changes were accompanied by splenomegaly and splenic erythroid hyperplasia. In conclusion, this animal model allowed us a better understanding of clinico-biological findings of the disease aplastic anemia following toxic exposure to the pesticide(s) used for agricultural and industrial purposes. PMID:24855541
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Differential growth factor control of bone formation through osteoprogenitor differentiation.
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.
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Jing, Xin; Mi, Hao-Yang; Wang, Xin-Chao; Peng, Xiang-Fang; Turng, Lih-Sheng
2015-04-01
In this work, scaffolds with a shish-kebab (SK) structure formed by poly(ε-caprolactone) (PCL) nanofibers and chitosan-PCL (CS-PCL) copolymers were prepared via electrospinning and subsequent crystallization for bone tissue engineering applications. The aim of this study was to introduce nanosized topography and the high biocompatibility of chitosan onto PCL nanofibers to enhance cell affinity to PCL scaffolds. CS-PCL copolymers with various ratios were synthesized, and then spontaneously crystallized as kebabs onto the electrospun PCL fibers, which acted as shishes. Scanning electron microscopy (SEM) results demonstrated that the copolymer with PCL to chitosan ratio of 8.8 could hierarchically decorate the PCL nanofibers and formed well-shaped kebabs on the PCL nanofiber surface. Water contact angle tests and biomimetic activity experiments revealed that the shish-kebab scaffolds with CS-PCL kebabs (PCL-SK(CS-PCL(8.8))) showed enhanced hydrophilicity and mineralization ability compared with smooth PCL and PCL-SK(PCL) shish-kebab scaffolds. Osteoblast-like MG63 cells cultured on the PCL-SK(CS-PCL(8.8)) scaffolds showed optimizing cell attachment, cell viability, and metabolic activity, demonstrating that this kind of scaffold has potential applications in bone tissue engineering.
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Vahabi, Surena; Torshabi, Maryam; Esmaeil Nejad, Azadeh
2016-12-01
Predictable regeneration of alveolar bone defects has always been a challenge in implant dentistry. Bone allografts are widely used bone substitutes with controversial osteoinductive activity. This in vitro study aimed to assess the osteogenic potential of some commercially available freeze-dried bone allografts supplemented with human recombinant platelet-derived growth factor-BB and transforming growth factor beta-1. Cell viability, mineralization, and osteogenic gene expression of MG-63 osteoblast-like cells were compared among the allograft alone, allograft/platelet-derived growth factor-BB, allograft/transforming growth factor beta-1, and allograft/platelet-derived growth factor-BB/transforming growth factor beta-1 groups. The methyl thiazol tetrazolium assay, real-time quantitative reverse transcription polymerase chain reaction and alizarin red staining were performed, respectively, for assessment of cell viability, differentiation, and mineralization at 24-72 h post treatment. The allograft with greater cytotoxic effect on MG-63 cells caused the lowest differentiation among the groups. In comparison with allograft alone, allograft/transforming growth factor beta-1, and allograft/transforming growth factor beta-1/platelet-derived growth factor-BB caused significant upregulation of bone sialoprotein and osteocalcin osteogenic mid-late marker genes, and resulted in significantly higher amounts of calcified nodules especially in mineralized non-cytotoxic allograft group. Supplementation of platelet-derived growth factor-BB alone in 5 ng/mL concentration had no significant effect on differentiation or mineralization markers. According to the results, transforming growth factor beta-1 acts synergistically with bone allografts to enhance the osteogenic differentiation potential. Therefore, this combination may be useful for rapid transformation of undifferentiated cells into bone-forming cells for bone regeneration. However, platelet-derived growth factor-BB supplementation did not support this synergistic ability to enhance osteogenic differentiation and thus, further investigations are required.
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Tencerova, Michaela; Figeac, Florence; Ditzel, Nicholas; Taipaleenmäki, Hanna; Nielsen, Tina Kamilla; Kassem, Moustapha
2018-06-01
Obesity represents a risk factor for development of insulin resistance and type 2 diabetes. In addition, it has been associated with increased adipocyte formation in the bone marrow (BM) along with increased risk for bone fragility fractures. However, little is known on the cellular mechanisms that link obesity, BM adiposity, and bone fragility. Thus, in an obesity intervention study in C57BL/6J mice fed with a high-fat diet (HFD) for 12 weeks, we investigated the molecular and cellular phenotype of bone marrow adipose tissue (BMAT), BM progenitor cells, and BM microenvironment in comparison to peripheral adipose tissue (AT). HFD decreased trabecular bone mass by 29%, cortical thickness by 5%, and increased BM adiposity by 184%. In contrast to peripheral AT, BMAT did not exhibit pro-inflammatory phenotype. BM progenitor cells isolated from HFD mice exhibited decreased mRNA levels of inflammatory genes (Tnfα, IL1β, Lcn2) and did not manifest an insulin resistant phenotype evidenced by normal levels of pAKT after insulin stimulation as well as normal levels of insulin signaling genes. In addition, BM progenitor cells manifested enhanced adipocyte differentiation in HFD condition. Thus, our data demonstrate that BMAT expansion in response to HFD exerts a deleterious effect on the skeleton. Continuous recruitment of progenitor cells to adipogenesis leads to progenitor cell exhaustion, decreased recruitment to osteoblastic cells, and decreased bone formation. In addition, the absence of insulin resistance and inflammation in the BM suggest that BMAT buffers extra energy in the form of triglycerides and thus plays a role in whole-body energy homeostasis. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.
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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.
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The functional activity of bone tissue cells under space flight conditions.
NASA Astrophysics Data System (ADS)
Rodionova, N. V.; Polkovenko, O. V.; Oganov, V. S.; Nesterenko, O. N.
The space flight conditions affect considerably the state of bone tissue leading to the development of osteoporosis and osteopenia Many aspects of reactions of bone tissue cells still remain unclear until now With the use of electron microscopy we studied the samples gathered from the femoral bone epiphyses and metaphyses of rats flown on board the space laboratory Spacelab -- 2 during 2 weeks It was established that under microgravity conditions there occur remodelling processes in a spongy bone related with a deficit of support load In this work the main attention is focused on studying the ultrastructure of osteogenetic cells and osteoclasts The degree of differentiation and functional state are evaluated according to the degree of development of organelles for specific biosynthesis rough endoplasmic reticulum RER Golgy complex GC as well as the state of mitochondria and cell nucleus As compared with a synchronous control the population of osteogenetic cells from zones of bone reconstruction shows a decrease in the number of functionally active forms We can judge of this from the reduction of a specific volume of RER GC mitochondria in osteoblasts RER loses architectonics typical for osteoblasts and as against the control is represented by short narrow canaliculi distributed throughout the cytoplasm some canals disintegrate GC is slightly pronounced mitochondria become smaller in size and acquire an optically dark matrix These phenomena are supposed to be associated with the desorganization of microtubules and
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Bär, Christian; Povedano, Juan Manuel; Serrano, Rosa; Benitez-Buelga, Carlos; Popkes, Miriam; Formentini, Ivan; Bobadilla, Maria; Bosch, Fatima; Blasco, Maria A
2016-04-07
Aplastic anemia is a fatal bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia. The disease can be hereditary or acquired and develops at any stage of life. A subgroup of the inherited form is caused by replicative impairment of hematopoietic stem and progenitor cells due to very short telomeres as a result of mutations in telomerase and other telomere components. Abnormal telomere shortening is also described in cases of acquired aplastic anemia, most likely secondary to increased turnover of bone marrow stem and progenitor cells. Here, we test the therapeutic efficacy of telomerase activation by using adeno-associated virus (AAV)9 gene therapy vectors carrying the telomerase Tert gene in 2 independent mouse models of aplastic anemia due to short telomeres (Trf1- and Tert-deficient mice). We find that a high dose of AAV9-Tert targets the bone marrow compartment, including hematopoietic stem cells. AAV9-Tert treatment after telomere attrition in bone marrow cells rescues aplastic anemia and mouse survival compared with mice treated with the empty vector. Improved survival is associated with a significant increase in telomere length in peripheral blood and bone marrow cells, as well as improved blood counts. These findings indicate that telomerase gene therapy represents a novel therapeutic strategy to treat aplastic anemia provoked or associated with short telomeres. © 2016 by The American Society of Hematology.
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Controlled release of drugs in electrosprayed nanoparticles for bone tissue engineering.
Jayaraman, Praveena; Gandhimathi, Chinnasamy; Venugopal, Jayarama Reddy; Becker, David Laurence; Ramakrishna, Seeram; Srinivasan, Dinesh Kumar
2015-11-01
Generating porous topographic substrates, by mimicking the native extracellular matrix (ECM) to promote the regeneration of damaged bone tissues, is a challenging process. Generally, scaffolds developed for bone tissue regeneration support bone cell growth and induce bone-forming cells by natural proteins and growth factors. Limitations are often associated with these approaches such as improper scaffold stability, and insufficient cell adhesion, proliferation, differentiation, and mineralization with less growth factor expression. Therefore, the use of engineered nanoparticles has been rapidly increasing in bone tissue engineering (BTE) applications. The electrospray technique is advantageous over other conventional methods as it generates nanomaterials of particle sizes in the micro/nanoscale range. The size and charge of the particles are controlled by regulating the polymer solution flow rate and electric voltage. The unique properties of nanoparticles such as large surface area-to-volume ratio, small size, and higher reactivity make them promising candidates in the field of biomedical engineering. These nanomaterials are extensively used as therapeutic agents and for drug delivery, mimicking ECM, and restoring and improving the functions of damaged organs. The controlled and sustained release of encapsulated drugs, proteins, vaccines, growth factors, cells, and nucleotides from nanoparticles has been well developed in nanomedicine. This review provides an insight into the preparation of nanoparticles by electrospraying technique and illustrates the use of nanoparticles in drug delivery for promoting bone tissue regeneration. Copyright © 2015 Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Ilgenstein, Bernd; Deyhle, Hans; Jaquiery, Claude; Kunz, Christoph; Stalder, Anja; Stübinger, Stefan; Jundt, Gernot; Beckmann, Felix; Müller, Bert; Hieber, Simone E.
2012-10-01
Bone augmentation is a vital part of surgical interventions of the oral and maxillofacial area including dental implantology. Prior to implant placement, sufficient bone volume is needed to reduce the risk of peri-implantitis. While augmentation using harvested autologous bone is still considered as gold standard, many surgeons prefer bone substitutes to reduce operation time and to avoid donor site morbidity. To assess the osteogenic efficacy of commercially available augmentation materials we analyzed drill cores extracted before implant insertion. In younger patients, distraction osteogenesis is successfully applied to correct craniofacial deformities through targeted bone formation. To study the influence of mesenchymal stem cells on bone regeneration during distraction osteogenesis, human mesenchymal stem cells were injected into the distraction gap of nude rat mandibles immediately after osteotomy. The distraction was performed over eleven days to reach a distraction gap of 6 mm. Both the rat mandibles and the drill cores were scanned using synchrotron radiation-based micro computed tomography. The three-dimensional data were manually registered and compared with corresponding two-dimensional histological sections to assess bone regeneration and its morphology. The analysis of the rat mandibles indicates that bone formation is enhanced by mesenchymal stem cells injected before distraction. The bone substitutes yielded a wide range of bone volume and degree of resorption. The volume fraction of the newly formed bone was determined to 34.4% in the computed tomography dataset for the augmentation material Geistlich Bio-Oss®. The combination of computed tomography and histology allowed a complementary assessment for both bone augmentation and distraction osteogenesis.
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Development of bioactive porous α-TCP/HAp beads for bone tissue engineering.
Asaoka, Teruo; Ohtake, Shoji; Furukawa, Katsuko S; Tamura, Akito; Ushida, Takashi
2013-11-01
Porous beads of bioactive ceramics such as hydroxyapatite (HAp) and tribasic calcium phosphate (TCP) are considered a promising scaffold for cultivating bone cells. To realize this, α-TCP/HAp functionally graded porous beads are fabricated with two main purposes: to maintain the function of the scaffold with sufficient strength up to the growth of new bone, and is absorbed completely after the growth. HAp is a bioactive material that has both high strength and strong tissue-adhesive properties, but is not readily absorbed by the human body. On the contrary, α-TCP is highly bioabsorbable, resulting in a scaffold that is absorbed before it is completely replaced by bone. In this study, we produced porous, bead-shaped carriers as scaffolds for osteoblast culture. To control the solubility in vivo, the fabricated beads contained α-TCP at the center and HAp at the surface. Cell adaptability of these beads for bone tissue engineering was confirmed in vitro. It was found that α-TCP/HAp bead carriers exhibit low toxicity in the initial stages of cell seeding and cell adhesion. The presence of HAp in the composite bead form effectively increased ALP activity. In conclusion, it is suggested that these newly developed α-TCP/HAp beads are a promising tool for bone tissue engineering. Copyright © 2013 Wiley Periodicals, Inc.
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Biomimetic nanoclay scaffolds for bone tissue engineering
NASA Astrophysics Data System (ADS)
Ambre, Avinash Harishchandra
Tissue engineering offers a significant potential alternative to conventional methods for rectifying tissue defects by evoking natural regeneration process via interactions between cells and 3D porous scaffolds. Imparting adequate mechanical properties to biodegradable scaffolds for bone tissue engineering is an important challenge and extends from molecular to macroscale. This work focuses on the use of sodium montmorillonite (Na-MMT) to design polymer composite scaffolds having enhanced mechanical properties along with multiple interdependent properties. Materials design beginning at the molecular level was used in which Na-MMT clay was modified with three different unnatural amino acids and further characterized using Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD). Based on improved bicompatibility with human osteoblasts (bone cells) and intermediate increase in d-spacing of MMT clay (shown by XRD), 5-aminovaleric acid modified clay was further used to prepare biopolymer (chitosan-polygalacturonic acid complex) scaffolds. Osteoblast proliferation in biopolymer scaffolds containing 5-aminovaleric acid modified clay was similar to biopolymer scaffolds containing hydroxyapatite (HAP). A novel process based on biomineralization in bone was designed to prepare 5-aminovaleric acid modified clay capable of imparting multiple properties to the scaffolds. Bone-like apatite was mineralized in modified clay and a novel nanoclay-HAP hybrid (in situ HAPclay) was obtained. FTIR spectroscopy indicated a molecular level organic-inorganic association between the intercalated 5-aminovaleric acid and mineralized HAP. Osteoblasts formed clusters on biopolymer composite films prepared with different weight percent compositions of in situ HAPclay. Human MSCs formed mineralized nodules on composite films and mineralized extracellular matrix (ECM) in composite scaffolds without the use of osteogenic supplements. Polycaprolactone (PCL), a synthetic polymer, was used for preparing composites (films and scaffolds) containing in situ HAPclay. Composite films showed significantly improved nanomechanical properties. Human MSCs formed mineralized ECM on films in absence of osteogenic supplements and were able to infiltrate the scaffolds. Atomic force microscopy imaging of mineralized ECM formed on composite films showed similarities in dimensions, arrangement of collagen and apatite with their natural bone counterparts. This work indicates the potential of in situ HAPclay to impart polymeric scaffolds with osteoinductive, osteoconductive abilities and improve their mechanical properties besides emphasizing nanoclays as cell-instructive materials.
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Pauksch, Linda; Hartmann, Sonja; Szalay, Gabor; Alt, Volker; Lips, Katrin S
2014-01-01
Peri-prosthetic infections caused by multidrug resistant bacteria have become a serious problem in surgery and orthopedics. The aim is to introduce biomaterials that avoid implant-related infections caused by multiresistant bacteria. The efficacy of silver nanoparticles (AgNP) against a broad spectrum of bacteria and against multiresistant pathogens has been repeatedly described. In the present study polymethylmethacrylate (PMMA) bone cement functionalized with AgNP and/or gentamicin were tested regarding their biocompatibility with bone forming cells. Therefore, influences on viability, cell number and differentiation of primary human mesenchymal stem cells (MSCs) and MSCs cultured in osteogenic differentiation media (MSC-OM) caused by the implant materials were studied. Furthermore, the growth behavior and the morphology of the cells on the testing material were observed. Finally, we examined the induction of cell stress, regarding antioxidative defense and endoplasmatic reticulum stress. We demonstrated similar cytocompatibility of PMMA loaded with AgNP compared to plain PMMA or PMMA loaded with gentamicin. There was no decrease in cell number, viability and osteogenic differentiation and no induction of cell stress for all three PMMA variants after 21 days. Addition of gentamicin to AgNP-loaded PMMA led to a slight decrease in osteogenic differentiation. Also an increase in cell stress was detectable for PMMA loaded with gentamicin and AgNP. In conclusion, supplementation of PMMA bone cement with gentamicin, AgNP, and both results in bone implants with an antibacterial potency and suitable cytocompatibility in MSCs and MSC-OM.
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The physiopathology of avascular necrosis of the femoral head: an update.
Guerado, Enrique; Caso, Enrique
2016-12-01
The physiopathology of the femoral head bone necrosis is similar for children and for adults. The disease is characterized by apoptosis of bone cells - bone marrow and bone forming cells-resulting in head collapse with a subsequent lesion of the overlying cartilage, and therefore flattening of the rounded surface shape of the head articulating with the acetabulum, provoking, eventually, secondary osteoarthritis. When the disease becomes clinically evident already destructive phenomena have occurred and collapse will eventually ensue. In children, because epiphyseal cartilage has growth capabilities, lost epiphyseal height can be recovered, however in adults collapse is irreversible. In this paper the physiopathology of this disease is examined as well as its implication for treatment. Prevention by genetic studies is discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Development and evaluation of tetrapod-shaped granular artificial bones.
Choi, Sungjin; Liu, I-li; Yamamoto, Kenichi; Igawa, Kazuyo; Mochizuki, Manabu; Sakai, Takamasa; Echigo, Ryosuke; Honnami, Muneki; Suzuki, Shigeki; Chung, Ung-il; Sasaki, Nobuo
2012-07-01
We have developed a novel form of granular artificial bone "Tetrabones" with a homogeneous tetrapod shape and uniform size. Tetrabones are four armed structures that accumulate to form the intergranular pores that allow invasion of cells and blood vessels. In this study we evaluated the physicochemical characteristics of Tetrabones in vitro, and compared their biological and biomechanical properties in vivo to those of conventional β-tricalcium phosphate (β-TCP) granule artificial bone. Both the rupture strength and elastic modulus of Tetrabone particles were higher than those of β-TCP granules in vitro. The connectivity of intergranular pores 100, 300, and 400 μm in size were higher in Tetrabones than in the β-TCP granules. Tetrabones showed similar osteoconductivity and biomechanical stiffness to β-TCP at 2 months after implantation in an in vivo study of canine bone defects. These results suggest that Tetrabones may be a good bone graft material in bone reconstruction. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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Roszkiewicz, J; Roszkiewicz, A
1977-01-01
The degranulation of the C cells, their hypertrophy and hyperplasia leading to the formation adenoma-like nodules were observed under conditions of prolonged hypervitaminosis A which causes bone damage without associated hypercalcemia. These changes which are probably the morphologic manifestation of the increased requirement of the body for calcitonin, connected with the damage of the bone tissue seem to indicate that calcitonin is an important factor essential for the protection of skeleton from its excessive mineralization.
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Miyazaki, H; Inoue, H; Yanagida, M; Horie, K; Mikayama, T; Ohashi, H; Nishikawa, M; Suzuki, T; Sudo, T
1992-08-01
We recently reported the production and characterization of four monoclonal antibodies (MoAbs) against rat platelet glycoprotein IIb/IIIa (GPIIb/IIIa). In this study we developed a simple and efficient three-step procedure, based on positive selection by immunoadsorption (panning) using one MoAb, P55, to purify rat megakaryocyte colony-forming cells (megakaryocyte colony-forming units, CFU-MK) from normal bone marrow. Cells obtained after each step were assayed for their ability to form megakaryocyte colonies in the presence of Concanavalin A (Con A)-stimulated rat spleen cell-conditioned medium in soft agar cultures. Marrow cells were first separated on discontinuous Percoll gradients. Cells sedimented at densities between 1.063 and 1.082 g/ml were depleted of cells adherent to plastic tissue culture dishes. The nonadherent cells were further incubated on dishes coated with P55 MoAb. CFU-MK were enriched about 50-fold in the adsorbed cell fraction. This sequential fractionation procedure resulted in a 345-fold (range 276 to 412-fold) enrichment of rat CFU-MK over whole bone marrow cells. The average cloning efficiency of CFU-MK in the final fraction was about 7% (range 5%-9.2%) of the nucleated cells. The overall recovery of CFU-MK averaged 20% (range 9%-29%). The panning step provided a 46-fold enrichment of megakaryocyte burst-forming cells (megakaryocyte burst-forming units, BFU-MK), whose average cloning efficiency in the post-panning fraction was 0.14% (range 0.07%-0.2%). In addition, erythroid burst-forming cells (erythroid burst-forming units, BFU-E) were also significantly enriched by panning, but to a lesser degree than BFU-MK and CFU-MK. By contrast, granulocyte-macrophage colony-forming cells (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid colony-forming cells (erythroid colony-forming units, CFU-E) were not enriched by panning. CFU-MK obtained after panning formed megakaryocyte colonies in the presence of recombinant rat interleukin 3 (rIL-3), mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF), or human erythropoietin (hEPO), as has been reported for murine CFU-MK in whole marrow cells. The highly enriched populations of rat CFU-MK should thus provide a basis for the further study of the regulation of megakaryocytopoiesis.
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Isolation of osteogenic cells from the trauma-activated periosteum
NASA Astrophysics Data System (ADS)
Wu, Chang-Hsiao; Bullock, John
1987-12-01
Closed, greenstick type fractures were created in adult male white New Zealand rabbits. After a waiting period of 5 days the developing callous and bone approximately 1 cm to each side of the callous was harvested and cell cultures established. Biochemical assays for total protein, alkaline phosphatase activity and glycosamino-glycan content were performed on spent media collected at each change and upon the cells after their termination, in an attempt to more fully characterize the osteoblast population. Since little is known about bone forming cells isolated from this source it is important to establish baseline data so as to be able to relate reactions of these cells to altered environmental conditions.
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Plasma Cell Neoplasms (Including Multiple Myeloma) Treatment (PDQ®)—Patient Version
Plasma cell neoplasms occur when abnormal plasma cells or myeloma cells form tumors in the bones or soft tissues of the body. Multiple myeloma, plasmacytoma, lymphoplasmacytic lymphoma, and monoclonal gammopathy of undetermined significance (MGUS) are different types of plasma cell neoplasms. Find out about risk factors, symptoms, diagnostic tests, prognosis, and treatment for these diseases.
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Silva, Gerluza Aparecida Borges; Bertassoli, Bruno Machado; Sousa, Cristiane Aparecida; Albergaria, Juliano Douglas; de Paula, Rayan Silva; Jorge, Erika Cristina
2018-01-01
Blocks of Bovine bone have shown promising results as implantable scaffolds to promote bone regeneration. Strontium ranelate (SrR) is both an antiresorptive and an anabolic drug that has been indicated for oral administration to treat osteoporosis. Few studies, however, have investigated the local effects of SrR and its use in association with biomaterials thus far. In this work, we investigated SrR effects in cultures of primary osteoblasts (PO, from Wistar rats calvaria) and immortalized osteoblasts (IO, from MC3T3-E1 cell line) cultivated as a monolayer or in association with scaffolds of bovine bone in mineralized (MBB) and demineralized (DBB) forms. The optimum dose to induce SrR effects on cell viability was established as 0.1 mM. Our results suggested that the local administration of SrR is biocompatible and non-cytotoxic. In addition, SrR appeared to accelerate primary osteoblast cell differentiation by enhancing alkaline phosphatase activity, the expression of osteogenic differentiation markers, the synthesis of the organic matrix, and a decrease of Ca 2+ ions in mineralized nodules. DBB was found to be a better scaffold material to promote PO and IO cell proliferation. Exposing the proteins of the demineralized bone matrix might improve scaffold osteoconductive properties. Our results indicated the importance of further investigation of the administration of SrR at sites of bone repair. The association of SrR and bone grafts suggests the possibility of using SrR as a co-adjuvant for bone tissue bioengineering and in bone regeneration therapies.
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LEE, CHANYOUNG; RICHTSMEIER, JOAN T.; KRAFT, REUBEN H.
2017-01-01
Bones of the murine cranial vault are formed by differentiation of mesenchymal cells into osteoblasts, a process that is primarily understood to be controlled by a cascade of reactions between extracellular molecules and cells. We assume that the process can be modeled using Turing’s reaction-diffusion equations, a mathematical model describing the pattern formation controlled by two interacting molecules (activator and inhibitor). In addition to the processes modeled by reaction-diffusion equations, we hypothesize that mechanical stimuli of the cells due to growth of the underlying brain contribute significantly to the process of cell differentiation in cranial vault development. Structural analysis of the surface of the brain was conducted to explore the effects of the mechanical strain on bone formation. We propose a mechanobiological model for the formation of cranial vault bones by coupling the reaction-diffusion model with structural mechanics. The mathematical formulation was solved using the finite volume method. The computational domain and model parameters are determined using a large collection of experimental data that provide precise three dimensional (3D) measures of murine cranial geometry and cranial vault bone formation for specific embryonic time points. The results of this study suggest that mechanical strain contributes information to specific aspects of bone formation. Our mechanobiological model predicts some key features of cranial vault bone formation that were verified by experimental observations including the relative location of ossification centers of individual vault bones, the pattern of cranial vault bone growth over time, and the position of cranial vault sutures. PMID:29225392
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Protective Effects of Selected Botanical Agents on Bone.
Jolly, James Jam; Chin, Kok-Yong; Alias, Ekram; Chua, Kien Hui; Soelaiman, Ima Nirwana
2018-05-11
Osteoporosis is a serious health problem affecting more than 200 million elderly people worldwide. The early symptoms of this disease are hardly detectable. It causes progressive bone loss, which ultimately renders the patients susceptible to fractures. Osteoporosis must be prevented because the associated fragility fractures result in high morbidity, mortality, and healthcare costs. Many plants used in herbal medicine contain bioactive compounds possessing skeletal protective effects. This paper explores the anti-osteoporotic properties of selected herbal plants, including their actions on osteoblasts (bone forming cells), osteoclasts (bone resorbing cells), and bone remodelling. Some of the herbal plant families included in this review are Berberidaceae, Fabaceae, Arecaceae, Labiatae, Simaroubaceaea, and Myrsinaceae. Their active constituents, mechanisms of action, and pharmaceutical applications were discussed. The literature shows that very few herbal plants have undergone human clinical trials to evaluate their pharmacological effects on bone to date. Therefore, more intensive research should be performed on these plants to validate their anti-osteoporotic properties so that they can complement the currently available conventional drugs in the battle against osteoporosis.
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Janardhanan, Sathyanarayana; Wang, Martha O; Fisher, John P
2012-08-01
The use of pluripotent stem cell populations for bone tissue regeneration provides many opportunities and challenges within the bone tissue engineering field. For example, coculture strategies have been utilized to mimic embryological development of bone tissue, and particularly the critical intercellular signaling pathways. While research in bone biology over the last 20 years has expanded our understanding of these intercellular signaling pathways, we still do not fully understand the impact of the system's physical characteristics (orientation, geometry, and morphology). This review of coculture literature delineates the various forms of coculture systems and their respective outcomes when applied to bone tissue engineering. To understand fully the key differences between the different coculture methods, we must appreciate the underlying paradigms of physiological interactions. Recent advances have enabled us to extrapolate these techniques to larger dimensions and higher geometric resolutions. Finally, the contributions of bioreactors, micropatterned biomaterials, and biomaterial interaction platforms are evaluated to give a sense of the sophistication established by a combination of these concepts with coculture systems.
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Sieber, Stefan; Wirth, Lorenz; Cavak, Nino; Koenigsmark, Marielle; Marx, Uwe; Lauster, Roland; Rosowski, Mark
2018-02-01
Multipotent haematopoietic stem and progenitor cells (HSPCs) are the source for all blood cell types. The bone marrow stem cell niche in which the HSPCs are maintained is known to be vital for their maintenance. Unfortunately, to date, no in vitro model exists that accurately mimics the aspects of the bone marrow niche and simultaneously allows the long-term culture of HSPCs. In this study, a novel three-dimensional coculture model is presented, based on a hydroxyapatite coated zirconium oxide scaffold, comprising of human mesenchymal stromal cells (MSCs) and cord blood derived HSPCs, enabling successful HSPC culture for a time span of 28 days within the microfluidic multiorgan chip. The HSPCs were found to stay in their primitive state (CD34 + CD38 - ) and capable of granulocyte, erythrocyte, macrophage, megakaryocyte colony formation. Furthermore, a microenvironment was formed bearing molecular and structural similarity to the in vivo bone marrow niche containing extracellular matrix and signalling molecules known to play an important role in HSPC homeostasis. Here, a novel human in vitro bone marrow model is presented for the first time, capable of long-term culture of primitive HSPCs in a microfluidic environment. Copyright © 2017 John Wiley & Sons, Ltd.
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Regeneration of subcutaneous tissue-engineered mandibular condyle in nude mice.
Wang, Feiyu; Hu, Yihui; He, Dongmei; Zhou, Guangdong; Yang, Xiujuan; Ellis, Edward
2017-06-01
To explore the feasibility of regenerating mandibular condyles based on cartilage cell sheet with cell bone-phase scaffold compared with cell-biphasic scaffolds. Tissue-engineered mandibular condyles were regenerated by the following: 1) cartilage cell sheet + bone-phase scaffold (PCL/HA) seeded with bone marrow stem cells (BMSCs) from minipigs (cell sheet group), and 2) cartilage phase scaffold (PGA/PLA) seeded with auricular chondrocytes + bone-phase scaffold seeded with BMSCs from minipigs (biphasic scaffold group). They were implanted subcutaneously in nude mice after being cultured in vitro for different periods of time. After 12 weeks, the mice were sacrificed, and the specimens were harvested and evaluated based on gross appearance and histopathologic observations with hematoxylin and eosin, safranin O-fast green and immumohistochemical staining for collagen I and II. The histopathologic assessment score of condylar cartilage and bone density were compared between the 2 groups using SPSS 17.0 software. The 2 groups' specimens all formed mature cartilage-like tissues with numerous chondrocytes, typical cartilage lacuna and abundant cartilage-specific extracellular matrix. The regenerated cartilage was instant, continuous, homogeneous and avascular. In the biphasic scaffold group, there were still a few residual PGA fibers in the cartilage layer. The cartilage and bone interface was established in the 2 groups, and the microchannels of the bone-phase scaffolds were filled with bone tissue. The score of cartilage regeneration in the cell sheet group was a little higher than that in the biphasic scaffold group, but the difference was not significant (p > 0.05). There was no significant difference in bone tissue formation between the 2 groups (p > 0.05). Both the cartilage cell sheet group and the biphasic scaffold group of nude mice underwent regeneration of condyle-shaped osteochondral composite. Without residual PGA fibers, the cell sheet group might have less chance of immunological rejection compared to biphasic scaffold group. Copyright © 2017 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
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Liao, Junyi; Wei, Qiang; Zou, Yulong; Fan, Jiaming; Song, Dongzhe; Cui, Jing; Zhang, Wenwen; Zhu, Yunxiao; Ma, Chao; Hu, Xue; Qu, Xiangyang; Chen, Liqun; Yu, Xinyi; Zhang, Zhicai; Wang, Claire; Zhao, Chen; Zeng, Zongyue; Zhang, Ruyi; Yan, Shujuan; Wu, Tingting; Wu, Xingye; Shu, Yi; Lei, Jiayan; Li, Yasha; Luu, Hue H; Lee, Michael J; Reid, Russell R; Ameer, Guillermo A; Wolf, Jennifer Moriatis; He, Tong-Chuan; Huang, Wei
2017-01-01
Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into several lineages including bone. Successful bone formation requires osteogenesis and angiogenesis coupling of MSCs. Here, we investigate if simultaneous activation of BMP9 and Notch signaling yields effective osteogenesis-angiogenesis coupling in MSCs. Recently-characterized immortalized mouse adipose-derived progenitors (iMADs) were used as MSC source. Transgenes BMP9, NICD and dnNotch1 were expressed by adenoviral vectors. Gene expression was determined by qPCR and immunohistochem¡stry. Osteogenic activity was assessed by in vitro assays and in vivo ectopic bone formation model. BMP9 upregulated expression of Notch receptors and ligands in iMADs. Constitutively-active form of Notch1 NICD1 enhanced BMP9-induced osteogenic differentiation both in vitro and in vivo, which was effectively inhibited by dominant-negative form of Notch1 dnNotch1. BMP9- and NICD1-transduced MSCs implanted with a biocompatible scaffold yielded highly mature bone with extensive vascularization. NICD1 enhanced BMP9-induced expression of key angiogenic regulators in iMADs and Vegfa in ectopic bone, which was blunted by dnNotch1. Notch signaling may play an important role in BMP9-induced osteogenesis and angiogenesis. It's conceivable that simultaneous activation of the BMP9 and Notch pathways should efficiently couple osteogenesis and angiogenesis of MSCs for successful bone tissue engineering. © 2017 The Author(s)Published by S. Karger AG, Basel.
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NASA Technical Reports Server (NTRS)
Doty, S. B.
1984-01-01
Rats which were subjected to spaceflight for 2-4 weeks showed considerable loss in ability to form new bone. Animals which are placed into nonweight bearing positions, as a model to simulate the absence of gravity here on the Earth's surface. Show a similar decline in new bone formation. It is suggested that the mechanisms underlying these changes may be the result of reduced transmission of gravitational force to the skeletal cells.
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Phage nanofibers induce vascularized osteogenesis in 3D printed bone scaffolds.
Wang, Jianglin; Yang, Mingying; Zhu, Ye; Wang, Lin; Tomsia, Antoni P; Mao, Chuanbin
2014-08-06
A virus-activated matrix is developed to overcome the challenge of forming vascularized bone tissue. It is generated by filling a 3D printed bioceramic scaffold with phage nanofibers displaying high-density RGD peptide. After it is seeded with mesenchymal stem cells (MSCs) and implanted into a bone defect, the phage nanofibers induce osteogenesis and angiogenesis by activating endothelialization and osteogenic differentiation of MSCs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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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.
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Xiong, Jimin; Mrozik, Krzysztof; Gronthos, Stan
2012-01-01
The epithelial cell rests of Malassez (ERM) are odontogenic epithelial cells located within the periodontal ligament matrix. While their function is unknown, they may support tissue homeostasis and maintain periodontal ligament space or even contribute to periodontal regeneration. We investigated the notion that ERM contain a subpopulation of stem cells that could undergo epithelial–mesenchymal transition and differentiate into mesenchymal stem-like cells with multilineage potential. For this purpose, ERM collected from ovine incisors were subjected to different inductive conditions in vitro, previously developed for the characterization of bone marrow mesenchymal stromal/stem cells (BMSC). We found that ex vivo-expanded ERM expressed both epithelial (cytokeratin-8, E-cadherin, and epithelial membrane protein-1) and BMSC markers (CD44, CD29, and heat shock protein-90β). Integrin α6/CD49f could be used for the enrichment of clonogenic cell clusters [colony-forming units-epithelial cells (CFU-Epi)]. Integrin α6/CD49f-positive-selected epithelial cells demonstrated over 50- and 7-fold greater CFU-Epi than integrin α6/CD49f-negative cells and unfractionated cells, respectively. Importantly, ERM demonstrated stem cell-like properties in their differentiation capacity to form bone, fat, cartilage, and neural cells in vitro. When transplanted into immunocompromised mice, ERM generated bone, cementum-like and Sharpey's fiber-like structures. Additionally, gene expression studies showed that osteogenic induction of ERM triggered an epithelial–mesenchymal transition. In conclusion, ERM are unusual cells that display the morphological and phenotypic characteristics of ectoderm-derived epithelial cells; however, they also have the capacity to differentiate into a mesenchymal phenotype and thus represent a unique stem cell population within the periodontal ligament. PMID:22122577
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Pathophysiology of immobilization osteoporosis
NASA Technical Reports Server (NTRS)
Doty, S. B.; DiCarlo, E. F.
1995-01-01
The reduction of gravity-related forces on the skeleton creates a type of osteoporosis that is unique because its severity is dependent on the mechanical stress bearing function of the skeleton as well as the length of time that the forces are absent or reduced. Bones that bear weight under normal conditions are more affected than bones that normally do not bear weight. The cytokine environment and the cells in the affected bones are altered in time so that stem cells produce fewer new cells and the differentiated cells tend to be less active. These alterations in the local environment of the affected parts appear to resemble those of age- and disease-associated systemic forms of osteoporosis. The osteoporosis produced as a result of the loss of normal activity however, appears to be at least partially reversible through remobilization, strenuous exercise, and--possibly in the future--cytokine therapy.
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T and B cells participate in bone repair by infiltrating the fracture callus in a two-wave fashion.
Könnecke, Ireen; Serra, Alessandro; El Khassawna, Thaqif; Schlundt, Claudia; Schell, Hanna; Hauser, Anja; Ellinghaus, Agnes; Volk, Hans-Dieter; Radbruch, Andreas; Duda, Georg N; Schmidt-Bleek, Katharina
2014-07-01
Fracture healing is a regenerative process in which bone is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process by recruiting accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. In order to answer this question, we analyzed femoral fracture healing in mice by confocal microscopy. Surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site and were detectable predominantly at the femoral neck and knee. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralization. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby the newly forming bone. During healing B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation. Both, osteoblasts and osteoclasts were found to have direct cell-cell contact with lymphocytes, strongly suggesting a regulatory role of the immune cells specifically in the later stages of fracture healing. Copyright © 2014 Elsevier Inc. All rights reserved.
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Goto, Koichi; Goto, Mayumi; Ando-Imaoka, Masako; Kai, Kiyonori; Mori, Kazuhiko
2017-01-01
In order to evaluate drug-induced hematotoxicity in monkey cells in vitro, colony-forming unit-granulocyte, macrophage (CFU-GM), and burst-forming unit-erythroid (BFU-E) colony assays were established using mononuclear cells in the bone marrow collected from male cynomolgus monkeys. Furthermore, the effects of doxorubicin, chloramphenicol, and linezolid on CFU-GM and BFU-E colony formation were investigated using established monkey CFU-GM and BFU-E colony assays in comparison with those on human CFU-GM and BFU-E colonies acquired from human umbilical cord blood cells. Bone marrow mononuclear cells were collected from the ischial or iliac bone of male cynomolgus monkeys. The cells were subsequently processed by density gradient separation at 1.067, 1.070, or 1.077 g/mL for CFU-GM or 1.077 g/mL for BFU-E, and then cultured in methylcellulose medium for 9 or 13 days, respectively. A sufficient number of CFU-GM colonies were formed from mononuclear cells processed at a density of 1.070 g/mL. Moreover, the number of BFU-E colonies from the cells processed at a density of 1.077 g/mL was sufficient for the colony assay. The number of CFU-GM or BFU-E colonies decreased after treatment with the drugs of interest in a concentration-dependent manner. Compared with human CFU-GM, monkey CFU-GM were more sensitive to chloramphenicol and resistant to doxorubicin, whereas monkey BFU-E were more sensitive to all compounds in comparison to the sensitivity of human BFU-E. In conclusion, monkey CFU-GM and BFU-E colony assays were established and considered useful tools to evaluate the differences in drug-induced hematotoxicity between species.
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Composites structures for bone tissue reconstruction
DOE Office of Scientific and Technical Information (OSTI.GOV)
Neto, W.; Santos, João; Avérous, L.
2015-05-22
The search for new biomaterials in the bone reconstitution field is growing continuously as humane life expectation and bone fractures increase. For this purpose, composite materials with biodegradable polymers and hydroxyapatite (HA) have been used. A composite material formed by a film, nanofibers and HA has been made. Both, the films and the non-woven mats of nanofibers were formed by nanocomposites made of butylene adipate-co-terephthalate (PBAT) and HA. The techniques used to produce the films and nanofibers were spin coating and electrospinning, respectively. The composite production and morphology were evaluated. The composite showed an adequate morphology and fibers size tomore » be used as scaffold for cell growth.« less
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Polyurethane foam/nano hydroxyapatite composite as a suitable scaffold for bone tissue regeneration.
Meskinfam, M; Bertoldi, S; Albanese, N; Cerri, A; Tanzi, M C; Imani, R; Baheiraei, N; Farokhi, M; Farè, S
2018-01-01
In bone tissue regeneration, the use of biomineralized scaffolds to create the 3D porous structure needed for well-fitting with defect size and appropriate cell interactions, is a promising alternative to autologous and heterologous bone grafts. Biomineralized polyurethane (PU) foams are here investigated as scaffold for bone tissue regeneration. Biomineralization of the foams was carried out by activation of PU surface by a two steps procedure performed for different times (1 to 4 weeks). Scaffolds were investigated for morphological, chemico-physical and mechanical properties, as well as for in vitro interaction with rat Bone Marrow Mesenchymal Stem Cells (BMSCs). Untreated and biomineralized PU samples showed a homogenous morphology and regular pore size (average Ø=407μm). Phase and structure of formed calcium phosphates (CaPs) layer onto the PU foam were analyzed by Fourier Transform Infrared spectroscopy and X-ray diffraction, proving the formation of bone-like nano hydroxyapatite. Biomineralization caused a significant increase of mechanical properties of treated foams compared to untreated ones. Biomineralization also affected the PU scaffold cytocompatibility providing a more appropriate surface for cell attachment and proliferation. Considering the obtained results, the proposed scaffold can be considered suitable for bone tissue regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.
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Role of TGF-β in breast cancer bone metastases
Chiechi, Antonella; Waning, David L.; Stayrook, Keith R.; Buijs, Jeroen T.; Guise, Theresa A.; Mohammad, Khalid S.
2014-01-01
Breast cancer is the most prevalent cancer among females worldwide leading to approximately 350,000 deaths each year. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in ~70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by increased fracture risk, pain, nerve compression and hypercalcemia, causing severe morbidity. In the bone matrix, transforming growth factor-β (TGF-β) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-β, in turn, stimulates bone metastatic tumor cells to secrete factors that further drive osteolytic bone destruction adjacent to the tumor. Thus, TGF-β is a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-β activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-β signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-β in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-β inhibitors in clinical practice to treat breast cancer bone metastases. PMID:24558636
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Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass
Smoum, Reem; Bar, Arik; Tan, Bo; Milman, Garry; Attar-Namdar, Malka; Ofek, Orr; Stuart, Jordyn M.; Bajayo, Alon; Tam, Joseph; Kram, Vardit; O'Dell, David; Walker, Michael J.; Bradshaw, Heather B.; Bab, Itai; Mechoulam, Raphael
2010-01-01
Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive. PMID:20876113
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Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass.
Smoum, Reem; Bar, Arik; Tan, Bo; Milman, Garry; Attar-Namdar, Malka; Ofek, Orr; Stuart, Jordyn M; Bajayo, Alon; Tam, Joseph; Kram, Vardit; O'Dell, David; Walker, Michael J; Bradshaw, Heather B; Bab, Itai; Mechoulam, Raphael
2010-10-12
Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive.
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Engineering anatomically shaped vascularized bone grafts with hASCs and 3D-printed PCL scaffolds.
Temple, Joshua P; Hutton, Daphne L; Hung, Ben P; Huri, Pinar Yilgor; Cook, Colin A; Kondragunta, Renu; Jia, Xiaofeng; Grayson, Warren L
2014-12-01
The treatment of large craniomaxillofacial bone defects is clinically challenging due to the limited availability of transplantable autologous bone grafts and the complex geometry of the bones. The ability to regenerate new bone tissues that faithfully replicate the anatomy would revolutionize treatment options. Advances in the field of bone tissue engineering over the past few decades offer promising new treatment alternatives using biocompatible scaffold materials and autologous cells. This approach combined with recent advances in three-dimensional (3D) printing technologies may soon allow the generation of large, bioartificial bone grafts with custom, patient-specific architecture. In this study, we use a custom-built 3D printer to develop anatomically shaped polycaprolactone (PCL) scaffolds with varying internal porosities. These scaffolds are assessed for their ability to support induction of human adipose-derived stem cells (hASCs) to form vasculature and bone, two essential components of functional bone tissue. The development of functional tissues is assessed in vitro and in vivo. Finally, we demonstrate the ability to print large mandibular and maxillary bone scaffolds that replicate fine details extracted from patient's computed tomography scans. The findings of this study illustrate the capabilities and potential of 3D printed scaffolds to be used for engineering autologous, anatomically shaped, vascularized bone grafts. © 2014 Wiley Periodicals, Inc.
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An essential role for the association of CD47 to SHPS-1 in skeletal remodeling.
Maile, Laura A; DeMambro, Victoria E; Wai, Christine; Lotinun, Sutada; Aday, Ariel W; Capps, Byron E; Beamer, Wesley G; Rosen, Clifford J; Clemmons, David R
2011-09-01
Integrin-associated protein (IAP/CD47) has been implicated in macrophage-macrophage fusion. To understand the actions of CD47 on skeletal remodeling, we compared Cd47(-/-) mice with Cd47(+/+) controls. Cd47(-/-) mice weighed less and had decreased areal bone mineral density compared with controls. Cd47(-/-) femurs were shorter in length with thinner cortices and exhibited lower trabecular bone volume owing to decreased trabecular number and thickness. Histomorphometry revealed reduced bone-formation and mineral apposition rates, accompanied by decreased osteoblast numbers. No differences in osteoclast number were observed despite a nonsignificant but 40% decrease in eroded surface/bone surface in Cd47(-/-) mice. In vitro, the number of functional osteoclasts formed by differentiating Cd47(-/-) bone marrow cells was significantly decreased compared with wild-type cultures and was associated with a decrease in bone-resorption capacity. Furthermore, by disrupting the CD47-SHPS-1 association, we found that osteoclastogenesis was markedly impaired. Assays for markers of osteoclast maturation suggested that the defect was at the point of fusion and not differentiation and was associated with a lack of SHPS-1 phosphorylation, SHP-1 phosphatase recruitment, and subsequent dephosphorylation of non-muscle cell myosin IIA. We also demonstrated a significant decrease in osteoblastogenesis in bone marrow stromal cells derived from Cd47(-/-) mice. Our finding of cell-autonomous defects in Cd47(-/-) osteoblast and osteoclast differentiation coupled with the pronounced skeletal phenotype of Cd47(-/-) mice support the conclusion that CD47 plays an important role in regulating skeletal acquisition and maintenance through its actions on both bone formation and bone resorption. Copyright © 2011 American Society for Bone and Mineral Research.
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Serum albumin coating of demineralized bone matrix results in stronger new bone formation.
Horváthy, Dénes B; Vácz, Gabriella; Szabó, Tamás; Szigyártó, Imola C; Toró, Ildikó; Vámos, Boglárka; Hornyák, István; Renner, Károly; Klára, Tamás; Szabó, Bence T; Dobó-Nagy, Csaba; Doros, Attila; Lacza, Zsombor
2016-01-01
Blood serum fractions are hotly debated adjuvants in bone replacement therapies. In the present experiment, we coated demineralized bone matrices (DBM) with serum albumin and investigated stem cell attachment in vitro and bone formation in a rat calvaria defect model. In the in vitro experiments, we observed that significantly more cells adhere to the serum albumin coated DBMs at every time point. In vivo bone formation with albumin coated and uncoated DBM was monitored biweekly by computed tomography until 11 weeks postoperatively while empty defects served as controls. By the seventh week, the bone defect in the albumin group was almost completely closed (remaining defect 3.0 ± 2.3%), while uncoated DBM and unfilled control groups still had significant defects (uncoated: 40.2 ± 9.1%, control: 52.4 ± 8.9%). Higher density values were also observed in the albumin coated DBM group. In addition, the serum albumin enhanced group showed significantly higher volume of newly formed bone in the microCT analysis and produced significantly higher breaking force and stiffness compared to the uncoated grafts (peak breaking force: uncoated: 15.7 ± 4 N, albumin 46.1 ± 11 N). In conclusion, this investigation shows that implanting serum albumin coated DBM significantly reduces healing period in nonhealing defects and results in mechanically stronger bone. These results also support the idea that serum albumin coating provides a convenient milieu for stem cell function, and a much improved bone grafting success can be achieved without the use of exogenous stem cells. © 2015 Wiley Periodicals, Inc.
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Development of a Novel Synthetic Drug for Osteoporosis and Fracture Healing
2012-09-01
application to chondrosarcoma , and preparation for bone fracture study. Body Aim 1: Development of salubrinal formulations and determination of...development of bone marrow derived cells, osteoclasts, osteoblasts, and chondrocytes. Application to chondrosarcoma : Besides osteoporosis and...osteoarthritis, we considered a potential application of salubrinal to skeletal malignancies. Approximately one-third of skeletal cancers form chondrosarcoma
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Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa
2009-04-01
The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.
2011-08-05
Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides themore » reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.« less
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Bone-Inspired Spatially Specific Piezoelectricity Induces Bone Regeneration
Yu, Peng; Ning, Chengyun; Zhang, Yu; Tan, Guoxin; Lin, Zefeng; Liu, Shaoxiang; Wang, Xiaolan; Yang, Haoqi; Li, Kang; Yi, Xin; Zhu, Ye; Mao, Chuanbin
2017-01-01
The extracellular matrix of bone can be pictured as a material made of parallel interspersed domains of fibrous piezoelectric collagenous materials and non-piezoelectric non-collagenous materials. To mimic this feature for enhanced bone regeneration, a material made of two parallel interspersed domains, with higher and lower piezoelectricity, respectively, is constructed to form microscale piezoelectric zones (MPZs). The MPZs are produced using a versatile and effective laser-irradiation technique in which K0.5Na0.5NbO3 (KNN) ceramics are selectively irradiated to achieve microzone phase transitions. The phase structure of the laser-irradiated microzones is changed from a mixture of orthorhombic and tetragonal phases (with higher piezoelectricity) to a tetragonal dominant phase (with lower piezoelectricity). The microzoned piezoelectricity distribution results in spatially specific surface charge distribution, enabling the MPZs to bear bone-like microscale electric cues. Hence, the MPZs induce osteogenic differentiation of stem cells in vitro and bone regeneration in vivo even without being seeded with stem cells. The concept of mimicking the spatially specific piezoelectricity in bone will facilitate future research on the rational design of tissue regenerative materials. PMID:28900517
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NASA Astrophysics Data System (ADS)
Tang, Anming; Qian, Yu; Liu, Shuang; Wang, Weijuan; Xu, Bing; Qin, An; Liang, Gaolin
2016-05-01
Osteoporosis (OP) is an important aging-related disease and the effective prevention/treatment of this disease remains challenging. Considering the acidic microenvironment of bone resorption lacunae, herein, we rationally designed two pamidronate (Pami)-derivative and alendronate (Alen)-derivative hydrogelators Pami-D and Alen-D which self-assemble into nanofibers to form supramolecular hydrogels under acidic conditions. Cell viability assay, osteoclastogenesis, osteoclastic gene expression, and in vitro bone resorption results indicated that both Pami-D and Alen-D have better inhibitory effects on osteoclastic formation and bone resorption than Pami and Alen, respectively. We anticipate that our new drugs Pami-D and Alen-D could ``smartly'' self-assemble and locally concentrate the drugs at bone resorption lacunae in vivo and subsequently prevent/treat osteoporosis more efficiently.Osteoporosis (OP) is an important aging-related disease and the effective prevention/treatment of this disease remains challenging. Considering the acidic microenvironment of bone resorption lacunae, herein, we rationally designed two pamidronate (Pami)-derivative and alendronate (Alen)-derivative hydrogelators Pami-D and Alen-D which self-assemble into nanofibers to form supramolecular hydrogels under acidic conditions. Cell viability assay, osteoclastogenesis, osteoclastic gene expression, and in vitro bone resorption results indicated that both Pami-D and Alen-D have better inhibitory effects on osteoclastic formation and bone resorption than Pami and Alen, respectively. We anticipate that our new drugs Pami-D and Alen-D could ``smartly'' self-assemble and locally concentrate the drugs at bone resorption lacunae in vivo and subsequently prevent/treat osteoporosis more efficiently. Electronic supplementary information (ESI) available: Experiment methods and details; syntheses and characterization of Pami-D and Alen-D; HPLC conditions; Fig. S1-S15, Schemes S1 and S2, Tables S1 and S2. See DOI: 10.1039/c6nr00843g
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Liao, Han-Tsung; Jiang, Cho-Pei
2014-01-01
The study described a novel bone tissue scaffold fabricated by computer-aided, air pressure-aided deposition system to control the macro- and microstructure precisely. The porcine bone marrow stem cells (PBMSCs) seeded on either mPEG-PCL-mPEG (PCL) or mPEG-PCL-mPEG/hydroxyapatite (PCL/HA) composite scaffold were cultured under osteogenic medium to test the ability of osteogenesis in vitro. The experimental outcomes indicated that both scaffolds possessed adequate pore size, porosity, and hydrophilicity for the attachment and proliferation of PBMSCs and the PBMSCs expressed upregulated genes of osteogensis and angiogenesis in similar manner on both scaffolds. The major differences between these two types of the scaffolds were the addition of HA leading to higher hardness of PCL/HA scaffold, cell proliferation, and VEGF gene expression in PCL/HA scaffold. However, the in vivo bone forming efficacy between PBMSCs seeded PCL and PCL/HA scaffold was different from the in vitro results. The outcome indicated that the PCL/HA scaffold which had bone-mimetic environment due to the addition of HA resulted in better bone regeneration and mechanical strength than those of PCL scaffold. Therefore, providing a bone-mimetic scaffold is another crucial factor for bone tissue engineering in addition to the biocompatibility, 3D architecture with high porosity, and interpored connection. PMID:24868523
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Derivation of Stromal (Skeletal and Mesenchymal) Stem-Like Cells from Human Embryonic Stem Cells
Harkness, Linda; Abdallah, Basem M.; Elsafadi, Mona; Al-Nbaheen, May S.; Aldahmash, Abdullah; Kassem, Moustapha
2012-01-01
Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESCs) is a prerequisite for their use in clinical applications. However, there is no standard protocol for differentiating hESCs into osteoblastic cells. The aim of this study was to identify the emergence of a human stromal (mesenchymal and skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESCs in a feeder-free environment using serum replacement and as suspension aggregates (embryoid bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106, and CD166 as revealed by immunohistochemical staining and flow cytometry (fluorescence-activated cell sorting) analysis. Ex vivo differentiation of hEBs using bone morphogenic protein 2 (BMP2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold, revealed bone and cartilage, and fibrous tissue elements after 8 weeks. These tissues were of human origin and there was no evidence of differentiation to nonmesodermal tissues. hEBs implanted in the absence of HA/TCP formed vacuolated tissue containing glandular, fibrous and muscle-like tissue elements. Conversely, implantation of undifferentiated hESCs resulted in the formation of a teratoma containing a mixture of endodermal, mesodermal, and ectodermal tissues. Our study demonstrates that hMSC-like cells can be obtained from hESCs and they can be induced to form skeletal tissues in vivo when combined with HA/TCP. These findings are relevant for tissue engineering and suggest that differentiated hEBs can provide an unlimited source for functional osteogenic cells. PMID:22612317
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Al-Hezaimi, Khalid; Ramalingam, Sundar; Al-Askar, Mansour; ArRejaie, Aws S; Nooh, Nasser; Jawad, Fawad; Aldahmash, Abdullah; Atteya, Muhammad; Wang, Cun-Yu
2016-03-30
The aim of the present real time in vivo micro-computed tomography (µCT) and histologic experiment was to assess the efficacy of guided bone regeneration (GBR) around standardized calvarial critical size defects (CSD) using bone marrow-derived mesenchymal stem cells (BMSCs), and collagen membrane (CM) with and without tricalcium phosphate (TCP) graft material. In the calvaria of nine female Sprague-Dawley rats, full-thickness CSD (diameter 4.6 mm) were created under general anesthesia. Treatment-wise, rats were divided into three groups. In group 1, CSD was covered with a resorbable CM; in group 2, BMSCs were filled in CSD and covered with CM; and in group 3, TCP soaked in BMSCs was placed in CSD and covered with CM. All defects were closed using resorbable sutures. Bone volume and bone mineral density of newly formed bone (NFB) and remaining TCP particles and rate of new bone formation was determined at baseline, 2, 4, 6, and 10 weeks using in vivo µCT. At the 10th week, the rats were killed and calvarial segments were assessed histologically. The results showed that the hardness of NFB was similar to that of the native bone in groups 1 and 2 as compared to the NFB in group 3. Likewise, values for the modulus of elasticity were also significantly higher in group 3 compared to groups 1 and 2. This suggests that TCP when used in combination with BMSCs and without CM was unable to form bone of significant strength that could possibly provide mechanical "lock" between the natural bone and NFB. The use of BMSCs as adjuncts to conventional GBR initiated new bone formation as early as 2 weeks of treatment compared to when GBR is attempted without adjunct BMSC therapy.
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Low temperature setting polymer-ceramic composites for bone tissue engineering
NASA Astrophysics Data System (ADS)
Sethuraman, Swaminathan
Tissue engineering is defined as "the application of biological, chemical and engineering principles towards the repair, restoration or regeneration of tissues using scaffolds, cells, factors alone or in combination". The hypothesis of this thesis is that a matrix made of a synthetic biocompatible, biodegradable composite can be designed to mimic the properties of bone, which itself is a composite. The overall goal was to design and develop biodegradable, biocompatible polymer-ceramic composites that will be a practical alternative to current bone repair materials. The first specific aim was to develop and evaluate the osteocompatibility of low temperature self setting calcium deficient apatites for bone tissue engineering. The four different calcium deficient hydroxyapatites evaluated were osteocompatible and expressed the characteristic genes for osteoblast proliferation, maturation, and differentiation. Our next objective was to develop and evaluate the osteocompatibility of biodegradable amino acid ester polyphosphazene in vitro as candidates for forming composites with low temperature apatites. We determined the structure-property relationship, the cellular adhesion, proliferation, and differentiation of primary rat osteoblast cells on two dimensional amino acid ester based polyphosphazene films. Our next goal was to evaluate the amino acid ester based polyphosphazenes in a subcutaneous rat model and our results demonstrated that the polyphosphazenes evaluated in the study were biocompatible. The physio-chemical property characterization, cellular response and gene expression on the composite surfaces were evaluated. The results demonstrated that the precursors formed calcium deficient hydroxyapatite in the presence of biodegradable polyphosphazenes. In addition, cells on the surface of the composites expressed normal phenotype and characteristic genes such as type I collagen, alkaline phosphatase, osteocalcin, osteopontin, and bone sialoprotein. The in vivo study of these novel bone cements in a 5mm unicortical defect in New Zealand white rabbits showed that the implants were osteoconductive, and osteointegrative. In conclusion, the various studies that have been carried out in this thesis to study the feasibility of a bone cement system have shown that these materials are promising candidates for various orthopaedic applications. Overall I believe that these next generation bone cements are promising bone graft substitutes in the armamentarium to treat bone defects.
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Chitosan Composites for Bone Tissue Engineering—An Overview
Venkatesan, Jayachandran; Kim, Se-Kwon
2010-01-01
Bone contains considerable amounts of minerals and proteins. Hydroxyapatite [Ca10(PO4)6(OH)2] is one of the most stable forms of calcium phosphate and it occurs in bones as major component (60 to 65%), along with other materials including collagen, chondroitin sulfate, keratin sulfate and lipids. In recent years, significant progress has been made in organ transplantation, surgical reconstruction and the use of artificial protheses to treat the loss or failure of an organ or bone tissue. Chitosan has played a major role in bone tissue engineering over the last two decades, being a natural polymer obtained from chitin, which forms a major component of crustacean exoskeleton. In recent years, considerable attention has been given to chitosan composite materials and their applications in the field of bone tissue engineering due to its minimal foreign body reactions, an intrinsic antibacterial nature, biocompatibility, biodegradability, and the ability to be molded into various geometries and forms such as porous structures, suitable for cell ingrowth and osteoconduction. The composite of chitosan including hydroxyapatite is very popular because of the biodegradability and biocompatibility in nature. Recently, grafted chitosan natural polymer with carbon nanotubes has been incorporated to increase the mechanical strength of these composites. Chitosan composites are thus emerging as potential materials for artificial bone and bone regeneration in tissue engineering. Herein, the preparation, mechanical properties, chemical interactions and in vitro activity of chitosan composites for bone tissue engineering will be discussed. PMID:20948907
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Expression of Essential B Cell Development Genes in Horses with Common Variable Immunodeficiency
Tallmadge, R.L.; Such, K.A.; Miller, K.C.; Matychak, M.B.; Felippe, M.J.B.
2012-01-01
Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p < 0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p < 0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients. PMID:22464097
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Gambardella, Alessandra; Nagaraju, Chandan K; O'Shea, Patrick J; Mohanty, Sindhu T; Kottam, Lucksy; Pilling, James; Sullivan, Michael; Djerbi, Mounira; Koopmann, Witte; Croucher, Peter I; Bellantuono, Ilaria
2011-04-01
Small molecules are attractive therapeutics to amplify and direct differentiation of stem cells. They also can be used to understand the regulation of their fate by interfering with specific signaling pathways. Mesenchymal stem cells (MSCs) have the potential to proliferate and differentiate into several cell types, including osteoblasts. Activation of canonical Wnt signaling by inhibition of glycogen synthase kinase 3 (GSK-3) has been shown to enhance bone mass, possibly by involving a number of mechanisms ranging from amplification of the mesenchymal stem cell pool to the commitment and differentiation of osteoblasts. Here we have used a highly specific novel inhibitor of GSK-3, AR28, capable of inducing β-catenin nuclear translocation and enhanced bone mass after 14 days of treatment in BALB/c mice. We have shown a temporally regulated increase in the number of colony-forming units-osteoblast (CFU-O) and -adipocyte (CFU-A) but not colony-forming units-fibroblast (CFU-F) in mice treated for 3 days. However, the number of CFU-O and CFU-A returned to normal levels after 14 days of treatment, and the number of CFU-F was decreased significantly. In contrast, the number of osteoblasts increased significantly only after 14 days of treatment, and this was seen together with a significant decrease in bone marrow adiposity. These data suggest that the increased bone mass is the result of an early temporal wave of amplification of a subpopulation of MSCs with both osteogenic and adipogenic potential, which is driven to osteoblast differentiation at the expense of adipogenesis. Copyright © 2011 American Society for Bone and Mineral Research.
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Barger, Anne M; Fan, Timothy M; de Lorimier, Louis-Philippe; Sprandel, Ian T; O'Dell-Anderson, Kristen
2007-01-01
Receptor activator of nuclear factor kappa-B (RANK), RANK-ligand (RANKL), and the soluble decoy receptor osteoprotegerin (OPG) form a key axis modulating osteoclastogenesis. In health, RANKL-expressing bone stromal cells and osteoblasts activate osteoclasts through RANK ligation, resulting in homeostatic bone resorption. Skeletal tumors of dogs and cats, whether primary or metastatic, may express RANKL and directly induce malignant osteolysis. Bone malignancies of dogs and cats may express RANKL, thereby contributing to pathologic bone resorption and pain. Furthermore, relative RANKL expression in bone tumors may correlate with radiographic characteristics of bone pathology. Forty-two dogs and 6 cats with spontaneously-occurring tumors involving bones or soft tissues were evaluated. A polyclonal anti-human RANKL antibody was validated for use in canine and feline cells by flow cytometry and immunocytochemistry. Fifty cytologic specimens were collected from bone and soft tissue tumors of 48 tumor-bearing animals and assessed for RANKL expression. In 15 canine osteosarcoma (OSA) samples, relative RANKL expression was correlated with radiographic characteristics of bone pathology. Expression of RANKL by neoplastic cells was identified in 32/44 canine and 5/6 feline tumor samples. In 15 dogs with OSA, relative RANKL expression did not correlate with either radiographic osteolysis or bone mineral density as assessed by dual energy x-ray absorptiometry. In dogs and cats, tumors classically involving bone and causing pain, often may express RANKL. Confirming RANKL expression in tumors is a necessary step toward the rational institution of novel therapies targeting malignant osteolysis via RANKL antagonism.
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Human Perivascular Stem Cell-Based Bone Graft Substitute Induces Rat Spinal Fusion
Chung, Choon G.; James, Aaron W.; Asatrian, Greg; Chang, Le; Nguyen, Alan; Le, Khoi; Bayani, Georgina; Lee, Robert; Stoker, David; Zhang, Xinli
2014-01-01
Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) because of its abundance and accessibility. We have previously defined a population of native MSCs termed perivascular stem cells (PSCs), purified from diverse human tissues, including adipose tissue. Human PSCs (hPSCs) are a bipartite cell population composed of pericytes (CD146+CD34−CD45−) and adventitial cells (CD146−CD34+CD45−), isolated by fluorescence-activated cell sorting and with properties identical to those of culture identified MSCs. Our previous studies showed that hPSCs exhibit improved bone formation compared with a sample-matched unpurified population (termed stromal vascular fraction); however, it is not known whether hPSCs would be efficacious in a spinal fusion model. To investigate, we evaluated the osteogenic potential of freshly sorted hPSCs without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. We compared increasing dosages of implanted hPSCs to assess for dose-dependent efficacy. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography. Computerized biomechanical simulation (finite element analysis) further demonstrated bone fusion with hPSC treatment. Histological analyses showed robust endochondral ossification in hPSC-treated samples. Finally, we confirmed that implanted hPSCs indeed differentiated into osteoblasts and osteocytes; however, the majority of the new bone formation was of host origin. These results suggest that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. In summary, hPSCs are a readily available MSC population that effectively forms bone without requirements for culture or predifferentiation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair. PMID:25154782
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Hu, Yi-cheng; Liu, Xin; Shen, Ji-jia; He, Jia-cai; Chen, Qiao-er
2014-08-01
To evaluate the effects of bone marrow mesenchymal stem cells (BMSCs) combined with calcium phosphate cement (CPC) scaffold for repair of mandibular defect in Beagle dogs. BMSCs were isolated from Beagle dogs and cultured in DMEM plus 10% FBS. The induction effect was determined using alizarin red staining or alkaline phosphate staining at 14-day of culture. BMSCs were added to the CPC scaffold for animal experiments. In vivo, three critical size bone defects were surgically created in each side of the mandible. The bone defects were repaired with BMSCs-CPC (scaffolds with composite seeding cells), CPC (scaffold alone) or no materials (blank group). Two dogs were sacrificed at 4-week and 8-week after operation. Gross observation, X-ray imaging, histologic and histometric analyses were performed to evaluate the level of bone formation. Newly formed bones were detected within all defect sites after operation. The BMSCs-CPC group and CPC group showed increased bone formation compared with the blank group. The BMSCs-CPC group exhibited more bone formation and degradation of the material than the CPC group. The percentage of new bone in the BMSCs-CPC and CPC treated group were significantly higher than that in the control group (P<0.05), while the percentage of new bone in the BMSCs-CPC sites was higher than that in the CPC sites (P<0.01); the percentage of residual material in the BMSCs-CPC sites was lower than that in the CPC sites (P<0.01) 4 weeks and 8 weeks after operation. Using the theory of tissue engineering, BMSCs composite CPC compound is an effective method in promoting new bone regeneration, which has a positive influence on the bone space preservation.
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Madhu, Vedavathi; Li, Ching-Ju; Dighe, Abhijit S; Balian, Gary; Cui, Quanjun
2014-01-01
Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.
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Development of a Three-Dimensional Bone-Like Construct in a Soft Self-Assembling Peptide Matrix
Marí-Buyé, Núria; Luque, Tomás; Navajas, Daniel
2013-01-01
This work describes the development of a three-dimensional (3D) model of osteogenesis using mouse preosteoblastic MC3T3-E1 cells and a soft synthetic matrix made out of self-assembling peptide nanofibers. By adjusting the matrix stiffness to very low values (around 120 Pa), cells were found to migrate within the matrix, interact forming a cell–cell network, and create a contracted and stiffer structure. Interestingly, during this process, cells spontaneously upregulate the expression of bone-related proteins such as collagen type I, bone sialoprotein, and osteocalcin, indicating that the 3D environment enhances their osteogenic potential. However, unlike MC3T3-E1 cultures in 2D, the addition of dexamethasone is required to acquire a final mature phenotype characterized by features such as matrix mineralization. Moreover, a slight increase in the hydrogel stiffness (threefold) or the addition of a cell contractility inhibitor (Rho kinase inhibitor) abrogates cell elongation, migration, and 3D culture contraction. However, this mechanical inhibition does not seem to noticeably affect the osteogenic process, at least at early culture times. This 3D bone model intends to emphasize cell–cell interactions, which have a critical role during tissue formation, by using a compliant unrestricted synthetic matrix. PMID:23157379
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Beta-catenin-dependent Wnt signaling in mandibular bone regeneration.
Leucht, Philipp; Kim, Jae-Beom; Helms, Jill A
2008-02-01
Osteoblasts are derived from two distinct embryonic lineages: cranial neural crest, and mesoderm. Both populations of cells are capable of forming bone and cartilage during fetal development and during adult bone repair, but whether they use equivalent molecular pathways to achieve osteoblast differentiation is unknown. We addressed this question in the context of cranial repair and focused on the role of Wnt signaling in mandibular skeletal healing. Transgenic Wnt reporter mice were used to pinpoint Wnt-responsive cells in the injury callus, and in situ hybridization was used to identify some of the Wnt ligands expressed by cells during the repair process. A gene transfer technique was employed to abrogate Wnt signaling during mandibular healing, and we found that reparative intramembranous ossification requires a functional Wnt pathway. Finally, we evaluated how constitutive activation of the Wnt pathway, caused by mutation of the LRP5 receptor, affected bone repair in the mandible. Taken together, these data underscore the functional requirement for Wnt signaling in cranial skeletal healing.
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Spontaneous osteosarcoma of the femur in a non-obese diabetic mouse
Hong, Sunhwa; Lee, Hyun-A; Choe, Ohmok; Chung, Youngho
2011-01-01
An abnormal swelling was identified in the distal portion of the right femur in a 1-year-old non-obese diabetic (NOD) mouse. Grossly, a large mass of the distal femur was observed in the right femur. Lesions were poorly marginated, associated with destruction of the cancellous and cortical elements of the bone, and showed ossification within the soft tissue component. Histologically, the tumor was identified as a poorly differentiated sarcoma. Histopathologic examination of the bone masses revealed invasive proliferation of poorly differentiated neoplastic mesenchymal cells forming streams, bundles, and nests, which resulted in destruction of normal bone. Neoplastic cells exhibited random variation in cellular appearance and arrangement, as well as matrix composition and abundance. Haphazard and often intermingling patterns of osteogenic, chondroblastic, lipoblastic, and angiogenic tissues were present. Larger areas of neoplastic bone and hyaline cartilage contained multiple large areas of hemorrhage and necrosis bordered by neoplastic cells. The mass was diagnosed as an osteosarcoma. To our knowledge, this is the first spontaneous osteosarcoma in an NOD mouse. PMID:21998615
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Piemontese, Marilina; Onal, Melda; Xiong, Jinhu; Han, Li; Thostenson, Jeff D.; Almeida, Maria; O’Brien, Charles A.
2016-01-01
Autophagy maintains cell function and homeostasis by recycling intracellular components. This process is also required for morphological changes associated with maturation of some cell types. Osteoblasts are bone forming cells some of which become embedded in bone and differentiate into osteocytes. This transformation includes development of long cellular projections and a reduction in endoplasmic reticulum and mitochondria. We examined the role of autophagy in osteoblasts by deleting Atg7 using an Osterix1-Cre transgene, which causes recombination in osteoblast progenitors and their descendants. Mice lacking Atg7 in the entire osteoblast lineage had low bone mass and fractures associated with reduced numbers of osteoclasts and osteoblasts. Suppression of autophagy also reduced the amount of osteocyte cellular projections and led to retention of endoplasmic reticulum and mitochondria in osteocytes. These results demonstrate that autophagy in osteoblasts contributes to skeletal homeostasis and to the morphological changes associated with osteocyte formation. PMID:27064143
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[Synthesis and characteristics of porous hydroxyapatite bioceramics].
Niu, Jinlong; Zhang, Zhenxi; Jiang, Dazong
2002-06-01
The macroporous structure of human bone allows the ingrowth of the soft tissues and organic cells into the bone matrix, profits the development and metabolism of bone tissue, and adapts the bone to the change of load. There is great requirement for artificial biomimic porous bioactive ceramics with the similar structure of bone tissue that can be used clinically for repairing lost bone. Fine hydroxyapatite (HAp) powder produced by wet chemical reaction was mixed with hydrogen peroxide (H2O2), polyvinyl alcohol, methyl cellulose or other pores-making materials to form green cake. After drying at low temperature (below 100 degrees C) and decarbonizing at about 300 degrees C-400 degrees C, the spongy ceramic block was sintered at high temperature, thus, macroporous HAp bioceramic with interconnected pores and reasonable porosity and pore-diameter was manufactured. This kind of porous HAp bioceramics were intrinsically osteoinductive to a certain degree, but its outstanding property was that they can absorb human bone morphogenetic proteins and other bone growth factors to form composites, so that the macroporous HAp bioactive ceramic has appropriate feasibility for clinical application. From the point of biomedical application, the recent developments in synthesis and characteristics investigation of macroporous HAp are reviewed in this paper.
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Osteogenic differentiation of human dental papilla mesenchymal cells
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ikeda, Etsuko; Hirose, Motohiro; Kotobuki, Noriko
We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of {beta}-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate thatmore » mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.« less
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Single-cell analysis of the fate of c-kit-positive bone marrow cells
NASA Astrophysics Data System (ADS)
Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D.; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa
2017-10-01
The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.
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Single-cell analysis of the fate of c-kit-positive bone marrow cells.
Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa
2017-01-01
The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.
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Zhang, Songjie; Zhou, Min; Ye, Zhaoyang; Zhou, Yan; Tan, Wen-Song
2017-08-01
Slow vascularization often impedes the viability and function of engineered bone replacements. Prevascularization is a promising way to solve this problem. In this study, a new process was developed by integrating microcarrier culture and coculture to fabricate pre-vascularized bone microtissues with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs). Initially, coculture medium and cell ratio between MSCs and HUVECs were optimized in tissue culture plates concerning cell proliferation, osteogenesis and angiogenesis. Subsequently, cells were seeded onto CultiSpher S microcarriers in spinner flasks and subjected to a two-stage (proliferative-osteogenic) culture process for four weeks. Both cells proliferated and functioned well in chosen medium and a 1 : 1 ratio between MSCs and HUVECs was chosen for better angiogenesis. After four weeks of culture in spinner flasks, the microtissues were formed with high cellularity, evenly distributed cells and tube formation ability. While coculture with HUVECs exerted an inhibitory effect on osteogenic differentiation of MSCs, with downregulated alkaline phosphatase activity, mineralization and gene expression of COLI, RUNX2 and OCN, this could be attenuated by employing a delayed seeding strategy of HUVECs against MSCs during the microtissue fabrication process. Collectively, this work established an effective method to fabricate pre-vascularized bone microtissues, which would lay a solid foundation for subsequent development of vascularized tissue grafts for bone regeneration. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation.
Hwang, Queena; Cheifetz, Sela; Overall, Christopher M; McCulloch, Christopher A; Sodek, Jaro
2009-04-22
A recent model for activation of the zymogen form of matrix metalloproteinase 2 (MMP-2, also known as gelatinase A) has suggested that interactions between the SIBLING protein bone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates the conversion of the pro-enzyme into a catalytically active form. This model is particularly relevant to cancer cell metastasis to bone since BSP, bound to the alphavbeta3 integrin through its arginine-glycine-aspartic acid motif, could recruit MMP-2 to the cell surface. We critically assessed the relationship between BSP and proMMP-2 and its activation using various forms of recombinant and purified BSP and MMP-2. Gelatinase and collagenase assays, fluorescence binding assays, real-time PCR, cell culture and pull-down assays were employed to test the model. Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 by BSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. While BSP-mediated invasiveness has been shown to depend on its integrin-binding RGD sequence, analysis of proMMP-2 activation and the level of membrane type 1 (MT1)-MMP in cells grown on a BSP substratum showed that the BSP-alphavbeta3 integrin interaction does not induce the expression of MT1-MMP. These studies do not support a role for BSP in promoting metastasis through interactions with pro-MMP-2.
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Assessment of a new biomimetic scaffold and its effects on bone formation by OCT
NASA Astrophysics Data System (ADS)
Yang, Ying; Aydin, Halil M.; Piskin, Erhan; El Haj, Alicia J.
2009-02-01
The ultimate target of bone tissue engineering is to generate functional load bearing bone. By nature, the porous volume in the trabecular bone is occupied by osseous medulla. The natural bone matrix consists of hydroxyapatite (HA) crystals precipitated along the collagen type I fibres. The mineral phase renders bone strength while collagen provides flexibility. Without mineral component, bone is very flexible and can not bear loads, whereas it is brittle in the case of mineral phase without the collagen presence. In this study, we designed and prepared a new type of scaffold which mimics the features of natural bone. The scaffold consists of three different components, a biphasic polymeric base composed of two different biodegradable polymers prepared by using dual porogen approach and bioactive agents, i.e., collagen and HA particles which are distributed throughout the matrix only in the pore surfaces. Interaction of the bioactive scaffolds possessing very high porosity and interconnected pore structures with cells were investigated in a prolonged culture period by using an osteoblastic cell line. The mineral HA particles have a slight different refractive index from the other elements such as polymeric scaffolds and cell/matrix in a tissue engineering constructs, exhibiting brighter images in OCT. Thus, OCT renders a convenient means to assess the morphology and architecture of the blank biomimetic scaffolds. This study also takes a close observation of OCT images for the cultured cell-scaffold constructs in order to assess neo-formed minerals and matrix. The OCT assessments have been compared with the results from confocal and SEM analysis.
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NASA Astrophysics Data System (ADS)
Rodionova, N.; Oganov, V.; Nosova, L.
The development and differentiation of osteogenic cells in organism happen in closely topographical and functional connection with blood capillaries. We formerly proofed, that small-differentiated cells, which are in the population of perivascular cells are osteogenic cells -precursors . At the present time it is actually to clear up, how these biostructures react on conditions of less of biomechanical load on skeleton bones. We researched peculiarities of blood-bed structure and perivascular cells in metaphises of thighbones and tibial bones in rats, which were onboard the American space station SLS-2 and in experiments of modeling hypokinesia. There were used methods of cytochemistry, histology and electron microscopy. We established, that under the support and functional load decreasing in zones of bones adaptive remodeling, comparatively to control, on histosections the own volume of sinusoid capillaries reduces. The small vessels prevail here. The spaces of sinusoid capillaries are limited by 1 2 cells of the endothelia. Endotheliocytes in- general have the typical ultrastructure. Basal membranes are expressed not-distinctly. Perivascular cells don't create the unbroken layer. The population of these cells is not-homogeneous. It includes enclosed to endothelia small-differentiated forms and separating cells with sings of fibroblastic differentiation (the own volume of rough endoplasmic reticulum in cytoplasm induces). The part of these cells reacts on the alkaline phosphatase (the marker of the osteogenic differentiation). Under the conditions of support load decreasing (especially under the microgravity) there is a tendency to reducing of separating osteogenic cells number. We noted the priority of differentiating fibroblasts. It leads to further development in zones of bone remodeling of hearths of fibrous tissue, that doesn't mineralize. The obtained data are seen as one of mechanisms of osteoporosis and osteopenia development under the deficite of support load.
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Zhang, Hengwei; Sun, Wen; Li, Xing; Wang, Mengmeng; Boyce, Brendan F; Hilton, Matthew J; Xing, Lianping
2016-01-01
Notch signaling plays a critical role in maintaining bone homeostasis partially by controlling the formation of osteoblasts from mesenchymal stem cells (MSCs). We reported that TNF activates Notch signaling in MSCs which inhibits osteoblast differentiation in TNF transgenic (TNF-Tg) mice, a mouse model of chronic inflammatory arthritis. In the current study, we used Hes1-GFP and Hes1-GFP/TNF-Tg mice to study the distribution and dynamic change of Notch active cells in normal and inflammatory bone loss and mechanisms mediating their enhanced proliferation. We found that Hes1-GFP+ cells are composed of cells expressing mesenchymal, hematopoietic and endothelial surface markers. CD45−/Hes1-GFP+ cells express high levels of mesenchymal markers and form CFU-F and CFU-ALP colonies. Expansion of CFU-F colonies is associated with a rapid increase in Hes1-GFP+ cell numbers and their GFP intensity. The GFP signal is lost when a CFU-F colony differentiates into an ALP+ osteoblast colony. TNF increases the numbers of CD45−/Hes1-GFP+ cells, which are stained negatively for osteoblast marker osteocalcin and localized adjacent to endosteal and trabecular bone surfaces. CD45−/Hes1-GFP+ cells in Hes1-GFP/TNF-Tg mice have increased BrdU incorporation and PDGFRβ levels. TNF increases the number of proliferating Hes1-GFP+ cells, which is prevented by a specific PDGFRβ inhibitor. Notch inhibition blocks TNF-mediated PDGFRβ expression and cell proliferation. Thus, TNF-induced MSC proliferation is mediated by PDGFRβ signal, which works at downstream of Notch. Hes1-GFP mice can be used to assess the activation status of Notch in bone cells. PMID:27269414
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Zhang, Hengwei; Sun, Wen; Li, Xing; Wang, Mengmeng; Boyce, Brendan F; Hilton, Matthew J; Xing, Lianping
2016-09-01
Notch signaling plays a critical role in maintaining bone homeostasis partially by controlling the formation of osteoblasts from mesenchymal stem cells (MSCs). We reported that TNF activates Notch signaling in MSCs which inhibits osteoblast differentiation in TNF transgenic (TNF-Tg) mice, a mouse model of chronic inflammatory arthritis. In the current study, we used Hes1-GFP and Hes1-GFP/TNF-Tg mice to study the distribution and dynamic change of Notch active cells in normal and inflammatory bone loss and mechanisms mediating their enhanced proliferation. We found that Hes1-GFP+ cells are composed of cells expressing mesenchymal, hematopoietic and endothelial surface markers. CD45-/Hes1-GFP+ cells express high levels of mesenchymal markers and form CFU-F and CFU-ALP colonies. Expansion of CFU-F colonies is associated with a rapid increase in Hes1-GFP+ cell numbers and their GFP intensity. The GFP signal is lost when a CFU-F colony differentiates into an ALP+ osteoblast colony. TNF increases the numbers of CD45-/Hes1-GFP+ cells, which are stained negatively for osteoblast marker osteocalcin and localized adjacent to endosteal and trabecular bone surfaces. CD45-/Hes1-GFP+ cells in Hes1-GFP/TNF-Tg mice have increased BrdU incorporation and PDGFRβ levels. TNF increases the number of proliferating Hes1-GFP+ cells, which is prevented by a specific PDGFRβ inhibitor. Notch inhibition blocks TNF-mediated PDGFRβ expression and cell proliferation. Thus, TNF-induced MSC proliferation is mediated by PDGFRβ signal, which works at downstream of Notch. Hes1-GFP mice can be used to assess the activation status of Notch in bone cells. Copyright © 2016 Elsevier Inc. All rights reserved.
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Tani-Ishii, N; Wang, C Y; Stashenko, P
1995-08-01
The bone-resorptive cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) have been implicated in the pathogenesis of many chronic inflammatory diseases, including pulpitis and apical periodontitis.To further elucidate their role in these disorders, we have identified cells that express IL-1 alpha and TNF alpha in infected pulps and in developing rat periapical lesions after surgical pulp exposure. As detected by immunohistochemistry, IL-1 alpha- and TNF alpha-positive cells were present as early as 2 days after pulp exposure in both the pulp and periapical region. The numbers of cytokine-expressing cells increased up to day 4 in the pulp and up to day 30 in the periapex. In contrast, cells expressing IL-1 beta and TNF beta, the homologous forms of these mediators, were not found in pulp or periapical lesions during this period. Cells expressing IL-1 alpha and TNF alpha were identified primarily as macrophages and fibroblasts, with occasional staining of polymorphonuclear leukocytes. Osteoblasts and osteoclasts were also positive, whereas lymphocytes were negative. In general, cytokine-expressing cells were located proximal to abscesses and the root apex. These findings demonstrate that cells that express bone-resorptive cytokines IL-1 alpha and TNF alpha are present immediately after pulp exposure in this model, which supports the hypothesis that these mediators play a key role in pulpal and periapical pathogenesis, including the concomitant bone destruction. They also indicate that both resident connective tissue cells as well as infiltrating cells express bone-resorptive cytokines in response to infection in these lesions.
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RIA fractions contain mesenchymal stroma cells with high osteogenic potency.
Kuehlfluck, Pamela; Moghaddam, Arash; Helbig, Lars; Child, Christopher; Wildemann, Britt; Schmidmaier, Gerhard
2015-12-01
The gold standard for treatment of non-union is the transplantation of autologous bone from iliac crest. As an alternative, material can be harvested by femoral reaming with the Reamer-Irrigator-Aspirator(®) (RIA)-System. This material might be a source for human mesenchymal stroma cells (MSCs) with osteogenic potency. The aim of this study was the characterisation of cells harvested with the RIA system and the comparison of their properties with cells isolated from bone marrow ("BM") and fat tissue ("adipose"). The RIA material was separated into the liquid aspiration fraction ("liquid") and the solid RIA fraction. From the solid RIA fraction the cells were cultured either directly ("native") or after collagenase digestion and filtration ("filtrate"). Stem cell characteristics were analysed and the osteogenic potential was investigated in vitro and in vivo. Fat tissue and bone marrow were harvested from nine patients (three women, six males, with a mean of 48.1 years) with atrophic non-union RIA material. The cells were isolated and characterised by flow cytometry, three lineage differentiation capacities and colony-forming unit fibroblast assay. Gene expression profiles were performed and osteogenic differentiation in vivo was analysed. All three RIA fractions contained mesenchymal stromal cells (MSCs) as demonstrated by CFU-F assay, three linage differentiation and surface marker analysis. The RIA-MSCs exhibited a significantly higher osteogenic potential in vitro compared to adipose-MSCs, whereas no difference was seen compared to BM-MSCs. Quantitative RT-PCR analysis revealed an expression of osteogenic markers in all isolated cells. The implantation of MSCs with β-TCP scaffolds into the mice muscle showed significantly higher bone formation for the filtrate RIA-MSC, native RIA-MSC and BM-MSC groups compared to the adipose-MSC group. The filtrate RIA-MSCs formed twice as much new bone in vivo compared to BM-MSCs. The present study showed high potency of cells isolated by reaming. Even in the irrigation fluid, which is normally discarded, cells with the characteristics of stromal stem cells were isolated. In comparison to adipose-MSCs and BM-MSCs, the RIA-MSCs showed a similar or even better osteogenic potential in vitro and in vivo and this supports their usability in orthopaedic surgery. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Matsuda, Saeka; Shoumura, Masahito; Osuga, Naoto; Tsujigiwa, Hidetsugu; Nakano, Keisuke; Okafuji, Norimasa; Ochiai, Takanaga; Hasegawa, Hiromasa; Kawakami, Toshiyuki
2016-01-01
Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantation mouse model. The floor of the pulp chamber in maxillary first molar was perforated using ½ dental round bur. Morphological assessment was carried out by micro CT and microscopy and GFP cell mechanism was further assessed by immunohistochemistry using double fluorescent staining with GFP-S100A4; GFP-Runx2 and GFP-CD31. Results of micro CT revealed alveolar bone resorption and widening of periodontal ligament. Histopathological examination showed proliferation of fibroblasts with some round cells and blood vessels in the granulation tissue. At 2 weeks, the outermost layer of the granulation tissue was lined by squamous cells with distinct intercellular bridges. At 4 weeks, the granulation tissue became larger than the perforation and the outermost layer was lined by relatively typical stratified squamous epithelium. Double immunofluorescent staining of GFP and Runx2 revealed that both proteins were expressed in spindle-shaped cells. Double immunofluorescent staining of GFP and CD31 revealed that both proteins were expressed in vascular endothelial cells in morphologically distinct vessels. The results suggest that fibroblasts, periodontal ligament fibroblasts and blood vessels in granulation tissue were derived from transplanted-bone marrow cells. Thus, essential growth of granulation tissue in periodontal polyp was caused by the migration of undifferentiated mesenchymal cells derived from bone marrow, which differentiated into fibroblasts and later on differentiated into other cells in response to injury.
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In vivo demonstration of cell types in bone that harbor epidermal growth factor receptors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Martineau-Doize, B.; Lai, W.H.; Warshawsky, H.
1988-08-01
The binding and internalization of (/sup 125/I)iodoepidermal growth factor (EGF) by bone cells of the rat was demonstrated in situ by quantitative radioautography. Specific binding sites were observed on a cell profile enriched in endocytic components, including lysosome-like structures, a rough endoplasmic reticulum-rich cell profile, and a cell profile that histologically resembles an undifferentiated precursor cell. By the criteria of gel filtration and precipitability by trichloroacetic acid, most of the bound (/sup 125/I)iodo-EGF was considered intact. By morphological criteria none of the cell profiles that bound (/sup 125/I)iodo-EGF corresponded to fully formed osteoclasts or osteoblasts. The endocytic cell was foundmore » in the epiphyseal plate between the invading capillary and the transverse and longitudinal cartilage septa as well as near osteoclasts in the zone of mixed spicules. The rough endoplasmic reticulum-rich cell was present in vacated chondrocyte lacunae of the epiphyseal plate close to the metaphysis, and the poorly differentiated cell was observed between the mixed spicules of the metaphysis. Similar cell types were also found in the alveolar bone surrounding the incisors. These cells may be the origin of established bone cell lines that harbor high concentrations of EGF receptors and may also be responsible for the humoral hypercalcemia in response to the reported actions of injected EGF or transforming growth factor-alpha as well as that of malignancy.« less
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Zhang, Wenjie; Li, Zihui; Huang, Qingfeng; Xu, Ling; Li, Jinhua; Jin, Yuqin; Wang, Guifang; Liu, Xuanyong; Jiang, Xinquan
2013-01-01
Various methods have been used to modify titanium implant surfaces with the aim of achieving better osseointegration. In this study, we fabricated a clustered nanorod structure on an acid-etched, microstructured titanium plate surface using hydrogen peroxide. We also evaluated biofunctionalization of the hybrid micro/nanorod topography on rat bone marrow mesenchymal stem cells. Scanning electron microscopy and x-ray diffraction were used to investigate the surface topography and phase composition of the modified titanium plate. Rat bone marrow mesenchymal stem cells were cultured and seeded on the plate. The adhesion ability of the cells was then assayed by cell counting at one, 4, and 24 hours after cell seeding, and expression of adhesion-related protein integrin β1 was detected by immunofluorescence. In addition, a polymerase chain reaction assay, alkaline phosphatase and Alizarin Red S staining assays, and osteopontin and osteocalcin immunofluorescence analyses were used to evaluate the osteogenic differentiation behavior of the cells. The hybrid micro/nanoscale texture formed on the titanium surface enhanced the initial adhesion activity of the rat bone marrow mesenchymal stem cells. Importantly, the hierarchical structure promoted osteogenic differentiation of these cells. This study suggests that a hybrid micro/nanorod topography on a titanium surface fabricated by treatment with hydrogen peroxide followed by acid etching might facilitate osseointegration of a titanium implant in vivo.
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Douglas, Timothy E L; Vandrovcová, Marta; Kročilová, Nikola; Keppler, Julia K; Zárubová, Jana; Skirtach, Andre G; Bačáková, Lucie
2018-01-01
Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component β-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
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Biofabrication of tissue constructs by 3D bioprinting of cell-laden microcarriers.
Levato, Riccardo; Visser, Jetze; Planell, Josep A; Engel, Elisabeth; Malda, Jos; Mateos-Timoneda, Miguel A
2014-09-01
Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.
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Synthetic octacalcium phosphate: a possible carrier for mesenchymal stem cells in bone regeneration.
Suzuki, Osamu; Anada, Takahisa
2013-01-01
The present paper reviews biomaterial studies of synthetic octacalcium phosphate (OCP) as a scaffold of osteoblastic cells. OCP crystals have been suggested to be one of precursor phases in hydroxyapatite (HA) crystal formation in bone and tooth. The recent intensive biomaterials and tissue engineering studies using synthetic OCP disclosed the potential function of OCP as a bioactive material as well as synthetic HA materials due to its highly osteoconductive and biodegradable properties. In vitro studies showed that OCP crystals exhibit a positive effect on osteoblastic cell differentiation. In vivo studies confirmed that the materials of OCP in a granule forms and OCP-based composite materials with natural polymers, such as gelatin and collagen, enhance bone regeneration if implanted in various model bone defects with critical-sized diameters, defined as a defect which does not heal spontaneously throughout the lifetime of the animals. One of particular characteristics of OCP, found as a mechanism to enhance bone regeneration in vivo, is a process of progressive conversion from OCP to HA at physiological conditions. The OCP-HA conversion is accompanied by progressive physicochemical changes of the material properties, which affects the tissue reaction around the crystals where osteoblastic cells are encountered. Mesenchymal stem cells (MSCs) seeded in an OCP-based material enhanced bone regeneration in the rat critical-sized calvaria defect more than that by the material alone. The overall results reveal that OCP crystals have an effect on osteoblastic cell differentiation including the differentiation of MSCs in vivo. The evidence collected experimentally in the laboratory was presented.
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Lu, Shun; Wang, Jing; Ye, Jixing; Zou, Yulong; Zhu, Yunxiao; Wei, Qiang; Wang, Xin; Tang, Shengli; Liu, Hao; Fan, Jiaming; Zhang, Fugui; Farina, Evan M; Mohammed, Maryam M; Song, Dongzhe; Liao, Junyi; Huang, Jiayi; Guo, Dan; Lu, Minpeng; Liu, Feng; Liu, Jianxiang; Li, Li; Ma, Chao; Hu, Xue; Lee, Michael J; Reid, Russell R; Ameer, Guillermo A; Zhou, Dongsheng; He, Tongchuan
2016-01-01
Regenerative medicine and bone tissue engineering using mesenchymal stem cells (MSCs) hold great promise as an effective approach to bone and skeletal reconstruction. While adipose tissue harbors MSC-like progenitors, or multipotent adipose-derived cells (MADs), it is important to identify and characterize potential biological factors that can effectively induce osteogenic differentiation of MADs. To overcome the time-consuming and technically challenging process of isolating and culturing primary MADs, here we establish and characterize the reversibly immortalized mouse multipotent adipose-derived cells (iMADs). The isolated mouse primary inguinal MAD cells are reversibly immortalized via the retrovirus-mediated expression of SV40 T antigen flanked with FRT sites. The iMADs are shown to express most common MSC markers. FLP-mediated removal of SV40 T antigen effectively reduces the proliferative activity and cell survival of iMADs, indicating the immortalization is reversible. Using the highly osteogenic BMP9, we find that the iMADs are highly responsive to BMP9 stimulation, express multiple lineage regulators, and undergo osteogenic differentiation in vitro upon BMP9 stimulation. Furthermore, we demonstrate that BMP9-stimulated iMADs form robust ectopic bone with a thermoresponsive biodegradable scaffold material. Collectively, our results demonstrate that the reversibly immortalized iMADs exhibit the characteristics of multipotent MSCs and are highly responsive to BMP9-induced osteogenic differentiation. Thus, the iMADs should provide a valuable resource for the study of MAD biology, which would ultimately enable us to develop novel and efficacious strategies for MAD-based bone tissue engineering.
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Kolk, Andreas; Handschel, Jörg; Drescher, Wolf; Rothamel, Daniel; Kloss, Frank; Blessmann, Marco; Heiland, Max; Wolff, Klaus-Dietrich; Smeets, Ralf
2012-12-01
An autologous bone graft is still the ideal material for the repair of craniofacial defects, but its availability is limited and harvesting can be associated with complications. Bone replacement materials as an alternative have a long history of success. With increasing technological advances the spectrum of grafting materials has broadened to allografts, xenografts, and synthetic materials, providing material specific advantages. A large number of bone-graft substitutes are available including allograft bone preparations such as demineralized bone matrix and calcium-based materials. More and more replacement materials consist of one or more components: an osteoconductive matrix, which supports the ingrowth of new bone; and osteoinductive proteins, which sustain mitogenesis of undifferentiated cells; and osteogenic cells (osteoblasts or osteoblast precursors), which are capable of forming bone in the proper environment. All substitutes can either replace autologous bone or expand an existing amount of autologous bone graft. Because an understanding of the properties of each material enables individual treatment concepts this review presents an overview of the principles of bone replacement, the types of graft materials available, and considers future perspectives. Bone substitutes are undergoing a change from a simple replacement material to an individually created composite biomaterial with osteoinductive properties to enable enhanced defect bridging. Copyright © 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
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Abshagen, K; Schrodi, I; Gerber, T; Vollmar, B
2009-11-01
One of the major challenges in the application of bone substitutes is adequate vascularization and biocompatibility of the implant. Thus, the temporal course of neovascularization and the microvascular inflammatory response of implants of NanoBone (fully synthetic nanocrystalline bone grafting material) were studied in vivo by using the mouse dorsal skinfold chamber model. Angiogenesis, microhemodynamics, and leukocyte-endothelial cell interaction were analyzed repetitively after implantation in the center and in the border zone of the implant up to 15 days. Both NanoBone granules and plates exhibited high biocompatibility comparable to that of cancellous bone, as indicated by a lack of venular leukocyte activation after implantation. In both synthetic NanoBone groups, signs of angiogenesis could be observed even at day 5 after implantation, whereas granules showed higher functional vessel density compared with NanoBone plates. The angiogenic response of the cancellous bone was markedly accelerated in the center of the implant tissue. Histologically, implant tissue showed an ingrowth of vascularized fibrous tissue into the material combined with an increased number of foreign-body giant cells. In conclusion, NanoBone, particularly in granular form, showed high biocompatibility and high angiogenic response, thus improving the healing of bone defects. Our results underline that, beside the composition and nanostructure, the macrostructure is also of importance for the incorporation of the biomaterial by the host tissue. (c) 2008 Wiley Periodicals, Inc.
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Xu, Wen Wen; Li, Bin; Guan, Xin Yuan; Chung, Sookja K.; Wang, Yang; Yip, Yim Ling; Law, Simon Y. K.; Chan, Kin Tak; Lee, Nikki P. Y.; Chan, Kwok Wah; Xu, Li Yan; Li, En Min; Tsao, Sai Wah; He, Qing-Yu; Cheung, Annie L. M.
2017-01-01
Local interactions between cancer cells and stroma can produce systemic effects on distant organs to govern cancer progression. Here we show that IGF2 secreted by inhibitor of differentiation (Id1)-overexpressing oesophageal cancer cells instigates VEGFR1-positive bone marrow cells in the tumour macroenvironment to form pre-metastatic niches at distant sites by increasing VEGF secretion from cancer-associated fibroblasts. Cancer cells are then attracted to the metastatic site via the CXCL5/CXCR2 axis. Bone marrow cells transplanted from nude mice bearing Id1-overexpressing oesophageal tumours enhance tumour growth and metastasis in recipient mice, whereas systemic administration of VEGFR1 antibody abrogates these effects. Mechanistically, IGF2 regulates VEGF in fibroblasts via miR-29c in a p53-dependent manner. Analysis of patient serum samples showed that concurrent elevation of IGF2 and VEGF levels may serve as a prognostic biomarker for oesophageal cancer. These findings suggest that the Id1/IGF2/VEGF/VEGFR1 cascade plays a critical role in tumour-driven pathophysiological processes underlying cancer progression. PMID:28186102
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Xu, Wen Wen; Li, Bin; Guan, Xin Yuan; Chung, Sookja K; Wang, Yang; Yip, Yim Ling; Law, Simon Y K; Chan, Kin Tak; Lee, Nikki P Y; Chan, Kwok Wah; Xu, Li Yan; Li, En Min; Tsao, Sai Wah; He, Qing-Yu; Cheung, Annie L M
2017-02-10
Local interactions between cancer cells and stroma can produce systemic effects on distant organs to govern cancer progression. Here we show that IGF2 secreted by inhibitor of differentiation (Id1)-overexpressing oesophageal cancer cells instigates VEGFR1-positive bone marrow cells in the tumour macroenvironment to form pre-metastatic niches at distant sites by increasing VEGF secretion from cancer-associated fibroblasts. Cancer cells are then attracted to the metastatic site via the CXCL5/CXCR2 axis. Bone marrow cells transplanted from nude mice bearing Id1-overexpressing oesophageal tumours enhance tumour growth and metastasis in recipient mice, whereas systemic administration of VEGFR1 antibody abrogates these effects. Mechanistically, IGF2 regulates VEGF in fibroblasts via miR-29c in a p53-dependent manner. Analysis of patient serum samples showed that concurrent elevation of IGF2 and VEGF levels may serve as a prognostic biomarker for oesophageal cancer. These findings suggest that the Id1/IGF2/VEGF/VEGFR1 cascade plays a critical role in tumour-driven pathophysiological processes underlying cancer progression.
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Tobita, Morikuni; Uysal, Cagri A; Guo, Xin; Hyakusoku, Hiko; Mizuno, Hiroshi
2013-12-01
One goal of periodontal therapy is to regenerate periodontal tissues. Stem cells, growth factors and scaffolds and biomaterials are vital for the restoration of the architecture and function of complex tissues. Adipose tissue-derived stem cells (ASCs) are an ideal population of stem cells for practical regenerative medicine. In addition, platelet-rich plasma (PRP) can be useful for its ability to stimulate tissue regeneration. PRP contains various growth factors and may be useful as a cell carrier in stem cell therapies. The purpose of this study was to determine whether a mixture of ASCs and PRP promoted periodontal tissue regeneration in a canine model. Autologous ASCs and PRP were implanted into areas with periodontal tissue defects. Periodontal tissue defects that received PRP alone or non-implantation were also examined. Histologic, immunohistologic and x-ray studies were performed 1 or 2 months after implantation. The amount of newly formed bone and the scale of newly formed cementum in the region of the periodontal tissue defect were analyzed on tissue sections. The areas of newly formed bone and cementum were greater 2 months after implantation of ASCs and PRP than at 1 month after implantation, and the radiopacity in the region of the periodontal tissue defect increased markedly by 2 months after implantation. The ASCs and PRP group exhibited periodontal tissue with the correct architecture, including alveolar bone, cementum-like structures and periodontal ligament-like structures, by 2 months after implantation. These findings suggest that a combination of autologous ASCs and PRP promotes periodontal tissue regeneration that develops the appropriate architecture for this complex tissue. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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Alluri, Ram; Jakus, Adam; Bougioukli, Sofia; Pannell, William; Sugiyama, Osamu; Tang, Amy; Shah, Ramille; Lieberman, Jay R
2018-04-01
The purpose of this study was to evaluate the viability of human adipose-derived stem cells (ADSCs) transduced with a lentiviral (LV) vector to overexpress bone morphogenetic protein-2 (BMP-2) loaded onto a novel 3D printed scaffold. Human ADSCs were transduced with a LV vector carrying the cDNA for BMP-2. The transduced cells were loaded onto a 3D printed Hyperelastic "Bone" (HB) scaffold. In vitro BMP-2 production was assessed using enzyme-linked immunosorbent assay analysis. The ability of ADSCs loaded on the HB scaffold to induce in vivo bone formation in a hind limb muscle pouch model was assessed in the following groups: ADSCs transduced with LV-BMP-2, LV-green fluorescent protein, ADSCs alone, and empty HB scaffolds. Bone formation was assessed using radiographs, histology and histomorphometry. Transduced ADSCs BMP-2 production on the HB scaffold at 24 hours was similar on 3D printed HB scaffolds versus control wells with transduced cells alone, and continued to increase after 1 and 2 weeks of culture. Bone formation was noted in LV-BMP-2 animals on plain radiographs at 2 and 4 weeks after implantation; no bone formation was noted in the other groups. Histology demonstrated that the LV-BMP-2 group was the only group that formed woven bone and the mean bone area/tissue area was significantly greater when compared with the other groups. 3D printed HB scaffolds are effective carriers for transduced ADSCs to promote bone repair. The combination of gene therapy and tissue engineered scaffolds is a promising multidisciplinary approach to bone repair with significant clinical potential. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1104-1110, 2018. © 2018 Wiley Periodicals, Inc.
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Qu, Xue; Cui, Wenjin; Yang, Fei; Min, Changchun; Shen, Hong; Bei, Jianzhong; Wang, Shenguo
2007-01-01
In this study, biodegradable poly(lactide-co-glycolide) (PLGA) (70/30) films and scaffolds were first treated with oxygen plasma and then incubated in a modified simulated body fluid 1.5SBF0 to prepare a bone-like apatite layer. The formation of the apatite and its influence on osteoblast-like cells growth were investigated. It was found that the bone-like apatite formability of PLGA(70/30) was enhanced by plasma pretreatment. The changes of surface chemistry and surface topography induced by oxygen plasma treatment were both effective for apatite formation. The apatite formability increased with increasing plasma-treating time. Under a treating condition of 20 W for 30 min, oxygen plasma treatment could penetrate into the inner scaffold. After 6 days incubation, the apatite formed in plasma-treated scaffold was better distributed than in untreated scaffold, and the weight and mechanical strength of the plasma-treated scaffold were both enhanced. Compared with PLGA(70/30), the apatite layer formed on oxygen plasma-treated PLGA(70/30) surface enhanced adhesion and proliferation of OCT-1 osteoblast-like cell, but had no significant effect on cell's ALP activity at day 7. A prolonged investigation is being in process to further verify the bone-like apatite effects on osteogenic differentiation.
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Long, Mian; Li, Shun-xiang; Xiao, Jiang-feng; Wang, Jian; Lozanoff, Scott; Zhang, Zhi-guang; Luft, Benjamin J; Johnson, Francis
2014-09-01
To study, at the cytological level, the basic concept of Chinese medicine that "the Kidney (Shen) controls the bone". Kaempferol was isolated form Rhizoma Drynariae (Gu Sui Bu, GSB) and at several concentrations was incubated with opossum kidney (OK) cells, osteoblasts (MC3T3 E1) and human fibroblasts (HF) at cell concentrations of 2×10(4)/mL. Opossum kidney cell-conditioned culture media with kaempferol at 70 nmol/L (70kaeOKM) and without kaempferol (0OKM) were used to stimulate MC3T3 E1 and HF proliferation. The bone morphological protein receptors I and II (BMPR I and II) in OK cells were identified by immune-fluorescence staining and Western blot analysis. Kaempferol was found to increase OK cell growth (P<0.05), but alone did not promote MC3T3 E1 or HF cell proliferation. However, although OKM by itself increased MC3T3 E1 growth by 198% (P<0.01), the 70kaeOKM further increased the growth of these cells by an additional 127% (P<0.01). It indicates that the kidney cell generates a previously unknown osteoblast growth factor (OGF) and kaempferol increases kidney cell secretion of OGF. Neither of these media had any significant effect on HF growth. Kaempferol also was found to increase the level of the BMPR II in OK cells. This lends strong support to the original idea that the Kidney has a significant influence over bone-formation, as suggested by some long-standing Chinese medical beliefs, kaempferol may also serve to stimulate kidney repair and indirectly stimulate bone formation.
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Son, Jun-Sik; Kim, Su-Gwan; Oh, Ji-Su; Appleford, Mark; Oh, Sunho; Ong, Joo L; Lee, Kyu-Bok
2011-12-15
This study presents a novel design of a ceramic/polymer biphasic combination scaffold that mimics natural bone structures and is used as a bone graft substitute. To mimic the natural bone structures, the outside cortical-like shells were composed of porous hydroxyapatite (HA) with a hollow interior using a polymeric template-coating technique; the inner trabecular-like core consisted of porous poly(D,L-lactic acid) (PLA) that was loaded with dexamethasone (DEX) and was directly produced using a particle leaching/gas forming technique to create the inner diameter of the HA scaffold. It was observed that the HA and PLA parts of the fabricated HA/PLA biphasic scaffold contained open and interconnected pore structures, and the boundary between both parts was tightly connected without any gaps. It was found that the structure of the combination scaffold was analogous to that of natural bone based on micro-computed tomography analysis. Additionally, the dense, uniform apatite layer was formed on the surface of the HA/PLA biphasic scaffold through a biomimetic process, and DEX was successfully released from the PLA of the biphasic scaffold over a 1-month period. This release caused human embryonic palatal mesenchyme cells to proliferate, differentiate, produce ECM, and form tissue in vitro. Therefore, it was concluded that this functionally graded scaffold is similar to natural bone and represents a potential bone-substitute material. Copyright © 2011 Wiley Periodicals, Inc.
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E, Ling-Ling; Xu, Wen-Huan; Feng, Lin; Liu, Yi; Cai, Dong-Qing; Wen, Ning; Zheng, Wen-Jie
2016-06-01
This study investigated the effects of estrogen on the bone regeneration potential of periodontal ligament stem cells (PDLSCs) derived from osteoporotic rats and seeded on a collagen-based composite scaffold [nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA)]. For this purpose, 48 healthy 3‑month-old Sprague-Dawley female rats were divided into 2 groups as follows: the bilaterally ovariectomized (OVX) rats and sham‑operated rats. The PDLSCs were isolated at 3 months after surgery (by which time postmenopausal osteoporosis had developed). The effects of estrogen on the characteristics of these cells seeded in a culture plate and of the cells seeded on nHAC/PLA were then investigated. The PDLSC + nHAC/PLA constructs were implanted subcutaneously into the backs of severe combined immunodeficient (SCID) mice for 12 weeks in order to examine the role of estrogen in the bone formation ability of PDLSCs derived from osteoporotic rats. The results from methyl thiazolyl tetrazolium (MTT) assay revealed that the proliferation of the cells derived from the rats in the OVX group was significantly higher than that of the cells derived from the rats in the sham-operated group at the stage of logarithmic growth. The staining intensity of alkaline phosphatase (ALP) and the mineralization of the cells derived from the rats in the OVX group was significantly weaker than that of the cells from the rats in the sham-operated group. When the PDLSCs were seeded on nHAC/PLA, ALP activity, osteocalcin (OCN) secretion, mineral formation and the mRNA expression levels of ALP, OCN, estrogen receptor (ER)α and ERβ in the cells derived from the rats in the OVX group were markedly decreased. Treatment with 17β-estradiol (E2) significantly weakened the proliferative ability of the cells derived from the OVX group rats, and enhanced their osteogenic differentiation ability and the mRNA expression levels of ALP, OCN, ERα and ERβ. When the constructs were implanted into the backs of SCID mice for 12 weeks, the results of histological analysis indicated that the constructs derived from the OVX group rats had a few newly formed bones and osteoids; however, a great number of newly formed bones and osteoids were present in the ones from the sham-operated group and the OVX + E2 group rats. Our findings further indicate that estrogen deficiency impairs the osteogenic differentiation potential of PDLSCs, and that ER plays an important role in the bone regeneration ability of PDLSCs. Estrogen enhances the bone regeneration potential of PDLSCs derived from osteoporotic rats and seeded on nHAC/PLA. This study may provide insight into the clinical management of periodontal bone tissue repair in postmenopausal women with the use of estrogen-mediated PDLSCs seeded on nHAC/PLA.
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E, LING-LING; XU, WEN-HUAN; FENG, LIN; LIU, YI; CAI, DONG-QING; WEN, NING; ZHENG, WEN-JIE
2016-01-01
This study investigated the effects of estrogen on the bone regeneration potential of periodontal ligament stem cells (PDLSCs) derived from osteoporotic rats and seeded on a collagen-based composite scaffold [nano-hydroxyapatite/collagen/poly(L-lactide) (nHAC/PLA)]. For this purpose, 48 healthy 3-month-old Sprague-Dawley female rats were divided into 2 groups as follows: the bilaterally ovariectomized (OVX) rats and sham-operated rats. The PDLSCs were isolated at 3 months after surgery (by which time postmenopausal osteoporosis had developed). The effects of estrogen on the characteristics of these cells seeded in a culture plate and of the cells seeded on nHAC/PLA were then investigated. The PDLSC + nHAC/PLA constructs were implanted subcutaneously into the backs of severe combined immunodeficient (SCID) mice for 12 weeks in order to examine the role of estrogen in the bone formation ability of PDLSCs derived from osteoporotic rats. The results from methyl thiazolyl tetrazolium (MTT) assay revealed that the proliferation of the cells derived from the rats in the OVX group was significantly higher than that of the cells derived from the rats in the sham-operated group at the stage of logarithmic growth. The staining intensity of alkaline phosphatase (ALP) and the mineralization of the cells derived from the rats in the OVX group was significantly weaker than that of the cells from the rats in the sham-operated group. When the PDLSCs were seeded on nHAC/PLA, ALP activity, osteocalcin (OCN) secretion, mineral formation and the mRNA expression levels of ALP, OCN, estrogen receptor (ER)α and ERβ in the cells derived from the rats in the OVX group were markedly decreased. Treatment with 17β-estradiol (E2) significantly weakened the proliferative ability of the cells derived from the OVX group rats, and enhanced their osteogenic differentiation ability and the mRNA expression levels of ALP, OCN, ERα and ERβ. When the constructs were implanted into the backs of SCID mice for 12 weeks, the results of histological analysis indicated that the constructs derived from the OVX group rats had a few newly formed bones and osteoids; however, a great number of newly formed bones and osteoids were present in the ones from the sham-operated group and the OVX + E2 group rats. Our findings further indicate that estrogen deficiency impairs the osteogenic differentiation potential of PDLSCs, and that ER plays an important role in the bone regeneration ability of PDLSCs. Estrogen enhances the bone regeneration potential of PDLSCs derived from osteoporotic rats and seeded on nHAC/PLA. This study may provide insight into the clinical management of periodontal bone tissue repair in postmenopausal women with the use of estrogen-mediated PDLSCs seeded on nHAC/PLA. PMID:27082697
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Redondo, Juliana; Sarkar, Pamela; Kemp, Kevin; Virgo, Paul F; Pawade, Joya; Norton, Aimie; Emery, David C; Guttridge, Martin G; Marks, David I; Wilkins, Alastair; Scolding, Neil J; Rice, Claire M
2017-05-01
Autologous bone-marrow-derived cells are currently employed in clinical studies of cell-based therapy in multiple sclerosis (MS) although the bone marrow microenvironment and marrow-derived cells isolated from patients with MS have not been extensively characterised. To examine the bone marrow microenvironment and assess the proliferative potential of multipotent mesenchymal stromal cells (MSCs) in progressive MS. Comparative phenotypic analysis of bone marrow and marrow-derived MSCs isolated from patients with progressive MS and control subjects was undertaken. In MS marrow, there was an interstitial infiltrate of inflammatory cells with lymphoid (predominantly T-cell) nodules although total cellularity was reduced. Controlling for age, MSCs isolated from patients with MS had reduced in vitro expansion potential as determined by population doubling time, colony-forming unit assay, and expression of β-galactosidase. MS MSCs expressed reduced levels of Stro-1 and displayed accelerated shortening of telomere terminal restriction fragments (TRF) in vitro. Our results are consistent with reduced proliferative capacity and ex vivo premature ageing of bone-marrow-derived cells, particularly MSCs, in MS. They have significant implication for MSC-based therapies for MS and suggest that accelerated cellular ageing and senescence may contribute to the pathophysiology of progressive MS. The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding for this study was provided by the Medical Research Council, UK (grant no. MR/K004166/1). The ACTiMuS study is sup-ported by the Silverman Family Foundation, Multiple Sclerosis Trust, Rosetree’s Trust, Catholic Bishops of England and Wales and Friends of Frenchay and SIAMMS-II by the Sir Halley Stewart Trust. C.M.R., P.S., and K.K. received support from the Burden Neurological Institute.
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NASA Astrophysics Data System (ADS)
Bortel, Emely L.; Langer, Max; Rack, Alexander; Forien, Jean-Baptiste; Duda, Georg N.; Fratzl, Peter; Zaslansky, Paul
2017-11-01
Holotomography, a phase sensitive synchrotron-based μCT modality, is a quantitative 3D imaging method. By exploiting partial spatial X-ray coherence, bones can be imaged volumetrically with high resolution coupled with impressive density sensitivity. This tomographic method reveals the main characteristics of the important tissue compartments in forming bones, including the rapidly-changing soft tissue and the partially or fully mineralized bone regions, while revealing subtle density differences in 3D. Here we show typical results observed within the growing femur bone midshafts of healthy mice that are 1, 3, 7, 10 and 14 days old (postpartum). Our results make use of partially-coherent synchrotron radiation employing inline Fresnel-propagation in multiple tomographic datasets obtained in the imaging beamline ID19 of the ESRF. The exquisite detail creates maps of the juxtaposed soft, partially mineralized and highly mineralized bone revealing the environment in which bone cells create and shape the matrix. This high resolution 3D data is a step towards creating realistic computational models that may be used to study the dynamic processes involved in bone tissue formation and adaptation. Such data will enhance our understanding of the important biomechanical interactions directing maturation and shaping of the bone micro- and macro-geometries.
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Guided bone regeneration using individualized ceramic sheets.
Malmström, J; Anderud, J; Abrahamsson, P; Wälivaara, D-Å; Isaksson, S G; Adolfsson, E
2016-10-01
Guided bone regeneration (GBR) describes the use of membranes to regenerate bony defects. A membrane for GBR needs to be biocompatible, cell-occlusive, non-toxic, and mouldable, and possess space-maintaining properties including stability. The purpose of this pilot study was to describe a new method of GBR using individualized ceramic sheets to perfect bone regeneration prior to implant placement; bone regeneration was assessed using traditional histology and three-dimensional (3D) volumetric changes in the bone and soft tissue. Three patients were included. After full-thickness flap reflection, the individualized ceramic sheets were fixed. The sites were left to heal for 7 months. All patients were evaluated preoperatively and at 7 months postoperative using cone beam computed tomography and 3D optical equipment. Samples of the regenerated bone and soft tissue were collected and analyzed. The bone regenerated in the entire interior volume of all sheets. Bone biopsies revealed newly formed trabecular bone with a lamellar structure. Soft tissue biopsies showed connective tissue with no signs of an inflammatory response. This was considered to be newly formed periosteum. Thus ceramic individualized sheets can be used to regenerate large volumes of bone in both vertical and horizontal directions independent of the bone defect and with good biological acceptance of the material. Copyright © 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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Treatment outcomes of temporal bone osteoradionecrosis.
Kammeijer, Quinten; van Spronsen, Erik; Mirck, Piet G B; Dreschler, Wouter A
2015-04-01
To investigate the clinical relevance of the classification systems used for temporal bone osteoradionecrosis (ORN) and to define a treatment protocol for temporal bone ORN. Retrospective case series. Amsterdam, department of otorhinolaryngology and head and neck surgery. Classification of temporal bone ORN was performed through use of clinical data and radiologic imaging. Outcomes of conservative and surgical treatment were investigated and compared for different grades of ORN. Of the 49 ears included in this study, 35 were primarily treated conservatively. At start of conservative treatment, 23 were classified as a localized and 8 as a diffuse form of ORN; 4 could not be classified. There was a significant difference in clinical outcome between the localized and diffuse forms of ORN (χ(2) = 5.862, P = .015), and mastoid air cell destruction on preoperative computed tomography scan was found to be a significant predictor for a negative outcome of conservative treatment (χ(2) = 4.34, P = .037). Fourteen ears with diffuse ORN were primarily treated surgically, and 11 were secondarily treated surgically following a period of conservative treatment. Twenty-two patients were treated with subtotal petrosectomy, of which 20 were cured. Three patients were treated with canal wall down mastoidectomy, and 2 had recurrence of disease. Ramsden's classification system is clinically relevant in predicting conservative treatment outcomes. Mastoid air cell destruction on computed tomography differentiates between the localized and diffuse forms of ORN. Given our results and experience with treating temporal bone ORN, we propose a treatment protocol. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.
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Bioprinted Osteogenic and Vasculogenic Patterns for Engineering 3D Bone Tissue.
Byambaa, Batzaya; Annabi, Nasim; Yue, Kan; Trujillo-de Santiago, Grissel; Alvarez, Mario Moisés; Jia, Weitao; Kazemzadeh-Narbat, Mehdi; Shin, Su Ryon; Tamayol, Ali; Khademhosseini, Ali
2017-08-01
Fabricating 3D large-scale bone tissue constructs with functional vasculature has been a particular challenge in engineering tissues suitable for repairing large bone defects. To address this challenge, an extrusion-based direct-writing bioprinting strategy is utilized to fabricate microstructured bone-like tissue constructs containing a perfusable vascular lumen. The bioprinted constructs are used as biomimetic in vitro matrices to co-culture human umbilical vein endothelial cells and bone marrow derived human mesenchymal stem cells in a naturally derived hydrogel. To form the perfusable blood vessel inside the bioprinted construct, a central cylinder with 5% gelatin methacryloyl (GelMA) hydrogel at low methacryloyl substitution (GelMA LOW ) was printed. We also develop cell-laden cylinder elements made of GelMA hydrogel loaded with silicate nanoplatelets to induce osteogenesis, and synthesized hydrogel formulations with chemically conjugated vascular endothelial growth factor to promote vascular spreading. It was found that the engineered construct is able to support cell survival and proliferation during maturation in vitro. Additionally, the whole construct demonstrates high structural stability during the in vitro culture for 21 days. This method enables the local control of physical and chemical microniches and the establishment of gradients in the bioprinted constructs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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NASA Technical Reports Server (NTRS)
Irons, R. D.; Stillman, W. S.; Colagiovanni, D. B.; Henry, V. A.; Clarkson, T. W. (Principal Investigator)
1992-01-01
The effects of in vitro pretreatment with benzene metabolites on colony-forming response of murine bone marrow cells stimulated with recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF) were examined. Pretreatment with hydroquinone (HQ) at concentrations ranging from picomolar to micromolar for 30 min resulted in a 1.5- to 4.6-fold enhancement in colonies formed in response to rGM-CSF that was due to an increase in granulocyte/macrophage colonies. The synergism equaled or exceeded that reported for the effects of interleukin 1, interleukin 3, or interleukin 6 with GM-CSF. Optimal enhancement was obtained with 1 microM HQ and was largely independent of the concentration of rGM-CSF. Pretreatment with other authentic benzene metabolites, phenol and catechol, and the putative metabolite trans, trans-muconaldehyde did not enhance growth factor response. Coadministration of phenol and HQ did not enhance the maximal rGM-CSF response obtained with HQ alone but shifted the optimal concentration to 100 pM. Synergism between HQ and rGM-CSF was observed with nonadherent bone marrow cells and lineage-depleted bone marrow cells, suggesting an intrinsic effect on recruitment of myeloid progenitor cells not normally responsive to rGM-CSF. Alterations in differentiation in a myeloid progenitor cell population may be of relevance in the pathogenesis of acute myelogenous leukemia secondary to drug or chemical exposure.
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Enamel: Molecular identity of its transepithelial ion transport system.
Lacruz, Rodrigo S
2017-07-01
Enamel is the most calcified tissue in vertebrates. It differs from bone in a number of characteristics including its origin from ectodermal epithelium, lack of remodeling capacity by the enamel forming cells, and absence of collagen. The enamel-forming cells known as ameloblasts, choreograph first the synthesis of a unique protein-rich matrix, followed by the mineralization of this matrix into a tissue that is ∼95% mineral. To do this, ameloblasts arrange the coordinated movement of ions across a cell barrier while removing matrix proteins and monitoring extracellular pH using a variety of buffering systems to enable the growth of carbonated apatite crystals. Although our knowledge of these processes and the molecular identity of the proteins involved in transepithelial ion transport has increased in the last decade, it remains limited compared to other cells. Here we present an overview of the evolution and development of enamel, its differences with bone, and describe the ion transport systems associated with ameloblasts. Copyright © 2017 The Author. Published by Elsevier Ltd.. All rights reserved.
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Babur, Betul Kul; Futrega, Kathryn; Lott, William B; Klein, Travis Jacob; Cooper-White, Justin; Doran, Michael Robert
2015-09-01
Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like and cartilage-like tissues requires two unique medium formulations and this presents a challenge both in achieving initial MSC differentiation and in maintaining tissue stability when the unified osteochondral tissue is subsequently cultured in a single medium formulation. In this proof-of-principle study, we used an in-house fabricated microwell platform to manufacture thousands of micropellets formed from 166 MSC each. We then characterized the development of bone-like and cartilage-like tissue formation in the micropellets maintained for 8-14 days in sequential combinations of osteogenic or chondrogenic induction medium. When bone-like or cartilage-like micropellets were induced for only 8 days, they displayed significant phenotypic changes when the osteogenic or chondrogenic induction medium, respectively, was swapped. Based on these data, we developed an extended 14-day protocol for the pre-culture of bone-like and cartilage-like micropellets in their respective induction medium. Unified osteochondral tissues were formed by layering 12,000 osteogenic micropellets and 12,000 chondrogenic micropellets into a biphasic structure and then further culture in chondrogenic induction medium. The assembled tissue was cultured for a further 8 days and characterized via histology. The micropellets had amalgamated into a continuous structure with distinctive bone-like and cartilage-like regions. This proof-of-concept study demonstrates the feasibility of micropellet assembly for the formation of osteochondral-like tissues for possible use in osteochondral defect repair.
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Further Analysis of the Crouzon Mouse, Effects of the FGFR2C342Y Mutation are Cranial Bone Dependent
Liu, Jin; Nam, Hwa Kyung; Wang, Estee; Hatch, Nan E.
2013-01-01
Crouzon syndrome is a debilitating congenital disorder involving abnormal craniofacial skeletal development caused by mutations in Fibroblast Growth Factor Receptor-2 (FGFR2). Phenotypic expression in humans exhibits an autosomal dominant pattern that commonly involves premature fusion of the coronal suture (craniosynostosis) and severe midface hypoplasia. To further investigate biologic mechanisms by which the Crouzon syndrome associated FGFR2C342Y mutation leads to abnormal craniofacial skeletal development we created congenic BALB/c FGFR2C342Y/+ mice. Here we show that BALB/c FGFR2C342Y/+ mice have a consistent craniofacial phenotype including partial fusion of the coronal and lambdoid sutures, intersphenoidal synchondrosis and multiple facial bones, with minimal fusion of other craniofacial sutures. This phenotype is similar to the classic and less severe form of Crouzon syndrome that involves significant midface hypoplasia with limited craniosynostosis. Linear and morphometric analyses demonstrate that FGFR2C342Y/+ mice on the BALB/c genetic background differ significantly in form and shape from their wild type littermates, and that in this genetic background the FGFR2C342Y mutation preferentially effects some craniofacial bones and sutures over others. Analysis of cranial bone cells indicates that the FGFR2C342Y mutation promotes aberrant osteoblast differentiation and increased apoptosis that is more severe in frontal than parietal bone cells. Additionally, FGFR2C342Y/+ frontal but not parietal bones exhibit significantly diminished bone volume and density compared to wild type mice. These results confirm that FGFR2-associated craniosynostosis occurs in association with diminished cranial bone tissue and may provide a potential biologic explanation for the clinical finding of phenotype consistency that exists between many Crouzon syndrome patients. PMID:23358860
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Adult T-cell leukemia: a report on two white patients.
Tricot, G J; Broeckaert-Van Orshoven, A; den Ottolander, G J; de Wolf-Peeters, C; Meyer, C J; Verwilghen, R L; Jansen, J
1983-01-01
Two white European males are reported with adult T-cell leukemia (ATL), a disease first described in Japan, but recently also in the U.K. and U.S.A. Both patients presented with lymphadenopathy, but without a mediastinal mass. In addition, one patient had skin infiltrates and the other had hepatosplenomegaly. Morphologic and ultrastructural examination of the blasts in bone marrow and lymph node biopsy revealed a predominance of polymorphic lymphoid cells with pronounced nuclear irregularities and a semi-mature chromatine pattern. Histopathology of the lymph nodes showed a diffuse infiltration with medium-sized lymphoblasts with irregular nuclei. The blasts in the bone marrow formed E rosettes with sheep erythrocytes, lacked terminal deoxynucleotidyl transferase (Tdt) activity but expressed the Ia-like antigen; although the majority of the cells reacted with a polyclonal anti-T-cell serum, they were negative for OKT3. In one patient a helper/inducer phenotype (OKT4+) was found in the lymphoblasts of bone marrow and lymph node, while in the other only in the lymph node. The difference between bone marrow and lymph node phenotype is discussed. To our knowledge, these are the first two European patients reported with ATL, a disease clearly different from convoluted T-cell acute lymphocytic leukemia.
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Zhou, X Z; Leung, V Y; Dong, Q R; Cheung, K M; Chan, D; Lu, W W
2008-06-01
This study investigates the capacity of a composite scaffold composed of polyglycolic acid-hydroxyapatite (PGA-HA) and autologous mesenchymal stem cells (MSCs) to promote repair of osteochondral defects. MSCs from culture-expanded rabbits were seeded onto a PGA and HA scaffold. After a 72-hour co-culture period, the cell-adhered PGA and HA were joined together, forming an MSCs-PGA-HA composite. Full-thickness cartilage defects in the intercondylar fossa of the femur were then implanted with the MSC-PGA-HA composite, the PGA-HA scaffold only, or they were left empty (n=20). Animals were sacrificed 16 or 32 weeks after surgery and the gross appearance of the defects was evaluated. The specimens were examined histologically for morphologic features, and stained immunohistochemically for type 2 collagen. Specimens of the MSCs-PGA-HA composite implantation group demonstrated hyaline cartilage and a complete subchondral bone formation. At 16 weeks post-implantation, significant integration of the newly formed tissue with surrounding normal cartilage and subchondral bone was observed when compared to the two control groups. At 32 weeks, no sign of progressive degeneration of the newly formed tissue was found. A significant difference in histological grading score was found compared with the control groups. The novel MSCs-seeded, PGA-HA biphasic graft facilitated both articular cartilage and subchondral bone regeneration in an animal model and might serve as a new approach for clinical applications.
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Szymczyk, Patrycja; Ziółkowski, Grzegorz; Junka, Adam; Chlebus, Edward
2018-06-08
Unlike conventional manufacturing techniques, additive manufacturing (AM) can form objects of complex shape and geometry in an almost unrestricted manner. AM’s advantages include higher control of local process parameters and a possibility to use two or more various materials during manufacture. In this work, we applied one of AM technologies, selective laser melting, using Ti6Al7Nb alloy to produce biomedical functional structures (BFS) in the form of bone implants. Five types of BFS structures (A1, A2, A3, B, C) were manufactured for the research. The aim of this study was to investigate such technological aspects as architecture, manufacturing methods, process parameters, surface modification, and to compare them with such functional properties such as accuracy, mechanical, and biological in manufactured implants. Initial in vitro studies were performed using osteoblast cell line hFOB 1.19 (ATCC CRL-11372) (American Type Culture Collection). The results of the presented study confirm high applicative potential of AM to produce bone implants of high accuracy and geometric complexity, displaying desired mechanical properties. The experimental tests, as well as geometrical accuracy analysis, showed that the square shaped (A3) BFS structures were characterized by the lowest deviation range and smallestanisotropy of mechanical properties. Moreover, cell culture experiments performed in this study proved that the designed and obtained implant’s internal porosity (A3) enhances the growth of bone cells (osteoblasts) and can obtain predesigned biomechanical characteristics comparable to those of the bone tissue.
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Zhou, Jian; Rogers, Jason H; Lee, Scott H; Sun, DongMing; Yao, Hai; Mao, Jeremy J; Kong, Kimi Y
2017-01-15
Endothelial progenitor cells/endothelial cells (EPCs/ECs) have great potential to treat pathological conditions such as cardiac infarction, muscle ischemia, and bone fractures, but isolation of EPC/ECs from existing cell sources is challenging due to their low EC frequency. We have isolated endothelial progenitor (EP)-like cells from rat oral mucosa and characterized their yield, immunophenotype, growth, and in vivo angiogenic potential. The frequency of EP-like cells derived from oral mucosa is thousands of folds higher than EPCs derived from donor-match bone marrow samples. EP-like cells from oral mucosa were positive for EC markers CD31, VE-Cadherin, and VEGFR2. Oral mucosa-derived EP-like cells displayed robust uptake of acetylated low-density lipoprotein and formed stable capillary networks in Matrigel. Subcutaneously implanted oral mucosa-derived EP-like cells anastomosed with host blood vessels, implicating their ability to elicit angiogenesis. Similar to endothelial colony-forming cells, EP-like cells from oral mucosa have a significantly higher proliferative rate than human umbilical vein endothelial cells. These findings identify a putative EPC source that is easily accessible in the oral cavity, potentially from discarded tissue specimens, and yet with robust yield and potency for angiogenesis in tissue and organ regeneration.
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Zhou, Jian; Rogers, Jason H.; Lee, Scott H.; Sun, DongMing; Yao, Hai; Mao, Jeremy J.
2017-01-01
Endothelial progenitor cells/endothelial cells (EPCs/ECs) have great potential to treat pathological conditions such as cardiac infarction, muscle ischemia, and bone fractures, but isolation of EPC/ECs from existing cell sources is challenging due to their low EC frequency. We have isolated endothelial progenitor (EP)-like cells from rat oral mucosa and characterized their yield, immunophenotype, growth, and in vivo angiogenic potential. The frequency of EP-like cells derived from oral mucosa is thousands of folds higher than EPCs derived from donor-match bone marrow samples. EP-like cells from oral mucosa were positive for EC markers CD31, VE-Cadherin, and VEGFR2. Oral mucosa-derived EP-like cells displayed robust uptake of acetylated low-density lipoprotein and formed stable capillary networks in Matrigel. Subcutaneously implanted oral mucosa-derived EP-like cells anastomosed with host blood vessels, implicating their ability to elicit angiogenesis. Similar to endothelial colony-forming cells, EP-like cells from oral mucosa have a significantly higher proliferative rate than human umbilical vein endothelial cells. These findings identify a putative EPC source that is easily accessible in the oral cavity, potentially from discarded tissue specimens, and yet with robust yield and potency for angiogenesis in tissue and organ regeneration. PMID:27832737
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Self-Setting Calcium Orthophosphate Formulations
Dorozhkin, Sergey V.
2013-01-01
In early 1980s, researchers discovered self-setting calcium orthophosphate cements, which are bioactive and biodegradable grafting bioceramics in the form of a powder and a liquid. After mixing, both phases form pastes, which set and harden forming either a non-stoichiometric calcium deficient hydroxyapatite or brushite. Since both of them are remarkably biocompartible, bioresorbable and osteoconductive, self-setting calcium orthophosphate formulations appear to be promising bioceramics for bone grafting. Furthermore, such formulations possess excellent molding capabilities, easy manipulation and nearly perfect adaptation to the complex shapes of bone defects, followed by gradual bioresorption and new bone formation. In addition, reinforced formulations have been introduced, which might be described as calcium orthophosphate concretes. The discovery of self-setting properties opened up a new era in the medical application of calcium orthophosphates and many commercial trademarks have been introduced as a result. Currently such formulations are widely used as synthetic bone grafts, with several advantages, such as pourability and injectability. Moreover, their low-temperature setting reactions and intrinsic porosity allow loading by drugs, biomolecules and even cells for tissue engineering purposes. In this review, an insight into the self-setting calcium orthophosphate formulations, as excellent bioceramics suitable for both dental and bone grafting applications, has been provided. PMID:24956191
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NF-κB as a Therapeutic Target in Inflammatory-Associated Bone Diseases.
Lin, T-H; Pajarinen, J; Lu, L; Nabeshima, A; Cordova, L A; Yao, Z; Goodman, S B
Inflammation is a defensive mechanism for pathogen clearance and maintaining tissue homeostasis. In the skeletal system, inflammation is closely associated with many bone disorders including fractures, nonunions, periprosthetic osteolysis (bone loss around orthopedic implants), and osteoporosis. Acute inflammation is a critical step for proper bone-healing and bone-remodeling processes. On the other hand, chronic inflammation with excessive proinflammatory cytokines disrupts the balance of skeletal homeostasis involving osteoblastic (bone formation) and osteoclastic (bone resorption) activities. NF-κB is a transcriptional factor that regulates the inflammatory response and bone-remodeling processes in both bone-forming and bone-resorption cells. In vitro and in vivo evidences suggest that NF-κB is an important potential therapeutic target for inflammation-associated bone disorders by modulating inflammation and bone-remodeling process simultaneously. The challenges of NF-κB-targeting therapy in bone disorders include: (1) the complexity of canonical and noncanonical NF-κB pathways; (2) the fundamental roles of NF-κB-mediated signaling for bone regeneration at earlier phases of tissue damage and acute inflammation; and (3) the potential toxic effects on nontargeted cells such as lymphocytes. Recent developments of novel inhibitors with differential approaches to modulate NF-κB activity, and the controlled release (local) or bone-targeting drug delivery (systemic) strategies, have largely increased the translational application of NF-κB therapy in bone disorders. Taken together, temporal modulation of NF-κB pathways with the combination of recent advanced bone-targeting drug delivery techniques is a highly translational strategy to reestablish homeostasis in the skeletal system. © 2017 Elsevier Inc. All rights reserved.
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NF-κB as a Therapeutic Target in Inflammatory-Associated Bone Diseases
Lin, T.-h.; Pajarinen, J.; Lu, L.; Nabeshima, A.; Cordova, L.A.; Yao, Z.; Goodman, S.B.
2017-01-01
Inflammation is a defensive mechanism for pathogen clearance and maintaining tissue homeostasis. In the skeletal system, inflammation is closely associated with many bone disorders including fractures, nonunions, periprosthetic osteolysis (bone loss around orthopedic implants), and osteoporosis. Acute inflammation is a critical step for proper bone-healing and bone-remodeling processes. On the other hand, chronic inflammation with excessive proinflammatory cytokines disrupts the balance of skeletal homeostasis involving osteoblastic (bone formation) and osteoclastic (bone resorption) activities. NF-κB is a transcriptional factor that regulates the inflammatory response and bone-remodeling processes in both bone-forming and bone-resorption cells. In vitro and in vivo evidences suggest that NF-κB is an important potential therapeutic target for inflammation-associated bone disorders by modulating inflammation and bone-remodeling process simultaneously. The challenges of NF-κB-targeting therapy in bone disorders include: (1) the complexity of canonical and noncanonical NF-κB pathways; (2) the fundamental roles of NF-κB-mediated signaling for bone regeneration at earlier phases of tissue damage and acute inflammation; and (3) the potential toxic effects on nontargeted cells such as lymphocytes. Recent developments of novel inhibitors with differential approaches to modulate NF-κB activity, and the controlled release (local) or bone-targeting drug delivery (systemic) strategies, have largely increased the translational application of NF-κB therapy in bone disorders. Taken together, temporal modulation of NF-κB pathways with the combination of recent advanced bone-targeting drug delivery techniques is a highly translational strategy to reestablish homeostasis in the skeletal system. PMID:28215222
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Hirasawa, Hideyuki; Tanaka, Shinya; Sakai, Akinori; Tsutsui, Masato; Shimokawa, Hiroaki; Miyata, Hironori; Moriwaki, Sawako; Niida, Shumpei; Ito, Masako; Nakamura, Toshitaka
2007-07-01
Osteoblast apoptosis increased in the tibias of apoE(-/-) mice fed with a high-fat diet, decreasing bone formation. The expression of p53 mRNA in marrow adherent cells increased. LDL or oxidized LDL increased apoptosis in the calvarial cells of apoE(-/-) mice. The increase in p53-mediated apoptosis is apparently related to a high-fat diet-induced osteopenia in apoE(-/-) mice. The effects of high-fat loading and the apolipoprotein E (apoE) gene on bones have not been elucidated. We hypothesized that apoE gene deficiency (apoE(-/-)) modulates the effects of high-fat loading on bones. We assessed this hypothesis using wildtype (WT) and apoE(-/-) mice fed a standard (WTS and ApoES groups) or a high-fat diet (WTHf and ApoEHf groups). The concentration of serum lipid levels and bone chemical markers were measured. Histomorphometry of the femurs was performed using microCT and a microscope. Bone marrow adherent cells from the femurs were used for colony-forming unit (CFU)-fibroblastic (CFU-f) assay and mRNA expressions analysis. The apoptotic cells in the tibias were counted. TUNEL fluorescein assay and Western analysis were performed in cultures of calvarial cells by the addition of low-density lipoprotein (LDL) or oxidized LDL. In the ApoEHf group, the values of cortical bone volume and trabecular and endocortical bone formation of the femurs decreased, and urinary deoxypyridinoline increased. Subsequent analysis revealed that the number of apoptotic cells in the tibias of the ApoES group increased, and more so in the ApoEHf group. The ratio of alkaline phosphatase-positive CFU-f to total CFU-f was decreased in the ApoEHf group. p53 mRNA expression in adherent cells of the apoE(-/-) mice increased and had a significantly strong positive correlation with serum LDL. TUNEL fluorescein assay of osteoblastic cells revealed an increase of apoptotic cells in the apoE(-/-) mice. The number of apoptotic cells in the apoE(-/-) mice increased with the addition of 100 microg/ml LDL or oxidized LDL. The p53 protein expression in apoE(-/-) cells exposed to 100 microg/ml LDL or oxidized LDL increased. We concluded that apoE gene deficiency enhances the reduction of bone formation induced by a high-fat diet through the stimulation of p53-mediated apoptosis in osteoblastic cells.
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Qiao, Peng-yan; Li, Fang-fang; Dong, Li-min; Xu, Tao; Xie, Qiu-fei
2014-01-01
Objective: To deliver cells deep into injectable calcium phosphate cement (CPC) through alginate-chitosan (AC) microcapsules and investigate the biological behavior of the cells released from microcapsules into the CPC. Methods: Mouse osteoblastic MC3T3-E1 cells were embedded in alginate and AC microcapsules using an electrostatic droplet generator. The two types of cell-encapsulating microcapsules were then mixed with a CPC paste. MC3T3-E1 cell viability was investigated using a Wst-8 kit, and osteogenic differentiation was demonstrated by an alkaline phosphatase (ALP) activity assay. Cell attachment in CPC was observed by an environment scanning electron microscopy. Results: Both alginate and AC microcapsules were able to release the encapsulated MC3T3-E1 cells when mixed with CPC paste. The released cells attached to the setting CPC scaffolds, survived, differentiated, and formed mineralized nodules. Cells grew in the pores concomitantly created by the AC microcapsules in situ within the CPC. At Day 21, cellular ALP activity in the AC group was approximately four times that at Day 7 and exceeded that of the alginate microcapsule group (P<0.05). Pores formed by the AC microcapsules had a diameter of several hundred microns and were spherical compared with those formed by alginate microcapsules. Conclusions: AC microcapsule is a promising carrier to release seeding cells deep into an injectable CPC scaffold for bone engineering. PMID:24711359
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Sieh, Shirly; Taubenberger, Anna V; Lehman, Melanie L; Clements, Judith A; Nelson, Colleen C; Hutmacher, Dietmar W
2014-06-01
As microenvironmental factors such as three-dimensionality and cell-matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Pyo, S. J.; Song, W. W.; Kim, I. R.; Park, B. S.; Kim, C. H.; Kim, S. S.; Chung, I. K.; Kim, Y. D.
2012-03-01
Previous research has investigated the effects of LLLT during titanium implantation, tooth movement and bone graft using deproteinized bovine bone and recognized that these circumstances were nothing more than intentional controlled overpressure against static cells since this controlled trauma could affect cell function/malfunction, or cell recovery/apoptosis. The present preliminary study was conducted to prove if LLL would influence cell viability and cell function after excessive damage, which is enough to diminish cell numbers and distort the features of cells. Our aim is to evaluate whether low level laser irradiation (LLLi) could be helpful in the recovery of traumatized osteoblasts (pressure damaged cells) by observing the morphology and the survival rate of those cells. This model used bone cell cultures which were traumatized by a pressure with 250 G of centripetal force and observed their response to such trauma and low level laser irradiation. In this experiment, a Ga-Al-As diode LLL (IMPRA-ORT, NDLux, Seoul, KOREA) was used with a wavelength of 808 nm, a focus of 14 × 24 mm, which was wide enough to cover the whole dish surface or well within at least 2 times radiation, and an output of 100 mW. Statistical analysis showed a higher recovery rate of damaged osteoblasts in the radiation group than the non-radiation group ( p < 0.05). The nonradiation group had a very poor proliferation rate in comparison to the control group ( p < 0.05) in every time period. In the control group, actin filaments showed a random orientation and cell process branched variously around each cell. In contrast, compressed cells, these patterns were turned into thicker and shorter cytoskeletons. As time progressed, every living cell recovered from the severe stress and recovered both form and function. In summary, the present study showed the capacity of LLLT to aid the recovery of the cell skeleton and affect cell viability on overpressured osteoblasts. These results may contribute toward a better understanding of the effect of LLLT on the recovery of cells after trauma. In addition, our results demonstrated that LLLT could be used in the field of bone tissue engineering to traumatized bone conditions and repair large bone defects such as bone graft and implant installation.
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Bone tissue engineering scaffolding: computer-aided scaffolding techniques.
Thavornyutikarn, Boonlom; Chantarapanich, Nattapon; Sitthiseripratip, Kriskrai; Thouas, George A; Chen, Qizhi
Tissue engineering is essentially a technique for imitating nature. Natural tissues consist of three components: cells, signalling systems (e.g. growth factors) and extracellular matrix (ECM). The ECM forms a scaffold for its cells. Hence, the engineered tissue construct is an artificial scaffold populated with living cells and signalling molecules. A huge effort has been invested in bone tissue engineering, in which a highly porous scaffold plays a critical role in guiding bone and vascular tissue growth and regeneration in three dimensions. In the last two decades, numerous scaffolding techniques have been developed to fabricate highly interconnective, porous scaffolds for bone tissue engineering applications. This review provides an update on the progress of foaming technology of biomaterials, with a special attention being focused on computer-aided manufacturing (Andrade et al. 2002) techniques. This article starts with a brief introduction of tissue engineering (Bone tissue engineering and scaffolds) and scaffolding materials (Biomaterials used in bone tissue engineering). After a brief reviews on conventional scaffolding techniques (Conventional scaffolding techniques), a number of CAM techniques are reviewed in great detail. For each technique, the structure and mechanical integrity of fabricated scaffolds are discussed in detail. Finally, the advantaged and disadvantage of these techniques are compared (Comparison of scaffolding techniques) and summarised (Summary).
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Xia, Yan; Zhou, Panyu; Wang, Fei; Qiu, Chao; Wang, Panfeng; Zhang, Yuntong; Zhao, Liming; Xu, Shuogui
2016-01-01
In this study, bioactive scaffold of nano magnesium phosphate (nMP)/wheat protein (WP) composite (MWC) was fabricated. The results revealed that the MWC scaffolds had interconnected not only macropores (sized 400-600 μm) but also micropores (sized 10-20 μm) on the walls of macropores. The MWC scaffolds containing 40 w% nMP had an appropriate degradability in phosphate-buffered saline and produced a weak alkaline microenvironment. In cell culture experiments, the results revealed that the MWC scaffolds significantly promoted the MC3T3-E1 cell proliferation, differentiation, and growth into the scaffolds. The results of synchrotron radiation microcomputed tomography and analysis of the histological sections of the in vivo implantation revealed that the MWC scaffolds evidently improved the new bone formation and bone defects repair as compared with WP scaffolds. Moreover, it was found that newly formed bone tissue continued to increase with the gradual reduction of materials residual in the MWC scaffolds. Furthermore, the immunohistochemical analysis further offered the evidence of the stimulatory effects of MWC scaffolds on osteogenic-related cell differentiation and new bone regeneration. The results indicated that MWC scaffolds with good biocompability and degradability could promote osteogenesis in vivo, which would have potential for bone tissue repair.
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Bernards, Matthew T; Qin, Chunlin; Jiang, Shaoyi
2008-07-15
Native bone tissue is composed of a complex matrix of collagen, non-collagenous proteins, and hydroxyapatite (HAP). Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the non-collagenous protein family termed the SIBLING (small integrin-binding ligand, N-linked glycoproteins) proteins, which are primarily found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen, while the MC3T3-E1 cell adhesion was shown to be dependant on the conformational flexibility of BSP specifically bound to collagen. Additionally, OPN was shown to play a greater role than BSP for cell binding to collagen. In this work, the orientations and conformations of BSP and OPN specifically bound to HAP are probed under similar conditions. Radiolabeled adsorption isotherms were obtained for BSP and OPN on HAP formed from a simulated body fluid, and the results show that HAP has the capacity to bind significantly more BSP than OPN. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of adsorbed BSP and OPN specifically bound to HAP. It was found that there is a preference for cell binding to HAP with adsorbed BSP as compared to OPN, but not to a statistically significant level. However, the maximum cell binding was observed on HAP substrates with adsorbed heat denatured bovine serum albumin (BSA). The influence of BSA on cell binding was shown to be concentration dependant and it is believed that the adsorbed BSA modulates the proliferation state of the bound cells.
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Bernards, Matthew T.; Qin, Chunlin; Jiang, Shaoyi
2008-01-01
Native bone tissue is composed of a complex matrix of collagen, non-collagenous proteins, and hydroxyapatite (HAP). Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the non-collagenous protein family termed the SIBLING (small integrin-binding ligand, N-linked glycoproteins) proteins, which are primarily found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen, while the MC3T3-E1 cell adhesion was shown to be dependant on the conformational flexibility of BSP specifically bound to collagen. Additionally, OPN was shown to play a greater role than BSP for cell binding to collagen. In this work, the orientations and conformations of BSP and OPN specifically bound to HAP are probed under similar conditions. Radiolabeled adsorption isotherms were obtained for BSP and OPN on HAP formed from a simulated body fluid, and the results show that HAP has the capacity to bind significantly more BSP than OPN. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of adsorbed BSP and OPN specifically bound to HAP. It was found that there is a preference for cell binding to HAP with adsorbed BSP as compared to OPN, but not to a statistically significant level. However, the maximum cell binding was observed on HAP substrates with adsorbed heat denatured bovine serum albumin (BSA). The influence of BSA on cell binding was shown to be concentration dependant and it is believed that the adsorbed BSA modulates the proliferation state of the bound cells. PMID:18420388
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Chen, Xia; Wang, Jian; Yu, Li; Zhou, Jia; Zheng, Danning; Zhang, Bo
2018-04-12
The therapeutic method traditionally used in bone defect reconstruction is autologous bone grafting. The most common problems affecting this type of repair approach are bone absorption and donor trauma. The approach taken in this study overcomes these problems. Bone marrow stromal cells (BMSCs) provided the crucial seed cells. Fibrin biological scaffolds were formed by combining the BMSCs with concentrated growth factor (CGF). BMSCs were isolated from Wistar rat femurs; CGF was prepared from rat heart blood. Five repair groups were created for comparative purposes: (A) CGF + BMSCs; (B) CGF; (C) collagen + BMSCs; (D) collagen; (E) blank. After three months, the rats were sacrificed, and histopathology and three-dimensional CT images produced. Bone regeneration was significantly higher in the (A) CGF + BMSC group; osteogenesis was lower in the (B) CGF and (C) collagen + BMSC groups, at very similar levels; the (D) collagen and (E) blank groups scored the lowest results. Our research suggests that combining CGF with BMSCs leads to the formation of fibrin scaffolds that have a powerful effect on osteogenesis as well as a subsidiary angiogenic effect. SEM images of the CGF scaffolds cultured with BMSCs confirmed good CGF biocompatibility. The superior osteoinductive activity of the CGF + BMSC combination makes it an excellent biomaterial for bone regeneration.
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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.
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Eskinazi-Budge, Aaron; Manickavasagam, Dharani; Czech, Tori; Novak, Kimberly; Kunzler, James; Oyewumi, Moses O
2018-05-30
Simvastatin (Sim) is a widely known drug in the treatment of hyperlipidemia that has attracted so much attention in bone regeneration based on its potential osteoanabolic effect. However, repurposing of Sim in bone regeneration will require suitable delivery systems that can negate undesirable off-target/side effects. In this study, we have investigated a new lipid nanoparticle (NP) platform that was fabricated using a binary blend of emulsifying wax (Ewax) and glyceryl monooleate (GMO). Using the binary matrix materials, NPs loaded with Sim (0-500 µg/mL) were prepared and showed an average particle size of about 150 nm. NP size stability was dependent on Sim concentration loaded in NPs. The suitability of NPs prepared with the binary matrix materials in Sim delivery for potential application in bone regeneration was supported by biocompatibility in pre-osteoclastic and pre-osteoblastic cells. Additional data demonstrated that biofunctional Sim was released from NPs that facilitated differentiation of osteoblasts (cells that form bones) while inhibiting differentiation of osteoclasts (cells that resorb bones). The overall work demonstrated the preparation of NPs from Ewax/GMO blends and characterization to ascertain potential suitability in Sim delivery for bone regeneration. Additional studies on osteoblast and osteoclast functions are warranted to fully evaluate the efficacy simvastatin-loaded Ewax/GMO NPs using in-vitro and in-vivo approaches.
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Protective Effects of Selected Botanical Agents on Bone
Jolly, James Jam; Alias, Ekram; Chua, Kien Hui; Soelaiman, Ima Nirwana
2018-01-01
Osteoporosis is a serious health problem affecting more than 200 million elderly people worldwide. The early symptoms of this disease are hardly detectable. It causes progressive bone loss, which ultimately renders the patients susceptible to fractures. Osteoporosis must be prevented because the associated fragility fractures result in high morbidity, mortality, and healthcare costs. Many plants used in herbal medicine contain bioactive compounds possessing skeletal protective effects. This paper explores the anti-osteoporotic properties of selected herbal plants, including their actions on osteoblasts (bone forming cells), osteoclasts (bone resorbing cells), and bone remodelling. Some of the herbal plant families included in this review are Berberidaceae, Fabaceae, Arecaceae, Labiatae, Simaroubaceaea, and Myrsinaceae. Their active constituents, mechanisms of action, and pharmaceutical applications were discussed. The literature shows that very few herbal plants have undergone human clinical trials to evaluate their pharmacological effects on bone to date. Therefore, more intensive research should be performed on these plants to validate their anti-osteoporotic properties so that they can complement the currently available conventional drugs in the battle against osteoporosis. PMID:29751644
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Ulm, Christian; Bertl, Kristina; Strbac, Georg D; Esfandeyari, Azadeh; Stavropoulos, Andreas; Zechner, Werner
2017-12-01
Sinus floor augmentation is a routinely used surgical technique for increasing the bone height/volume of the atrophic posterior maxilla. Optimal integration of the implanted augmentation material within the newly formed bone will-at least partly-depend on adequate vascularization to ensure sufficient recruitment of osteoblast and osteoclast precursor cells. The present technical note describes a modification intended to facilitate increased blood inflow into the augmented space. After preparation of the lateral window and elevation of the Schneiderian membrane, the cortical bone of the sinus floor is perforated several times either by using a piezoelectric device or a microsurgical handpiece with the corresponding tip or bur; these perforations should extend into the trabecular bone. The experiences with this modified technique after 12 patients are presented and discussed. It is expected that by means of this relatively simple technique, increased blood and cell inflow into the augmented space is achieved. This may, in turn, enhance new bone formation and improve the integration of the augmentation material.
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Differentiations and Functional State of Osteogenic Cells in Conditions of Microgravity
NASA Astrophysics Data System (ADS)
Onishchenko, Ganna; Rodionova, Natalia; Markevich, Ganna; Markevich, Ganna
The space flight factors (space radiation, magnetic fields etc.) affect considerably the state of bone tissue, leading to the development of osteoporosis and osteopenia in the bone skeleton. Many aspects of reactions of bone tissue cells still remain unclear until now. With the use of electron microscopy and autoradiography with 3H-thymidine we studied the samples gathered from the femoral bone epiphyses and metaphyses of rats flown on board American Spacelab -2 and in experiments with modeling of microgravity ("tail suspension" method). In our work the main attention is focused on studying the ultrastructure and metabolism of osteogenetic cells. The degree of differentiation and functional state are evaluated according to the degree of development of organelles for specific biosynthesis: rough endoplasmic reticulum (RER), Golgy complex (GC), as well as the state of mitochondria and cell nucleus. As compared with a control, the population of osteogenetic cells from zones of bone reconstruction shows a decrease in the number of functionally active forms. We can judge of this from the reduction volume of RER, GC, mitochondria in osteoblasts. RER loses architectonics typical for osteoblasts and, as against the control, is represented by short narrow canaliculi distributed throughout the cy-toplasm; some canals disintegrate. GC is slightly pronounced, mitochondria become smaller in size and acquire an optically dark matrix. These phenomena are supposed to be associated with the desorganization of microtubules and microfilaments in the cells under microgravity condi-tions. The number of degrading and apoptotic cells increases in the population of osteoblasts. The dynamics of labeled cells following various intervals after 3H-thymidine injection testifies to a delay in the rates of osteoblasts' differentiation and their transformation to osteocytes in the experiment animals. A lower 3H-glycine uptake by the osteogenic cells and bone matrix as compared with a control is indicative of a decrease of the osteoplastic process under hypokinesia and modeling microgravity. We concluded that microgravity results in low differentiations and reduction specific functions of osteogenic cells.
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Klotz, B J; Lim, K S; Chang, Y X; Soliman, B G; Pennings, I; Melchels, F P W; Woodfield, T B F; Rosenberg, A J; Malda, J; Gawlitta, D
2018-05-30
In engineering of tissue analogues, upscaling to clinically-relevant sized constructs remains a significant challenge. The successful integration of a vascular network throughout the engineered tissue is anticipated to overcome the lack of nutrient and oxygen supply to residing cells. This work aimed at developing a multiscale bone-tissue-specific vascularisation strategy. Engineering pre-vascularised bone leads to biological and fabrication dilemmas. To fabricate channels endowed with an endothelium and suitable for osteogenesis, rather stiff materials are preferable, while capillarisation requires soft matrices. To overcome this challenge, gelatine-methacryloyl hydrogels were tailored by changing the degree of functionalisation to allow for cell spreading within the hydrogel, while still enabling endothelialisation on the hydrogel surface. An additional challenge was the combination of the multiple required cell-types within one biomaterial, sharing the same culture medium. Consequently, a new medium composition was investigated that simultaneously allowed for endothelialisation, capillarisation and osteogenesis. Integrated multipotent mesenchymal stromal cells, which give rise to pericyte-like and osteogenic cells, and endothelial-colony-forming cells (ECFCs) which form capillaries and endothelium, were used. Based on the aforementioned optimisation, a construct of 8 × 8 × 3 mm, with a central channel of 600 µm in diameter, was engineered. In this construct, ECFCs covered the channel with endothelium and osteogenic cells resided in the hydrogel, adjacent to self-assembled capillary-like networks. This study showed the promise of engineering complex tissue constructs by means of human primary cells, paving the way for scaling-up and finally overcoming the challenge of engineering vascularised tissues.
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Pederson, Larry; Ruan, Ming; Westendorf, Jennifer J.; Khosla, Sundeep; Oursler, Merry Jo
2008-01-01
Under most conditions, resorbed bone is nearly precisely replaced in location and amount by new bone. Thus, it has long been recognized that bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblast-mediated bone formation are tightly coupled processes. Abundant data conclusively demonstrate that osteoblasts direct osteoclast differentiation. Key questions remain, however, as to how osteoblasts are recruited to the resorption site and how the amount of bone produced is so precisely controlled. We hypothesized that osteoclasts play a crucial role in the promotion of bone formation. We found that osteoclast conditioned medium stimulates human mesenchymal stem (hMS) cell migration and differentiation toward the osteoblast lineage as measured by mineralized nodule formation in vitro. We identified candidate osteoclast-derived coupling factors using the Affymetrix microarray. We observed significant induction of sphingosine kinase 1 (SPHK1), which catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), in mature multinucleated osteoclasts as compared with preosteoclasts. S1P induces osteoblast precursor recruitment and promotes mature cell survival. Wnt10b and BMP6 also were significantly increased in mature osteoclasts, whereas sclerostin levels decreased during differentiation. Stimulation of hMS cell nodule formation by osteoclast conditioned media was attenuated by the Wnt antagonist Dkk1, a BMP6-neutralizing antibody, and by a S1P antagonist. BMP6 antibodies and the S1P antagonist, but not Dkk1, reduced osteoclast conditioned media-induced hMS chemokinesis. In summary, our findings indicate that osteoclasts may recruit osteoprogenitors to the site of bone remodeling through SIP and BMP6 and stimulate bone formation through increased activation of Wnt/BMP pathways. PMID:19075223
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Yao, Wei; Evan Lay, Yu-An; Kot, Alexander; Liu, Ruiwu; Zhang, Hongliang; Chen, Haiyan; Lam, Kit; Lane, Nancy E.
2017-01-01
Mesenchymal stem cell (MSC) transplantation has been tested in animal and clinical fracture studies. We have developed a bone-seeking compound, LLP2A-Alendronate (LLP2A-Ale) that augments MSC homing to bone. The purpose of this study was to determine whether treatment with LLP2A-Ale or a combination of LLP2A-Ale and MSCs would accelerate bone healing in a mouse closed fracture model and if the effects are sex dependent. A right mid-femur fracture was induced in two-month-old osterix-mCherry (Osx-mCherry) male and female reporter mice. The mice were subsequently treated with placebo, LLP2A-Ale (500 µg/kg, IV), MSCs derived from wild-type female Osx-mCherry adipose tissue (ADSC, 3 × 105, IV) or ADSC + LLP2A-Ale. In phosphate buffered saline-treated mice, females had higher systemic and surface-based bone formation than males. However, male mice formed a larger callus and had higher volumetric bone mineral density and bone strength than females. LLP2A-Ale treatment increased exogenous MSC homing to the fracture gaps, enhanced incorporation of these cells into callus formation, and stimulated endochondral bone formation. Additionally, higher engraftment of exogenous MSCs in fracture gaps seemed to contribute to overall fracture healing and improved bone strength. These effects were sex-independent. There was a sex-difference in the rate of fracture healing. ADSC and LLP2A-Ale combination treatment was superior to on callus formation, which was independent of sex. Increased mobilization of exogenous MSCs to fracture sites accelerated endochondral bone formation and enhanced bone tissue regeneration. PMID:27334693
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Chackalaparampil, I.; Mukherjee, B.B.; Peri, A.
1994-09-01
Osteopetrosis, affecting mice and humans alike, arises from reduced or impaired bone resorption, causing abnormally dense bone formation. Normal bone differentiation requires continuous resorption and remodeling by osteoclasts which are derived from monocyte/macrophage lineage in the bone marrow. It has been reported that targeted homozygous disruption of c-src proto-oncogene in mice results in the development of osteopetrosis due to impaired bone-resorbing function of osteoclast cells. However, the molecular mechanism(s) which leads to osteoclast dysfunction in c-src deficient (src{sup -/-}) mice remains unclear. Here, we report that in embryonic fibroblasts derived from homozygous Src{sup -/-} mice, the expression of the genemore » coding for osteopontin (OP), a phosphorylated glycoprotein involved in bone differentiation, is drastically repressed. OP gene expression is not, however, affected in the heterozygous (Src{sup +/-}) mutant cells of identical origin, or in the c-src expression and OP production. Moreover, OP expression in c-src-deficient cells could be rescued upon treatment with 12-0-tetradecanoyl phorbol-13-myristate-acetate or okadaic acid. These observations indicate that OP expression is regulated via an src-mediated protein kinase C signaling pathway. Since it is known that OP mediates osteoclast adherence to the bone matrix, a key event in bone differentiation, our data is most significant in that they strongly suggest that drastic inhibition of synthesis of OP prevents osteoclasts in Src{sup -/-} mice from anchoring to the bone matrix. Consequently, this disruption of osteoclast adherence impairs their ability to form bone-resorbing ruffled border, causing osteopetrosis.« less
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Kawasaki, Haruhisa; Guan, Jianjun; Tamama, Kenichi
2010-07-02
Cell therapy with bone marrow multipotential stromal cells/mesenchymal stem cells (MSCs) represents a promising approach in the field of regenerative medicine. Low frequency of MSCs in adult bone marrow necessitates ex vivo expansion of MSCs after harvest; however, such a manipulation causes cellular senescence with loss of differentiation, proliferative, and therapeutic potentials of MSCs. Hydrogen molecules have been shown to exert organ protective effects through selective reduction of hydroxyl radicals. As oxidative stress is one of the key insults promoting cell senescence in vivo as well as in vitro, we hypothesized that hydrogen molecules prevent senescent process during MSC expansion. Addition of 3% hydrogen gas enhanced preservation of colony forming early progenitor cells within MSC preparation and prolonged the in vitro replicative lifespan of MSCs without losing differentiation potentials and paracrine capabilities. Interestingly, 3% hydrogen gas treatment did not decrease hydroxyl radical, protein carbonyl, and 8-hydroxydeoxyguanosine, suggesting that scavenging hydroxyl radical might not be responsible for these effects of hydrogen gas in this study. Copyright 2010 Elsevier Inc. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kawasaki, Haruhisa; Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210; Guan, Jianjun
2010-07-02
Cell therapy with bone marrow multipotential stromal cells/mesenchymal stem cells (MSCs) represents a promising approach in the field of regenerative medicine. Low frequency of MSCs in adult bone marrow necessitates ex vivo expansion of MSCs after harvest; however, such a manipulation causes cellular senescence with loss of differentiation, proliferative, and therapeutic potentials of MSCs. Hydrogen molecules have been shown to exert organ protective effects through selective reduction of hydroxyl radicals. As oxidative stress is one of the key insults promoting cell senescence in vivo as well as in vitro, we hypothesized that hydrogen molecules prevent senescent process during MSC expansion.more » Addition of 3% hydrogen gas enhanced preservation of colony forming early progenitor cells within MSC preparation and prolonged the in vitro replicative lifespan of MSCs without losing differentiation potentials and paracrine capabilities. Interestingly, 3% hydrogen gas treatment did not decrease hydroxyl radical, protein carbonyl, and 8-hydroxydeoxyguanosine, suggesting that scavenging hydroxyl radical might not be responsible for these effects of hydrogen gas in this study.« less
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Hou, Tao; Liu, Yanshuang; Kolba, Nikolai; Guo, Danjun; He, Hui
2017-05-12
Desalted duck egg white peptides (DPs) have been proven to promote calcium uptake in Caco-2 cells and rats treated with a calcium-deficient diet. The retinoic acid-induced bone loss model was used to evaluate the effect of DPs on calcium absorption and bone formation. Three-month-old Wistar female rats were treated with 0.9% saline, DPs (800 mg/kg), or alendronate (5 mg/kg) for three weeks immediately after retinoic acid treatment (80 mg/kg) once daily for two weeks. The model group was significantly higher in serum bone alkaline phosphatase than the other three groups ( p < 0.05), but lower in calcium absorption rate, serum osteocalcin, bone weight index, bone calcium content, bone mineral density, and bone max load. After treatment with DPs or alendronate, the absorption rate increased and some serum and bone indices recovered. The morphology results indicated bone tissue form were ameliorated and numbers of osteoclasts decreased after supplementation with DPs or alendronate. The in vitro study showed that the transient receptor potential vanilloid 6 (TRPV6) calcium channel was the main transport pathway of both DPs and Val-Ser-Glu-Glu peptitde (VSEE), which was identified from DPs. Our results indicated that DPs could be a promising alternative to current therapeutic agents for bone loss because of the promotion of calcium uptake and regulation of bone formation.
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... by scar tissue and causes decreased blood cell production). Ruxolitinib is in a class of medications called kinase inhibitors. It works by blocking an enzyme that causes scar tissue to form in the bone marrow.
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Ali-Osman, F; Giblin, J; Berger, M; Murphy, M J; Rosenblum, M L
1985-09-01
Although the antitumor effects of chloroethylnitrosoureas have been shown to be due primarily to DNA-DNA cross-linking by the alkylating moieties of these agents, the basis of the often accompanying bone marrow toxicity has been more controversial. We report on the relative bone marrow toxicity of four model nitrosoureas with different alkylating and carbamoylating activities: 1,3-bis(2-chloroethyl)-1-nitrosourea; 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea; chlorozotozin, (2-[3-(2-chloroethyl)-3 -nitrosoureido]-2-deoxy-D-glucopyranose); and -3-(beta-D-glucopyranosyl)-1-nitrosourea. Inhibitions of DNA, RNA, and protein synthesis in murine bone marrow cells and of colony growth of myeloid precursor cells (granulocyte-macrophage colony-forming units) were used as in vitro end points of myelotoxicity. Further, we determined the antiglioma activity of the four nitrosoureas on two human gliomas in a clonogenic tumor cell assay and studied the effect of the non-nitrosourea carbamoylators potassium cyanate, chloroethyl isocyanate, cyclohexyl isocyanate, ethyl isocyanate, and ethyl isothiocyanate on granulocyte-macrophage colony-forming units. The results show that, at equivalent drug exposures, clonogenic glioma cell kill was significant and comparative for 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, and chlorozotocin; 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea showed little activity. In contrast, granulocyte-macrophage colony-forming unit toxicity was low with chlorozotocin and 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea and very high with 1,3-bis(2-chloroethyl)-1-nitrosourea and 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea. Of the isocyanates, bone marrow toxicity was highest with chloroethyl isocyanate and cyclohexyl isocyanate, intermediate with ethyl isocyanate, and lowest with KOCN and ethyl isothiocyanate. Our results indicate that (a) bifunctional alkylation is essential for antiglioma activity of nitrosoureas and (b) myelosuppression is at least partly linked with carbamoylation but that structural entities in the carbamoylating isocyanate rather than a quantitative degree of carbamoylation determine the degree of potential myelotoxicity.
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Mebarek, Saida; Abousalham, Abdelkarim; Magne, David; Do, Le Duy; Bandorowicz-Pikula, Joanna; Pikula, Slawomir; Buchet, René
2013-01-01
The present review aims to systematically and critically analyze the current knowledge on phospholipases and their role in physiological and pathological mineralization undertaken by mineralization competent cells. Cellular lipid metabolism plays an important role in biological mineralization. The physiological mechanisms of mineralization are likely to take place in tissues other than in bones and teeth under specific pathological conditions. For instance, vascular calcification in arteries of patients with renal failure, diabetes mellitus or atherosclerosis recapitulates the mechanisms of bone formation. Osteoporosis—a bone resorbing disease—and rheumatoid arthritis originating from the inflammation in the synovium are also affected by cellular lipid metabolism. The focus is on the lipid metabolism due to the effects of dietary lipids on bone health. These and other phenomena indicate that phospholipases may participate in bone remodelling as evidenced by their expression in smooth muscle cells, in bone forming osteoblasts, chondrocytes and in bone resorbing osteoclasts. Among various enzymes involved, phospholipases A1 or A2, phospholipase C, phospholipase D, autotaxin and sphingomyelinase are engaged in membrane lipid remodelling during early stages of mineralization and cell maturation in mineralization-competent cells. Numerous experimental evidences suggested that phospholipases exert their action at various stages of mineralization by affecting intracellular signaling and cell differentiation. The lipid metabolites—such as arachidonic acid, lysophospholipids, and sphingosine-1-phosphate are involved in cell signaling and inflammation reactions. Phospholipases are also important members of the cellular machinery engaged in matrix vesicle (MV) biogenesis and exocytosis. They may favour mineral formation inside MVs, may catalyse MV membrane breakdown necessary for the release of mineral deposits into extracellular matrix (ECM), or participate in hydrolysis of ECM. The biological functions of phospholipases are discussed from the perspective of animal and cellular knockout models, as well as disease implications, development of potent inhibitors and therapeutic interventions. PMID:23455471
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Bioactive scaffold for bone tissue engineering: An in vivo study
NASA Astrophysics Data System (ADS)
Livingston, Treena Lynne
Massive bone loss of the proximal femur is a common problem in revision cases of total hip implants. Allograft is typically used to reconstruct the site for insertion of the new prosthesis. However, for long term fixation and function, it is desirable that the allograft becomes fully replaced by bone tissue and aids in the regeneration of bone to that site. However, allograft use is typically associated with delayed incorporation and poor remodeling. Due to these profound limitations, alternative approaches are needed. Tissue engineering is an attractive approach to designing improved graft materials. By combining osteogenic activity with a resorbable scaffold, bone formation can be stimulated while providing structure and stability to the limb during incorporation and remodeling of the scaffold. Porous, surface modified bioactive ceramic scaffolds (pSMC) have been developed which stimulate the expression of the osteoblastic phenotype and production of bone-like tissue in vitro. The scaffold and two tissue-engineered constructs, osteoprogenitor cells seeded onto scaffolds or cells expanded in culture to form bone tissue on the scaffolds prior to implantation, were investigated in a long bone defect model. The rate of incorporation was assessed. Both tissue-engineered constructs stimulated bone formation and comparable repair at 2 weeks. In a rat femoral window defect model, bone formation increased over time for all groups in concert with scaffold resorption, leading to a 40% increase in bone and 40% reduction of the scaffold in the defect by 12 weeks. Both tissue-engineered constructs enhanced the rate of mechanical repair of long bones due to better bony union with the host cortex. Long bones treated with tissue engineered constructs demonstrated a return in normal torsional properties by 4 weeks as compared to 12 weeks for long bones treated with pSMC. Culture expansion of cells to produce bone tissue in vitro did not accelerate incorporation over the treatment with cells seeded at the time of surgery. Porous, surface modified bioactive ceramic is a promising scaffold material for tissue-engineered bone repair. Bone formation and scaffold resorption act in concert for maintenance and improvement of the structural properties of the long bones over time. As determined histomorphometrically and mechanically, the rate of incorporation of the scaffold was enhanced with the tissue-engineered constructs.
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Kira, Tsutomu; Akahane, Manabu; Omokawa, Shohei; Shimizu, Takamasa; Kawate, Kenji; Onishi, Tadanobu; Tanaka, Yasuhito
2017-10-18
To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate (TCP) on osteogenesis. Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium (MEM) containing ascorbic acid phosphate (AscP) and dexamethasone (Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase (ALP) activity and osteocalcin (OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs were implanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk. In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group ( P < 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group ( P > 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.
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NASA Astrophysics Data System (ADS)
Lau, Patrick; Hellweg, Christine E.; Kirchner, Simone; Baumstark-Khan, Christa
During longterm space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. In addition to weightlessness, exposure to cosmic ionization radiation is another space related factor endangering health and productivity of astronauts. In order to elucidate changes in bone cell metabolism induced by ionizing radiation, ground-based bone cell models have been developed. The differentiation level of the bone cells may influence their radiation sensitivity. Therefore, our cell model comprises a collection of immortalized murine pre-osteoblast, osteoblast and osteocyte cell lines representing discrete stages of differentiation: the subclones 4 and 24 of the osteoblast cell line MC3T3-E1, the osteoblast cell line OCT-1 and the osteocyte cell line MLO-Y4 display varying potential to produce mineralized bone matrix upon incubation with ascorbic acid and β-glycerophosphate (osteogenic medium). The MLO-Y4 cells showed the highest and subclone 24 the lowest proliferation rate. The most intense von Kossa reaction after culture in osteogenic medium was observed in subclone 4, indicating mineralized bone matrix. The bone cell markers alkaline phosphatase and osteocalcin were determined to further characterize the differentiation stage. All cell lines expressed osteocalcin, as determined by reverse transcriptase polymerase chain reaction. The activity of alkaline phosphatase was highest in the cell line OCT-1 and very low in MLO-Y4 and S4. The peculiarity of the markers suggests a characterization of OCT-1 and S24 as preosteoblast, S4 as (mature) osteoblast, and MLO-Y4 as osteocyte. Survival after exposure to X-rays was determined using the colony forming ability test. The resulting dose-effect relationships revealed normal radiation sensitivity (compared to human fibroblasts). Cell clone specific variations (subclones 4 and 24) in the radiation sensitivity may be due to the differentiation level. The survival curve of MLO-Y4 shows a broad shoulder, suggesting a high repair capacity or a high DNA damage or misrepair tolerance. The quantitative acquisition of DNA-strand breaks was performed by fluorescent analysis of DNA unwinding and revealed a high level of DNA damage immediately after X-irradiation, which increases dose dependently. In conclusion, the cell line with the highest differentiation level (MLO-Y4) displays lower radiation sensitivity, regarding the shoulder width of the dose-effect curve, compared to the less differentiated osteoblast cell lines.
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Elvenes, Jan; Knutsen, Gunnar; Johansen, Oddmund; Moe, Bjørn T; Martinez, Inigo
2009-07-01
Mesenchymal progenitor cells from bone marrow hold great potential as a cell source for cartilage repair. Aspiration from the iliac crest is the most widely used method to harvest bone marrow cells for cartilage repair. The objective of our study was to establish a new method to isolate mesenchymal progenitor cells by direct aspiration of bone marrow from the subchondral spongious bone underneath cartilage defects during microfracture treatment and to confirm the chondrogenic potential of the resulting cell cultures. Bone marrow was aspirated arthroscopically from patients treated for isolated cartilage defects. Adherent stromal cells were isolated, expanded in monolayer cultures, and characterized by flow cytometry. Chondrogenic induction of cells was achieved by combination of spheroid cultures in hanging drops and the concomitant use of transforming growth factor-beta (TGFbeta). Articular chondrocytes established in three-dimensional (3D) cultures were used as positive cartilage-forming units, and skin fibroblasts were used as negative controls. Three-dimensional constructs were stained for immunohistochemical and histological examination, and a real-time polymerase chain reaction (PCR) was performed to quantify the expression of aggrecan, collagen types 1 and 2, and Sox9. Mesenchymal stem cell-like progenitor cells (MSCs) displaying chondrogenic differentiation capacity were harvested arthroscopically from underneath cartilage lesions on distal femurs using the one-hole technique. Stem cell-related surface antigens analyzed by flow cytometry confirmed the nature of the isolated adherent cells. MSC spheroids stained positive for glycosaminoglycans and collagen type 2. Realtime PCR showed that MSCs in 3D spheroids significantly increased gene expression of collagen type 2, aggrecan, and Sox 9 and down-regulated expression of collagen type 1 when compared to the mRNA levels measured in MSCs monolayers. We describe a new technique that may be applied for harvesting bone marrow cells from cartilage defects during arthroscopic intervention of the knee. Cells harvested in this way hold full chondrogenic differentiation potential. Our data imply that MSC storage may be established by using marrow from this approach, bypassing the need for cell aspiration from the iliac crest.
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NASA Astrophysics Data System (ADS)
Milgram, S.; Carrière, M.; Thiebault, C.; Berger, P.; Khodja, H.; Gouget, B.
2007-07-01
Uranium acute intoxication has been documented to induce nephrotoxicity. Kidneys are the main target organs after short term exposures to high concentrations of the toxic, while chronic exposures lead to its accumulation in the skeleton. In this paper, chemical toxicity of uranium is investigated for rat osteoblastic bone cells and compared to results previously obtained on renal cells. We show that bone cells are less sensitive to uranium than renal cells. The influence of the chemical form on U cytotoxicity is demonstrated. For both cell types, a comparison of uranium toxicity with other metals or metalloids toxicities (Mn, Ni, Co, Cu, Zn, Se and Cd) permits classification of Cd, Zn, Se IV and Cu as the most toxic and Ni, Se VI, Mn and U as the least toxic. Chemical toxicity of natural uranium proves to be far less than that of cadmium. To try to explain the differences in sensitivities observed between metals and different cell types, cellular accumulations in cell monolayers are quantified by inductively coupled plasma-mass spectroscopy (ICP-MS), function of time or function of dose: lethal doses which simulate acute intoxications and sub-lethal doses which are more realistic with regard to environmentally metals concentrations. In addition to being more resistant, bone cells accumulated much more uranium than did renal cells. Moreover, for both cell models, Mn, U-citrate and U-bicarbonate are strongly accumulated whereas Cu, Zn and Ni are weakly accumulated. On the other hand, a strong difference in Cd behaviour between the two cell types is shown: whereas Cd is very weakly accumulated in bone cells, it is very strongly accumulated in renal cells. Finally, elemental distribution of the toxics is determined on a cellular scale using nuclear microprobe analysis. For both renal and osteoblastic cells, uranium was accumulated in as intracellular precipitates similar to those observed previously by SEM/EDS.
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Wang, Zhi-qiang; Li, Qi-jia; Wang, Qi
2002-11-01
To observe the difference of the fracture reparation using autogeneic-iliac bone and allogenic bone. Comminuted fracture of humerus in two sides were made in rabbits. Autogeneic-iliac bone was implanted in one side, while allogenic bone of equal capacity was implanted in the other side. General observation, X-ray, and HE histologic section were done when the rabbits were put to death in different stages. One week after implantation, the graft had been enclosed by connective tissue without infiltration of the inflammatory cells. At the 2nd week, the graft had been enclosed in osteoplastic granulation tissue, and the cartilage callus had formed. At the 3rd week, there had been broken sequestrum among the callus; the cartilage had actively formed the bone; and the medulla had been making. At the 4th week, the sequestrum had disappeared, and the mature callus had appeared; the osteoblasts had arranged in a line around the edge of the mature callus. At the 5th week, the callus was strong, compact and approached mature bones. At the 6th week, there had been the compact lamellar structures and the complete haversian's systems. There was no significant difference between callus of two sides by using image quantitative analysis in the 3rd, 4th week (P > 0.05). The allogenic bone has good histocompatibility and bone conduction effect, and can be used for bone transplantation substitute with autogenous-iliac bone.
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Dvorak, Melita M; De Joussineau, Cyrille; Carter, D Howard; Pisitkun, Trairak; Knepper, Mark A; Gamba, Gerardo; Kemp, Paul J; Riccardi, Daniela
2008-01-01
Thiazide diuretics are used, worldwide, as the first-choice drug for patients with uncomplicated hypertension. In addition to their anti-hypertensive actions, they increase bone mineral density and reduce the prevalence of fractures, indicating that thiazides may have a role in the management of postmenopausal osteoporosis. Traditionally, the bone-protective effects of thiazides have been attributed to an increase in renal calcium reabsorption, secondary to the inhibition of the sodium chloride cotransporter, NCC, expressed in the kidney distal tubule. Whether thiazides exert a direct osteoanabolic effect independently of their renal action is controversial. Here we demonstrate that freshly frozen sections of human and rat bone express NCC, principally in bone-forming cells, the osteoblasts. In primary and established culture models of osteoblasts, fetal rat calvarial (FRC) and human MG63 cells, NCC protein is virtually absent in proliferating cells while its expression is dramatically increased during differentiation. Thiazides directly stimulate the production of osteoblast markers, runt-related transcription factor 2 (runx2) and osteopontin, in the absence of a proliferative effect. Using overexpression/knockdown studies in FRC cells, we show that thiazides, but not loop diuretics, increase mineralized nodule formation acting on NCC. Overall, our study demonstrates that thiazides stimulate osteoblast differentiation and bone mineral formation independently of their renal actions. In addition to their use as part of a therapeutic treatment plan for elderly, hypertensive individuals, our discovery opens up the possibility that bone-specific drug targeting by thiazides may be developed for the prevention and treatment of osteoporosis in the patient population as a whole. PMID:17656470
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Peripheral cannabinoid receptor, CB2, regulates bone mass
Ofek, Orr; Karsak, Meliha; Leclerc, Nathalie; Fogel, Meirav; Frenkel, Baruch; Wright, Karen; Tam, Joseph; Attar-Namdar, Malka; Kram, Vardit; Shohami, Esther; Mechoulam, Raphael; Zimmer, Andreas; Bab, Itai
2006-01-01
The endogenous cannabinoids bind to and activate two G protein-coupled receptors, the predominantly central cannabinoid receptor type 1 (CB1) and peripheral cannabinoid receptor type 2 (CB2). Whereas CB1 mediates the cannabinoid psychotropic, analgesic, and orectic effects, CB2 has been implicated recently in the regulation of liver fibrosis and atherosclerosis. Here we show that CB2-deficient mice have a markedly accelerated age-related trabecular bone loss and cortical expansion, although cortical thickness remains unaltered. These changes are reminiscent of human osteoporosis and may result from differential regulation of trabecular and cortical bone remodeling. The CB2–/– phenotype is also characterized by increased activity of trabecular osteoblasts (bone-forming cells), increased osteoclast (the bone-resorbing cell) number, and a markedly decreased number of diaphyseal osteoblast precursors. CB2 is expressed in osteoblasts, osteocytes, and osteoclasts. A CB2-specific agonist that does not have any psychotropic effects enhances endocortical osteoblast number and activity and restrains trabecular osteoclastogenesis, apparently by inhibiting proliferation of osteoclast precursors and receptor activator of NF-κB ligand expression in bone marrow-derived osteoblasts/stromal cells. The same agonist attenuates ovariectomy-induced bone loss and markedly stimulates cortical thickness through the respective suppression of osteoclast number and stimulation of endocortical bone formation. These results demonstrate that the endocannabinoid system is essential for the maintenance of normal bone mass by osteoblastic and osteoclastic CB2 signaling. Hence, CB2 offers a molecular target for the diagnosis and treatment of osteoporosis, the most prevalent degenerative disease in developed countries. PMID:16407142
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Takano, H; Takahashi, T; Nakata, A; Nogami, S; Yusa, K; Kuwajima, S; Yamazaki, M; Fukuda, M
2016-05-01
The aim of this study was to investigate the bone resorption effect of the mediators delivered in joint cavity of patients with mandibular condyle fractures by detecting osteoclast markers using cellular biochemistry methods, and by analysing bone resorption activities via inducing osteoclast differentiation of the infiltrated cells from arthrocentesis. Sixteen joints in 10 patients with mandibular condyle fractures were evaluated. The control group consisted of synovial fluid (SF) samples from seven joints of four volunteers who had no clinical signs or symptoms involving the temporomandibular joint (TMJ) or disc displacement. We collected SF cells from all patients during therapeutic arthrocentesis. The infiltrating cells from TMJ SF were cultured, differentiated into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells and examined bone resorption activities. We also investigated factors related to osteoclast induction of SF, using ELISA procedures. Osteoclast-like cells were induced from the SF cells obtained from all patients with condylar fractures. These multinucleated giant cells were positive for TRAP and actin, and had the ability to absorb dentin slices. The levels of macrophage colony-stimulating factor (M-CSF), prostaglandin E2 (PGE2), soluble form of receptor activator of nuclear factor kappa-B ligand (sRANKL) and osteoprotegerin (OPG), in SF samples from the patients, were significantly higher than in the controls. These findings indicate that bone resorption activities in SF from patients with mandibular condyle fractures were upregulated and may participate in the pathogenesis and wound healing. © 2016 The Authors. Journal of Oral Rehabilitation Published by John Wiley & Sons Ltd.
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Weber, Kristy L; Doucet, Michele; Price, Janet E; Baker, Cheryl; Kim, Sun Jin; Fidler, Isaiah J
2003-06-01
Renal cell carcinoma (RCC) frequently produces metastases to the musculoskeletal system that are a major source of morbidity in the form of pain, immobilization, fractures, neurological compromise, and a decreased ability to perform activities of daily living. Patients with metastatic RCC therefore have a dismal prognosis because there is no effective adjuvant treatment for this disease. Because the epidermal growth factor receptor (EGF-R) signaling cascade is important in the growth and metastasis of RCC, its blockade has been hypothesized to inhibit tumor growth and hence prevent resultant bone destruction. We determined whether blockade of EGF-R by the tyrosine kinase inhibitor PKI 166 inhibited the growth of RCC in bone. We use a novel cell line, RBM1-IT4, established from a human RCC bone metastasis. Protein and mRNA expression of the ligands and receptors was assessed by Western and Northern blots. The stimulation of RBM1-IT4 cells with epidermal growth factor or transforming growth factor alpha resulted in increased cellular proliferation and tyrosine kinase autophosphorylation. PKI 166 prevented these effects. First, RBM1-IT4 cells were implanted into the tibia of nude mice, where they established lytic, progressively growing lesions, after which the mice were treated with PKI 166 alone or in combination with paclitaxel (Taxol). Immunohistochemical analysis revealed that tumor cells and tumor-associated endothelial cells in control mice expressed activated EGF-R. Treatment of mice with PKI 166 alone or in combination with Taxol produced a significant decrease in the incidence and size of bone lesions as compared with the results in control or Taxol-treated mice (P < 0.001). Treatment with PKI 166 also decreased the expression of phosphorylated EGF-R by tumor cells and tumor-associated endothelial cells, and this was even more pronounced with PKI 166 plus Taxol treatment. The PKI 166 plus Taxol combination produced apoptosis of tumor cells and tumor-associated endothelial cells. Tumor cell proliferation, shown by proliferating cell nuclear antigen positivity, was decreased in all treatment groups. In addition, the integrity of the bone was maintained in mice treated with PKI 166 or PKI 166 plus Taxol, whereas massive bone destruction was seen in control and Taxol-treated mice. These results suggest that blockade of EGF-R signaling inhibits growth of RCC in the bone by its effect on tumor cells and tumor-associated endothelial cells.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Witasp, Erika; Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm; Gustafsson, Ann-Catrin
2005-05-13
Previous studies have suggested that 1,25(OH){sub 2}D{sub 3}, the active form of vitamin D{sub 3}, may increase the survival of bone-forming osteoblasts through an inhibition of apoptosis. On the other hand, vitamin D{sub 3} has also been shown to trigger apoptosis in human cancer cells, including osteosarcoma-derived cell lines. In the present study, we show that 1,25(OH){sub 2}D{sub 3} induces a time- and dose-dependent loss of cell viability in the rat osteosarcoma cell line, UMR-106, and the human osteosarcoma cell line, TE-85. We were unable, however, to detect nuclear condensation, phosphatidylserine externalization, or other typical signs of apoptosis in thismore » model. Moreover, 1,25(OH){sub 2}D{sub 3} failed to protect against apoptosis induced by serum starvation or incubation with the protein kinase inhibitor, staurosporine. These in vitro findings are thus at variance with several previous reports in the literature and suggest that induction of or protection against apoptosis of bone-derived cells may not be a primary function of vitamin D{sub 3}.« less
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Role of T cells in sex differences in syngeneic bone marrow transfers.
Raveche, E S; Santoro, T; Brecher, G; Tjio, J H
1985-11-01
Transferred marrow cells will proliferate in normal mice not exposed to irradiation or any other type of stem cell depletion when five consecutive transfers of 40 million cells are given. Approximately 25% of the mitotic cells are of male donor origin observed cytogenetically in all of the female recipient spleens and marrow analyzed from two weeks to one and one-half years after transfusions. Male donor stem cells are accepted and form a stable component of the self-renewing stem cell pool. In contrast, only 5% female cells are found in male recipients. This sex difference in engraftment is not hormonal since castration of recipients does not alter the percentage of donor cells. Rigorous T depletion of female donor bone marrow, however, increases the percentage of donor engraftment to the level observed when male marrow, either whole or T depleted, is transferred to female recipients. The success of T-depleted female stem cells to seed male recipients is observed in both C57BL/6, a responder strain in which females readily respond to the H-Y antigen as manifest by skin graft rejection, and CBA/J, a strain in which females do not readily respond to H-Y. In addition, recipient nude BALB/c males, which lack a thymus, fail to accept whole bone marrow from BALB/c females. However, male bone marrow cells seed BALB/c nude females. These studies demonstrate that the poor engraftment of female cells in transfused male recipients is abrogated by the removal of T cells from the donor female marrow.
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Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping
2012-01-01
Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption. PMID:23139637
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Messina, Valeria; Valtieri, Mauro; Rubio, Mercedes; Falchi, Mario; Mancini, Francesca; Mayor, Alfredo; Alano, Pietro; Silvestrini, Francesco
2018-01-01
The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs). Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages. PMID:29546035
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Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping
2012-01-01
Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption.
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Gou, Shanmiao; Liu, Tao; Li, Xiangsheng; Cui, Jing; Wan, Chidan; Wang, Chunyou
2012-01-01
Bone marrow-derived mesenchymal stem cells (bMSCs) contribute to tissue repair and regeneration. Cell fusion between somatic cells and bMSCs to form hybrid cells may have an important role in tissue repair through the subsequent reprogramming of the somatic cell nucleus. Few studies have assessed the mesenchymal characteristics of fusion-induced hybrid cells and their survival mechanisms. In this study, we investigated the effect of cell fusion on the biological characteristics of pancreatic ductal cells (PDCs) and on the survival mechanism of hybrid cells. To this end, we generated mouse-mouse hybrid cells in vitro by polyethylene glycol-mediated fusion of primary mouse bMSCs with primary mouse PDCs. Hybrid cells showed an enhanced capacity for proliferation and self-renewal compared with PDCs. No PDC had the capacity for anchorage-independent growth or invasion into Matrigel, but some hybrid cells were able to form colonies in soft agar and invade Matrigel. Expression of the tumor suppressor protein p53, which initiates apoptosis, was detected in hybrid cells but not in PDCs or bMSCs. However, the p53 deacetylase, sirtuin 1 (SIRT1), was also detected in hybrid cells, and the level of acetylated p53, the active form, was low. The addition of nicotinamide (Nam) inhibited the deacetylation activity of SIRT1 on p53 and induced cell apoptosis in hybrid cells. This study demonstrated that PDCs could obtain high proliferation rates, self-renewal capabilities, and mesenchymal characteristics by fusion with bMSCs. SIRT1 expression in the hybrid cells attenuated their apoptosis. Copyright © 2012 S. Karger AG, Basel.
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Self-Assembling Nanoclay Diffusion Gels for Bioactive Osteogenic Microenvironments.
Shi, Pujiang; Kim, Yang-Hee; Mousa, Mohamed; Sanchez, Roxanna Ramnarine; Oreffo, Richard O C; Dawson, Jonathan I
2018-06-17
Laponite nanoparticles have attracted attention in the tissue engineering field for their protein interactions, gel-forming properties, and, more recently, osteogenic bioactivity. Despite growing interest in the osteogenic properties of Laponite, the application of Laponite colloidal gels to host the osteogenic differentiation of responsive stem cell populations remains unexplored. Here, the potential to harness the gel-forming properties of Laponite to generate injectable bioactive microenvironments for osteogenesis is demonstrated. A diffusion/dialysis gelation method allows the rapid formation of stable transparent gels from injectable, thixotropic Laponite suspensions in physiological fluids. Upon contact with buffered saline or blood serum, nanoporous gel networks exhibiting, respectively, fivefold and tenfold increases in gel stiffness are formed due to the reorganization of nanoparticle interactions. Laponite diffusion gels are explored as osteogenic microenvironments for skeletal stem cell containing populations. Laponite films support cell adhesion, proliferation, and differentiation of human bone marrow stromal cells in 2D. Laponite gel encapsulation significantly enhances osteogenic protein expression compared with 3D pellet culture controls. In both 2D and 3D conditions, cell associated mineralization is strongly enhanced. This study demonstrates that Laponite diffusion gels offer considerable potential as biologically active and clinically relevant bone tissue engineering scaffolds. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Nanostructured magnesium increases bone cell density.
Weng, Lucy; Webster, Thomas J
2012-12-07
Magnesium has attracted some attention in orthopedics due to its biodegradability and mechanical properties. Since magnesium is an essential natural mineral for bone growth, it can be expected that as a biomaterial, it would support bone formation. However, upon degradation in the body, magnesium releases OH(-) which results in an alkaline pH that can be detrimental to cell density (for example, osteoblasts or bone forming cells). For this reason, modification of magnesium may be necessary to compensate for such detrimental effects to cells. This study created biologically inspired nanoscale surface features on magnesium by soaking magnesium in various concentrations of NaOH (from 1 to 10 N) and for various periods of time (from 10 to 30 min). The results provided the first evidence of increased roughness, surface energy, and consequently greater osteoblast adhesion, after 4 h as well as density up to 7 days on magnesium treated with any concentration of NaOH for any length of time compared to untreated controls. For these reasons, this study suggests that soaking magnesium in NaOH could be an inexpensive, simple and effective manner to promote osteoblast functions for numerous orthopedic applications and, thus, should be further studied.
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Leite, Yulla Klinger de Carvalho; de Carvalho, Camila Ernanda Sousa; Feitosa, Matheus Levi Tajra; Alves, Michel Muálem de Moraes; Carvalho, Fernando Aécio de Amorim; Neto, Bartolomeu Cruz Viana; Miglino, Maria Angélica
2018-01-01
Background Tissue engineering has been shown to exhibit great potential for the creation of biomaterials capable of developing into functional tissues. Cellular expansion and integration depends on the quality and surface-determinant factors of the scaffold, which are required for successful biological implants. The objective of this research was to characterize and evaluate the in vitro characteristics of rabbit bone marrow mesenchymal stem cells (BM-MSCs) associated with a bacterial cellulose membrane (BCM). We assessed the adhesion, expansion, and integration of the biomaterial as well as its ability to induce macrophage activation. Finally, we evaluated the cytotoxicity and toxicity of the BCM. Methods Samples of rabbit bone marrow were collected. Mesenchymal stem cells were isolated from medullary aspirates to establish fibroblast colony-forming unit assay. Osteogenic, chondrogenic, and adipogenic differentiation was performed. Integration with the BCM was assessed by scanning electron microscopy at 1, 7, and 14 days. Cytotoxicity was assessed via the production of nitric oxide, and BCM toxicity was assessed with the MTT assay; phagocytic activity was also determined. Results The fibroblastoid colony-forming unit (CFU-F) assay showed cells with a fibroblastoid morphology organized into colonies, and distributed across the culture area surface. In the growth curve, two distinct phases, lag and log phase, were observed at 15 days. Multipotentiality of the cells was evident after induction of osteogenic, chondrogenic, and adipogenic lineages. Regarding the BM-MSCs’ bioelectrical integration with the BCM, BM-MSCs were anchored in the BCM in the first 24 h. On day 7 of culture, the cytoplasm was scattered, and on day 14, the cells were fully integrated with the biomaterial. We also observed significant macrophage activation; analysis of the MTT assay and the concentration of nitric oxide revealed no cytotoxicity of the biomaterial. Conclusion The BCM allowed the expansion and biointegration of bone marrow progenitor cells with a stable cytotoxic profile, thus presenting itself as a biomaterial with potential for tissue engineering. PMID:29736332
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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.
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Bioelectric modulation of wound healing in a 3D in vitro model of tissue-engineered bone.
Sundelacruz, Sarah; Li, Chunmei; Choi, Young Jun; Levin, Michael; Kaplan, David L
2013-09-01
Long-standing interest in bioelectric regulation of bone fracture healing has primarily focused on exogenous stimulation of bone using applied electromagnetic fields. Endogenous electric signals, such as spatial gradients of resting potential among non-excitable cells in vivo, have also been shown to be important in cell proliferation, differentiation, migration, and tissue regeneration, and may therefore have as-yet unexplored therapeutic potential for regulating wound healing in bone tissue. To study this form of bioelectric regulation, there is a need for three-dimensional (3D) in vitro wound tissue models that can overcome limitations of current in vivo models. We present a 3D wound healing model in engineered bone tissue that serves as a pre-clinical experimental platform for studying electrophysiological regulation of wound healing. Using this system, we identified two electrophysiology-modulating compounds, glibenclamide and monensin, that augmented osteoblast mineralization. Of particular interest, these compounds displayed differential effects in the wound area compared to the surrounding tissue. Several hypotheses are proposed to account for these observations, including the existence of heterogeneous subpopulations of osteoblasts that respond differently to bioelectric signals, or the capacity of the wound-specific biochemical and biomechanical environment to alter cell responses to electrophysiological treatments. These data indicate that a comprehensive characterization of the cellular, biochemical, biomechanical, and bioelectrical components of in vitro wound models is needed to develop bioelectric strategies to control cell functions for improved bone regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation
2009-01-01
Background A recent model for activation of the zymogen form of matrix metalloproteinase 2 (MMP-2, also known as gelatinase A) has suggested that interactions between the SIBLING protein bone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates the conversion of the pro-enzyme into a catalytically active form. This model is particularly relevant to cancer cell metastasis to bone since BSP, bound to the αvβ3 integrin through its arginine-glycine-aspartic acid motif, could recruit MMP-2 to the cell surface. Methods We critically assessed the relationship between BSP and proMMP-2 and its activation using various forms of recombinant and purified BSP and MMP-2. Gelatinase and collagenase assays, fluorescence binding assays, real-time PCR, cell culture and pull-down assays were employed to test the model. Results Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 by BSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. While BSP-mediated invasiveness has been shown to depend on its integrin-binding RGD sequence, analysis of proMMP-2 activation and the level of membrane type 1 (MT1)-MMP in cells grown on a BSP substratum showed that the BSP-αvβ3 integrin interaction does not induce the expression of MT1-MMP. Conclusion These studies do not support a role for BSP in promoting metastasis through interactions with pro-MMP-2. PMID:19386107
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The Molecular and Cellular Events That Take Place during Craniofacial Distraction Osteogenesis
Rachmiel, Adi
2014-01-01
Summary: Gradual bone lengthening using distraction osteogenesis principles is the gold standard for the treatment of hypoplastic facial bones. However, the long treatment time is a major disadvantage of the lengthening procedures. The aim of this study is to review the current literature and summarize the cellular and molecular events occurring during membranous craniofacial distraction osteogenesis. Mechanical stimulation by distraction induces biological responses of skeletal regeneration that is accomplished by a cascade of biological processes that may include differentiation of pluripotential tissue, angiogenesis, osteogenesis, mineralization, and remodeling. There are complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Studies have implicated number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (transforming growth factor-β, bone morphogenetic proteins, insulin-like growth factor-1, and fibroblast growth factor-2) and extracellular matrix proteins (osteonectin, osteopontin) during distraction osteogenesis has been best characterized and discussed. Understanding the biomolecular mechanisms that mediate membranous distraction osteogenesis may guide the development of targeted strategies designed to improve distraction osteogenesis and accelerate bone regeneration that may lead to shorten the treatment duration. PMID:25289295
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Biomimetic component coating on 3D scaffolds using high bioactivity of mesoporous bioactive ceramics
Yun, Hui-suk; Kim, Sang-Hyun; Khang, Dongwoo; Choi, Jungil; Kim, Hui-hoon; Kang, Minji
2011-01-01
Background Mesoporous bioactive glasses (MBGs) are very attractive materials for use in bone tissue regeneration because of their extraordinarily high bone-forming bioactivity in vitro. That is, MBGs may induce the rapid formation of hydroxy apatite (HA) in simulated body fluid (SBF), which is a major inorganic component of bone extracellular matrix (ECM) and comes with both good osteoconductivity and high affinity to adsorb proteins. Meanwhile, the high bioactivity of MBGs may lead to an abrupt initial local pH variation during the initial Ca ion-leaching from MBGs at the initial transplant stage, which may induce unexpected negative effects on using them in in vivo application. In this study we suggest a new way of using MBGs in bone tissue regeneration that can improve the strength and make up for the weakness of MBGs. We applied the outstanding bone-forming bioactivity of MBG to coat the main ECM components HA and collagen on the MBG-polycarplolactone (PCL) composite scaffolds for improving their function as bone scaffolds in tissue regeneration. This precoating process can also expect to reduce initial local pH variation of MBGs. Methods and materials The MBG-PCL scaffolds were immersed in the mixed solution of the collagen and SBF at 37°C for 24 hours. The coating of ECM components on the MBG-PCL scaffolds and the effect of ECM coating on in vitro cell behaviors were confirmed. Results The ECM components were fully coated on MBG-PCL scaffolds after immersing in SBF containing dilute collagen-I solution only for 24 hours due to the high bone-forming bioactivity of MBG. Both cell affinity and osteoconductivity of MBG-PCL scaffolds were dramatically enhanced by this precoating process. Conclusion The precoating process of ECM components on MBG-PCL scaffold using a high bioactivity of MBG was not only effective in enhancing the functionality of scaffolds but also effective in eliminating the unexpected side effect. The MBG-PCL scaffold-coated ECM components ideally satisfied the required conditions of scaffold in tissue engineering, including 3D well-interconnected pore structures with high porosity, good bioactivity, enhanced cell affinity, biocompatibility, osteoconductivity, and sufficient mechanical properties, and promise excellent potential application in the field of biomaterials. PMID:22072886
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[Quantitative data analysis for live imaging of bone.
Seno, Shigeto
Bone tissue is a hard tissue, it was difficult to observe the interior of the bone tissue alive. With the progress of microscopic technology and fluorescent probe technology in recent years, it becomes possible to observe various activities of various cells forming bone society. On the other hand, the quantitative increase in data and the diversification and complexity of the images makes it difficult to perform quantitative analysis by visual inspection. It has been expected to develop a methodology for processing microscopic images and data analysis. In this article, we introduce the research field of bioimage informatics which is the boundary area of biology and information science, and then outline the basic image processing technology for quantitative analysis of live imaging data of bone.
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FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting
Riminucci, Mara; Collins, Michael T.; Fedarko, Neal S.; Cherman, Natasha; Corsi, Alessandro; White, Kenneth E.; Waguespack, Steven; Gupta, Anurag; Hannon, Tamara; Econs, Michael J.; Bianco, Paolo; Gehron Robey, Pamela
2003-01-01
FGF-23, a novel member of the FGF family, is the product of the gene mutated in autosomal dominant hypophosphatemic rickets (ADHR). FGF-23 has been proposed as a circulating factor causing renal phosphate wasting not only in ADHR (as a result of inadequate degradation), but also in tumor-induced osteomalacia (as a result of excess synthesis by tumor cells). Renal phosphate wasting occurs in approximately 50% of patients with McCune-Albright syndrome (MAS) and fibrous dysplasia of bone (FD), which result from postzygotic mutations of the GNAS1 gene. We found that FGF-23 is produced by normal and FD osteoprogenitors and bone-forming cells in vivo and in vitro. In situ hybridization analysis of FGF-23 mRNA expression identified “fibrous” cells, osteogenic cells, and cells associated with microvascular walls as specific cellular sources of FGF-23 in FD. Serum levels of FGF-23 were increased in FD/MAS patients compared with normal age-matched controls and significantly higher in FD/MAS patients with renal phosphate wasting compared with those without, and correlated with disease burden bone turnover markers commonly used to assess disease activity. Production of FGF-23 by FD tissue may play an important role in the renal phosphate–wasting syndrome associated with FD/MAS. PMID:12952917
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Effects of microwaves on the colony-forming capacity of haemopoietic stem cells in mice.
Rotkovská, D; Vacek, A; Bartonícková, A
1987-01-01
A suspension of bone marrow cells from femurs of female (CBA X C57Bl)F1 mice was exposed to 2450 MHz CW microwaves in a specially designed waveguide exposure system. The temperature of the suspension rose, during exposure to microwaves, from 20 degrees C to 45 degrees C, and at an interval within 20 degrees C to 45 degrees C the number of haemopoietic stem cells (CFUs) was determined by the spleen exocolony method. The time of exposure of bone marrow cells to each temperature studied was 20 s. Control suspensions of bone marrow cells were exposed to a water bath temperature. There were no significant effects of the CFUs with the water bath temperature, while after exposure to microwaves the number of spleen colonies was elevated with a nadir at the temperature of 37 degrees C. With a microwave-induced increase of the temperature above 41 degrees C the number of CFUs in the bone marrow suspension decreased. The increase in the number of colonies was related to the rise in the seeding rate of the CFUs as well as to a rise in their proliferative activity, while the drop in the number of colonies was influenced also by heat-killing of the CFUs by microwave exposure.
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Sun, Bin; Ma, Wei; Su, Fang; Wang, Yi; Liu, Jiaqiang; Wang, Dongshen; Liu, Hongchen
2011-09-01
Type I collagen was added to the composite chitosan solution in a ratio of 1:2 to build a physical cross-linked self-forming chitosan/collagen/β-GP hydrogel. Osteogenic properties of this novel injectable hydrogel were evaluated. Gelation time was about 8 min which offered enough time for handling a mixture containing cells and the subsequent injection. Scanning electronic microscopy (SEM) observations indicated good spreading of bone marrow mesenchymal stem cells (BMSCs) in this hydrogel scaffold. Mineral nodules were found in the dog-BMSCs inoculated hydrogel by SEM after 28 days. After subcutaneous injection into nude mouse dorsum for 4 weeks, partial bone formation was observed in the chitosan/collagen/β-GP hydrogel loaded with pre-osteodifferentiated dog-BMSCs, which indicated that chitosan/collagen/β-GP hydrogel composite could induce osteodifferentiation in BMSCs without exposure to a continual supply of external osteogenic factors. In conclusion, the novel chitosan/collagen/β-GP hydrogel composite should prove useful as a bone regeneration scaffold.
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Pajovich, Harrison T.; Banerjee, Ipsita A.
2017-01-01
Fucoidan (Fuc), a natural polysaccharide derived from brown seaweed algae, and gelatin (Gel) were conjugated to form a template for preparation of biomimetic scaffolds for potential applications in bone tissue regeneration. To the Fuc–Gel we then incorporated the peptide sequence MTNYDEAAMAIASLN (MTN) derived from the E-F hand domain, known for its calcium binding properties. To mimic the components of the extracellular matrix of bone tissue, the Fuc–Gel–MTN assemblies were incubated in simulated body fluid (SBF) to induce biomineralization, resulting in the formation of β-tricalcium phosphate, and hydroxyapatite (HAp). The formed Fuc–Gel–MTN–beta–TCP/HAP scaffolds were found to display an average Young’s Modulus value of 0.32 GPa (n = 5) with an average surface roughness of 91 nm. Rheological studies show that the biomineralized scaffold exhibited higher storage and loss modulus compared to the composites formed before biomineralization. Thermal phase changes were studied through DSC and TGA analysis. XRD and EDS analyses indicated a biphasic mixture of β-tricalcium phosphate and hydroxyapatite and the composition of the scaffold. The scaffold promoted cell proliferation, differentiation and displayed actin stress fibers indicating the formation of cell-scaffold matrices in the presence of MT3C3-E1 mouse preosteoblasts. Osteogenesis and mineralization were found to increase with Fuc–Gel–MTN–beta–TCP/HAP scaffolds. Thus, we have developed a novel scaffold for possible applications in bone tissue engineering. PMID:29036882
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Biology of Bone: The Vasculature of the Skeletal System.
Watson, Emma C; Adams, Ralf H
2017-09-11
Blood vessels are essential for the distribution of oxygen, nutrients, and immune cells, as well as the removal of waste products. In addition to this conventional role as a versatile conduit system, the endothelial cells forming the innermost layer of the vessel wall also possess important signaling capabilities and can control growth, patterning, homeostasis, and regeneration of the surrounding organ. In the skeletal system, blood vessels regulate developmental and regenerative bone formation as well as hematopoiesis by providing vascular niches for hematopoietic stem cells. Here we provide an overview of blood vessel architecture, growth and properties in the healthy, aging, and diseased skeletal system. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.
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Purification and Cultivation of Human Pituitary Growth Hormones Secreting Cells
NASA Technical Reports Server (NTRS)
Hymer, W. C.; Todd, P.; Grindeland, R.; Lanham, W.; Morrison, D.
1985-01-01
The rat and human pituitary gland contains a mixture of hormone producing cell types. The separation of cells which make growth hormone (GH) is attempted for the purpose of understanding how the hormone molecule is made within the pituitary cell; what form(s) it takes within the cell; and what form(s) GH assumes as it leaves the cell. Since GH has a number of biological targets (e.g., muscle, liver, bone), the assessment of the activities of the intracellular/extracellular GH by new and sensitive bioassays. GH cells contained in the mixture was separated by free flow electrophoresis. These experiments show that GH cells have different electrophoretic mobilities. This is relevant to NASA since a lack of GH could be a prime causative factor in muscle atrophy. Further, GH has recently been implicated in the etiology of motion sickness in space. Continous flow electrophoresis experiment on STS-8 showed that GH cells could be partially separated in microgravity. However, definitive cell culture studies could not be done due to insufficient cell recoveries.
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Yang, Yang; Lan, Ding; Huang, Yan; Li, Yanming; Wang, Yuren; Sun, Lianwen; Fan, Yubo
2014-06-01
Polydimethylsiloxane (PDMS) and hydroxyapatite (HA) were combined in our laboratory to fabricate an elastic porous cell scaffold with pore-forming agent, and then the scaffold was used as culture media for rat bone marrow derived mesenchymal stem cells (rBMSCs). Different porous materials (square and circular in shape) were prepared by different pore-forming agents (NaCl or paraffin spheres) with adjustable porosity (62%-76%). The HA crystals grew on the wall of hole when the material was exposed to SBF solutions, showing its biocompatibility and ability to support the cells to attach on the materials.
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High levels of circulating triiodothyronine induce plasma cell differentiation.
Bloise, Flavia Fonseca; Oliveira, Felipe Leite de; Nobrega, Alberto Félix; Vasconcellos, Rita; Cordeiro, Aline; Paiva, Luciana Souza de; Taub, Dennis D; Borojevic, Radovan; Pazos-Moura, Carmen Cabanelas; Mello-Coelho, Valéria de
2014-03-01
The effects of hyperthyroidism on B-cell physiology are still poorly known. In this study, we evaluated the influence of high-circulating levels of 3,5,3'-triiodothyronine (T3) on bone marrow, blood, and spleen B-cell subsets, more specifically on B-cell differentiation into plasma cells, in C57BL/6 mice receiving daily injections of T3 for 14 days. As analyzed by flow cytometry, T3-treated mice exhibited increased frequencies of pre-B and immature B-cells and decreased percentages of mature B-cells in the bone marrow, accompanied by an increased frequency of blood B-cells, splenic newly formed B-cells, and total CD19(+)B-cells. T3 administration also promoted an increase in the size and cellularity of the spleen as well as in the white pulp areas of the organ, as evidenced by histological analyses. In addition, a decreased frequency of splenic B220(+) cells correlating with an increased percentage of CD138(+) plasma cells was observed in the spleen and bone marrow of T3-treated mice. Using enzyme-linked immunospot assay, an increased number of splenic immunoglobulin-secreting B-cells from T3-treated mice was detected ex vivo. Similar results were observed in mice immunized with hen egg lysozyme and aluminum adjuvant alone or together with treatment with T3. In conclusion, we provide evidence that high-circulating levels of T3 stimulate plasma cytogenesis favoring an increase in plasma cells in the bone marrow, a long-lived plasma cell survival niche. These findings indicate that a stimulatory effect on plasma cell differentiation could occur in untreated patients with Graves' disease.
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Mandibular Repair in Rats with Premineralized Silk Scaffolds and BMP-2-modified bMSCs
Jiang, Xinquan; Zhao, Jun; Wang, Shaoyi; Sun, Xiaojuan; Zhang, Xiuli; Chen, Jake; Kaplan, David L.; Zhang, Zhiyuan
2010-01-01
Premineralized silk fibroin protein scaffolds (mSS) were prepared to combine the osteoconductive properties of biological apatite with aqueous-derived silk scaffold (SS) as a composite scaffold for bone regeneration. The aim of present study was to evaluate the effect of premineralized silk scaffolds combined with bone morphogenetic protein-2 (BMP-2) modified bone marrow stromal cells (bMSCs) to repair mandibular bony defects in a rat model. bMSCs were expanded and transduced with adenovirus AdBMP-2, AdLacZ gene in vitro. These genetically modified bMSCs were then combined with premineralized silk scaffolds to form tissue engineered bone. Mandibular repairs with AdBMP-2 transduced bMSCs/mSS constructs were compared with those treated with AdLacZ transduced bMSCs/mSS constructs, native (nontransduced) bMSCs/mSS constructs and mSS alone. Eight weeks post-operation, the mandibles were explanted and evaluated by radiographic observation, micro-CT, histological analysis and immunohistochemistry. The presence of BMP-2 gene enhanced tissue engineered bone in terms of the most new bone formed and the highest local bone mineral densities (BMD) found. These results demonstrated that premineralized silk scaffold could serve as a potential substrate for bMSCs to construct tissue engineered bone for mandibular bony defects. BMP-2 gene therapy and tissue engineering techniques could be used in mandibular repair and bone regeneration. PMID:19501905
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Gan, Xueqi; Zhang, Ling; Liu, Beilei; Zhu, Zhuoli; He, Yuting; Chen, Junsheng; Zhu, Junfei; Yu, Haiyang
2018-04-20
Bone is a dynamic organ, the bone-forming osteoblasts and bone-resorbing osteoclasts form the physiological basis of bone remodeling process. During pathological process of numerous inflammatory diseases, these two aspects are uncoupled and the balance is usually tipped in favor of bone destruction. Evidence suggests that the inflammatory destruction of bone is mainly attributed to oxidative stress and is closely related to mitochondrial dysfunction. The mechanisms underlying osteogenic dysfunction in inflammation still need further investigation. Reactive oxygen species (ROS) is associated with mitochondrial dysfunction and cellular damage. Here, we reported an unexplored role of cyclophilin D (CypD), the major modulator of mitochondrial permeability transition pore (mPTP), and the CypD-mPTP axis in inflammation-induced mitochondrial dysfunction and bone damage. And the protective effects of knocking down CypD by siRNA interference or the addition of cyclosporin A (CsA), an inhibitor of CypD, were evidenced by rescued mitochondrial function and osteogenic function of osteoblast under tumor necrosis factor-α (TNF-α) treatment. These findings provide new insights into the role of CypD-mPTP-dependent mitochondrial pathway in the inflammatory bone injury. The protective effect of CsA or other moleculars affecting the mPTP formation may hold promise as a potential novel therapeutic strategy for inflammation-induced bone damage via mitochondrial pathways.
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Effects of spaceflight on rat peripheral blood leukocytes and bone marrow progenitor cells
NASA Technical Reports Server (NTRS)
Ichiki, A. T.; Gibson, L. A.; Jago, T. L.; Strickland, K. M.; Johnson, D. L.; Lange, R. D.; Allebban, Z.
1996-01-01
The white blood cell (WBC) elements and the bone marrow myeloid progenitor cell populations were analyzed to ascertain adaptation to micro-gravity and subsequent readaptation to 1 G in rats flown on the 14-day Spacelab Life Sciences-2 (SLS-2) mission. Bone marrow cells were harvested from one group of rats killed inflight (FD13) and blood was drawn from three other groups at various times. The WBC level was normal on FD14 with the exception of neutrophilia. On FD13, numbers of colony-forming units-granulocyte (CFU-G), CFU-GM, and CFU-M from flight animals were decreased compared with ground controls when incubated with recombinant rat interleukin-3 (rrIL-3) alone or in combination with recombinant human erythropoietin (rhEpo). On recovery (R + 0), flight rats had decreased numbers of total leukocytes and absolute numbers of lymphocytes and monocytes with elevated neutrophils compared with control rats. They had lower numbers of CD4, CD8, CD2, CD3, and B cells in the peripheral blood but no differences in spleen lymphocytes.
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Generating trunk neural crest from human pluripotent stem cells
Huang, Miller; Miller, Matthew L.; McHenry, Lauren K.; Zheng, Tina; Zhen, Qiqi; Ilkhanizadeh, Shirin; Conklin, Bruce R.; Bronner, Marianne E.; Weiss, William A.
2016-01-01
Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior “cranial” NCC form craniofacial bone, whereas solely posterior “trunk” NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages. PMID:26812940
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Le Nail, Louis-Romée; Brennan, Meadhbh; Rosset, Philippe; Piloquet, Philippe; Pichon, Olivier; Le Caignec, Cédric; Crenn, Vincent; Layrolle, Pierre; Hérault, Olivier; De Pinieux, Gonzague
2018-01-01
Osteosarcoma (OS) is suspected to originate from dysfunctional mesenchymal stromal/stem cells (MSC). We sought to identify OS-derived cells (OSDC) with potential cancer stem cell (CSC) properties by comparing OSDC to MSC derived from bone marrow of patients. This study included in vitro characterization with sphere forming assays, differentiation assays, cytogenetic analysis, and in vivo investigations of their tumorigenicity and tumor supportive capacities. Primary cell lines were isolated from nine high-grade OS samples. All primary cell lines demonstrated stromal cell characteristics. Compared to MSC, OSDC presented a higher ability to form sphere clones, indicating a potential CSC phenotype, and were more efficient at differentiation towards osteoblasts. None of the OSDC displayed the complex chromosome rearrangements typical of high grade OS and none of them induced tumors in immunodeficient mice. However, two OSDC demonstrated focused genomic abnormalities. Three out of seven, and six out of seven OSDC showed a supportive role on local tumor development, and on metastatic progression to the lungs, respectively, when co-injected with OS cells in nude mice. The observation of OS-associated stromal cells with rare genetic abnormalities and with the capacity to sustain tumor progression may have implications for future tumor treatments. PMID:29494553
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Svet-Moldavskaya, I A; Zinzar, S N; Svet-Moldavsky, G J; Arlin, Z; Vergara, C; Koziner, B; Clarkson, B D; Holland, J F
1983-08-01
Normal human sera induce the formation of fat-containing cells (FCC) in human bone marrow cultures. A nearly complete monolayer of FCC is formed after 7-14 days of cultivation with 20% human sera in the medium. FCC-inducing activity (FCCIA) is nondialyzable through 14,900-dalton cutoff membrane and is stable at 56 degrees C for 30 min. Abundant FCCIA was found in 83% of normal human sera but in only 20% of sera from untreated patients with different hemopoietic disorders and in 32% of treated leukemic patients. It is suggested that FCCIA may be involved in regulation of the bone marrow microenvironment an that it varies in normal individuals and in patients with different diseases.
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Vesicular delivery of crystalline calcium minerals to ECM in biomineralized nanoclay composites
NASA Astrophysics Data System (ADS)
Katti, Kalpana S.; Ambre, Avinash H.; Payne, Scott; Katti, Dinesh R.
2015-04-01
The mechanisms of mineralization and new bone formation were explored in newly formed extracellular matrix in a nanoclay based composite. Nanoclay films were prepared by intercalating the clays with amino acids and using the amino acids for mineralization of hydroxyapatite. The biomineralized hydroxyapatite (HAP) inside nanoclay galleries or in situ HAP/clay was further used to make films (substrates) using polycaprolactone (PCL) that were seeded with mesenchymal stem cells in a two-stage seeding process. SEM imaging experiments performed on PCL/in situ HAPclay composite films seeded with human MSCs indicated formation of matrix vesicles. The vesicles appear to emerge from the cells that are adhered to the nanoclay HAP films and also deposited in the extracellular space. Vesicles are also observed to be embedded in the cells or under the surface of cells. Crystalline structures with Ca and P were found inside vesicles. The Ca/P ratios obtained using energy dispersive spectroscopy indicate values ranging from below 0.7 to the stoichiometric HAP value of 1.67. The Ca/P ratios were obtained to be closer to the stoichiometric value for single seeding experiments as compared to the double seeding experiments indicating more new bone formation in double seeding experiments. New bone formation with bone mimetic mineralization is thus observed on the in situ HAP nanoclay PCL samples. Hence the PCL/in situ HAPclay composites besides being osteoinductive are also capable of providing a favorable micro-environment for cell dependent processes involved in bone mineral formation.
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High-Density Spot Seeding for Tissue Model Formation
NASA Technical Reports Server (NTRS)
Marquette, Michele L. (Inventor); Sognier, Marguerite A. (Inventor)
2014-01-01
A method for making a tissue includes seeding cells at a selected concentration on a support to form a cell spot, incubating the cells to allow the cells to partially attach, rinsing the cells to remove any unattached cells, adding culture medium to enable the cells to proliferate at a periphery of the cell spot and to differentiate toward a center of the cell spot, and further incubating the cells to form the tissue. The cells may be C2C12 cells or other subclones of the C2 cell line, H9c2(2-1) cells, L6 cells, L8 cells, QM7 cells, Sol8 cells, G-7 cells, G-8 cells, other myoblast cells, cells from other tissues, or stem cells. The selected concentration is in a range from about 1 x 10(exp 5) cells/ml to about 1 x 10(exp 6) cells/ml. The tissue formed may be a skeletal muscle tissue, a cardiac muscle tissue, nerve tissue, or a bone tissue.
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NASA Technical Reports Server (NTRS)
Globus, R. K.; Moursi, A.; Zimmerman, D.; Lull, J.; Damsky, C.
1995-01-01
The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.
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Prevascularization of 3D printed bone scaffolds by bioactive hydrogels and cell co-culture.
Kuss, Mitchell A; Wu, Shaohua; Wang, Ying; Untrauer, Jason B; Li, Wenlong; Lim, Jung Yul; Duan, Bin
2017-09-13
Vascularization is a fundamental prerequisite for large bone construct development and remains one of the main challenges of bone tissue engineering. Our current study presents the combination of 3D printing technique with a hydrogel-based prevascularization strategy to generate prevascularized bone constructs. Human adipose derived mesenchymal stem cells (ADMSC) and human umbilical vein endothelial cells (HUVEC) were encapsulated within our bioactive hydrogels, and the effects of culture conditions on in vitro vascularization were determined. We further generated composite constructs by forming 3D printed polycaprolactone/hydroxyapatite scaffolds coated with cell-laden hydrogels and determined how the co-culture affected vascularization and osteogenesis. It was demonstrated that 3D co-cultured ADMSC-HUVEC generated capillary-like networks within the porous 3D printed scaffold. The co-culture systems promoted in vitro vascularization, but had no significant effects on osteogenesis. The prevascularized constructs were subcutaneously implanted into nude mice to evaluate the in vivo vascularization capacity and the functionality of engineered vessels. The hydrogel systems facilitated microvessel and lumen formation and promoted anastomosis of vascular networks of human origin with host murine vasculature. These findings demonstrate the potential of prevascularized 3D printed scaffolds with anatomical shape for the healing of larger bone defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.
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Pilipchuk, Sophia P; Monje, Alberto; Jiao, Yizu; Hao, Jie; Kruger, Laura; Flanagan, Colleen L; Hollister, Scott J; Giannobile, William V
2016-03-01
Scaffold design incorporating multiscale cues for clinically relevant, aligned tissue regeneration has potential to improve structural and functional integrity of multitissue interfaces. The objective of this preclinical study is to develop poly(ε-caprolactone) (PCL) scaffolds with mesoscale and microscale architectural cues specific to human ligament progenitor cells and assess their ability to form aligned bone-ligament-cementum complexes in vivo. PCL scaffolds are designed to integrate a 3D printed bone region with a micropatterned PCL thin film consisting of grooved pillars. The patterned film region is seeded with human ligament cells, fibroblasts transduced with bone morphogenetic protein-7 genes seeded within the bone region, and a tooth dentin segment positioned on the ligament region prior to subcutaneous implantation into a murine model. Results indicate increased tissue alignment in vivo using micropatterned PCL films, compared to random-porous PCL. At week 6, 30 μm groove depth significantly enhances oriented collagen fiber thickness, overall cell alignment, and nuclear elongation relative to 10 μm groove depth. This study demonstrates for the first time that scaffolds with combined hierarchical mesoscale and microscale features can align cells in vivo for oral tissue repair with potential for improving the regenerative response of other bone-ligament complexes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Unique cell culture systems for ground based research
NASA Technical Reports Server (NTRS)
Lewis, Marian L.
1990-01-01
The horizontally rotating fluid-filled, membrane oxygenated bioreactors developed at NASA Johnson for spacecraft applications provide a powerful tool for ground-based research. Three-dimensional aggregates formed by cells cultured on microcarrier beads are useful for study of cell-cell interactions and tissue development. By comparing electron micrographs of plant seedlings germinated during Shuttle flight 61-C and in an earth-based rotating bioreactor it is shown that some effects of microgravity are mimicked. Bioreactors used in the UAH Bioreactor Laboratory will make it possible to determine some of the effects of altered gravity at the cellular level. Bioreactors can be valuable for performing critical, preliminary-to-spaceflight experiments as well as medical investigations such as in vitro tumor cell growth and chemotherapeutic drug response; the enrichment of stem cells from bone marrow; and the effect of altered gravity on bone and muscle cell growth and function and immune response depression.
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Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function
Kara, Firas M.; Chitu, Violeta; Sloane, Jennifer; Axelrod, Matthew; Fredholm, Bertil B.; Stanley, E. Richard; Cronstein, Bruce N.
2010-01-01
Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A1R, A2AR, A2BR, and A3R). Because A1R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A1R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A1R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the presence of colony stimulating factor-1 and receptor activator of NF-κB ligand in the presence or absence of the A1R antagonist 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Osteoclast morphology was analyzed in tartrate-resistant acid phosphatase or F-actin-stained samples, and bone resorption was evaluated by toluidine blue staining of dentin. BMCs from A1R-knockout mice form fewer osteoclasts than BMCs from WT mice, and the A1R antagonist DPCPX inhibits osteoclast formation (IC50=1 nM), with altered morphology and reduced ability to resorb bone. A1R blockade increased ubiquitination and degradation of TRAF6 in RAW264.7 cells induced to differentiate into osteoclasts. These studies suggest a critical role for adenosine in bone homeostasis via interaction with adenosine A1R and further suggest that A1R may be a novel pharmacologic target to prevent the bone loss associated with inflammatory diseases and menopause.—Kara, F. M., Chitu, V., Sloane, J., Axelrod, M., Fredholm, B. B., Stanley, R., Cronstein, B. N. Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function. PMID:20181934
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Tuzlakoglu, Kadriye; Santos, Marina I; Neves, Nuno; Reis, Rui L
2011-02-01
Mimicking the structural organization and biologic function of natural extracellular matrix has been one of the main goals of tissue engineering. Nevertheless, the majority of scaffolding materials for bone regeneration highlights biochemical functionality in detriment of mechanical properties. In this work we present a rather innovative construct that combines in the same structure electrospun type I collagen nanofibers with starch-based microfibers. These combined structures were obtained by a two-step methodology and structurally consist in a type I collagen nano-network incorporated on a macro starch-based support. The morphology of the developed structures was assessed by several microscopy techniques and the collagenous nature of the nano-network was confirmed by immunohistochemistry. In addition, and especially regarding the requirements of large bone defects, we also successfully introduced the concept of layer by layer, as a way to produce thicker structures. In an attempt to recreate bone microenvironment, the design and biochemical composition of the combined structures also envisioned bone-forming cells and endothelial cells (ECs). The inclusion of a type I collagen nano-network induced a stretched morphology and improved the metabolic activity of osteoblasts. Regarding ECs, the presence of type I collagen on the combined structures provided adhesive support and obviated the need of precoating with fibronectin. It was also importantly observed that ECs on the nano-network organized into circular structures, a three-dimensional arrangement distinct from that observed for osteoblasts and resembling the microcappillary-like organizations formed during angiogenesis. By providing simultaneously physical and chemical cues for cells, the herein-proposed combined structures hold a great potential in bone regeneration as a man-made equivalent of extracellular matrix.
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Sorokina, Tamara; Shipounova, Irina; Bigildeev, Alexey; Petinati, Nataliya; Drize, Nina; Turkina, Anna; Chelysheva, Ekaterina; Shukhov, Oleg; Kuzmina, Larisa; Parovichnikova, Elena; Savchenko, Valery
2016-09-01
The development of leukemia impairs normal hematopoiesis and marrow stromal microenvironment. The aim of the investigation was to study the ability of multipotent mesenchymal stromal cells (MSCs) derived from the bone marrow of patients with leukemia to maintain normal hematopoietic progenitor cells. MSCs were obtained from the bone marrow of 14 patients with acute lymphoblastic (ALL), 25 with myeloid (AML), and 15 with chronic myeloid (CML) leukemia. As a control, MSCs from 22 healthy donors were used. The incidence of cobblestone area forming cells (CAFC 7-8 d) in the bone marrow of healthy donor cultivated on the supportive layer of patients MSCs was measured. The ability of MSCs from AML and ALL patients at the moment of diagnosis to maintain normal CAFC was significantly decreased when compared to donors. After chemotherapy, the restoration of ALL patients' MSCs functions was slower than that of AML. CML MSCs maintained CAFC better than donors' at the moment of diagnosis and this ability increased with treatment. The ability of patients' MSCs to maintain normal hematopoietic progenitor cells was shown to change in comparison with MSCs from healthy donors and depended on nosology. During treatment, the functional capacity of patients' MSCs had been partially restored. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Pericytes as a supplementary source of osteoblasts in periosteal osteogenesis.
Diaz-Flores, L; Gutierrez, R; Lopez-Alonso, A; Gonzalez, R; Varela, H
1992-02-01
In the adult rat femur, lifting a periosteum strip with microscopic bone flakes on its deep surface, if performed without damaging the surrounding microcirculation, rapidly leads to new bone formation and angiogenesis. Using vascular labeling, the pericytes and endothelial cells (ECs) were labeled with monastral blue (MB) in the preformed, preexisting postcapillary venules of the periosteal microcirculation. MB was detectable by light and electron microscopy and it persisted in some of the daughter cells. Between one and 21 hours, the MB labeling was restricted to the pericytes and ECs of postcapillary venules. Immediately afterward, both pericytes and ECs of these vessels were activated and continued to show MB. The phenomenon of pericyte activation includes enlargement, disruption of their basal lamina, separation from the walls of the preformed vessels, and the presence of mitotic figures. At this stage, activated pericytes with MB in their cytoplasm, fibroblast-like cells, and transitional cell forms between them were seen in interstitial areas. After 27 hours, vascular buds appeared and MB was detected in some ECs and pericytes. Between three and six days, when bone-tissue development was observed, some osteoblasts were MB labeled. Previous findings support the hypothesis that when the periosteum is activated, the process of bone formation from cells already present in the periosteum is augmented by proliferation and differentiation of pericytes, which contribute a supplementary population of osteoprogenitor cells.
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Azami, Mahmoud; Tavakol, Shima; Samadikuchaksaraei, Ali; Hashjin, Mehran Solati; Baheiraei, Nafiseh; Kamali, Mehdi; Nourani, Mohammad Reza
2012-01-01
In this study, a nano-structured scaffold was designed for bone repair using hydroxapatite and gelatin as its main components. The scaffold was prepared via layer solvent casting combined with freeze-drying and lamination techniques and characterized by the commonly used bulk techniques. The biocompatibility and osteoconductivity of this scaffold and its capacity to promote bone healing were also evaluated. Osteoblast-like cells were seeded on these scaffolds and their proliferation rate, intracellular alkaline phosphatase (ALP) activity and ability to form mineralized bone nodules were compared with those osteoblasts grown on cell culture plastic surfaces. Also, the scaffolds were implanted in a critical bone defect created on rat calvarium. Engineering analyses show that the scaffold posses a three dimensional interconnected homogenous porous structure with a porosity of about 82% and pore sizes ranging from 300 to 500 μm. Mechanical indices are in the range of spongy bones. The results obtained from biological assessment show that this scaffold does not negatively affect osteoblasts proliferation rate and improves osteoblasts function as shown by increasing the ALP activity and calcium deposition and formation of mineralized bone nodules. In addition, the scaffold promoted healing of critical size calvarial bone defect in rats. It could be concluded that this scaffold fulfills all the main requirements to be considered as a bone substitute.