Immune response

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... These cells develop into two groups in the bone marrow. From the bone marrow, one group of lymphocytes migrates to a ... or B cells, mature and develop within the bone marrow itself. In that process, they achieve the ...

Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism

PubMed Central

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

Bone morphogenetic protein-mediated interaction of periosteum and diaphysis. Citric acid and other factors influencing the generation of parosteal bone.

PubMed

Kübler, N; Urist, M R

1990-09-01

In rabbits, after long-bone growth is complete and the cambium layer regresses, mesenchymal-type cells with embryonic potential (competence) for bone development persist in the adventitial layer of periosteum. These cells are not determined osteoprogenitor cells (stem cells) because bone tissue differentiation does not occur when adult periosteum is transplanted into a heterotopic site. In this respect, adventitial cells differ from bone marrow stroma cells. In a parosteal orthotopic site in the space between the adult periosteum and diaphysis, implants of bone morphogenetic protein (BMP) and associated noncollagenous proteins (BMP/NCP) induce adventitia and adjacent muscle connective-tissue-derived cells to switch from a fibrogenetic to a chondroosteoprogenetic pattern of bone development. The quantity of induced bone is proportional to the dose of BMP/NCP in the range from 10 to 50 mg; immature rabbits produced larger deposits than mature rabbits in response to BMP/NCP. Preoperative local intramuscular injections of citric, edetic, or hyaluronic acids in specified concentrations markedly enhanced subperiosteal BMP/NCP-induced bone formation. The quantity of bovine or human BMP/NCP-induced bone formation in rabbits is also increased by very low-dose immunosuppression but not by bone mineral, tricalcium phosphate ceramic, inorganic calcium salts, or various space-occupying, unspecific chemical irritants. Although composities of BMP/NCP and allogeneic rabbit tendon collagen increased the quantity of bone in a parosteal site, in a heterotopic site the composite failed to induce bone formation. In a parosteal site, the conditions permitting BMP/NCP-induced bone formation develop, and the end product of the morphogenetic response is a duplicate diaphysis. How BMP reactivates the morphogenetic process in postfetal mesenchymal-type adventitial cells persisting in adult periosteum (including adjacent muscle attachments) is not known.

[Research advances of fluid bio-mechanics in bone].

PubMed

Chen, Zebin; Huo, Bo

2017-04-01

It has been found for more than one century that when experiencing mechanical loading, the structure of bone will adapt to the changing mechanical environment, which is called bone remodeling. Bone remodeling is charaterized as two processes of bone formation and bone resorption. A large number of studies have confirmed that the shear stress is resulted from interstitial fluid flow within bone cavities under mechanical loading and it is the key factor of stimulating the biological responses of bone cells. This review summarizes the major research progress during the past years, including the biological response of bone cells under fluid flow, the pressure within bone cavities, the theoretical modeling, numerical simulation and experiments about fluid flow within bone, and finally analyzes and predicts the possible tendency in this field in the future.

Impact of mechanical stretch on the cell behaviors of bone and surrounding tissues

PubMed Central

Yu, Hye-Sun; Kim, Jung-Ju; Kim, Hae-Won; Lewis, Mark P; Wall, Ivan

2016-01-01

Mechanical loading is recognized to play an important role in regulating the behaviors of cells in bone and surrounding tissues in vivo. Many in vitro studies have been conducted to determine the effects of mechanical loading on individual cell types of the tissues. In this review, we focus specifically on the use of the Flexercell system as a tool for studying cellular responses to mechanical stretch. We assess the literature describing the impact of mechanical stretch on different cell types from bone, muscle, tendon, ligament, and cartilage, describing individual cell phenotype responses. In addition, we review evidence regarding the mechanotransduction pathways that are activated to potentiate these phenotype responses in different cell populations. PMID:26977284

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis.

PubMed

Clines, Katrina L; Clines, Gregory A

2018-05-14

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaP M cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis. Published by Elsevier Inc.

Experiment K-6-23. Effect of spaceflight on levels and function of immune cells

NASA Technical Reports Server (NTRS)

Mandel, A. D.; Sonnenfeld, G.; Berry, W.; Taylor, G.; Wellhausen, S. R.; Konstantinova, I.; Lesnyak, A.; Fuchs, B.

1990-01-01

Two different immunology experiments were performed on samples received from rats flown on Cosmos 1887. In the first experiment, rat bone marrow cells were examined in Moscow for their response to colony stimulating factor-M. In the second experiment, rat spleen and bone marrow cells were stained in Moscow with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and shipped to the United States where they were subjected to analysis on a flow cytometer. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor than did bone marrow cells from control rats. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell and innate interleukin-2 receptor antigens than from control animals. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin than did equivalent cells from control rats.

Origins of bone repair in the armour of fossil fish: response to a deep wound by cells depositing dentine instead of dermal bone.

PubMed

Johanson, Zerina; Smith, Moya; Kearsley, Anton; Pilecki, Peter; Mark-Kurik, Elga; Howard, Charles

2013-10-23

The outer armour of fossil jawless fishes (Heterostraci) is, predominantly, a bone with a superficial ornament of dentine tubercles surrounded by pores leading to flask-shaped crypts (ampullae). However, despite the extensive bone present in these early dermal skeletons, damage was repaired almost exclusively with dentine. Consolidation of bone, by dentine invading and filling the vascular spaces, was previously recognized in Psammolepis and other heterostracans but was associated with ageing and dermal shield wear (reparative). Here, we describe wound repair by deposition of dentine directly onto a bony scaffold of fragmented bone. An extensive wound response occurred from massive deposition of dentine (reactionary), traced from tubercle pulp cavities and surrounding ampullae. These structures may provide the cells to make reparative and reactionary dentine, as in mammalian teeth today in response to stimuli (functional wear or damage). We suggest in Psammolepis, repair involved mobilization of these cells in response to a local stimulatory mechanism, for example, predator damage. By comparison, almost no new bone is detected in repair of the Psammolepis shield. Dentine infilling bone vascular tissue spaces of both abraded dentine and wounded bone suggests that recruitment of this process has been evolutionarily conserved over 380 Myr and precedes osteogenic skeletal repair.

Bone metastasis target redox-responsive micell for the treatment of lung cancer bone metastasis and anti-bone resorption.

PubMed

Ye, Wei-Liang; Zhao, Yi-Pu; Cheng, Ying; Liu, Dao-Zhou; Cui, Han; Liu, Miao; Zhang, Bang-Le; Mei, Qi-Bing; Zhou, Si-Yuan

2018-01-16

In order to inhibit the growth of lung cancer bone metastasis and reduce the bone resorption at bone metastasis sites, a bone metastasis target micelle DOX@DBMs-ALN was prepared. The size and the zeta potential of DOX@DBNs-ALN were about 60 nm and -15 mV, respectively. DOX@DBMs-ALN exhibited high binding affinity with hydroxyapatite and released DOX in redox-responsive manner. DOX@DBMs-ALN was effectively up taken by A549 cells and delivered DOX to the nucleus of A549 cells, which resulted in strong cytotoxicity on A549 cells. The in vivo experimental results indicated that DOX@DBMs-ALN specifically delivered DOX to bone metastasis site and obviously prolonged the retention time of DOX in bone metastasis site. Moreover, DOX@DBMs-ALN not only significantly inhibited the growth of bone metastasis tumour but also obviously reduced the bone resorption at bone metastasis sites without causing marked systemic toxicity. Thus, DOX@DBMs-ALN has great potential in the treatment of lung cancer bone metastasis.

Biophotonics and Bone Biology

NASA Technical Reports Server (NTRS)

Zimmerli, Gregory; Fischer, David; Asipauskas, Marius; Chauhan, Chirag; Compitello, Nicole; Burke, Jamie; Tate, Melissa Knothe

2004-01-01

One of the more serious side effects of extended space flight is an accelerated bone loss. Rates of bone loss are highest in the weight-bearing bones of the hip and spine regions, and the average rate of bone loss as measured by bone mineral density measurements is around 1.2% per month for persons in a microgravity environment. It is well known that bone remodeling responds to mechanical forces. We are developing two-photon microscopy techniques to study bone tissue and bone cell cultures to better understand the fundamental response mechanism in bone remodeling. Osteoblast and osteoclast cell cultures are being studied, and the goal is to use molecular biology techniques in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) to study the physiology of in-vitro cell cultures in response to various stimuli, such as fluid flow induced shear stress and mechanical stress. We have constructed a two-photon fluorescence microscope for these studies, and are currently incorporating FLIM detection. Current progress will be reviewed. This work is supported by the NASA John Glenn Biomedical Engineering Consortium.

Host cell recruitment patterns by bone morphogenetic protein-2 releasing hyaluronic acid hydrogels in a mouse subcutaneous environment.

PubMed

Todeschi, Maria R; El Backly, Rania M; Varghese, Oommen P; Hilborn, Jöns; Cancedda, Ranieri; Mastrogiacomo, Maddalena

2017-07-01

This study aimed to identify host cell recruitment patterns in a mouse model in response to rhBMP-2 releasing hyaluronic acid hydrogels and influence of added nano-hydroxyapatite particles on rhBMP-2 release and pattern of bone formation. Implanted gels were retrieved after implantation and cells were enzymatically dissociated for flow cytometric analysis. Percentages of macrophages, progenitor endothelial cells and putative mesenchymal stem cells were measured. Implants were evaluated for BMP-2 release by ELISA and by histology to monitor tissue formation. Hyaluronic acid+BMP-2 gels influenced the inflammatory response in the bone healing microenvironment. Host-derived putative mesenchymal stem cells were major contributors. Addition of hydroxyapatite nanoparticles modified the release pattern of rhBMP-2, resulting in enhanced bone formation.

Growth plate-derived hedgehog-signal-responsive cells provide skeletal tissue components in growing bone.

PubMed

Haraguchi, Ryuma; Kitazawa, Riko; Imai, Yuuki; Kitazawa, Sohei

2018-04-01

Longitudinal bone growth progresses by continuous bone replacement of epiphyseal cartilaginous tissue, known as "growth plate", produced by columnar proliferated- and differentiated-epiphyseal chondrocytes. The endochondral ossification process at the growth plate is governed by paracrine signals secreted from terminally differentiated chondrocytes (hypertrophic chondrocytes), and hedgehog signaling is one of the best known regulatory signaling pathways in this process. Here, to investigate the developmental relationship between longitudinal endochondral bone formation and osteogenic progenitors under the influence of hedgehog signaling at the growth plate, genetic lineage tracing was carried out with the use of Gli1 CreERT2 mice line to follow the fate of hedgehog-signal-responsive cells during endochondral bone formation. Gli1 CreERT2 genetically labeled cells are detected in hypertrophic chondrocytes and osteo-progenitors at the chondro-osseous junction (COJ); these progeny then commit to the osteogenic lineage in periosteum, trabecular and cortical bone along the developing longitudinal axis. Furthermore, in ageing bone, where longitudinal bone growth ceases, hedgehog-signal responsiveness and its implication in osteogenic lineage commitment is significantly weakened. These results show, for the first time, evidence of the developmental contribution of endochondral progenitors under the influence of epiphyseal chondrocyte-derived secretory signals in longitudinally growing bone. This study provides a precise outline for assessing the skeletal lineage commitment of osteo-progenitors in response to growth-plate-derived regulatory signals during endochondral bone formation.

Genetic response and morphologic characterization of chicken bone-marrow derived dendritic cells during infection with high and low pathogenic avian influenza viruses

USDA-ARS?s Scientific Manuscript database

Dendritic cells (DC) are professional antigen-presenting cells of the immune system that function to initiate primary immune responses. Progenitors of DCs are derived from haematopoietic stem cells in the bone marrow (BM) that migrate in non-lymphoid tissues to develop into immature DCs. Here, they ...

IGF-1 Receptor Expression on Circulating Osteoblast Progenitor Cells Predicts Tissue-Based Bone Formation Rate and Response to Teriparatide in Premenopausal Women With Idiopathic Osteoporosis.

PubMed

Cohen, Adi; Kousteni, Stavroula; Bisikirska, Brygida; Shah, Jayesh G; Manavalan, J Sanil; Recker, Robert R; Lappe, Joan; Dempster, David W; Zhou, Hua; McMahon, Donald J; Bucovsky, Mariana; Kamanda-Kosseh, Mafo; Stubby, Julie; Shane, Elizabeth

2017-06-01

We have previously reported that premenopausal women with idiopathic osteoporosis (IOP) have profound microarchitectural deficiencies and heterogeneous bone remodeling. Those with the lowest bone formation rate have higher baseline serum insulin-like growth factor-1 (IGF-1) levels and less robust response to teriparatide. Because IGF-1 stimulates bone formation and is critical for teriparatide action on osteoblasts, these findings suggest a state of IGF-1 resistance in some IOP women. To further investigate the hypothesis that osteoblast and IGF-1-related mechanisms mediate differential responsiveness to teriparatide in IOP, we studied circulating osteoblast progenitor (COP) cells and their IGF-1 receptor (IGF-1R) expression. In premenopausal women with IOP, peripheral blood mononuclear cells (PBMCs) were obtained at baseline (n = 25) and over 24 months of teriparatide treatment (n = 11). Flow cytometry was used to identify and quantify COPs (non-hematopoetic lineage cells expressing osteocalcin and RUNX2) and to quantify IGF-1R expression levels. At baseline, both the percent of PBMCs that were COPs (%COP) and COP cell-surface IGF-1R expression correlated directly with several histomorphometric indices of bone formation in tetracycline-labeled transiliac biopsies. In treated subjects, both %COP and IGF-1R expression increased promptly after teriparatide, returning toward baseline by 18 months. Although neither baseline %COP nor increase in %COP after 3 months predicted the bone mineral density (BMD) response to teriparatide, the percent increase in IGF-1R expression on COPs at 3 months correlated directly with the BMD response to teriparatide. Additionally, lower IGF-1R expression after teriparatide was associated with higher body fat, suggesting links between teriparatide resistance, body composition, and the GH/IGF-1 axis. In conclusion, these assays may be useful to characterize bone remodeling noninvasively and may serve to predict early response to teriparatide and possibly other bone formation-stimulating medications. These new tools may also have utility in the mechanistic investigation of teriparatide resistance in premenopausal IOP and perhaps in other populations. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

[Endogenous pyrogen formation by bone marrow cells].

PubMed

Efremov, O M; Sorokin, A V; El'kina, O A

1978-01-01

The cells of the rabbit bone marrow produced endogenous pyrogen in response to stimulation with bacterial lipopolysaccharide. Incubation of the cells in medium No 199 containing a 15% homologous serum is optimal for the release of pyrogen. It is supposed that the cells of the bone marrow take part in the formation of endgenous pyrogen and in the mechanism of pyrexia in the organism.

DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH.

PubMed

Liu, Zhongbo; Kennedy, Oran D; Cardoso, Luis; Basta-Pljakic, Jelena; Partridge, Nicola C; Schaffler, Mitchell B; Rosen, Clifford J; Yakar, Shoshana

2016-02-01

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF-1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF-1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)-1-mediated Ghr knockout (DMP-GHRKO) mice to address the role of the GH/IGF axis in osteocytes. We found that DMP-GHRKO did not affect linear growth but compromised overall bone accrual. DMP-GHRKO mice exhibited reduced serum inorganic phosphate and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte-like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase-2 and IGF-1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth. © FASEB.

The Association of Endothelin-1 Signaling with Bone Alkaline Phosphatase Expression and Protumorigenic Activities in Canine Osteosarcoma.

PubMed

Neumann, Z L; Pondenis, H C; Masyr, A; Byrum, M L; Wycislo, K L; Fan, T M

2015-01-01

Canine osteosarcoma (OS) is an aggressive sarcoma characterized by pathologic skeletal resorption and pulmonary metastases. A number of negative prognostic factors, including bone alkaline phosphatase, have been identified in dogs with OS, but the underlying biologic factors responsible for such observations have not been thoroughly investigated. Endothelin-1-mediated signaling is active during bone repair, and is responsible for osteoblast migration, survival, proliferation, and bone alkaline phosphatase expression. The endothelin-1 signaling axis is active in canine OS cells, and this pathway is utilized by malignant osteoblasts for promoting cellular migration, survival, proliferation, and bone alkaline phosphatase activities. 45 dogs with appendicular OS. The expressions of endothelin-1 and endothelin A receptor were studied in OS cell lines and in samples from spontaneously occurring tumors. Activities mediated by endothelin-1 signaling were investigated by characterizing responses in 3 OS cell lines. In 45 dogs with OS, bone alkaline phosphatase concentrations were correlated with primary tumor osteoproductivity. Canine OS cells express endothelin-1 and endothelin A receptor, and this signaling axis mediates OS migration, survival, proliferation, and bone alkaline phosphatase activities. In OS-bearing dogs, circulating bone alkaline phosphatase activities were positively correlated with primary tumor relative bone mineral densities. Canine OS cells express endothelin-1 and functional endothelin A receptors, with the potential for a protumorigenic signaling loop. Increases in bone alkaline phosphatase activity are associated with osteoblastic OS lesions, and might be an epiphenomenon of active endothelin-1 signaling or excessive osteoproduction within the localized bone microenvironment. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Mesenchymal stem cells for bone repair and metabolic bone diseases.

PubMed

Undale, Anita H; Westendorf, Jennifer J; Yaszemski, Michael J; Khosla, Sundeep

2009-10-01

Human mesenchymal stem cells offer a potential alternative to embryonic stem cells in clinical applications. The ability of these cells to self-renew and differentiate into multiple tissues, including bone, cartilage, fat, and other tissues of mesenchymal origin, makes them an attractive candidate for clinical applications. Patients who experience fracture nonunion and metabolic bone diseases, such as osteogenesis imperfecta and hypophosphatasia, have benefited from human mesenchymal stem cell therapy. Because of their ability to modulate immune responses, allogeneic transplant of these cells may be feasible without a substantial risk of immune rejection. The field of regenerative medicine is still facing considerable challenges; however, with the progress achieved thus far, the promise of stem cell therapy as a viable option for fracture nonunion and metabolic bone diseases is closer to reality. In this review, we update the biology and clinical applicability of human mesenchymal stem cells for bone repair and metabolic bone diseases.

[Bone marrow stromal damage mediated by immune response activity].

PubMed

Vojinović, J; Kamenov, B; Najman, S; Branković, Lj; Dimitrijević, H

1994-01-01

The aim of this work was to estimate influence of activated immune response on hematopoiesis in vitro, using the experimental model of BCG immunized BALB/c mice and in patients with chronic immunoactivation: long-lasting infections, autoimmunity or malignancy. We correlated changes in long term bone marrow cultures (Dexter) and NBT reduction with appearance of anemia in patients and experimental model of immunization by BCG. Increased spontaneous NBT reduction pointed out role of macrophage activation in bone marrow stroma damage. Long-term bone marrow cultures showed reduced number of hematopoietic cells, with predomination of fibroblasts and loss of fat cells. This results correlated with anemia and leucocytosis with stimulated myelopoiesis in peripheral blood. Activation of immune response, or acting of any agent that directly changes extracellular matrix and cellularity of bone marrow, may result in microenviroment bone marrow damage that modify hematopoiesis.

Allogeneic adipose-derived stem cells regenerate bone in a critical-sized ulna segmental defect

PubMed Central

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

Modeling Hematopoiesis and Responses to Radiation Countermeasures in a Bone Marrow-on-a-Chip.

PubMed

Torisawa, Yu-Suke; Mammoto, Tadanori; Jiang, Elisabeth; Jiang, Amanda; Mammoto, Akiko; Watters, Alexander L; Bahinski, Anthony; Ingber, Donald E

2016-05-01

Studies on hematopoiesis currently rely on animal models because in vitro culture methods do not accurately recapitulate complex bone marrow physiology. We recently described a bone marrow-on-a-chip microfluidic device that enables the culture of living hematopoietic bone marrow and mimics radiation toxicity in vitro. In the present study, we used this microdevice to demonstrate continuous blood cell production in vitro and model bone marrow responses to potential radiation countermeasure drugs. The device maintained mouse hematopoietic stem and progenitor cells in normal proportions for at least 2 weeks in culture. Increases in the number of leukocytes and red blood cells into the microfluidic circulation also could be detected over time, and addition of erythropoietin induced a significant increase in erythrocyte production. Exposure of the bone marrow chip to gamma radiation resulted in reduction of leukocyte production, and treatment of the chips with two potential therapeutics, granulocyte-colony stimulating factor or bactericidal/permeability-increasing protein (BPI), induced significant increases in the number of hematopoietic stem cells and myeloid cells in the fluidic outflow. In contrast, BPI was not found to have any effect when analyzed using static marrow cultures, even though it has been previously shown to accelerate recovery from radiation-induced toxicity in vivo. These findings demonstrate the potential value of the bone marrow-on-a-chip for modeling blood cell production, monitoring responses to hematopoiesis-modulating drugs, and testing radiation countermeasures in vitro.

Metabolic Acidosis Increases Intracellular Calcium in Bone Cells Through Activation of the Proton Receptor OGR1

PubMed Central

Frick, Kevin K; Krieger, Nancy S; Nehrke, Keith; Bushinsky, David A

2009-01-01

Metabolic acidosis increases urine Ca without increasing intestinal absorption, leading to bone Ca loss. It is unclear how bone cells detect the increase in proton concentration. To determine which G protein-coupled proton sensing receptors are expressed in bone, PCR was performed, and products were detected for OGR1, TDAG8, G2A, and GPR4. We tested the hypothesis that the G protein-coupled proton sensor, OGR1, is an H+-sensing receptor in bone. To determine whether acid-induced bone resorption involves OGR1, we incubated mouse calvariae in neutral pH (NTL) or acidic (MET) medium ± the OGR1 inhibitor CuCl2. CuCl2 decreased MET-induced Ca efflux. We used fluorescent imaging of perfused bone cells to determine whether MET increases Cai. Perfusion with MET induced a rapid, flow-independent, increase in Cai in individual bone cells. To determine whether transfection of OGR1 into a heterologous cell type would increase Cai in response to H+, we perfused Chinese hamster ovary (CHO) cells transfected with mouse OGR1 cDNA. Perfusion with MET induced a rapid increase in Cai in OGR1-transfected CHO cells. These data indicate that OGR1 induces an increase in Cai in response to MET and is a prime candidate for an osteoblast proton sensor. PMID:18847331

Metabolic acidosis increases intracellular calcium in bone cells through activation of the proton receptor OGR1.

PubMed

Frick, Kevin K; Krieger, Nancy S; Nehrke, Keith; Bushinsky, David A

2009-02-01

Metabolic acidosis increases urine Ca without increasing intestinal absorption, leading to bone Ca loss. It is unclear how bone cells detect the increase in proton concentration. To determine which G protein-coupled proton sensing receptors are expressed in bone, PCR was performed, and products were detected for OGR1, TDAG8, G2A, and GPR4. We tested the hypothesis that the G protein-coupled proton sensor, OGR1, is an H(+)-sensing receptor in bone. To determine whether acid-induced bone resorption involves OGR1, we incubated mouse calvariae in neutral pH (NTL) or acidic (MET) medium +/- the OGR1 inhibitor CuCl(2). CuCl(2) decreased MET-induced Ca efflux. We used fluorescent imaging of perfused bone cells to determine whether MET increases Ca(i). Perfusion with MET induced a rapid, flow-independent, increase in Ca(i) in individual bone cells. To determine whether transfection of OGR1 into a heterologous cell type would increase Ca(i) in response to H(+), we perfused Chinese hamster ovary (CHO) cells transfected with mouse OGR1 cDNA. Perfusion with MET induced a rapid increase in Ca(i) in OGR1-transfected CHO cells. These data indicate that OGR1 induces an increase in Ca(i) in response to MET and is a prime candidate for an osteoblast proton sensor.

Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrow-derived preosteoclast-like cells

NASA Technical Reports Server (NTRS)

McAllister, T. N.; Du, T.; Frangos, J. A.

2000-01-01

Bone is a porous tissue that is continuously perfused by interstitial fluid. Fluid flow, driven by both vascular pressure and mechanical loading, may generate significant shear stresses through the canaliculi as well as along the bone lining at the endosteal surface. Both osteoblasts and osteocytes produce signaling factors such as prostaglandins and nitric in response to fluid shear stress (FSS); however, these humoral agents appear to have more profound affects on osteoclast activity at the endosteal surface. We hypothesized that osteoclasts and preosteoclasts may also be mechanosensitive and that osteoclast-mediated autocrine signaling may be important in bone remodeling. In this study, we investigated the effect of FSS on nitric oxide (NO), prostaglandin E(2) (PGE(2)), and prostacyclin (PGI(2)) release by neonatal rat bone marrow-derived preosteoclast-like cells. These cells were tartrate-resistant acid phosphatase (TRAP) positive, weakly nonspecific esterase (NSE) positive, and capable of fusing into calcitonin-responsive, bone-resorbing, multinucleated cells. Bone marrow-derived preosteoclast-like cells exposed for 6 h to a well-defined FSS of 16 dynes/cm(2) produced NO at a rate of 7.5 nmol/mg protein/h, which was 10-fold that of static controls. This response was completely abolished by 100 microM N(G)-amino-L-arginine (L-NAA). Flow also stimulated PGE(2) production (3.9 microg/mg protein/h) and PGI(2) production (220 pg/mg protein/h). L-NAA attenuated flow-induced PGE(2) production by 30%, suggesting that NO may partially modulate PGE(2) production. This is the first report demonstrating that marrow derived cells are sensitive to FSS and that autocrine signaling in these cells may play an important role in load-induced remodeling and signal transduction in bone. Copyright 2000 Academic Press.

The response of bone to unloading

NASA Technical Reports Server (NTRS)

Bikle, D. D.; Halloran, B. P.

1999-01-01

Skeletal unloading leads to decreased bone formation and decreased bone mass. Bone resorption is uncoupled from bone formation, contributing to the bone loss. During spaceflight bone is lost principally from the bones most loaded in the 1-g environment, and some redistribution of bone from the lower extremities to the head appears to take place. Although changes in calcitropic hormones have been demonstrated during skeletal unloading (PTH and 1,25(OH)2D decrease), it remains unclear whether such changes account for or are in response to the changes in bone formation and resorption. Bed rest studies with human volunteers and hindlimb elevation studies with rats have provided useful data to help explain the changes in bone formation during spaceflight. These models of skeletal unloading reproduce a number of the conditions associated with microgravity, and the findings from such studies confirm many of the observations made during spaceflight. Determining the mechanism(s) by which loading of bone is sensed and translated into a signal(s) controlling bone formation remains the holy grail in this field. Such investigations couple biophysics to biochemistry to cell and molecular biology. Although studies with cell cultures have revealed biochemical responses to mechanical loads comparable to that seen in intact bone, it seems likely that matrix-cell interactions underlie much of the mechanocoupling. The role for systemic hormones such as PTH, GH, and 1,25(OH)2D compared to locally produced factors such as IGF-I, PTHrP, BMPs, and TGF-beta in modulating the cellular response to load remains unclear. As the mechanism(s) by which bone responds to mechanical load with increased bone formation are further elucidated, applications of this knowledge to other etiologies of osteoporosis are likely to develop. Skeletal unloading provides a perturbation in bone mineral homeostasis that can be used to understand the mechanisms by which bone mineral homeostasis is maintained, with the expectation that such understanding will lead to effective treatment for disuse osteoporosis.

CELLS INVOLVED IN THE IMMUNE RESPONSE

PubMed Central

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

Centrifugation of Cultured Osteoblasts And Macrophages as a Model To Study How Gravity Regulates The Function of Skeletal Cells

NASA Technical Reports Server (NTRS)

Globus, Ruth K.; Searby, Nancy D.; Almeida, Eduardo A. C.; Sutijono, Darrell; Yu, Joon-Ho; Malouvier, Alexander; Doty, Steven B.; Morey-Holton, Emily; Weinstein, Steven L.; Dalton, Bonnie P. (Technical Monitor)

2000-01-01

Mechanical loading helps define the architecture of weight-bearing bone via the tightly regulated process of skeletal turnover. Turnover occurs by the concerted activity of osteoblasts, responsible for bone formation. and osteoclasts, responsible for bone resorption. Osteoclasts are specialized megakaryon macrophages, which differentiate from monocytes in response to resorption stimuli, such as reduced weight-bearing. Habitation in space dramatically alters musculoskeletal loading, which modulates both cell function and bone structure. Our long-term objective is to define the molecular and cellular mechanisms that mediate skeletal adaptations to altered gravity environments. Our experimental approach is to apply hypergravity loads by centrifugation to rodents and cultured cells. As a first step, we examined the influence of centrifugation on the structure of cancellous bone in rats to test the ability of hypergravity to change skeletal architecture. Since cancellous bone undergoes rapid turnover we expected the most dramatic structural changes to occur in the shape of trabeculae of weight-bearing, cancellous bone. To define the cellular responses to hypergravity loads, we exposed cultured osteoblasts and macrophages to centrifugation. The intraosseous and intramedullary pressures within long bones in vivo reportedly range from 12-40 mm Hg, which would correspond to 18-59 gravity (g) in our cultures. We assumed that hydrostatic pressure from the medium above the cell layer is at least one major component of the mechanical load generated by centrifuging cultured cells. and therefore we exposed the cells to 10-50g. In osteoblasts, we examined the structure of their actin and microtubule networks, production of prostaglandin E2 (PGE2), and cell survival. Analysis of the shape of the cytoskeletal networks provides evidence for the ability of centrifugation to affect cell structure, while the production of PGE2 serves as a convenient marker for mechanical stimulation. We examined cell survival, reasoning that osteoblasts might mold skeletal structure in a hypergravity environment in part by regulating apoptosis and thus the duration of osteoblast productivity. Finally, we tested the influence of centrifugation on microbial activation of a macrophage cell line (RAW264.7). In response to the appropriate hormonal stimulation, this cell line is reportedly capable of undergoing differentiation to express osteoclast markers. In addition, a component of the cell wall of gram-negative bacteria, lipopolysaccaride (LPS), stimulates the formation of osteoclasts in vivo. Thus we tested the influence on centrifugation on RAW264.7 cells stimulated with LPS to provide an index of the function of osteoclast precursors.

Quantitative MRI and spectroscopy of bone marrow

PubMed Central

Ruschke, Stefan; Dieckmeyer, Michael; Diefenbach, Maximilian; Franz, Daniela; Gersing, Alexandra S.; Krug, Roland; Baum, Thomas

2017-01-01

Bone marrow is one of the largest organs in the human body, enclosing adipocytes, hematopoietic stem cells, which are responsible for blood cell production, and mesenchymal stem cells, which are responsible for the production of adipocytes and bone cells. Magnetic resonance imaging (MRI) is the ideal imaging modality to monitor bone marrow changes in healthy and pathological states, thanks to its inherent rich soft‐tissue contrast. Quantitative bone marrow MRI and magnetic resonance spectroscopy (MRS) techniques have been also developed in order to quantify changes in bone marrow water–fat composition, cellularity and perfusion in different pathologies, and to assist in understanding the role of bone marrow in the pathophysiology of systemic diseases (e.g. osteoporosis). The present review summarizes a large selection of studies published until March 2017 in proton‐based quantitative MRI and MRS of bone marrow. Some basic knowledge about bone marrow anatomy and physiology is first reviewed. The most important technical aspects of quantitative MR methods measuring bone marrow water–fat composition, fatty acid composition, perfusion, and diffusion are then described. Finally, previous MR studies are reviewed on the application of quantitative MR techniques in both healthy aging and diseased bone marrow affected by osteoporosis, fractures, metabolic diseases, multiple myeloma, and bone metastases. Level of Evidence: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;47:332–353. PMID:28570033

Bone modeling and cell-material interface responses induced by nickel-titanium shape memory alloy after periosteal implantation.

PubMed

Ryhänen, J; Kallioinen, M; Tuukkanen, J; Lehenkari, P; Junila, J; Niemelä, E; Sandvik, P; Serlo, W

1999-07-01

The purpose of this study was to evaluate the new bone formation, modeling and cell-material interface responses induced by nickel-titanium shape memory alloy after periosteal implantation. We used a regional acceleratory phenomenon (RAP) model, in which a periosteal contact stimulus provokes an adaptive modelling response. NiTi has thermal shape memory and superelasticity properties uncommon in other implant alloys. So far, there are insufficient data concerning the biocompatibility of NiTi as a bone implant. NiTi was compared to stainless steel (stst) and Ti-6Al-4V. The test implant was placed in contact with the intact femur periosteum, but it was not fixed inside the bone. Histomorphometry with digital image analysis was used to determine the bone formation and resorption parameters. The ultrastructural features of cell-material adhesion were analysed with scanning electron microscopy (FESEM). A typical peri-implant bone wall modelation was seen due to the normal RAP. The maximum new woven bone formation started earlier (2 weeks) in the Ti-6Al-4V group than in the NiTi (P < 0.01) group, but also decreased earlier, and at 8 weeks the NiTi (P < 0.05) and stst (P < 0.005) groups had greater cortical bone width. At 12 and 26 weeks no statistical differences were seen in the histomorphometric values. The histological response of the soft tissues around the NiTi implant was also clearly non-toxic and non-irritating. Cell adhesion and focal contacts were similar between the materials studied by FESEM. We conclude that NiTi had no negative effect on total new bone formation or normal RAP after periosteal implantation during a 26-week follow-up.

The effects of tumour necrosis factor-α on bone cells involved in periodontal alveolar bone loss; osteoclasts, osteoblasts and osteocytes.

PubMed

Algate, K; Haynes, D R; Bartold, P M; Crotti, T N; Cantley, M D

2016-10-01

Periodontitis is the most common bone loss pathology in adults and if left untreated is responsible for premature tooth loss. Cytokines, such as tumour necrosis factor-α (TNFα), involved in the chronic inflammatory response within the periodontal gingiva, significantly influence the normal bone remodelling processes. In this review, the effects of TNFα on bone metabolism in periodontitis are evaluated in relation to its direct and indirect actions on bone cells including osteoclasts, osteoblasts and osteocytes. Evidence published to date suggests a potent catabolic role for TNFα through the stimulation of osteoclastic bone resorption as well as the suppression of osteoblastic bone formation and osteocytic survival. However, the extent and timing of TNFα exposure in vitro and in vivo greatly influences its effect on skeletal cells, with contradictory anabolic activity observed with TNFα in a number of studies. None the less, it is evident that managing the chronic inflammatory response in addition to the deregulated bone metabolism is required to improve periodontal and inflammatory bone loss treatments‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

PubMed

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

2012-08-03

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

Gamma Radiation Induces Micronucleated Reticulocytes in 3-D Bone Marrow Bioreactors in Vitro

PubMed Central

Sun, Hongliang; Dertinger, Stephen D.; Hyrien, Ollivier; David Wu, J. H.; Chen, Yuhchyau

2009-01-01

Radiation injury to the bone marrow is potentially lethal due to the potent DNA-damaging effects on cells of the hematopoietic system, including bone marrow stem cell, progenitor, and the precursor cell populations. Investigation of radiation genotoxic effects on bone marrow progenitor/precursor cells has been challenged by the lack of optimal in vitro surrogate organ culture systems, and the overall difficulty to sustain lineage-specific proliferation and differentiation of hematopoiesis in vitro. We report the investigation of radiation genotoxic effects in bone marrow cultures of C57Bl/6 mice established in 3-D bioreactors, which sustain long-term bone marrow cultures. For these studies, genotoxicity is measured by the induction of micronucleated reticulocytes (MN-RET). The kinetics and dose-response relationship of MN-RET induction in response to gamma-radiation of bioreactor-maintained bone marrow cultures are presented. Our data showed that 3-D long-term bone marrow cultures had sustained erythropoiesis capable of generating reticulocytes up to 8 weeks. The peak time-interval of viable cell output and percentage of reticulocytes increased steadily and reached the initial peak between the 14th to 21st days after inoculations. This was followed by a rebound or staying relatively constant until week 8. The percentage of MN-RET reached the maximum between 24 and 32 hours post 1 Gy gamma-ray. There was a near linear MN-RET induction by gamma radiation from 0 Gy to 1.0 Gy, followed by an attenuated increase to 1.5 – 2.0 Gy. The MN-RET response showed a downtrend beyond 2 Gy. Our data suggest that bone marrow culture in the 3-D bioreactor may be a useful organ culture system for the investigation of radiation genotoxic effect in vitro. PMID:19786117

Spaceflight alters immune cell function and distribution

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Mandel, Adrian D.; Konstantinova, Irina V.; Berry, Wallace D.; Taylor, Gerald R.; Lesniak, A. T.; Fuchs, Boris B.; Rakhmilevich, Alexander L.

1992-01-01

Experiments are described which were performed onboard Cosmos 2044 to determine spaceflight effects on immunologically important cell function and distribution. Results indicate that bone marrow cells from flown and suspended rats exhibited a decreased response to a granulocyte/monocyte colony-stimulating factor compared with the bone marrow cells from control rats. Bone marrow cells showed an increase in the percentage of cells expressing markers for helper T-cells in the myelogenous population and increased percentages of anti-asialo granulocyte/monocyte-1-bearing interleulin-2 receptor bearing pan T- and helper T-cells in the lymphocytic population.

Bone microenvironment-mediated resistance of cancer cells to bisphosphonates and impact on bone osteocytes/stem cells.

PubMed

Alasmari, Abeer; Lin, Shih-Chun; Dibart, Serge; Salih, Erdjan

2016-08-01

Anti-resorptive bisphosphonates (BPs) have been clinically used to prevent cancer-bone metastasis and cancer-induced bone pathologies despite the fact that the phenotypic response of the cancer-bone interactions to BP exposure is "uncharted territory". This study offers unique insights into the interplay between cancer stem cells and osteocytes/osteoblasts and mesenchymal stem cells using a three-dimensional (3D) live cancer-bone interactive model. We provide extraordinary cryptic details of the biological events that occur as a result of alendronate (ALN) treatment using 3D live cancer-bone model systems under specific bone remodeling stages. While cancer cells are susceptible to BP treatment in the absence of bone, they are totally unaffected in the presence of bone. Cancer cells colonize live bone irrespective of whether the bone is committed to bone resorption or formation and hence, cancer-bone metastasis/interactions are though to be "independent of bone remodeling stages". In our 3D live bone model systems, ALN inhibited bone resorption at the osteoclast differentiation level through effects of mineral-bound ALN on osteocytes and osteoblasts. The mineral-bound ALN rendered bone incapable of osteoblast differentiation, while cancer cells colonize the bone with striking morphological adaptations which led to a conclusion that a direct anti-cancer effect of BPs in a "live or in vivo" bone microenvironment is implausible. The above studies were complemented with mass spectrometric analysis of the media from cancer-bone organ cultures in the absence and presence of ALN. The mineral-bound ALN impacts the bone organs by limiting transformation of mesenchymal stem cells to osteoblasts and leads to diminished endosteal cell population and degenerated osteocytes within the mineralized bone matrix.

Estrogen prevents bone loss through transforming growth factor β signaling in T cells

PubMed Central

Gao, Yuhao; Qian, Wei-Ping; Dark, Kimberly; Toraldo, Gianluca; Lin, Angela S. P.; Guldberg, Robert E.; Flavell, Richard A.; Weitzmann, M. Neale; Pacifici, Roberto

2004-01-01

Estrogen (E) deficiency leads to an expansion of the pool of tumor necrosis factor (TNF)-producing T cells through an IFN-γ-dependent pathway that results in increased levels of the osteoclastogenic cytokine TNF in the bone marrow. Disregulated IFN-γ production is instrumental for the bone loss induced by ovariectomy (ovx), but the responsible mechanism is unknown. We now show that mice with T cell-specific blockade of type β transforming growth factor (TGFβ) signaling are completely insensitive to the bone-sparing effect of E. This phenotype results from a failure of E to repress IFN-γ production, which, in turn, leads to increased T cell activation and T cell TNF production. Furthermore, ovx blunts TGFβ levels in the bone marrow, and overexpression of TGFβ in vivo prevents ovx-induced bone loss. These findings demonstrate that E prevents bone loss through a TGFβ-dependent mechanism, and that TGFβ signaling in T cells preserves bone homeostasis by blunting T cell activation. Thus, stimulation of TGFβ production in the bone marrow is a critical “upstream” mechanism by which E prevents bone loss, and enhancement of TGFβ levels in vivo may constitute a previously undescribed therapeutic approach for preventing bone loss. PMID:15531637

Morphological and functional changes in RAW264 macrophage-like cells in response to a hydrated layer of carbonate-substituted hydroxyapatite.

PubMed

Igeta, Kazuki; Kuwamura, Yuta; Horiuchi, Naohiro; Nozaki, Kosuke; Shiraishi, Daichi; Aizawa, Mamoru; Hashimoto, Kazuaki; Yamashita, Kimihiro; Nagai, Akiko

2017-04-01

Synthetic hydroxyapatite (HAp) is used clinically as a material for bone prostheses owing to its good bone-bonding ability; however, it does not contribute to bone remodeling. Carbonate-substituted hydroxyapatite (CAp) has greater bioresorption capacity than HAp while having similar bone-bonding potential, and is therefore considered as a next promising material for bone prostheses. However, the effects of the CAp instability on inflammatory and immune responses are unknown in detail. Here, we show that the surface layer of CAp is more hydrated than that of HAp and induces changes in the shape and function of macrophage-like cells. HAp and CAp were synthesized by wet method and molded into disks. The carbonate content of CAp disks was 6.2% as determined by Fourier transform (FT) infrared spectral analysis. Diffuse reflectance infrared FT analysis confirmed that physisorbed water and surface hydroxyl groups (OH - ) were increased whereas structural OH - was decreased on the CAp as compared to the HAp surface. The degree of hydroxylation in CAp was comparable to that in bone-apatite structures, and the CAp surface exhibited greater hydrophilicity and solubility than HAp. We investigated immune responses to these materials by culturing RAW264 cells (macrophage precursors) on their surfaces. Cell spreading on the CAp disk was suppressed and the secretion level of inflammatory cytokines was reduced as compared to cells grown on HAp. These results indicate that the greater surface hydration of CAp surface can attenuate adverse inflammatory responses to implanted bone prostheses composed of this material. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1063-1070, 2017. © 2017 Wiley Periodicals, Inc.

Autologous implantation of BMP2-expressing dermal fibroblasts to improve bone mineral density and architecture in rabbit long bones.

PubMed

Ishihara, Akikazu; Weisbrode, Steve E; Bertone, Alicia L

2015-10-01

Cell-mediated gene therapy may treat bone fragility disorders. Dermal fibroblasts (DFb) may be an alternative cell source to stem cells for orthopedic gene therapy because of their rapid cell yield and excellent plasticity with bone morphogenetic protein-2 (BMP2) gene transduction. Autologous DFb or BMP2-expressing autologous DFb were administered in twelve rabbits by two delivery routes; a transcortical intra-medullar infusion into tibiae and delayed intra-osseous injection into femoral drill defects. Both delivery methods of DFb-BMP2 resulted in a successful cell engraftment, increased bone volume, bone mineral density, improved trabecular bone microarchitecture, greater bone defect filling, external callus formation, and trabecular surface area, compared to non-transduced DFb or no cells. Cell engraftment within trabecular bone and bone marrow tissue was most efficiently achieved by intra-osseous injection of DFb-BMP2. Our results suggested that BMP2-expressing autologous DFb have enhanced efficiency of engraftment in target bones resulting in a measurable biologic response by the bone of improved bone mineral density and bone microarchitecture. These results support that autologous implantation of DFb-BMP2 warrants further study on animal models of bone fragility disorders, such as osteogenesis imperfecta and osteoporosis to potentially enhance bone quality, particularly along with other gene modification of these diseases. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Improved bone marrow stromal cell adhesion on micropatterned titanium surfaces.

PubMed

Iskandar, Maria E; Cipriano, Aaron F; Lock, Jaclyn; Gott, Shannon C; Rao, Masaru P; Liu, Huinan

2012-01-01

Implant longevity is desired for all bone replacements and fixatives. Titanium (Ti) implants fail due to lack of juxtaposed bone formation, resulting in implant loosening. Implant surface modifications have shown to affect the interactions between the implant and bone. In clinical applications, it is crucial to improve osseointegration and implant fixation at the implant and bone interface. Moreover, bone marrow derived cells play a significant role for implant and tissue integration. Therefore, the objective of this study is to investigate how surface micropatterning on Ti influences its interactions with bone marrow derived cells containing mesenchymal and hematopoietic stem cells. Bone marrow derived mesenchymal stem cells (BMSC) have the capability of differentiating into osteoblasts that contribute to bone growth, and therefore implant/bone integration. Hematopoietic stem cell derivatives are precursor cells that contribute to inflammatory response. By using all three cells naturally contained within bone marrow, we mimic the physiological environment to which an implant is exposed. Primary rat bone marrow derived cells were seeded onto Ti with surfaces composed of arrays of grooves of equal width and spacing ranging from 0.5 to 50 µm, fabricated using a novel plasma-based dry etching technique. Results demonstrated enhanced total cell adhesion on smaller micrometer-scale Ti patterns compared with larger micrometer-scale Ti patterns, after 24-hr culture. Further studies are needed to determine bone marrow derived cell proliferation and osteogenic differentiation potential on micropatterned Ti, and eventually nanopatterned Ti.

Bone marrow support of the heart in pressure overload is lost with aging.

PubMed

Sopko, Nikolai A; Turturice, Benjamin A; Becker, Mitchell E; Brown, Chase R; Dong, Feng; Popović, Zoran B; Penn, Marc S

2010-12-21

Exogenous stem cell delivery is under investigation to prevent and treat cardiac dysfunction. It is less studied as to the extent endogenous bone marrow derived stem cells contribute to cardiac homeostais in response to stress and the affects of aging on this stress response. To determine the role of bone marrow (BM) derived stem cells on cardiac homeostasis in response to pressure overload (PO) and how this response is altered by aging. Young (8 weeks) and old (>40 weeks) C57/b6 mice underwent homo- and heterochronic BM transplantation prior to transverse aortic constriction (TAC). We found that older BM is associated with decreased cardiac function following TAC. This decreased function is associated with decrease in BM cell engraftment, increased myocyte apoptosis, decreased myocyte hypertrophy, increased myocardial fibrosis and decreased cardiac function. Additionally, there is a decrease in activation of resident cells within the heart in response to PO in old mice. Interestingly, these effects are not due to alterations in vascular density or inflammation in response to PO or differences in ex vivo stem cell migration between young and old mice. BM derived stem cells are activated in response to cardiac PO, and the recruitment of BM derived cells are involved in cardiac myocyte hypertrophy and maintenance of function in response to PO which is lost with aging.

Infusion of freshly isolated autologous bone marrow derived mononuclear cells prevents endotoxin-induced lung injury in an ex-vivo perfused swine model

PubMed Central

2013-01-01

Introduction The acute respiratory distress syndrome (ARDS), affects up to 150,000 patients per year in the United States. We and other groups have demonstrated that bone marrow derived mesenchymal stromal stem cells prevent ARDS induced by systemic and local administration of endotoxin (lipopolysaccharide (LPS)) in mice. Methods A study was undertaken to determine the effects of the diverse populations of bone marrow derived cells on the pathophysiology of ARDS, using a unique ex-vivo swine preparation, in which only the ventilated lung and the liver are perfused with autologous blood. Six experimental groups were designated as: 1) endotoxin alone, 2) endotoxin + total fresh whole bone marrow nuclear cells (BMC), 3) endotoxin + non-hematopoietic bone marrow cells (CD45 neg), 4) endotoxin + hematopoietic bone marrow cells (CD45 positive), 5) endotoxin + buffy coat and 6) endotoxin + in vitro expanded swine CD45 negative adherent allogeneic bone marrow cells (cultured CD45neg). We measured at different levels the biological consequences of the infusion of the different subsets of cells. The measured parameters were: pulmonary vascular resistance (PVR), gas exchange (PO2), lung edema (lung wet/dry weight), gene expression and serum concentrations of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6. Results Infusion of freshly purified autologous total BMCs, as well as non-hematopoietic CD45(-) bone marrow cells significantly reduced endotoxin-induced pulmonary hypertension and hypoxemia and reduced the lung edema. Also, in the groups that received BMCs and cultured CD45neg we observed a decrease in the levels of IL-1β and TNF-α in plasma. Infusion of hematopoietic CD45(+) bone marrow cells or peripheral blood buffy coat cells did not protect against LPS-induced lung injury. Conclusions We conclude that infusion of freshly isolated autologous whole bone marrow cells and the subset of non-hematopoietic cells can suppress the acute humoral and physiologic responses induced by endotoxemia by modulating the inflammatory response, mechanisms that do not involve engraftment or trans-differentiation of the cells. These observations may have important implications for the design of future cell therapies for ARDS. PMID:23497755

T Lymphocytes Influence the Mineralization Process of Bone

PubMed Central

El Khassawna, Thaqif; Serra, Alessandro; Bucher, Christian H.; Petersen, Ansgar; Schlundt, Claudia; Könnecke, Ireen; Malhan, Deeksha; Wendler, Sebastian; Schell, Hanna; Volk, Hans-Dieter; Schmidt-Bleek, Katharina; Duda, Georg N.

2017-01-01

Bone is a unique organ able to regenerate itself after injuries. This regeneration requires the local interplay between different biological systems such as inflammation and matrix formation. Structural reconstitution is initiated by an inflammatory response orchestrated by the host immune system. However, the individual role of T cells and B cells in regeneration and their relationship to bone tissue reconstitution remain unknown. Comparing bone and fracture healing in animals with and without mature T and B cells revealed the essential role of these immune cells in determining the tissue mineralization and thus the bone quality. Bone without mature T and B cells is stiffer when compared to wild-type bone thus lacking the elasticity that helps to absorb forces, thus preventing fractures. In-depth analysis showed dysregulations in collagen deposition and osteoblast distribution upon lack of mature T and B cells. These changes in matrix deposition have been correlated with T cells rather than B cells within this study. This work presents, for the first time, a direct link between immune cells and matrix formation during bone healing after fracture. It illustrates specifically the role of T cells in the collagen organization process and the lack thereof in the absence of T cells. PMID:28596766

Connecting mechanics and bone cell activities in the bone remodeling process: an integrated finite element modeling.

PubMed

Hambli, Ridha

2014-01-01

Bone adaptation occurs as a response to external loadings and involves bone resorption by osteoclasts followed by the formation of new bone by osteoblasts. It is directly triggered by the transduction phase by osteocytes embedded within the bone matrix. The bone remodeling process is governed by the interactions between osteoblasts and osteoclasts through the expression of several autocrine and paracrine factors that control bone cell populations and their relative rate of differentiation and proliferation. A review of the literature shows that despite the progress in bone remodeling simulation using the finite element (FE) method, there is still a lack of predictive models that explicitly consider the interaction between osteoblasts and osteoclasts combined with the mechanical response of bone. The current study attempts to develop an FE model to describe the bone remodeling process, taking into consideration the activities of osteoclasts and osteoblasts. The mechanical behavior of bone is described by taking into account the bone material fatigue damage accumulation and mineralization. A coupled strain-damage stimulus function is proposed, which controls the level of autocrine and paracrine factors. The cellular behavior is based on Komarova et al.'s (2003) dynamic law, which describes the autocrine and paracrine interactions between osteoblasts and osteoclasts and computes cell population dynamics and changes in bone mass at a discrete site of bone remodeling. Therefore, when an external mechanical stress is applied, bone formation and resorption is governed by cells dynamic rather than adaptive elasticity approaches. The proposed FE model has been implemented in the FE code Abaqus (UMAT routine). An example of human proximal femur is investigated using the model developed. The model was able to predict final human proximal femur adaptation similar to the patterns observed in a human proximal femur. The results obtained reveal complex spatio-temporal bone adaptation. The proposed FEM model gives insight into how bone cells adapt their architecture to the mechanical and biological environment.

Asthma progression to airway remodeling and bone marrow eosinophil responses in genetically distinct strains of mice.

PubMed

Hogan, Mary Beth; Piktel, Debra; Hubbs, Ann F; McPherson, Leslie E; Landreth, Kenneth S

2008-12-01

Patient factors that cause long-term airway remodeling are largely unidentified. This suggests that genetic differences may determine which asthmatic patients develop airway remodeling. A murine model with repeated allergen exposure leading to peribronchial fibrosis in complement factor 5 (C5)-deficient A/J mice has been used to study asthma progression. No studies have addressed the systemic effects of allergen sensitization or chronic allergen exposure on bone marrow eosinophilopoiesis in this mouse strain. To investigate bone marrow eosinophil responses during acute sensitization and chronic allergen exposure using genetically distinct mouse strains differing in persistent airway reactivity and remodeling. The C5-sufficient BALB/c and C5-deficient A/J mice were repetitively exposed to intranasal ovalbumin for 12 weeks. Subsequently, the mice were evaluated for airway eosinophilia, mucus-containing goblet cells, and peribronchial fibrosis. Both strains of mice were also acutely sensitized to ovalbumin. Bone marrow eosinophil progenitor cells and mature eosinophils were enumerated. BALB/c and A/J mice have similar bone marrow responses after acute allergen exposure, with elevations in bone marrow eosinophil progenitor cell and eosinophil numbers. After chronic allergen exposure, only C5-deficient A/J mice that developed peribronchial fibrosis exhibited bone marrow eosinophilia. BALB/c mice lacked peribronchial fibrosis and extinguished accelerated eosinophil production after long-term allergen challenge. Chronic airway remodeling after repeated allergen exposure in genetically different mice correlated with differences in long-term bone marrow eosinophilopoiesis. Preventing asthma from progressing to chronic airway remodeling with fibrosis may involve identifying genetically determined influences on bone marrow responses to chronic allergen exposure.

Survival of Free and Encapsulated Human and Rat Islet Xenografts Transplanted into the Mouse Bone Marrow

PubMed Central

Meier, Raphael P. H.; Seebach, Jörg D.; Morel, Philippe; Mahou, Redouan; Borot, Sophie; Giovannoni, Laurianne; Parnaud, Geraldine; Montanari, Elisa; Bosco, Domenico; Wandrey, Christine; Berney, Thierry; Bühler, Leo H.; Muller, Yannick D.

2014-01-01

Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation. PMID:24625569

Does Simulated Spaceflight Modify Epigenetic Status During Bone Remodeling?

NASA Technical Reports Server (NTRS)

Thomas, Nicholas J.; Stevick, Rebecca J.; Tran, Luan H.; Nalavadi, Mohit O.; Almeida, Eduardo A.C.; Globus, Ruth K.; Alwood, Joshua S.

2015-01-01

Little is known about the effects of spaceflight conditions on epigenetics. The term epigenetics describes changes to the genome that can affect expression of a gene without changes to the sequence of DNA. Epigenetic processes are thought to underlie cellular differentiation, where transcription of specific genes occurs in response to key stimuli, and may be heritable - passing from one cell to its daughter cell. We hypothesize that the mechanical environment during spaceflight, namely microgravity-induced weightlessness or exercise regulate gene expression in the osteoblast-lineage cells both to control bone formation by osteoblasts and bone resorption by osteoclasts, which continually shapes bone structure throughout life. Similarly we intend to evaluate how radiation regulates these same bone cell activity and differentiation related genes. We further hypothesize that the regulation in bone cell gene expression is at least partially controlled through epigenetic mechanisms of methylation or small non-coding RNA (microRNAs). We have acquired preliminary data suggesting that global genome methylation is modified in response to axial compression of the tibia - a model of exercise. We intend to pursue these hypotheses wherein we will evaluate changes in gene expression and, congruently, changes in epigenetic state in bones from mice subjected to the aforementioned conditions: hindlimb unloading to simulate weightlessness, axial compression of the tibia, or radiation exposure in order to gain insight into the role of epigenetics in spaceflight-induced bone loss.

Cell interactions in bone tissue engineering.

PubMed

Pirraco, R P; Marques, A P; Reis, R L

2010-01-01

Bone fractures, where the innate regenerative bone response is compromised, represent between 4 and 8 hundred thousands of the total fracture cases, just in the United States. Bone tissue engineering (TE) brought the notion that, in cases such as those, it was preferable to boost the healing process of bone tissue instead of just adding artificial parts that could never properly replace the native tissue. However, despite the hype, bone TE so far could not live up to its promises and new bottom-up approaches are needed. The study of the cellular interactions between the cells relevant for bone biology can be of essential importance to that. In living bone, cells are in a context where communication with adjacent cells is almost permanent. Many fundamental works have been addressing these communications nonetheless, in a bone TE approach, the 3D perspective, being part of the microenvironment of a bone cell, is as crucial. Works combining the study of cell-to-cell interactions in a 3D environment are not as many as expected. Therefore, the bone TE field should not only gain knowledge from the field of fundamental Biology but also contribute for further understanding the biology of bone. In this review, a summary of the main works in the field of bone TE, aiming at studying cellular interactions in a 3D environment, and how they contributed towards the development of a functional engineered bone tissue, is presented.

BMP-non-responsive Sca1+ CD73+ CD44+ mouse bone marrow derived osteoprogenitor cells respond to combination of VEGF and BMP-6 to display enhanced osteoblastic differentiation and ectopic bone formation.

PubMed

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.

Stem cell niches and other factors that influence the sensitivity of bone marrow to radiation-induced bone cancer and leukaemia in children and adults

PubMed Central

Richardson, Richard B

2011-01-01

Purpose: This paper reviews and reassesses the internationally accepted niches or ‘targets’ in bone marrow that are sensitive to the induction of leukaemia and primary bone cancer by radiation. Conclusions: The hypoxic conditions of the 10 μm thick endosteal/osteoblastic niche where preleukemic stem cells and hematopoietic stem cells (HSC) reside provides a radioprotective microenvironment that is 2-to 3-fold less radiosensitive than vascular niches. This supports partitioning the whole marrow target between the low haematological cancer risk of irradiating HSC in the endosteum and the vascular niches within central marrow. There is a greater risk of induced bone cancer when irradiating a 50 μm thick peripheral marrow adjacent to the remodelling/reforming portion of the trabecular bone surface, rather than marrow next to the quiescent bone surface. This choice of partitioned bone cancer target is substantiated by the greater radiosensitivity of: (i) Bone with high remodelling rates, (ii) the young, (iii) individuals with hypermetabolic benign diseases of bone, and (iv) the epidemiology of alpha-emitting exposures. Evidence is given to show that the absence of excess bone-cancer in atomic-bomb survivors may be partially related to the extremely low prevalence among Japanese of Paget's disease of bone. Radiation-induced fibrosis and the wound healing response may be implicated in not only radiogenic bone cancers but also leukaemia. A novel biological mechanism for adaptive response, and possibility of dynamic targets, is advocated whereby stem cells migrate from vascular niches to stress-mitigated, hypoxic niches. PMID:21204614

Hematopoietic Responses to Lipopolysaccharide in C57BL/10Sn and C57BL/10ScN Strain Mice

DTIC Science & Technology

1982-12-01

Responses of endogenous (E-CFU) stem cells as well as bone marrow and spleen-derived exogenous (CFU-s) stem cells, granulocyte-macrophage (GM;-CFC... endogenous (E-CFU) stem cells as well as bone marrow and spleen-derived exogenous (CFU-s) stem cells, granulocyte-macrophage (GM-CFC) and macrophage (M...IOScN in comparison to the normal C57BL/1OSn strain mice, as measured by endogenous (E-CFU) and exogenous (CFU-s) stem cells and committed granulocyte

Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

PubMed Central

Yang, Jing; He, Jin; Wang, Ji; Cao, Yabing; Ling, Jianhua; Qian, Jianfei; Lu, Yong; Li, Haiyan; Zheng, Yuhuan; Lan, Yongsheng; Hong, Sungyoul; Matthews, Jairo; Starbuck, Michael W; Navone, Nora M; Orlowski, Robert Z.; Lin, Pei; Kwak, Larry W.; Yi, Qing

2012-01-01

Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into SCID or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism—sactivation of p38 signaling in myeloma cells—by which myeloma cells induce osteolytic bone lesions and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease. PMID:22425892

Aging of marrow stromal (skeletal) stem cells and their contribution to age-related bone loss.

PubMed

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.

Fate of bone marrow stromal cells in a syngenic model of bone formation.

PubMed

Boukhechba, Florian; Balaguer, Thierry; Bouvet-Gerbettaz, Sébastien; Michiels, Jean-François; Bouler, Jean-Michel; Carle, Georges F; Scimeca, Jean-Claude; Rochet, Nathalie

2011-09-01

Bone marrow stromal cells (BMSCs) have been demonstrated to induce bone formation when associated to osteoconductive biomaterials and implanted in vivo. Nevertheless, their role in bone reconstruction is not fully understood and rare studies have been conducted to follow their destiny after implantation in syngenic models. The aim of the present work was to use sensitive and quantitative methods to track donor and recipient cells after implantation of BMSCs in a syngenic model of ectopic bone formation. Using polymerase chain reaction (PCR) amplification of the Sex determining Region Y (Sry) gene and in situ hybridization of the Y chromosome in parallel to histological analysis, we have quantified within the implants the survival of the donor cells and the colonization by the recipient cells. The putative migration of the BMSCs in peripheral organs was also analyzed. We show here that grafted cells do not survive more than 3 weeks after implantation and might migrate in peripheral lymphoid organs. These cells are responsible for the attraction of host cells within the implants, leading to the centripetal colonization of the biomaterial by new bone.

Differences in responses to X-ray exposure between osteoclast and osteoblast cells

PubMed Central

Zhang, Jian; Wang, Ziyang; Wu, Anqing; Nie, Jing; Pei, Hailong; Hu, Wentao; Wang, Bing; Shang, Peng; Li, Bingyan

2017-01-01

Abstract Radiation-induced bone loss is a potential health concern for cancer patients undergoing radiotherapy. Enhanced bone resorption by osteoclasts and decreased bone formation by osteoblasts were thought to be the main reasons. In this study, we showed that both pre-differentiating and differentiating osteoclasts were relatively sensitive to X-rays compared with osteoblasts. X-rays decreased cell viability to a greater degree in RAW264.7 cells and in differentiating cells than than in osteoblastic MC3T3-E1 cells. X-rays at up to 8 Gy had little effects on osteoblast mineralization. In contrast, X-rays at 1 Gy induced enhanced osteoclastogenesis by enhanced cell fusion, but had no effects on bone resorption. A higher dose of X-rays at 8 Gy, however, had an inhibitory effect on bone resorption. In addition, actin ring formation was disrupted by 8 Gy of X-rays and reorganized into clusters. An increased activity of Caspase 3 was found after X-ray exposure. Actin disorganization and increased apoptosis may be the potential effects of X-rays at high doses, by inhibiting osteoclast differentiation. Taken together, our data indicate high radiosensitivity of osteoclasts. X-ray irradiation at relatively low doses can activate osteoclastogenesis, but not osteogenic differentiation. The radiosensitive osteoclasts are the potentially responsive cells for X-ray-induced bone loss. PMID:28541506

Immune Humanization of Immunodeficient Mice Using Diagnostic Bone Marrow Aspirates from Carcinoma Patients

PubMed Central

Werner-Klein, Melanie; Proske, Judith; Werno, Christian; Schneider, Katharina; Hofmann, Hans-Stefan; Rack, Brigitte; Buchholz, Stefan; Ganzer, Roman; Blana, Andreas; Seelbach-Göbel, Birgit; Nitsche, Ulrich

2014-01-01

Tumor xenografts in immunodeficient mice, while routinely used in cancer research, preclude studying interactions of immune and cancer cells or, if humanized by allogeneic immune cells, are of limited use for tumor-immunological questions. Here, we explore a novel way to generate cancer models with an autologous humanized immune system. We demonstrate that hematopoietic stem and progenitor cells (HSPCs) from bone marrow aspirates of non-metastasized carcinoma patients, which are taken at specialized centers for diagnostic purposes, can be used to generate a human immune system in NOD-scid IL2rγ(null) (NSG) and HLA-I expressing NSG mice (NSG-HLA-A2/HHD) comprising both, lymphoid and myeloid cell lineages. Using NSG-HLA-A2/HHD mice, we show that responsive and self-tolerant human T cells develop and human antigen presenting cells can activate human T cells. As critical factors we identified the low potential of bone marrow HSPCs to engraft, generally low HSPC numbers in patient-derived bone marrow samples, cryopreservation and routes of cell administration. We provide here an optimized protocol that uses a minimum number of HSPCs, preselects high-quality bone marrow samples defined by the number of initially isolated leukocytes and intra-femoral or intra-venous injection. In conclusion, the use of diagnostic bone marrow aspirates from non-metastasized carcinoma patients for the immunological humanization of immunodeficient mice is feasible and opens the chance for individualized analyses of anti-tumoral T cell responses. PMID:24830425

Experiment K-7-23: Effect of Spaceflight on Level and Function of Immune Cells. Part 1; Immunology Studies

NASA Technical Reports Server (NTRS)

Sonnenfeld, G.; Mandel, A.; Konstantinova, I. V.; Berry, W. D.; Taylor, G. R.; Lesnyak, A. T.; Fuchs, B. B.; Rakhmilevich, A. L.

1994-01-01

Two different experiments were carried out in this segment of the immunology protocol for samples received from rats flown on Cosmos 2044. Control groups included vivarium, synchronous and antiorthostatically suspended rats. In the first experiment, rat bone marrow cells were examined in Moscow for their response to recombinant murine colony stimulating factor-granulocyte / monocyte (CSF-GM). In the second experiment, rat spleen and bone marrow cells were stained in Moscow with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and shipped to the United States for analysis on a flow cytometer. The results of the studies indicated that bone marrow cells from flown and suspended rats showed a decreased response to CSF-GM as compared to bone marrow cells from control rats. Spleen cells from flown rats showed increased percentages of suppressor-cytotoxic-T and helper-T cells amongst the entire cell population. Bone marrow cells showed an increase in the percentage of helper-T cells in the myelogenous population and increased percentages of anti-asialo GM-1 bearing, interleukin-2 receptor bearing, pan-T and helper-T cells in the lymphocytic population. Cell populations from rats suspended antiorthostatically did not follow the same pattern of distribution of leukocytes as cell populations for flown rats. These results are similar, but not identical to, earlier results from Cosmos 1887, and confirm that space flight can have profound effects on immune system components and activities.

Effects of hypergravity on immunologic function

NASA Technical Reports Server (NTRS)

Sonnenfeld, G.; Koebel, D. A.; Davis, S.

1995-01-01

The purpose of this study was to compare the effects of hypergravity exposure (2g) with those of exposure to space flight in the Cosmos 2044 flight. To do so, rats were centrifuged continuously for 14 days. Two different experiments were carried out on tissue obtained from the centrifuged rats. In the first experiment, rat bone marrow cells were examined for their response to recombinant murine colony stimulating factor-granulocyte/monocyte (GM-CSF). In the second experiment, rat spleen and bone marrow cells were stained in with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and analyzed on a flow cytometer. The results of the studies indicated that bone marrow cells from centrifuged rats showed no significant change in response to GM-CSF as compared to bone marrow cells from control rats. Spleen cells from flown rats showed some statistically significant changes in leukocytes subset distribution, but no differences that appeared to be of biological significance. These results indicate that hypergravity did not greatly affect the same immunological parameters affected by space flight in the Cosmos 2044 mission.

Effects of hypergravity on immunologic function

NASA Technical Reports Server (NTRS)

Sonnenfeld, G.; Koebel, D. A.; Davis, S.

1994-01-01

The purpose of this study was to compare the effects of hypergravity exposure (2g) with those of exposure to space flight in the Cosmos 2044 flight. To do so, rats were centrifuged continuously for 14 days. Two different experiments were carried out on tissue obtained from the centrifuged rats. In the first experiment, rat bone marrow cells were examined for their response to recombinant murine colony stimulating factor-granulocyte/monocyte (GM-CSF). In the second experiment, rate spleen and bone marrow cells were stained in with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and analyzed on a flow cytometer. The results of the studies indicated that bone marrow cells from centrifuged rats showed no significant change in response to GM-CSF as compared to bone marrow cells from control rats. Spleen cells from flown rats showed some statistically significant changes in leukocytes subset distribution, but no differences that appeared to be of biological significance. These results indicate that hypergravity did not greatly affect the same immunological parameters affected by space flight in the Cosmos 2044 mission.

Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain

NASA Technical Reports Server (NTRS)

Owan, I.; Burr, D. B.; Turner, C. H.; Qiu, J.; Tu, Y.; Onyia, J. E.; Duncan, R. L.

1997-01-01

Mechanical force applied to bone produces two localized mechanical signals on the cell: deformation of the extracellular matrix (substrate strain) and extracellular fluid flow. To study the effects of these stimuli on osteoblasts, MC3T3-E1 cells were grown on type I collagen-coated plastic plates and subjected to four-point bending. This technique produces uniform levels of physiological strain and fluid forces on the cells. Each of these parameters can be varied independently. Osteopontin (OPN) mRNA expression was used to assess the anabolic response of MC3T3-E1 cells. When fluid forces were low, neither strain magnitude nor strain rate was correlated with OPN expression. However, higher-magnitude fluid forces significantly increased OPN message levels independently of the strain magnitude or rate. These data indicate that fluid forces, and not mechanical stretch, influence OPN expression in osteoblasts and suggest that fluid forces induced by extracellular fluid flow within the bone matrix may play an important role in bone formation in response to mechanical loading.

Androgen receptor-negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms.

PubMed

Li, Zhi Gang; Mathew, Paul; Yang, Jun; Starbuck, Michael W; Zurita, Amado J; Liu, Jie; Sikes, Charles; Multani, Asha S; Efstathiou, Eleni; Lopez, Adriana; Wang, Jing; Fanning, Tina V; Prieto, Victor G; Kundra, Vikas; Vazquez, Elba S; Troncoso, Patricia; Raymond, Austin K; Logothetis, Christopher J; Lin, Sue-Hwa; Maity, Sankar; Navone, Nora M

2008-08-01

In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor-negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer-induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor-null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells.

Androgen receptor–negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms

PubMed Central

Li, Zhi Gang; Mathew, Paul; Yang, Jun; Starbuck, Michael W.; Zurita, Amado J.; Liu, Jie; Sikes, Charles; Multani, Asha S.; Efstathiou, Eleni; Lopez, Adriana; Wang, Jing; Fanning, Tina V.; Prieto, Victor G.; Kundra, Vikas; Vazquez, Elba S.; Troncoso, Patricia; Raymond, Austin K.; Logothetis, Christopher J.; Lin, Sue-Hwa; Maity, Sankar; Navone, Nora M.

2008-01-01

In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor–negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer–induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor–null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells. PMID:18618013

Change in Mouse Bone Turnover in Response to Microgravity on RR-1

NASA Technical Reports Server (NTRS)

Cheng-Campbell, Margareth A.; Blaber, Elizabeth A.; Almeida, Eduardo A. C.

2016-01-01

Mechanical unloading during spaceflight is known to adversely affect mammalian physiology. Our previous studies using the Animal Enclosure Module on short duration Shuttle missions enabled us to identify a deficit in stem cell based-tissue regeneration as being a significant concern for long-duration spaceflight. Specifically, we found that mechanical unloading in microgravity resulted in inhibition of differentiation of mesenchymal and hematopoietic stem cells in the bone marrow compartment. Also, we observed overexpression of a cell cycle arrest molecule, CDKN1ap21, in osteoprecursor cells on the bone surface, chondroprogenitors in the articular cartilage, and in myofibers attached to bone tissue. Specifically in bone tissue during both short (15-day) and long (30-day) microgravity experiments, we observed significant loss of bone tissue and structure in both the pelvis and the femur. After 15-days of microgravity on STS-131, pelvic ischium displayed a 6.23 decrease in bone fraction (p0.005) and 11.91 decrease in bone thickness (p0.002). Furthermore, during long-duration spaceflight we observed onset of an accelerated aging-like phenotype and osteoarthritic disease state indicating that stem cells within the bone tissue fail to repair and regenerate tissues in a normal manner, leading to drastic tissue alterations in response to microgravity. The Rodent Research Hardware System provides the capability to investigate these effects during long-duration experiments on the International Space Station. During the Rodent Research-1 mission 10 16-week-old female C57Bl6J mice were exposed to 37-days of microgravity. All flight animals were euthanized and frozen on orbit for future dissection. Ground (n10) and vivarium controls (n10) were housed and processed to match the flight animal timeline. During this study we collected pelvis, femur, and tibia from all animal groups to test the hypothesis that stem cell-based tissue regeneration is significantly altered after 37-days of spaceflight. To do this, we will analyze differences in bone morphometric parameters using MicroCT. The pelvis, femur, and tibia are key in supporting and distributing weight under normal conditions. Therefore, we expect to see altered remodeling in flight animals in response to microgravity with respect to ground controls. In combination with histomorphometry, these results will help elucidate the complex mechanisms underlying bone tissue maintenance and stem cell regeneration.

Changes in Mouse Bone Turnover in Response to Microgravity

NASA Technical Reports Server (NTRS)

Cheng-Campbell, M.; Blaber, E.; Almeida, E.

2016-01-01

Mechanical unloading during spaceflight is known to adversely affect mammalian physiology. Our previous studies using the Animal Enclosure Module on short duration Shuttle missions enabled us to identify a deficit in stem cell based-tissue regeneration as being a significant concern for long-duration spaceflight. Specifically, we found that mechanical unloading in microgravity resulted in inhibition of differentiation of mesenchymal and hematopoietic stem cells in the bone marrow compartment. Also, we observed overexpression of a cell cycle arrest molecule, CDKN1a/p21, in osteoprecursor cells on the bone surface, chondroprogenitors in the articular cartilage, and in myofibers attached to bone tissue. Specifically in bone tissue during both short (15-day) and long (30-day) microgravity experiments, we observed significant loss of bone tissue and structure in both the pelvis and the femur. After 15-days of microgravity on STS-131, pelvic ischium displayed a 6.23% decrease in bone fraction (p=0.005) and 11.91% decrease in bone thickness (p=0.002). Furthermore, during long-duration spaceflight we observed onset of an accelerated aging-like phenotype and osteoarthritic disease state indicating that stem cells within the bone tissue fail to repair and regenerate tissues in a normal manner, leading to drastic tissue alterations in response to microgravity. The Rodent Research Hardware System provides the capability to investigate these effects during long-duration experiments on the International Space Station. During the Rodent Research-1 mission 10 16-week-old female C57Bl/6J mice were exposed to 37-days of microgravity. All flight animals were euthanized and frozen on orbit for future dissection. Ground (n=10) and vivarium controls (n=10) were housed and processed to match the flight animal timeline. During this study we collected pelvis, femur, and tibia from all animal groups to test the hypothesis that stem cell-based tissue regeneration is significantly altered after 37-days of spaceflight. To do this, we will analyze differences in bone morphometric parameters using MicroCT. The pelvis, femur, and tibia are key in supporting and distributing weight under normal conditions. Therefore, we expect to see altered remodeling in flight animals in response to microgravity with respect to ground controls. In combination with histomorphometry, these results will help elucidate the complex mechanisms underlying bone tissue maintenance and stem cell regeneration.

Osteocyte physiology and response to fluid shear stress are impaired following exposure to cobalt and chromium: Implications for bone health following joint replacement

PubMed Central

Shah, Karan M.; Orton, Peter; Mani, Nick

2016-01-01

ABSTRACT The effects of metal ion exposure on osteocytes, the most abundant cell type in bone and responsible for coordinating bone remodeling, remain unclear. However, several studies have previously shown that exposure to cobalt (Co2+) and chromium (Cr3+), at concentrations equivalent to those found clinically, affect osteoblast and osteoclast survival and function. In this study, we tested the hypothesis that metal ions would similarly impair the normal physiology of osteocytes. The survival, dendritic morphology, and response to fluid shear stress of the mature osteocyte‐like cell‐line MLO‐Y4 following exposure to clinically relevant concentrations and combinations of Co and Cr ions were measured in 2D‐culture. Exposure of MLO‐Y4 cells to metal ions reduced cell number, increased dendrites per cell and increased dendrite length. We found that combinations of metal ions had a greater effect than the individual ions alone, and that Co2+ had a predominate effect on changes to cell numbers and dendrites. Combined metal ion exposure blunted the responses of the MLO‐Y4 cells to fluid shear stress, including reducing the intracellular calcium responses and modulation of genes for the osteocyte markers Cx43 and Gp38, and the signaling molecules RANKL and Dkk‐1. Finally, we demonstrated that in the late osteoblasts/early osteocytes cell line MLO‐A5 that Co2+ exposure had no effect on mineralization, but Cr3+ treatment inhibited mineralization in a dose‐dependent manner, without affecting cell viability. Taken together, these data indicate that metal exposure can directly affect osteocyte physiology, with potential implications for bone health including osseointegration of cementless components, and periprosthetic bone remodeling. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1716–1723, 2017. PMID:27673573

In vivo analysis of biocompatibility and vascularization of the synthetic bone grafting substitute NanoBone.

PubMed

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.

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.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis.

PubMed

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.

Infection of chicken bone marrow mononuclear cells with subgroup J avian leukosis virus inhibits dendritic cell differentiation and alters cytokine expression.

PubMed

Liu, Di; Qiu, Qianqian; Zhang, Xu; Dai, Manman; Qin, Jianru; Hao, Jianjong; Liao, Ming; Cao, Weisheng

2016-10-01

Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus known to induce tumor formation and immunosuppression in infected chickens. One of the organs susceptible to ALV-J is the bone marrow, from which specialized antigen-presenting cells named dendritic cells (BM-DCs) are derived. Notably, these cells possess the unique ability to induce primary immune responses. In the present study, a method of cultivating and purifying DCs from chicken bone marrow in vitro was established to investigate the effects of ALV-J infection on BM-DC differentiation or generation. The results indicated that ALV-J not only infects the chicken bone marrow mononuclear cells but also appears to inhibit the differentiation and maturation of BM-DCs and to trigger apoptosis. Moreover, substantial reductions in the mRNA expression of TLR1, TLR2, TLR3, MHCI, and MHCII and in cytokine production were detected in the surviving BM-DCs following ALV-J infection. These findings indicate that ALV-J infection disrupts the process of bone marrow mononuclear cell differentiation into BM-DCs likely via altered antigen presentation, resulting in a downstream immune response in affected chickens. Copyright © 2016 Elsevier B.V. All rights reserved.

Pore size regulates cell and tissue interactions with PLGA-CaP scaffolds used for bone engineering.

PubMed

Sicchieri, Luciana Gonçalves; Crippa, Grasiele Edilaine; de Oliveira, Paulo Tambasco; Beloti, Marcio Mateus; Rosa, Adalberto Luiz

2012-02-01

A common subject in bone tissue engineering is the need for porous scaffolds to support cell and tissue interactions aiming at repairing bone tissue. As poly(lactide-co-glycolide)-calcium phosphate (PLGA-CaP) scaffolds can be manufactured with different pore sizes, the aim of this study was to evaluate the effect of pore diameter on osteoblastic cell responses and bone tissue formation. Scaffolds were prepared with 85% porosity, with pore diameters in the ranges 470-590, 590-850 and 850-1200 µm. Rat bone marrow stem cells differentiated into osteoblasts were cultured on the scaffolds for up to 10 days to evaluate cell growth, alkaline phosphatase (ALP) activity and the gene expression of the osteoblast markers RUNX2, OSX, COL, MSX2, ALP, OC and BSP by real-time PCR. Scaffolds were implanted in critical size rat calvarial defects for 2, 4, and 8 weeks for histomorphometric analysis. Cell growth and ALP activity were not affected by the pore size; however, there was an increase in the gene expression of osteoblastic markers with the increase in the pore sizes. At 2 weeks all scaffolds displayed a similar amount of bone and blood vessels formation. At 4 and 8 weeks much more bone formation and an increased number of blood vessels were observed in scaffolds with pores of 470-590 µm. These results show that PLGA-CaP is a promising biomaterial for bone engineering. However, ideally, combinations of larger (-1000 µm) and smaller (-500 µm) pores in a single scaffold would optimize cellular and tissue responses during bone healing. Copyright © 2011 John Wiley & Sons, Ltd.

Analysing the bioactive makeup of demineralised dentine matrix on bone marrow mesenchymal stem cells for enhanced bone repair.

PubMed

Avery, S J; Sadaghiani, L; Sloan, A J; Waddington, R J

2017-07-10

Dentine matrix has proposed roles for directing mineralised tissue repair in dentine and bone; however, the range of bioactive components in dentine and specific biological effects on bone-derived mesenchymal stem cells (MSCs) in humans are less well understood. The aims of this study were to further elucidate the biological response of MSCs to demineralised dentine matrix (DDM) in enhancing wound repair responses and ascertain key contributing components. Dentine was obtained from human teeth and DDM proteins solubilised with ethylenediaminetetraacetic acid (EDTA). Bone marrow derived MSCs were commercially obtained. Cells with a more immature phenotype were then selected by preferential fibronectin adhesion (FN-BMMSCs) for use in subsequent in vitro assays. DDM at 10 µg/mL reduced cell expansion, attenuated apoptosis and was the minimal concentration capable of inducing osteoblastic differentiation. Enzyme-linked immunosorbent assay (ELISA) quantification of growth factors indicated physiological levels produced the above responses; transforming growth factor β (TGF-β1) was predominant (15.6 ng/mg DDM), with relatively lower concentrations of BMP-2, FGF, VEGF and PDGF (6.2-4.7 ng/mg DDM). Fractionation of growth factors from other DDM components by heparin affinity chromatography diminished osteogenic responses. Depletion of biglycan from DDM also attenuated osteogenic potency, which was partially rescued by the isolated biglycan. Decorin depletion from DDM had no influence on osteogenic potency. Collectively, these results demonstrate the potential of DDM for the delivery of physiological levels of growth factors for bone repair processes, and substantiate a role for biglycan as an additional adjuvant for driving osteogenic pathways.

Human regulatory T cells do not suppress the antitumor immunity in the bone marrow: a role for bone marrow stromal cells in neutralizing regulatory T cells.

PubMed

Guichelaar, Teun; Emmelot, Maarten E; Rozemuller, Henk; Martini, Bianka; Groen, Richard W J; Storm, Gert; Lokhorst, Henk M; Martens, Anton C; Mutis, Tuna

2013-03-15

Regulatory T cells (Tregs) are potent tools to prevent graft-versus-host disease (GVHD) induced after allogeneic stem cell transplantation or donor lymphocyte infusions. Toward clinical application of Tregs for GVHD treatment, we investigated the impact of Tregs on the therapeutic graft-versus-tumor (GVT) effect against human multiple myeloma tumors with various immunogenicities, progression rates, and localizations in a humanized murine model. Immunodeficient Rag2(-/-)γc(-/-) mice, bearing various human multiple myeloma tumors, were treated with human peripheral blood mononuclear cell (PBMC) alone or together with autologous ex vivo cultured Tregs. Mice were analyzed for the in vivo engraftment, homing of T-cell subsets, development of GVHD and GVT. In additional in vitro assays, Tregs that were cultured together with bone marrow stromal cells were analyzed for phenotype and functions. Treatment with PBMC alone induced variable degrees of antitumor response, depending on the immunogenicity and the growth rate of the tumor. Coinfusion of Tregs did not impair the antitumor response against tumors residing within the bone marrow, irrespective of their immunogenicity or growth rates. In contrast, Tregs readily inhibited the antitumor effect against tumors growing outside the bone marrow. Exploring this remarkable phenomenon, we discovered that bone marrow stroma neutralizes the suppressive activity of Tregs in part via production of interleukin (IL)-1β/IL-6. We furthermore found in vitro and in vivo evidence of conversion of Tregs into IL-17-producing T cells in the bone marrow environment. These results provide new insights into the Treg immunobiology and indicate the conditional benefits of future Treg-based therapies.

Overexpression of Insulin-Like Growth Factor 1 Enhanced the Osteogenic Capability of Aging Bone Marrow Mesenchymal Stem Cells.

PubMed

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.

Regulation of Prostate Cancer Bone Metastasis by DKK1

DTIC Science & Technology

2012-09-01

information is estimated to average 1 hour per response , including the time for reviewing instructions, searching existing data sources, gathering and...ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U c. THIS PAGE U UU 13 19b. TELEPHONE NUMBER...cells into bone irrevocably alters the bone microenvironment and initiates a skeletal response that is dependent on the type of tumor (1). Breast

Interactions between bone cells and biomaterials: An update.

PubMed

Beauvais, Sabrina; Drevelle, Olivier; Jann, Jessica; Lauzon, Marc-Antoine; Foruzanmehr, Mohammadreza; Grenier, Guillaume; Roux, Sophie; Faucheux, Nathalie

2016-06-01

As the populations of the Western world become older, they will suffer more and more from bone defects related to osteoporosis (non-union fractures, vertebral damages), cancers (malignant osteolysis) and infections (osteomyelitis). Autografts are usually used to fill these defects, but they have several drawbacks such as morbidity at the donor site and the amount and quality of bone that can be harvested. Recent scientific milestones made in biomaterials development were shown to be promising to overcome these limitations. Cell interactions with biomaterials can be improved by adding at their surface functional groups such as adhesive peptides and/or growth factors. The development of such biomimetic materials able to control bone cell responses can only proceed if it is based on a sound understanding of bone cell behavior and regulation. This review focuses on bone physiology and the regulation of bone cell differentiation and function, and how the latest advances in biomimetic materials can be translated within promising clinical outcomes.

Effects of deletion of ER-alpha in osteoblast-lineage cells on bone mass and adaptation to mechanical loading differs in female and male mice

PubMed Central

Melville, Katherine M.; Kelly, Natalie H.; Surita, Gina; Buchalter, Daniel B.; Schimenti, John C.; Main, Russell P.; Ross, F. Patrick; van der Meulen, Marjolein C. H.

2015-01-01

Estrogen receptor alpha (ERα) has been implicated in bone’s response to mechanical loading in both males and females. ERα in osteoblast lineage cells is important for determining bone mass, but results depend on animal sex and the cellular stage at which ERα is deleted. We demonstrated previously that when ERα is deleted from mature osteoblasts and osteocytes in mixed background female mice, bone mass and strength are decreased. However, few studies exist examining the skeletal response to loading in bone cell-specific ERαKO mice. Therefore, we crossed ERα floxed (ERαfl/fl) and osteocalcin-Cre (OC-Cre) mice to generate animals lacking ERα in mature osteoblasts and osteocytes (pOC-ERαKO) and littermate controls (LC). At 10 weeks of age the left tibia was loaded in vivo for two weeks. We analyzed bone mass through microCT, bone formation rate by dynamic histomorphometry, bone strength from mechanical testing, and osteoblast and osteoclast activity by serum chemistry and immunohistochemistry. ERα in mature osteoblasts differentially regulated bone mass in males and females. Compared to LC, female pOC-ERαKO mice had decreased cortical and cancellous bone mass, while male pOC-ERαKO mice had equal or greater bone mass than LC. Bone mass results correlated with decreased compressive strength in pOC-ERαKO female L5 vertebrae, and with increased maximum moment in pOC-ERαKO male femora. Female pOC-ERαKO mice responded more to mechanical loading, while the response of pOC-ERαKO male animals was similar to their littermate controls. PMID:25707500

Pilot Study: Unique Response of Bone Tissue During an Investigation of Radio-Adaptive Effects in Mice

NASA Technical Reports Server (NTRS)

Sibonga, J. D.; Iwaniec, U.; Wu, H.

2011-01-01

PURPOSE: We obtained bone tissue to evaluate the collateral effects of experiments designed to investigate molecular mechanisms of radio-adaptation in a mouse model. Radio-adaptation describes a process by which the prior exposure to low dose radiation can protect against the toxic effect of a subsequent high dose exposure. In the radio-adaptation experiments, C57Bl/6 mice were exposed to either a Sham or a priming Low Dose (5 cGy) of Cs-137 gamma rays before being exposed to either a Sham or High Dose (6 Gy) 24 hours later. ANALYSIS: Bone tissue were obtained from two experiments where mice were sacrificed at 3 days (n=3/group, 12 total) and at 14 days (n=6/group, 24 total) following high dose exposure. Tissues were analyzed to 1) evaluate a radio-adaptive response in bone tissue and 2) describe cellular and microstructural effects for two skeletal sites with different rates of bone turnover. One tibia and one lumbar vertebrae (LV2), collected at the 3-day time-point, were analyzed by bone histomorphometry and micro-CT to evaluate the cellular response and any evidence of microarchitectural impact. Likewise, tibia and LV2, collected at the 14-day time-point, were analyzed by micro-CT alone to evaluate resulting changes to bone structure and microarchitecture. The data were analyzed by 2-way ANOVA to evaluate the effects of the priming low dose radiation, of the high dose radiation, and of any interaction between the priming low and high doses of radiation. Bone histomorphometry was performed in the cancellous bone (aka trabecular bone) compartments of the proximal tibial metaphysis and of LV2. RESULTS: Cellular Response @ 3 Days The priming Low Dose radiation decreased osteoblast-covered bone perimeter in the proximal tibia and the total cell density in the bone marrow in the LV2. High Dose radiation, regardless of prior exposure to priming dose, dramatically reduced total cell density in bone marrow of both the long bone and vertebra. However, in the proximal tibia, High Dose radiation increased the osteoclast-covered bone perimeters, the density of adipocytes in bone marrow, and the area of bone marrow occupied by fat cells -- while in the LV2, adipocytes were rare and not stimulated by High Dose radiation. In an unexpected response, High Dose radiation dramatically increased (10-fold) osteoblast-covered bone perimeter in the LV2.

Osteoimmunology: Influence of the Immune System on Bone Regeneration and Consumption.

PubMed

Limmer, Andreas; Wirtz, Dieter C

2017-06-01

Background Stimulating bone regeneration is a central aim in orthopaedic and trauma surgery. Although the replacement of bone with artificial materials like cement or apatite helps to keep up bone stability, new bone often cannot be regenerated. Increasing research efforts have led to the clinical application of growth factors stimulating bone growth (e.g. bone morphogenic protein, BMP) and inhibitors preventing bone consumption (e.g. RANKL blocking antibodies). These factors mostly concentrate on stimulating osteoblast or preventing osteoclast activity. Current Situation It is widely accepted that osteoblasts and osteoclasts are central players in bone regeneration. This concept assumes that osteoblasts are responsible for bone growth while osteoclasts cause bone consumption by secreting matrix-degrading enzymes such as cathepsin K and matrix metalloproteinases (MMP). However, according to new research results, bone growth or consumption are not regulated by single cell types. It is rather the interaction of various cell types that regulates bone metabolism. While factors secreted by osteoblasts are essential for osteoclast differentiation and activation, factors secreted by activated osteoclasts are essential for osteoblast activity. In addition, recent research results imply that the influence of the immune system on bone metabolism has long been neglected. Factors secreted by macrophages or T cells strongly influence bone growth or degradation, depending on the bone microenvironment. Infections, sterile inflammation or tumour metastases not only affect bone cells directly, but also influence immune cells such as T cells indirectly. Furthermore, immune cells and bone are mechanistically regulated by similar factors such as cytokines, chemokines and transcription factors, suggesting that the definition of bone and immune cells has to be thought over. Outlook Bone and the immune system are regulated by similar mechanisms. These newly identified similarities between bone and the immune system imply that medication developed for tumour and autoimmune patients could also be applied in bone diseases. Georg Thieme Verlag KG Stuttgart · New York.

Role of whole bone marrow, whole bone marrow cultured cells, and mesenchymal stem cells in chronic wound healing.

PubMed

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.

Human osteocalcin and bone sialoprotein mediating osteomimicry of prostate cancer cells: role of cAMP-dependent protein kinase A signaling pathway.

PubMed

Huang, Wen-Chin; Xie, Zhihui; Konaka, Hiroyuki; Sodek, Jaro; Zhau, Haiyen E; Chung, Leland W K

2005-03-15

Osteocalcin and bone sialoprotein are the most abundant noncollagenous bone matrix proteins expressed by osteoblasts. Surprisingly, osteocalcin and bone sialoprotein are also expressed by malignant but not normal prostate epithelial cells. The purpose of this study is to investigate how osteocalcin and bone sialoprotein expression is regulated in prostate cancer cells. Our investigation revealed that (a) human osteocalcin and bone sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b) Deletion analysis of human osteocalcin and bone sialoprotein promoter regions identified cyclic AMP (cAMP)-responsive elements (CRE) as the critical determinants for conditioned medium-mediated osteocalcin and bone sialoprotein gene expression in prostate cancer cells. Consistent with these results, the protein kinase A (PKA) pathway activators forskolin and dibutyryl cAMP and the PKA pathway inhibitor H-89, respectively, increased or repressed human osteocalcin and bone sialoprotein promoter activities. (c) Electrophoretic mobility shift assay showed that conditioned medium-mediated stimulation of human osteocalcin and bone sialoprotein promoter activities occurs through increased interaction between CRE and CRE-binding protein. (d) Conditioned medium was found to induce human osteocalcin and bone sialoprotein promoter activities via increased CRE/CRE-binding protein interaction in a cell background-dependent manner, with marked stimulation in selected prostate cancer but not bone stromal cells. Collectively, these results suggest that osteocalcin and bone sialoprotein expression is coordinated and regulated through cAMP-dependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.

Demineralized dentin matrix composite collagen material for bone tissue regeneration.

PubMed

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.

Development of Short Term Immunotoxicological Assays for Prediction of Chronic Toxicological Responses Induced by Environmental Chemicals

DTIC Science & Technology

1986-11-25

Time 16 El E 4. IUdR Incorporation into Sp*,een Cells as .Function of Preincubationr :ime.II....... ... ...... ........ I...... I........ I...trichloroethylene B) Bone marrow parameters 1) Bone marrow cell number 3) Bone marrow stem cell number (CFU-GM) Y . ,.= z , 0 .2 Cc-C 02 @13 .~ LL 0 CL uj 0...lipopolysac- • .4 -C.- * 50 Figure 21 S pleen Cell Number In Cultures 8- Exposed to S3. Mixes .............. 6 2. 0 60 180 Exposure Time (Minutes) El No S9

OSTEOCLAST-INDUCED FOXP3+ CD8 T-CELLS LIMIT BONE LOSS IN MICE

PubMed Central

Buchwald, Zachary S.; Kiesel, Jennifer R.; Yang, Chang; DiPaolo, Richard; Novack, Deborah V.; Aurora, Rajeev

2014-01-01

Osteoimmunology is the crosstalk between the skeletal and immune system. We have previously shown in vitro that osteoclasts (OC) crosspresent antigens to induce FoxP3 in CD8 T-cells (OCiTcREG), which then suppress osteoclast activity. Here we assessed the ability of OC-iTcREG to limit bone resorption in vivo. Mice lacking CD8 T-cells lose more bone in response to RANKL (Tnfsf11) administration. Using adoptive transfer experiments we demonstrate that FoxP3+ CD8 T-cells limit bone loss by RANKL administration. In ovariectomized mice, a murine model of postmenopausal osteoporosis, OC-iTcREG limited bone loss and increased bone density as assessed by serum markers, micro computed tomography (μCT) and histomorphometry. Indeed, OC-iTcREG—treated ovariectomized mice had decreased levels of effector T-cells in the bone marrow compared to untreated mice, and increased bone formation rates relative to bisphosphonate-treated mice. Our results provide the first in vivo evidence that OC-iTcREG have anti-resorptive activity and repress the immune system, thus extending the purview of osteoimmunology. PMID:23756229

In Vitro Impact of Conditioned Medium From Demineralized Freeze-Dried Bone on Human Umbilical Endothelial Cells.

PubMed

Harnik, Branko; Miron, Richard J; Buser, Daniel; Gruber, Reinhard

2017-03-01

Angiogenesis is essential for the consolidation of bone allografts. The underlying molecular mechanism, however, remains unclear. Soluble factors released from demineralized freeze-dried bone target mesenchymal cells; however, their effect on endothelial cells has not been investigated so far. The aim of the present study was therefore to examine the effect of conditioned medium from demineralized freeze-dried bone on human umbilical endothelial cells in vitro. Conditioned medium was first prepared from demineralized freeze-dried bone following 24 hours incubation at room temperature to produce demineralized bone conditioned media. Thereafter, conditioned medium was used to stimulate human umbilical vein endothelial cells in vitro by determining the cell response based on viability, proliferation, expression of apoptotic genes, a Boyden chamber to determine cell migration, and the formation of branches. The authors report here that conditioned medium decreased viability and proliferation of endothelial cells. Neither of the apoptotic marker genes was significantly altered when endothelial cells were exposed to conditioned medium. The Boyden chamber revealed that endothelial cells migrate toward conditioned medium. Moreover, conditioned medium moderately stimulated the formation of branches. These findings support the concept that conditioned medium from demineralized freeze-dried bone targets endothelial cells by decreasing their proliferation and enhancing their motility under these in vitro conditions.

DC-STAMP Is an Osteoclast Fusogen Engaged in Periodontal Bone Resorption

PubMed Central

Wisitrasameewong, W.; Kajiya, M.; Movila, A.; Rittling, S.; Ishii, T.; Suzuki, M.; Matsuda, S.; Mazda, Y.; Torruella, M.R.; Azuma, M.M.; Egashira, K.; Freire, M.O.; Sasaki, H.; Wang, C.Y.; Han, X.; Taubman, M.A.; Kawai, T.

2017-01-01

Dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in the induction of osteoclast (OC) cell fusion, as well as DC-mediated immune regulation. While DC-STAMP gene expression is upregulated in the gingival tissue with periodontitis, its pathophysiological roles in periodontitis remain unclear. To evaluate the effects of DC-STAMP in periodontitis, anti-DC-STAMP–monoclonal antibody (mAb) was tested in a mouse model of ligature-induced periodontitis (n = 6–7/group) where Pasteurella pneumotropica (Pp)-reactive immune response activated T cells to produce receptor activator of nuclear factor kappa-B ligand (RANKL), which, in turn, promotes the periodontal bone loss via upregulation of osteoclastogenesis. DC-STAMP was expressed on the cell surface of mature multinuclear OCs, as well as immature mononuclear OCs, in primary cultures of RANKL-stimulated bone marrow cells. Anti-DC-STAMP-mAb suppressed the emergence of large, but not small, multinuclear OCs, suggesting that DC-STAMP is engaged in the late stage of cell fusion. Anti-DC-STAMP-mAb also inhibited pit formation caused by RANKL-stimulated bone marrow cells. Attachment of ligature to a second maxillary molar induced DC-STAMP messenger RNA and protein, along with elevated tartrate-resistant acid phosphatase–positive (TRAP+) OCs and alveolar bone loss. As we expected, systemic administration of anti-DC-STAMP-mAb downregulated the ligature-induced alveolar bone loss. Importantly, local injection of anti-DC-STAMP-mAb also suppressed alveolar bone loss and reduced the total number of multinucleated TRAP+ cells in mice that received ligature attachment. Attachment of ligature induced significantly elevated tumor necrosis factor–α, interleukin-1β, and RANKL in the gingival tissue compared with the control site without ligature (P < 0.05), which was unaffected by local injection with either anti-DC-STAMP-mAb or control-mAb. Neither in vivo anti-Pp IgG antibody nor in vitro anti-Pp T-cell response and resultant production of RANKL was affected by anti-DC-STAMP-mAb. This study illustrated the roles of DC-STAMP in promoting local OC cell fusion without affecting adaptive immune responses to oral bacteria. Therefore, it is plausible that a novel therapeutic regimen targeting DC-STAMP could suppress periodontal bone loss. PMID:28199142

Small Molecule Protection of Bone Marrow Hematopoietic Stem Cells

DTIC Science & Technology

2017-12-01

using isogenic (mutant/complemented) human cell line pairs from patients with Fanconi anemia (FA), a heritable human bone marrow failure (BMF) syndrome ...small molecules could be therapeutically useful in reducing the risk of BMF in diseases such as Fanconi anemia, and perhaps after radiation exposure...damage-repair, DNA damage response, Fanconi anemia and associated bone marrow failure syndromes and environmental and molecular toxicology will all be

The osteogenic response of undifferentiated human mesenchymal stem cells (hMSCs) to mechanical strain is inversely related to body mass index of the donor.

PubMed

Friedl, Gerald; Windhager, Reinhard; Schmidt, Helena; Aigner, Reingard

2009-08-01

While the importance of physical factors in the maintenance and regeneration of bone tissue has been recognized for many years and the mechano-sensitivity of bone cells is well established, there is increasing evidence that body fat constitutes an independent risk factor for complications in bone fracture healing and aseptic loosening of implants. Although mechanical causes have been widely suggested, we hypothesized that the osteogenic mechano-response of human mesenchymal stem cells (hMSCs) may be altered in obese patients. We determined the phenotypic and genotypic response of undifferentiated hMSCs of 10 donors to cyclic tensile strain (CTS) under controlled in vitro conditions and analyzed the potential relationship relevant to the donor's anthropomorphometric and biochemical parameters related to donor's fat and bone metabolism. The osteogenic marker genes were all statistically significantly upregulated by CTS, which was accompanied by a significant increase in cell-based ALP activity. Linear correlation analysis revealed that there was a significant correlation between phenotypic CTS response and the body mass index of the donor (r = -0.91, p < 0.001) and phenotypic CTS response was also significantly related to leptin levels (r = -0.68) and estradiol levels (r = 0.67) within the bone marrow microenvironment of the donor. Such an upstream imprinting process mediated by factors tightly related to the donor's fat metabolism, which hampers the mechanosensitivity of hMSCs in obese patients, may be of pathogenetic relevance for the complications associated with obesity that are seen in orthopedic surgery.

Bone marrow mesenchymal stem cells from patients with aplastic anemia maintain functional and immune properties and do not contribute to the pathogenesis of the disease.

PubMed

Bueno, Clara; Roldan, Mar; Anguita, Eduardo; Romero-Moya, Damia; Martín-Antonio, Beatriz; Rosu-Myles, Michael; del Cañizo, Consuelo; Campos, Francisco; García, Regina; Gómez-Casares, Maite; Fuster, Jose Luis; Jurado, Manuel; Delgado, Mario; Menendez, Pablo

2014-07-01

Aplastic anemia is a life-threatening bone marrow failure disorder characterized by peripheral pancytopenia and marrow hypoplasia. The majority of cases of aplastic anemia remain idiopathic, although hematopoietic stem cell deficiency and impaired immune responses are hallmarks underlying the bone marrow failure in this condition. Mesenchymal stem/stromal cells constitute an essential component of the bone marrow hematopoietic microenvironment because of their immunomodulatory properties and their ability to support hematopoiesis, and they have been involved in the pathogenesis of several hematologic malignancies. We investigated whether bone marrow mesenchymal stem cells contribute, directly or indirectly, to the pathogenesis of aplastic anemia. We found that mesenchymal stem cell cultures can be established from the bone marrow of aplastic anemia patients and display the same phenotype and differentiation potential as their counterparts from normal bone marrow. Mesenchymal stem cells from aplastic anemia patients support the in vitro homeostasis and the in vivo repopulating function of CD34(+) cells, and maintain their immunosuppressive and anti-inflammatory properties. These data demonstrate that bone marrow mesenchymal stem cells from patients with aplastic anemia do not have impaired functional and immunological properties, suggesting that they do not contribute to the pathogenesis of the disease. Copyright© Ferrata Storti Foundation.

Th1 biased response to a novel Porphyromonas gingivalis protein aggravates bone resorption caused by this oral pathogen

PubMed Central

Leshem, Onir; Kashino, Suely S.; Gonçalves, Reginaldo B.; Suzuki, Noriyuki; Onodera, Masao; Fujimura, Akira; Sasaki, Hajime; Stashenko, Philip; Campos-Neto, Antonio

2013-01-01

In previous studies we showed that biasing the immune response to Porphyromonas gingivalis antigens to the Th1 phenotype increases inflammatory bone resorption caused by this organism. Using a T cell screening strategy we identified eight P. gingivalis genes coding for proteins that appear to be involved in T-helper cell responses. In the present study we characterized the protein, encoded by PG_1841 gene and evaluated its relevance in the in bone resorption caused by P. gingivalis because subcutaneous infection of mice with this organism resulted in the induction of Th1 biased response to the recombinant PG1841 antigen molecule. Using an immunization regime that strongly biases toward the Th1 phenotype followed by challenge with P. gingivalis in dental pulp tissue, we demonstrate that mice pre-immunized with rPG1841 developed severe bone loss compared with control immunized mice. Pre-immunization of mice with the antigen using a Th2 biasing regime resulted in no exacerbation of the disease. These results support the notion that selected antigens of P. gingivalis are involved in a biased Th1 host response that leads to the severe bone loss caused by this oral pathogen. PMID:18457976

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group.

PubMed

Burchill, Susan A; Beiske, Klaus; Shimada, Hiroyuki; Ambros, Peter F; Seeger, Robert; Tytgat, Godelieve A M; Brock, Penelope R; Haber, Michelle; Park, Julie R; Berthold, Frank

2017-04-01

The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design. Cancer 2017;123:1095-1105. © 2016 American Cancer Society. © 2016 American Cancer Society.

Recurrence of chronic active Epstein-Barr virus infection from donor cells after achieving complete response through allogeneic bone marrow transplantation.

PubMed

Arai, Ayako; Imadome, Ken-ichi; Wang, Ludan; Wu, Nan; Kurosu, Tetsuya; Wake, Atsushi; Yamamoto, Hisashi; Ota, Yasunori; Harigai, Masayoshi; Fujiwara, Shigeyoshi; Miura, Osamu

2012-01-01

We report the case of a 35-year-old woman with chronic active Epstein-Barr virus (EBV) infection (CAEBV). She underwent allogeneic bone marrow transplantation (BMT) from an unrelated male donor and achieved a complete response. However, her CAEBV relapsed one year after BMT. EBV-infected cells proliferated clonally and revealed a 46XY karyotype. In addition, the infecting EBV strain differed from that detected before BMT. These findings indicated that her disease had developed from donor cells. This is the first report of donor cell-derived CAEBV that recurred after transplantation, suggesting that host factors may be responsible for the development of this disease.

Subchondral pre-solidified chitosan/blood implants elicit reproducible early osteochondral wound-repair responses including neutrophil and stromal cell chemotaxis, bone resorption and repair, enhanced repair tissue integration and delayed matrix deposition

PubMed Central

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

* Calvarial Bone Regeneration Is Enhanced by Sequential Delivery of FGF-2 and BMP-2 from Layer-by-Layer Coatings with a Biomimetic Calcium Phosphate Barrier Layer.

PubMed

Gronowicz, Gloria; Jacobs, Emily; Peng, Tao; Zhu, Li; Hurley, Marja; Kuhn, Liisa T

2017-12-01

A drug delivery coating for synthetic bone grafts has been developed to provide sequential delivery of multiple osteoinductive factors to better mimic aspects of the natural regenerative process. The coating is composed of a biomimetic calcium phosphate (bCaP) layer that is applied to a synthetic bone graft and then covered with a poly-l-Lysine/poly-l-Glutamic acid polyelectrolyte multilayer (PEM) film. Bone morphogenetic protein-2 (BMP-2) was applied before the coating process directly on the synthetic bone graft and then, bCaP-PEM was deposited followed by adsorption of fibroblast growth factor-2 (FGF-2) into the PEM layer. Cells access the FGF-2 immediately, while the bCaP-PEM temporally delays the cell access to BMP-2. In vitro studies with cells derived from mouse calvarial bones demonstrated that Sca-1 and CD-166 positive osteoblast progenitor cells proliferated in response to media dosing with FGF-2. Coated scaffolds with BMP-2 and FGF-2 were implanted in mouse calvarial bone defects and harvested at 1 and 3 weeks. After 1 week in vivo, proliferation of cells, including Sca-1+ progenitors, was observed with low dose FGF-2 and BMP-2 compared to BMP-2 alone, indicating that in vivo delivery of FGF-2 activated a similar population of cells as shown by in vitro testing. At 3 weeks, FGF-2 and BMP-2 delivery increased bone formation more than BMP-2 alone, particularly in the center of the defect, confirming that the proliferation of the Sca-1 positive osteoprogenitors by FGF-2 was associated with increased bone healing. Areas of bone mineralization were positive for double fluorochrome labeling of calcium and alkaline phosphatase staining of osteoblasts, along with increased TRAP+ osteoclasts, demonstrating active bone formation distinct from the bone-like collagen/hydroxyapatite scaffold. In conclusion, the addition of a bCaP layer to PEM delayed access to BMP-2 and allowed the FGF-2 stimulated progenitors to populate the scaffold before differentiating in response to BMP-2, leading to improved bone defect healing.

Sphene ceramics for orthopedic coating applications: an in vitro and in vivo study.

PubMed

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.

Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice.

PubMed

Campbell, J Preston; Karolak, Matthew R; Ma, Yun; Perrien, Daniel S; Masood-Campbell, S Kathryn; Penner, Niki L; Munoz, Steve A; Zijlstra, Andries; Yang, Xiangli; Sterling, Julie A; Elefteriou, Florent

2012-07-01

Bone and lung metastases are responsible for the majority of deaths in patients with breast cancer. Following treatment of the primary cancer, emotional and psychosocial factors within this population precipitate time to recurrence and death, however the underlying mechanism(s) remain unclear. Using a mouse model of bone metastasis, we provide experimental evidence that activation of the sympathetic nervous system, which is one of many pathophysiological consequences of severe stress and depression, promotes MDA-231 breast cancer cell colonization of bone via a neurohormonal effect on the host bone marrow stroma. We demonstrate that induction of RANKL expression in bone marrow osteoblasts, following β2AR stimulation, increases the migration of metastatic MDA-231 cells in vitro, independently of SDF1-CXCR4 signaling. We also show that the stimulatory effect of endogenous (chronic stress) or pharmacologic sympathetic activation on breast cancer bone metastasis in vivo can be blocked with the β-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells. These findings indicate that RANKL promotes breast cancer cell metastasis to bone via its pro-migratory effect on breast cancer cells, independently of its effect on bone turnover. The emerging clinical implication, supported by recent epidemiological studies, is that βAR-blockers and drugs interfering with RANKL signaling, such as Denosumab, could increase patient survival if used as adjuvant therapy to inhibit both the early colonization of bone by metastatic breast cancer cells and the initiation of the "vicious cycle" of bone destruction induced by these cells.

The Effect of Spaceflight on Bone Cell Cultures

NASA Technical Reports Server (NTRS)

Landis, William J.

1999-01-01

Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural methods (conventional and high voltage electron microscopy, inununocytochemistry, stereomicroscopy, and 3D image reconstruction). The studies have provided new knowledge of aspects of bone cell development and structural regulation, extracellular matrix assembly, and mineralization during spaceflight and under normal gravity. The information has contributed to insights into the means in general by which cells respond and adapt to different conditions of gravity (loading). The data may as well have suggested an underlying basis for the observed loss of bone by vertebrates, including man, in microgravity; and these scientific results may have implications for understanding bone loss following fracture healing and extended periods of inactivity such as during long-term bedrest.

TOPICAL REVIEW: Stem cell technology using bioceramics: hard tissue regeneration towards clinical application

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.

Role of bone marrow-derived CD11c+ dendritic cells in systolic overload-induced left ventricular inflammation, fibrosis and hypertrophy.

PubMed

Wang, Huan; Kwak, Dongmin; Fassett, John; Liu, Xiaohong; Yao, Wu; Weng, Xinyu; Xu, Xin; Xu, Yawei; Bache, Robert J; Mueller, Daniel L; Chen, Yingjie

2017-05-01

Inflammatory responses play an important role in the development of left ventricular (LV) hypertrophy and dysfunction. Recent studies demonstrated that increased T-cell infiltration and T-cell activation contribute to LV hypertrophy and dysfunction. Dendritic cells (DCs) are professional antigen-presenting cells that orchestrate immune responses, especially by modulating T-cell function. In this study, we investigated the role of bone marrow-derived CD11c + DCs in transverse aortic constriction (TAC)-induced LV fibrosis and hypertrophy in mice. We observed that TAC increased the number of CD11c + cells and the percentage of CD11c + MHCII + (major histocompatibility complex class II molecule positive) DCs in the LV, spleen and peripheral blood in mice. Using bone marrow chimeras and an inducible CD11c + DC ablation model, we found that depletion of bone marrow-derived CD11c + DCs significantly attenuated LV fibrosis and hypertrophy in mice exposed to 24 weeks of moderate TAC. CD11c + DC ablation significantly reduced TAC-induced myocardial inflammation as indicated by reduced myocardial CD45 + cells, CD11b + cells, CD8 + T cells and activated effector CD8 + CD44 + T cells in LV tissues. Moreover, pulsing of autologous DCs with LV homogenates from TAC mice promoted T-cell proliferation. These data indicate that bone marrow-derived CD11c + DCs play a maladaptive role in hemodynamic overload-induced cardiac inflammation, hypertrophy and fibrosis through the presentation of cardiac self-antigens to T cells.

Behaviour of human mesenchymal stem cells on chemically synthesized HA-PCL scaffolds for hard tissue regeneration.

PubMed

D'Antò, Vincenzo; Raucci, Maria Grazia; Guarino, Vincenzo; Martina, Stefano; Valletta, Rosa; Ambrosio, Luigi

2016-02-01

Our goal was to characterize the response of human mesenchymal stem cells (hMSCs) to a novel composite scaffold for bone tissue engineering. The hydroxyapatite-polycaprolactone (HA-PCL) composite scaffolds were prepared by a sol-gel method at room temperature and the scaffold morphology was investigated by scanning electron microscopy (SEM)/energy-dispersive spectroscopy (EDS) to validate the synthesis process. The response of two different lines of hMSCs, bone-marrow-derived human mesenchymal stem cells (BMSCs) and dental pulp stem cells (DPSCs) in terms of cell proliferation and differentiation into the osteoblastic phenotype, was evaluated using Alamar blue assay, SEM, histology and alkaline phosphatase activity. Our results indicate that tissue engineering by means of composite HA-PCL scaffolds may represent a new therapeutic strategy to repair craniofacial bone defects. Copyright © 2013 John Wiley & Sons, Ltd.

The effect of cationically-modified phosphorylcholine polymers on human osteoblasts in vitro and their effect on bone formation in vivo.

PubMed

Lawton, Jonathan M; Habib, Mariam; Ma, Bingkui; Brooks, Roger A; Best, Serena M; Lewis, Andrew L; Rushton, Neil; Bonfield, William

2017-08-17

The effect of introducing cationic charge into phosphorylcholine (PC)-based polymers has been investigated in this study with a view to using these materials as coatings to improve bone formation and osseointegration at the bone-implant interface. PC-based polymers, which have been used in a variety of medical devices to improve biocompatibility, are associated with low protein adsorption resulting in reduced complement activation, inflammatory response and cell adhesion. However, in some applications, such as orthopaedics, good integration between the implant and bone is needed to allow the distribution of loading stresses and a bioactive response is required. It has previously been shown that the incorporation of cationic charge into PC-based polymers may increase protein adsorption that stimulates subsequent cell adhesion. In this paper, the effect of cationic charge in PC-based polymers on human osteoblasts (HObs) in vitro and the effect of these polymers on bone formation in the rat tibia was assessed. Increasing PC positive surface charge increased HOb cell adhesion and stimulated increased cell differentiation and the production of calcium phosphate deposits. However, when implanted in bone these materials were at best biotolerant, stimulating the production of fibrous tissue and areas of loosely associated matrix (LAM) around the implant. Their development, as formulated in this study, as bone interfacing implant coatings is therefore not warranted.

Effects of Amplitude and Frequency of Mechanical Vibration Stimulation on Cultured Osteoblasts

NASA Astrophysics Data System (ADS)

Shikata, Tetsuo; Shiraishi, Toshihiko; Morishita, Shin; Takeuchi, Ryohei; Saito, Tomoyuki

Mechanical stimulation to bones affects bone formation such as decrease of bone mass of astronauts under zero gravity, walking rehabilitation to bone fracture and fracture repair with ultrasound devices. Bone cells have been reported to sense and response to mechanical stimulation at cellular level morphologically and metabolically. In the view of mechanical vibrations, bone cells are deformed according to mechanical stimulation and their mechanical characteristics. In this study, sinusoidal inertia force was applied to cultured osteoblasts, which are a kind of bone cells, and effects of frequency and acceleration amplitude of mechanical vibration on the cells were investigated in respect of the cell proliferation, bone matrix generation and alkaline phosphatase (ALP) gene expression. The results to be obtained are as follows. The significant difference of cell density and bone mass generation between the non-vibrating and vibrating groups is found. ALP gene expression shows a peak to frequency at 50 Hz and the value of it is approximately 4.5 times as high as that of the non-vibrating group in the case of the acceleration amplitude of 0.5 G. ALP gene expression at 0.5 G is significantly larger than at 0, 0.125 or 0.25 G in the case of the frequency of 50 Hz.

The skeletal vascular system - Breathing life into bone tissue.

PubMed

Stegen, Steve; Carmeliet, Geert

2017-08-26

During bone development, homeostasis and repair, a dense vascular system provides oxygen and nutrients to highly anabolic skeletal cells. Characteristic for the vascular system in bone is the serial organization of two capillary systems, each typified by specific morphological and physiological features. Especially the arterial capillaries mediate the growth of the bone vascular system, serve as a niche for skeletal and hematopoietic progenitors and couple angiogenesis to osteogenesis. Endothelial cells and osteoprogenitor cells interact not only physically, but also communicate to each other by secretion of growth factors. A vital angiogenic growth factor is vascular endothelial growth factor and its expression in skeletal cells is controlled by osteogenic transcription factors and hypoxia signaling, whereas the secretion of angiocrine factors by endothelial cells is regulated by Notch signaling, blood flow and possibly hypoxia. Bone loss and impaired fracture repair are often associated with reduced and disorganized blood vessel network and therapeutic targeting of the angiogenic response may contribute to enhanced bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

The bone marrow is not only a primary lymphoid organ: The critical role for T lymphocyte migration and housing of long-term memory plasma cells.

PubMed

Pabst, Reinhard

2018-05-22

In immunology and anatomy textbooks the bone marrow is described as a typical "primary lymphoid organ" producing lymphoid cells independent of antigens. The hematopoietic bone marrow is largely age-dependent organ with great anatomical and functional differences among various species. There are estimates that about 12% of all lymphoid cells in the human body are found in the bone marrow at any given time (2% in the peripheral blood). Enormous numbers of T lymphocytes migrate to the bone marrow and partly return later to the blood. Many of these lymphocytes are memory CD4 + and CD8 + T cells. A few days after immunization a wave of plasma cells and their precursors migrate to the bone marrow where they lose their migratory response to CXCL-12 and CXCL9. There is a relative enrichment of CD19 + B cells in the bone marrow outnumbering those in the blood and secondary lymphoid organs. This is not due to local production. The proliferation and migration kinetics of these lymphoid cells in the bone marrow have to be studied in more detail as this is of major clinical relevance. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Parathyroid Hormone Directs Bone Marrow Mesenchymal Cell Fate.

PubMed

Fan, Yi; Hanai, Jun-Ichi; Le, Phuong T; Bi, Ruiye; Maridas, David; DeMambro, Victoria; Figueroa, Carolina A; Kir, Serkan; Zhou, Xuedong; Mannstadt, Michael; Baron, Roland; Bronson, Roderick T; Horowitz, Mark C; Wu, Joy Y; Bilezikian, John P; Dempster, David W; Rosen, Clifford J; Lanske, Beate

2017-03-07

Intermittent PTH administration builds bone mass and prevents fractures, but its mechanism of action is unclear. We genetically deleted the PTH/PTHrP receptor (PTH1R) in mesenchymal stem cells using Prx1Cre and found low bone formation, increased bone resorption, and high bone marrow adipose tissue (BMAT). Bone marrow adipocytes traced to Prx1 and expressed classic adipogenic markers and high receptor activator of nuclear factor kappa B ligand (Rankl) expression. RANKL levels were also elevated in bone marrow supernatant and serum, but undetectable in other adipose depots. By cell sorting, Pref1 + RANKL + marrow progenitors were twice as great in mutant versus control marrow. Intermittent PTH administration to control mice reduced BMAT significantly. A similar finding was noted in male osteoporotic patients. Thus, marrow adipocytes exhibit osteogenic and adipogenic characteristics, are uniquely responsive to PTH, and secrete RANKL. These studies reveal an important mechanism for PTH's therapeutic action through its ability to direct mesenchymal cell fate. Copyright © 2017 Elsevier Inc. All rights reserved.

Conception on the cell mechanisms of bone tissue loss under spase flight conditions

NASA Astrophysics Data System (ADS)

Rodionova, Natalia; Oganov, Victor; Kabitskaya, Olga

Basing on the analysis of available literature and the results of our own electron microscopic and radioautographic researches the data are presented about the morpho-functional peculiarities and succession of cellular interactions in adaptive remodeling of bone structures under normal conditions and after exposure of animals (rats, monkeys, mice) to microgravity (SLS-2, Bion-11, BionM-1). The probable cellular mechanisms of the development of osteopenia and osteoporosis are considered. Our conception on remodeling proposes the following sequence in the development of cellular interactions after decrease of the mechanical loading: a primary response of osteocytes (mechanosensory cells) to the mechanical stimulus; osteocytic remodeling (osteolysis); transmission of the mechanical signals through a system of canals and processes to functionally active osteoblasts and surface osteocytes as well as to the bone-marrow stromal cells and to those lying on bone surfaces. As a response to the mechanical stimulus (microgravity) the system of stromal cell-preosteoblast-osteoblast shows a delay in proliferation, differentiation and specific functioning of the osteogenetic cells, some of the osteoblasts undergo apoptosis. Then the osteoclastic reaction occurs (attraction of monocytes and formation of osteoclasts and bone matrix resorption in the loci of apoptosis of osteoblasts and osteocytes). The macrophagal reaction is followed by osteoblastogenesis, which appears to be a rehabilitating process. However, during prolonged absence of mechanical stimuli (microgravity, long-time immobilization) the adaptive activization of osteoblastogenesis doesn’t occur (as it is the case during the physiological remodeling of bone tissue) or it occurs to a smaller degree. The loading deficit leads to an adaptive differentiation of stromal cells to fibroblastic cells and adipocytes in these remodeling loci. These cell reactions are considered as adaptive-compensatory, but they don’t result in rehabilitation of the resorbed bone tissue. This sequence of events is considered as a mechanism of bone tissue loss which underlies the development of osteopenia and osteoporosis under the mechanical loading deficit.

The Application of Bone Marrow Transplantation to the Treatment of Genetic Diseases

NASA Astrophysics Data System (ADS)

Parkman, Robertson

1986-06-01

Genetic diseases can be treated by transplantation of either normal allogeneic bone marrow or, potentially, autologous bone marrow into which the normal gene has been inserted in vitro (gene therapy). Histocompatible allogeneic bone marrow transplantation is used for the treatment of genetic diseases whose clinical expression is restricted to lymphoid or hematopoietic cells. The therapeutic role of bone marrow transplantation in the treatment of generalized genetic diseases, especially those affecting the central nervous system, is under investigation. The response of a generalized genetic disease to allogeneic bone marrow transplantation may be predicted by experiments in vitro. Gene therapy can be used only when the gene responsible for the disease has been characterized. Success of gene therapy for a specific genetic disease may be predicted by its clinical response to allogeneic bone marrow transplantation.

Perivascular Stem Cells: A Prospectively Purified Mesenchymal Stem Cell Population for Bone Tissue Engineering

PubMed Central

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

Improved osteogenesis and angiogenesis of magnesium-doped calcium phosphate cement via macrophage immunomodulation.

PubMed

Wang, Meng; Yu, Yuanman; Dai, Kai; Ma, Zhengyu; Liu, Yang; Wang, Jing; Liu, Changsheng

2016-10-18

Immune responses are vital for bone regeneration and play an essential role in the fate of biomaterials after implantation. As a kind of plastic cell, macrophages are central regulators of the immune response during the infection and wound healing process including osteogenesis and angiogenesis. Magnesium-calcium phosphate cement (MCPC) has been reported as a promising candidate for bone repair with promoted osteogenesis both in vitro and in vivo. However, relatively little is known about the effects of MCPC on immune response and the following outcome. In this study, we investigated the interactions between macrophages and MCPC. Here we found that the pro-inflammatory cytokines including TNF-α and IL-6 were less expressed and the bone repair related cytokine of TGF-β1 was up-regulated by macrophages in MCPC extract. Furthermore, the enhanced osteogenic capacity of BMSCs and angiogenic potential of HUVECs were acquired in vitro by the MCPC-induced immune microenvironment. These findings suggest that MCPC is able to facilitate bone healing by endowing favorable osteoimmunomodulatory properties and influencing crosstalk behavior between immune cells and osteogenesis-related cells.

Alkaline biodegradable implants for osteoporotic bone defects--importance of microenvironment pH.

PubMed

Liu, W; Wang, T; Yang, C; Darvell, B W; Wu, J; Lin, K; Chang, J; Pan, H; Lu, W W

2016-01-01

Change of microenvironment pH by biodegradable implants may ameliorate unbalanced osteoporotic bone remodeling. The present work demonstrated that a weak alkaline condition stimulated osteoblasts differentiation while suppressed osteoclast generation. In vivo, implants with an alkaline microenvironment pH (monitored by a pH microelectrode) exhibited a promising healing effect for the repair of osteoporotic bone defects. Under osteoporotic conditions, the response of the bone microenvironment to an endosseous implant is significantly impaired, and this substantially increases the risk of fracture, non-union and aseptic implant loosening. Acid-base equilibrium is an important factor influencing bone cell behaviour. The present purpose was to study the effect of a series of alkaline biodegradable implant materials on regeneration of osteoporotic bone defect, monitoring the microenvironment pH (μe-pH) over time. The proliferation and differentiation potential of osteoporotic rat bone marrow stromal cells and RAW 264.7 cells were examined under various pH conditions. Ovariectomized rat bone defects were filled with specific biodegradable materials, and μe-pH was measured by pH microelectrode. New osteoid and tartrate-resistant acid phosphatase-positive osteoclast-like cells were examined by Goldner's trichrome and TRAP staining, respectively. The intermediate layer between implants and new bone were studied using energy-dispersive X-ray spectroscopy (EDX) linear scanning. In vitro, weak alkaline conditions stimulated osteoporotic rat bone marrow stromal cells (oBMSC) differentiation, while inhibiting the formation of osteoclasts. In vivo, μe-pH differs from that of the homogeneous peripheral blood and exhibits variations over time particular to each material. Higher initial μe-pH was associated with more new bone formation, late response of TRAP-positive osteoclast-like cells and the development of an intermediate 'apatitic' layer in vivo. EDX suggested that residual material may influence μe-pH even 9 weeks post-surgery. The pH microelectrode is suitable for in vivo μe-pH detection. Alkaline biodegradable materials generate an in vivo microenvironmental pH which is higher than the normal physiological value and show promising healing effects in the context of osteoporotic bone defects.

Bone marrow produces sufficient alloreactive natural killer (NK) cells in vivo to cure mice from subcutaneously and intravascularly injected 4T1 breast cancer.

PubMed

van Gelder, Michel; Vanclée, Ariane; van Elssen, Catharina H M J; Hupperets, Pierre; Wieten, Lotte; Bos, Gerard M

2017-02-01

Administration of 5 million alloreactive natural killer (NK) cells after low-dose chemo-irradiation cured mice of 4T1 breast cancer, supposedly dose dependent. We now explored the efficacy of bone marrow as alternative in vivo source of NK cells for anti-breast cancer treatment, as methods for in vitro clinical scale NK cell expansion are still in developmental phases. Progression-free survival (PFS) after treatment with different doses of spleen-derived alloreactive NK cells to 4T1-bearing Balb/c mice was measured to determine a dose-response relation. The potential of bone marrow as source of alloreactive NK cells was explored using MHC-mismatched mice as recipients of 4T1. Chemo-irradiation consisted of 2× 2 Gy total body irradiation and 200 mg/kg cyclophosphamide. Antibody-mediated in vivo NK cell depletion was applied to demonstrate the NK cell's role. Administration of 2.5 instead of 5 million alloreactive NK cells significantly reduced PFS, evidencing dose responsiveness. Compared to MHC-matched receivers of subcutaneous 4T1, fewer MHC-mismatched mice developed tumors, which was due to NK cell alloreactivity because in vivo NK cell depletion facilitated tumor growth. Application of low-dose chemo-irradiation increased plasma levels of NK cell-activating cytokines, NK cell activity and enhanced NK cell-dependent elimination of subcutaneous tumors. Intravenously injected 4T1 was eliminated by alloreactive NK cells in MHC-mismatched recipients without the need for chemo-irradiation. Bone marrow is a suitable source of sufficient alloreactive NK cells for the cure of 4T1 breast cancer. These results prompt clinical exploration of bone marrow transplantation from NK-alloreactive MHC-mismatched donors in patients with metastasized breast cancer.

Matrix Metalloproteinases in Bone Resorption, Remodeling, and Repair.

PubMed

Paiva, Katiucia B S; Granjeiro, José M

2017-01-01

Matrix metalloproteinases (MMPs) are the major protease family responsible for the cleavage of the matrisome (global composition of the extracellular matrix (ECM) proteome) and proteins unrelated to the ECM, generating bioactive molecules. These proteins drive ECM remodeling, in association with tissue-specific and cell-anchored inhibitors (TIMPs and RECK, respectively). In the bone, the ECM mediates cell adhesion, mechanotransduction, nucleation of mineralization, and the immobilization of growth factors to protect them from damage or degradation. Since the first description of an MMP in bone tissue, many other MMPs have been identified, as well as their inhibitors. Numerous functions have been assigned to these proteins, including osteoblast/osteocyte differentiation, bone formation, solubilization of the osteoid during bone resorption, osteoclast recruitment and migration, and as a coupling factor in bone remodeling under physiological conditions. In turn, a number of pathologies, associated with imbalanced bone remodeling, arise mainly from MMP overexpression and abnormalities of the ECM, leading to bone osteolysis or bone formation. In this review, we will discuss the functions of MMPs and their inhibitors in bone cells, during bone remodeling, pathological bone resorption (osteoporosis and bone metastasis), bone repair/regeneration, and emergent roles in bone bioengineering. © 2017 Elsevier Inc. All rights reserved.

TRAP-Positive Multinucleated Giant Cells Are Foreign Body Giant Cells Rather Than Osteoclasts: Results From a Split-Mouth Study in Humans.

PubMed

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.

Nanocrystallinity effects on osteoblast and osteoclast response to silicon substituted hydroxyapatite.

PubMed

Casarrubios, Laura; Matesanz, María Concepción; Sánchez-Salcedo, Sandra; Arcos, Daniel; Vallet-Regí, María; Portolés, María Teresa

2016-11-15

Silicon substituted hydroxyapatites (SiHA) are highly crystalline bioceramics treated at high temperatures (about 1200°C) which have been approved for clinical use with spinal, orthopedic, periodontal, oral and craniomaxillofacial applications. The preparation of SiHA with lower temperature methods (about 700°C) provides nanocrystalline SiHA (nano-SiHA) with enhanced bioreactivity due to higher surface area and smaller crystal size. The aim of this study has been to know the nanocrystallinity effects on the response of both osteoblasts and osteoclasts (the two main cell types involved in bone remodelling) to silicon substituted hydroxyapatite. Saos-2 osteoblasts and osteoclast-like cells (differentiated from RAW-264.7 macrophages) have been cultured on the surface of nano-SiHA and SiHA disks and different cell parameters have been evaluated: cell adhesion, proliferation, viability, intracellular content of reactive oxygen species, cell cycle phases, apoptosis, cell morphology, osteoclast-like cell differentiation and resorptive activity. This comparative in vitro study evidences that nanocrystallinity of SiHA affects the cell/biomaterial interface inducing bone cell apoptosis by loss of cell anchorage (anoikis), delaying osteoclast-like cell differentiation and decreasing the resorptive activity of this cell type. These results suggest the potential use of nano-SiHA biomaterial for preventing bone resorption in treatment of osteoporotic bone. Copyright © 2016 Elsevier Inc. All rights reserved.

Response of SAOS-2 cells to simulated microgravity and effect of biocompatible sol-gel hybrid coatings

NASA Astrophysics Data System (ADS)

Catauro, M.; Bollino, F.; Papale, F.

2016-05-01

The health of astronauts, during space flight, is threatened by bone loss induced by microgravity, mainly attributed to an imbalance in the bone remodeling process. In the present work, the response to the microgravity of bone cells has been studied using the SAOS-2 cell line grown under the condition of weightlessness, simulated by means of a Random Positioning Machine (RPM). Cell viability after 72 h of rotation has been evaluated by means of WST-8 assay and compared to that of control cells. Although no significant difference between the two cell groups has been observed in terms of viability, F-actin staining showed that microgravity environment induces cell apoptosis and altered F-actin organization. To investigate the possibility of hindering the trend of the cells towards the death, after 72 h of rotation the cells have been seeded onto biocompatible ZrO2/PCL hybrid coatings, previously obtained using a sol-gel dip coating procedure. WST-8 assay, carried out after 24 h, showed that the materials are able to inhibit the pro-apoptotic effect of microgravity on cells.

Decreased heterotopic osteogenesis in vitamin-D-deficient, but normocalcemic guinea pigs

NASA Technical Reports Server (NTRS)

Dziedzic-Goclawska, A.; Toverud, S. U.; Kaminski, A.; Boass, A.; Yamauchi, M.

1992-01-01

The effect of vitamin D deficiency unhampered by hypocalcemia on de novo bone formation was studied in guinea pigs. Heterotopic induction of osteogenesis was evaluated 4 weeks after intramuscular transplantation of allogenic urinary bladder transitional epithelium from vitamin-D-repleted (+D) donors into +D and -D recipients. In -D recipients the frequency of osteogenesis and the amount of induced bone were significantly diminished; induced bone was less mature, scantly cellular woven bone poorly repopulated with bone marrow. No effect of vitamin D deficiency on orthotopic bone growth and on mineralization of orthotopic and heterotopically induced bone was observed. It is proposed that in addition to inducing factors (BMPs, growth factors) which may be responsible for transformation of mesenchymal cells to osteoprogenitor cells, normal concentrations of 1,25-(OH)2D3 may be required for proliferation and further differentiation of these cells into osteoblasts and for expression of genes engaged in extracellular matrix formation and maturation.

Mechanical Regulation of Signaling Pathways in Bone

PubMed Central

Thompson, William R.; Rubin, Clinton T.; Rubin, Janet

2012-01-01

A wide range of cell types depend on mechanically induced signals to enable appropriate physiological responses. The skeleton is particularly dependent on mechanical information to guide the resident cell population towards adaptation, maintenance and repair. Research at the organ, tissue, cell and molecular levels has improved our understanding of how the skeleton can recognize the functional environment, and how these challenges are translated into cellular information that can site-specifically alter phenotype. This review first considers those cells within the skeleton that are responsive to mechanical signals, including osteoblasts, osteoclasts, osteocytes and osteoprogenitors. This is discussed in light of a range of experimental approaches that can vary parameters such as strain, fluid shear stress, and pressure. The identity of mechanoreceptor candidates is approached, with consideration of integrins, pericellular tethers, focal adhesions, ion channels, cadherins, connexins, and the plasma membrane including caveolar and non-caveolar lipid rafts and their influence on integral signaling protein interactions. Several mechanically regulated intracellular signaling cascades are detailed including activation of kinases (Akt, MAPK, FAK), β-catenin, GTPases, and calcium signaling events. While the interaction of bone cells with their mechanical environment is complex, an understanding of mechanical regulation of bone signaling is crucial to understanding bone physiology, the etiology of diseases such as osteoporosis, and to the development of interventions to improve bone strength. PMID:22575727

Solid freeform fabrication and in-vitro response of osteoblast cells of mPEG-PCL-mPEG bone scaffolds.

PubMed

Jiang, Cho-Pei; Chen, Yo-Yu; Hsieh, Ming-Fa; Lee, Hung-Maan

2013-04-01

Bone tissue engineering is an emerging approach to provide viable substitutes for bone regeneration. Poly(ethylene glycol) (PEG) is a good candidate of bone scaffold because of several advantages such as hydrophilicity, biocompatibility, and intrinsic resistance to protein adsorption and cell adhesion. However, its low compressive strength limits application for bone regeneration. Poly(ε-caprolactone) (PCL), a hydrophobic nonionic polymer, is adopted to enhance the compressive strength of PEG alone.We aimed to investigate the in-vitro response of osteoblast-like cells cultured with porous scaffolds of triblock PEG-PCL-PEG copolymer fabricated by an air pressure-aided deposition system. A desktop air pressure-aided deposition system that involves melting and plotting PEG-PCL-PEG was used to fabricate three-dimensional scaffolds having rectangular pores. The experimental results showed that PEG-PCL-PEG with a molecular weight of 25,000 can be melted and stably deposited through a heating nozzle at an air pressure of 0.3 MPa and no crack occurs after it solidifies. The scaffolds with pre-determined pore size of 400× 420 μm and a porosity of 79 % were fabricated, and their average compressive strength was found to be 18.2 MPa. Osteoblast-like cells, MC3T3-E1, were seeded on fabricated scaffolds to investigate the in-vitro response of cells including toxicity and cellular locomotion. In a culture period of 28 days, the neutral-red stained osteoblasts were found to well distributed in the interior of the scaffold. Furthermore, the cellular attachment and movement in the first 10 h of cell culture were observed with time-lapse microscopy indicating that the porous PEG-PCL-PEG scaffolds fabricated by air pressure-aided deposition system is non-toxicity for osteoblast-like cells.

Aging Periosteal Progenitor Cells have Reduced Regenerative Responsiveness to Bone Injury and to the Anabolic Actions of PTH 1-34 Treatment

PubMed Central

Yukata, Kiminori; Xie, Chao; Li, Tian-Fang; Takahata, Masahiko; Hoak, Donna; Kondabolu, Sirish; Zhang, Xinping; Awad, Hani A.; Schwarz, Edward M.; Beck, Christopher A.; Jonason, Jennifer H.; O’Keefe, Regis J.

2014-01-01

A stabilized tibia fracture model was used in young (8-week old) and aged (1-year old) mice to define the relative bone regenerative potential and the relative responsiveness of the periosteal progenitor population with aging and PTH 1-34 (PTH) systemic therapy. Bone regeneration was assessed through gene expressions, radiographic imaging, histology/histomorphometry, and biomechanical testing. Radiographs and microCT showed increased calcified callus tissue and enhanced bone healing in young compared to aged mice. A key mechanism involved reduced proliferation, expansion, and differentiation of periosteal progenitor cell populations in aged mice. The experiments showed that PTH increased calcified callus tissue and torsional strength with a greater response in young mice. Histology and quantitative histomorphometry confirmed that PTH increased callus tissue area due primarily to an increase in bone formation, since minimal changes in cartilage and mesenchyme tissue area occurred. Periosteum examined at 3, 5, and 7 days showed that PTH increased cyclin D1 expression, the total number of cells in the periosteum, and width of the periosteal regenerative tissue. Gene expression showed that aging delayed differentiation of both bone and cartilage tissues during fracture healing. PTH resulted in sustained Col10a1 expression consistent with delayed chondrocyte maturation, but otherwise minimally altered cartilage gene expression. In contrast, PTH 1-34 stimulated expression of Runx2 and Osterix, but resulted in reduced Osteocalcin. β-catenin staining was present in mesenchymal chondroprogenitors and chondrocytes in early fracture healing, but was most intense in osteoblastic cells at later times. PTH increased active β-catenin staining in the osteoblast populations of both young and aged mice, but had a lesser effect in cartilage. Altogether the findings show that reduced fracture healing in aging involves decreased proliferation and differentiation of stem cells lining the bone surface. While PTH 1-34 enhances the proliferation and expansion of the periosteal stem cell population and accelerates bone formation and fracture healing, the effects are proportionately reduced in aged mice compared to young mice. β-catenin is induced by PTH in early and late fracture healing and is a potential target of PTH 1-34 effects. PMID:24530870

Adult Rat Bones Maintain Distinct Regionalized Expression of Markers Associated with Their Development

PubMed Central

Rawlinson, Simon C. F.; McKay, Ian J.; Ghuman, Mandeep; Wellmann, Claudia; Ryan, Paul; Prajaneh, Saengsome; Zaman, Gul; Hughes, Francis J.; Kingsmill, Virginia J.

2009-01-01

The incidence of limb bone fracture and subsequent morbidity and mortality due to excessive bone loss is increasing in the progressively ageing populations of both men and women. In contrast to bone loss in the weight-bearing limb, bone mass in the protective skull vault is maintained. One explanation for this could be anatomically diverse bone matrix characteristics generated by heterogeneous osteoblast populations. We have tested the hypothesis that adult bones demonstrate site-specific characteristics, and report differences at the organ, cell and transcriptome levels. Limb bones contain greater amounts of polysulphated glycosaminoglycan stained with Alcian Blue and have significantly higher osteocyte densities than skull bone. Site-specific patterns persist in cultured adult bone-derived cells both phenotypically (proliferation rate, response to estrogen and cell volumes), and at the level of specific gene expression (collagen triple helix repeat containing 1, reelin and ras-like and estrogen-regulated growth inhibitor). Based on genome-wide mRNA expression and cluster analysis, we demonstrate that bones and cultured adult bone-derived cells segregate according to site of derivation. We also find the differential expression of genes associated with embryological development (Skull: Zic, Dlx, Irx, Twist1 and Cart1; Limb: Hox, Shox2, and Tbx genes) in both adult bones and isolated adult bone-derived cells. Together, these site-specific differences support the view that, analogous to different muscle types (cardiac, smooth and skeletal), skull and limb bones represent separate classes of bone. We assign these differences, not to mode of primary ossification, but to the embryological cell lineage; the basis and implications of this division are discussed. PMID:20027296

Is bone transplantation the gold standard for repair of alveolar bone defects?

PubMed

Raposo-Amaral, Cassio Eduardo; Bueno, Daniela Franco; Almeida, Ana Beatriz; Jorgetti, Vanda; Costa, Cristiane Cabral; Gouveia, Cecília Helena; Vulcano, Luiz Carlos; Fanganiello, Roberto D; Passos-Bueno, Maria Rita; Alonso, Nivaldo

2014-01-01

New strategies to fulfill craniofacial bone defects have gained attention in recent years due to the morbidity of autologous bone graft harvesting. We aimed to evaluate the in vivo efficacy of bone tissue engineering strategy using mesenchymal stem cells associated with two matrices (bovine bone mineral and α-tricalcium phosphate), compared to an autologous bone transfer. A total of 28 adult, male, non-immunosuppressed Wistar rats underwent a critical-sized osseous defect of 5 mm diameter in the alveolar region. Animals were divided into five groups. Group 1 (n = 7) defects were repaired with autogenous bone grafts; Group 2 (n = 5) defects were repaired with bovine bone mineral free of cells; Group 3 (n = 5) defects were repaired with bovine bone mineral loaded with mesenchymal stem cells; Group 4 (n = 5) defects were repaired with α-tricalcium phosphate free of cells; and Group 5 (n = 6) defects were repaired with α-tricalcium phosphate loaded with mesenchymal stem cells. Groups 2-5 were compared to Group 1, the reference group. Healing response was evaluated by histomorphometry and computerized tomography. Histomorphometrically, Group 1 showed 60.27% ± 16.13% of bone in the defect. Groups 2 and 3 showed 23.02% ± 8.6% (p = 0.01) and 38.35% ± 19.59% (p = 0.06) of bone in the defect, respectively. Groups 4 and 5 showed 51.48% ± 11.7% (p = 0.30) and 61.80% ± 2.14% (p = 0.88) of bone in the defect, respectively. Animals whose bone defects were repaired with α-tricalcium phosphate and mesenchymal stem cells presented the highest bone volume filling the defects; both were not statistically different from autogenous bone.

Osteoblasts Protect AML Cells from SDF-1-Induced Apoptosis

PubMed Central

Kremer, Kimberly N.; Dudakovic, Amel; McGee-Lawrence, Meghan E.; Philips, Rachael L.; Hess, Allan D.; Smith, B. Douglas; van Wijnen, Andre J.; Karp, Judith E.; Kaufmann, Scott H.; Westendorf, Jennifer J.; Hedin, Karen E.

2014-01-01

The bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF-1 naturally present in that location. In co-culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF-1-induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W-20-17, mediated this protection via a cell contact-independent mechanism. In contrast, bone marrow-derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4-dependent mechanism and failed to protect AML cells from exogenously added SDF-1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF-1-driven apoptotic pathway of CXCR4-expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF-1-induced apoptosis of AML cells residing within the bone marrow microenvironment. PMID:24851270

Improvement of anemia in W/WV mice by recombinant human erythropoietin (rHuEPO) mediated through EPO receptors with lowered affinity.

PubMed

Kabaya, K; Akiyama, H; Nishi, N; Misaizu, T; Okada, Y; Kawagishi, M; Amano, K; Kusaka, M; Seki, M; Uzumaki, H

1995-01-01

We studied the effects of recombinant human erythropoietin (rHuEPO) on anemic W/WV mice which manifested severe anemia accompanied by mutation of the W gene encoding tyrosine kinase type receptor (c-kit gene) of bone marrow hematopoietic cells. Nine-week-old male W/WV mice or normal littermates (+/+) were used. Since serum EPO concentration in W/WV mice increased in proportion to severity of anemia, EPO production in the kidneys of these animals was found to be regulated normally. Hematocrit in +/+ mice increased and a maximal response was also obtained with 2,000 IU/kg of rHuEPO. On the other hand, hematocrit in W/WV mice increased in a dose-responsive manner by administration with 2,000 and 10,000 IU/kg, showing different responses to rHuEPO in these two types of mice. The responsiveness of W/WV mice to rHuEPO was low in terms of increases in erythroblastic precursor cells (CFU-E), and immature cells in the bone marrow. Scatchard analysis of the specific binding of 125I-rHuEPO against bone marrow cells revealed that the different responsiveness to rHuEPO between W/WV and +/+ mice may be correlated with differences in affinity of EPO receptor of bone marrow cells in these mice. From these results, a high dose of rHuEPO is capable of improving the anemia in W/WV mice that had EPO receptors with lowered affinity, indicating the possible effectiveness of rHuEPO in anemic patients with EPO receptor abnormality.

Skeletal stem cell isolation: A review on the state-of-the-art microfluidic label-free sorting techniques.

PubMed

Xavier, Miguel; Oreffo, Richard O C; Morgan, Hywel

2016-01-01

Skeletal stem cells (SSC) are a sub-population of bone marrow stromal cells that reside in postnatal bone marrow with osteogenic, chondrogenic and adipogenic differentiation potential. SSCs reside only in the bone marrow and have organisational and regulatory functions in the bone marrow microenvironment and give rise to the haematopoiesis-supportive stroma. Their differentiation capacity is restricted to skeletal lineages and therefore the term SSC should be clearly distinguished from mesenchymal stem cells which are reported to exist in extra-skeletal tissues and, critically, do not contribute to skeletal development. SSCs are responsible for the unique regeneration capacity of bone and offer unlimited potential for application in bone regenerative therapies. A current unmet challenge is the isolation of homogeneous populations of SSCs, in vitro, with homogeneous regeneration and differentiation capacities. Challenges that limit SSC isolation include a) the scarcity of SSCs in bone marrow aspirates, estimated at between 1 in 10-100,000 mononuclear cells; b) the absence of specific markers and thus the phenotypic ambiguity of the SSC and c) the complexity of bone marrow tissue. Microfluidics provides innovative approaches for cell separation based on bio-physical features of single cells. Here we review the physical principles underlying label-free microfluidic sorting techniques and review their capacity for stem cell selection/sorting from complex (heterogeneous) samples. Copyright © 2016 Elsevier Inc. All rights reserved.

It May Seem Inflammatory, but Some T Cells Are Innately Healing to the Bone.

PubMed

Kalyan, Shirin

2016-11-01

Among the most significant developments to have taken place in osteology over the last few decades is an evolution from treating and viewing bone disorders primarily through an endocrine lens to instead seeing them as metabolic disorders that interface at the molecular and cellular level with the immune system. Osteoimmunology was officially born in response to accumulating evidence that the immune system is integrally involved in bone remodeling, but much of the early work focused on the role of conventional αβ T cells in driving bone loss. There is, however, emerging data indicating that innate lymphocytes, in particular γδ T cells, may in fact be important for bone regeneration. We first observed that bisphosphonate-associated osteonecrosis of the jaw (ONJ), a rare but serious adverse drug effect characterized by nonhealing necrotic bone tissue of the mandible or maxilla, was linked to a deficiency in a subset of γδ T cells found in human peripheral blood. Patients who developed ONJ while on bisphosphonate therapy not only lacked the main subset of circulating γδ T cells, but they also all had underlying conditions that compromised their immune integrity. A number of recent studies have unraveled the role of γδ T cells (and lymphocytes sharing their characteristics) in bone regeneration-particularly for fracture healing. These findings seem to contradict the prevailing view of such "inflammatory" T cells as being bone degenerative rather than restorative. This viewpoint melds together the emerging evidence of these so-called inflammatory T cells in bone remodeling and healing-showing that they are not in fact "all bad to the bone." © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

Nanotopographical control of human osteoprogenitor differentiation.

PubMed

Dalby, Matthew J; Gadegaard, Nikolaj; Curtis, Adam S G; Oreffo, Richard O C

2007-05-01

Current load-bearing orthopaedic implants are produced in 'bio-inert' materials such as titanium alloys. When inserted into the reamed bone during hip or knee replacement surgery the implants interact with mesenchymal populations including the bone marrow. Bio-inert materials are shielded from the body by differentiation of the cells along the fibroblastic lineage producing scar tissue and inferior healing. This is exacerbated by implant micromotion, which can lead to capsule formation. Thus, next-generation implant materials will have to elicit influence over osteoprogenitor differentiation and mesenchymal populations in order to recruit osteoblastic cells and produce direct bone apposition onto the implant. A powerful method of delivering cues to cells is via topography. Micro-scale topography has been shown to affect cell adhesion, migration, cytoskeleton, proliferation and differentiation of a large range of cell types (thus far all cell types tested have been shown to be responsive to topographical cues). More recent research with nanotopography has also shown a broad range of cell response, with fibroblastic cells sensing down to 10 nm in height. Initial studies with human mesenchymal populations and osteoprogenitor populations have again shown strong cell responses to nanofeatures with increased levels of osteocalcin and osteopontin production from the cells on certain topographies. This is indicative of increased osteoblastic activity on the nanotextured materials. Looking at preliminary data, it is tempting to speculate that progenitor cells are, in fact, more responsive to topography than more mature cell types and that they are actively seeking cues from their environment. This review will investigate the range of nanotopographies available to researchers and our present understanding of mechanisms of progenitor cell response. Finally, it will make some speculations of the future of nanomaterials and progenitor cells in tissue engineering.

Direct comparison of progenitor cells derived from adipose, muscle, and bone marrow from wild-type or craniosynostotic rabbits

PubMed Central

GM, Cooper; EL, Lensie; JJ, Cray; MR, Bykowski; GE, DeCesare; MA, Smalley; MP, Mooney; PG, Campbell; JE, Losee

2010-01-01

Background Reports have identified cells capable of osteogenic differentiation in bone marrow, muscle, and adipose tissues, but there are few direct comparisons of these different cell-types. Also, few have investigated the potential connection between a tissue-specific pathology and cells derived from seemingly unrelated tissues. Here, we compare cells isolated from wild-type rabbits or rabbits with nonsyndromic craniosynostosis, defined as the premature fusion of one or more of the cranial sutures. Methods Cells were derived from bone marrow, adipose, and muscle of 10 day-old wild-type rabbits (WT; n=17) or from age-matched rabbits with familial nonsyndromic craniosynostosis (CS; n=18). Cells were stimulated with bone morphogenetic protein 4 (BMP4) and alkaline phosphatase expression and cell proliferation were assessed. Results In WT rabbits, cells derived from muscle had more alkaline phosphatase activity than cells derived from either adipose or bone marrow. The cells derived from CS rabbit bone marrow and muscle were significantly more osteogenic than WT. Adipose-derived cells demonstrated no significant differences. While muscle-derived cells were most osteogenic in WT rabbits, bone marrow-derived cells were most osteogenic in CS rabbits. Conclusions Results suggest that cells from different tissues have different potentials for differentiation. Furthermore, cells derived from rabbits with craniosynostosis were different from wild-type derived cells. Interestingly, cells derived from the craniosynostotic rabbits were not uniformly more responsive compared with wild-type cells, suggesting that specific tissue-derived cells may react differently in individuals with craniosynostosis. PMID:20871482

A fluid–structure interaction model to characterize bone cell stimulation in parallel-plate flow chamber systems

PubMed Central

Vaughan, T. J.; Haugh, M. G.; McNamara, L. M.

2013-01-01

Bone continuously adapts its internal structure to accommodate the functional demands of its mechanical environment and strain-induced flow of interstitial fluid is believed to be the primary mediator of mechanical stimuli to bone cells in vivo. In vitro investigations have shown that bone cells produce important biochemical signals in response to fluid flow applied using parallel-plate flow chamber (PPFC) systems. However, the exact mechanical stimulus experienced by the cells within these systems remains unclear. To fully understand this behaviour represents a most challenging multi-physics problem involving the interaction between deformable cellular structures and adjacent fluid flows. In this study, we use a fluid–structure interaction computational approach to investigate the nature of the mechanical stimulus being applied to a single osteoblast cell under fluid flow within a PPFC system. The analysis decouples the contribution of pressure and shear stress on cellular deformation and for the first time highlights that cell strain under flow is dominated by the pressure in the PPFC system rather than the applied shear stress. Furthermore, it was found that strains imparted on the cell membrane were relatively low whereas significant strain amplification occurred at the cell–substrate interface. These results suggest that strain transfer through focal attachments at the base of the cell are the primary mediators of mechanical signals to the cell under flow in a PPFC system. Such information is vital in order to correctly interpret biological responses of bone cells under in vitro stimulation and elucidate the mechanisms associated with mechanotransduction in vivo. PMID:23365189

Cell-specific paracrine actions of IL-6 family cytokines from bone, marrow and muscle that control bone formation and resorption.

PubMed

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.

Osteocyte calcium signals encode strain magnitude and loading frequency in vivo.

PubMed

Lewis, Karl J; Frikha-Benayed, Dorra; Louie, Joyce; Stephen, Samuel; Spray, David C; Thi, Mia M; Seref-Ferlengez, Zeynep; Majeska, Robert J; Weinbaum, Sheldon; Schaffler, Mitchell B

2017-10-31

Osteocytes are considered to be the major mechanosensory cells of bone, but how osteocytes in vivo process, perceive, and respond to mechanical loading remains poorly understood. Intracellular calcium (Ca 2+ ) signaling resulting from mechanical stimulation has been widely studied in osteocytes in vitro and in bone explants, but has yet to be examined in vivo. This is achieved herein by using a three-point bending device which is capable of delivering well-defined mechanical loads to metatarsal bones of living mice while simultaneously monitoring the intracellular Ca 2+ responses of individual osteocytes by using a genetically encoded fluorescent Ca 2+ indicator. Osteocyte responses are imaged by using multiphoton fluorescence microscopy. We investigated the in vivo responses of osteocytes to strains ranging from 250 to 3,000 [Formula: see text] and frequencies from 0.5 to 2 Hz, which are characteristic of physiological conditions reported for bone. At all loading frequencies examined, the number of responding osteocytes increased strongly with applied strain magnitude. However, Ca 2+ intensity within responding osteocytes did not change significantly with physiological loading magnitudes. Our studies offer a glimpse into how these critical bone cells respond to mechanical load in vivo, as well as provide a technique to determine how the cells encode magnitude and frequency of loading. Published under the PNAS license.

Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT).

PubMed

Alvarez, Marta B; Childress, Paul; Philip, Binu K; Gerard-O'Riley, Rita; Hanlon, Michael; Herbert, Brittney-Shea; Robling, Alexander G; Pavalko, Fredrick M; Bidwell, Joseph P

2012-05-01

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH-induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4-knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH-responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH-induced c-fos femoral mRNA expression is enhanced in Nmp4-KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c-fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism. Copyright © 2011 Wiley Periodicals, Inc.

A Novel 3 Dimensional Stromal-based Model for In Vitro Chemotherapy Sensitivity Testing of Leukemia Cells

PubMed Central

Aljitawi, Omar S.; Li, Dandan; Xiao, Yinghua; Zhang, Da; Ramachandran, Karthik; Stehno-Bittel, Lisa; Van Veldhuizen, Peter; Lin, Tara L.; Kambhampati, Suman; Garimella, Rama

2014-01-01

The disparate responses of leukemia cells to chemotherapy in vivo, compared to in vitro, is partly related to the interactions of leukemic cells and the 3 dimensional (3D) bone marrow stromal microenvironment. We investigated the effects of chemotherapy agents on leukemic cell lines co-cultured with human bone marrow mesenchymal stem cell (hu-BM-MSC) in 3D. Comparison was made to leukemic cells treated in suspension, or grown on a hu-BM-MSC monolayer (2D conditions). We demonstrated that leukemic cells cultured in 3D were more resistant to drug-induced apoptosis compared to cells cultured in 2D or in suspension. We also demonstrated significant differences in leukemic cell response to chemotherapy using different leukemic cell lines cultured in 3D. We suggest that the differential responses to chemotherapy in 3D may be related to the expression of N-cadherin in the co-culture system. This unique model provides an opportunity to study leukemic cell responses to chemotherapy in 3D. PMID:23566162

In vivo response to starch-based scaffolds designed for bone tissue engineering applications.

PubMed

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.

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources.

PubMed

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.

Bone vs. fat: Embryonic origin of progenitors determines response to androgen in adipocytes and osteoblasts

PubMed Central

Wiren, Kristine M.; Hashimoto, Joel G.; Semirale, Anthony A.; Zhang, Xiao-Wei

2011-01-01

Although androgen is considered an anabolic hormone, the consequences of androgen receptor (AR) overexpression in skeletally-targeted AR-transgenic lines highlight the detrimental effect of enhanced androgen sensitivity on cortical bone quality. A compartment-specific anabolic response is observed only in male but not female AR3.6-transgenic (tg) mice, with increased periosteal bone formation and calvarial thickening. To identify anabolic signaling cascades that have the potential to increase bone formation, qPCR array analysis was employed to define expression differences between AR3.6-tg and wild-type (WT) periosteal tissue. Notably, categories that were significantly different between the two genotypes included axonal guidance, CNS development and negative regulation of Wnt signaling with a node centered on stem cell pathways. Further, fine mapping of AR3.6-tg calvaria revealed that anabolic thickening in vivo is not uniform across the calvaria, occurring only in frontal but not parietal bones. Multipotent fraction 1 progenitor populations from both genotypes were cultured separately as frontal bone neural crest stem-like cells (fNCSC) and parietal bone mesenchymal stem-like cells (pMSC). Both osteoblastic and adipogenic differentiation in these progenitor populations was influenced by embryonic lineage and by genotype. Adipogenesis was enhanced in WT fNCSC compared to pMSC, but transgenic cultures showed strong suppression of lipid accumulation only in fNCSC cells. Osteoblastogenesis was significantly increased in transgenic fNCSC cultures compared to WT, with elevated alkaline phosphatase (ALP) activity and induction of mineralization and nodule formation assessed by alizarin red and von Kossa staining. Osteocalcin (OC) and ALP mRNA levels were also increased in fNCSC cultures from AR3.6-tg vs. WT, but in pMSC cultures ALP mRNA levels, mineralization and nodule formation were decreased in AR3.6-tg cells. Expression differences identified by array in long bone periosteal tissue from AR3.6-tg vs. WT were recapitulated in the fNCSC samples while pMSCs profiles reflected cortical expression. These observations reveal the opposing effects of androgen signaling on lineage commitment and osteoblast differentiation that is enhanced in cells derived from a neural crest origin but inhibited in cells derived from a mesodermal origin, consistent with in vivo compartment-specific responses to androgen. Combined, these results highlight the complex action of androgen in the body that is dependent on the embryonic lineage and developmental origin of the cell. Further, these data these data suggest that the periosteum surrounding long bone is derived from neural crest. PMID:21704206

Crosstalk between bone marrow-derived mesenchymal stem cells and regulatory T cells through a glucocorticoid-induced leucine zipper/developmental endothelial locus-1-dependent mechanism.

PubMed

Yang, Nianlan; Baban, Babak; Isales, Carlos M; Shi, Xing-Ming

2015-09-01

Bone marrow is a reservoir for regulatory T (T(reg)) cells, but how T(reg) cells are regulated in that environment remains poorly understood. We show that expression of glucocorticoid (GC)-induced leucine zipper (GILZ) in bone marrow mesenchymal lineage cells or bone marrow-derived mesenchymal stem cells (BMSCs) increases the production of T(reg) cells via a mechanism involving the up-regulation of developmental endothelial locus-1 (Del-1), an endogenous leukocyte-endothelial adhesion inhibitor. We found that the expression of Del-1 is increased ∼4-fold in the bone tissues of GILZ transgenic (Tg) mice, and this increase is coupled with a significant increase in the production of IL-10 (2.80 vs. 0.83) and decrease in the production of IL-6 (0.80 vs. 2.33) and IL-12 (0.25 vs. 1.67). We also show that GILZ-expressing BMSCs present antigen in a way that favors T(reg) cells. These results indicate that GILZ plays a critical role mediating the crosstalk between BMSCs and T(reg) in the bone marrow microenvironment. These data, together with our previous findings that overexpression of GILZ in BMSCs antagonizes TNF-α-elicited inflammatory responses, suggest that GILZ plays important roles in bone-immune cell communication and BMSC immune suppressive functions. © FASEB.

Reconciling the effects of inflammatory cytokines on mesenchymal cell osteogenic differentiation

PubMed Central

Deshpande, Sagar; James, Aaron W.; Blough, Jordan; Donneys, Alexis; Wang, Stewart C.; Cederna, Paul S.; Buchman, Steven R.; Levi, Benjamin

2015-01-01

Therapies using mesenchymal stem cells are a popular current avenue for development and utilization, especially in the fields of de novo tissue engineering (Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol 2000;164:247.) or tissue regeneration after physical injury (Kitoh H, Kitakoji T, Tsuchiya H, et al. Transplantation of marrow-derived mesenchymal stem cells and platelet-rich plasma during distraction osteogenesis—a preliminary result of three cases. Bone 2004;35:892; Shumakov VI, Onishchenko NA, Rasulov MF, Krasheninnikov ME, Zaidenov VA. Mesenchymal bone marrow stem cells more effectively stimulate regeneration of deep burn wounds than embryonic fibroblasts. Bull Exp Biol Med 2003;136:192; Bruder SP, Fink DJ, Caplan AI. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem 1994;56:283.). The osteogenic potential of these cells is of particular interest, given their recent usage for the closure of critical-sized bone defects and other nonhealing bone scenarios such as a nonunion. Recent literature suggests that inflammatory cytokines can significantly impact the osteogenic potential of these cells. A review of relevant, recent literature is presented regarding the impact of the inflammatory cascade on the osteogenic differentiation of these cells and how this varies across species. Finally, we identify areas of conflicting or absent evidence regarding the behavior of mesenchymal stem cells in response to inflammatory cytokines. PMID:23972621

Synergetic topography and chemistry cues guiding osteogenic differentiation in bone marrow stromal cells through ERK1/2 and p38 MAPK signaling pathway.

PubMed

Zhang, Xinran; Li, Haotian; Lin, Chucheng; Ning, Congqin; Lin, Kaili

2018-01-30

Both the topographic surface and chemical composition modification can enhance rapid osteogenic differentiation and bone formation. Till now, the synergetic effects of topography and chemistry cues guiding biological responses have been rarely reported. Herein, the ordered micro-patterned topography and classically essential trace element of strontium (Sr) ion doping were selected to imitate topography and chemistry cues, respectively. The ordered micro-patterned topography on Sr ion-doped bioceramics was successfully duplicated using the nylon sieve as the template. Biological response results revealed that the micro-patterned topography design or Sr doping could promote cell attachment, ALP activity, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Most importantly, the samples both with micro-patterned topography and Sr doping showed the highest promotion effects, and could synergistically activate the ERK1/2 and p38 MAPK signaling pathways. The results suggested that the grafts with both specific topography and chemistry cues have synergetic effects on osteogenic activity of BMSCs and provide an effective approach to design functional bone grafts and cell culture substrates.

What are the local and systemic biologic reactions and mediators to wear debris, and what host factors determine or modulate the biologic response to wear particles?

PubMed

Tuan, Rocky S; Lee, Francis Young-In; T Konttinen, Yrjö; Wilkinson, J Mark; Smith, Robert Lane

2008-01-01

New clinical and basic science data on the cellular and molecular mechanisms by which wear particles stimulate the host inflammatory response have provided deeper insight into the pathophysiology of periprosthetic bone loss. Interactions among wear particles, macrophages, osteoblasts, bone marrow-derived mesenchymal stem cells, fibroblasts, endothelial cells, and T cells contribute to the production of pro-inflammatory and pro-osteoclastogenic cytokines such as TNF-alpha, RANKL, M-SCF, PGE2, IL-1, IL-6, and IL-8. These cytokines not only promote osteoclastogenesis but interfere with osteogenesis led by osteoprogenitor cells. Recent studies indicate that genetic variations in TNF-alpha, IL-1, and FRZB can result in subtle changes in gene function, giving rise to altered susceptibility or severity for periprosthetic inflammation and bone loss. Continuing research on the biologic effects and mechanisms of action of wear particles will provide a rational basis for the development of novel and effective ways of diagnosis, prevention, and treatment of periprosthetic inflammatory bone loss.

Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

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

Tang, C.-H., E-mail: chtang@mail.cmu.edu.t; Graduate Institute of Basic Medical Science, China Medical University, Taichung Taiwan; Chiu, Y.-C.

Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromalmore » cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.« less

Persistent injury-associated anemia: the role of the bone marrow microenvironment.

PubMed

Millar, Jessica K; Kannan, Kolenkode B; Loftus, Tyler J; Alamo, Ines G; Plazas, Jessica; Efron, Philip A; Mohr, Alicia M

2017-06-15

The regulation of erythropoiesis involves hematopoietic progenitor cells, bone marrow stroma, and the microenvironment. Following severe injury, a hypercatecholamine state develops that is associated with increased mobilization of hematopoietic progenitor cells to peripheral blood and decreased growth of bone marrow erythroid progenitor cells that manifests clinically as a persistent injury-associated anemia. Changes within the bone marrow microenvironment influence the development of erythroid progenitor cells. Therefore, we sought to determine the effects of lung contusion, hemorrhagic shock, and chronic stress on the hematopoietic cytokine response. Bone marrow was obtained from male Sprague-Dawley rats (n = 6/group) killed 7 d after lung contusion followed by hemorrhagic shock (LCHS) or LCHS followed by daily chronic restraint stress (LCHS/CS). End point polymerase chain reaction was performed for interleukin-1β, interleukin-10, stem cell factor, transforming growth factor-β, high-mobility group box-1 (HMGB-1), and B-cell lymphoma-extra large. Seven days following LCHS and LCHS/CS, bone marrow expression of prohematopoietic cytokines (interleukin-1β, interleukin-10, stem cell factor, and transforming growth factor-β) was significantly decreased, and bone marrow expression of HMGB-1 was significantly increased. B-cell lymphoma-extra large bone marrow expression was not affected by LCHS or LCHS/CS (naïve: 44 ± 12, LCHS: 44 ± 12, LCHS/CS: 37 ± 1, all P > 0.05). The bone marrow microenvironment was significantly altered following severe trauma in a rodent model. Prohematopoietic cytokines were downregulated, and the proinflammatory cytokine HMGB-1 had increased bone marrow expression. Modulation of the bone marrow microenvironment may represent a therapeutic strategy following severe trauma to alleviate persistent injury-associated anemia. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Acute Exposure to High Dose γ-Radiation Results in Transient Activation of Bone Lining Cells

PubMed Central

Turner, Russell T.; Iwaniec, Urszula T.; Wong, Carmen P.; Lindenmaier, Laurence B.; Wagner, Lindsay A.; Branscum, Adam J.; Menn, Scott A.; Taylor, James; Zhang, Ye; Wu, Honglu; Sibonga, Jean D.

2014-01-01

The present studies investigated the cellular mechanisms for the detrimental effects of high dose whole body γ-irradiation on bone. In addition, radioadaptation and bone marrow transplantation were assessed as interventions to mitigate the skeletal complications of irradiation. Increased trabecular thickness and separation and reduced fractional cancellous bone volume, connectivity density, and trabecular number were detected in proximal tibia and lumbar vertebra 14 days following γ-irradiation with 6 Gy. To establish the cellular mechanism for the architectural changes, vertebrae were analyzed by histomorphometry 1, 3, and 14 days following irradiation. Marrow cell density decreased within 1 day (67% reduction, p<0.0001), reached a minimum value after 3 days (86% reduction, p<0.0001), and partially rebounded by 14 days (30% reduction, p=0.0025) following irradiation. In contrast, osteoblast-lined bone perimeter was increased by 290% (1 day, p=0.04), 1230% (3 days, p<0.0001), and 530% (14 days, p=0.003), respectively. There was a strong association between radiation-induced marrow cell death and activation of bone lining cells to express the osteoblast phenotype (Pearson correlation −0.85, p<0.0001). An increase (p=0.004) in osteoclast-lined bone perimeter was also detected with irradiation. A priming dose of γ-radiation (0.5 mGy), previously shown to reduce mortality, had minimal effect on the cellular responses to radiation and did not prevent detrimental changes in bone architecture. Bone marrow transplantation normalized marrow cell density, bone turnover, and most indices of bone architecture following irradiation. In summary, radiation-induced death of marrow cells is associated with 1) a transient increase in bone formation due, at least in part, to activation of bone lining cells, and 2) an increase in bone resorption due to increased osteoclast perimeter. Bone marrow transplantation is effective in mitigating the detrimental effects of acute exposure to high dose whole body γ-radiation on bone turnover. PMID:23954507

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

Bone as a source of organism vitality and regeneration.

PubMed

Mackiewicz, Zygmunt; Niklińska, Wiesława Ewa; Kowalewska, Jolanta; Chyczewski, Lech

2011-01-01

The most important features that determine the vital role of bone include: a) a continuous supply of calcium, which is indispensible for every cell of the entire organism at all times, and b) the delivery of circulating blood cells and some adult stem cells to keep the body vigorous, ready for self-reparation, and continuously rebuilding throughout life. These functions of bones are no less important than protecting the body cavities, serving as mechanical levers connected to the muscles, and determining the shape and dimensions of the entire organism. The aim of this review was to address some basic cellular and molecular knowledge to better understand the complex interactions of bone structural components. The apprehension of osteoblast differentiation and its local regulation has substantially increased in recent years. It has been suggested that osteocytes, cells within the bone matrix, act as regulatory mechanosensors. Therefore immobility as well as limited activity has a dramatic effect on bone structure and influences a broad spectrum of bone physiology-related functions as well as the functions of many other organs. Lifelong bone rebuilding is modulated through several pathways, including the Wnt pathway that regulates bone formation and resorption. In the adult skeleton, bone is continuously renewed in response to a variety of stimuli, such as the specific process of remodeling dependent on RANK/ /RANKL/OPG interactions. Better understanding of bone biology provides opportunities for the development of more effective prevention and treatment modalities for a variety of bone diseases, including new approaches to adult stem cell-based therapies.

Bone morphogenetic protein 9 (BMP9) induces effective bone formation from reversibly immortalized multipotent adipose-derived (iMAD) mesenchymal stem cells.

PubMed

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.

Dynamic Fluid Flow Mechanical Stimulation Modulates Bone Marrow Mesenchymal Stem Cells.

PubMed

Hu, Minyi; Yeh, Robbin; Lien, Michelle; Teeratananon, Morgan; Agarwal, Kunal; Qin, Yi-Xian

2013-03-01

Osteoblasts are derived from mesenchymal stem cells (MSCs), which initiate and regulate bone formation. New strategies for osteoporosis treatments have aimed to control the fate of MSCs. While functional disuse decreases MSC growth and osteogenic potentials, mechanical signals enhance MSC quantity and bias their differentiation toward osteoblastogenesis. Through a non-invasive dynamic hydraulic stimulation (DHS), we have found that DHS can mitigate trabecular bone loss in a functional disuse model via rat hindlimb suspension (HLS). To further elucidate the downstream cellular effect of DHS and its potential mechanism underlying the bone quality enhancement, a longitudinal in vivo study was designed to evaluate the MSC populations in response to DHS over 3, 7, 14, and 21 days. Five-month old female Sprague Dawley rats were divided into three groups for each time point: age-matched control, HLS, and HLS+DHS. DHS was delivered to the right mid-tibiae with a daily "10 min on-5 min off-10 min on" loading regime for five days/week. At each sacrifice time point, bone marrow MSCs of the stimulated and control tibiae were isolated through specific cell surface markers and quantified by flow cytometry analysis. A strong time-dependent manner of bone marrow MSC induction was observed in response to DHS, which peaked on day 14. After 21 days, this effect of DHS was diminished. This study indicates that the MSC pool is positively influenced by the mechanical signals driven by DHS. Coinciding with our previous findings of mitigation of disuse bone loss, DHS induced changes in MSC number may bias the differentiation of the MSC population towards osteoblastogenesis, thereby promoting bone formation under disuse conditions. This study provides insights into the mechanism of time-sensitive MSC induction in response to mechanical loading, and for the optimal design of osteoporosis treatments.

Basic Biology of Skeletal Aging: Role of Stress Response Pathways

PubMed Central

2013-01-01

Although a decline in bone formation and loss of bone mass are common features of human aging, the molecular mechanisms mediating these effects have remained unclear. Evidence from pharmacological and genetic studies in mice has provided support for a deleterious effect of oxidative stress in bone and has strengthened the idea that an increase in reactive oxygen species (ROS) with advancing age represents a pathophysiological mechanism underlying age-related bone loss. Mesenchymal stem cells and osteocytes are long-lived cells and, therefore, are more susceptible than other types of bone cells to the molecular changes caused by aging, including increased levels of ROS and decreased autophagy. However, short-lived cells like osteoblast progenitors and mature osteoblasts and osteoclasts are also affected by the altered aged environment characterized by lower levels of sex steroids, increased endogenous glucocorticoids, and higher oxidized lipids. This article reviews current knowledge on the effects of the aging process on bone, with particular emphasis on the role of ROS and autophagy in cells of the osteoblast lineage in mice. PMID:23825036

Histone deacetylase inhibitors reduce differentiating osteoblast-mediated protection of acute myeloid leukemia cells from cytarabine

PubMed Central

Sterner, Rosalie M.; Kremer, Kimberly N.; Al-Kali, Aref; Patnaik, Mrinal M.; Gangat, Naseema; Litzow, Mark R.; Kaufmann, Scott H.; Westendorf, Jennifer J.; van Wijnen, Andre J.; Hedin, Karen E.

2017-01-01

The bone marrow microenvironment protects acute myeloid leukemia (AML) cells during chemotherapy and is a major factor in relapse. Here, we examined which type(s) of bone marrow cells are responsible for the relapse of AML following treatment with cytarabine (Ara-C), and we identified a means to inhibit this protection. To determine the protective cell type(s), AML cells were treated with Ara-C, and AML cell survival in the presence or absence of osteoblast lineage cells was assessed. Cultured AML cells and patient bone marrow isolates were each significantly protected from Ara-C-induced apoptosis by co-culture with differentiating osteoblasts. Moreover, pretreating differentiating osteoblasts with the histone deacetylase inhibitors (HDACi) vorinostat and panobinostat abrogated the ability of the differentiating osteoblasts to protect AML cells. Together, our results indicate that differentiating osteoblasts have the potential to promote residual AML in the bone marrow following standard chemotherapy and act via a mechanism requiring HDACi-sensitive gene expression. Using HDACi to target the leukemic microenvironment in combination with Ara-C could potentially improve treatment of AML. Moreover, other strategies for manipulating bone marrow osteoblasts may also help eradicate AML cells and reduce relapse. PMID:29212250

IL-12p40 impairs mesenchymal stem cell-mediated bone regeneration via CD4+ T cells

PubMed Central

Xu, Jiajia; Wang, Yiyun; Li, Jing; Zhang, Xudong; Geng, Yiyun; Huang, Yan; Dai, Kerong; Zhang, Xiaoling

2016-01-01

Severe or prolonged inflammatory response caused by infection or biomaterials leads to delayed healing or bone repair failure. This study investigated the important roles of the proinflammatory cytokines of the interleukin-12 (IL-12) family, namely, IL-12 and IL-23, in the inflammation-mediated inhibition of bone formation in vivo. IL-12p40−/− mice lacking IL-12 and IL-23 exhibited enhanced bone formation. IL-12 and IL-23 indirectly inhibited bone marrow mesenchymal stem cell (BMMSC) differentiation by stimulating CD4+ T cells to increase interferon γ (IFN-γ) and IL-17 levels. Mechanistically, IL-17 synergistically enhanced IFN-γ-induced BMMSC apoptosis. Moreover, INF-γ and IL-17 exerted proapoptotic effects by upregulating the expression levels of Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as by activating the caspase cascade in BMMSCs. IL-12p40 depletion in mice could promote ectopic bone formation. Thus, IL-12p40 is an attractive therapeutic target to overcome the inflammation-mediated inhibition of bone formation in vivo. PMID:27472064

Long Term Maintenance of Polysaccharide-specific Antibodies by IgM Secreting Cells1

PubMed Central

Foote, Jeremy B.; Mahmoud, Tamer I.; Vale, Andre M.; Kearney, John F.

2011-01-01

Many bacteria-associated polysaccharides induce long-lived antibody responses that protect against pathogenic microorganisms. The maintenance of polysaccharide-specific antibody titers may be due to long-lived plasma cells or ongoing antigen-driven B cell activation due to polysaccharide persistence. BALB/c and VHJ558.3 transgenic (TG) mice respond to α 1→3-dextran (DEX) by generating a peak anti-DEX response at 7 days, followed by maintenance of serum antibody levels for up to 150 days. Analysis of the cellular response to DEX identified a population of short-lived, cyclophosphamide sensitive DEX-specific plasmablasts in the spleen, and a quiescent, cyclophosphamide resistant DEX-specific antibody-secreting population in the bone marrow. BrdU pulse-chase experiments demonstrated the longevity of the DEX-specific antibody-secreting population in the bone marrow. Splenic DEX-specific plasmablasts were located in the red pulp with persisting DEX-associated CD11c+ dendritic cells 90 days after immunization, whereas DEX was not detected in the bone marrow after 28 days. Selective depletion of short-lived DEX-specific plasmablasts and memory B1b B cells using cyclophosphamide and anti-CD20 treatment had a minimal impact on the maintenance of serum anti-DEX antibodies. Collectively, these findings demonstrate that the maintenance of serum polysaccharide-specific antibodies is the result of continuous antigen-driven formation of short-lived plasmablasts in the spleen and a quiescent population of antibody-secreting cells maintained in the bone marrow for a long duration. PMID:22116821

Fanca−/− hematopoietic stem cells demonstrate a mobilization defect which can be overcome by administration of the Rac inhibitor NSC23766

PubMed Central

Milsom, Michael D.; Lee, Andrew W.; Zheng, Yi; Cancelas, Jose A.

2009-01-01

Fanconi anemia is a severe bone marrow failure syndrome resulting from inactivating mutations of Fanconi anemia pathway genes. Gene and cell therapy trials using hematopoietic stem cells and progenitors have been hampered by poor mobilization of HSC to peripheral blood in response to G-CSF. Using a murine model of Fanconi anemia (Fanca−/− mice), we found that the Fanca deficiency was associated with a profound defect in hematopoietic stem cells and progenitors mobilization in response to G-CSF in absence of bone marrow failure, which correlates with the findings of clinical trials in Fanconi anemia patients. This mobilization defect was overcome by co-administration of the Rac inhibitor NSC23766, suggesting that Rac signaling is implicated in the retention of Fanca−/− hematopoietic stem cells and progenitors in the bone marrow. In view of these data, we propose that targeting Rac signaling may enhance G-CSF-induced HSC mobilization in Fanconi anemia. PMID:19491337

Fanca-/- hematopoietic stem cells demonstrate a mobilization defect which can be overcome by administration of the Rac inhibitor NSC23766.

PubMed

Milsom, Michael D; Lee, Andrew W; Zheng, Yi; Cancelas, Jose A

2009-07-01

Fanconi anemia is a severe bone marrow failure syndrome resulting from inactivating mutations of Fanconi anemia pathway genes. Gene and cell therapy trials using hematopoietic stem cells and progenitors have been hampered by poor mobilization of HSC to peripheral blood in response to G-CSF. Using a murine model of Fanconi anemia (Fanca(-/-) mice), we found that the Fanca deficiency was associated with a profound defect in hematopoietic stem cells and progenitors mobilization in response to G-CSF in absence of bone marrow failure, which correlates with the findings of clinical trials in Fanconi anemia patients. This mobilization defect was overcome by co-administration of the Rac inhibitor NSC23766, suggesting that Rac signaling is implicated in the retention of Fanca(-/-) hematopoietic stem cells and progenitors in the bone marrow. In view of these data, we propose that targeting Rac signaling may enhance G-CSF-induced HSC mobilization in Fanconi anemia.

Osteogenic differentiation capacity of human mesenchymal stromal cells in response to extracellular calcium with special regard to connexin 43.

PubMed

Wagner, Alena-Svenja; Glenske, Kristina; Wolf, Verena; Fietz, Daniela; Mazurek, Sybille; Hanke, Thomas; Moritz, Andreas; Arnhold, Stefan; Wenisch, Sabine

2017-01-01

The effects of extracellular calcium on osteogenic differentiation capacity of human bone-derived mesenchymal stromal cells with special regard to connexin 43 (cx43) have been investigated by means of cell culture experiments. Mesenchymal stromal cells isolated from human cancellous bone were cultured on tissue culture plates at different calcium ion (Ca 2+ ) concentrations (1.8mmoll -1 , 10mmoll -1 , 20mmoll -1 ). Cell responses were evaluated by quantitative RT-PCR, immunofluorescence staining, and Lucifer Yellow fluorescence uptake experiments. It could be shown that increasing Ca 2+ concentrations correlate with increasing cx43 and bone sialoprotein mRNA levels as well as with enhanced cx43 fluorescence signaling and matrix mineralization of the cultures as shown by von Kossa staining. Hemichannel gating - assessed by Lucifer Yellow uptake - increases with increasing extracellular Ca 2+ concentrations suggesting that regulatory effects at the hemichannel level are calcium-dependent. Copyright © 2016 Elsevier GmbH. All rights reserved.

Accentuated Osteoclastic Response to Parathyroid Hormone Undermines Bone Mass Acquisition in Osteonectin-null Mice

PubMed Central

do Reis, Luciene Machado; Kessler, Catherine B.; Adams, Douglas J.; Lorenzo, Joseph; Jorgetti, Vanda; Delany, Anne M.

2008-01-01

Matricellular proteins play a unique role in the skeleton as regulators of bone remodeling, and the matricellular protein osteonectin (SPARC, BM-40) is the most abundant non-collagenous protein in bone. In the absence of osteonectin, mice develop progressive low turnover osteopenia, particularly affecting trabecular bone. Polymorphisms in a regulatory region of the osteonectin gene are associated with bone mass in a subset of idiopathic osteoporosis patients, and these polymorphisms likely regulate osteonectin expression. Thus it is important to determine how osteonectin gene dosage affects skeletal function. Moreover, intermittent administration of parathyroid hormone (PTH) (1-34) is the only anabolic therapy approved for the treatment of osteoporosis, and it is critical to understand how modulators of bone remodeling, such as osteonectin, affect skeletal response to anabolic agents. In this study, 10 week old female wild type, osteonectin-haploinsufficient, and osteonectin-null mice (C57Bl/6 genetic background) were given 80 μg/kg body weight/day PTH(1-34) for 4 weeks. Osteonectin gene dosage had a profound effect on bone microarchitecture. The connectivity density of trabecular bone in osteonectin-haploinsufficient mice was substantially decreased compared with that of wild type mice, suggesting compromised mechanical properties. Whereas mice of each genotype had a similar osteoblastic response to PTH treatment, the osteoclastic response was accentuated in osteonectin-haploinsufficient and osteonectin-null mice. Eroded surface and osteoclast number were significantly higher in PTH-treated osteonectin-null mice, as was endosteal area. In vitro studies confirmed that PTH induced the formation of more osteoclast-like cells in marrow from osteonectin-null mice compared with wild type. PTH treated osteonectin-null bone marrow cells expressed more RANKL mRNA compared with wild type. However, the ratio of RANKL:OPG mRNA was somewhat lower in PTH treated osteonectin-null cultures. Increased expression of RANKL in response to PTH could contribute to the accentuated osteoclastic response in osteonectin-/- mice, but other mechanisms are also likely to be involved. The molecular mechanisms by which PTH elicits bone anabolic vs. bone catabolic effects remain poorly understood. Our results imply that osteonectin levels may play a role in modulating the balance of bone formation and resorption in response to PTH. PMID:18499553

Analogous cellular contribution and healing mechanisms following digit amputation and phalangeal fracture in mice

PubMed Central

Dawson, Lindsay A.; Simkin, Jennifer; Sauque, Michelle; Pela, Maegan; Palkowski, Teresa

2016-01-01

Abstract Regeneration of amputated structures is severely limited in humans and mice, with complete regeneration restricted to the distal portion of the terminal phalanx (P3). Here, we investigate the dynamic tissue repair response of the second phalangeal element (P2) post amputation in the adult mouse, and show that the repair response of the amputated bone is similar to the proximal P2 bone fragment in fracture healing. The regeneration‐incompetent P2 amputation response is characterized by periosteal endochondral ossification resulting in the deposition of new trabecular bone, corresponding to a significant increase in bone volume; however, this response is not associated with bone lengthening. We show that cells of the periosteum respond to amputation and fracture by contributing both chondrocytes and osteoblasts to the endochondral ossification response. Based on our studies, we suggest that the amputation response represents an attempt at regeneration that ultimately fails due to the lack of a distal organizing influence that is present in fracture healing. PMID:27499878

Stem cells and bone: a historical perspective.

PubMed

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.

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

PubMed Central

Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

2009-01-01

Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. Conclusions Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications. PMID:19773264

Synergistic action of the benzene metabolite hydroquinone on myelopoietic stimulating activity of granulocyte/macrophage colony-stimulating factor in vitro

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.

The interactions of the cells in the development of osteoporotic changes in bones under space flight conditions

NASA Astrophysics Data System (ADS)

Rodionova, Natalia; Kabitskaya, Olga

2016-07-01

Using the methods of electron microscopy and autoradiography with ³N-glycine and ³N-thymidine on biosatellites "Bion-11" (Macaca mulatta, the duration of the experiments -10 days), "Bion-M1" (mouse C57 Black, duration of the flight - 30 days) in the experiments with modeled hypokinesia (white rats, hind limbs unloading, the duration of the experiments 28 days) new data about the morpho-functional peculiarities of cellular interactions in adaptive remodeling zones of bone structures under normal conditions and after exposure of animals to microgravity. Our conception on remodeling proposes the following sequence in the development of cellular interactions after decrease of the mechanical loading: a primary response of osteocytes (mechanosensory cells) to the mechanical stimulus; osteocytic remodeling (osteolysis); transmission of the mechanical signals through a system of canals and processes to functionally active osteoblasts and paving endost one as well as to the bone-marrow stromal cells and perivascular cells. As a response to the mechanical stimulus (microgravity) the system of perivascular cell-stromal cell-preosteoblast-osteoblast shows a delay in proliferation, differentiation and specific functioning of the osteogenetic cells, the number of apoptotic osteoblasts increases. Then the osteoclastic reaction occurs (attraction of monocytes and formation of osteoclasts, bone matrix resorption in the loci of apoptosis of osteoblasts and osteocytes). The macrophagal reaction is followed by osteoblastogenesis, which appears to be a rehabilitating process. However, during prolonged absence of mechanical stimuli (microgravity, long-time immobilization) the adaptive activization of osteoblastogenesis doesn't occur (as it is the case during the physiological remodeling of bone tissue) or it occurs to a smaller degree. The loading deficit leads to an adaptive differentiation of stromal cells to fibroblastic cells and adipocytes in remodeling loci. These cell reactions are considered as adaptive-compensatory, but they don't result in rehabilitation of the resorbed bone tissue. This sequence of cells interactions is considered as a mechanism of bone tissue loss which underlies the development of osteopenia and osteoporosis under the mechanical loading deficit.

Predicting the impact of combined therapies on myeloma cell growth using a hybrid multi-scale agent-based model.

PubMed

Ji, Zhiwei; Su, Jing; Wu, Dan; Peng, Huiming; Zhao, Weiling; Nlong Zhao, Brian; Zhou, Xiaobo

2017-01-31

Multiple myeloma is a malignant still incurable plasma cell disorder. This is due to refractory disease relapse, immune impairment, and development of multi-drug resistance. The growth of malignant plasma cells is dependent on the bone marrow (BM) microenvironment and evasion of the host's anti-tumor immune response. Hence, we hypothesized that targeting tumor-stromal cell interaction and endogenous immune system in BM will potentially improve the response of multiple myeloma (MM). Therefore, we proposed a computational simulation of the myeloma development in the complicated microenvironment which includes immune cell components and bone marrow stromal cells and predicted the effects of combined treatment with multi-drugs on myeloma cell growth. We constructed a hybrid multi-scale agent-based model (HABM) that combines an ODE system and Agent-based model (ABM). The ODEs was used for modeling the dynamic changes of intracellular signal transductions and ABM for modeling the cell-cell interactions between stromal cells, tumor, and immune components in the BM. This model simulated myeloma growth in the bone marrow microenvironment and revealed the important role of immune system in this process. The predicted outcomes were consistent with the experimental observations from previous studies. Moreover, we applied this model to predict the treatment effects of three key therapeutic drugs used for MM, and found that the combination of these three drugs potentially suppress the growth of myeloma cells and reactivate the immune response. In summary, the proposed model may serve as a novel computational platform for simulating the formation of MM and evaluating the treatment response of MM to multiple drugs.

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).

T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments.

PubMed

Hawkins, Edwin D; Duarte, Delfim; Akinduro, Olufolake; Khorshed, Reema A; Passaro, Diana; Nowicka, Malgorzata; Straszkowski, Lenny; Scott, Mark K; Rothery, Steve; Ruivo, Nicola; Foster, Katie; Waibel, Michaela; Johnstone, Ricky W; Harrison, Simon J; Westerman, David A; Quach, Hang; Gribben, John; Robinson, Mark D; Purton, Louise E; Bonnet, Dominique; Lo Celso, Cristina

2016-10-27

It is widely accepted that complex interactions between cancer cells and their surrounding microenvironment contribute to disease development, chemo-resistance and disease relapse. In light of this observed interdependency, novel therapeutic interventions that target specific cancer stroma cell lineages and their interactions are being sought. Here we studied a mouse model of human T-cell acute lymphoblastic leukaemia (T-ALL) and used intravital microscopy to monitor the progression of disease within the bone marrow at both the tissue-wide and single-cell level over time, from bone marrow seeding to development/selection of chemo-resistance. We observed highly dynamic cellular interactions and promiscuous distribution of leukaemia cells that migrated across the bone marrow, without showing any preferential association with bone marrow sub-compartments. Unexpectedly, this behaviour was maintained throughout disease development, from the earliest bone marrow seeding to response and resistance to chemotherapy. Our results reveal that T-ALL cells do not depend on specific bone marrow microenvironments for propagation of disease, nor for the selection of chemo-resistant clones, suggesting that a stochastic mechanism underlies these processes. Yet, although T-ALL infiltration and progression are independent of the stroma, accumulated disease burden leads to rapid, selective remodelling of the endosteal space, resulting in a complete loss of mature osteoblastic cells while perivascular cells are maintained. This outcome leads to a shift in the balance of endogenous bone marrow stroma, towards a composition associated with less efficient haematopoietic stem cell function. This novel, dynamic analysis of T-ALL interactions with the bone marrow microenvironment in vivo, supported by evidence from human T-ALL samples, highlights that future therapeutic interventions should target the migration and promiscuous interactions of cancer cells with the surrounding microenvironment, rather than specific bone marrow stroma, to combat the invasion by and survival of chemo-resistant T-ALL cells.

The osteo-inductive activity of bone-marrow-derived mononuclear cells resides within the CD14+ population and is independent of the CD34+ population.

PubMed

Henrich, D; Seebach, C; Verboket, R; Schaible, A; Marzi, I; Bonig, H

2018-03-06

Bone marrow mononuclear cells (BMC) seeded on a scaffold of β-tricalcium phosphate (β-TCP) promote bone healing in a critical-size femur defect model. Being BMC a mixed population of predominantly mature haematopoietic cells, which cell type(s) is(are) instrumental for healing remains elusive. Although clinical therapies using BMC are often dubbed as stem cell therapies, whether stem cells are relevant for the therapeutic effects is unclear and, at least in the context of bone repair, seems dubious. Instead, in light of the critical contribution of monocytes and macrophages to tissue development, homeostasis and injury repair, in the current study it was hypothesised that BMC-mediated bone healing derived from the stem cell population. To test this hypothesis, bone remodelling studies were performed in an established athymic rats critical-size femoral defect model, with β-TCP scaffolds augmented with complete BMC or BMC immunomagnetically depleted of stem cells (CD34+) or monocytes/macrophages (CD14+). Bone healing was assessed 8 weeks after transplantation. Compared to BMC-augmented controls, when CD14- BMC, but not CD34- BMC were transplanted into the bone defect, femora possessed dramatically decreased biomechanical stability and new bone formation was markedly reduced, as measured by histology. The degree of vascularisation did not differ between the two groups. It was concluded that the monocyte fraction within the BMC provided critical osteo-inductive cues during fracture healing. Which factors were responsible at the molecular levels remained elusive. However, this study marked a significant progress towards elucidating the mechanisms by which BMC elicit their therapeutic effects, at least in bone regeneration.

Interactions between MSCs and Immune Cells: Implications for Bone Healing

PubMed Central

Kovach, Tracy K.; Dighe, Abhijit S.; Lobo, Peter I.; Cui, Quanjun

2015-01-01

It is estimated that, of the 7.9 million fractures sustained in the United States each year, 5% to 20% result in delayed or impaired healing requiring therapeutic intervention. Following fracture injury, there is an initial inflammatory response that plays a crucial role in bone healing; however, prolonged inflammation is inhibitory for fracture repair. The precise spatial and temporal impact of immune cells and their cytokines on fracture healing remains obscure. Some cytokines are reported to be proosteogenic while others inhibit bone healing. Cell-based therapy utilizing mesenchymal stromal cells (MSCs) is an attractive option for augmenting the fracture repair process. Osteoprogenitor MSCs not only differentiate into bone, but they also exert modulatory effects on immune cells via a variety of mechanisms. In this paper, we review the current literature on both in vitro and in vivo studies on the role of the immune system in fracture repair, the use of MSCs in the enhancement of fracture healing, and interactions between MSCs and immune cells. Insight into this paradigm can provide valuable clues in identifying cellular and noncellular targets that can potentially be modulated to enhance both natural bone healing and bone repair augmented by the exogenous addition of MSCs. PMID:26000315

Analyses of the combination of 6-MP and dasatinib in cell culture

PubMed Central

KAUR, GURMEET; BEHRSING, HOLGER; PARCHMENT, RALPH E.; MILLIN, MYRTLE DAVIS; TEICHER, BEVERLY A.

2013-01-01

A major tenet of cancer therapeutics is that combinations of anticancer agents with different mechanisms of action and different toxicities may be effective treatment regimens. Evaluation of additivity/synergy in cell culture may be used to identify drug combination opportunities and to assess risk of additive/synergistic toxicity. The combination of 6-mercaptopurine and dasatinib was assessed for additivity/synergy using the combination index (CI) method and a response surface method in six human tumor cell lines including MCF-7 and MDA-MB-468 breast cancer, NCI-H23 and NCI-H460 non-small cell lung cancer, and A498 and 786-O renal cell cancer, based on two experimental end-points: ATP content and colony formation. Clonal colony formation by human bone marrow CFU-GM was used to assess risk of enhanced toxicity. The concentration ranges tested for each drug were selected to encompass the clinical Cmax concentrations. The combination regimens were found to be additive to sub-additive by both methods of data analysis, but synergy was not detected. The non-small cell lung cancer cell lines were the most responsive among the tumor lines tested and the renal cell carcinoma lines were the least responsive. The bone marrows CFU-GM were more sensitive to the combination regimens than were the tumor cell lines. Based upon these data, it appears that the possibility of enhanced efficacy from combining 6-mercaptopurine (6-MP) and dasatinib would be associated with increased risk of severe bone marrow toxicity, so the combination is unlikely to provide a therapeutic advantage for treating solid tumor patients where adequate bone marrow function must be preserved. PMID:23652925

Regulation of proliferation of rat cartilage and bone by sex steroid hormones.

PubMed

Sömjen, D; Weisman, Y; Mor, Z; Harell, A; Kaye, A M

1991-01-01

We have demonstrated previously that 17 beta-estradiol (E2) stimulates proliferation of skeletal tissues, both in vivo and in vitro, as measured by increased DNA synthesis and creatine kinase (CK) specific activity. The effect of E2 on bone is sex specific. E2 is active only in females and androgens only in males. By contrast, in cartilage of both sexes, dihydrotestosterone (DHT) as well as E2 stimulates CK specific activity and DNA synthesis. In bone, we find that sex steroids stimulate skeletal cell proliferation in gonadectomized as well as in immature rats. Ovariectomized (OVX) rats, between 1 and 4 weeks after surgery, show stimulation of CK by E2. The basal activity and response of CK changes with the varying endogenous levels of E2 in cycling rats, in which the highest basal activity is at proestrus and estrus and the highest response is in diestrus. In rats of all ages tested, both the basal and stimulated specific activity of CK is higher in diaphysis and epiphysis than in the uterus, or in the adipose tissue adjacent to the uterus, which has a response similar to that of the uterus itself. The effect of E2 in vivo, and in chrondroblasts and osteoblasts in vitro, is inhibited by high levels of the antiestrogen tamoxifen which, by itself, in similar high concentrations, shows stimulatory effects. In addition to the sex steroids, skeletal cells are also stimulated by secosteroid and peptide calciotrophic hormones. The interactions of the sex steroids with these hormones modulate the response of cartilage and bone cells to both sex steroids and the other calciotrophic hormones. These results provide the first steps towards understanding the regulation of bone cell proliferation and growth by the concerted action of a variety of hormones and growth factors.

Defective B cell response to T-dependent immunization in lupus-prone mice

PubMed Central

Niu, Haitao; Sobel, Eric S.; Morel, Laurence

2009-01-01

Lupus anti-nuclear Abs show the characteristics of Ag-driven T cell-dependent (TD) humoral responses. If autoAgs elicit the same response as exogenous Ags, lupus should enhance humoral responses to immunization. Blunted responses to various immunizations have, however, been reported in a significant portion of lupus patients. In this study, we show that lupus-prone B6.Sle1.Sle2.Sle3 (B6.TC) mice produce significantly less Ab in response to TD immunization than congenic controls, while producing significantly more total Ig. This blunted Ab response to TD Ag could be reconstituted with B6.TC B and CD4+ T cells. Multiple defects were found in the B6.TC response to NP-KLH as compared to total Ig, including a smaller percentage of B cells participating to the NP-response, a reduced entry into germinal centers, and highly defective production of NP-specific long-lived plasma cells in the bone marrow. B6.TC plasma cells expressed reduced levels of FcγRIIb, which suggests that reduced apoptosis in resident plasma cells prevents the establishment of newly-formed NP-specific plasma cells in bone marrow niches. Overall, these results show that lupus-prone mice responded differently to auto- and exogenous antigens and suggest that low FcγRIIb, hypergammaglobulinemia and high autoantibody production would be predictive of a poor response to immunization in lupus patients. PMID:18924209

[Headache as a manifestation of SAPHO syndrome with a lesion extending to the dura mater, parietal bone, and temporal muscle].

PubMed

Uematsu, Miho; Tobisawa, Shinsuke; Nagao, Masahiro; Matsubara, Shiro; Mizutani, Toshio; Shibuya, Makoto

2012-01-01

A 50-year-old woman with a history of palmoplantar pustulosis, femur osteomyelitis, and sterno-costo-clavicular hyperostosis presented with a chronic severe left temporal headache that had progressed during the previous year. Her CRP level was elevated. Cranial images showed Gadolinium-enhancement of the left temporal muscle, left parietal bone and dura mater. (99m)Tc-HMDP scintigram showed increased uptake in the left parietal bone, left sterno-costo-clavicular joint, right femoral head and intervertebral joints. Biopsy of the lesion demonstrated 1) proliferation of connective tissue in both perimysium and endomysium of the temporal muscle with mild inflammatory cell infiltration within the interstitium, 2) marked infiltration of granulocytes to the bone marrow of the parietal bone, 3) necrosis and moderate fibrosis in the interstitium with inflammatory cell infiltration in the parietal bone, and 4) moderate fibrosis and slight infiltration of inflammatory cells in the dura mater. The patient was diagnosed with a cranial lesion of synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome. There was a moderate response to treatment with intravenous steroid pulse therapy and subsequent methotrexate. In a case of headache accompanied by inflammatory response, palmoplantar pustulosis and joint lesions such as hyperostosis, the possibility of a rare cranial manifestation of SAPHO syndrome should be considered.

External bone marrow cytological examination quality assurance (EQAhem)--summary after 6 years in Poland.

PubMed

Lewandowski, Krzysztof; Kurpierz, Katarzyna; Sledzinska, Anna

2015-10-01

Bone marrow macroscopic examination remains one of the most difficult and subjective laboratory assessments in hematology. Only a few external quality assurance programs in the field are present worldwide. We have developed an external quality assurance program EQAhem that allows assessment of the whole process of bone marrow examination. The program participants assess blood and bone marrow smears from the patient, identify selected cells from photographs provided to them, and interpret the microscopic results. In this article, the results of the EQAhem program in Poland from 6 years are summarized. During this time, 62 labs were assessed in total, and positive results were achieved by 89.25 % labs, taking into account all tests. Correct responses with respect to the percentage of cell count were provided by ca. 77.5 % labs. Slightly worse results were obtained when megakaryocyte count and cell identification from photographs were tested. The worst results were obtained in case of dysplasia assessment and clinical interpretation of microscopic examination (54.1 and 58.6 % correct responses, respectively). EQAhem delivers precise information about the quality of bone marrow examinations performed in Poland and has a substantial educational value. We believe that after 6 years, EQAhem has significantly improved the quality of bone marrow microscopic examinations performed in Poland.

Preventing painful age-related bone fractures: Anti-sclerostin therapy builds cortical bone and increases the proliferation of osteogenic cells in the periosteum of the geriatric mouse femur.

PubMed

Thompson, Michelle L; Chartier, Stephane R; Mitchell, Stefanie A; Mantyh, Patrick W

2016-01-01

Age-related bone fractures are usually painful and have highly negative effects on a geriatric patient's functional status, quality of life, and survival. Currently, there are few analgesic therapies that fully control bone fracture pain in the elderly without significant unwanted side effects. However, another way of controlling age-related fracture pain would be to preemptively administer an osteo-anabolic agent to geriatric patients with high risk of fracture, so as to build new cortical bone and prevent the fracture from occurring. A major question, however, is whether an osteo-anabolic agent can stimulate the proliferation of osteogenic cells and build significant amounts of new cortical bone in light of the decreased number and responsiveness of osteogenic cells in aging bone. To explore this question, geriatric and young mice, 20 and 4 months old, respectively, received either vehicle or a monoclonal antibody that sequesters sclerostin (anti-sclerostin) for 28 days. From days 21 to 28, animals also received sustained administration of the thymidine analog, bromodeoxyuridine (BrdU), which labels the DNA of dividing cells. Animals were then euthanized at day 28 and the femurs were examined for cortical bone formation, bone mineral density, and newly borne BrdU+ cells in the periosteum which is a tissue that is pivotally involved in the formation of new cortical bone. In both the geriatric and young mice, anti-sclerostin induced a significant increase in the thickness of the cortical bone, bone mineral density, and the proliferation of newly borne BrdU+ cells in the periosteum. These results suggest that even in geriatric animals, anti-sclerostin therapy can build new cortical bone and increase the proliferation of osteogenic cells and thus reduce the likelihood of painful age-related bone fractures. © The Author(s) 2016.

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

PubMed Central

Nollet, Marie; Santucci-Darmanin, Sabine; Breuil, Véronique; Al-Sahlanee, Rasha; Cros, Chantal; Topi, Majlinda; Momier, David; Samson, Michel; Pagnotta, Sophie; Cailleteau, Laurence; Battaglia, Séverine; Farlay, Delphine; Dacquin, Romain; Barois, Nicolas; Jurdic, Pierre; Boivin, Georges; Heymann, Dominique; Lafont, Frank; Lu, Shi Shou; Dempster, David W; Carle, Georges F; Pierrefite-Carle, Valérie

2014-01-01

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies. PMID:25484092

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.

Osteoclast Progenitors Reside in the Peroxisome Proliferator-Activated Receptor γ-Expressing Bone Marrow Cell Population ▿

PubMed Central

Wei, Wei; Zeve, Daniel; Wang, Xueqian; Du, Yang; Tang, Wei; Dechow, Paul C.; Graff, Jonathan M.; Wan, Yihong

2011-01-01

Osteoclasts are bone-resorbing cells essential for skeletal development, homeostasis, and regeneration. They derive from hematopoietic progenitors in the monocyte/macrophage lineage and differentiate in response to RANKL. However, the precise nature of osteoclast progenitors is a longstanding and important question. Using inducible peroxisome proliferator-activated receptor γ (PPARγ)-tTA TRE-GFP (green fluorescent protein) reporter mice, we show that osteoclast progenitors reside specifically in the PPARγ-expressing hematopoietic bone marrow population and identify the quiescent PPARγ+ cells as osteoclast progenitors. Importantly, two PPARγ-tTA TRE-Cre-controlled genetic models provide compelling functional evidence. First, Notch activation in PPARγ+ cells causes high bone mass due to impaired osteoclast precursor proliferation. Second, selective ablation of PPARγ+ cells by diphtheria toxin also causes high bone mass due to decreased osteoclast numbers. Furthermore, PPARγ+ cells respond to both pathological and pharmacological resorption-enhancing stimuli. Mechanistically, PPARγ promotes osteoclast progenitors by activating GATA2 transcription. These findings not only identify the long-sought-after osteoclast progenitors but also establish unprecedented tools for their visualization, isolation, characterization, and genetic manipulation. PMID:21947280

Effect of metabolic and respiratory acidosis on intracellular calcium in osteoblasts

PubMed Central

Bushinsky, David A.

2010-01-01

In vivo, metabolic acidosis {decreased pH from decreased bicarbonate concentration ([HCO3−])} increases urine calcium (Ca) without increased intestinal Ca absorption, resulting in a loss of bone Ca. Conversely, respiratory acidosis [decreased pH from increased partial pressure of carbon dioxide (Pco2)] does not appreciably alter Ca homeostasis. In cultured bone, chronic metabolic acidosis (Met) significantly increases cell-mediated net Ca efflux while isohydric respiratory acidosis (Resp) does not. The proton receptor, OGR1, appears critical for cell-mediated, metabolic acid-induced bone resorption. Perfusion of primary bone cells or OGR1-transfected Chinese hamster ovary (CHO) cells with Met induces transient peaks of intracellular Ca (Cai). To determine whether Resp increases Cai, as does Met, we imaged Cai in primary cultures of bone cells. pH for Met = 7.07 ([HCO3−] = 11.8 mM) and for Resp = 7.13 (Pco2 = 88.4 mmHg) were similar and lower than neutral (7.41). Both Met and Resp induced a marked, transient increase in Cai in individual bone cells; however, Met stimulated Cai to a greater extent than Resp. We used OGR1-transfected CHO cells to determine whether OGR1 was responsible for the greater increase in Cai in Met than Resp. Both Met and Resp induced a marked, transient increase in Cai in OGR1-transfected CHO cells; however, in these cells Met was not different than Resp. Thus, the greater induction of Cai by Met in primary bone cells is not a function of OGR1 alone, but must involve H+ receptors other than OGR1, or pathways sensitive to Pco2, HCO3−, or total CO2 that modify the effect of H+ in primary bone cells. PMID:20504884

Effect of metabolic and respiratory acidosis on intracellular calcium in osteoblasts.

PubMed

Frick, Kevin K; Bushinsky, David A

2010-08-01

In vivo, metabolic acidosis {decreased pH from decreased bicarbonate concentration ([HCO(3)(-)])} increases urine calcium (Ca) without increased intestinal Ca absorption, resulting in a loss of bone Ca. Conversely, respiratory acidosis [decreased pH from increased partial pressure of carbon dioxide (Pco(2))] does not appreciably alter Ca homeostasis. In cultured bone, chronic metabolic acidosis (Met) significantly increases cell-mediated net Ca efflux while isohydric respiratory acidosis (Resp) does not. The proton receptor, OGR1, appears critical for cell-mediated, metabolic acid-induced bone resorption. Perfusion of primary bone cells or OGR1-transfected Chinese hamster ovary (CHO) cells with Met induces transient peaks of intracellular Ca (Ca(i)). To determine whether Resp increases Ca(i), as does Met, we imaged Ca(i) in primary cultures of bone cells. pH for Met = 7.07 ([HCO(3)(-)] = 11.8 mM) and for Resp = 7.13 (Pco(2) = 88.4 mmHg) were similar and lower than neutral (7.41). Both Met and Resp induced a marked, transient increase in Ca(i) in individual bone cells; however, Met stimulated Ca(i) to a greater extent than Resp. We used OGR1-transfected CHO cells to determine whether OGR1 was responsible for the greater increase in Ca(i) in Met than Resp. Both Met and Resp induced a marked, transient increase in Ca(i) in OGR1-transfected CHO cells; however, in these cells Met was not different than Resp. Thus, the greater induction of Ca(i) by Met in primary bone cells is not a function of OGR1 alone, but must involve H(+) receptors other than OGR1, or pathways sensitive to Pco(2), HCO(3)(-), or total CO(2) that modify the effect of H(+) in primary bone cells.

Defective bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase.

PubMed

Armour, K E; Armour, K J; Gallagher, M E; Gödecke, A; Helfrich, M H; Reid, D M; Ralston, S H

2001-02-01

Nitric oxide (NO) is a pleiotropic signaling molecule that is produced by bone cells constitutively and in response to diverse stimuli such as proinflammatory cytokines, mechanical strain, and sex hormones. Endothelial nitric oxide synthase (eNOS) is the predominant NOS isoform expressed in bone, but its physiological role in regulating bone metabolism remains unclear. Here we studied various aspects of bone metabolism in female mice with targeted disruption of the eNOS gene. Mice with eNOS deficiency (eNOS KO) had reduced bone mineral density, and cortical thinning when compared with WT controls and histomorphometric analysis of bone revealed profound abnormalities of bone formation, with reduced osteoblast numbers, surfaces and mineral apposition rate. Studies in vitro showed that osteoblasts derived from eNOS KO mice had reduced rates of growth when compared with WT and were less well differentiated as reflected by lower levels of alkaline phosphatase activity. Mice with eNOS deficiency lost bone normally following ovariectomy but exhibited a significantly blunted anabolic response to high dose exogenous estrogen. We conclude that the eNOS pathway plays an essential role in regulating bone mass and bone turnover by modulating osteoblast function.

Dysregulated microRNA Activity in Shwachman-Diamond Syndrome

DTIC Science & Technology

2016-09-01

define transcriptional signatures of bone marrow failure in SDS using single cell RNA -seq of patient cells. We will analyze these datasets to test the...microRNA expression profiles from HSPCs to be overlaid onto mRNA profiles. 15. SUBJECT TERMS Single cell RNA -seq; bone marrow failure; hematopoiesis...myelopoiesis; targeted RNA -seq 16. SECURITY CLASSIFICATION OF: U 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON

Sanguiin H-6, a constituent of Rubus parvifolius L., inhibits receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis and bone resorption in vitro and prevents tumor necrosis factor-α-induced osteoclast formation in vivo.

PubMed

Sakai, Eiko; Aoki, Yuri; Yoshimatsu, Masako; Nishishita, Kazuhisa; Iwatake, Mayumi; Fukuma, Yutaka; Okamoto, Kuniaki; Tanaka, Takashi; Tsukuba, Takayuki

2016-07-15

Osteoclasts are multinucleated bone-resorbing cells that differentiate in response to receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). Enhanced osteoclastogenesis contributes to bone diseases, such as osteoporosis and rheumatoid arthritis. Rubus parvifolius L. is traditionally used as an herbal medicine for rheumatism; however, its detailed chemical composition and the molecular mechanisms responsible for its biological action have not been elucidated. To investigate the mechanisms by which R. parvifolius L. extract and its major constituent sanguiin H-6, inhibit osteoclastogenesis and bone resorption. Cell proliferation, cell differentiation, and bone resorption were detected in vitro. Inhibition of signaling pathways, marker protein expression, and protein nuclear translocation were evaluated by western blot analysis. Tumor necrosis factor-α (TNF-α)-mediated osteoclastogenesis was examined in vivo. R. parvifolius L. extract inhibited the bone-resorption activity of osteoclasts. In addition, sanguiin H-6 markedly inhibited RANKL-induced osteoclast differentiation and bone resorption, reduced reactive oxygen species production, and inhibited the phosphorylation of inhibitor of NF-κB alpha (IκBα) and p38 mitogen-activated protein kinase. Sanguiin H-6 also decreased the protein levels of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), cathepsin K, and c-Src. Moreover, sanguiin H-6 inhibited the nuclear translocation of NFATc1, c-Fos, and NF-κB in vitro, as well as TNF-α-mediated osteoclastogenesis in vivo. Our data revealed that R. parvifolius L. has anti-bone resorption activity and suggest that its constituent, sanguiin H-6, can potentially be used for the prevention and treatment of bone diseases associated with excessive osteoclast formation and subsequent bone destruction. Copyright © 2016 Elsevier GmbH. All rights reserved.

Impaired endothelial progenitor cell mobilization and dysfunctional bone marrow stroma in diabetes mellitus.

PubMed

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.

Tissue-engineered composite scaffold of poly(lactide-co-glycolide) and hydroxyapatite nanoparticles seeded with autologous mesenchymal stem cells for bone regeneration*

PubMed Central

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

Tissue-engineered composite scaffold of poly(lactide-co-glycolide) and hydroxyapatite nanoparticles seeded with autologous mesenchymal stem cells for bone regeneration.

PubMed

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.

Effects of microgravity on rat bone, cartlage and connective tissues

NASA Technical Reports Server (NTRS)

Doty, S.

1990-01-01

The response to hypogravity by the skeletal system was originally thought to be the result of a reduction in weight bearing. Thus a reduced rate of new bone formation in the weight-bearing bones was accepted, when found, as an obvious result of hypogravity. However, data on non-weight-bearing tissues have begun to show that other physiological changes can be expected to occur to animals during spaceflight. This overview of the Cosmos 1887 data discusses these results as they pertain to individual bones or tissues because the response seems to depend on the architecture and metabolism of each tissue under study. Various effects were seen in different tissues from the rats flown on Cosmos 1887. The femur showed a reduced bone mineral content but only in the central region of the diaphysis. This same region in the tibia showed changes in the vascularity of bone as well as some osteocytic cell death. The humerus demonstrated reduced morphometric characteristics plus a decrease in mechanical stiffness. Bone mineral crystals did not mature normally as a result of flight, suggesting a defect in the matrix mineralization process. Note that these changes relate directly to the matrix portion of the bone or some function of bone which slowly responds to changes in the environment. However, most cellular functions of bone are rapid responders. The stimulation of osteoblast precursor cells, the osteoblast function in collagen synthesis, a change in the proliferation rate of cells in the epiphyseal growth plate, the synthesis and secretion of osteocalcin, and the movement of water into or out of tissues, are all processes which respond to environmental change. These rapidly responding events produced results from Cosmos 1887 which were frequently quite different from previous space flight data.

Cellular Sites of Immunologic Unresponsiveness*

PubMed Central

Chiller, Jacques M.; Habicht, Gail S.; Weigle, William O.

1970-01-01

The reconstitution of the immune response of lethally irradiated mice to human γ-globulin is dependent on the synergistic action of bone marrow with thymus cells. Immunologic unresponsiveness appears to involve a functional defect at each of these cellular levels, inasmuch as neither bone marrow nor thymus cells from unresponsive donors are capable of demonstrating synergism in combination with their normal counterpart. PMID:4192271

A Cellular Automata Model of Bone Formation

PubMed Central

Van Scoy, Gabrielle K.; George, Estee L.; Asantewaa, Flora Opoku; Kerns, Lucy; Saunders, Marnie M.; Prieto-Langarica, Alicia

2017-01-01

Bone remodeling is an elegantly orchestrated process by which osteocytes, osteoblasts and osteoclasts function as a syncytium to maintain or modify bone. On the microscopic level, bone consists of cells that create, destroy and monitor the bone matrix. These cells interact in a coordinated manner to maintain a tightly regulated homeostasis. It is this regulation that is responsible for the observed increase in bone gain in the dominant arm of a tennis player and the observed increase in bone loss associated with spaceflight and osteoporosis. The manner in which these cells interact to bring about a change in bone quality and quantity has yet to be fully elucidated. But efforts to understand the multicellular complexity can ultimately lead to eradication of metabolic bone diseases such as osteoporosis and improved implant longevity. Experimentally validated mathematical models that simulate functional activity and offer eventual predictive capabilities offer tremendous potential in understanding multicellular bone remodeling. Here we undertake the initial challenge to develop a mathematical model of bone formation validated with in vitro data obtained from osteoblastic bone cells induced to mineralize and quantified at 26 days of culture. A cellular automata model was constructed to simulate the in vitro characterization. Permutation tests were performed to compare the distribution of the mineralization in the cultures and the distribution of the mineralization in the mathematical models. The results of the permutation test show the distribution of mineralization from the characterization and mathematical model come from the same probability distribution, therefore validating the cellular automata model. PMID:28189632

INF-γ encoding plasmid administration triggers bone loss and disrupts bone marrow microenvironment.

PubMed

Agas, Dimitrios; Gusmão Silva, Guilherme; Laus, Fulvio; Marchegiani, Andrea; Capitani, Melania; Vullo, Cecilia; Catone, Giuseppe; Lacava, Giovanna; Concetti, Antonio; Marchetti, Luigi; Sabbieti, Maria Giovanna

2017-02-01

IFN-γ is a pleotropic cytokine produced in the bone microenvironment. Although IFN-γ is known to play a critical role on bone remodeling, its function is not fully elucidated. Consistently, outcomes on the effects of IFN-γ recombinant protein on bone loss are contradictory among reports. In our work we explored, for the first time, the role of IFN-γ encoding plasmid (pIFN-γ) in a mouse model of osteopenia induced by ovariectomy and in the sham-operated counterpart to estimate its effects in skeletal homeostasis. Ovariectomy produced a dramatic decrease of bone mineral density (BMD). pINF-γ injected mice showed a pathologic bone and bone marrow phenotype; the disrupted cortical and trabecular bone microarchitecture was accompanied by an increased release of pro-inflammatory cytokine by bone marrow cells. Moreover, mesenchymal stem cells' (MSCs) commitment to osteoblast was found impaired, as evidenced by the decline of osterix-positive (Osx + ) cells within the mid-diaphyseal area of femurs. For instance, a reduction and redistribution of CXCL12 cells have been found, in accordance with bone marrow morphological alterations. As similar effects were observed both in sham-operated and in ovariectomized mice, our studies proved that an increased IFN-γ synthesis in bone marrow might be sufficient to induce inflammatory and catabolic responses even in the absence of pathologic predisposing substrates. In addition, the obtained data might raise questions about pIFN-γ's safety when it is used as vaccine adjuvant. © 2017 Society for Endocrinology.

From Milk to Bones, Moving Calcium Through the Body: Calcium Kinetics During Space Flight

NASA Technical Reports Server (NTRS)

Smith, Scott; Bloomberg, Jacob; Lee, Angie (Technical Monitor)

2002-01-01

Did you know that when astronauts are in space, their height increases about two inches? This happens because the weightlessness of space allows the spine, usually compressed in Earth's gravity, to expand. While this change is relatively harmless, other more serious things can happen with extended stays in weightlessness, notably bone loss. From previous experiments, scientists have observed that astronauts lose bone mass at a rate of about one percent per month during flight. Scientists know that bone is a dynamic tissue - continually being made and repaired by specialized bone cells throughout life. Certain cells produce new bone, while other cells are responsible for removing and replacing old bone. Research on the mechanisms of bone metabolism and the effects of space flight on its formation and repair are part of the exciting studies that will be performed during STS-107. Calcium plays a central role because 1) it gives strength and structure to bone and 2) all types of cells require it to function normally. Ninety-nine percent of calcium in the body is stored in the skeleton. However, calcium may be released, or resorbed, from bone to provide for other tissues when you are not eating. To better understand how and why weightlessness induces bone loss, astronauts will participate in a study of calcium kinetics - that is, the movement of calcium through the body, including absorption from food, and its role in the formation and breakdown of bone.

Evaluation of erythroblast macrophage protein related to erythroblastic islands in patients with hematopoietic stem cell transplantation

PubMed Central

2013-01-01

Background Hematopoietic evaluation of the patients after Hematopoietic stem cell transplantation (HSCT) is very important. Erythroblast macrophage protein (Emp) is a key protein with function in normal differentiation of erythroid cells and macrophages. Emp expression correlates with erythroblastic island formation, a process widely believed to be associated with hematopoiesis in bone marrow. We aimed to investigate the hematopoietic function of bone marrow from 46 HSCT patients and 16 inpatients with severe anemia applied to the treatment of EPO by measuring Emp expression level. Methods Emp mRNA and protein expression levels in mononuclear cells of bone marrow and peripheral blood samples were detected by RT-PCR and Western blotting method respectively. Results While hematopoiesis occurs in bone marrow, Emp expression level was elevated and more erythroblastic islands were found , and Emp is upregulated in bone marrow in response to erythropoietin (EPO) treatment. Conclusions Emp expression correlates with erythroblastic island formation and has an important function for bone marrow hematopoiesis. Emp could be a potential biomarker for hematopoietic evaluation of HSCT patients. PMID:23566571

ω-3 Fatty Acids Reduce Chemotherapy-Induced Hematological Toxicity by Bone Marrow Stimulation in Mice.

PubMed

Murakami, Kohei; Miyata, Hiroshi; Miyazaki, Yasuhiro; Makino, Tomoki; Takahashi, Tsuyoshi; Kurokawa, Yukinori; Yamasaki, Makoto; Nakajima, Kiyokazu; Takiguchi, Shuji; Mori, Masaki; Doki, Yuichiro

2017-07-01

ω-3 Fatty acids exert several benefits during chemotherapy, such as preventing intestinal mucosal damage and improving response to chemotherapy. However, little is known about the effect of ω-3 fatty acids on chemotherapy-induced hematological toxicities. Mice that had consumed either an ω-3-rich or an ω-3-poor diet for 2 weeks were intraperitoneally administered cisplatin. The resultant changes in blood cell count, bone marrow cell count, and cytokine levels in bone marrow supernatant were analyzed. The effect of ω-3 fatty acids on human peripheral blood mononuclear cells (PBMCs) exposed to cisplatin was also examined. Although peripheral blood cell counts decreased after cisplatin treatment in both groups of mice, the decrease in white blood cell count was significantly lower in mice that consumed the ω-3-rich diet. The decrease in bone marrow cells after cisplatin treatment was also reduced in mice that consumed the ω-3-rich diet. Levels of stem cell factor (SCF) and fibroblast growth factor 1 (FGF-1) were significantly higher in bone marrow supernatants from mice that consumed the ω-3-rich diet. The rate of apoptosis in PBMCs (after exposure to cisplatin) cultured in medium containing ω-3 fatty acids was significantly lower than in PBMCs cultured in control medium. ω-3-Rich diets reduced chemotherapy-induced leukopenia in mice. This may be the result of increased numbers of bone marrow cells due to higher levels of SCF and FGF-1 in the bone marrow.

Cellular and molecular mechanisms for the bone response to mechanical loading

NASA Technical Reports Server (NTRS)

Bloomfield, S. A.

2001-01-01

To define the cellular and molecular mechanisms for the osteogenic response of bone to increased loading, several key steps must be defined: sensing of the mechanical signal by cells in bone, transduction of the mechanical signal to a biochemical one, and transmission of that biochemical signal to effector cells. Osteocytes are likely to serve as sensors of loading, probably via interstitial fluid flow produced during loading. Evidence is presented for the role of integrins, the cell's actin cytoskeleton, G proteins, and various intracellular signaling pathways in transducing that mechanical signal to a biochemical one. Nitric oxide, prostaglandins, and insulin-like growth factors all play important roles in these pathways. There is growing evidence for modulation of these mechanotransduction steps by endocrine factors, particularly parathyroid hormone and estrogen. The efficiency of this process is also impaired in the aged animal, yet what remains undefined is at what step mechanotransduction is affected.

Research on dental implant and its industrialization stage

NASA Astrophysics Data System (ADS)

Dongjoon, Yang; Sukyoung, Kim

2017-02-01

Bone cell attachment to Ti implant surfaces is the most concerned issue in the clinical implant dentistry. Many attempts to achieve the fast and strong integration between bone and implant have been tried in many ways, such as selection of materials (for example, Ti, ZrO2), shape design of implant (for example, soft tissue level, bone level, taped or conical, etc), and surface modification of implants (for example, roughed. coated, hybrid), etc. Among them, a major consideration is the surface design of dental implants. The surface with proper structural characteristics promotes or induces the desirable responses of cells and tissues. To obtain such surface which has desirable cell and tissue response, a variety of surface modification techniques has been developed and employed for many years. In this review, the method and trend of surface modification will be introduced and explained in terms of the surface topography and chemistry of dental implants.

Bone marrow vascular endothelial growth factor level per platelet count might be a significant predictor for the treatment outcomes of patients with diffuse large B-cell lymphomas.

PubMed

Kim, Jung Sun; Gang, Ga Won; Lee, Se Ryun; Sung, Hwa Jung; Park, Young; Kim, Dae Sik; Choi, Chul Won; Kim, Byung Soo

2015-10-01

Developing a parameter to predict bone marrow invasion by non-Hodgkin's lymphoma is an important unmet medical need for treatment decisions. This study aimed to confirm the validity of the hypothesis that bone marrow plasma vascular endothelial growth factor level might be correlated with the risk of bone marrow involvement and the prognosis of patients with diffuse large B-cell non-Hodgkin's lymphoma. Forty-nine diffuse large B-cell lymphoma patients treated with rituximab, cyclophosphamide, daunorubicin, vincristine and prednisolone regimen were enrolled. Vascular endothelial growth factor level was measured with enzyme-linked immunosorbent assay. The validity of bone marrow plasma vascular endothelial growth factor level and bone marrow vascular endothelial growth factor level per platelet count for predicting treatment response and survival after initial rituximab, cyclophosphamide, daunorubicin, vincristine and prednisolone combined chemotherapy was assessed. Bone marrow plasma vascular endothelial growth factor level per platelet count was significantly associated with old age (≥ 65 years), poor performance score (≥ 2), high International prognosis index (≥ 3) and bone marrow invasion. The patients with high bone marrow plasma vascular endothelial growth factor level per platelet count (≥ 3.01) showed a significantly lower complete response rate than the others. On Kaplan-Meier survival curves, the patients with high bone marrow plasma vascular endothelial growth factor levels (≥ 655 pg/ml) or high bone marrow plasma vascular endothelial growth factor level per platelet count (≥ 3.01) demonstrated a significantly shorter overall survival and progression-free survival than the others. In the patients without bone marrow involvement, bone marrow plasma vascular endothelial growth factor level per platelet count had a significant relationship with overall survival and progression-free survival. Multivariate analysis revealed that the patients without BM invasion showing high level of bone marrow plasma vascular endothelial growth factor per platelet count had significantly shorter progression-free survival and overall survival. Bone marrow plasma vascular endothelial growth factor level per platelet count might be associated with bone marrow invasion by diffuse large B-cell lymphoma and is correlated with clinical outcomes after treatment. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The Transplantation of hBM-MSCs Increases Bone Neo-Formation and Preserves Hearing Function in the Treatment of Temporal Bone Defects - on the Experience of Two Month Follow Up.

PubMed

Školoudík, Lukáš; Chrobok, Viktor; Kočí, Zuzana; Popelář, Jiří; Syka, Josef; Laco, Jan; Filipová, Alžběta; Syková, Eva; Filip, Stanislav

2018-06-03

Temporal bone reconstruction is a persisting problem following middle ear cholesteatoma surgery. Seeking to advance the clinical transfer of stem cell therapy we attempted the reconstruction of temporal bone using a composite bioartificial graft based on a hydroxyapatite bone scaffold combined with human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). The aim of this study was to evaluate the effect of the combined biomaterial on the healing of postoperative temporal bone defects and the preservation of physiological hearing functions in a guinea pig model. The treatment's effect could be observed at 1 and 2 months after implantation of the biomaterial, as opposed to the control group. The clinical evaluation of our results included animal survival, clinical signs of an inflammatory response, and exploration of the tympanic bulla. Osteogenesis, angiogenesis, and inflammation were evaluated by histopathological analyses, whereas hBM-MSCs survival was evaluated by immunofluorescence assays. Hearing capacity was evaluated by objective audiometric methods, i.e. auditory brainstem responses and otoacoustic emission. Our study shows that hBM-MSCs, in combination with hydroxyapatite scaffolds, improves the repair of bone defects providing a safe and effective alternative in their treatment following middle ear surgery due to cholesteatoma.

Three-Dimensional Mechanical Loading Modulates the Osteogenic Response of Mesenchymal Stem Cells to Tumor-Derived Soluble Signals.

PubMed

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.

Esophageal squamous cell carcinoma with dural and bone marrow metastases.

PubMed

Chen, Yen-Hao; Huang, Cheng-Hua

2014-09-21

Patients with esophageal squamous cell carcinoma generally present at an advanced stage at the time of diagnosis. The most common sites of visceral metastasis are the lung, liver and bone, but brain and bone marrow involvement is exceedingly rare. Herein, we report a 62-year-old man with a 4-wk history of progressive low back pain with radiation to bilateral lower legs, dysphagia and body weight loss. Esophageal squamous cell carcinoma with regional lymph node, liver and bone metastases was diagnosed. He underwent concurrent chemoradiotherapy and got a partial response. Four months later, he complained of headache, diplopia and severe hearing impairment in the left ear. There was no evidence for bacterial, fungal, tuberculous infection or neoplastic infiltration. Magnetic resonance imaging of the brain demonstrated thickening and enhancement of bilateral pachymeninges and multiple enhancing masses in bilateral skull. Dural metastasis was diagnosed and he received whole brain irradiation. In addition, laboratory examination revealed severe thrombocytopenia and leucopenia, and bone marrow study confirmed the diagnosis of metastatic squamous cell carcinoma. This is the first described case of esophageal squamous cell carcinoma with dural and bone marrow metastases. We also discuss the pathogenesis of unusual metastatic diseases and differential diagnosis of pachymeningeal thickening.

A bioactive triphasic ceramic-coated hydroxyapatite promotes proliferation and osteogenic differentiation of human bone marrow stromal cells.

PubMed

Nair, Manitha B; Bernhardt, Anne; Lode, Anja; Heinemann, Christiane; Thieme, Sebastian; Hanke, Thomas; Varma, Harikrishna; Gelinsky, Michael; John, Annie

2009-08-01

Hydroxyapatite (HA) ceramics are widely used as bone graft substitutes because of their biocompatibility and osteoconductivity. However, to enhance the success of therapeutic application, many efforts are undertaken to improve the bioactivity of HA. We have developed a triphasic, silica-containing ceramic-coated hydroxyapatite (HASi) and evaluated its performance as a scaffold for cell-based tissue engineering applications. Human bone marrow stromal cells (hBMSCs) were seeded on both HASi and HA scaffolds and cultured with and without osteogenic supplements for a period of 4 weeks. Cellular responses were determined in vitro in terms of cell adhesion, viability, proliferation, and osteogenic differentiation, where both materials exhibited excellent cytocompatibility. Nevertheless, an enhanced rate of cell proliferation and higher levels of both alkaline phosphatase expression and activity were observed for cells cultured on HASi with osteogenic supplements. These findings indicate that the bioactivity of HA endowed with a silica-containing coating has definitely influenced the cellular activity, projecting HASi as a suitable candidate material for bone regenerative therapy.

Bone Marrow Mesenchymal Stem Cells Loaded With an Oncolytic Adenovirus Suppress the Anti-adenoviral Immune Response in the Cotton Rat Model

PubMed Central

Ahmed, Atique U; Rolle, Cleo E; Tyler, Matthew A; Han, Yu; Sengupta, Sadhak; Wainwright, Derek A; Balyasnikova, Irina V; Ulasov, Ilya V; Lesniak, Maciej S

2010-01-01

Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer. However, following intratumoral injections, oncolytic viruses fail to efficiently migrate away from the injection site and are rapidly cleared by the immune system. We have previously demonstrated enhanced viral delivery and replicative persistence in vivo using human bone marrow–derived mesenchymal stem cells (MSCs) as delivery vehicles. In this study, we evaluated the immune response to adenovirus (Ad)-loaded MSCs using the semipermissive cotton rat (CR) model. First, we isolated MSCs from CR bone marrow aspirates. Real-time quantitative PCR analysis revealed that CR MSCs supported the replication of Ads in vitro. Moreover, we observed similar levels of suppression of T-cell proliferation in response to mitogenic stimulation, by MSCs alone and virus-loaded MSCs. Additionally, we found that MSCs suppressed the production of interferon-γ (IFN-γ) by activated T cells. In our in vivo model, CR MSCs enhanced the dissemination and persistence of Ad, compared to virus injection alone. Collectively, our data suggest that the use of MSCs as a delivery strategy for oncolytic Ad potentially offers a myriad of benefits, including improved delivery, enhanced dissemination, and increased persistence of viruses via suppression of the antiviral immune response. PMID:20588259

A "Bony" Proposition: Pathways Mediating Responses to Simulated Weightlessness and Radiation

NASA Technical Reports Server (NTRS)

Tahimic, Candice; Globus, Ruth

2016-01-01

There is evidence that weightlessness and radiation, two elements of the spaceflight environment, can lead to detrimental changes in human musculoskeletal tissue, including bone loss and muscle atrophy. This bone loss is thought to be brought about by the increased activity of bone-resorbing osteoclasts and functional changes in bone-forming osteoblasts, cells that give rise to mature osteocytes. My current area of research focuses on understanding the mechanistic basis for the responses of bone to the spaceflight environment using earth-based animal and cellular models. The overarching goal is to identify molecular targets to prevent bone loss in space exploration and earth-based scenarios of radiotherapy, accidental radiation exposure and reduced mobility. In this talk, I will highlight two signaling pathways that potentially play a role in the response of bone to spaceflight-like conditions. Firstly, I will discuss the role of insulin-like growth factor 1 (IGF1) signaling as it pertains to the recovery of bone from simulated weightlessness (rodent hindlimb unloading model). Secondly, I will share recent findings from our study that aims to understand the emerging role of autophagy in maintaining the balance between bone formation and resorption (bone homeostasis) as well as normal skeletal structure.

DLC1-dependent parathyroid hormone-like hormone inhibition suppresses breast cancer bone metastasis.

PubMed

Wang, Yufeng; Lei, Rong; Zhuang, Xueqian; Zhang, Ning; Pan, Hong; Li, Gang; Hu, Jing; Pan, Xiaoqi; Tao, Qian; Fu, Da; Xiao, Jianru; Chin, Y Eugene; Kang, Yibin; Yang, Qifeng; Hu, Guohong

2014-04-01

Bone metastasis is a frequent complication of breast cancer that is often accelerated by TGF-β signaling; however, little is known about how the TGF-β pathway is regulated during bone metastasis. Here we report that deleted in liver cancer 1 (DLC1) is an important regulator of TGF-β responses and osteolytic metastasis of breast cancer cells. In murine models, breast cancer cells lacking DLC1 expression exhibited enhanced capabilities of bone metastasis. Knockdown of DLC1 in cancer cells promoted bone metastasis, leading to manifested osteolysis and accelerated death in mice, while DLC1 overexpression suppressed bone metastasis. Activation of Rho-ROCK signaling in the absence of DLC1 mediated SMAD3 linker region phosphorylation and TGF-β-induced expression of parathyroid hormone-like hormone (PTHLH), leading to osteoclast maturation for osteolytic colonization. Furthermore, pharmacological inhibition of Rho-ROCK effectively reduced PTHLH production and breast cancer bone metastasis in vitro and in vivo. Evaluation of clinical breast tumor samples revealed that reduced DLC1 expression was linked to elevated PTHLH expression and organ-specific metastasis to bone. Overall, our findings define a stroma-dependent paradigm of Rho signaling in cancer and implicate Rho-TGF-β crosstalk in osteolytic bone metastasis.

Two Distinct Processes of Bone-like Tissue Formation by Dental Pulp Cells after Tooth Transplantation

PubMed Central

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

Monocytes Induce STAT3 Activation in Human Mesenchymal Stem Cells to Promote Osteoblast Formation

PubMed Central

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

Inhibition of TGF–β signaling in subchondral bone mesenchymal stem cells attenuates osteoarthritis

PubMed Central

Zhen, Gehua; Wen, Chunyi; Jia, Xiaofeng; Li, Yu; Crane, Janet L.; Mears, Simon C.; Askin, Frederic B.; Frassica, Frank J.; Chang, Weizhong; Yao, Jie; Nayfeh, Tariq; Johnson, Carl; Artemov, Dmitri; Chen, Qianming; Zhao, Zhihe; Zhou, Xuedong; Cosgarea, Andrew; Carrino, John; Riley, Lee; Sponseller, Paul; Wan, Mei; Lu, William Weijia; Cao, Xu

2013-01-01

Osteoarthritis is a highly prevalent and debilitating joint disorder. There is no effective medical therapy for osteoarthritis due to limited understanding of osteoarthritis pathogenesis. We show that TGF–β1 is activated in the subchondral bone in response to altered mechanical loading in an anterior cruciate ligament transection (ACLT) osteoarthritis mouse model. TGF–β1 concentrations also increased in human osteoarthritis subchondral bone. High concentrations of TGF–β1 induced formation of nestin+ mesenchymal stem cell (MSC) clusters leading to aberrant bone formation accompanied by increased angiogenesis. Transgenic expression of active TGF–β1 in osteoblastic cells induced osteoarthritis. Inhibition of TGF–β activity in subchondral bone attenuated degeneration of osteoarthritis articular cartilage. Notably, knockout of the TGF–β type II receptor (TβRII) in nestin+ MSCs reduced development of osteoarthritis in ACLT mice. Thus, high concentrations of active TGF–β1 in the subchondral bone initiated the pathological changes of osteoarthritis, inhibition of which could be a potential therapeutic approach. PMID:23685840

Src Homology 2–containing 5-Inositol Phosphatase (SHIP) Suppresses an Early Stage of Lymphoid Cell Development through Elevated Interleukin-6 Production by Myeloid Cells in Bone Marrow

PubMed Central

Nakamura, Koji; Kouro, Taku; Kincade, Paul W.; Malykhin, Alexander; Maeda, Kazuhiko; Coggeshall, K. Mark

2004-01-01

The Src homology (SH)2–containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP−/− animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP−/− mice. Lineage-negative marrow (Lin−) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP−/− mice. Furthermore, culture supernatants of SHIP−/− Lin− cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP−/− Lin− cells contained more IL-6 transcripts than wild-type Lin− cells, and neutralizing anti–IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP−/− Lin− cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin− bone marrow cells reproduced the phenotype of SHIP−/− bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow. PMID:14718513

Mechanically induced alterations in cultured skeletal muscle growth

NASA Technical Reports Server (NTRS)

Vandenburgh, H. H.; Hatfaludy, S.; Karlisch, P.; Shansky, J.

1991-01-01

Model systems are available for mechanically stimulating cultured skeletal muscle cells by passive tensile forces which simulate those found in vivo. When applied to embryonic muscle cells in vitro these forces induce tissue organogenesis, metabolic adaptations, and muscle cell growth. The mechanical stimulation of muscle cell growth correlates with stretch-induced increases in the efflux of prostaglandins PGE2 and PGF2(alpha) in a time and frequency dependent manner. These prostaglandins act as mechanical 'second messengers' regulating skeletal muscle protein turnover rates. Since they also effect bone remodelling in response to tissue loading and unloading, secreted prostaglandins may serve as paracrine growth factors, coordinating the growth rates of muscle and bone in response to external mechanical forces. Cell culture model systems will supplement other models in understanding mechanical transduction processes at the molecular level.

Constant Applied Force Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

NASA Technical Reports Server (NTRS)

Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E. A. C.

2003-01-01

Reduced weight-bearing caused by immobilization, bed-rest or microgravity leads to atrophy in mechanosensitive tissue such as muscle and bone. We hypothesize that bone tissue requires earth s gravity (1-g) for the maintenance of extracellular matrix, integrin, and kinase-mediated cell growth and survival pathways. We investigate the role of matrix-integrin signaling in bone cells using cell culture centrifugation to provide different levels of hypergravity mechanostimulation. The 10-50-g range we use also mimics physiological intermedullary pressure (1.2 - 5 kPa). 24 hours at 50-g increased primary rat osteoblast proliferation on collagen Type I and fibronectin, but not laminin or uncoated plastic. BrdU incorporation in primary osteoblasts over 24 h showed hypergravity increased the number of cells actively synthesizing DNA from about 60% at 1-g to over 90% at 25-g. Primary rat fibroblasts grown at 50-g (24 h) showed no proliferation increase, suggesting this is a tissue-specific phenomenon. These results suggest that the betal and alpha4 integrins may be involved. To further test this, we used osteocytic-like MLO-Y4 cells that showed increased proliferation at 1-g with stable expression of a betal integrin cytoplasmic tail and transmembrane domain construct. At 50-g, MLO-Y4/betal cells showed greater MAPK activation than MLO-Y4 vector controls, suggesting that betal integrin is involved in transducing mitogenic signals in response to hypergravity. Preliminary results also show that interfering with the alpha4 integrin in primary osteoblasts grown on fibronectin blocked the proliferation response. These results indicate that cells from mechanosensitive bone tissue can respond to gravity-generated forces, and this response involves specific matrix and integrin-dependent signaling pathways.

CD31+ Cells From Peripheral Blood Facilitate Bone Regeneration in Biologically Impaired Conditions Through Combined Effects on Immunomodulation and Angiogenesis.

PubMed

Sass, F Andrea; Schmidt-Bleek, Katharina; Ellinghaus, Agnes; Filter, Sebastian; Rose, Alexander; Preininger, Bernd; Reinke, Simon; Geissler, Sven; Volk, Hans-Dieter; Duda, Georg N; Dienelt, Anke

2017-05-01

Controlled revascularization and inflammation are key elements regulating endogenous regeneration after (bone) tissue trauma. Peripheral blood-derived cell subsets, such as regulatory T-helper cells and circulating (endothelial) progenitor cells, respectively, can support endogenous tissue healing, whereas effector T cells that are associated with an aged immune system can hinder bone regeneration. CD31 is expressed by diverse leukocytes and is well recognized as a marker of circulating endothelial (precursor) cells; however, CD31 is absent from the surface of differentiated effector T cells. Thus, we hypothesized that by separating the inhibitory fractions from the supportive fractions of circulating cells within the peripheral blood (PB) using the CD31 marker, bone regeneration in biologically compromised conditions, such as those observed in aged patients, could be improved. In support of our hypothesis, we detected an inverse correlation between CD31+ cells and effector T cells in the hematomas of human fracture patients, dependent on the age of the patient. Furthermore, we demonstrated the regenerative capacity of human PB-CD31+ cells in vitro. These findings were translated to a clinically relevant rat model of impaired bone healing. The transplantation of rat PB-CD31+ cells advanced bone tissue restoration in vivo and was associated with an early anti-inflammatory response, the stimulation of (re)vascularization, and reduced fibrosis. Interestingly, the depletion or enrichment of the highly abundant CD31+/14+ monocytes from the mixed CD31+ cell population diminished tissue regeneration at different levels, suggesting combined effects within the PB-CD31+ subsets. In summary, an intraoperative enrichment of PB-CD31+ cells might be a novel option to facilitate endogenous regeneration under biologically impaired situations by supporting immunomodulation and vascularization. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

Cabozantinib Affects Osteosarcoma Growth Through A Direct Effect On Tumor Cells and Modifications In Bone Microenvironment.

PubMed

Fioramonti, M; Fausti, V; Pantano, F; Iuliani, M; Ribelli, G; Lotti, F; Pignochino, Y; Grignani, G; Santini, D; Tonini, G; Vincenzi, B

2018-03-08

Osteosarcoma (OS) is the most common primary malignant tumor of the bone. Due to its high heterogeneity and to survival signals from bone microenvironment, OS can resist to standard treatments, therefore novel therapies are needed. c-MET oncogene, a tyrosine-kinase receptor, plays a crucial role in OS initiation and progression. The present study aimed to evaluate the effect of c-MET inhibitor cabozantinib (CBZ) on OS both directly and through its action on bone microenvironment. We tested different doses of CBZ in in vitro models of OS alone or in co-culture with bone cells in order to reproduce OS-tumor microenvironment interactions. CBZ is able to decrease proliferation and migration of OS cells, inhibiting ERK and AKT signaling pathways. Furthermore, CBZ leads to the inhibition of the proliferation of OS cells expressing receptor activator of nuclear factor κB (RANK), due to its effect on bone microenvironment, where it causes an overproduction of osteoprotegerin and a decrease of production of RANK ligand by osteoblasts. Overall, our data demonstrate that CBZ might represent a new potential treatment against OS, affecting both OS cells and their microenvironment. In this scenario, RANK expression in OS cells could represent a predictive factor of better response to CBZ treatment.

Leukemia inhibitory factor: a novel bone-active cytokine.

PubMed

Reid, L R; Lowe, C; Cornish, J; Skinner, S J; Hilton, D J; Willson, T A; Gearing, D P; Martin, T J

1990-03-01

A number of cytokines have been found to be potent regulators of bone resorption and to share the properties originally attributed to osteoclast-activating factor. One such activity, differentiation-inducing factor (DIF, D-factor) from mouse spleen cells, shares a number of biological and biochemical properties with the recently characterized and cloned leukemia inhibitory factor (LIF). We have assessed the effects of recombinant LIF on bone resorption and other parameters in neonatal mouse calvaria. Both recombinant murine and human (h) LIFs stimulated 45Ca release from prelabeled calvaria in a dose-dependent manner. The increase in bone resorption was associated with an increase in the number of osteoclasts per mm2 bone. The osteolytic effect of hLIF were blocked by 10(-7) M indomethacin. hLIF also stimulated incorporation of [3H] thymidine into calvaria, but the dose-response relationship was distinct from that for bone resorption, and this effect was not blocked by indomethacin. Similarly, hLIF increased [3H]phenylalanine incorporation into calvaria, and this was also not inhibited by indomethacin. It is concluded that LIF stimulates bone resorption by a mechanism involving prostaglandin production, but that a distinct mechanism is responsible for its stimulation of DNA and protein synthesis. The primary structure of LIF differs from that of other fully characterized, bone-active cytokines, and it, thus, represents a novel factor which may be involved in the normal regulation of bone cell function.

Degeneration of the osteocyte network in the C57BL/6 mouse model of aging.

PubMed

Tiede-Lewis, LeAnn M; Xie, Yixia; Hulbert, Molly A; Campos, Richard; Dallas, Mark R; Dusevich, Vladimir; Bonewald, Lynda F; Dallas, Sarah L

2017-10-26

Age-related bone loss and associated fracture risk are major problems in musculoskeletal health. Osteocytes have emerged as key regulators of bone mass and as a therapeutic target for preventing bone loss. As aging is associated with changes in the osteocyte lacunocanalicular system, we focused on the responsible cellular mechanisms in osteocytes. Bone phenotypic analysis was performed in young-(5mo) and aged-(22mo) C57BL/6 mice and changes in bone structure/geometry correlated with alterations in osteocyte parameters determined using novel multiplexed-3D-confocal imaging techniques. Age-related bone changes analogous to those in humans were observed, including increased cortical diameter, decreased cortical thickness, reduced trabecular BV/TV and cortical porosities. This was associated with a dramatic reduction in osteocyte dendrite number and cell density, particularly in females, where osteocyte dendricity decreased linearly from 5, 12, 18 to 22mo and correlated significantly with cortical bone parameters. Reduced dendricity preceded decreased osteocyte number, suggesting dendrite loss may trigger loss of viability. Age-related degeneration of osteocyte networks may impair bone anabolic responses to loading and gender differences in osteocyte cell body and lacunar fluid volumes we observed in aged mice may lead to gender-related differences in mechanosensitivity. Therapies to preserve osteocyte dendricity and viability may be beneficial for bone health in aging.

Tumor Trp53 status and genotype affect the bone marrow microenvironment in acute myeloid leukemia

PubMed Central

Jacamo, Rodrigo; Davis, R. Eric; Ling, Xiaoyang; Sonnylal, Sonali; Wang, Zhiqiang; Ma, Wencai; Zhang, Min; Ruvolo, Peter; Ruvolo, Vivian; Wang, Rui-Yu; McQueen, Teresa; Lowe, Scott; Zuber, Johannes; Kornblau, Steven M.; Konopleva, Marina; Andreeff, Michael

2017-01-01

The genetic heterogeneity of acute myeloid leukemia (AML) and the variable responses of individual patients to therapy suggest that different AML genotypes may influence the bone marrow (BM) microenvironment in different ways. We performed gene expression profiling of bone marrow mesenchymal stromal cells (BM-MSC) isolated from normal C57BL/6 mice or mice inoculated with syngeneic murine leukemia cells carrying different human AML genotypes, developed in mice with Trp53 wild-type or nullgenetic backgrounds. We identified a set of genes whose expression in BM-MSC was modulated by all four AML genotypes tested. In addition, there were sets of differentially-expressed genes in AML-exposed BM-MSC that were unique to the particular AML genotype or Trp53 status. Our findings support the hypothesis that leukemia cells alter the transcriptome of surrounding BM stromal cells, in both common and genotype-specific ways. These changes are likely to be advantageous to AML cells, affecting disease progression and response to chemotherapy, and suggest opportunities for stroma-targeting therapy, including those based on AML genotype. PMID:29137349

Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles.

PubMed

Förster, Yvonne; Schmidt, Johannes R; Wissenbach, Dirk K; Pfeiffer, Susanne E M; Baumann, Sven; Hofbauer, Lorenz C; von Bergen, Martin; Kalkhof, Stefan; Rammelt, Stefan

2016-01-01

Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis.

Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles

PubMed Central

Wissenbach, Dirk K.; Pfeiffer, Susanne E. M.; Baumann, Sven; Hofbauer, Lorenz C.; von Bergen, Martin; Kalkhof, Stefan; Rammelt, Stefan

2016-01-01

Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis. PMID:27441377

Impaired Endothelial Progenitor Cell Mobilization and Dysfunctional Bone Marrow Stroma in Diabetes Mellitus

PubMed Central

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

Comparisons of the humoral and cellular immunity induced by live A16R attenuated spore and AVA-like anthrax vaccine in mice.

PubMed

Lv, Jin; Zhang, Ying-Ying; Lu, Xun; Zhang, Hao; Wei, Lin; Gao, Jun; Hu, Bin; Hu, Wen-Wei; Hu, Dun-Zhong; Jia, Na; Feng, Xin

2017-03-01

The live attenuated anthrax vaccine and anthrax vaccine adsorbed (AVA) are two main types of anthrax vaccines currently used in human. However, the immunoprotective mechanisms are not fully understood. In this study, we compared humoral and cellular immunity induced by live A16R spore vaccine and A16R strain derived AVA-like vaccine in mice peripheral blood, spleen and bone marrow. Both A16R spores and AVA-like vaccines induced a sustained IgG antibody response with IgG1/IgG2b subtype dominance. However, A16R spores vaccine induced higher titer of IgG2a compared with AVA-like vaccine, indicating a stronger Th1 response to A16R spores. Using antigen-specific ELISpot assay, we observed a significant response of ASCs (antibody secreting cells) and IL4-CSCs (cytokine secreting cells) in mice. Specially, there was a positive correlation between the frequencies of antigen specific ASCs and IL4-CSCs in bone marrow derived cells, either by A16R spore or AVA-like vaccine vaccination. Moreover, we also found A16R spore vaccine, not AVA-like vaccine, could induce sustained frequency of IFN-γ-CSCs in bone marrow derived cells. Collectively, both the vaccines induced a mixed Th1/Th2 response with Th2 dominance in mice and A16R spore vaccine might provide a more comprehensive protection because of humoral and cellular immunity induced in bone marrow. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Oxidative Stress and Autophagy Responses of Osteocytes Exposed to Spaceflight-like Radiation.

NASA Technical Reports Server (NTRS)

Tahimic, Candice; Rael, Victoria E.; Globus, Ruth K.

2015-01-01

Weightlessness and radiation, two of the unique elements of the space environment, causes a profound decrement in bone mass that mimics aging. This bone loss is thought to result from increased activity of bone-resorbing osteoclasts and functional changes in bone-forming osteoblasts, cells that give rise to mature osteocytes. Our current understanding of the signaling factors and mechanisms underlying bone loss is incomplete. However, it is known that oxidative stress, characterized by the excess production of free radicals, is elevated during radiation exposure. The goals of this study is to examine the response of osteocytes to spaceflight-like radiation and to identify signaling processes that may be targeted to mitigate bone loss in scenarios of space exploration, earth-based radiotherapy and accidental radiation exposure. We hypothesize that (1) oxidative stress, as induced by radiation, decreases osteocyte survival and increases pro-osteoclastogenic signals and that (2) autophagy is one of the key cellular defenses against oxidative stress. Autophagy is the process by which cellular components including organelles and proteins are broken down and recycled. To test our hypothesis, we exposed the osteocyte-like cell line, MLO-Y4, to 0.5, 1, and 2 Gy of simulated space radiation (Iron-56 radiation at 600 MeV/n) and assessed cell numbers, cell growth-associated molecules as well as markers of autophagy and oxidative stress at various time points post-irradiation. We observed a reduction in cell numbers in the groups exposed to 1 and 2 Gy of Iron-56 radiation. Collectively, flow cytometry and gene expression analysis revealed that radiation caused a shift in cell cycle distribution consistent with growth arrest. Compared to sham-treatment, 2 Gy of Iron-56 increased FoxO3, SOD1, and RANKL gene expression yet unexpectedly decreased LC3B-II protein levels at 4 and 24 hours post-IR. Taken together, these findings suggest that simulated space radiation invoke antioxidant, pro-osteoclastogenic, and growth arrest responses in osteocytes. The implications of reduced autophagy flux at the time points examined remain to be elucidated.

Role of hepatocyte growth factor in the development of dendritic cells from CD34+ bone marrow cells.

PubMed

Ovali, E; Ratip, S; Kibaroglu, A; Tekelioglu, Y; Cetiner, M; Karti, S; Aydin, F; Bayik, M; Akoglu, T

2000-05-01

Hepatocyte growth factor (HGF) is known to augment the effects of stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoetin, and granulocyte colony-stimulating factor, all of which are involved in hematopoiesis. HGF is also known to have a role in immune responses. The aim of this study was to investigate whether HGF is involved in the development of dendritic cells (DC) from CD34+ bone marrow cells. CD34+ cells obtained from three healthy donors were incubated in various combinations of HGF, GM-CSF, and tumor necrosis factor (TNF) for 12 days. Developing cell populations were analyzed for surface markers, morphology and functional capacities by flow cytometry, light microscopy and mixed lymphocyte reaction, respectively. Incubation with HGF alone generated greater number of dendritic cells from CD34+ bone marrow cells than incubation with GM-CSF, or a combination of GM-CSF with TNF. HGF was also found to potentiate the effect of GM-CSF on DC and monocyte development. The effects of HGF were inhibited by the concurrent use of TNF. HGF appears to be a significant factor in the development of dendritic cells from CD34+ bone marrow cells.

Erythropoietin in sickle cell disease: relation of erythropoietin levels to crisis and other complications.

PubMed

Haddy, T B

1982-01-01

Erythropoietin responsible for the hormonal regulation of red blood cell production. Its formation is largely controlled by the kidneys. A number of assay methods for erythropoietin are available. Asymptomatic patients with sickle cell disease have elevated erythropoietin levels, as expected with chronic hemolysis. When complicated by chronic renal failure, erythropoietin levels do not rise appropriately. Chronic infection has not been studied, but the erythropoietin response in acute infection does not seem to conform to a pattern. Aplastic crises are characterized by very high levels of erythropoietin, suggesting bone marrow suppression, but events that trigger the crises remain obscure. In vaso-occlusive crises, there is also some suggestion of mild and transient lack of bone marrow response. Patients with sickle cell disease, with their chronic high erythropoietin anemia and susceptibility to altered states, are uniquely suited for investigating the physiology of erythropoietin, especially under the constraints of present assay methods.

So You Want to Go to Mars: Bones and Matters of the Heart

NASA Technical Reports Server (NTRS)

Tahimic, Candice; Globus, Ruth; Torres, Samantha; Steczina, Sonette

2017-01-01

There is evidence that weightlessness and radiation, two elements of the spaceflight environment, can lead to detrimental changes in human musculoskeletal tissue, including bone loss and muscle atrophy. This bone loss is thought to be brought about by the increased activity of bone-resorbing osteoclasts and functional changes in bone-forming osteoblasts, cells that give rise to mature osteocytes. Collectively, our research team aims to understand the molecular mechanisms underlying the responses of mammalian tissue to the spaceflight environment using earth-based animal and cellular models. The overarching goal is to identify molecular targets to prevent tissue decrements induced by spaceflight and earth-based scenarios of radiotherapy, accidental radiation exposure and reduced mobility. In this talk, I will provide an overview of skeletal and cardiovascular responses to spaceflight and will highlight our research progress on understanding the role of reactive oxygen species (ROS) signaling in skeletal responses to radiation and simulated weightlessness.

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.

FGF23 is elevated in multiple myeloma and increases heparanase expression by tumor cells

PubMed Central

Suvannasankha, Attaya; Tompkins, Douglas R.; Edwards, Daniel F.; Petyaykina, Katarina V.; Crean, Colin D.; Fournier, Pierrick G.; Parker, Jamie M.; Sandusky, George E.; Ichikawa, Shoji; Imel, Erik A.; Chirgwin, John M.

2015-01-01

Multiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone. PMID:25944690

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells.

PubMed

Hong, Seok-Jung; Jeong, Ishik; Noh, Kyung-Tae; Yu, Hye-Sun; Lee, Gil-Su; Kim, Hae-Won

2009-09-01

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(epsilon-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA-PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA-PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA-PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA-PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering.

Bone marrow-on-a-chip replicates hematopoietic niche physiology in vitro.

PubMed

Torisawa, Yu-suke; Spina, Catherine S; Mammoto, Tadanori; Mammoto, Akiko; Weaver, James C; Tat, Tracy; Collins, James J; Ingber, Donald E

2014-06-01

Current in vitro hematopoiesis models fail to demonstrate the cellular diversity and complex functions of living bone marrow; hence, most translational studies relevant to the hematologic system are conducted in live animals. Here we describe a method for fabricating 'bone marrow-on-a-chip' that permits culture of living marrow with a functional hematopoietic niche in vitro by first engineering new bone in vivo, removing it whole and perfusing it with culture medium in a microfluidic device. The engineered bone marrow (eBM) retains hematopoietic stem and progenitor cells in normal in vivo-like proportions for at least 1 week in culture. eBM models organ-level marrow toxicity responses and protective effects of radiation countermeasure drugs, whereas conventional bone marrow culture methods do not. This biomimetic microdevice offers a new approach for analysis of drug responses and toxicities in bone marrow as well as for study of hematopoiesis and hematologic diseases in vitro.

Enthesis fibrocartilage cells originate from a population of Hedgehog-responsive cells modulated by the loading environment.

PubMed

Schwartz, Andrea G; Long, Fanxin; Thomopoulos, Stavros

2015-01-01

Tendon attaches to bone across a specialized tissue called the enthesis. This tissue modulates the transfer of muscle forces between two materials, i.e. tendon and bone, with vastly different mechanical properties. The enthesis for many tendons consists of a mineralized graded fibrocartilage that develops postnatally, concurrent with epiphyseal mineralization. Although it is well described that the mineralization and development of functional maturity requires muscle loading, the biological factors that modulate enthesis development are poorly understood. By genetically demarcating cells expressing Gli1 in response to Hedgehog (Hh) signaling, we discovered a unique population of Hh-responsive cells in the developing murine enthesis that were distinct from tendon fibroblasts and epiphyseal chondrocytes. Lineage-tracing experiments revealed that the Gli1 lineage cells that originate in utero eventually populate the entire mature enthesis. Muscle paralysis increased the number of Hh-responsive cells in the enthesis, demonstrating that responsiveness to Hh is modulated in part by muscle loading. Ablation of the Hh-responsive cells during the first week of postnatal development resulted in a loss of mineralized fibrocartilage, with very little tissue remodeling 5 weeks after cell ablation. Conditional deletion of smoothened, a molecule necessary for responsiveness to Ihh, from the developing tendon and enthesis altered the differentiation of enthesis progenitor cells, resulting in significantly reduced fibrocartilage mineralization and decreased biomechanical function. Taken together, these results demonstrate that Hh signaling within developing enthesis fibrocartilage cells is required for enthesis formation. © 2015. Published by The Company of Biologists Ltd.

Enthesis fibrocartilage cells originate from a population of Hedgehog-responsive cells modulated by the loading environment

PubMed Central

Schwartz, Andrea G.; Long, Fanxin; Thomopoulos, Stavros

2015-01-01

Tendon attaches to bone across a specialized tissue called the enthesis. This tissue modulates the transfer of muscle forces between two materials, i.e. tendon and bone, with vastly different mechanical properties. The enthesis for many tendons consists of a mineralized graded fibrocartilage that develops postnatally, concurrent with epiphyseal mineralization. Although it is well described that the mineralization and development of functional maturity requires muscle loading, the biological factors that modulate enthesis development are poorly understood. By genetically demarcating cells expressing Gli1 in response to Hedgehog (Hh) signaling, we discovered a unique population of Hh-responsive cells in the developing murine enthesis that were distinct from tendon fibroblasts and epiphyseal chondrocytes. Lineage-tracing experiments revealed that the Gli1 lineage cells that originate in utero eventually populate the entire mature enthesis. Muscle paralysis increased the number of Hh-responsive cells in the enthesis, demonstrating that responsiveness to Hh is modulated in part by muscle loading. Ablation of the Hh-responsive cells during the first week of postnatal development resulted in a loss of mineralized fibrocartilage, with very little tissue remodeling 5 weeks after cell ablation. Conditional deletion of smoothened, a molecule necessary for responsiveness to Ihh, from the developing tendon and enthesis altered the differentiation of enthesis progenitor cells, resulting in significantly reduced fibrocartilage mineralization and decreased biomechanical function. Taken together, these results demonstrate that Hh signaling within developing enthesis fibrocartilage cells is required for enthesis formation. PMID:25516975

Cell specific radiation dosimetry in skeleton from life-span carcinogenesis studies. Final report

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

Webster, S.S.J.

1993-04-05

The osteogenic sarcoma is the dominant life-threatening pathology in lifespan studies of beagles exposed to alpha-emitting bone-seeking radionuclides. It was deduced from these studies that certain skeletal sites are more prone to develop tumors. This project sought to determine the bone cells at risk and their cell-specific radiation dose. The cell-specific radiation dose values are related to loss and high Ra-226 and Pu-239 induced osteogenic sarcoma sites, to test different dose response hypothesis and predict the extent of effects in humans.

Cell specific radiation dosimetry in skeleton from life-span carcinogenesis studies

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

Webster, S.S.J.

1993-04-05

The osteogenic sarcoma is the dominant life-threatening pathology in lifespan studies of beagles exposed to alpha-emitting bone-seeking radionuclides. It was deduced from these studies that certain skeletal sites are more prone to develop tumors. This project sought to determine the bone cells at risk and their cell-specific radiation dose. The cell-specific radiation dose values are related to loss and high Ra-226 and Pu-239 induced osteogenic sarcoma sites, to test different dose response hypothesis and predict the extent of effects in humans.

A cellular automata model of bone formation.

PubMed

Van Scoy, Gabrielle K; George, Estee L; Opoku Asantewaa, Flora; Kerns, Lucy; Saunders, Marnie M; Prieto-Langarica, Alicia

2017-04-01

Bone remodeling is an elegantly orchestrated process by which osteocytes, osteoblasts and osteoclasts function as a syncytium to maintain or modify bone. On the microscopic level, bone consists of cells that create, destroy and monitor the bone matrix. These cells interact in a coordinated manner to maintain a tightly regulated homeostasis. It is this regulation that is responsible for the observed increase in bone gain in the dominant arm of a tennis player and the observed increase in bone loss associated with spaceflight and osteoporosis. The manner in which these cells interact to bring about a change in bone quality and quantity has yet to be fully elucidated. But efforts to understand the multicellular complexity can ultimately lead to eradication of metabolic bone diseases such as osteoporosis and improved implant longevity. Experimentally validated mathematical models that simulate functional activity and offer eventual predictive capabilities offer tremendous potential in understanding multicellular bone remodeling. Here we undertake the initial challenge to develop a mathematical model of bone formation validated with in vitro data obtained from osteoblastic bone cells induced to mineralize and quantified at 26 days of culture. A cellular automata model was constructed to simulate the in vitro characterization. Permutation tests were performed to compare the distribution of the mineralization in the cultures and the distribution of the mineralization in the mathematical models. The results of the permutation test show the distribution of mineralization from the characterization and mathematical model come from the same probability distribution, therefore validating the cellular automata model. Copyright © 2017 Elsevier Inc. All rights reserved.

cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

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

Son, Jieun; Lee, Jong-Ho; Kim, Ha-Neui

2010-07-23

Research highlights: {yields} CREB is highly expressed in advanced breast cancer cells. {yields} Tumor-related factors such as TGF-{beta} further elevate CREB expression. {yields} CREB upregulation stimulates metastatic potential of breast cancer cells. {yields} CREB signaling is required for breast cancer-induced bone destruction. -- Abstract: cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cellsmore » and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-{beta}. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.« less

Hard, soft tissue and in vitro cell response to porous nickel-titanium: a biocompatibility evaluation.

PubMed

Rhalmi, S; Odin, M; Assad, M; Tabrizian, M; Rivard, C H; Yahia, L H

1999-01-01

Porous nickel-titanium (NiTi) alloys have demonstrated bone attachment as well as tissue ingrowth in the past. However, very few studies have compared porous NiTi soft and hard tissue reactions, and in vitro cell response. We therefore have evaluated the general muscle and bone reaction to porous nickel-titanium. The latter material was implanted in rabbit tibias and back muscle, and assessed after three, six and twelve weeks of implantation. Porous NiTi specimens did not cause any adverse effect regardless of both implantation site and post-surgery recovery time. Muscle tissue exhibited thin tightly adherent fibrous capsules with fibers penetrating into implant pores. We observed that attachment strength of the soft tissue to the porous implant seemed to increase with post-implantation time. Bone tissue demonstrated good healing of the osteotomy. There was bone remodeling characterized by osteoclastic and osteoblastic activity in the cortex. This general good in vivo biocompatibility with muscle and bone tissue corresponded very well with the in vitro cell culture results we obtained. Fibroblasts seeded on porous nickel-titanium sheets managed to grow into the pores and all around specimen edges showing an another interesting cytocompatibility behavior. These results indicate good biocompatibility acceptance of porous nickel-titanium and are very promising towards eventual NiTi medical device approbation.

Characterization of neutrophils and macrophages from ex vivo cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry

PubMed Central

Pelletier, Margery G. H.; Szymczak, Klaudia; Barbeau, Anna M.; Prata, Gianna N.; O’Fallon, Kevin S.; Gaines, Peter

2016-01-01

Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis. PMID:27663441

Gain-of-function mutation in FGFR3 in mice leads to decreased bone mass by affecting both osteoblastogenesis and osteoclastogenesis

PubMed Central

Su, Nan; Sun, Qidi; Li, Can; Lu, Xiumin; Qi, Huabing; Chen, Siyu; Yang, Jing; Du, Xiaolan; Zhao, Ling; He, Qifen; Jin, Min; Shen, Yue; Chen, Di; Chen, Lin

2010-01-01

Achondroplasia (ACH) is a short-limbed dwarfism resulting from gain-of-function mutations in fibroblast growth factor receptor 3 (FGFR3). Previous studies have shown that ACH patients have impaired chondrogenesis, but the effects of FGFR3 on bone formation and bone remodeling at adult stages of ACH have not been fully investigated. Using micro-computed tomography and histomorphometric analyses, we found that 2-month-old Fgfr3G369C/+ mice (mouse model mimicking human ACH) showed decreased bone mass due to reduced trabecular bone volume and bone mineral density, defect in bone mineralization and increased osteoclast numbers and activity. Compared with primary cultures of bone marrow stromal cells (BMSCs) from wild-type mice, Fgfr3G369C/+ cultures showed decreased cell proliferation, increased osteogenic differentiation including up-regulation of alkaline phosphatase activity and expressions of osteoblast marker genes, and reduced bone matrix mineralization. Furthermore, our studies also suggest that decreased cell proliferation and enhanced osteogenic differentiation observed in Fgfr3G369C/+ BMSCs are caused by up-regulation of p38 phosphorylation and that enhanced Erk1/2 activity is responsible for the impaired bone matrix mineralization. In addition, in vitro osteoclast formation and bone resorption assays demonstrated that osteoclast numbers and bone resorption area were increased in cultured bone marrow cells derived from Fgfr3G369C/+ mice. These findings demonstrate that gain-of-function mutation in FGFR3 leads to decreased bone mass by regulating both osteoblast and osteoclast activities. Our studies provide new insight into the mechanism underlying the development of ACH. PMID:20053668

Immature myeloid cells are critical for enhancing bone fracture healing through angiogenic cascade

PubMed Central

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

Immature myeloid cells are critical for enhancing bone fracture healing through angiogenic cascade.

PubMed

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.

Safety Evaluation of a Bioglass–Polylactic Acid Composite Scaffold Seeded with Progenitor Cells in a Rat Skull Critical-Size Bone Defect

PubMed Central

El-Kady, Abeer M.; Arbid, Mahmoud S.; Abd El-Hady, Bothaina M.; Marzi, Ingo; Seebach, Caroline

2014-01-01

Treating large bone defects represents a major challenge in traumatic and orthopedic surgery. Bone tissue engineering provides a promising therapeutic option to improve the local bone healing response. In the present study tissue biocompatibility, systemic toxicity and tumorigenicity of a newly developed composite material consisting of polylactic acid (PLA) and 20% or 40% bioglass (BG20 and BG40), respectively, were analyzed. These materials were seeded with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) and tested in a rat calvarial critical size defect model for 3 months and compared to a scaffold consisting only of PLA. Serum was analyzed for organ damage markers such as GOT and creatinine. Leukocyte count, temperature and free radical indicators were measured to determine the degree of systemic inflammation. Possible tumor occurrence was assessed macroscopically and histologically in slides of liver, kidney and spleen. Furthermore, the concentrations of serum malondialdehyde (MDA) and sodium oxide dismutase (SOD) were assessed as indicators of tumor progression. Qualitative tissue response towards the implants and new bone mass formation was histologically investigated. BG20 and BG40, with or without progenitor cells, did not cause organ damage, long-term systemic inflammatory reactions or tumor formation. BG20 and BG40 supported bone formation, which was further enhanced in the presence of EPCs and MSCs. This investigation reflects good biocompatibility of the biomaterials BG20 and BG40 and provides evidence that additionally seeding EPCs and MSCs onto the scaffold does not induce tumor formation. PMID:24498345

Studies of Intercellular Communication and Intracellular Metabolic Responses by Bone Cells to Simulated Weightlessness

NASA Technical Reports Server (NTRS)

Doty, Stephen B.

1997-01-01

Spaceflight affects the weight bearing skeletal tissues by reducing the rate of new bone formation. This effect on the long bones of flown rats has been quantitated but the effect at the cellular level and the mechanism(s) involved are not understood. We are applying electron microscopy, coupled with histochemistry and immunocytochemistry to determine the cellular functions most affected by spaceflight. The emphasis for study of these samples from SLS-1, a 9-day mission, is on the histochemical and structural changes of the endosteal and perivascular osteoblasts found in diaphyseal bone of femur and tibia. Work is still in progress but some findings are described: (1) An expected decrease in alkaline phosphatase activity in osteoblasts from flight animals, but an increase in enzyme activity in the stromal stem cells adjacent to the osteoblast. (2) An increase in osteoclastic TRAP activity in the trabecular bone region in response to spaceflight. (3) A large increase in procollagen containing secretory granules in osteoblasts in the recovery group, and a significant decrease in granule numbers in the flight group.

Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages.

PubMed

da Silva, Bruno José Martins; Rodrigues, Ana Paula D; Farias, Luis Henrique S; Hage, Amanda Anastácia P; Do Nascimento, Jose Luiz M; Silva, Edilene O

2014-10-03

The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent.

Physalis angulata induces in vitro differentiation of murine bone marrow cells into macrophages

PubMed Central

2014-01-01

Background The bone marrow is a hematopoietic tissue that, in the presence of cytokines and growth factors, generates all of the circulating blood cells. These cells are important for protecting the organism against pathogens and for establishing an effective immune response. Previous studies have shown immunomodulatory effects of different products isolated from plant extracts. This study aimed to evaluate the immunomodulatory properties of aqueous Physalis angulata (AEPa) extract on the differentiation of bone marrow cells. Results Increased cellular area, higher spreading ability and several cytoplasmatic projections were observed in the treated cells, using optical microscopy, suggesting cell differentiation. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by Transmission Electron Microscopy of treated cells showed spreading ability, high number of cytoplasmatic projections and increase of autophagic vacuoles. Moreover, a high level of LC3b expression by treated cells was detected by flow cytometry, suggesting an autophagic process. Cell surface expression of F4/80 and CD11b also indicated that AEPa may stimulate differentiation of bone marrow cells mainly into macrophages. In addition, AEPa did not differentiate cells into dendritic cells, as assessed by CD11c analysis. Furthermore, no cytotoxic effects were observed in the cells treated with AEPa. Conclusion Results demonstrate that AEPa promotes the differentiation of bone marrow cells, particularly into macrophages and may hold promise as an immunomodulating agent. PMID:25281406

Three-Dimensional Mechanical Loading Modulates the Osteogenic Response of Mesenchymal Stem Cells to Tumor-Derived Soluble Signals

PubMed Central

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

Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

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.

Bone regeneration performance of surface-treated porous titanium.

PubMed

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.

In vitro characterization of MG-63 osteoblast-like cells cultured on organic-inorganic lyophilized gelatin sponges for early bone healing.

PubMed

Rodriguez, Isaac A; Saxena, Gunjan; Hixon, Katherine R; Sell, Scott A; Bowlin, Gary L

2016-08-01

The development of three-dimensional porous scaffolds with enhanced osteogenic and angiogenic potential would be beneficial for inducing early-stage bone regeneration. Previous studies have demonstrated the advantages of mineralized and nonmineralized acellular 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) cross-linked gelatin sponges enhanced with preparations rich in growth factors, hydroxyapatite, and chitin whiskers. In this study, those same scaffolds were mineralized and dynamically seeded with MG-63 cells. Cell proliferation, protein/cytokine secretion, and compressive mechanical properties of scaffolds were evaluated. It was found that mineralization and the addition of growth factors increased cell proliferation compared to gelatin controls. Cells on all scaffolds responded in an appropriate bone regenerative fashion as shown through osteocalcin secretion and little to no secretion of bone resorbing markers. However, compressive mechanical properties of cellularized scaffolds were not significantly different from acellular scaffolds. The combined results of increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion on scaffolds support the need for the addition of a bone-like mineral surface. Cellularized scaffolds containing growth factors reported similar advantages and mechanical values in the range of native tissues present in the early stages of bone healing. These results suggest that the developed composite sponges exhibited cellular responses and mechanical properties appropriate for promoting early bone healing in various applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2011-2019, 2016. © 2016 Wiley Periodicals, Inc.

Interleukin (IL)-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts1

PubMed Central

Iida, Shoichi; Tsuda, Hidetoshi; Tanaka, Toshiaki; Kish, Danielle D.; Abe, Toyofumi; Su, Charles A.; Abe, Ryo; Tanabe, Kazunari; Valujskikh, Anna; Baldwin, William M.; Fairchild, Robert L.

2016-01-01

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared to complete MHC-mismatched wild type cardiac allografts, IL-1R−/− allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R−/− allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R−/− cardiac allografts took 3 weeks longer than wild type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R−/−/wild type chimeric donors indicated that IL-1R signaling on graft non-hematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli provoking development and elicitation of optimal alloimmune responses to the grafts. PMID:26856697

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.

The secreted factors responsible for pre-metastatic niche formation: old sayings and new thoughts.

PubMed

Peinado, Héctor; Lavotshkin, Simon; Lyden, David

2011-04-01

Metastasis is a multistep process that requires acquisition of malignant cell phenotypes which allow tumor cells to escape from the primary tumor site. Each of the steps during metastatic progression involves co-evolution of the tumor and its microenvironment. Although tumor cells are the driving force of metastasis, new findings suggest that the host cells within the tumor microenvironment play a key role in influencing metastatic behavior. Many of these contributing cells are derived from the bone marrow; in particular, recruited bone marrow progenitor cells generate the "pre-metastatic niche" to which the tumor cells metastasize. Analysis of the molecular mechanisms involved in pre-metastatic niche formation has revealed that secreted soluble factors are key players in bone marrow cell mobilization during metastasis. In addition, membrane vesicles derived from both tumor and host cells have recently been recognized as new candidates with important roles in the promotion of tumor growth and metastasis. This review describes old ideas and presents new insights into the role of tumor and bone marrow-derived microvesicles and exosomes in pre-metastatic niche formation and metastasis. Copyright © 2011 Elsevier Ltd. All rights reserved.

Histomorphologic findings on human bone samples six months after bone augmentation of the maxillary sinus with Algipore.

PubMed

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.

Reciprocal Relation between Marrow Adiposity and the Amount of Bone in the Axial and Appendicular Skeleton of Young Adults

PubMed Central

Di Iorgi, Natascia; Rosol, Michael; Mittelman, Steven D.; Gilsanz, Vicente

2008-01-01

Background: Studies in the elderly suggest a reciprocal relation between increased marrow adiposity and bone loss, supporting basic research data indicating that osteoblasts and adipocytes share a common progenitor cell. However, whether this relation represents a preferential differentiation of stromal cells from osteoblasts to adipocytes or whether a passive accumulation of fat as bone is lost and marrow space increases with aging is unknown. To address this question and avoid the confounding effect of bone loss, we examined teenagers and young adults. Methods: Using computed tomography, we obtained measurements of bone density and cross-sectional area of the lumbar vertebral bodies and cortical bone area, cross-sectional area, marrow canal area, and fat density in the marrow of the femurs in 255 sexually mature subjects (126 females, 129 males; 15–24.9 yr of age). Additionally, values for total body fat were obtained with dual-energy x-ray absorptiometry. Results: Regardless of gender, reciprocal relations were found between fat density and measures of vertebral bone density and femoral cortical bone area (r = 0.19–0.39; all P values ≤ .03). In contrast, there was no relation between marrow canal area and cortical bone area in the femurs, neither between fat density and the cross-sectional dimensions of the bones. We also found no relation between anthropometric or dual-energy x-ray absorptiometry fat values and measures for marrow fat density. Conclusions: Our results indicate an inverse relation between bone marrow adiposity and the amount of bone in the axial and appendicular skeleton and support the notion of a common progenitor cell capable of mutually exclusive differentiation into the cell lineages responsible for bone and fat formation. PMID:18381577

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

PubMed

Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

2009-01-01

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion

PubMed Central

Carbonaro, Denise A.; Jin, Xiangyang; Cotoi, Daniel; Mi, Tiejuan; Yu, Xiao-Jin; Skelton, Dianne C.; Dorey, Frederick; Kellems, Rodney E.; Blackburn, Michael R.

2008-01-01

Adenosine deaminase (ADA)–deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose–dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy. PMID:18356486

Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion.

PubMed

Carbonaro, Denise A; Jin, Xiangyang; Cotoi, Daniel; Mi, Tiejuan; Yu, Xiao-Jin; Skelton, Dianne C; Dorey, Frederick; Kellems, Rodney E; Blackburn, Michael R; Kohn, Donald B

2008-06-15

Adenosine deaminase (ADA)-deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose-dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy.

DLC1-dependent parathyroid hormone–like hormone inhibition suppresses breast cancer bone metastasis

PubMed Central

Wang, Yufeng; Lei, Rong; Zhuang, Xueqian; Zhang, Ning; Pan, Hong; Li, Gang; Hu, Jing; Pan, Xiaoqi; Tao, Qian; Fu, Da; Xiao, Jianru; Chin, Y. Eugene; Kang, Yibin; Yang, Qifeng; Hu, Guohong

2014-01-01

Bone metastasis is a frequent complication of breast cancer that is often accelerated by TGF-β signaling; however, little is known about how the TGF-β pathway is regulated during bone metastasis. Here we report that deleted in liver cancer 1 (DLC1) is an important regulator of TGF-β responses and osteolytic metastasis of breast cancer cells. In murine models, breast cancer cells lacking DLC1 expression exhibited enhanced capabilities of bone metastasis. Knockdown of DLC1 in cancer cells promoted bone metastasis, leading to manifested osteolysis and accelerated death in mice, while DLC1 overexpression suppressed bone metastasis. Activation of Rho-ROCK signaling in the absence of DLC1 mediated SMAD3 linker region phosphorylation and TGF-β–induced expression of parathyroid hormone–like hormone (PTHLH), leading to osteoclast maturation for osteolytic colonization. Furthermore, pharmacological inhibition of Rho-ROCK effectively reduced PTHLH production and breast cancer bone metastasis in vitro and in vivo. Evaluation of clinical breast tumor samples revealed that reduced DLC1 expression was linked to elevated PTHLH expression and organ-specific metastasis to bone. Overall, our findings define a stroma-dependent paradigm of Rho signaling in cancer and implicate Rho–TGF-β crosstalk in osteolytic bone metastasis. PMID:24590291

Bone regeneration with biomaterials and active molecules delivery.

PubMed

D' Este, Matteo; Eglin, David; Alini, Mauro; Kyllonen, Laura

2015-01-01

The combination of biomaterials and drug delivery strategies is a promising avenue towards improved synthetic bone substitutes. With the delivery of active species biomaterials can be provided with the bioactivity they still lack for improved bone regeneration. Recently, a lot of research efforts have been put towards this direction. Biomaterials for bone regeneration have been supplemented with small or biological molecules for improved osteoprogenitor cell recruitment, osteoinductivity, anabolic or angiogenic response, regulation of bone metabolism and others. The scope of this review is to summarize the most recent results in this field.

Effect of transforming growth factor beta (TGF-β) receptor I kinase inhibitor on prostate cancer bone growth.

PubMed

Wan, Xinhai; Li, Zhi-Gang; Yingling, Jonathan M; Yang, Jun; Starbuck, Michael W; Ravoori, Murali K; Kundra, Vikas; Vazquez, Elba; Navone, Nora M

2012-03-01

Transforming growth factor beta 1 (TGF-β1) has been implicated in the pathogenesis of prostate cancer (PCa) bone metastasis. In this study, we tested the antitumor efficacy of a selective TGF-β receptor I kinase inhibitor, LY2109761, in preclinical models. The effect of LY2109761 on the growth of MDA PCa 2b and PC-3 human PCa cells and primary mouse osteoblasts (PMOs) was assessed in vitro by measuring radiolabeled thymidine incorporation into DNA. In vivo, the right femurs of male SCID mice were injected with PCa cells. We monitored the tumor burden in control- and LY2109761-treated mice with MRI analysis and the PCa-induced bone response with X-ray and micro-CT analyses. Histologic changes in bone were studied by performing bone histomorphometric evaluations. PCa cells and PMOs expressed TGF-β receptor I. TGF-β1 induced pathway activation (as assessed by induced expression of p-Smad2) and inhibited cell growth in PC-3 cells and PMOs but not in MDA PCa 2b cells. LY2109761 had no effect on PCa cells but induced PMO proliferation in vitro. As expected, LY2109761 reversed the TGF-β1-induced pathway activation and growth inhibition in PC-3 cells and PMOs. In vivo, LY2109761 treatment for 6weeks resulted in increased volume in normal bone and increased osteoblast and osteoclast parameters. In addition, LY2109761 treatment significantly inhibited the growth of MDA PCa 2b and PC-3 in the bone of SCID mice (p<0.05); moreover, it resulted in significantly less bone loss and change in osteoclast-associated parameters in the PC-3 tumor-bearing bones than in the untreated mice. In summary, we report for the first time that targeting TGF-β receptors with LY2109761 can control PCa bone growth while increasing the mass of normal bone. This increased bone mass in nontumorous bone may be a desirable side effect of LY2109761 treatment for men with osteopenia or osteoporosis secondary to androgen-ablation therapy, reinforcing the benefit of effectively controlling PCa growth in bone. Thus, targeting TGF-β receptor I is a valuable intervention in men with advanced PCa. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of transforming growth factor beta (TGF-β) receptor I kinase inhibitor on prostate cancer bone growth

PubMed Central

Wan, Xinhai; Li, Zhi-Gang; Yingling, Jonathan M.; Yang, Jun; Starbuck, Michael W.; Ravoori, Murali K.; Kundra, Vikas; Vazquez, Elba; Navone, Nora M.

2012-01-01

Transforming growth factor beta 1 (TGF-β1) has been implicated in the pathogenesis of prostate cancer (PCa) bone metastasis. In this study, we tested the antitumor efficacy of a selective TGF-β receptor I kinase inhibitor, LY2109761, in preclinical models. The effect of LY2109761 on the growth of MDA PCa 2b and PC-3 human PCa cells and primary mouse osteoblasts (PMOs) was assessed in vitro by measuring radiolabeled thymidine incorporation into DNA. In vivo, the right femurs of male SCID mice were injected with PCa cells. We monitored the tumor burden in control- and LY2109761-treated mice with MRI analysis and the PCa-induced bone response with x-ray and micro-CT analyses. Histologic changes in bone were studied by performing bone histomorphometric evaluations. PCa cells and PMOs expressed TGF-β receptor I. TGF-β1 induced pathway activation (as assessed by induced expression of p-Smad2) and inhibited cell growth in PC-3 cells and PMOs but not in MDA PCa 2b cells. LY2109761 had no effect on PCa cells but induced PMO proliferation in vitro. As expected, LY2109761 reversed the TGF-β1–induced pathway activation and growth inhibition in PC-3 cells and PMOs. In vivo, LY2109761 treatment for 6 weeks resulted in increased volume in normal bone and increased osteoblast and osteoclast parameters. In addition, LY2109761 treatment significantly inhibited the growth of MDA PCa 2b and PC-3 in the bone of SCID mice (p < 0.05); moreover, it resulted in significantly less bone loss and change in osteoclast-associated parameters in the PC-3 tumor–bearing bones than in the untreated mice. In summary, we report for the first time that targeting TGF-β receptors with LY2109761 can control PCa bone growth while increasing the mass of normal bone. This increased bone mass in nontumorous bone may be a desirable side effect of LY2109761 treatment for men with osteopenia or osteoporosis secondary to androgen-ablation therapy, reinforcing the benefit of effectively controlling PCa growth in bone. Thus, targeting TGF-β receptor I is a valuable intervention in men with advanced PCa. PMID:22173053

Effect of autogenous and fresh-frozen bone grafts on osteoblast differentiation.

PubMed

Ferraz, E P; Xavier, S P; Azevedo, F G; de Oliveira, F S; Beloti, M M; Rosa, A L

2015-01-01

Fresh-frozen bone allograft (FFBA) is an alternative to autogenous bone (AB) for reconstructing maxillary bone. Despite the promising clinical results, cell responses to FFBA and AB were not evaluated. Thus, our aim was to compare cells harvested from maxillary reconstructed sites with either AB or FFBA in terms of osteoblast differentiation and to evaluate the effect of culturing cells in contact with FFBA. Cells harvested from three patients submitted to bilateral maxillary reconstruction with AB and FFBA were cultured to evaluate: proliferation, alkaline phosphatase activity, extracellular matrix mineralization and gene expression of osteoblastic markers. The effect of FFBA on osteoblast differentiation was studied by culturing cells harvested from AB in contact with FFBA and evaluating the same parameters. Data were compared using either two-way ANOVA followed by Tukey-b test or Student's t test (p≤0.05). Cell proliferation was higher in cultures from AB grafted sites and extracellular matrix mineralization was higher in cultures derived from FFBA grafted sites. The gene expression of alkaline phosphatase, RUNX2, bone sialoprotein and osteocalcin was higher in cells derived from FFBA compared with cells from AB grafted sites. However, the exposure of cells derived from AB to FFBA particles did not have any remarkable effect on osteoblast differentiation. These results indicate the higher osteogenic activity of cells derived from FFBA compared with AB reconstructed sites, offering an explanation at cellular level of why FFBA could be a suitable alternative to AB for reconstructing maxillary bone defects. Copyright © 2014 Elsevier Ltd. All rights reserved.

Preclinical studies in support of defibrotide for the treatment of multiple myeloma and other neoplasias.

PubMed

Mitsiades, Constantine S; Rouleau, Cecile; Echart, Cinara; Menon, Krishna; Teicher, Beverly; Distaso, Maria; Palumbo, Antonio; Boccadoro, Mario; Anderson, Kenneth C; Iacobelli, Massimo; Richardson, Paul G

2009-02-15

Defibrotide, an orally bioavailable polydisperse oligonucleotide, has promising activity in hepatic veno-occlusive disease, a stem cell transplantation-related toxicity characterized by microangiopathy. The antithrombotic properties of defibrotide and its minimal hemorrhagic risk could serve for treatment of cancer-associated thrombotic complications. Given its cytoprotective effect on endothelium, we investigated whether defibrotide protects tumor cells from cytotoxic antitumor agents. Further, given its antiadhesive properties, we evaluated whether defibrotide modulates the protection conferred to multiple myeloma cells by bone marrow stromal cells. Defibrotide lacks significant single-agent in vitro cytotoxicity on multiple myeloma or solid tumor cells and does not attenuate their in vitro response to dexamethasone, bortezomib, immunomodulatory thalidomide derivatives, and conventional chemotherapeutics, including melphalan and cyclophosphamide. Importantly, defibrotide enhances in vivo chemosensitivity of multiple myeloma and mammary carcinoma xenografts in animal models. In cocultures of multiple myeloma cells with bone marrow stromal cells in vitro, defibrotide enhances the multiple myeloma cell sensitivity to melphalan and dexamethasone, and decreases multiple myeloma-bone marrow stromal cell adhesion and its sequelae, including nuclear factor-kappaB activation in multiple myeloma and bone marrow stromal cells, and associated cytokine production. Moreover, defibrotide inhibits expression and/or function of key mediators of multiple myeloma interaction with bone marrow stromal cell and endothelium, including heparanase, angiogenic cytokines, and adhesion molecules. Defibrotide's in vivo chemosensitizing properties and lack of direct in vitro activity against tumor cells suggest that it favorably modulates antitumor interactions between bone marrow stromal cells and endothelia in the tumor microenvironment. These data support clinical studies of defibrotide in combination with conventional and novel therapies to potentially improve patient outcome in multiple myeloma and other malignancies.

Evaluating acetate metabolism for imaging and targeting in multiple myeloma

PubMed Central

Fontana, Francesca; Ge, Xia; Su, Xinming; Hathi, Deep; Xiang, Jingyu; Cenci, Simone; Civitelli, Roberto; Shoghi, Kooresh I.; Akers, Walter J.; D’avignon, Andre

2016-01-01

Purpose We hypothesized that in multiple myeloma cells (MMC), high membrane biosynthesis will induce acetate uptake in vitro and in vivo. Here, we studied acetate metabolism and targeting in MMC in vitro and tested the efficacy of 11C-acetate-PET (positron emission tomography) to detect and quantitatively image myeloma treatment response in vivo. Experimental design Acetate fate tracking using 13C-edited-1H NMR (nuclear magnetic resonance) was performed to study in vitro acetate uptake and metabolism in MMC. Effects of pharmacological modulation of acetate transport or acetate incorporation into lipids on MMC cell survival and viability were assessed. Preclinical mouse MM models of subcutaneous and bone tumors were evaluated using 11C-acetate-PET/CT imaging and tissue biodistribution. Results In vitro, NMR showed significant uptake of acetate by MMC, and acetate incorporation into intracellular metabolites and membrane lipids. Inhibition of lipid synthesis and acetate transport was toxic to MMC, while sparing resident bone cells or normal B cells. In vivo, 11C-acetate uptake by PET imaging was significantly enhanced in subcutaneous and bone MMC tumors compared to unaffected bone or muscle tissue. Likewise, 11C-acetate uptake was significantly reduced in MM tumors after treatment. Conclusions Uptake of acetate from the extracellular environment was enhanced in MMC and was critical to cellular viability. 11C-acetate-PET detected the presence of myeloma cells in vivo, including uptake in intramedullary bone disease. 11C-acetate-PET also detected response to therapy in vivo. Our data suggested that acetate metabolism and incorporation into lipids was crucial to MM cell biology and that 11C-acetate-PET is a promising imaging modality for MM. PMID:27486177

Biophysical regulation of stem cell differentiation.

PubMed

Govey, Peter M; Loiselle, Alayna E; Donahue, Henry J

2013-06-01

Bone adaptation to its mechanical environment, from embryonic through adult life, is thought to be the product of increased osteoblastic differentiation from mesenchymal stem cells. In parallel with tissue-scale loading, these heterogeneous populations of multipotent stem cells are subject to a variety of biophysical cues within their native microenvironments. Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro. Further studies are needed to confirm both direct and indirect mechanisms of biophysical regulation within the in vivo stem cell niche.

Osteoblast and osteocyte: games without frontiers.

PubMed

Capulli, Mattia; Paone, Riccardo; Rucci, Nadia

2014-11-01

The portrait of osteoblasts and osteocytes has been subjected to a revision, since a large body of evidence is attributing these cells amazing roles both inside and outside the bone. The osteoblast, long confined to its bone building function, is actually a very eclectic cell, actively regulating osteoclast formation and function as well as hematopoietic stem cells homeostasis. It is also an endocrine cell, affecting energy metabolism, male fertility and cognition through the release of osteocalcin, a perfect definition-fitting hormone in its uncarboxylated state. As for the osteocytes, many evidence shows that they do not merely represent the final destination of the osteoblasts, but they are instead very active cells that, besides a mechanosensorial function, actively contribute to the bone remodelling by regulating bone formation and resorption. The regulation is exerted by the production of sclerostin (SOST), which in turn inhibits osteoblast differentiation by blocking Wnt/beta-catenin pathway. At the same time, osteocytes influence bone resorption both indirectly, by producing RANKL, which stimulates osteoclastogenesis, and directly by means of a local osteolysis, which is observed especially under pathological conditions. The great versatility of both these cells reflects the complexity of the bone tissue, which has not only a structural role, but influences and is influenced by different organs, taking part in homeostatic and adaptive responses affecting the whole organism. Copyright © 2014. Published by Elsevier Inc.

Nicotine suppresses bone sialoprotein gene expression.

PubMed

Nakayama, Y; Mezawa, M; Araki, S; Sasaki, Y; Wang, S; Han, J; Li, X; Takai, H; Ogata, Y

2009-10-01

Tobacco smoking is a risk factor for periodontitis and osteoporosis. Nicotine is a major component of tobacco, and has been reported to inhibit proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. The purpose of this study was to determine the effects of nicotine on bone metabolism. We used rat osteobast-like UMR106 and ROS 17/2.8 cells and rat stromal bone marrow RBMC-D8 cells. To determine the molecular basis of the transcriptional regulation of the BSP gene by nicotine, we conducted Northern hybridization, transient transfection analyses with chimeric constructs of the BSP gene promoter linked to a luciferase reporter gene and gel mobility shift assays. Nicotine (250 microg/mL) decreased the BSP mRNA levels at 12 and 24 h in UMR106 and ROS 17/2.8 cells. From transient transfection assays using various sized BSP promoter-luciferase constructs, nicotine decreased the luciferase activities of the construct, including the promoter sequence nucleotides -116 to +60, in UMR106 and RBMC-D8 cells. Nicotine decreased the nuclear protein binding to the cAMP response element (CRE), fibroblast growth factor 2 response element (FRE) and homeodomain protein-binding site (HOX) at 12 and 24 h. This study indicates that nicotine suppresses BSP transcription mediated through CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene.

Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance and Lead to Significant Bone Tissue Formation

PubMed Central

Cusella-De Angelis, Maria Gabriella; Laino, Gregorio; Piattelli, Adriano; Pacifici, Maurizio; De Rosa, Alfredo; Papaccio, Gianpaolo

2007-01-01

Background Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear. Methodology and Findings In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80–120 µm in diameter and 40–100 µm in depth, which we termed primary; and (ii) smaller microcavities of 10–20 µm in diameter and 3–10 µm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold. Conclusion In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and bioengineering applications in the future. PMID:17551577

Methods for Studying the Role of RAAS in the Modulation of Vascular Repair-Relevant Functions of Stem/Progenitor Cells.

PubMed

Jarajapu, Yagna P R

2017-01-01

In recent years, previously unknown functions have been conferred to the RAAS and have been explored in mechanistic studies and disease models. Implication of bone marrow stem/progenitor cells in the cardiovascular protective or detrimental effects of RAAS is a prominent advancement because of the translational significance. Selected members of RAAS are now known to modulate migration, proliferation, and mobilization of bone marrow cells in response to ischemic insult, which are sensitive indicators of vascular repair-relevant functions. In this Chapter, protocols for most frequently used, in vitro, ex vivo, and in vivo assays to explore the potential of RAAS members to stimulate vascular repair-relevant functions of bone marrow stem/progenitor cells of human and murine origin.

Comparison of cellular responses of mesenchymal stem cells derived from bone marrow and synovium on combined silk scaffolds.

PubMed

Liu, Haifeng; Wei, Xing; Ding, Xili; Li, Xiaoming; Zhou, Gang; Li, Ping; Fan, Yubo

2015-01-01

As a brand new member in mesenchymal stem cells (MSCs) families, synovium-derived mesenchymal stem cells (SMSCs) have been increasingly regarded as a promising therapeutic cell species for musculoskeletal regeneration. However, there are few reports mentioning ligamentogenesis of SMSCs and especially null for their engineering use towards ligament regeneration. The aim of this study was to investigate and compare the cellular responses of MSCs derived from bone marrow and synovium on combined silk scaffolds that can be used to determine the cell source most appropriate for tissue-engineered ligament. Rabbit SMSCs and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and cultured in vitro for two weeks after seeding on the combined silk scaffolds. Samples were studied and compared for their cellular morphology, proliferation, collagen production, gene, and protein expression of ligament-related extracellular matrix (ECM) markers. In addition, the two cell types were transfected with green fluorescent protein to evaluate their fate after implantation in an intraarticular environment of the knee joint. After 14 days of culturing, SMSCs showed a significant increase in proliferation as compared with BMSCs. The transcript and protein expression levels of ligament-related ECM markers in SMSCs were significantly higher than those in BMSCs. Moreover, 6 weeks postoperatively, more viable cells were presented in SMSC-loaded constructs than in BMSC-loaded constructs. Therefore, based on the cellular response in vitro and in vivo, SMSCs may represent a more suitable cell source than BMSCs for further study and development of tissue-engineered ligament. © 2014 Wiley Periodicals, Inc.

Central Nervous System Fibrosis Is Associated with Fibrocyte-Like Infiltrates

PubMed Central

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

Bone marrow transplantation alters lung antigen presenting cells to promote TH17 response and the development of pneumonitis and fibrosis following gammaherpesvirus infection

PubMed Central

Zhou, Xiaofeng; Loomis-King, Hillary; Gurczynski, Stephen J.; Wilke, Carol A.; Konopka, Kristine E.; Ptaschinski, Catherine; Coomes, Stephanie M; Iwakura, Yoichiro; van Dyk, Linda F.; Lukacs, Nicholas W.; Moore, Bethany B.

2015-01-01

Hematopoietic stem cell transplantation (HSCT) efficacy is limited by numerous pulmonary complications. We developed a model of syngeneic bone marrow transplant (BMT) followed by infection with murine gamma herpesvirus (γHV-68) that results in pneumonitis and fibrosis and mimics human “non-infectious” HSCT complications. BMT mice experience increased early lytic replication, but establish viral latency by 21 days post infection (dpi). CD4 T cells in BMT mice are skewed towards IL-17A rather than IFN-γ production. Transplantation of bone marrow from Il-17a−/− donors or treatment with anti-IL-17A neutralization antibodies at late stages attenuates pneumonitis and fibrosis in infected BMT mice, suggesting that hematopoietic-derived IL-17A is essential for development of pathology. IL-17A directly influences activation and extracellular matrix production by lung mesenchymal cells. Lung CD11c+ cells of BMT mice secrete more TGF-β1, and pro-TH17 mRNAs for IL-23 and IL-6, and less TH1-promoting cytokine mRNA for IFN-γ but slightly more IL-12 mRNA in response to viral infection. Adoptive transfer of non-BMT lung CD11c-enriched cells restores robust TH1 response and suppresses aberrant TH17 response in BMT mice to improve lung pathology. Our data suggest “non-infectious” HSCT lung complications may reflect preceding viral infections and demonstrate that IL-17A neutralization may offer therapeutic advantage even after disease onset. PMID:26376362

Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells

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

Liu, P.-S., E-mail: pslediting@mail.scu.edu.t; Chen, C.-Y.

2010-05-01

Butyl benzyl phthalate (BBP), an endocrine disruptor present in the environment, exerts its genomic effects via intracellular steroid receptors and elicits non-genomic effects by interfering with membrane ion-channel receptors. We previously found that BBP blocks the calcium signaling coupled with P2X receptors in PC12 cells (Liu and Chen, 2006). Osteoblast P2X receptors were recently reported to play a role in cell proliferation and bone remodeling. In this present study, the effects of BBP on ATP-induced responses were investigated in human osteosarcoma HOS cells. These receptors mRNA had been detected, named P2X4, P2X7, P2Y2, P2Y4, P2Y5, P2Y9, and P2Y11, in humanmore » osteosarcoma HOS cells by RT-PCR. The enhancement of cell proliferation and the decrease of cytoviability had both been shown to be coupled to stimulation via different concentrations of ATP. BBP suppressed the ATP-induced calcium influx (mainly coupled with P2X) and cell proliferation but not the ATP-induced intracellular calcium release (mainly coupled with P2Y) and cytotoxicity in human osteosarcoma HOS cells. Suramin, a common P2 receptor's antagonist, blocked the ATP-induced calcium signaling, cell proliferation, and cytotoxicity. We suggest that P2X is mainly responsible for cell proliferation, and P2Y might be partially responsible for the observed cytotoxicity. BBP suppressed the calcium signaling coupled with P2X, suppressing cell proliferation. Since the importance of P2X receptors during bone metastasis has recently become apparent, the possible toxic risk of environmental BBP during bone remodeling is a public problem of concern.« less

Autophagy: a new player in skeletal maintenance?

PubMed

Hocking, Lynne J; Whitehouse, Caroline; Helfrich, Miep H

2012-07-01

Imbalances between bone resorption and formation lie at the root of disorders such as osteoporosis, Paget's disease of bone (PDB), and osteopetrosis. Recently, genetic and functional studies have implicated proteins involved in autophagic protein degradation as important mediators of bone cell function in normal physiology and in pathology. Autophagy is the conserved process whereby aggregated proteins, intracellular pathogens, and damaged organelles are degraded and recycled. This process is important both for normal cellular quality control and in response to environmental or internal stressors, particularly in terminally-differentiated cells. Autophagic structures can also act as hubs for the spatial organization of recycling and synthetic process in secretory cells. Alterations to autophagy (reduction, hyperactivation, or impairment) are associated with a number of disorders, including neurodegenerative diseases and cancers, and are now being implicated in maintenance of skeletal homoeostasis. Here, we introduce the topic of autophagy, describe the new findings that are starting to emerge from the bone field, and consider the therapeutic potential of modifying this pathway for the treatment of age-related bone disorders. Copyright © 2012 American Society for Bone and Mineral Research.

Fluoride’s Effects on the Formation of Teeth and Bones, and the Influence of Genetics

PubMed Central

Everett, E.T.

2011-01-01

Fluorides are present in the environment. Excessive systemic exposure to fluorides can lead to disturbances of bone homeostasis (skeletal fluorosis) and enamel development (dental/enamel fluorosis). The severity of dental fluorosis is also dependent upon fluoride dose and the timing and duration of fluoride exposure. Fluoride’s actions on bone cells predominate as anabolic effects both in vitro and in vivo. More recently, fluoride has been shown to induce osteoclastogenesis in mice. Fluorides appear to mediate their actions through the MAPK signaling pathway and can lead to changes in gene expression, cell stress, and cell death. Different strains of inbred mice demonstrate differential physiological responses to ingested fluoride. Genetic studies in mice are capable of identifying and characterizing fluoride-responsive genetic variations. Ultimately, this can lead to the identification of at-risk human populations who are susceptible to the unwanted or potentially adverse effects of fluoride action and to the elucidation of fundamental mechanisms by which fluoride affects biomineralization. PMID:20929720

Cellular Responses Evoked by Different Surface Characteristics of Intraosseous Titanium Implants

PubMed Central

Feller, Liviu; Jadwat, Yusuf; Khammissa, Razia A. G.; Meyerov, Robin; Lemmer, Johan

2015-01-01

The properties of biomaterials, including their surface microstructural topography and their surface chemistry or surface energy/wettability, affect cellular responses such as cell adhesion, proliferation, and migration. The nanotopography of moderately rough implant surfaces enhances the production of biological mediators in the peri-implant microenvironment with consequent recruitment of differentiating osteogenic cells to the implant surface and stimulates osteogenic maturation. Implant surfaces with moderately rough topography and with high surface energy promote osteogenesis, increase the ratio of bone-to-implant contact, and increase the bonding strength of the bone to the implant at the interface. Certain features of implant surface chemistry are also important in enhancing peri-implant bone wound healing. It is the purpose of this paper to review some of the more important features of titanium implant surfaces which have an impact on osseointegration. PMID:25767803

Review of osteoimmunology and the host response in endodontic and periodontal lesions

PubMed Central

Graves, Dana T.; Oates, Thomas; Garlet, Gustavo P.

2011-01-01

Both lesions of endodontic origin and periodontal diseases involve the host response to bacteria and the formation of osteolytic lesions. Important for both is the upregulation of inflammatory cytokines that initiate and sustain the inflammatory response. Also important are chemokines that induce recruitment of leukocyte subsets and bone-resorptive factors that are largely produced by recruited inflammatory cells. However, there are differences also. Lesions of endodontic origin pose a particular challenge since that bacteria persist in a protected reservoir that is not readily accessible to the immune defenses. Thus, experiments in which the host response is inhibited in endodontic lesions tend to aggravate the formation of osteolytic lesions. In contrast, bacteria that invade the periodontium appear to be less problematic so that blocking arms of the host response tend to reduce the disease process. Interestingly, both lesions of endodontic origin and periodontitis exhibit inflammation that appears to inhibit bone formation. In periodontitis, the spatial location of the inflammation is likely to be important so that a host response that is restricted to a subepithelial space is associated with gingivitis, while a host response closer to bone is linked to bone resorption and periodontitis. However, the persistence of inflammation is also thought to be important in periodontitis since inflammation present during coupled bone formation may limit the capacity to repair the resorbed bone. PMID:21547019

From Osteoimmunology to Osteomicrobiology: How the Microbiota and the Immune System Regulate Bone.

PubMed

Hsu, Emory; Pacifici, Roberto

2018-05-01

Osteomicrobiology refers to the role of microbiota in bone health and the mechanisms by which the microbiota regulates post-natal skeletal development, bone aging, and pathologic bone loss. Here, we review recent reports linking gut microbiota to changes in bone phenotype. A pro-inflammatory cytokine milieu drives bone resorption in conditions such as sex steroid hormone deficiency. The response of the immune system to activation by the microbiome results in increased circulating osteoclastogenic cytokines in a T cell-dependent mechanism. Additionally, gut microbiota affect bone homeostasis through nutrient absorption, mediation of the IGF-1 pathway, and short chain fatty acid and metabolic products. Manipulation of microbiota through prebiotics or probiotics reduces inflammatory cytokine production, leading to changes in bone density. One mechanism of probiotic action is through upregulating tight junction proteins, increasing the strength of the gut epithelial layer, and leading to less antigen presentation and less activation of intestinal immune cells. Thus, prebiotics or probiotics may represent a future therapeutic avenue for ameliorating the risk of postmenopausal bone loss in humans.

Decursin from Angelica gigas suppresses RANKL-induced osteoclast formation and bone loss.

PubMed

Wang, Xin; Zheng, Ting; Kang, Ju-Hee; Li, Hua; Cho, Hyewon; Jeon, Raok; Ryu, Jae-Ha; Yim, Mijung

2016-03-05

Osteoclasts are the only cells capable of breaking down bone matrix, and excessive activation of osteoclasts is responsible for bone-destructive diseases. In this study, we investigated the effects of decursin from extract of Angelica gigas root on receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast formation using mouse bone marrow-derived macrophages (BMMs). Decursin inhibited RANKL-induced osteoclast formation without cytotoxicity. In particular, decursin maintains the characteristics of macrophages by blocking osteoclast differentiation by RANKL. Furthermore, the RANKL-stimulated bone resorption was diminished by decursin. Mechanistically, decursin blocked the RANKL-triggered ERK mitogen-activated protein kinases (MAPK) phosphorylation, which results in suppression of c-Fos and the nuclear factor of activated T cells (NFATc1) expression. In accordance with the in vitro study, decursin reduced lipopolysaccharide (LPS)- or ovariectomy (OVX)-induced bone loss in vivo. Therefore, decursin exerted an inhibitory effect on osteoclast formation and bone loss in vitro and in vivo. Decursin could be useful for the treatment of bone diseases associated with excessive bone resorption. Copyright © 2016 Elsevier B.V. All rights reserved.

Altered paracrine signaling from the injured knee joint impairs postnatal long bone growth.

PubMed

Roselló-Díez, Alberto; Stephen, Daniel; Joyner, Alexandra L

2017-07-25

Regulation of organ growth is a poorly understood process. In the long bones, the growth plates (GPs) drive elongation by generating a scaffold progressively replaced by bone. Although studies have focused on intrinsic GP regulation, classic and recent experiments suggest that local signals also modulate GP function. We devised a genetic mouse model to study extrinsic long bone growth modulation, in which injury is specifically induced in the left hindlimb, such that the right hindlimb serves as an internal control. Remarkably, when only mesenchyme cells surrounding postnatal GPs were killed, left bone growth was nevertheless reduced. GP signaling was impaired by altered paracrine signals from the knee joint, including activation of the injury response and, in neonates, dampened IGF1 production. Importantly, only the combined prevention of both responses rescued neonatal growth. Thus, we identified signals from the knee joint that modulate bone growth and could underlie establishment of body proportions.

Effects of microgravity on the immune system

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Taylor, Gerald R.

1991-01-01

Changes in resistance to bacterial and viral infections in Apollo crew members has stimulated interest in the study of immunity and space flight. Results of studies from several laboratories in both humans and rodents have indicated alterations after space flight that include the following immunological parameters: thymus size, lymphocyte blastogenesis, interferon and interleukin production, natural killer cell activity, cytotoxic T-cell activity, leukocyte subset population distribution, response of bone marrow cells to colony stimulating factors, and delayed hypersensitivity skin test reactivity. The interactions of the immune system with other physiological systems, including muscle, bone, and the nervous system, may play a major role in the development of these immunological parameters during and after flight. There may also be direct effects of space flight on immune responses.

Macrophages – Key Cells in the Response to Wear Debris from Joint Replacements

PubMed Central

Nich, Christophe; Takakubo, Yuya; Pajarinen, Jukka; Ainola, Mari; Salem, Abdelhakim; Sillat, Tarvo; Rao, Allison J.; Raska, Milan; Tamaki, Yasunobu; Takagi, Michiaki; Konttinen, Yrjö T.; Goodman, Stuart B.; Gallo, Jiri

2013-01-01

The generation of wear debris is an inevitable result of normal usage of joint replacements. Wear debris particles stimulate local and systemic biological reactions resulting in chronic inflammation, periprosthetic bone destruction, and eventually, implant loosening and revision surgery. The latter may be indicated in up to 15% patients in the decade following the arthroplasty using conventional polyethylene. Macrophages play multiple roles in both inflammation and in maintaining tissue homeostasis. As sentinels of the innate immune system, they are central to the initiation of this inflammatory cascade, characterized by the release of pro-inflammatory and pro-osteoclastic factors. Similar to the response to pathogens, wear particles elicit a macrophage response, based on the unique properties of the cells belonging to this lineage, including sensing, chemotaxis, phagocytosis, and adaptive stimulation. The biological processes involved are complex, redundant, both local and systemic, and highly adaptive. Cells of the monocyte/macrophage lineage are implicated in this phenomenon, ultimately resulting in differentiation and activation of bone resorbing osteoclasts. Simultaneously, other distinct macrophage populations inhibit inflammation and protect the bone-implant interface from osteolysis. Here, the current knowledge about the physiology of monocyte/macrophage lineage cells is reviewed. In addition, the pattern and consequences of their interaction with wear debris and the recent developments in this field are presented. PMID:23568608

[Biocompatibility research of true bone ceramics].

PubMed

Qiao, Wei; Ren, Xiaoqi; Shi, Hao; Li, Jing; Yang, Ting; Ma, Shaoying; Zhao, Yaping; Su, Chengzhong; Li, Baoxing

2017-10-01

To investigate the biocompatibility of true bone ceramic (TBC) and provide experimental basis for clinic application. TBC was prepared from healthy adult bovine cancellous bone by deproteinization and high temperature calcinations. Mouse fibroblast cell line (L929 cells) were cultured with the leaching liquor of TBC in vitro , and the cytotoxicity was evaluated at 2nd, 4th, and 7th days. L929 cells were inoculated into the TBC and cultured for 4 days. The cell adhesion and proliferation on the surface of the TBC were observed by scanning electron microscopy, and evaluated the cell compatibility of TBC. Ten New Zealand white rabbits were divided into 2 groups, and drilled holes at the tibia of both hind limbs. TBC and hydroxyapatite (HA) were implanted into the left side (experimental group) and the right side (control group), respectively. And the biocompatibility of TBC was evaluated by general observation and histological observation at 4 and 26 weeks after implantation. Cytotoxicity test showed that the cytotoxicity level of leaching liquor of TBC was grade 0-1. Cell compatibility experiments showed that the L929 cells adhered well on the surface of TBC and migrated into the pores. The implantation test in vivo showed that experimental group and control group both had mild or moderate inflammatory response at 4 weeks, and new bone formation occurred. At 26 weeks, there was no inflammatory reaction observed in both groups, and new bone formation was observed in varying degrees. TBC have good biocompatibility and can be used to repair bone defect in clinic.

Ultra-Porous Nanoparticle Networks: A Biomimetic Coating Morphology for Enhanced Cellular Response and Infiltration

PubMed Central

Nasiri, Noushin; Ceramidas, Anthony; Mukherjee, Shayanti; Panneerselvan, Anitha; Nisbet, David R.; Tricoli, Antonio

2016-01-01

Orthopedic treatments are amongst the most common cause of surgery and are responsible for a large share of global healthcare expenditures. Engineering materials that can hasten bone integration will improve the quality of life of millions of patients per year and reduce associated medical costs. Here, we present a novel hierarchical biomimetic coating that mimics the inorganic constituent of mammalian bones with the aim of improving osseointegration of metallic implants. We exploit the thermally-driven self-organization of metastable core-shell nanoparticles during their aerosol self-assembly to rapidly fabricate robust, ultra-porous nanoparticle networks (UNN) of crystalline hydroxyapatite (HAp). Comparative analysis of the response of osteoblast cells to the ultra-porous nanostructured HAp surfaces and to the spin coated HAp surfaces revealed superior osseointegrative properties of the UNN coatings with significant cell and filopodia infiltration. This flexible synthesis approach for the engineering of UNN HAp coatings on titanium implants provides a platform technology to study the bone-implant interface for improved osseointegration and osteoconduction. PMID:27076035

Cytokine-induced killer cells eradicate bone and soft-tissue sarcomas.

PubMed

Sangiolo, Dario; Mesiano, Giulia; Gammaitoni, Loretta; Leuci, Valeria; Todorovic, Maja; Giraudo, Lidia; Cammarata, Cristina; Dell'Aglio, Carmine; D'Ambrosio, Lorenzo; Pisacane, Alberto; Sarotto, Ivana; Miano, Sara; Ferrero, Ivana; Carnevale-Schianca, Fabrizio; Pignochino, Ymera; Sassi, Francesco; Bertotti, Andrea; Piacibello, Wanda; Fagioli, Franca; Aglietta, Massimo; Grignani, Giovanni

2014-01-01

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease.

Mechanical Loading Attenuates Radiation-Induced Bone Loss in Bone Marrow Transplanted Mice.

PubMed

Govey, Peter M; Zhang, Yue; Donahue, Henry J

2016-01-01

Exposure of bone to ionizing radiation, as occurs during radiotherapy for some localized malignancies and blood or bone marrow cancers, as well as during space travel, incites dose-dependent bone morbidity and increased fracture risk. Rapid trabecular and endosteal bone loss reflects acutely increased osteoclastic resorption as well as decreased bone formation due to depletion of osteoprogenitors. Because of this dysregulation of bone turnover, bone's capacity to respond to a mechanical loading stimulus in the aftermath of irradiation is unknown. We employed a mouse model of total body irradiation and bone marrow transplantation simulating treatment of hematologic cancers, hypothesizing that compression loading would attenuate bone loss. Furthermore, we hypothesized that loading would upregulate donor cell presence in loaded tibias due to increased engraftment and proliferation. We lethally irradiated 16 female C57Bl/6J mice at age 16 wks with 10.75 Gy, then IV-injected 20 million GFP(+) total bone marrow cells. That same day, we initiated 3 wks compression loading (1200 cycles 5x/wk, 10 N) in the right tibia of 10 of these mice while 6 mice were irradiated, non-mechanically-loaded controls. As anticipated, before-and-after microCT scans demonstrated loss of trabecular bone (-48.2% Tb.BV/TV) and cortical thickness (-8.3%) at 3 wks following irradiation. However, loaded bones lost 31% less Tb.BV/TV and 8% less cortical thickness (both p<0.001). Loaded bones also had significant increases in trabecular thickness and tissue mineral densities from baseline. Mechanical loading did not affect donor cell engraftment. Importantly, these results demonstrate that both cortical and trabecular bone exposed to high-dose therapeutic radiation remain capable of an anabolic response to mechanical loading. These findings inform our management of bone health in cases of radiation exposure.

Phenotypic research on senile osteoporosis caused by SIRT6 deficiency

PubMed Central

Zhang, De-Mao; Cui, Di-Xin; Xu, Ruo-Shi; Zhou, Ya-Chuan; Zheng, Li-Wei; Liu, Peng; Zhou, Xue-Dong

2016-01-01

Osteoporosis is a serious public bone metabolic disease. However, the mechanisms underlying bone loss combined with ageing, which is known as senile osteoporosis, remains unknown. Here we show the detailed phenotype of this disease caused by SIRT6 knock out (KO) in mice. To the best of our knowledge, this is the first study to reveal that SIRT6 is expressed in both bone marrow stroma cells and bone-related cells in both mouse and human models, which suggests that SIRT6 is an important regulator in bone metabolism. SIRT6-KO mice exhibit a significant decrease in body weight and remarkable dwarfism. The skeleton of the SIRT6-KO mouse is deficient in cartilage and mineralized bone tissue. Moreover, the osteocalcin concentration in blood is lower, which suggests that bone mass is markedly lost. Besides, the tartrate-resistant acid phosphatase 5b (TRAP5b) concentration is much higher, which suggests that bone resorption is overactive. Both trabecular and cortical bones exhibit severe osteopenia, and the bone mineral density is decreased. Moreover, double-labelling analysis shows that bone formation is much slower. To determine whether SIRT6 directly regulates bone metabolism, we cultured primary bone marrow stromal cells for osteogenesis and osteoclastogenesis separately to avoid indirect interference in vivo responses such as inflammation. Taken together, these results show that SIRT6 can directly regulate osteoblast proliferation and differentiation, resulting in attenuation in mineralization. Furthermore, SIRT6 can directly regulate osteoclast differentiation and results in a higher number of small osteoclasts, which may be related to overactive bone resorption. PMID:27357320

Hybrid micro/nano-topography of a TiO2 nanotube-coated commercial zirconia femoral knee implant promotes bone cell adhesion in vitro.

PubMed

Frandsen, Christine J; Noh, Kunbae; Brammer, Karla S; Johnston, Gary; Jin, Sungho

2013-07-01

Various approaches have been studied to engineer the implant surface to enhance bone in-growth properties, particularly using micro- and nano-topography. In this study, the behavior of osteoblast (bone) cells was analyzed in response to a titanium oxide (TiO2) nanotube-coated commercial zirconia femoral knee implant consisting of a combined surface structure of a micro-roughened surface with the nanotube coating. The osteoblast cells demonstrated high degrees of adhesion and integration into the surface of the nanotube-coated implant material, indicating preferential cell behavior on this surface when compared to the bare implant. The results of this brief study provide sufficient evidence to encourage future studies. The development of such hierarchical micro- and nano-topographical features, as demonstrated in this work, can provide insightful designs for advanced bone-inducing material coatings on ceramic orthopedic implant surfaces. Copyright © 2013 Elsevier B.V. All rights reserved.

Mesenchymal progenitor cells for the osteogenic lineage.

PubMed

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.

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration.

PubMed

Leucht, Philipp; Kim, Jae-Beom; Amasha, Raimy; James, Aaron W; Girod, Sabine; Helms, Jill A

2008-09-01

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.

Effects of electric fields on human mesenchymal stem cell behaviour and morphology using a novel multichannel device.

PubMed

Banks, T A; Luckman, P S B; Frith, J E; Cooper-White, J J

2015-06-01

The intrinsic piezoelectric nature of collagenous-rich tissues, such as bone and cartilage, can result in the production of small, endogenous electric fields (EFs) during applied mechanical stresses. In vivo, these EFs may influence cell migration, a vital component of wound healing. As a result, the application of small external EFs to bone fractures and cutaneous wounds is actively practiced clinically. Due to the significant regenerative potential of stem cells in bone and cartilage healing, and their potential role in the observed improved healing in vivo post applied EFs, using a novel medium throughput device, we investigated the impacts of physiological and aphysiological EFs on human bone marrow-derived mesenchymal stem cells (hBM-MSCs) for up to 15 hours. The applied EFs had significant impacts on hBM-MSC morphology and migration; cells displayed varying degrees of conversion to a highly elongated phenotype dependent on the EF strength, consistent perpendicular alignment to the EF vector, and definitive cathodal migration in response to EF strengths ≥0.5 V cm(-1), with the fastest migration speeds observed at between 1.7 and 3 V cm(-1). We observed variability in hBM-MSC donor-to-donor responses and overall tolerances to applied EFs. This study thus confirms hBM-MSCs are responsive to applied EFs, and their rate of migration towards the cathode is controllable depending on the EF strength, providing new insight into the physiology of hBM-MSCs and possibly a significant opportunity for the utilisation of EFs in directed scaffold colonisation in vitro for tissue engineering applications or in vivo post implantation.

Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones

NASA Astrophysics Data System (ADS)

Riddell, Stanley R.; Watanabe, Kathe S.; Goodrich, James M.; Li, Cheng R.; Agha, Mounzer E.; Greenberg, Philip D.

1992-07-01

The adoptive transfer of antigen-specific T cells to establish immunity is an effective therapy for viral infections and tumors in animal models. The application of this approach to human disease would require the isolation and in vitro expansion of human antigen-specific T cells and evidence that such T cells persist and function in vivo after transfer. Cytomegalovirus-specific CD8^+ cytotoxic T cell (CTL) clones could be isolated from bone marrow donors, propagated in vitro, and adoptively transferred to immunodeficient bone marrow transplant recipients. No toxicity developed and the clones provided persistent reconstitution of CD8^+ cytomegalovirus-specific CTL responses.

Effect of space flight on cytokine production and other immunologic parameters of rhesus monkeys

NASA Technical Reports Server (NTRS)

Sonnenfeld, G.; Davis, S.; Taylor, G. R.; Mandel, A. D.; Konstantinova, I. V.; Lesnyak, A.; Fuchs, B. B.; Peres, C.; Tkackzuk, J.; Schmitt, D. A.

1996-01-01

During a recent flight of a Russian satellite (Cosmos #2229), initial experiments examining the effects of space flight on immunologic responses of rhesus monkeys were performed to gain insight into the effect of space flight on resistance to infection. Experiments were performed on tissue samples taken from the monkeys before and immediately after flight. Additional samples were obtained approximately 1 month after flight for a postflight restraint study. Two types of experiments were carried out throughout this study. The first experiment determined the ability of leukocytes to produce interleukin-1 and to express interleukin-2 receptors. The second experiment examined the responsiveness of rhesus bone marrow cells to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Human reagents that cross-reacted with monkey tissue were utilized for the bulk of the studies. Results from both studies indicated that there were changes in immunologic function attributable to space flight. Interleukin-1 production and the expression of interleukin-2 receptors was decreased after space flight. Bone marrow cells from flight monkeys showed a significant decrease in their response to GM-CSF compared with the response of bone marrow cells from nonflight control monkeys. These results suggest that the rhesus monkey may be a useful surrogate for humans in future studies that examine the effect of space flight on immune response, particularly when conditions do not readily permit human study.

R-Spondin 1 promotes vibration-induced bone formation in mouse models of osteoporosis

PubMed Central

Wang, Haitao; Brennan, Tracy A.; Russell, Elizabeth; Kim, Jung-Hoon; Egan, Kevin P.; Chen, Qijun; Israelite, Craig; Schultz, David C.; Johnson, Frederick B.; Pignolo, Robert J.

2013-01-01

Bone tissue adapts to its functional environment by optimizing its morphology for mechanical demand. Among the mechanosensitive cells that recognize and respond to forces in the skeleton are osteocytes, osteoblasts, and mesenchymal progenitor cells (MPCs). Therefore, the ability to use mechanical signals to improve bone health through exercise and devices that deliver mechanical signals is an attractive approach to age-related bone loss; however, the extracellular or circulating mediators of such signals are largely unknown. Using SDS-PAGE separation of proteins secreted by MPCs in response to low magnitude mechanical signals and in-gel trypsin digestion followed by HPLC and mass spectroscopy, we identified secreted proteins up-regulated by vibratory stimulation. We exploited a cell senescence-associated secretory phenotype screen, and reasoned that a subset of vibration-induced proteins with diminished secretion by senescent MPCs will have the capacity to promote bone formation in vivo. We identified one such vibration-induced bone-enhancing (vibe) gene as R-Spondin 1, a Wnt pathway modulator, and demonstrated that it has the capacity to promote bone formation in three mouse models of age-related bone loss. By virtue of their secretory status, some vibe proteins may be candidates for pre-clinical development as anabolic agents for the treatment of osteoporosis. PMID:23974989

Mature osteoblasts dedifferentiate in response to traumatic bone injury in the zebrafish fin and skull.

PubMed

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.

Foreign Body Giant Cell-Related Encapsulation of a Synthetic Material Three Years After Augmentation.

PubMed

Lorenz, Jonas; Barbeck, Mike; Sader, Robert A; Kirkpatrick, Charles J; Russe, Philippe; Choukroun, Joseph; Ghanaati, Shahram

2016-06-01

Bone substitute materials of different origin and chemical compositions are frequently used in augmentation procedures to enlarge the local bone amount. However, relatively little data exist on the long-term tissue reactions. The presented case reports for the first time histological and histomorphometrical analyses of a nanocrystaline hydroxyapatite-based bone substitute material implanted in the human sinus cavity after an integration period of 3 years. The extracted biopsy was analyzed histologically and histomorphometrically with focus on the tissue reactions, vascularization, new bone formation, and the induction of a foreign body reaction. A comparably high rate of connective tissue (48.25%) surrounding the remaining bone substitute granules (42.13%) was observed. Accordingly, the amount of bone tissue (9.62%) built the smallest fraction within the biopsy. Further, tartrate-resistant acid phosphatase-positive and -negative multinucleated giant cells (4.35 and 3.93 cells/mm(2), respectively) were detected on the material-tissue interfaces. The implantation bed showed a mild vascularization of 10.03 vessels/mm(2) and 0.78%. The present case report shows that after 3 years, a comparable small amount of bone tissue was observable. Thus, the foreign body response to the bone substitute seems to be folded without further degradation or regeneration.

Adoptive transfer of ex vivo expanded Vγ9Vδ2 T cells in combination with zoledronic acid inhibits cancer growth and limits osteolysis in a murine model of osteolytic breast cancer.

PubMed

Zysk, Aneta; DeNichilo, Mark O; Panagopoulos, Vasilios; Zinonos, Irene; Liapis, Vasilios; Hay, Shelley; Ingman, Wendy; Ponomarev, Vladimir; Atkins, Gerald; Findlay, David; Zannettino, Andrew; Evdokiou, Andreas

2017-02-01

Bone metastases occur in over 75% of patients with advanced breast cancer and are responsible for high levels of morbidity and mortality. In this study, ex vivo expanded cytotoxic Vγ9Vδ2 T cells isolated from human peripheral blood were tested for their anti-cancer efficacy in combination with zoledronic acid (ZOL), using a mouse model of osteolytic breast cancer. In vitro, expanded Vγ9Vδ2 T cells were cytotoxic against a panel of human breast cancer cell lines, and ZOL pre-treatment further sensitised breast cancer cells to killing by Vγ9Vδ2 T cells. Vγ9Vδ2 T cells adoptively transferred into NOD/SCID mice localised to osteolytic breast cancer lesions in the bone, and multiple infusions of Vγ9Vδ2 T cells reduced tumour growth in the bone. ZOL pre-treatment potentiated the anti-cancer efficacy of Vγ9Vδ2 T cells, with mice showing further reductions in tumour burden. Mice treated with the combination also had reduced tumour burden of secondary pulmonary metastases, and decreased bone degradation. Our data suggests that adoptive transfer of Vγ9Vδ2 T cell in combination with ZOL may prove an effective immunotherapeutic approach for the treatment of breast cancer bone metastases. Copyright © 2016. Published by Elsevier Ireland Ltd.

In vivo immunogenicity of bovine bone removed by a novel decellularization protocol based on supercritical carbon dioxide.

PubMed

You, Ling; Weikang, Xu; Lifeng, Yang; Changyan, Liang; Yongliang, Lin; Xiaohui, Wei; Bin, Xu

2018-05-04

Trauma or infections associated critical bone defects lead to a huge economic burden in the healthcare system worldwide. Recent advances in tissue engineering have led to potential new strategies for the repair, replacement, and regeneration of bone defects, especially in biomaterials and decellularization protocols from xenogenic tissues. However, the complexity in bone structure and mechanical environment limits the synthesis of artificial bone with biomaterials. Thus, the purpose of our study is to develop a natural bone scaffold with great immunocompatibility. We combined decellularization techniques base on SC-CO 2 to decellularize bovine bone. In order to study the immune response of mice to materials, the histology, spleen index, immune cells contents and in vitro proliferative performance, cytokine and immunoglobulin light chain expression of mice were characterized. Compared with the fresh bone group, the immune responses of decellularized group were significantly reduced. In conclusion, decellularization via this method can achieve a decellularized scaffold with great immunocompatibility. Our findings suggest the potential of using decellularized BB as a scaffold for bone bioengineering.

Bone Marrow Mesenchymal Stem Cells Enhance the Differentiation of Human Switched Memory B Lymphocytes into Plasma Cells in Serum-Free Medium

PubMed Central

Gervais-St-Amour, Catherine

2016-01-01

The differentiation of human B lymphocytes into plasma cells is one of the most stirring questions with regard to adaptive immunity. However, the terminal differentiation and survival of plasma cells are still topics with much to be discovered, especially when targeting switched memory B lymphocytes. Plasma cells can migrate to the bone marrow in response to a CXCL12 gradient and survive for several years while secreting antibodies. In this study, we aimed to get closer to niches favoring plasma cell survival. We tested low oxygen concentrations and coculture with mesenchymal stem cells (MSC) from human bone marrow. Besides, all cultures were performed using an animal protein-free medium. Overall, our model enables the generation of high proportions of CD38+CD138+CD31+ plasma cells (≥50%) when CD40-activated switched memory B lymphocytes were cultured in direct contact with mesenchymal stem cells. In these cultures, the secretion of CXCL12 and TGF-β, usually found in the bone marrow, was linked to the presence of MSC. The level of oxygen appeared less impactful than the contact with MSC. This study shows for the first time that expanded switched memory B lymphocytes can be differentiated into plasma cells using exclusively a serum-free medium. PMID:27872867

Rapid prototyped porous nickel–titanium scaffolds as bone substitutes

PubMed Central

Hoffmann, Waldemar; Bormann, Therese; Rossi, Antonella; Müller, Bert; Schumacher, Ralf; Martin, Ivan; Wendt, David

2014-01-01

While calcium phosphate–based ceramics are currently the most widely used materials in bone repair, they generally lack tensile strength for initial load bearing. Bulk titanium is the gold standard of metallic implant materials, but does not match the mechanical properties of the surrounding bone, potentially leading to problems of fixation and bone resorption. As an alternative, nickel–titanium alloys possess a unique combination of mechanical properties including a relatively low elastic modulus, pseudoelasticity, and high damping capacity, matching the properties of bone better than any other metallic material. With the ultimate goal of fabricating porous implants for spinal, orthopedic and dental applications, nickel–titanium substrates were fabricated by means of selective laser melting. The response of human mesenchymal stromal cells to the nickel–titanium substrates was compared to mesenchymal stromal cells cultured on clinically used titanium. Selective laser melted titanium as well as surface-treated nickel–titanium and titanium served as controls. Mesenchymal stromal cells had similar proliferation rates when cultured on selective laser melted nickel–titanium, clinically used titanium, or controls. Osteogenic differentiation was similar for mesenchymal stromal cells cultured on the selected materials, as indicated by similar gene expression levels of bone sialoprotein and osteocalcin. Mesenchymal stromal cells seeded and cultured on porous three-dimensional selective laser melted nickel–titanium scaffolds homogeneously colonized the scaffold, and following osteogenic induction, filled the scaffold’s pore volume with extracellular matrix. The combination of bone-related mechanical properties of selective laser melted nickel–titanium with its cytocompatibility and support of osteogenic differentiation of mesenchymal stromal cells highlights its potential as a superior bone substitute as compared to clinically used titanium. PMID:25383165

Non-tumour bone marrow lymphocytes correlate with improved overall survival in childhood acute lymphoblastic leukaemia.

PubMed

Edwin, Claire; Dean, Joanne; Bonnett, Laura; Phillips, Kate; Keenan, Russell

2016-10-01

Composition of tumour immune cell infiltrates correlates with response to treatment and overall survival (OS) in several cancer settings. We retrospectively examined immune cells present in diagnostic bone marrow aspirates from paediatric patients with B-cell acute lymphoblastic leukaemia. Our analysis identified a sub-group (∼30% of patients) with >2.37% CD20 and >6.05% CD7 expression, which had 100% OS, and a sub-group (∼30% of patients) with ≤2.37% CD20 and ≤6.05% CD7 expression at increased risk of treatment failure (66.7% OS, P < 0.05). Immune cell infiltrate at diagnosis may predict treatment response and could provide a means to enhance immediate treatment risk stratification. © 2016 The Authors. Pediatric Blood & Cancer, published by Wiley Periodicals, Inc.

Peripheral blood biomarkers of solid tumor angiogenesis in dogs: A polychromatic flow cytometry pilot study

PubMed Central

Bentley, R. Timothy; Mund, Julie A.; Pollok, Karen E.; Childress, Michael O.; Case, Jamie

2012-01-01

A subset of peripheral blood hematopoietic stem and progenitor cells of bone marrow origin is elevated in humans with solid cancers before treatment and declines with therapy. This biomarker of angiogenesis is not specific to tumor type and has great potential in the objective assessment of treatment response in clinical trials. This pilot study was designed to develop a biomarker of neoangiogenesis in dogs for the diagnosis of cancer, the measurement of treatment response, and the provision of objective data in clinical trials. Polychromatic flow cytometry was used to quantify two subsets of circulating hematopoietic stem and progenitor cells in dogs with spontaneous solid tumors before (n = 8) and after (n = 3) treatment, and normal controls (n = 6). Pro-angiogenic peripheral blood cells of bone marrow origin were detected in all eight cases and the six normal controls; however, there was no statistically significant difference between the two groups. Interestingly, an apparent decline in pro-angiogenic cells was observed after treatment. Bone marrow derived hematopoietic cells appear to contribute to tumor angiogenesis in dogs, as has been previously reported in humans. While the methodology for pro-angiogenic cell quantification in a small number of dogs in the current study did not result in a significant difference from normal controls, an optimized canine polychromatic flow cytometry protocol holds great promise in the development of a canine cancer model and for the objective measurements of treatment response in clinical trials. PMID:23063489

Gelatine modified monetite as a bone substitute material: An in vitro assessment of bone biocompatibility.

PubMed

Kruppke, Benjamin; Farack, Jana; Wagner, Alena-Svenja; Beckmann, Sarah; Heinemann, Christiane; Glenske, Kristina; Rößler, Sina; Wiesmann, Hans-Peter; Wenisch, Sabine; Hanke, Thomas

2016-03-01

Calcium phosphate phases are increasingly used for bone tissue substitution, and the load bearing properties of these inherently brittle biomaterials are increased by inclusion of organic components. Monetite prepared using mineralization of gelatine pre-structured through phosphate leads to a significantly increased biaxial strength and indirect tensile strength compared to gelatine-free monetite. Besides the mechanical properties, degradation in physiological solutions and osteoblast and osteoclast cell response were investigated. Human bone marrow stromal cells (hBMSCs) showed considerably higher proliferation rates on the gelatine modified monetite than on polystyrene reference material in calcium-free as well as standard cell culture medium (α-MEM). Osteogenic differentiation on the material was comparable to polystyrene in both medium types. Osteoclast-like cells derived from monocytes were able to actively resorb the biomaterial. Osteoblastic differentiation and perhaps even more important the cellular resorption of the biomaterial indicate that it can be actively involved in the bone remodeling process. Thus the behavior of osteoblasts and osteoclasts as well as the adequate degradation and mechanical properties are strong indicators for bone biocompatibility, although in vivo studies are still required to prove this. New and unique? A low temperature precipitationprocessforcalcium anhydrous hydrogen phosphateallows for the first time to produce monolithic compact composites of monetite and gelatine. The composite is degradable and resorbable. To prove that, the question arises: what is bone biocompatibility? The reaction of both mayor cell types of bone represents this biocompatibility. Therefore, human bone marrow stromal cells were seeded revealing the materials pro-osteogenic properties. Monocyte cultivation, becoming recently focus of interest, revealed the capability of the biomaterial to be actively resorbed by derived osteoclast-like cells. Not new but necessary ismechanical characterization, which is often only investigated as uniaxial property. Here, a biaxial method is applied, to characterize the materials properties closer to its application loads. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

High-Fat Diet-Induced Obesity Promotes Expansion of Bone Marrow Adipose Tissue and Impairs Skeletal Stem Cell Functions in Mice.

PubMed

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.

Mechanical Loading Attenuates Radiation-Induced Bone Loss in Bone Marrow Transplanted Mice

PubMed Central

Govey, Peter M.; Zhang, Yue; Donahue, Henry J.

2016-01-01

Exposure of bone to ionizing radiation, as occurs during radiotherapy for some localized malignancies and blood or bone marrow cancers, as well as during space travel, incites dose-dependent bone morbidity and increased fracture risk. Rapid trabecular and endosteal bone loss reflects acutely increased osteoclastic resorption as well as decreased bone formation due to depletion of osteoprogenitors. Because of this dysregulation of bone turnover, bone’s capacity to respond to a mechanical loading stimulus in the aftermath of irradiation is unknown. We employed a mouse model of total body irradiation and bone marrow transplantation simulating treatment of hematologic cancers, hypothesizing that compression loading would attenuate bone loss. Furthermore, we hypothesized that loading would upregulate donor cell presence in loaded tibias due to increased engraftment and proliferation. We lethally irradiated 16 female C57Bl/6J mice at age 16 wks with 10.75 Gy, then IV-injected 20 million GFP(+) total bone marrow cells. That same day, we initiated 3 wks compression loading (1200 cycles 5x/wk, 10 N) in the right tibia of 10 of these mice while 6 mice were irradiated, non-mechanically-loaded controls. As anticipated, before-and-after microCT scans demonstrated loss of trabecular bone (-48.2% Tb.BV/TV) and cortical thickness (-8.3%) at 3 wks following irradiation. However, loaded bones lost 31% less Tb.BV/TV and 8% less cortical thickness (both p<0.001). Loaded bones also had significant increases in trabecular thickness and tissue mineral densities from baseline. Mechanical loading did not affect donor cell engraftment. Importantly, these results demonstrate that both cortical and trabecular bone exposed to high-dose therapeutic radiation remain capable of an anabolic response to mechanical loading. These findings inform our management of bone health in cases of radiation exposure. PMID:27936104

Differential osteogenic activity of osteoprogenitor cells on HA and TCP/HA scaffold of tissue engineered bone.

PubMed

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.

Transcriptional Activation by NFκB Increases Perlecan/HSPG2 Expression in the Desmoplastic Prostate Tumor Microenvironment

PubMed Central

Warren, Curtis R.; Grindel, Brian J.; Francis, Lewis; Carson, Daniel D.; Farach-Carson, Mary C.

2014-01-01

Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF-α and/or TGFβ1 identified TNF-α as a major cytokine regulator of perlecan production. TNF-α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix-secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine-mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co-opted by prostate cancer cells. PMID:24700612

IL-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts.

PubMed

Iida, Shoichi; Tsuda, Hidetoshi; Tanaka, Toshiaki; Kish, Danielle D; Abe, Toyofumi; Su, Charles A; Abe, Ryo; Tanabe, Kazunari; Valujskikh, Anna; Baldwin, William M; Fairchild, Robert L

2016-03-15

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil, and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor (IL-1R) signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared with complete MHC-mismatched wild-type cardiac allografts, IL-1R(-/-) allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant, whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R(-/-) allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R(-/-) cardiac allografts took 3 wk longer than wild-type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R(-/-)/wild-type chimeric donors indicated that IL-1R signaling on graft nonhematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild-type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli-provoking development and elicitation of optimal alloimmune responses to the grafts. Copyright © 2016 by The American Association of Immunologists, Inc.

In vitro degradation and cell response of calcium carbonate composite ceramic in comparison with other synthetic bone substitute materials.

PubMed

He, Fupo; Zhang, Jing; Yang, Fanwen; Zhu, Jixiang; Tian, Xiumei; Chen, Xiaoming

2015-05-01

The robust calcium carbonate composite ceramics (CC/PG) can be acquired by fast sintering calcium carbonate at a low temperature (650 °C) using a biocompatible, degradable phosphate-based glass (PG) as sintering agent. In the present study, the in vitro degradation and cell response of CC/PG were assessed and compared with 4 synthetic bone substitute materials, calcium carbonate ceramic (CC), PG, hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics. The degradation rates in decreasing order were as follows: PG, CC, CC/PG, β-TCP, and HA. The proliferation of rat bone mesenchymal stem cells (rMSCs) cultured on the CC/PG was comparable with that on CC and PG, but inferior to HA and β-TCP. The alkaline phosphatase (ALP) activity of rMSCs on CC/PG was lower than PG, comparable with β-TCP, but higher than HA. The rMSCs on CC/PG and PG had enhanced gene expression in specific osteogenic markers, respectively. Compared to HA and β-TCP, the rMSCs on the CC/PG expressed relatively lower level of collagen I and runt-related transcription factor 2, but showed more considerable expression of osteopontin. Although CC, PG, HA, and β-TCP possessed impressive performances in some specific aspects, they faced extant intrinsic drawbacks in either degradation rate or mechanical strength. Based on considerable compressive strength, moderate degradation rate, good cell response, and being free of obvious shortcoming, the CC/PG is promising as another choice for bone substitute materials. Copyright © 2015 Elsevier B.V. All rights reserved.

A New Regulator of Osteoclastogenesis: Estrogen Response Element–Binding Protein in Bone

PubMed Central

Chen, Hong; Gilbert, Linda C; Lu, X; Liu, Zhaofan; You, Shaojin; Weitzmann, M Neale; Nanes, Mark S; Adams, John

2012-01-01

The heterogeneous nuclear ribonucleoprotein (hnRNP)–like estrogen response element–binding protein (ERE-BP) competes with estrogen receptor α (ERα) for occupancy of estrogen response elements (EREs). Here we report that ERE-BP potently stimulates osteoclastogenesis. ERE-BP mRNA and protein were found to be expressed ubiquitously in bone. Overexpression of ERE-BP in cultured osteoblasts stimulated expression of the receptor activator of NF-κB ligand (RANKL) and decreased osteoprotegerin (OPG). The effect of ERE-BP on RANKL was shown to be transcriptional in transient transfection assay and competed with via the ER. Constitutive expression of ERE-BP increased the sensitivity of cells toward 1,25-dihydroxyvitamin D3 stimulation of RANKL expression. In contrast, knockdown of ERE-BP in stromal ST-2 cells decreased basal RANKL promoter activity. Cocultures of ERE-BP lentivirus–transduced ST-2 cells with spleen monocytes induced formation of multinucleated osteoclasts (OCs) characterized by tartrate-resistant acid phosphatase, calcitonin receptors, and functional calcium resorption from bone slices. Although ERα competed with ERE-BP for an ERE in a dose-dependent manner, ERE-BP was an independent and potent regulator of RANKL and osteoclastogenesis. In preosteoclastic RAW cells, overexpression of ERE-BP increased RANK, upregulated NF-κB signaling, and enhanced differentiation toward a mature OC phenotype independent of RANKL. These results identify ERE-BP as a potent modulator of osteoclastogenesis. We hypothesize that ERE-BP may play a critical role in the regulation of bone homeostasis as a modulator of estrogen sensitivity as well as by direct action on the transcription of critical osteoclastogenic genes. PMID:21773989

Aromatase expression in a human osteoblastic cell line increases in response to prostaglandin E(2) in a dexamethasone-dependent fashion.

PubMed

Watanabe, M; Noda, M; Nakajin, S

2007-09-01

Recent progress supports the importance of local estrogen secretion in human bone tissue to increase and maintain bone-mineral density. In a previous report, we found that forskolin (FSK) synergistically induces aromatase (CYP19: a rate-limiting enzyme for estrogen synthesis) expression in dexamethasone (Dex) dependent manner in a human osteoblastic cell line, SV-HFO [Watanabe M, Ohno S, Nakajin S. Forskolin and dexamethasone synergistically induce aromatase (CYP19) expression in the human osteoblastic cell line SV-HFO. Eur J Endocrinol 2005;152:619-24]. In this report, we investigated whether prostaglandin (PG) E(2) induces estrogen production, in other words, if PGE(2) exerts the same effect as FSK because PGE(2) is the major prostanoid in the bone and is one of the key molecules in the osteoblast. We found PGE(2) up-regulates aromatase activity synergistically, but this up-regulation depends on Dex. CYP19 gene expression was also increased synergistically by Dex and PGE(2). Promoter I.4 was activated synergistically by PGE(2) and Dex. PGE(2) receptor, EP(1), EP(2) and EP(4) were involved in the up-regulation of aromatase activity in response to PGE(2) in a Dex-dependent manner. The cAMP-PKA pathway and Ca(2+) signaling pathway were involved in the up-regulation of aromatase activity in response to PGE(2). Furthermore, glucocorticoid response element on promoter I.4 sequence was an essential minimum requirement for its activity and synergism of PGE(2) and Dex. These findings are the first report on osteoblastic cell line which uses predominantly promoter I.4 to drive aromatase expression. These findings also suggest that endogenous PGE(2) produced in bone mainly may synergistically support local estrogen production in osteoblastic cells in the presence of glucocorticoid.

Complete metabolic remission with Gefitinib in a hemodialysis patient with bone metastases from non-small cell lung cancer.

PubMed

Del Conte, Alessandro; Minatel, Emilio; Schinella, Domenico; Baresic, Tanja; Basso, Stefano M M; Lumachi, Franco

2014-01-01

Gefitinib is highly active in patients with advanced or metastatic non-small cell lung cancer (NSCLC) harboring activating mutation of the epidermal growth factor receptor (EGFR) gene. The feasibility and the degree of response to treatment with gefitinib in patients with chronic renal failure (CRF) undergoing hemodialysis has not yet been fully described in literature. We describe the case of a 70-year-old man with CRF undergoing hemodialysis three times-a-week who developed vertebral and rib bone metastasis three years after lobectomy. The bone biopsy confirmed the pulmonary origin and pyrosequencing analysis revealed deletion in E746-E750 of exon 19. We started daily administration of 250 mg gefitinib with no changes in the hemodialysis schedule. Gefitinib was well-tolerated without any adverse event. After three months, the (18)F-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography (FDG PET/CT) showed complete metabolic remission of bone lesions. The patient is still under treatment and maintains response (30 months to date). To our knowledge, this is the first description of complete metabolic remission in this type of patient. In conclusion, gefitinib has been safely administered to a patient with NSCLC with EGFR-activating mutation undergoing chronic hemodialysis and its use has achieved an excellent and prolonged response on bone metastases.

INDUCTION OF IMMUNOLOGIC TOLERANCE IN OLDER NEW ZEALAND MICE REPOPULATED WITH YOUNG SPLEEN, BONE MARROW, OR THYMUS

PubMed Central

Staples, Parker J.; Steinberg, Alfred D.; Talal, Norman

1970-01-01

Newborn, 7–9 day, and 16–18 day old NZB and B/W mice were, unlike older New Zealand mice, rendered tolerant to single doses of 8–10 mg of soluble BGG. After challenge, this tolerance was of short duration and escape occurred rapidly. Age-matched and similarly treated C3H, Balb/c and C57Bl mice did not escape from tolerance. Partial tolerance could be maintained by repeated injections of BGG. Biofiltration ruled out hyperphagocytosis as an explanation for this resistance to tolerance. Tolerance could be induced in older B/W mice if they were thymectomized, irradiated, and repopulated with young (12–15 day), but not old (2–3 month), spleen or bone marrow cells. Old bone marrow cells gave a non-tolerant response even when combined with young thymic grafts. Young bone marrow gave a tolerant response which was followed by the expected rapid escape only if a young thymus graft was also present. Escape was retarded if old thymus, or old irradiated thymus, was combined with young bone marrow. These results are best explained by abnormalities of both lymphoid precursors and thymic regulation. PMID:4192570

Three-dimensional nano-architected scaffolds with tunable stiffness for efficient bone tissue growth.

PubMed

Maggi, Alessandro; Li, Hanqing; Greer, Julia R

2017-11-01

The precise mechanisms that lead to orthopedic implant failure are not well understood; it is believed that the micromechanical environment at the bone-implant interface regulates structural stability of an implant. In this work, we seek to understand how the 3D mechanical environment of an implant affects bone formation during early osteointegration. We employed two-photon lithography (TPL) direct laser writing to fabricate 3-dimensional rigid polymer scaffolds with tetrakaidecahedral periodic geometry, herewith referred to as nanolattices, whose strut dimensions were on the same order as osteoblasts' focal adhesions (∼2μm) and pore sizes on the order of cell size, ∼10μm. Some of these nanolattices were subsequently coated with thin conformal layers of Ti or W, and a final outer layer of 18nm-thick TiO 2 was deposited on all samples to ensure biocompatibility. Nanomechanical experiments on each type of nanolattice revealed the range of stiffnesses of 0.7-100MPa. Osteoblast-like cells (SAOS-2) were seeded on each nanolattice, and their mechanosensitve response was explored by tracking mineral secretions and intracellular f-actin and vinculin concentrations after 2, 8 and 12days of cell culture in mineralization media. Experiments revealed that the most compliant nanolattices had ∼20% more intracellular f-actin and ∼40% more Ca and P secreted onto them than the stiffer nanolattices, where such cellular response was virtually indistinguishable. We constructed a simple phenomenological model that appears to capture the observed relation between scaffold stiffness and f-actin concentration. This model predicts a range of optimal scaffold stiffnesses for maximum f-actin concentration, which appears to be directly correlated with osteoblast-driven mineral deposition. This work suggests that three-dimensional scaffolds with titania-coated surfaces may provide an optimal microenvironment for cell growth when their stiffness is similar to that of cartilage (∼0.5-3MPa). These findings help provide a greater understanding of osteoblast mechanosensitivity and may have profound implications in developing more effective and safer bone prostheses. Creating prostheses that lead to optimal bone remodeling has been a challenge for more than two decades because of a lack of thorough knowledge of cell behavior in three-dimensional (3D) environments. Literature has shown that 2D substrate stiffness plays a significant role in determining cell behavior, however, limitations in fabrication techniques and difficulties in characterizing cell-scaffold interactions have limited our understanding of how 3D scaffolds' stiffness affects cell response. The present study shows that scaffold structural stiffness affects osteoblasts cellular response. Specifically this work shows that the cells grown on the most compliant nanolattices with a stiffness of 0.7MPa expressed ∼20% higher concentration of intracellular f-actin and secreted ∼40% more Ca and P compared with all other nanolattices. This suggests that bone scaffolds with a stiffness close to that of cartilage may serve as optimal 3D scaffolds for new synthetic bone graft materials. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Surface microtopography modulates sealing zone development in osteoclasts cultured on bone

PubMed Central

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

The effects of prostaglandin E2 in growing rats - Increased metaphyseal hard tissue and cortico-endosteal bone formation

NASA Technical Reports Server (NTRS)

Jee, W. S. S.; Ueno, K.; Deng, Y. P.; Woodbury, D. M.

1985-01-01

The role of in vivo prostaglandin E2 (PGE2) in bone formation is investigated. Twenty-five male Sprague-Dawley rats weighing between 223-267 g were injected subcutaneously with 0.3, 1.0, 3.0, and 6.0 mg of PGE2-kg daily for 21 days. The processing of the tibiae for observation is described. Radiographs and histomorphometric analyses are also utilized to study bone formation. Body weight, weights of soft tissues and bones morphometry are evaluated. It is observed that PGE2 depressed longitudinal bone growth, increased growth cartilage thickness, decreased degenerative cartilage cell size and cartilage cell production, and significantly increased proximal tibial metaphyseal hard tissue mass. The data reveal that periosteal bone formation is slowed down at higher doses of PGE2 and endosteal bone formation is slightly depressed less than 10 days post injection; however, here is a late increase (10 days after post injection) in endosteal bone formation and in the formation of trabecular bone in the marrow cavity of the tibial shaft. It is noted that the effects of PGE2 on bone formation are similar to the responses of weaning rats to PGE2.

Effects of hibernation on bone marrow transcriptome in thirteen-lined ground squirrels.

PubMed

Cooper, Scott T; Sell, Shawn S; Fahrenkrog, Molly; Wilkinson, Kory; Howard, David R; Bergen, Hannah; Cruz, Estefania; Cash, Steve E; Andrews, Matthew T; Hampton, Marshall

2016-07-01

Mammalian hibernators adapt to prolonged periods of immobility, hypometabolism, hypothermia, and oxidative stress, each capable of reducing bone marrow activity. In this study bone marrow transcriptomes were compared among thirteen-lined ground squirrels collected in July, winter torpor, and winter interbout arousal (IBA). The results were consistent with a suppression of acquired immune responses, and a shift to innate immune responses during hibernation through higher complement expression. Consistent with the increase in adipocytes found in bone marrow of hibernators, expression of genes associated with white adipose tissue are higher during hibernation. Genes that should strengthen the bone by increasing extracellular matrix were higher during hibernation, especially the collagen genes. Finally, expression of heat shock proteins were lower, and cold-response genes were higher, during hibernation. No differential expression of hematopoietic genes involved in erythrocyte or megakaryocyte production was observed. This global view of the changes in the bone marrow transcriptome over both short term (torpor vs. IBA) and long term (torpor vs. July) hypothermia can explain several observations made about circulating blood cells and the structure and strength of the bone during hibernation. Copyright © 2016 the American Physiological Society.

Cell behavior of human mesenchymal stromal cells in response to silica/collagen based xerogels and calcium deficient culture conditions.

PubMed

Wagner, Alena-Svenja; Glenske, Kristina; Henß, Anja; Kruppke, Benjamin; Rößler, Sina; Hanke, Thomas; Moritz, Andreas; Rohnke, Marcus; Kressin, Monika; Arnhold, Stefan; Schnettler, Reinhard; Wenisch, Sabine

2017-07-04

Herein, we aim to elucidate osteogenic effects of two silica-based xerogels with different degrees of bioactivity on human bone-derived mesenchymal stromal cells by means of scanning electron microscopy, quantitative PCR enhanced osteogenic effects and the formation of an extracellular matrix which could be ascribed to the sample with lower bioactivity. Given the high levels of bioactivity, the cells revealed remarkable sensitivity to extremely low calcium levels of the media. Therefore, additional experiments were performed to elucidate cell behavior under calcium deficient conditions. The results refer to capacity of the bone-derived stromal cells to overcome calcium deficiency even though proliferation, migration and osteogenic differentiation capabilities were diminished. One reason for the differences of the cellular response (on tissue culture plates versus xerogels) to calcium deficiency seems to be the positive effect of silica. The silica could be detected intracellularly as shown by time of flight-secondary ion mass spectrometry after cultivation of primary cells for 21 days on the surfaces of the xerogels. Thus, the present findings refer to different osteogenic differentiation potentials of the xerogels according to the different degrees of bioactivity, and to the role of silica as a stimulator of osteogenesis. Finally, the observed pattern of connexin-based hemichannel gating supports the assumption that connexin 43 is a key factor for calcium-mediated osteogenesis in bone-derived mesenchymal stromal cells.

Biological effectiveness of nuclear fragments produced by high-energy protons interacting in tissues near the bone- soft tissue interface

NASA Astrophysics Data System (ADS)

Shavers, Mark Randall

1999-12-01

High-energy protons in the galactic cosmic rays (GCR)-or generated by nuclear interactions of GCR heavy-ions with material-are capable of penetrating great thicknesses of shielding to irradiate humans in spacecraft or in lunar or Martian habitats. As protons interact with the nuclei of the elemental constituents of soft tissue and bone, low energy nuclei-target fragments-are emitted into the cells responsible for bone development and maintenance and for hematopoiesis. Leukemogenesis is the principal endpoint of concern because it is the most likely deleterious effect, and it has a short latency period and comparatively low survival rate, although other myelo- proliferative disorders and osteosarcoma also may be induced. A one-dimensional proton-target fragment transport model was used to calculate the energy spectra of fragments produced in bone and soft tissue, and present in marrow cavities at distances from a bone interface. In terms of dose equivalent, the target fragments are as significant as the incident protons. An average radiation quality factor was found to be between 1.8 and 2.6. Biological response to the highly non- uniform energy deposition of the target fragments is such that an alternative approach to conventional predictive risk assessment is needed. Alternative procedures are presented. In vitro cell response and relative biological effectiveness were calculated from the radial dose distribution of each fragment produced by 1-GeV protons using parameters of a modified Ion-Gamma- Kill (IGK) model of radiation action. The modelled endpoints were survival of C3H10t 1/2 and V79 cells, neoplastic transformation of C3H10t1/2 cells, and mutation of the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in V79 cells. The dose equivalent and cell responses increased by 10% or less near the interface. Since RBE increases with decreasing dose in the IGK model, comparisons with quality factors were made at dose levels 0.01 <= D [Gy] <= 2. Applying average quality factors derived herein to GCR exposures results in a <= 5% increase of in average quality. Calculated RBEs indicate that accepted quality factors for high-energy protons may be too low due to the relatively high effectiveness of the low-charged target fragments. Derived RBEs for target fragments increase the calculated biological effectiveness of GCR by 20% to 180%.

Transplantation of stem cells from human exfoliated deciduous teeth for bone regeneration in the dog mandibular defect

PubMed Central

Behnia, Ali; Haghighat, Abbas; Talebi, Ardeshir; Nourbakhsh, Nosrat; Heidari, Fariba

2014-01-01

AIM: To investigate the effect of stem cells from human exfoliated deciduous teeth (SHED) transplanted for bone regeneration in the dog mandibular defect. METHODS: In this prospective comparative study, SHEDs had been isolated 5 years ago from human exfoliated deciduous teeth. The undifferentiated stem cells were seeded into mandibular bone through-and-through defects of 4 dogs. Similar defects in control group were filled with cell-free collagen scaffold. After 12 wk, biopsies were taken and morphometric analysis was performed. The percentage of new bone formation and foreign body reaction were measured in each case. The data were subject to statistical analysis using the Mann-Whitney U and Kruskalwalis statistical tests. Differences at P < 0.05 was considered as significant level. RESULTS: There were no significant differences between control and SHED-seeded groups in connective tissue (P = 0.248), woven bone (P = 0.248) and compact bone (P = 0.082). There were not any side effects in transplanted SHED group such as teratoma or malignancy and abnormalities in this period. CONCLUSION: SHEDs which had been isolated and characterized 5 years ago and stored with cryopreservation banking were capable of proliferation and osteogenesis after 5 years, and no immune response was observed after three months of seeded SHEDs. PMID:25258673

Transplanted Umbilical Cord Mesenchymal Stem Cells Modify the In Vivo Microenvironment Enhancing Angiogenesis and Leading to Bone Regeneration

PubMed Central

Todeschi, Maria Rosa; El Backly, Rania; Capelli, Chiara; Daga, Antonio; Patrone, Eugenio; Introna, Martino; Cancedda, Ranieri

2015-01-01

Umbilical cord mesenchymal stem cells (UC-MSCs) show properties similar to bone marrow mesenchymal stem cells (BM-MSCs), although controversial data exist regarding their osteogenic potential. We prepared clinical-grade UC-MSCs from Wharton's Jelly and we investigated if UC-MSCs could be used as substitutes for BM-MSCs in muscoloskeletal regeneration as a more readily available and functional source of MSCs. UC-MSCs were loaded onto scaffolds and implanted subcutaneously (ectopically) and in critical-sized calvarial defects (orthotopically) in mice. For live cell-tracking experiments, UC-MSCs were first transduced with the luciferase gene. Angiogenic properties of UC-MSCs were tested using the mouse metatarsal angiogenesis assay. Cell secretomes were screened for the presence of various cytokines using an array assay. Analysis of implanted scaffolds showed that UC-MSCs, contrary to BM-MSCs, remained detectable in the implants for 3 weeks at most and did not induce bone formation in an ectopic location. Instead, they induced a significant increase of blood vessel ingrowth. In agreement with these observations, UC-MSC-conditioned medium presented a distinct and stronger proinflammatory/chemotactic cytokine profile than BM-MSCs and a significantly enhanced angiogenic activity. When UC-MSCs were orthotopically transplanted in a calvarial defect, they promoted increased bone formation as well as BM-MSCs. However, at variance with BM-MSCs, the new bone was deposited through the activity of stimulated host cells, highlighting the importance of the microenvironment on determining cell commitment and response. Therefore, we propose, as therapy for bone lesions, the use of allogeneic UC-MSCs by not depositing bone matrix directly, but acting through the activation of endogenous repair mechanisms. PMID:25685989

Steroid and xenobiotic receptor-mediated effects of bisphenol A on human osteoblasts.

PubMed

Miki, Yasuhiro; Hata, Shuko; Nagasaki, Shuji; Suzuki, Takashi; Ito, Kiyoshi; Kumamoto, Hiroyuki; Sasano, Hironobu

2016-06-15

Bisphenol A, one of the industrial chemicals used in plastics and in the coating of dishes and medical equipment, behaves as an endocrine disruptor in the human body. Bisphenol A can bind directly to several types of nuclear receptors, including steroid and xenobiotic receptor (SXR). SXR plays an important role in bone metabolism through the activation of osteoblasts in vitro, but SXR protein localization has not been reported in bone tissues. Additionally, it is not known whether bisphenol A acts on osteoblasts through SXR activation. Therefore, in this study, we first examined the immunolocalization of the SXR protein in human adult and fetal bone tissues. We then examined the effects of bisphenol A on human osteoblasts in vitro. SXR immunoreactivity was detected in osteoblasts, but not in osteoclasts, of both adult and fetal bone tissues. In fetal bone tissues, the mesenchymal cells or fetal connective tissue were also positive for SXR immunoreactivity. Expression of SXR target genes (tsukushi, matrilin-2, and CYP3A4) and SXR response element-luciferase activity were increased by bisphenol A treatment in normal osteoblasts transfected with SXR (hFOB/SXR) and in osteoblast-like cells (MG-63). Bisphenol A also stimulated cell proliferation and collagen accumulation in hFOB/SXR cells. These results suggest that, as in other tissues, SXR plays important roles in bone metabolism and fetal bone development and that bisphenol A may disturb bone homeostasis in both adult and fetus through SXR. Copyright © 2016 Elsevier Inc. All rights reserved.

BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor, CTGF) expression.

PubMed

Ren, Wei; Sun, Xiaoxiao; Wang, Ke; Feng, Honglei; Liu, Yuehong; Fei, Chang; Wan, Shaoheng; Wang, Wei; Luo, Jinyong; Shi, Qiong; Tang, Min; Zuo, Guowei; Weng, Yaguang; He, Tongchuan; Zhang, Yan

2014-03-01

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, regulate a wide range of cellular responses including cell proliferation, differentiation, adhesion, migration, and apoptosis. BMP9, the latest BMP to be discovered, is reportedly expressed in a variety of human carcinoma cell lines, but the role of BMP9 in breast cancer has not been fully clarified. In a previous study, BMP9 was found to inhibit the growth, migration, and invasiveness of MDA-MB-231 breast cancer cells. In the current study, the effect of BMP9 on the bone metastasis of breast cancer cells was investigated. After absent or low expression of BMP9 was detected in the MDA-MB-231 breast cancer cells and breast non-tumor adjacent tissues using Western blot and immunohistochemistry, In our previous study, BMP9 could inhibit the proliferation and invasiveness of breast cancer cells MDA-MB-231 in vitro and in vivo. This paper shows that BMP9 inhibit the bone metastasis of breast cancer cells by activating the BMP/Smad signaling pathway and downregulating connective tissue growth factor (CTGF); however, when CTGF expression was maintained, the inhibitory effect of BMP9 on the MDA-MB-231 cells was abolished. Together, these observations indicate that BMP9 is an important mediator of breast cancer bone metastasis and a potential therapeutic target for treating this deadly disease.

The potential role of the osteoblast in the development of periprosthetic osteolysis: review of in vitro osteoblast responses to wear debris, corrosion products, and cytokines and growth factors.

PubMed

Vermes, C; Glant, T T; Hallab, N J; Fritz, E A; Roebuck, K A; Jacobs, J J

2001-12-01

Limited information is available on the responses of osteoblasts to wear debris, corrosion products, and cytokines and on the roles of altered osteoblast functions in the development of periprosthetic bone loss. Wear debris-challenged osteoblasts exhibit altered functions resulting in the loss of their capacity to produce bone matrix and to replace the resorbed bone. Also, osteoblasts may secrete cytokines, which act in a paracrine fashion to recruit inflammatory cells into the periprosthetic space and to stimulate osteoclastic bone resorption. These effects may be mediated in part by ionic metal dissolution products. We review the mechanisms by which altered osteoblast functions, in response to particulate wear debris, corrosion products, and cytokines and growth factors, may contribute to the development and the progression of periprosthetic osteolysis.

Effect of SCG, 1,3-beta-D-glucan from Sparassis crispa on the hematopoietic response in cyclophosphamide induced leukopenic mice.

PubMed

Harada, Toshie; Miura, Noriko; Adachi, Yoshiyuki; Nakajima, Mitsuhiro; Yadomae, Toshiro; Ohn, Naohito

2002-07-01

Sparassis crispa Fr. is an edible mushroom recently cultivable in Japan. It contains a remarkably high content of 6-branched 1,3-beta-D-glucan showing antitumor activity. Using ion-exchange chromatography, a purified beta-glucan preparation, SCG, was prepared. In this study, we examined the hematopoietic response by SCG in cyclophosphamide (CY)-induced leukopenic mice. SCG enhanced the hematopoietic response in CY induced leukopenic mice by intraperitoneal routes over a wide range of concentrations. SCG enhanced the hematopoietic response in CY-treated mice by prior or post administration. Analyzing the leukocyte population by flow cytometry, monocytes and granulocytes in the peritoneal cavity, liver, spleen and bone marrow (BM) recovered faster than in the control group. The ratio of natural killer cells and gammadelta T cells in the liver, spleen and peritoneal cavity was also increased. In contrast, CD4+ CD8+ cells in the thymus were temporarily significantly decreased by the administration of SCG. Interleukin-6 (IL-6) production of CY+SCG-treated peritoneal exdated cells (PECs), spleen cells and bone marrow cells (BMCs) were higher than that of the CY-treated group. By in vitro culture of CY-treated PEC and spleen cells, IL-6 production was enhanced by the addition of SCG. These facts suggested the possibility that IL-6 might be a key cytokine for the enhanced hematopoietic response by SCG.

Coating of VEGF-releasing scaffolds with bioactive glass for angiogenesis and bone regeneration.

PubMed

Leach, J Kent; Kaigler, Darnell; Wang, Zhuo; Krebsbach, Paul H; Mooney, David J

2006-06-01

Bioactive glasses are potentially useful as bone defect fillers, and vascular endothelial growth factor (VEGF) has demonstrated benefit in bone regeneration as well. We hypothesized that the specific combination of prolonged localized VEGF presentation from a matrix coated with a bioactive glass may enhance bone regeneration. To test this hypothesis, the capacity of VEGF-releasing polymeric scaffolds with a bioactive glass coating was examined in vitro and in vivo using a rat critical-sized defect model. In the presence of a bioactive glass coating, we did not detect pronounced differences in the differentiation of human mesenchymal stem cells in vitro. However, we observed significantly enhanced mitogenic stimulation of endothelial cells in the presence of the bioactive glass coating, with an additive effect with VEGF release. This trend was maintained in vivo, where coated VEGF-releasing scaffolds demonstrated significant improvements in blood vessel density at 2 weeks versus coated control scaffolds. At 12 weeks, bone mineral density was significantly increased in coated VEGF-releasing scaffolds versus coated controls, while only a slight increase in bone volume fraction was observed. The results of this study suggest that a bioactive glass coating on a polymeric substrate participates in bone healing through indirect processes which enhance angiogenesis and bone maturation and not directly on osteoprogenitor differentiation and bone formation. The mass of bioactive glass used in this study provides a comparable and potentially additive, response to localized VEGF delivery over early time points. These studies demonstrate a materials approach to achieve an angiogenic response formerly limited to the delivery of inductive growth factors.

Differentiating zones at periodontal ligament-bone and periodontal ligament-cementum entheses.

PubMed

Lee, J-H; Pryce, B A; Schweitzer, R; Ryder, M I; Ho, S P

2015-12-01

The structural and functional integrity of bone-periodontal ligament (PDL)-cementum complex stems from the load-bearing attachment sites (entheses) between soft (PDL) and hard (bone, cementum) tissues. These attachment sites are responsible for the maintenance of a bone-PDL-cementum complex biomechanical function. The objective was to investigate changes in spatiotemporal expression of key biomolecules in developing and functionally active entheses. Multilabeling technique was performed on hemimandibles of 3 wk and 3 mo-old scleraxis-GFP transgenic mice for CD146, CD31, NG2, osterix and bone sialoprotein. Regions of dominant stretch within the PDL were evaluated by identifying directionality of collagen fibrils, PDL fibroblasts and PDL cell cytoskeleton. CD146+ cells adjacent to CD31+ vasculature were identified at PDL-bone enthesis. NG2+ cells were located at coronal bone-PDL and apical cementum-PDL entheses in the 3-wk-old group, but at 3 mo, NG2 was positive at the entheses of the apical region and alveolar crest. NG2 and osterix were colocalized at the osteoid and cementoid regions of the PDL-bone and PDL-cementum entheses. Bone sialoprotein was prominent at the apical region of 3-wk-old mice. The directionality of collagen fibers, fibroblasts and their cytoskeleton overlapped, except in the apical region of 3 wk. Colocalization of biomolecules at zones of the PDL adjacent to attachment sites may be essential for the formation of precementum and osteoid interfaces at a load-bearing bone-PDL-tooth fibrous joint. Biophysical cues resulting from development and function can regulate recruitment and differentiation of stem cells potentially from a vascular origin toward osteo- and cemento-blastic lineages at the PDL-bone and PDL-cementum entheses. Investigating the coupled effect of biophysical and biochemical stimuli leading to cell differentiation at the functional attachment sites is critical for developing regeneration strategies to enable functional reconstruction of the periodontal complex. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Synthetic Bone Substitute Engineered with Amniotic Epithelial Cells Enhances Bone Regeneration after Maxillary Sinus Augmentation

PubMed Central

Barboni, Barbara; Mangano, Carlo; Valbonetti, Luca; Marruchella, Giuseppe; Berardinelli, Paolo; Martelli, Alessandra; Muttini, Aurelio; Mauro, Annunziata; Bedini, Rossella; Turriani, Maura; Pecci, Raffaella; Nardinocchi, Delia; Zizzari, Vincenzo Luca; Tetè, Stefano; Piattelli, Adriano; Mattioli, Mauro

2013-01-01

Background Evidence has been provided that a cell-based therapy combined with the use of bioactive materials may significantly improve bone regeneration prior to dental implant, although the identification of an ideal source of progenitor/stem cells remains to be determined. Aim In the present research, the bone regenerative property of an emerging source of progenitor cells, the amniotic epithelial cells (AEC), loaded on a calcium-phosphate synthetic bone substitute, made by direct rapid prototyping (rPT) technique, was evaluated in an animal study. Material And Methods Two blocks of synthetic bone substitute (∼0.14 cm3), alone or engineered with 1×106 ovine AEC (oAEC), were grafted bilaterally into maxillary sinuses of six adult sheep, an animal model chosen for its high translational value in dentistry. The sheep were then randomly divided into two groups and sacrificed at 45 and 90 days post implantation (p.i.). Tissue regeneration was evaluated in the sinus explants by micro-computer tomography (micro-CT), morphological, morphometric and biochemical analyses. Results And Conclusions The obtained data suggest that scaffold integration and bone deposition are positively influenced by allotransplantated oAEC. Sinus explants derived from sheep grafted with oAEC engineered scaffolds displayed a reduced fibrotic reaction, a limited inflammatory response and an accelerated process of angiogenesis. In addition, the presence of oAEC significantly stimulated osteogenesis either by enhancing bone deposition or making more extent the foci of bone nucleation. Besides the modulatory role played by oAEC in the crucial events successfully guiding tissue regeneration (angiogenesis, vascular endothelial growth factor expression and inflammation), data provided herein show that oAEC were also able to directly participate in the process of bone deposition, as suggested by the presence of oAEC entrapped within the newly deposited osteoid matrix and by their ability to switch-on the expression of a specific bone-related protein (osteocalcin, OCN) when transplanted into host tissues. PMID:23696804

TRAF family member-associated NF-κB activator (TANK) is a negative regulator of osteoclastogenesis and bone formation.

PubMed

Maruyama, Kenta; Kawagoe, Tatsukata; Kondo, Takeshi; Akira, Shizuo; Takeuchi, Osamu

2012-08-17

The differentiation of bone-resorbing osteoclasts is induced by RANKL signaling, and leads to the activation of NF-κB via TRAF6 activation. TRAF family member-associated NF-κB activator (TANK) acts as a negative regulator of Toll-like receptors (TLRs) and B-cell receptor (BCR) signaling by inhibiting TRAF6 activation. Tank(-/-) mice spontaneously develop autoimmune glomerular nephritis in an IL-6-dependent manner. Despite its importance in the TCRs and BCR-activated TRAF6 inhibition, the involvement of TANK in RANKL signaling is poorly understood. Here, we report that TANK is a negative regulator of osteoclast differentiation. The expression levels of TANK mRNA and protein were up-regulated during RANKL-induced osteoclastogenesis, and overexpression of TANK in vitro led to a decrease in osteoclast formation. The in vitro osteoclastogenesis of Tank(-/-) cells was significantly increased, accompanied by increased ubiquitination of TRAF6 and enhanced canonical NF-κB activation in response to RANKL stimulation. Tank(-/-) mice showed severe trabecular bone loss, but increased cortical bone mineral density, because of enhanced bone erosion and formation. TANK mRNA expression was induced during osteoblast differentiation and Tank(-/-) osteoblasts exhibited enhaced NF-κB activation, IL-11 expression, and bone nodule formation than wild-type control cells. Finally, wild-type mice transplanted with bone marrow cells from Tank(-/-) mice showed trabecular bone loss analogous to that in Tank(-/-) mice. These findings demonstrate that TANK is critical for osteoclastogenesis by regulating NF-κB, and is also important for proper bone remodeling.

TRAF Family Member-associated NF-κB Activator (TANK) Is a Negative Regulator of Osteoclastogenesis and Bone Formation*

PubMed Central

Maruyama, Kenta; Kawagoe, Tatsukata; Kondo, Takeshi; Akira, Shizuo; Takeuchi, Osamu

2012-01-01

The differentiation of bone-resorbing osteoclasts is induced by RANKL signaling, and leads to the activation of NF-κB via TRAF6 activation. TRAF family member-associated NF-κB activator (TANK) acts as a negative regulator of Toll-like receptors (TLRs) and B-cell receptor (BCR) signaling by inhibiting TRAF6 activation. Tank−/− mice spontaneously develop autoimmune glomerular nephritis in an IL-6-dependent manner. Despite its importance in the TCRs and BCR-activated TRAF6 inhibition, the involvement of TANK in RANKL signaling is poorly understood. Here, we report that TANK is a negative regulator of osteoclast differentiation. The expression levels of TANK mRNA and protein were up-regulated during RANKL-induced osteoclastogenesis, and overexpression of TANK in vitro led to a decrease in osteoclast formation. The in vitro osteoclastogenesis of Tank−/− cells was significantly increased, accompanied by increased ubiquitination of TRAF6 and enhanced canonical NF-κB activation in response to RANKL stimulation. Tank−/− mice showed severe trabecular bone loss, but increased cortical bone mineral density, because of enhanced bone erosion and formation. TANK mRNA expression was induced during osteoblast differentiation and Tank−/− osteoblasts exhibited enhaced NF-κB activation, IL-11 expression, and bone nodule formation than wild-type control cells. Finally, wild-type mice transplanted with bone marrow cells from Tank−/− mice showed trabecular bone loss analogous to that in Tank−/− mice. These findings demonstrate that TANK is critical for osteoclastogenesis by regulating NF-κB, and is also important for proper bone remodeling. PMID:22773835

Cellular Architecture of Spinal Granulomas and the Immunological Response in Tuberculosis Patients Coinfected with HIV.

PubMed

Bhattacharya, Debapriya; Danaviah, Siva; Muema, Daniel M; Akilimali, Ngomu Akeem; Moodley, Prashini; Ndung'u, Thumbi; Das, Gobardhan

2017-01-01

Mycobacterium tuberculosis ( M.tb ) and HIV are individually responsible for the most deaths worldwide among all infectious agents, and coinfection with M.tb and HIV is a significant public health challenge in the developing world. Although the lung is the primary target organ for tuberculosis (TB), M.tb can also cause extrapulmonary tuberculosis (EPTB) such as in the bones and joints. Treatment of EPTB is much more challenging than treatment of pulmonary TB. The hallmark of the host immune response against TB is the formation of organized structures called granulomas that are infiltrated with immune cells and are rich in cytokines and chemokines. Inside granulomas, the host confines the M.tb bacteria to a particular region of the organ and avoids dispersion. In this study, we analyzed immune cells in bone granulomas of patients with EPTB that are also coinfected with HIV. We found that HIV-infected TB patients have dispersed bone granulomas, with reduced T cell numbers and a concomitant increase in plasma cells. Additionally, HIV-infected patients exhibited dramatically increased serum levels of IgM and IgG1 antibodies, which is indicative of T-cell-independent B-cell activation and mucosal T-cell activation, respectively. Interestingly, we also observed that CD29 + stem cells are increased in HIV-TB coinfection, suggesting a link with HIV infection. Therefore, our work provides new insights into the architecture of spinal TB granulomas and the role of B-cells and humoral immunity against a highly infectious intracellular pathogen. We propose that our findings will inform biomarker identification for EPTB and possibly the development of related therapeutics and/or vaccines to protect HIV-infected patients against disseminated TB.

Responsiveness of mouse calvaria to parathyroid hormone after explant cryopreservation: 45Ca release in vitro

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

Wezeman, F.H.; Dungan, D.D.

1986-08-01

Newborn mouse calvaria prelabeled with /sup 45/Ca and cryopreserved at -196 degrees C in serum-free medium containing dimethylsulfoxide were compared to unpreserved explants for response to parathyroid hormone during subsequent culture. After short-term cryopreservation followed by rapid thawing, the viable explants continued to release /sup 45/Ca to the culture medium but additions of parathyroid hormone to the medium did not cause increased bone resorption. The data suggest that cryopreservation and thawing impairs mechanisms responsible for parathyroid hormone action on bone cells.

Biological response of laser macrostructured and oxidized titanium alloy: an in vitro and in vivo study.

PubMed

Paz, María Dolores; Álava, J Iñaki; Goikoetxea, Leire; Chiussi, Stefano; Díaz-Güemes, Idoia; Usón, Jesus; Sánchez, Francisco; León, Betty

2011-01-01

To assess both the in vitro and in vivo biological response of a laser modified surface in an integrated manner. A combined innovative approach applies lasers to macrostructure as well as to oxidize the surface of titanium alloy implants. A Nd:YAG marking and ArF excimer lasers were used for macrostructuring and UV-oxidizing the surface of Ti6Al4V discs, respectively. Human fetal osteoblastic cell culture and a sheep tibia model were used to assess the cell response and the osseogeneration capability of as-machined, laser macrostructured and laser macrostructured and oxidized surfaces. In vitro: Laser macrostructuration alone did not promote cell response. Cellular proliferation was enhanced by the additional UV laser oxidation. In vivo: A greater significant percentage of bone-implant contact was obtained for both laser treated surfaces compared to machine-turned control samples, three months after implantation, in spite of the low cellular response for macrostructured samples. The use of sheep model for six months appears to be less adequate for a comparison because of the high level of bone integration in all samples. In spite of the often reported positive effect of titanium oxidation on the triggering of faster osseointegration, in this experiment the additional UV laser oxidation did not lead to a significant in vivo improvement. Laser macrostructuration of titanium alloy surfaces appears to promote bone apposition and may therefore constitute a promising surface modification strategy. In animal models, the natural process of titanium surface oxidation, because of physiologic fluids, alters properties observed in vitro with cells.

Investigation of biomineralization by Raman spectroscopy

NASA Astrophysics Data System (ADS)

Fatscher, Robert William

Biomineralization is a process in which living organism grow composite materials consisting of inorganic and organic materials. This produces a composite material consisting of both inorganic and organic components, with superior mechanical properties. In the human body bone and dentin are both examples of biominerals. In this research Raman spectroscopy was used to characterize dentin from mice and human teeth, to determine composition. In the mouse tooth samples areas of irregular dentin were found, along the inside of the tooth, to be in the process of mineralization. By analyzing the samples along these areas we were able to determine the composition of dentin and track how it changed in these area. By analysis of the mineral to matrix ratio the areas of irregular dentin were determined to have less mineral present. Observations of other organic components and collagen in increased concentrations in this area suggested these area were in the process of biomineralization. The understanding of the structure of dentin and its biomineralization process is of crucial importance when trying reproduce dentin. Scientists and engineers are able to produce dentin minerals in vitro by culturing various dental stem cells. The ability to create dentin mineral from cells could lead to methods of repairing dentin in patients, or even lead to the creation of a completely engineered tooth. While dentin-like materials can be produced in a laboratory environment, analysis and comparison of the composition of these materials must be performed to ensure the mineral produced is consistent with dentin. Mineralized nodules from six different dental stem cell lines were cultured to produce a mineralized deposit. Utilizing Raman spectroscopy, we were able to determine cell source dependent differences in a variety of dental stem cells, and compare the mineral produced to native dentin. Orthopedic implants are implants used to replace damaged bone, examples include knee, hip and dental implants. These implants are designed to osteointegrate with the native healthy tissues in order to create a functionally stable and structural interface. Biomaterials such as hydroxyapatite and titania are known to increase the rate of bone regeneration in vivo.1 By accelerating the early response of bone forming cells to these implants, better fixation is achieved between the implant and the bone, shortening recovery times and increasing the viability of these implants. In the last part of this research an investigation of osteoblasts cultured at 14 days on five different heat-treated titania substrates was investigated by Raman spectroscopy, in order to observe the initial cellular response to the titania substrates. The heat-treatment of titania changes the amount of oxygen on it's surface which in turn effects the surface energy. A change in the surface energy of a material will affect the cellular response, by culturing cells on various heat-treated titania substrates a relationship between the surface energy and cellular response can be investigated. A faster cellular response would lead to an increased rate of bone regeneration shortening healing times and allowing for better fixation of the implant.

Deletion of the Distal Tnfsf11 RL-D2 Enhancer That Contributes to PTH-Mediated RANKL Expression in Osteoblast Lineage Cells Results in a High Bone Mass Phenotype in Mice.

PubMed

Onal, Melda; St John, Hillary C; Danielson, Allison L; Pike, J Wesley

2016-02-01

Receptor activator of nuclear factor-κB ligand (RANKL) is a tumor necrosis factor (TNF)-like cytokine that is necessary for osteoclast formation and survival. Elevated RANKL synthesis is associated with both increased osteoclast number and bone resorption. Earlier studies identified an enhancer 76 kb upstream of the Tnfsf11 transcriptional start site (TSS) termed RL-D5 or the distal control region (DCR) that modulates RANKL expression in response to PTH, 1,25(OH)2D3,, and an array of cytokines. Mice lacking RL-D5 exhibit high bone mass associated with decreased RANKL expression in bone, spleen, and thymus. In addition to RL-D5, genome-wide studies have identified 9 additional Tnfsf11 enhancers residing upstream of the gene's TSS, which provide RANKL cell type-specificity and responsiveness to local and systemic factors. ChIP-chip analyses has revealed inducible vitamin D receptor (VDR) and cAMP response element-binding protein (CREB) binding at an enhancer termed RL-D2 23 kb upstream of the Tnfsf11 TSS in osteoblastic ST2 cells. Herein, we use ChIP-seq analyses to confirm this finding and then delete this enhancer from the mouse genome to determine its physiological role in vivo. RL-D2(-/-) primary stromal cells showed decreased RANKL-induction by both forskolin and 1,25(OH)2D3 ex vivo. Consistent with this, the parathyroid hormone (PTH) induction of RANKL expression was significantly blunted in RL-D2(-/-) mice in vivo. In contrast, lack of RL-D2 had no effect on 1,25(OH)2D3 induction of RANKL in vivo. Similar to the results found in RL-D5(-/-) mice, lack of RL-D2 led to decreased skeletal RANKL expression, resulting in decreased osteoclast numbers and a progressive increase in bone mineral density. Lack of RL-D2 increased cancellous bone mass in femur and spine but did not alter femoral cortical bone thickness. These results highlight the role of distal enhancers in the regulation of RANKL expression by PTH and perhaps 1,25(OH)2D3 and suggest that the RL-D2 and RL-D5 enhancers contribute in either an additive or synergistic manner to regulate bone remodeling. © 2015 American Society for Bone and Mineral Research.

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

Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.

The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despitemore » this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased focal adhesion kinase activity. • Shb is critical for the long-term maintenance of the hematopoietic stem cell pool.« less

Major Histocompatibilty Complex-Restricted Adaptive Immune Responses to CT26 Colon Cancer Cell Line in Mixed Allogeneic Chimera.

PubMed

Lee, K W; Choi, B; Kim, Y M; Cho, C W; Park, H; Moon, J I; Choi, G-S; Park, J B; Kim, S J

2017-06-01

Although the induction of mixed allogeneic chimera shows promising clinical tolerance results in organ transplantation, its clinical relevance as an anti-cancer therapy is yet unknown. We introduced a mixed allogenic chimera setting with the use of a murine colon cancer cell line, CT26, by performing double bone marrow transplantation. We analyzed donor- and recipient-restricted anti-cancer T-cell responses, and phenotypes of subpopulations of T cells. The protocol involves challenging 1 × 10 5 cells of CT26 cells intra-hepatically on day 50 after bone marrow transplantation, and, by use of CT26 lysates and an H-2L d -restricted AH1 pentamer, flow cytometric analysis was performed to detect the generation of cancer-specific CD4 + and CD8 + T cells at various time points. We found that immunocompetence against tumors depends heavily on cancer-specific CD8 + T-cell responses in a major histocompatibility complex-restricted manner; the evidence was further supported by the increase of interferon-γ-secreting CD4 + T cells. Moreover, we demonstrated that during the effector immune response to CT26 cancer challenge, there was a presence of central memory cells (CD62L hi CCR7 + ) as well as effector memory cells (CD62L lo CCR7 - ). Moreover, mixed allogeneic chimeras (BALB/c to C56BL/6 or vice versa) showed similar or heightened immune responses to CT26 cells compared with that of wild-type mice. Our results suggest that the responses of primary immunocompetency and of pre-existing memory T cells against allogeneic cancer are sustained and preserved long-term in a mixed allogeneic chimeric environment. Copyright © 2017 Elsevier Inc. All rights reserved.

Osteoblast response to zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate

PubMed Central

Whited, Bryce M.; Skrtic, Drago; Love, Brian J.

2006-01-01

Calcium phosphate bioceramics, such as hydroxyapatite, have long been used as bone substitutes because of their proven biocompatibility and bone binding properties in vivo. Recently, a zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized, which is more soluble than hydroxyapatite and allows for controlled release of calcium and phosphate ions. These ions have been postulated to increase osteoblast differentiation and mineralization in vitro. The focus of this work is to elucidate the physicochemical properties of Zr-ACP and to measure cell response to Zr-ACP in vitro using a MC3T3-E1 mouse calvarial-derived osteoprogenitor cell line. Cells were cultured in osteogenic medium and mineral was added to culture at different stages in cell maturation. Culture in the presence of Zr-ACP showed significant increases in cell proliferation, alkaline phosphatase activity (ALP), and osteopontin (OPN) synthesis, whereas collagen synthesis was unaffected. In addition, calcium and phosphate ion concentrations and medium pH were found to transiently increase with the addition of Zr-ACP, and are hypothesized to be responsible for the osteogenic effect of Zr-ACP. PMID:16278876

Osteoblast-Secreted Factors Mediate Dormancy of Metastatic Prostate Cancer in the Bone via Activation of the TGFβRIII-p38MAPK-pS249/T252RB Pathway.

PubMed

Yu-Lee, Li-Yuan; Yu, Guoyu; Lee, Yu-Chen; Lin, Song-Chang; Pan, Jing; Pan, Tianhong; Yu, Kai-Jie; Liu, Bin; Creighton, Chad J; Rodriguez-Canales, Jaime; Villalobos, Pamela A; Wistuba, Ignacio I; de Nadal, Eulalia; Posas, Francesc; Gallick, Gary E; Lin, Sue-Hwa

2018-06-01

Bone metastasis from prostate cancer can occur years after prostatectomy, due to reactivation of dormant disseminated tumor cells (DTC) in the bone, yet the mechanism by which DTCs are initially induced into a dormant state in the bone remains to be elucidated. We show here that the bone microenvironment confers dormancy to C4-2B4 prostate cancer cells, as they become dormant when injected into mouse femurs but not under the skin. Live-cell imaging of dormant cells at the single-cell level revealed that conditioned medium from differentiated, but not undifferentiated, osteoblasts induced C4-2B4 cellular quiescence, suggesting that differentiated osteoblasts present locally around the tumor cells in the bone conferred dormancy to prostate cancer cells. Gene array analyses identified GDF10 and TGFβ2 among osteoblast-secreted proteins that induced quiescence of C4-2B4, C4-2b, and PC3-mm2, but not 22RV1 or BPH-1 cells, indicating prostate cancer tumor cells differ in their dormancy response. TGFβ2 and GDF10 induced dormancy through TGFβRIII to activate phospho-p38MAPK, which phosphorylates retinoblastoma (RB) at the novel N-terminal S249/T252 sites to block prostate cancer cell proliferation. Consistently, expression of dominant-negative p38MAPK in C4-2b and C4-2B4 prostate cancer cell lines abolished tumor cell dormancy both in vitro and in vivo Lower TGFβRIII expression in patients with prostate cancer correlated with increased metastatic potential and decreased survival rates. Together, our results identify a dormancy mechanism by which DTCs are induced into a dormant state through TGFβRIII-p38MAPK-pS249/pT252-RB signaling and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. Significance: These findings provide mechanistic insights into the dormancy of metastatic prostate cancer in the bone and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. Cancer Res; 78(11); 2911-24. ©2018 AACR . ©2018 American Association for Cancer Research.

Rictor is required for optimal bone accrual in response to anti-sclerostin therapy in the mouse.

PubMed

Sun, Weiwei; Shi, Yu; Lee, Wen-Chih; Lee, Seung-Yon; Long, Fanxin

2016-04-01

Wnt signaling has emerged as a major target pathway for the development of novel bone anabolic therapies. Neutralizing antibodies against the secreted Wnt antagonist sclerostin (Scl-Ab) increase bone mass in both animal models and humans. Because we have previously shown that Rictor-dependent mTORC2 activity contributes to Wnt signaling, we test here whether Rictor is required for Scl-Ab to promote bone anabolism. Mice with Rictor deleted in the early embryonic limb mesenchyme (Prx1-Cre;Rictor(f/f), hereafter RiCKO) were subjected to Scl-Ab treatment for 5weeks starting at 4months of age. In vivo micro-computed tomography (μCT) analyses before the treatment showed that the RiCKO mice displayed normal trabecular, but less cortical bone mass than the littermate controls. After 5weeks of treatment, Scl-Ab dose-dependently increased trabecular and cortical bone mass in both control and RiCKO mice, but the increase was significantly blunted in the latter. Dynamic histomorphometry revealed that the RiCKO mice formed less bone than the control in response to Scl-Ab. In addition, the RiCKO mice possessed fewer osteoclasts than normal under the basal condition and exhibited lesser suppression in osteoclast number by Scl-Ab. Consistent with the fewer osteoclasts in vivo, bone marrow stromal cells (BMSC) from the RiCKO mice expressed less Rankl but normal levels of Opg or M-CSF, and were less effective than the control cells in supporting osteoclastogenesis in vitro. The reliance of Rankl on Rictor appeared to be independent of Wnt-β-catenin or Wnt-mTORC2 signaling as Wnt3a had no effect on Rankl expression by BMSC from either control or RICKO mice. Overall, Rictor in the limb mesenchymal lineage is required for the normal response to the anti-sclerostin therapy in both bone formation and resorption. Copyright © 2016 Elsevier Inc. All rights reserved.

Roles of Stat3 and ERK in G-CSF signaling.

PubMed

Kamezaki, Kenjirou; Shimoda, Kazuya; Numata, Akihiko; Haro, Takashi; Kakumitsu, Haruko; Yoshie, Masumi; Yamamoto, Masahiro; Takeda, Kiyoshi; Matsuda, Tadashi; Akira, Shizuo; Ogawa, Katsuhiro; Harada, Mine

2005-02-01

G-CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil-granulocyte lineage. Although Stat3 was thought to be essential for the transduction of G-CSF-induced cell proliferation and differentiation signals, mice deficient for Stat3 in hematopoietic cells show neutrocytosis and infiltration of cells into the digestive tract. The number of progenitor cells in the neutrophil lineage is not changed, and G-CSF-induced proliferation of progenitor cells and prolonged neutrophil survival were observed in Stat3-deficient mice. In hematopoietic cells from Stat3-deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression was not induced by G-CSF stimulation. Stat3-null bone marrow cells displayed a significant activation of extra-cellular regulated kinase 1 (ERK1)/ERK2 under basal conditions, and the activation of ERK was enhanced and sustained by G-CSF stimulation. Furthermore, the augmented proliferation of Stat3-deficient bone marrow cells in response to G-CSF was dramatically decreased by addition of a MEK1 inhibitor. These results indicate that Stat3 functions as a negative regulator of G-CSF signaling by inducing SOCS3 expression and that ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G-CSF.

Mesenchymal Stem Cell Benefits Observed in Bone Marrow Failure and Acquired Aplastic Anemia

PubMed Central

Gonzaga, Vivian Fonseca; Lisboa, Gustavo Sabino; Frare, Eduardo Osório

2017-01-01

Acquired aplastic anemia (AA) is a type of bone marrow failure (BMF) syndrome characterized by partial or total bone marrow (BM) destruction resulting in peripheral blood (PB) pancytopenia, which is the reduction in the number of red blood cells (RBC) and white blood cells (WBC), as well as platelets (PLT). The first-line treatment option of AA is given by hematopoietic stem cell (HSCs) transplant and/or immunosuppressive (IS) drug administration. Some patients did not respond to the treatment and remain pancytopenic following IS drugs. The studies are in progress to test the efficacy of adoptive cellular therapies as mesenchymal stem cells (MSCs), which confer low immunogenicity and are reliable allogeneic transplants in refractory severe aplastic anemia (SAA) cases. Moreover, bone marrow stromal cells (BMSC) constitute an essential component of the hematopoietic niche, responsible for stimulating and enhancing the proliferation of HSCs by secreting regulatory molecules and cytokines, providing stimulus to natural BM microenvironment for hematopoiesis. This review summarizes scientific evidences of the hematopoiesis improvements after MSC transplant, observed in acquired AA/BMF animal models as well as in patients with acquired AA. Additionally, we discuss the direct and indirect contribution of MSCs to the pathogenesis of acquired AA. PMID:29333168

Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption.

PubMed

Canalis, Ernesto; Schilling, Lauren; Yee, Siu-Pok; Lee, Sun-Kyeong; Zanotti, Stefano

2016-01-22

Notch receptors are determinants of cell fate and function and play a central role in skeletal development and bone remodeling. Hajdu Cheney syndrome, a disease characterized by osteoporosis and fractures, is associated with NOTCH2 mutations resulting in a truncated stable protein and gain-of-function. We created a mouse model reproducing the Hajdu Cheney syndrome by introducing a 6955C→T mutation in the Notch2 locus leading to a Q2319X change at the amino acid level. Notch2(Q2319X) heterozygous mutants were smaller and had shorter femurs than controls; and at 1 month of age they exhibited cancellous and cortical bone osteopenia. As the mice matured, cancellous bone volume was restored partially in male but not female mice, whereas cortical osteopenia persisted in both sexes. Cancellous bone histomorphometry revealed an increased number of osteoclasts and bone resorption, without a decrease in osteoblast number or bone formation. Osteoblast differentiation and function were not affected in Notch2(Q2319X) cells. The pre-osteoclast cell pool, osteoclast differentiation, and bone resorption in response to receptor activator of nuclear factor κB ligand in vitro were increased in Notch2(Q2319X) mutants. These effects were suppressed by the γ-secretase inhibitor LY450139. In conclusion, Notch2(Q2319X) mice exhibit cancellous and cortical bone osteopenia, enhanced osteoclastogenesis, and increased bone resorption. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone

PubMed Central

Tu, Xiaolin; Delgado-Calle, Jesus; Condon, Keith W.; Maycas, Marta; Zhang, Huajia; Carlesso, Nadia; Taketo, Makoto M.; Burr, David B.; Plotkin, Lilian I.; Bellido, Teresita

2015-01-01

Osteocytes, >90% of the cells in bone, lie embedded within the mineralized matrix and coordinate osteoclast and osteoblast activity on bone surfaces by mechanisms still unclear. Bone anabolic stimuli activate Wnt signaling, and human mutations of components along this pathway underscore its crucial role in bone accrual and maintenance. However, the cell responsible for orchestrating Wnt anabolic actions has remained elusive. We show herein that activation of canonical Wnt signaling exclusively in osteocytes [dominant active (da)βcatOt mice] induces bone anabolism and triggers Notch signaling without affecting survival. These features contrast with those of mice expressing the same daß-catenin in osteoblasts, which exhibit decreased resorption and perinatal death from leukemia. daßcatOt mice exhibit increased bone mineral density in the axial and appendicular skeleton, and marked increase in bone volume in cancellous/trabecular and cortical compartments compared with littermate controls. daßcatOt mice display increased resorption and formation markers, high number of osteoclasts and osteoblasts in cancellous and cortical bone, increased bone matrix production, and markedly elevated periosteal bone formation rate. Wnt and Notch signaling target genes, osteoblast and osteocyte markers, and proosteoclastogenic and antiosteoclastogenic cytokines are elevated in bones of daßcatOt mice. Further, the increase in RANKL depends on Sost/sclerostin. Thus, activation of osteocytic β-catenin signaling increases both osteoclasts and osteoblasts, leading to bone gain, and is sufficient to activate the Notch pathway. These findings demonstrate disparate outcomes of β-catenin activation in osteocytes versus osteoblasts and identify osteocytes as central target cells of the anabolic actions of canonical Wnt/β-catenin signaling in bone. PMID:25605937

The CardiAMP Heart Failure trial: A randomized controlled pivotal trial of high-dose autologous bone marrow mononuclear cells using the CardiAMP cell therapy system in patients with post-myocardial infarction heart failure: Trial rationale and study design.

PubMed

Raval, Amish N; Cook, Thomas D; Duckers, Henricus J; Johnston, Peter V; Traverse, Jay H; Abraham, William T; Altman, Peter A; Pepine, Carl J

2018-07-01

Heart failure following myocardial infarction is a common, disabling, and deadly condition. Direct injection of autologous bone marrow mononuclear cells into the myocardium may result in improved functional recovery, relieve symptoms, and improve other cardiovascular outcomes. CardiAMP-HF is a randomized, double-blind, sham-controlled, pivotal trial designed to investigate the safety and efficacy of autologous bone marrow mononuclear cells treatment for patients with medically refractory and symptomatic ischemic cardiomyopathy. The primary end point is change in 6-minute walk distance adjusted for major adverse cardiovascular events at 12 months following treatment. Particularly novel aspects of this trial include a cell potency assay to screen subjects who have bone marrow cell characteristics that suggest a favorable response to treatment, a point-of-care treatment method, a high target dose of 200 million cells, and an efficient transcatheter intramyocardial delivery method that is associated with high cell retention. This novel approach may lead to a new treatment for those with ischemic heart disease suffering from medically refractory heart failure. Copyright © 2018 Elsevier Inc. All rights reserved.

Mechanical Unloading of Mouse Bone in Microgravity Significantly Alters Cell Cycle Gene Set Expression

NASA Astrophysics Data System (ADS)

Blaber, Elizabeth; Dvorochkin, Natalya; Almeida, Eduardo; Kaplan, Warren; Burns, Brnedan

2012-07-01

Spaceflight factors, including microgravity and space radiation, have many detrimental short-term effects on human physiology, including muscle and bone degradation, and immune system dysfunction. The long-term progression of these physiological effects is still poorly understood, and a serious concern for long duration spaceflight missions. We hypothesized that some of the degenerative effects of spaceflight may be caused in part by an inability of stem cells to proliferate and differentiate normally resulting in an impairment of tissue regenerative processes. Furthermore, we hypothesized that long-term bone tissue degeneration in space may be mediated by activation of the p53 signaling network resulting in cell cycle arrest and/or apoptosis in osteoprogenitors. In our analyses we found that spaceflight caused significant bone loss in the weight-bearing bones of mice with a 6.3% reduction in bone volume and 11.9% decrease in bone thickness associated with increased osteoclastic activity. Along with this rapid bone loss we also observed alterations in the cell cycle characterized by an increase in the Cdkn1a/p21 cell cycle arrest molecule independent of Trp53. Overexpression of Cdkn1a/p21 was localized to osteoblasts lining the periosteal surface of the femur and chondrocytes in the head of the femur, suggesting an inhibition of proliferation in two key regenerative cell types of the femur in response to spaceflight. Additionally we found overexpression of several matrix degradation molecules including MMP-1a, 3 and 10, of which MMP-10 was localized to osteocytes within the shaft of the femur. This, in conjunction with 40 nm resolution synchrotron nano-Computed Tomography (nano-CT) observations of an increase in osteocyte lacunae cross-sectional area, perimeter and a decrease in circularity indicates a potential role for osteocytic osteolysis in the observed bone degeneration in spaceflight. To further investigate the genetic response of bone to mechanical unloading in spaceflight, we conducted genome wide microarray analysis of total RNA isolated from the mouse pelvis. Specifically, 16 week old mice were subjected to 15 days spaceflight onboard NASA's STS-131 space shuttle mission. The pelvis of the mice was dissected, the bone marrow was flushed and the bones were briefly stored in RNAlater. The pelvii were then homogenized, and RNA was isolated using TRIzol. RNA concentration and quality was measured using a Nanodrop spectrometer, and 0.8% agarose gel electrophoresis. Samples of cDNA were analyzed using an Affymetrix GeneChip\\S Gene 1.0 ST (Sense Target) Array System for Mouse and GenePattern Software. We normalized the ST gene arrays using Robust Multichip Average (RMA) normalization, which summarizes perfectly matched spots on the array through the median polish algorithm, rather than normalizing according to mismatched spots. We also used Limma for statistical analysis, using the BioConductor Limma Library by Gordon Smyth, and differential expression analysis to identify genes with significant changes in expression between the two experimental conditions. Finally we used GSEApreRanked for Gene Set Enrichment Analysis (GSEA), with Kolmogorov-Smirnov style statistics to identify groups of genes that are regulated together using the t-statistics derived from Limma. Preliminary results show that 6,603 genes expressed in pelvic bone had statistically significant alterations in spaceflight compared to ground controls. These prominently included cell cycle arrest molecules p21, and p18, cell survival molecule Crbp1, and cell cycle molecules cyclin D1, and Cdk1. Additionally, GSEA results indicated alterations in molecular targets of cyclin D1 and Cdk4, senescence pathways resulting from abnormal laminin maturation, cell-cell contacts via E-cadherin, and several pathways relating to protein translation and metabolism. In total 111 gene sets out of 2,488, about 4%, showed statistically significant set alterations. These alterations indicate significant impairment of normal cellular function in the mechanically unloaded environment of space and could provide important genetic insight into the observed uncoupling of bone formation and resorption in space.

Primitive Sca-1 Positive Bone Marrow HSC in Mouse Model of Aplastic Anemia: A Comparative Study through Flowcytometric Analysis and Scanning Electron Microscopy

PubMed Central

Chatterjee, Sumanta; Basak, Pratima; Das, Prosun; Das, Madhurima; Pereira, Jacintha Archana; Dutta, Ranjan Kumar; Chaklader, Malay; Chaudhuri, Samaresh; Law, Sujata

2010-01-01

Self-renewing Hematopoietic Stem Cells (HSCs) are responsible for reconstitution of all blood cell lineages. Sca-1 is the “stem cell antigen” marker used to identify the primitive murine HSC population, the expression of which decreases upon differentiation to other mature cell types. Sca-1+ HSCs maintain the bone marrow stem cell pool throughout the life. Aplastic anemia is a disease considered to involve primary stem cell deficiency and is characterized by severe pancytopenia and a decline in healthy blood cell generation system. Studies conducted in our laboratory revealed that the primitive Sca-1+ BM-HSCs (bone marrow hematopoietic stem cell) are significantly affected in experimental Aplastic animals pretreated with chemotherapeutic drugs (Busulfan and Cyclophosphamide) and there is increased Caspase-3 activity with consecutive high Annexin-V positivity leading to premature apoptosis in the bone marrow hematopoietic stem cell population in Aplastic condition. The Sca-1bright, that is, “more primitive” BM-HSC population was more affected than the “less primitive” BM-HSC Sca-1dim  population. The decreased cell population and the receptor expression were directly associated with an empty and deranged marrow microenvironment, which is evident from scanning electron microscopy (SEM). The above experimental evidences hint toward the manipulation of receptor expression for the benefit of cytotherapy by primitive stem cell population in Aplastic anemia cases. PMID:21048851

Knee loading inhibits osteoclast lineage in a mouse model of osteoarthritis

PubMed Central

Li, Xinle; Yang, Jing; Liu, Daquan; Li, Jie; Niu, Kaijun; Feng, Shiqing; Yokota, Hiroki; Zhang, Ping

2016-01-01

Osteoarthritis (OA) is a whole joint disorder that involves cartilage degradation and periarticular bone response. Changes of cartilage and subchondral bone are associated with development and activity of osteoclasts from subchondral bone. Knee loading promotes bone formation, but its effects on OA have not been well investigated. Here, we hypothesized that knee loading regulates subchondral bone remodeling by suppressing osteoclast development, and prevents degradation of cartilage through crosstalk of bone-cartilage in osteoarthritic mice. Surgery-induced mouse model of OA was used. Two weeks application of daily dynamic knee loading significantly reduced OARSI scores and CC/TAC (calcified cartilage to total articular cartilage), but increased SBP (subchondral bone plate) and B.Ar/T.Ar (trabecular bone area to total tissue area). Bone resorption of osteoclasts from subchondral bone and the differentiation of osteoclasts from bone marrow-derived cells were completely suppressed by knee loading. The osteoclast activity was positively correlated with OARSI scores and negatively correlated with SBP and B.Ar/T.Ar. Furthermore, knee loading exerted protective effects by suppressing osteoclastogenesis through Wnt signaling. Overall, osteoclast lineage is the hyper responsiveness of knee loading in osteoarthritic mice. Mechanical stimulation prevents OA-induced cartilage degeneration through crosstalk with subchondral bone. Knee loading might be a new potential therapy for osteoarthritis patients. PMID:27087498

Influence of irradiation on the osteoinductive potential of demineralized bone matrix.

PubMed

Wientroub, S; Reddi, A H

1988-04-01

Samples of demineralized bone matrix (DBM) were exposed to graduated doses of radiation (1-15 Megarad) (Mrad) utilizing a linear accelerator and then implanted into the thoracic region of Long-Evans rats. Subcutaneous implantation of DBM into allogenic rats induces endochondral bone. In response to matrix implantation, a cascade of events ensues; mesenchymal cell proliferation on day 3 postimplantation, chondrogenesis on day 7, calcification of the cartilagenous matrix and chondrolysis on day 9, and osteogenesis on day 11 resulting in formation of an ossicle containing active hemopoietic tissue. Bone formation was assessed by measuring alkaline phosphatase activity, the rate of mineralization was determined by measuring 45Ca incorporation to bone mineral, and 40Ca content measured the extent of mineralization; acid phosphatase activity was used as a parameter for bone resorption. The dose of radiation (2.5 Mrad) currently used by bone banks for sterilization of bone tissue did not destroy the bone induction properties of DBM. Furthermore, radiation of 3-5 Mrad even enhanced bone induction, insofar as it produced more bone at the same interval of time than was obtained from unirradiated control samples. None of the radiation doses used in these experiments abolished bone induction, although the response induced by matrix irradiated with doses higher than 5 Mrad was delayed.

Successful Treatment of Aggressive Mature B-cell Lymphoma Mimicking Immune Thrombocytopenic Purpura.

PubMed

Ono, Koya; Onishi, Yasushi; Kobayashi, Masahiro; Ichikawa, Satoshi; Hatta, Shunsuke; Watanabe, Shotaro; Okitsu, Yoko; Fukuhara, Noriko; Ichinohasama, Ryo; Harigae, Hideo

2018-03-30

A 55-year-old woman suffered from hemorrhagic tendency. She had severe thrombocytopenia without any hematological or coagulatory abnormalities, and a bone marrow examination revealed an increased number of megakaryocytes without any abnormal cells or blasts. No lymphadenopathy or hepatosplenomegaly was observed on computed tomography. She was initially diagnosed with immune thrombocytopenic purpura (ITP). None of the treatments administered for ITP produced a response. However, abnormal cells were eventually found during the third bone marrow examination. The pathological diagnosis was mature B-cell lymphoma. Rituximab-containing chemotherapy produced a marked increase in the patient's platelet count, and her lymphoma went into complete remission.

Human mesenchymal stromal cells are mechanosensitive to vibration stimuli.

PubMed

Kim, I S; Song, Y M; Lee, B; Hwang, S J

2012-12-01

Low-magnitude high-frequency (LMHF) vibrations have the ability to stimulate bone formation and reduce bone loss. However, the anabolic mechanisms that are mediated by vibration in human bone cells at the cellular level remain unclear. We hypothesized that human mesenchymal stromal cells (hMSCs) display direct osteoblastic responses to LMHF vibration signals. Daily exposure to vibrations increased the proliferation of hMSCs, with the highest efficiency occurring at a peak acceleration of 0.3 g and vibrations at 30 to 40 Hz. Specifically, these conditions promoted osteoblast differentiation through an increase in alkaline phosphatase activity and in vitro matrix mineralization. The effect of vibration on the expression of osteogenesis-related factors differed depending on culture method. hMSCs that underwent vibration in a monolayer culture did not exhibit any changes in the expressions of these genes, while cells in three-dimensional culture showed increased expression of type I collagen, osteoprotegerin, or VEGF, and VEGF induction appeared in 2 different hMSC lines. These results are among the first to demonstrate a dose-response effect upon LMHF stimulation, thereby demonstrating that hMSCs are mechanosensitive to LMHF vibration signals such that they could facilitate the osteogenic process.

Aging alters bone-fat reciprocity by shifting in vivo mesenchymal precursor cell fate towards an adipogenic lineage

PubMed Central

Singh, Lakshman; Brennan, Tracy A.; Russell, Elizabeth; Kim, Jung-Hoon; Chen, Qijun; Johnson, F. Brad; Pignolo, Robert J.

2016-01-01

Bone marrow derived mesenchymal progenitor cells (MPCs) play an important role in bone homeostasis. Age-related changes occur in bone resulting in a decrease in bone density and a relative increase in adipocity. Although in vitro studies suggest the existence of an age-related lineage switch between osteogenic and adipogenic fates, stem cell and microenvironmental contributions to this process have not been elucidated in vivo. In order to study the effects of MPC and microenvironmental aging on functional engraftment and lineage switching, transplantation studies were performed under non-myeloablative conditions in old recipients, with donor MPCs derived from young and old green fluorescent protein (GFP) transgenic mice. Robust engraftment by young MPCs or their progeny was observed in the marrow, bone-lining region and in the matrix of young recipients; however, significantly lower engraftment was seen at the same sites in old recipients transplanted with old MPCs. Differentiation of transplanted MPCs strongly favored adipogenesis over osteogenesis in old recipients irrespective of MPC donor age, suggesting that microenvironmental alterations that occur with in vivo aging are predominately responsible for MPC lineage switching. These data indicate that aging alters bone-fat reciprocity and differentiation of mesenchymal progenitors toward an adipogenic fate. PMID:26805026

Aging alters bone-fat reciprocity by shifting in vivo mesenchymal precursor cell fate towards an adipogenic lineage.

PubMed

Singh, Lakshman; Brennan, Tracy A; Russell, Elizabeth; Kim, Jung-Hoon; Chen, Qijun; Brad Johnson, F; Pignolo, Robert J

2016-04-01

Bone marrow derived mesenchymal progenitor cells (MPCs) play an important role in bone homeostasis. Age-related changes occur in bone resulting in a decrease in bone density and a relative increase in adipocity. Although in vitro studies suggest the existence of an age-related lineage switch between osteogenic and adipogenic fates, stem cell and microenvironmental contributions to this process have not been elucidated in vivo. In order to study the effects of MPC and microenvironmental aging on functional engraftment and lineage switching, transplantation studies were performed under non-myeloablative conditions in old recipients, with donor MPCs derived from young and old green fluorescent protein (GFP) transgenic mice. Robust engraftment by young MPCs or their progeny was observed in the marrow, bone-lining region and in the matrix of young recipients; however, significantly lower engraftment was seen at the same sites in old recipients transplanted with old MPCs. Differentiation of transplanted MPCs strongly favored adipogenesis over osteogenesis in old recipients irrespective of MPC donor age, suggesting that microenvironmental alterations that occur with in vivo aging are predominately responsible for MPC lineage switching. These data indicate that aging alters bone-fat reciprocity and differentiation of mesenchymal progenitors towards an adipogenic fate. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of bone-cartilage-stromal progenitor populations in trauma induced and genetic models of heterotopic ossification

PubMed Central

Agarwal, Shailesh; Loder, Shawn; Li, Shuli; Shrestha, Swati; Li, Jon; Zhao, Bin; Mishina, Yuji; James, Aaron; Levi, Benjamin

2016-01-01

Heterotopic ossification (HO), the formation of extra-skeletal bone in soft tissues, is a pathologic process occurring after substantial burns or trauma, or in patients with type I bone morphogenetic protein (BMP) receptor hyperactivating mutations. Identifying the cells responsible for de novo bone formation during adulthood is of critical importance for therapeutic and regenerative purposes. Using a model of trauma-induced HO with hindlimb Achilles’ tenotomy and dorsal burn injury and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt), we demonstrate enrichment of previously defined bone-cartilage-stromal progenitor cells (BCSP: AlphaV+/CD105+/Tie2-/CD45-/Thy1-/6C3-) at the site of HO formation when compared with marrow isolated from the ipsilateral hindlimb, or from tissue of the contralateral, uninjured hindlimb. Upon transplantation into tenotomy sites soon after injury, BCSPs isolated from neonatal mice or developing HO incorporate into the developing lesion in cartilage and bone and express chondrogenic and osteogenic transcription factors. Additionally, BCSPs isolated from developing HO similarly incorporate into new HO lesions upon transplantation. Finally, adventitial cells, but not pericytes, appear to play a supportive role in HO formation. Our findings indicate that BCSPs contribute to de novo bone formation during adulthood and may hold substantial regenerative potential. PMID:27068890

Role of TGF-β in breast cancer bone metastases

PubMed Central

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

Osteogenic Response to BMP-2 of hMSCs Grown on Apatite-Coated Scaffolds

PubMed Central

Davis, Hillary E.; Case, Erin M.; Miller, Stephanie L.; Genetos, Damian C.; Leach, J. Kent

2011-01-01

Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to nonmineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration. PMID:21656707

Fluid flow in the osteocyte mechanical environment: a fluid-structure interaction approach.

PubMed

Verbruggen, Stefaan W; Vaughan, Ted J; McNamara, Laoise M

2014-01-01

Osteocytes are believed to be the primary sensor of mechanical stimuli in bone, which orchestrate osteoblasts and osteoclasts to adapt bone structure and composition to meet physiological loading demands. Experimental studies to quantify the mechanical environment surrounding bone cells are challenging, and as such, computational and theoretical approaches have modelled either the solid or fluid environment of osteocytes to predict how these cells are stimulated in vivo. Osteocytes are an elastic cellular structure that deforms in response to the external fluid flow imposed by mechanical loading. This represents a most challenging multi-physics problem in which fluid and solid domains interact, and as such, no previous study has accounted for this complex behaviour. The objective of this study is to employ fluid-structure interaction (FSI) modelling to investigate the complex mechanical environment of osteocytes in vivo. Fluorescent staining of osteocytes was performed in order to visualise their native environment and develop geometrically accurate models of the osteocyte in vivo. By simulating loading levels representative of vigorous physiological activity ([Formula: see text] compression and 300 Pa pressure gradient), we predict average interstitial fluid velocities [Formula: see text] and average maximum shear stresses [Formula: see text] surrounding osteocytes in vivo. Interestingly, these values occur in the canaliculi around the osteocyte cell processes and are within the range of stimuli known to stimulate osteogenic responses by osteoblastic cells in vitro. Significantly our results suggest that the greatest mechanical stimulation of the osteocyte occurs in the cell processes, which, cell culture studies have indicated, is the most mechanosensitive area of the cell. These are the first computational FSI models to simulate the complex multi-physics mechanical environment of osteocyte in vivo and provide a deeper understanding of bone mechanobiology.

Anti-Idiotype Probes for Toxin Detection.

DTIC Science & Technology

1994-11-08

Immunol. 133:187-195. 2. Fleming, S.D., L.S. Edelman and S.K. Chapes. 1991. Effects of corticosterone and microgravity on inflammatory cell ...Leuk. Biol. 44:551-558. Stevenson, J., J. Kreiling, and R. Taylor. 1989. Effects of corticosterone on responses of murine splenic B and T cells to...cytometric analysis of bone marrow cell ysis indicates that corticosterone is not responsible for subpopulations the differential effects of antiorthostatic

Effects of the activin A-myostatin-follistatin system on aging bone and muscle progenitor cells

PubMed Central

Bowser, Matthew; Herberg, Samuel; Arounleut, Phonepasong; Shi, Xingming; Fulzele, Sadanand; Hill, William D.; Isales, Carlos M.; Hamrick, Mark W.

2013-01-01

The activin A-myostatin-follistatin system is thought to play an important role in the regulation of muscle and bone mass throughout growth, development, and aging; however, the effects of these ligands on progenitor cell proliferation and differentiation in muscle and bone are not well understood. In addition, age-associated changes in the relative expression of these factors in musculoskeletal tissues have not been described. We therefore examined changes in protein levels of activin A, follistatin, and myostatin (GDF-8) in both muscle and bone with age in C57BL6 mice using ELISA. We then investigated the effects of activin A, myostatin and follistatin on the proliferation and differentiation of primary myoblasts and mouse bone marrow stromal cells (BMSCs) in vitro. Myostatin levels and the myostatin:follistatin ratio increased with age in the primarily slow-twitch mouse soleus muscle, whereas the pattern was reversed with age in the fast-twitch extensor digitorum longus muscle. Myostatin levels and the myostatin: follistatin ratio increased significantly (+75%) in mouse bone marrow with age, as did activin A levels (+17%). Follistatin increased the proliferation of primary myoblasts from both young and aged mice, whereas myostatin increased proliferation of younger myoblasts but decreased proliferation of older myoblasts. Myostatin reduced proliferation of both young and aged BMSCs in a dose-dependent fashion, and activin A increased mineralization in both young and aged BMSCs. Together these data suggest that aging in mice is accompanied by changes in the expression of activin A and myostatin, as well as changes in the response of bone and muscle progenitor cells to these factors. Myostatin appears to play a particularly important role in the impaired proliferative capacity of muscle and bone progenitor cells from aged mice. PMID:23178301

Recruitment of bone marrow CD11b+Gr-1+ cells by polymeric nanoparticles for antigen cross-presentation

NASA Astrophysics Data System (ADS)

Yang, Ya-Wun; Luo, Wen-Hui

2017-03-01

The objective of this study was to investigate the function of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on the activation of antigen-specific CD8+ T cell responses via the CD11b+Gr-1+ myeloid subpopulations in murine bone marrow (BM). PLGA NPs containing ovalbumin (OVA) were fabricated by the double-emulsion method. The CD11b+Gr-1lowLy-6Chigh and CD11b+Gr-1highLy-6Clow subsets from mice bone marrow were sorted and treated with the PLGA/OVA NPs, followed by co-culture with the carboxyfluorescein succinimidyl ester (CFSE)-labelled OT-I CD8+ cells. Co-culture of OT-I CD8+ T cells with PLGA/OVA NPs-primed CD11b+Gr-1+ subsets upregulated the expression of IL-2, TNF-α, INF-γ, granzyme B, and perforin, resulting in proliferation of CD8+ T cells and differentiation into effector cytotoxic T lymphocytes (CTLs). In vivo proliferation of CFSE-labelled OT-I CD8+ cells in response to OVA was also obtained in the animals immunized with PLGA/OVA NPs. The results presented in this study demonstrate the ability of polymeric NPs to recruit two CD11b+Gr-1+ myeloid subsets for effective presentation of exogenous antigen to OT-I CD8+ T cells in the context of major histocompatibility complex (MHC) class I, leading to an induction of antigen-specific cell proliferation and differentiation into effector cells.

Fast epi-detected broadband multiplex CARS and SHG imaging of mouse skull cells

PubMed Central

Capitaine, Erwan; Moussa, Nawel Ould; Louot, Christophe; Bardet, Sylvia M.; Kano, Hideaki; Duponchel, Ludovic; Lévêque, Philippe; Couderc, Vincent; Leproux, Philippe

2017-01-01

We present a bimodal imaging system able to obtain epi-detected mutiplex coherent anti-Stokes Raman scattering (M-CARS) and second harmonic generation (SHG) signals coming from biological samples. We studied a fragment of mouse parietal bone and could detect broadband anti-Stokes and SHG responses originating from bone cells and collagen respectively. In addition we compared two post-processing methods to retrieve the imaginary part of the third-order nonlinear susceptibility related to the spontaneous Raman scattering. PMID:29359100

Interleukin-2 therapy reverses some immunosuppressive effects of skeletal unloading

NASA Technical Reports Server (NTRS)

Armstrong, Jason W.; Balch, Signe; Chapes, Stephen K.

1994-01-01

Using antiorthostatic suspension, we characterized hematopoietic changes that may be responsible for the detrimental effect of skeletal unloading on macrophage development. Skeletally unloaded mice had suppressed macrophage development in unloaded and loaded bones, which indicated a systemic effect. Bone marrow cells from unloaded mice secreted less macrophage colony-stimulating factor and interleukin-6 than control mice. Additionally, T-lymphocyte proliferation was reduced after skeletal unloading. We show that polyethylene glycol-interleukin-2 therapy reversed the effects of skeletal unloading on macrophage development and cell proliferation.

Mesenchymal stem cells: Emerging mechanisms of immunomodulation and therapy

PubMed Central

Glenn, Justin D; Whartenby, Katharine A

2014-01-01

Mesenchymal stem cells (MSCs) are a pleiotropic population of cells that are self-renewing and capable of differentiating into canonical cells of the mesenchyme, including adipocytes, chondrocytes, and osteocytes. They employ multi-faceted approaches to maintain bone marrow niche homeostasis and promote wound healing during injury. Biomedical research has long sought to exploit their pleiotropic properties as a basis for cell therapy for a variety of diseases and to facilitate hematopoietic stem cell establishment and stromal reconstruction in bone marrow transplantation. Early results demonstrated their usage as safe, and there was little host response to these cells. The discovery of their immunosuppressive functions ushered in a new interest in MSCs as a promising therapeutic tool to suppress inflammation and down-regulate pathogenic immune responses in graft-versus-host and autoimmune diseases such as multiple sclerosis, autoimmune diabetes, and rheumatoid arthritis. MSCs produce a large number of soluble and membrane-bound factors, some of which inhibit immune responses. However, the full range of MSC-mediated immune-modulation remains incompletely understood, as emerging reports also reveal that MSCs can adopt an immunogenic phenotype, stimulate immune cells, and yield seemingly contradictory results in experimental animal models of inflammatory disease. The present review describes the large body of literature that has been accumulated on the fascinating biology of MSCs and their complex effects on immune responses. PMID:25426250

Role of CXCR4-mediated bone marrow colonization in CNS infiltration by T cell acute lymphoblastic leukemia.

PubMed

Jost, Tanja Rezzonico; Borga, Chiara; Radaelli, Enrico; Romagnani, Andrea; Perruzza, Lisa; Omodho, Lorna; Cazzaniga, Giovanni; Biondi, Andrea; Indraccolo, Stefano; Thelen, Marcus; Te Kronnie, Geertruy; Grassi, Fabio

2016-06-01

Infiltration of the central nervous system is a severe trait of T cell acute lymphoblastic leukemia. Inhibition of CXC chemokine receptor 4 significantly ameliorates T cell acute lymphoblastic leukemia in murine models of the disease; however, signaling by CXC chemokine receptor 4 is important in limiting the divagation of peripheral blood mononuclear cells out of the perivascular space into the central nervous system parenchyma. Therefore, Inhibition of CXC chemokine receptor 4 potentially may untangle T cell acute lymphoblastic leukemia cells from retention outside the brain. Here, we show that leukemic lymphoblasts massively infiltrate cranial bone marrow, with diffusion to the meninges without invasion of the brain parenchyma, in mice that underwent xenotransplantation with human T cell acute lymphoblastic leukemia cells or that developed leukemia from transformed hematopoietic progenitors. We tested the hypothesis that T cell acute lymphoblastic leukemia neuropathology results from meningeal infiltration through CXC chemokine receptor 4-mediated bone marrow colonization. Inhibition of leukemia engraftment in the bone marrow by pharmacologic CXC chemokine receptor 4 antagonism significantly ameliorated neuropathologic aspects of the disease. Genetic deletion of CXCR4 in murine hematopoietic progenitors abrogated leukemogenesis induced by constitutively active Notch1, whereas lack of CCR6 and CCR7, which have been shown to be involved in T cell and leukemia extravasation into the central nervous system, respectively, did not influence T cell acute lymphoblastic leukemia development. We hypothesize that lymphoblastic meningeal infiltration as a result of bone marrow colonization is responsible for the degenerative alterations of the neuroparenchyma as well as the alteration of cerebrospinal fluid drainage in T cell acute lymphoblastic leukemia xenografts. Therefore, CXC chemokine receptor 4 may constitute a pharmacologic target for T cell acute lymphoblastic leukemia neuropathology. © Society for Leukocyte Biology.

Adhesive interactions of human multiple myeloma cell lines with different extracellular matrix molecules.

PubMed

Kibler, C; Schermutzki, F; Waller, H D; Timpl, R; Müller, C A; Klein, G

1998-06-01

Multiple myeloma represents a human B cell malignancy which is characterized by a predominant localization of the malignant cell clone within the bone marrow. With the exception of the terminal stage of the disease the myeloma tumor cells do not circulate in the peripheral blood. The bone marrow microenvironment is believed to play an important role in homing, proliferation and terminal differentiation of myeloma cells. Here we have studied the expression of several extracellular matrix (ECM) molecules in the bone marrow of multiple myeloma patients and analyzed their adhesive capacities with four different human myeloma-derived cell lines. All ECM molecules analyzed (tenascin, laminin, fibronectin, collagen types I, III, V and VI) could be detected in bone marrow cryostat sections of multiple myeloma patients. Adhesion assays showed that only laminin, the microfibrillar collagen type VI and fibronectin were strong adhesive components for the myeloma cell lines U266, IM-9, OPM-2 and NCI-H929. Tenascin and collagen type I were only weak adhesive substrates for these myeloma cells. Adhesion to laminin and fibronectin was beta 1-integrin-mediated since addition of anti-beta 1-integrin antibodies could inhibit the binding of the four different cell types to both matrix molecules. In contrast, integrins do not seem to be involved in binding of the myeloma cells to collagen type VI. Instead, inhibition of binding by heparin suggested that membrane-bound heparan sulfate proteoglycans are responsible ligands for binding to collagen type VI. Adhesion assays with several B-cell lines resembling earlier differentiation stages revealed only weak interactions with tenascin and no interactions with collagen type VI, laminin or fibronectin. In summary, the interactions of human myeloma cells with the extracellular matrix may explain the specific retention of the plasma cells within the bone marrow.

In vitro and in vivo evidence for orphan nuclear receptor RORα function in bone metabolism

PubMed Central

Meyer, Thomas; Kneissel, Michaela; Mariani, Jean; Fournier, Brigitte

2000-01-01

Bone is a major target site for steroid hormone action. Steroid hormones like cortisol, vitamin D, and estradiol are responsible for principal events associated with bone formation and resorption. Over the past decade, new members of the nuclear hormone gene family have been identified that lack known ligands. These orphan receptors can be used to uncover signaling molecules that regulate yet unidentified physiological networks. In the present study the function of retinoic acid receptor-related orphan receptor (ROR) α in bone metabolism has been examined. We showed that RORα and RORγ, but not RORβ, are expressed in mesenchymal stem cells derived from bone marrow. Interestingly, for RORα we observed an increased messenger signal expression between control cells and cells undergoing osteogenic differentiation. Furthermore, the direct activation of mouse bone sialoprotein by RORα, typically 7-fold, has been shown. In contrast, transient overexpression of RORα overrides the activation of the osteocalcin promoter by 1α,25-dihydroxyvitamin D3. In addition, we have investigated bone mass parameters and bone geometry in the mouse mutant staggerer (sg/sg), a mouse strain that carries a deletion within the RORα gene. Homozygote mutants have thin long bones compared with the heterozygote animals and wild-type littermates. More interestingly, the bones of the sg/sg animals are osteopenic as indicated by the comparison of bone mineral contents of sg/sg animals to the heterozygote and wild-type animals. We conclude that these in vitro and in vivo results suggest a function for RORα in bone biology. RORα most likely acts by direct modulation of a bone matrix component. PMID:10900268

The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis

PubMed Central

Xia, Yunfeng; Yan, Jingyin; Jin, Xiaogao; Entman, Mark L.; Wang, Yanlin

2014-01-01

Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. Since chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly fewer bone marrow-derived fibroblasts accumulated in the kidney of CXCR6 knockout mice in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type mice. CXCR6 deficiency inhibited total collagen deposition and suppressed expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, wild type mice engrafted with CXCR6−/− bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with wild-type mice engrafted with CXCR6+/+ bone marrow cells. Transplant of wild type bone marrow into CXCR6−/− recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may play important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis. PMID:24646857

The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis.

PubMed

Xia, Yunfeng; Yan, Jingyin; Jin, Xiaogao; Entman, Mark L; Wang, Yanlin

2014-08-01

Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, and they proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. As chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly, the kidney of CXCR6 knockout mice accumulated fewer bone marrow-derived fibroblasts in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type (WT) mice. CXCR6 deficiency inhibited total collagen deposition and suppressed the expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, WT mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with WT mice engrafted with CXCR6(+/+) bone marrow cells. Transplant of WT bone marrow into CXCR6(-/-) recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may have important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis.

The effect of a slow mode of BMP-2 delivery on the inflammatory response provoked by bone-defect-filling polymeric scaffolds.

PubMed

Wu, Gang; Liu, Yuelian; Iizuka, Tateyuki; Hunziker, Ernst Bruno

2010-10-01

We investigated the inflammatory response to, and the osteoinductive efficacies of, four polymers (collagen, Ethisorb, PLGA and Polyactive) that bore either an adsorbed (fast-release kinetics) or a calcium-phosphate-coating-incorporated (slow-release kinetics) depot of BMP-2. Titanium-plate-supported discs of each polymer (n = 6 per group) were implanted at an ectopic (subcutaneous) ossification site in rats (n = 48). Five weeks later, they were retrieved for a histomorphometric analysis of the volumes of ectopic bone and foreign-body giant cells (a gauge of inflammatory reactivity), and the degree of polymer degradation. For each polymer, the osteoinductive efficacy of BMP-2 was higher when it was incorporated into a coating than when it was directly adsorbed onto the material. This mode of BMP-2 carriage was consistently associated with an attenuation of the inflammatory response. For coated materials, the volume density of foreign-body giant cells was inversely correlated with the volume density of bone (r(2) = 0.96), and the volume density of bone was directly proportional to the surface-area density of the polymer (r(2) = 0.97). Following coating degradation, other competitive factors, such as the biocompatibility and the biodegradability of the polymer itself, came into play. 2010 Elsevier Ltd. All rights reserved.

Modulation by vitamin D status of the responsiveness of rat bone to gonadal steroids.

PubMed

Sömjen, D; Kaye, A M; Harell, A; Weisman, Y

1989-10-01

We have previously demonstrated that gonadal steroids stimulate [3H]thymidine incorporation and creatine kinase specific activity in skeletal tissues. In the present study we report that in 20-day-old vitamin D-deficient Wistar-derived rats, 17 beta-estradiol (E2; 5 micrograms/rat) or testosterone (50 micrograms/rat) failed to stimulate [3H]thymidine incorporation into diaphyses of long bones and that the response to these hormones in terms of increased creatine kinase specific activity was less than half the value in normally fed rats. Two daily ip injections of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3; 0.5 ng/g BW], but not 24,25-(OH)2D3 (5 ng/g BW), partially restored the biological responses to E2 in bone of 21-day-old vitamin D-deficient female rats. Vitamin D deficiency did not impair the responsiveness to gonadal steroids in the epiphysis of long bones, uterus, or prostate, in contrast to its effect on diaphysis. In 21-day-old normally fed female rats, neither vitamin D metabolite enhanced the response to E2. When cultures of rat epiphyseal cells were treated daily for 5 days with either 1,25-(OH)2D3 (1 nM) or 24,25-(OH)2D3 (10 nM), followed by E2 (30 nM) for 24 h, creatine kinase activity was significantly higher than in cultures treated daily for 5 days with vehicle alone, and then with E2. The same treatment of rat embryo calvaria bone cells showed that 1,25-(OH)2D3, but not 24,25-(OH)2D3, significantly increased the creatine kinase activity response to E2. These findings suggest that vitamin D metabolites selectively affect the biological responses of skeletal tissues to gonadal steroids.

Mesenchymal Stem Cells From Bone Marrow, Adipose Tissue, and Lung Tissue Differentially Mitigate Lung and Distal Organ Damage in Experimental Acute Respiratory Distress Syndrome.

PubMed

Silva, Johnatas D; Lopes-Pacheco, Miquéias; Paz, Ana H R; Cruz, Fernanda F; Melo, Elga B; de Oliveira, Milena V; Xisto, Débora G; Capelozzi, Vera L; Morales, Marcelo M; Pelosi, Paolo; Cirne-Lima, Elizabeth; Rocco, Patricia R M

2018-02-01

Mesenchymal stem cells-based therapies have shown promising effects in experimental acute respiratory distress syndrome. Different mesenchymal stem cells sources may result in diverse effects in respiratory diseases; however, there is no information regarding the best source of mesenchymal stem cells to treat pulmonary acute respiratory distress syndrome. We tested the hypothesis that mesenchymal stem cells derived from bone marrow, adipose tissue, and lung tissue would lead to different beneficial effects on lung and distal organ damage in experimental pulmonary acute respiratory distress syndrome. Animal study and primary cell culture. Laboratory investigation. Seventy-five Wistar rats. Wistar rats received saline (control) or Escherichia coli lipopolysaccharide (acute respiratory distress syndrome) intratracheally. On day 2, acute respiratory distress syndrome animals were further randomized to receive saline or bone marrow, adipose tissue, or lung tissue mesenchymal stem cells (1 × 10 cells) IV. Lung mechanics, histology, and protein levels of inflammatory mediators and growth factors were analyzed 5 days after mesenchymal stem cells administration. RAW 264.7 cells (a macrophage cell line) were incubated with lipopolysaccharide followed by coculture or not with bone marrow, adipose tissue, and lung tissue mesenchymal stem cells (10 cells/mL medium). Regardless of mesenchymal stem cells source, cells administration improved lung function and reduced alveolar collapse, tissue cellularity, collagen, and elastic fiber content in lung tissue, as well as decreased apoptotic cell counts in liver. Bone marrow and adipose tissue mesenchymal stem cells administration also reduced levels of tumor necrosis factor-α, interleukin-1β, keratinocyte-derived chemokine, transforming growth factor-β, and vascular endothelial growth factor, as well as apoptotic cell counts in lung and kidney, while increasing expression of keratinocyte growth factor in lung tissue. Additionally, mesenchymal stem cells differently modulated the secretion of biomarkers by macrophages depending on their source. Mesenchymal stem cells from different sources led to variable responses in lungs and distal organs. Bone marrow and adipose tissue mesenchymal stem cells yielded greater beneficial effects than lung tissue mesenchymal stem cells. These findings may be regarded as promising in clinical trials.

Altered paracrine signaling from the injured knee joint impairs postnatal long bone growth

PubMed Central

Roselló-Díez, Alberto; Stephen, Daniel; Joyner, Alexandra L

2017-01-01

Regulation of organ growth is a poorly understood process. In the long bones, the growth plates (GPs) drive elongation by generating a scaffold progressively replaced by bone. Although studies have focused on intrinsic GP regulation, classic and recent experiments suggest that local signals also modulate GP function. We devised a genetic mouse model to study extrinsic long bone growth modulation, in which injury is specifically induced in the left hindlimb, such that the right hindlimb serves as an internal control. Remarkably, when only mesenchyme cells surrounding postnatal GPs were killed, left bone growth was nevertheless reduced. GP signaling was impaired by altered paracrine signals from the knee joint, including activation of the injury response and, in neonates, dampened IGF1 production. Importantly, only the combined prevention of both responses rescued neonatal growth. Thus, we identified signals from the knee joint that modulate bone growth and could underlie establishment of body proportions. DOI: http://dx.doi.org/10.7554/eLife.27210.001 PMID:28741471

Tissue-dependent differences in the asynchronous appearance of mast cells in normal mice and in congenic mast cell-deficient mice after infusion of normal bone marrow cells

PubMed Central

DU, T; FRIEND, D S; AUSTEN, K F; KATZ, H R

1996-01-01

The time courses of the appearance of tissue mast cells in six sites were compared in normal WBB6F1-+/+ mice (+/+) and in congenic mast cell-deficient WBB6F1-W/Wv mice (W/Wv) that received an intravenous infusion of bone marrow cells from +/+mice (BM→W/Wv). As assessed by morphometric analysis of Carnoy's solution-fixed, methylene blue-stained tissue sections, the density of mast cells in the stomach mucosa, stomach submucosa, and spleen of +/+ mice reached maximal levels by 8 weeks of age, whereas the density of mast cells in the skin, extraparenchymal airway walls, and lung parenchyma did not reach maximal levels until 18 weeks of age. When 8-week-old W/Wv mice were infused with 2×107 bone marrow cells from +/+ mice, mast cells appeared in the stomach mucosa and submucosa after 2.5 weeks, in the spleen and extraparenchymal airway walls after 5 weeks, and in the lung parenchyma after 10 weeks. Twenty weeks after bone marrow infusion, the mast cell densities in the spleen, stomach mucosa, and stomach submucosa were seven-, 13-, and five-fold greater, respectively, than those in age-matched +/+ mice, but were eight-, two-, and five-fold lower in the skin, extraparenchymal airway walls, and lung parenchyma, respectively. Thus, those tissues that in +/+ mice reached maximal mast cell densities earlier exhibited abnormally high mast cell densities in BM→W/Wv mice, and those that reached maximal mast cell densities later in +/+ mice had abnormally low mast cell densities in BM→W/Wv mice. Immunological and inflammatory responses are often compared in W/Wv and BM→W/Wv mice to assess mast cell dependency. Our results indicate that the capacity to restore a mast cell-dependent response in a particular tissue of the latter mice may relate to the local mast cell density and whether the immunological challenge activates mast cells only in that tissue or systematically with attendant widespread release of proinflammatory mediators. PMID:8565318

Population control of resident and immigrant microglia by mitosis and apoptosis.

PubMed

Wirenfeldt, Martin; Dissing-Olesen, Lasse; Anne Babcock, Alicia; Nielsen, Marianne; Meldgaard, Michael; Zimmer, Jens; Azcoitia, Iñigo; Leslie, Robert Graham Quinton; Dagnaes-Hansen, Frederik; Finsen, Bente

2007-08-01

Microglial population expansion occurs in response to neural damage via processes that involve mitosis and immigration of bone marrow-derived cells. However, little is known of the mechanisms that regulate clearance of reactive microglia, when microgliosis diminishes days to weeks later. We have investigated the mechanisms of microglial population control in a well-defined model of reactive microgliosis in the mouse dentate gyrus after perforant pathway axonal lesion. Unbiased stereological methods and flow cytometry demonstrate significant lesion-induced increases in microglial numbers. Reactive microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced in bone marrow chimeric mice. These results broaden our understanding of the microglial response to neural damage by demonstrating that simultaneously occurring mitosis and apoptosis regulate expansion and reduction of both resident and immigrant microglial cell populations.

In Silico Investigations of the Anti-Catabolic Effects of Pamidronate and Denosumab on Multiple Myeloma-Induced Bone Disease

PubMed Central

Wang, Yan; Lin, Bo

2012-01-01

It is unclear whether the new anti-catabolic agent denosumab represents a viable alternative to the widely used anti-catabolic agent pamidronate in the treatment of Multiple Myeloma (MM)-induced bone disease. This lack of clarity primarily stems from the lack of sufficient clinical investigations, which are costly and time consuming. However, in silico investigations require less time and expense, suggesting that they may be a useful complement to traditional clinical investigations. In this paper, we aim to (i) develop integrated computational models that are suitable for investigating the effects of pamidronate and denosumab on MM-induced bone disease and (ii) evaluate the responses to pamidronate and denosumab treatments using these integrated models. To achieve these goals, pharmacokinetic models of pamidronate and denosumab are first developed and then calibrated and validated using different clinical datasets. Next, the integrated computational models are developed by incorporating the simulated transient concentrations of pamidronate and denosumab and simulations of their actions on the MM-bone compartment into the previously proposed MM-bone model. These integrated models are further calibrated and validated by different clinical datasets so that they are suitable to be applied to investigate the responses to the pamidronate and denosumab treatments. Finally, these responses are evaluated by quantifying the bone volume, bone turnover, and MM-cell density. This evaluation identifies four denosumab regimes that potentially produce an overall improved bone-related response compared with the recommended pamidronate regime. This in silico investigation supports the idea that denosumab represents an appropriate alternative to pamidronate in the treatment of MM-induced bone disease. PMID:23028650

GAS6/Mer axis regulates the homing and survival of the E2A/PBX1 positive B-cell precursor acute lymphoblastic leukemia in the bone marrow niche

PubMed Central

Shiozawa, Yusuke; Pedersen, Elisabeth A.; Taichman, Russell S.

2009-01-01

Despite improvements in current combinational chemotherapy regimens, the prognosis of the (1;19)(q23;p13) translocation (E2A/PBX1) positive B-cell precursor acute lymphoblastic leukemia (ALL) is poor in pediatric leukemia patients. In this study, we examined the roles of GAS6/Mer axis in the interactions between E2A/PBX1 positive B-cell precursor ALL cells and the osteoblastic niche in the bone marrow. The data show that primary human osteoblasts secrete GAS6 in response to the Mer-over-expressed E2A/PBX1 positive ALL cells through MAPK signaling pathway and that leukemia cells migrate toward GAS6 using pathways activated by Mer. Importantly, GAS6 supports the survival and prevents apoptosis from chemotherapy of E2A/PBX1 positive ALL cells by inducing dormancy. Together, these data suggest that GAS6/Mer axis regulates the homing and survival of the E2A/PBX1 positive B-cell precursor ALL in the bone marrow niche. PMID:19922767

Modulation of bone remodeling via mechanically activated ion channels

NASA Technical Reports Server (NTRS)

Duncan, Randall L. (Principal Investigator)

1996-01-01

A critical factor in the maintenance of bone mass is the physical forces imposed upon the skeleton. Removal of these forces, such as in a weightless environment, results in a rapid loss of bone, whereas application of exogenous mechanical strain has been shown to increase bone formation. Numerous flight and ground-based experiments indicate that the osteoblast is the key bone cell influenced by mechanical stimulation. Aside from early transient fluctuations in response to unloading, osteoclast number and activity seem unaffected by removal of strain. However, bone formation is drastically reduced in weightlessness and osteoblasts respond to mechanical strain with an increase in the activity of a number of second messenger pathways resulting in increased anabolic activity. Unfortunately, the mechanism by which the osteoblast converts physical stimuli into a biochemical message, a process we have termed biochemical coupling, remains elusive. Prior to the application of this grant, we had characterized a mechanosensitive, cation nonselective channel (SA-cat) in osteoblast-like osteosarcoma cells that we proposed is the initial signalling mechanism for mechanotransduction. During the execution of this grant, we have made considerable progress to further characterize this channel as well as to determine its role in the osteoblastic response to mechanical strain. To achieve these goals, we combined electrophysiologic techniques with cellular and molecular biology methods to examine the role of these channels in the normal function of the osteoblast in vitro.

Genomic variation in the MMP-1 promoter influences estrogen receptor mediated activity in a mechanically activated environment: potential implications for microgravity risk assessment

NASA Astrophysics Data System (ADS)

Thaler, John; Myers, Ken; Lu, Ting; Hart, David

Background: Mechanotransduction, the conversion of mechanical forces (tensile, compression, shear etc.) into cellular signals is a significant response mechanism in bone that contributes to the balance between formation and resorption and helps maintain bone density. In microgravity the lack of mechanical signals can lead to a loss of bone density, however the signaling pathways responsible are not well understood. For women, sex-specific hormones are also important in maintaining bone density since estrogen deficiency is a major factor in the etiology of osteoporosis in postmenopausal women. Estrogen Receptors (ERs) are present in human connective tissue cells such as osteoblasts and may play a role in mechanotransduction responses. The two ER isoforms, alpha (ER-α) and beta (ER-β) differentially regulate expression of matrixmetalloproteinases (MMPs) which degrade extracellular matrix components found in connective tissues. Mechanical stimulation is known to affect the expression of MMP-1, a collagenase involved in the bone resorption process. The MMP-1 promoter region contains a single nucleotide polymorphism of an additional guanine (G) at position -1607 bp which creates a binding site for a member of the Ets family of mammalian transcription factors. The 2G allele is known to be present in 45-70% of healthy populations and has been associated with higher MMP-1 expression. The 2G allele has been linked to higher risk of several types of cancer but a link to osteoporosis or microgravity induced bone loss has not been explored. The purpose of the present study was to conduct a case-study to determine whether small genetic variations can influence cellular and tissue responses to mechanical loading. Specifically we examined the potential of the 1G/2G -1607 MMP-1 promoter SNP to alter the interplay between mechanical shear stress and estrogen receptors in controlling MMP-1 expression. Methods: Rabbit synovial cells (HIG-82) were used as an in vitro model system to examine the potential impact of the 1G/2G SNP on the cellular response to mechanical loading. HIG-82 cells are estrogen receptor (ER) negative and were transiently transfected with SV40 expression vectors for either ER-α or ER-β isoforms. Cells grown on glass slides were also co-transfected with either a 1G or 2G MMP-1 promoter-luciferase construct. Transfected cells were subjected to dynamic shear stress in a Flexcell Streamer Shear Stress Device. The dynamic loading regime was 0.5 Hz, 10 dyn/cm2 shear for 1 minute followed by 14 minutes rest and repeated for 8 hrs. A Promega Dual Luciferase Reporter Assay System was used to assess MMP-1 promoter activity. Results: Shear stress loading increased both 1G and 2G MMP-1 promoter activity compared to unloaded controls, however the 2G promoter had significantly higher rates of expression than the 1G promoter across all loading regimes and ER co-transfections. Transfection with ER-β resulted in higher MMP-1 promoter activity than that in cells expressing ER-α or in ER-neg cells. Conclusions: Specific genomic variations can lead to differences in cellular responses to changes in mechanical loading environments such as are encountered in microgravity environments or earth-based analogs. These genomic differences may predispose individuals to greater risk of bone loss. It is important to understand the combined effects of mechanical loading, genetic variation and sex hormones on bone maintenance so that risks can be identified for microgravity or analog environments, and specific interventions developed to counteract such risk or even exclude some individuals from prolonged space environments due to the extent of the risk.

Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice.

PubMed

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.

Bone mechanobiology, gravity and tissue engineering: effects and insights.

PubMed

Ruggiu, Alessandra; Cancedda, Ranieri

2015-12-01

Bone homeostasis strongly depends on fine tuned mechanosensitive regulation signals from environmental forces into biochemical responses. Similar to the ageing process, during spaceflights an altered mechanotransduction occurs as a result of the effects of bone unloading, eventually leading to loss of functional tissue. Although spaceflights represent the best environment to investigate near-zero gravity effects, there are major limitations for setting up experimental analysis. A more feasible approach to analyse the effects of reduced mechanostimulation on the bone is represented by the 'simulated microgravity' experiments based on: (1) in vitro studies, involving cell cultures studies and the use of bioreactors with tissue engineering approaches; (2) in vivo studies, based on animal models; and (3) direct analysis on human beings, as in the case of the bed rest tests. At present, advanced tissue engineering methods allow investigators to recreate bone microenvironment in vitro for mechanobiology studies. This group and others have generated tissue 'organoids' to mimic in vitro the in vivo bone environment and to study the alteration cells can go through when subjected to unloading. Understanding the molecular mechanisms underlying the bone tissue response to mechanostimuli will help developing new strategies to prevent loss of tissue caused by altered mechanotransduction, as well as identifying new approaches for the treatment of diseases via drug testing. This review focuses on the effects of reduced gravity on bone mechanobiology by providing the up-to-date and state of the art on the available data by drawing a parallel with the suitable tissue engineering systems. Copyright © 2014 John Wiley & Sons, Ltd.

The anti-tumour effects of zoledronic acid

PubMed Central

Zekri, Jamal; Mansour, Maged; Karim, Syed Mustafa

2014-01-01

Bone is the most common site for metastasis in patients with solid tumours. Bisphosphonates are an effective treatment for preventing skeletal related events and preserving quality of life in these patients. Zoledronic acid (ZA) is the most potent osteoclast inhibitor and is licensed for the treatment of bone metastases. Clodronate and pamidronate are also licensed for this indication. In addition, ZA has been demonstrated to exhibit antitumour effect. Direct and indirect mechanisms of anti-tumour effect have been postulated and at many times proven. Evidence exists that ZA antitumour effect is mediated through inhibition of tumour cells proliferation, induction of apoptosis, synergistic/additive to inhibitory effect of cytotoxic agents, inhibition of angiogenesis, decrease tumour cells adhesion to bone, decrease tumour cells invasion and migration, disorganization of cell cytoskeleton and activation of specific cellular antitumour immune response. There is also clinical evidence from clinical trials that ZA improved long term survival outcome in cancer patients with and without bone metastases. In this review we highlight the preclinical and clinical studies investigating the antitumour effect of bisphosphonates with particular reference to ZA. PMID:26909294

Prolonged pancytopenia in a gene therapy patient with ADA-deficient SCID and trisomy 8 mosaicism: a case report.

PubMed

Engel, Barbara C; Podsakoff, Greg M; Ireland, Joanna L; Smogorzewska, E Monika; Carbonaro, Denise A; Wilson, Kathy; Shah, Ami; Kapoor, Neena; Sweeney, Mirna; Borchert, Mark; Crooks, Gay M; Weinberg, Kenneth I; Parkman, Robertson; Rosenblatt, Howard M; Wu, Shi-Qi; Hershfield, Michael S; Candotti, Fabio; Kohn, Donald B

2007-01-15

A patient with adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) was enrolled in a study of retroviral-mediated ADA gene transfer to bone marrow hematopoietic stem cells. After the discontinuation of ADA enzyme replacement, busulfan (75 mg/m2) was administered for bone marrow cytoreduction, followed by infusion of autologous, gene-modified CD34+ cells. The expected myelosuppression developed after busulfan but then persisted, necessitating the administration of untransduced autologous bone marrow back-up at day 40. Because of sustained pancytopenia and negligible gene marking, diagnostic bone marrow biopsy and aspirate were performed at day 88. Analyses revealed hypocellular marrow and, unexpectedly, evidence of trisomy 8 in 21.6% of cells. Trisomy 8 mosaicism (T8M) was subsequently diagnosed by retrospective analysis of a pretreatment marrow sample that might have caused the lack of hematopoietic reconstitution. The confounding effects of this preexisting marrow cytogenetic abnormality on the response to gene transfer highlights another challenge of gene therapy with the use of autologous hematopoietic stem cells.

Prolonged pancytopenia in a gene therapy patient with ADA-deficient SCID and trisomy 8 mosaicism: a case report

PubMed Central

Engel, Barbara C.; Podsakoff, Greg M.; Ireland, Joanna L.; Smogorzewska, E. Monika; Carbonaro, Denise A.; Wilson, Kathy; Shah, Ami; Kapoor, Neena; Sweeney, Mirna; Borchert, Mark; Crooks, Gay M.; Weinberg, Kenneth I.; Parkman, Robertson; Rosenblatt, Howard M.; Wu, Shi-Qi; Hershfield, Michael S.; Candotti, Fabio; Kohn, Donald B.

2007-01-01

A patient with adenosine deaminase–deficient severe combined immune deficiency (ADA-SCID) was enrolled in a study of retroviral-mediated ADA gene transfer to bone marrow hematopoietic stem cells. After the discontinuation of ADA enzyme replacement, busulfan (75 mg/m2) was administered for bone marrow cytoreduction, followed by infusion of autologous, gene-modified CD34+ cells. The expected myelosuppression developed after busulfan but then persisted, necessitating the administration of untransduced autologous bone marrow back-up at day 40. Because of sustained pancytopenia and negligible gene marking, diagnostic bone marrow biopsy and aspirate were performed at day 88. Analyses revealed hypocellular marrow and, unexpectedly, evidence of trisomy 8 in 21.6% of cells. Trisomy 8 mosaicism (T8M) was subsequently diagnosed by retrospective analysis of a pretreatment marrow sample that might have caused the lack of hematopoietic reconstitution. The confounding effects of this preexisting marrow cytogenetic abnormality on the response to gene transfer highlights another challenge of gene therapy with the use of autologous hematopoietic stem cells. PMID:16973956

Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344----Lewis and in Lewis----F344 bone marrow chimeras

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

van Bruggen, M.C.; van den Broek, M.F.; van den Berg, W.B.

1991-09-01

Streptococcal cell wall (SCW)-induced arthritis and adjuvant arthritis (AA) are rat models for chronic, erosive polyarthritis. Both models can be induced in susceptible Lewis rats, whereas F344 rats are resistant. In AA as well as in SCW arthritis, antigen-specific T lymphocytes have been demonstrated to be crucial for chronic disease. In this communication the authors describe their studies to probe the cellular mechanism responsible for the difference in susceptibility of Lewis and F344, using bone marrow chimeras. By transplanting bone marrow cells from F344 into lethally irradiated Lewis recipients, Lewis rats were rendered resistant to SCW arthritis induction. F344 ratsmore » reconstituted with Lewis bone marrow, i.e., Lewis----F344 chimeras, develop an arthritis upon SCW injection. For AA comparable results were obtained. These data suggest that both resistance and susceptibility to bacterium-induced chronic arthritis are mediated by hemopoietic/immune cells and that the recipiental environment does not influence the susceptibility to chronic joint inflammation.« less

Polymer-Ceramic Spiral Structured Scaffolds for Bone Tissue Engineering: Effect of Hydroxyapatite Composition on Human Fetal Osteoblasts

PubMed Central

Zhang, Xiaojun; Chang, Wei; Lee, Paul; Wang, Yuhao; Yang, Min; Li, Jun; Kumbar, Sangamesh G.; Yu, Xiaojun

2014-01-01

For successful bone tissue engineering, a scaffold needs to be osteoconductive, porous, and biodegradable, thus able to support attachment and proliferation of bone cells and guide bone formation. Recently, hydroxyapatites (HA), a major inorganic component of natural bone, and biodegrade polymers have drawn much attention as bone scaffolds. The present study was designed to investigate whether the bone regenerative properties of nano-HA/polycaprolactone (PCL) spiral scaffolds are augmented in an HA dose dependent manner, thereby establishing a suitable composition as a bone formation material. Nano-HA/PCL spiral scaffolds were prepared with different weight ratios of HA and PCL, while porosity was introduced by a modified salt leaching technique. Human fetal osteoblasts (hFOBs) were cultured on the nano-HA/PCL spiral scaffolds up to 14 days. Cellular responses in terms of cell adhesion, viability, proliferation, differentiation, and the expression of bone-related genes were investigated. These scaffolds supported hFOBs adhesion, viability and proliferation. Cell proliferation trend was quite similar on polymer-ceramic and neat polymer spiral scaffolds on days 1, 7, and 14. However, the significantly increased amount of alkaline phosphatase (ALP) activity and mineralized matrix synthesis was evident on the nano-HA/PCL spiral scaffolds. The HA composition in the scaffolds showed a significant effect on ALP and mineralization. Bone phenotypic markers such as bone sialoprotein (BSP), osteonectin (ON), osteocalcin (OC), and type I collagen (Col-1) were semi-quantitatively estimated by reverse transcriptase polymerase chain reaction analysis. All of these results suggested the osteoconductive characteristics of HA/PCL nanocomposite and cell maturation were HA dose dependent. For instance, HA∶PCL = 1∶4 group showed significantly higher ALP mineralization and elevated levels of BSP, ON, OC and Col-I expression as compared other lower or higher ceramic ratios. Amongst the different nano-HA/PCL spiral scaffolds, the 1∶4 weight ratio of HA and PCL is shown to be the most optimal composition for bone tissue regeneration. PMID:24475056

Osteoblasts are target cells for transformation in c-fos transgenic mice

PubMed Central

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

Mouse Bone Marrow VSELs Exhibit Differentiation into Three Embryonic Germ Lineages and Germ & Hematopoietic Cells in Culture.

PubMed

Shaikh, Ambreen; Anand, Sandhya; Kapoor, Sona; Ganguly, Ranita; Bhartiya, Deepa

2017-04-01

Very small embryonic-like stem cells (VSELs) have been reported in various adult tissues, express pluripotent and primordial germ cells (PGCs) specific markers, are mobilized under stress/disease conditions, give rise to tissue committed progenitors and thus help regenerate and maintain homeostasis. The aim of the present study was to evaluate in vitro differentiation potential of VSELs using a quantitative approach. VSELs were collected from mouse bone marrow after 4 days of 5-fluorouracil (5-FU, 150 mg/Kg) treatment, further enriched by size based filtration and cultured on a feeder support in the presence of specific differentiation media. Cultured VSELs were found to differentiate into all three embryonic germ cell lineages, germ and hematopoietic cells after 14 days in culture. This was confirmed by studying Nestin, PDX-1, NKX2.5, DAZL, CD45 and other markers expression by various approaches. Very small, CD45 negative cells collected and enriched from GFP positive 5-FU treated mice bone marrow transitioned into CD45 positive cells in vitro thus demonstrating that VSELs can give rise to hematopoietic stem cells (HSCs). We envision that VSELs may be responsible for plasticity and ability of bone marrow cells to give rise to non-hematopoietic tissue progenitors of all 3 germ layers. Moreover the ability of VSELs to differentiate into germ cells as well as all the three lineages provides further evidence to support their pluripotent state and confirms developmental link between bone marrow VSELs and PGCs. The property of quiescence, no risk of teratoma formation and autologus source, make pluripotent VSELs a potential candidate to facilitate endogenous regeneration compared to cell replacement strategy envisioned using embryonic and induced pluripotent stem cells.

p53-Based Strategy for Protection of Bone Marrow From Y-90 Ibritumomab Tiuxetan

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

Su, Hang, E-mail: suh3@uthscsa.edu; Ganapathy, Suthakar; Li, Xiaolei

Purpose: The main drawbacks of radioimmunotherapy have been severe hematological toxicity and potential development of myelodysplastic syndrome and secondary leukemia. Activation of p53 follows a major pathway by which normal tissues respond to DNA-damaging agents, such as chemotherapy and radiation therapy, that result in injuries and pathological consequences. This pathway is separate from the tumor suppressor pathway of p53. We have previously reported that use of low-dose arsenic (LDA) temporarily and reversibly suppresses p53 activation, thereby ameliorating normal tissue toxicity from exposure to 5-fluorouracil and X rays. We have also demonstrated that LDA-mediated protection requires functional p53 and thus ismore » selective to normal tissues, as essentially every cancer cell has dysfunctional p53. Here we tested the protective efficacy of LDA for bone marrow tissue against radioimmunotherapy through animal experiments. Methods and Materials: Mice were subjected to LDA pretreatment for 3 days, followed by treatment with Y-90 ibritumomab tiuxetan. Both dose course (10, 25, 50, 100, and 200 μCi) and time course (6, 24, and 72 hours and 1 and 2 weeks) experiments were performed. The response of bone marrow cells to LDA was determined by examining the expression of NFκB, Glut1, and Glut3. Staining with hematoxylin and eosin, γ-H2AX, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to examine morphology, DNA damage response, and apoptotic cell populations. Results: Elevated levels of NFκB, Glut1, and Glut3 were observed in bone marrow cells after LDA treatment. Bone marrow damage levels induced by Y-90 ibritumomab tiuxetan were greatly reduced by LDA pretreatment. Consistent with this observation, significantly less DNA damage and fewer apoptotic cells were accumulated after Y-90 ibritumomab tiuxetan treatment in LDA-pretreated mice. Furthermore, in the mouse xenograft model implanted with human Karpas-422 lymphoma cells, LDA pretreatment did not have any detectable effect on either tumor growth or Y-90 ibritumomab tiuxetan (200 μCi)-induced tumor suppression. Conclusions: LDA pretreatment protected bone marrow without compromising tumor control caused by Y-90 ibritumomab tiuxetan.« less

Flow velocity-driven differentiation of human mesenchymal stromal cells in silk fibroin scaffolds: A combined experimental and computational approach.

PubMed

Vetsch, Jolanda Rita; Betts, Duncan Colin; Müller, Ralph; Hofmann, Sandra

2017-01-01

Mechanical loading plays a major role in bone remodeling and fracture healing. Mimicking the concept of mechanical loading of bone has been widely studied in bone tissue engineering by perfusion cultures. Nevertheless, there is still debate regarding the in-vitro mechanical stimulation regime. This study aims at investigating the effect of two different flow rates (vlow = 0.001m/s and vhigh = 0.061m/s) on the growth of mineralized tissue produced by human mesenchymal stromal cells cultured on 3-D silk fibroin scaffolds. The flow rates applied were chosen to mimic the mechanical environment during early fracture healing or during bone remodeling, respectively. Scaffolds cultured under static conditions served as a control. Time-lapsed micro-computed tomography showed that mineralized extracellular matrix formation was completely inhibited at vlow compared to vhigh and the static group. Biochemical assays and histology confirmed these results and showed enhanced osteogenic differentiation at vhigh whereas the amount of DNA was increased at vlow. The biological response at vlow might correspond to the early stage of fracture healing, where cell proliferation and matrix production is prominent. Visual mapping of shear stresses, simulated by computational fluid dynamics, to 3-D micro-computed tomography data revealed that shear stresses up to 0.39mPa induced a higher DNA amount and shear stresses between 0.55mPa and 24mPa induced osteogenic differentiation. This study demonstrates the feasibility to drive cell behavior of human mesenchymal stromal cells by the flow velocity applied in agreement with mechanical loading mimicking early fracture healing (vlow) or bone remodeling (vhigh). These results can be used in the future to tightly control the behavior of human mesenchymal stromal cells towards proliferation or differentiation. Additionally, the combination of experiment and simulation presented is a strong tool to link biological responses to mechanical stimulation and can be applied to various in-vitro cultures to improve the understanding of the cause-effect relationship of mechanical loading.

Patient-specific 3D microfluidic tissue model for multiple myeloma.

PubMed

Zhang, Wenting; Lee, Woo Y; Siegel, David S; Tolias, Peter; Zilberberg, Jenny

2014-08-01

In vitro culturing of primary multiple myeloma cells (MMC) has been a major challenge as this plasma cell malignancy depends on the bone marrow environment for its survival. Using a microfluidic platform to emulate the dynamic physiology of the bone marrow microenvironment, we report here a new approach for culturing difficult to preserve primary human MMC. The system uses a three-dimensional ossified tissue to mimic the tumor niche and recapitulate interactions between bone marrow cells and osteoblasts (OSB). To this end, the human fetal OSB cell line hFOB 1.19 was cultured in an eight-chamber microfluidic culture device to facilitate the seeding of mononuclear cells from bone marrow aspirates from three multiple myeloma patients. Optical microscopy, used for real-time monitoring of mononuclear cell interactions with the ossified tissue, confirmed that these are drawn toward the OSB layer. After 3 weeks, cocultures were characterized by flow cytometry to evaluate the amount of expansion of primary MMC (with CD138(+) and CD38(+)CD56(+) phenotypes) in this system. For each of the three patients analyzed, bone marrow mononuclear cells underwent, on an average, 2 to 5 expansions; CD38(+)CD56(+) cells underwent 1 to 3 expansions and CD138(+) cells underwent 2.5 to 4.6 expansions. This approach is expected to provide a new avenue that can facilitate: (1) testing of personalized therapeutics for multiple myeloma patients; (2) evaluation of new drugs without the need for costly animal models; and (3) studying the biology of multiple myeloma, and in particular, the mechanisms responsible for drug resistance and relapse.

Immunolocalization of bone-resorptive cytokines in rat pulp and periapical lesions following surgical pulp exposure.

PubMed

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.

Immune transfer studies in canine allogeneic marrow graft donor-recipient pairs

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

Grosse-Wilde, H.; Krumbacher, K.; Schuening, F.D.

1986-07-01

Transfer of immunity occurring with bone marrow grafting was studied using the dog as a preclinical model. Allogeneic bone marrow transplantation (BMT) was performed between DLA-identical beagle litter-mates. The donors were immunized with tetanus toxoid (TT) or sheep red blood cells (SRBC), and their humoral response was monitored by hemagglutination. The recipients of bone marrow from TT-immunized donors showed a marked increase of antibody titer one week posttransplantation, while in the recipients of marrow from SRBC immunized donors the antibody titers were considerably lower. Within the following 60 days the antibody titers in both groups diminished gradually to pregrafting levels.more » Control experiments in which cell-free plasma from donors immunized with TT and SRBC respectively was transfused indicated that the initial rise of specific antibody titers after marrow grafting is likely to be due to a passive transfer of humoral immunity. A single challenge of these marrow graft recipients with the respective antigen 15-18 weeks posttransplantation led to a secondary type of humoral immune response. It could be demonstrated that transfer of memory against TT or SRBC was independent from the actual antibody titer and the time of vaccination of the donor. One dog was immunized with TT after serving as marrow donor. When the donor had shown an antibody response, a peripheral blood leukocytes (PBL) transfusion was given to his chimera. Subsequent challenge of the latter resulted in a secondary type of specific antibody response. This indicates that specific cellular-bound immunological memory can be transferred after BMT from the donor to his allogeneic bone marrow chimera by transfusion of peripheral blood leukocytes. The data may be of importance in clinical BMT to protect patients during the phase of reduced immune reactivity by transfer of memory cells.« less

Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging.

PubMed

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.

Computational modelling of the mechanics of trabecular bone and marrow using fluid structure interaction techniques.

PubMed

Birmingham, E; Grogan, J A; Niebur, G L; McNamara, L M; McHugh, P E

2013-04-01

Bone marrow found within the porous structure of trabecular bone provides a specialized environment for numerous cell types, including mesenchymal stem cells (MSCs). Studies have sought to characterize the mechanical environment imposed on MSCs, however, a particular challenge is that marrow displays the characteristics of a fluid, while surrounded by bone that is subject to deformation, and previous experimental and computational studies have been unable to fully capture the resulting complex mechanical environment. The objective of this study was to develop a fluid structure interaction (FSI) model of trabecular bone and marrow to predict the mechanical environment of MSCs in vivo and to examine how this environment changes during osteoporosis. An idealized repeating unit was used to compare FSI techniques to a computational fluid dynamics only approach. These techniques were used to determine the effect of lower bone mass and different marrow viscosities, representative of osteoporosis, on the shear stress generated within bone marrow. Results report that shear stresses generated within bone marrow under physiological loading conditions are within the range known to stimulate a mechanobiological response in MSCs in vitro. Additionally, lower bone mass leads to an increase in the shear stress generated within the marrow, while a decrease in bone marrow viscosity reduces this generated shear stress.

Beta-catenin-dependent Wnt signaling in mandibular bone regeneration.

PubMed

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.

Success of Maxillary Alveolar Defect Repair in Rats Using Osteoblast-Differentiated Human Deciduous Dental Pulp Stem Cells.

PubMed

Jahanbin, Arezoo; Rashed, Roozbeh; Alamdari, Daryoush Hamidi; Koohestanian, Niloufar; Ezzati, Atefeh; Kazemian, Mojgan; Saghafi, Shadi; Raisolsadat, Mohammad Ali

2016-04-01

The use of cell-based therapies represents one of the most advanced methods for enhancing the regenerative response in craniofacial abnormalities. The main aim of this study was to evaluate the regenerative potential of human dental pulp stem cells, isolated from deciduous teeth, for reconstructing maxillary alveolar defects in Wistar rats. Human deciduous dental pulp stem cells were isolated and stimulated to differentiate into osteoblasts in culture media. Maxillary alveolar defects were created in 60 Wistar rats by a surgical procedure. Then, on the basis of the type of graft used to repair the bone defect, the rats were divided into 6 equal groups: groups 1 and 2, transplantation of iliac bone graft; groups 3 and 4, transplantation of stem cells derived from deciduous dental pulp in addition to collagen matrix; groups 5 and 6, transplantation of just collagen matrix. Then, fetal bone formation, granulation tissue, fibrous tissue, and inflammatory tissue were evaluated by hematoxylin-eosin staining at 1 month (groups 1, 3, and 5) and 2 months (groups 2, 4, and 6) after surgery, and data were analyzed and compared using the Fisher exact test. Maximum fetal bone formation occurred in group 2, in which iliac bone graft was inserted into the defect area for 2 months; there also were significant differences among the groups for bone formation (P = .009). In the 1-month groups, there were no significant differences between the control and stem cell-plus-scaffold groups. There were significant differences between the 2-month groups for fetal bone formation only between the control and scaffold groups (P = .026). The study showed that human dental pulp stem cells are an additional cell resource for repairing maxillary alveolar defects in rats and constitute a promising model for reconstruction of human maxillary alveolar defects in patients with cleft lip and palate. Copyright © 2016 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Impaired immune function in children and adults with Fanconi anemia.

PubMed

Myers, Kasiani C; Sauter, Sharon; Zhang, Xue; Bleesing, Jacob J; Davies, Stella M; Wells, Susanne I; Mehta, Parinda A; Kumar, Ashish; Marmer, Daniel; Marsh, Rebecca; Brown, Darron; Butsch Kovacic, Melinda

2017-11-01

Fanconi anemia (FA) is a rare genetic disorder characterized by genome instability, bone marrow failure, and cancer predisposition. Previously, small studies have reported heterogeneous immune dysfunction in FA. We performed a detailed immunologic assessment in a large FA cohort who have not undergone bone marrow transplantation or developed malignancies. Comprehensive quantitative and functional immunologic assessment of 29 FA individuals was compared to healthy age-matched controls. Compared to non-FA persons of similar ages, FA individuals showed lower absolute total B cells (P < 0.001), lower memory B cells (P < 0.001), and decreased IgM (P < 0.001) but normal IgG. NK cells (P < 0.001) and NK cytotoxicity (P < 0.001) were decreased. CD4 + T cells were decreased (P = 0.022), while CD8 + T cell and absolute T-cell numbers were comparable. Cytotoxic T cells (P < 0.003), and antigen proliferation response to tetanus (P = 0.019) and candida (P = 0.019), were diminished in FA. Phytohemagglutinin responses and plasma cytokines were normal. Within FA subjects, adults and older children (≥10 years) exhibited higher CD8 + T cells than younger children (P = 0.004). Documented atypical infections were infrequent, although oral human papilloma virus (HPV) prevalence was higher (31% positive) in FA. Overall, these results demonstrate a high rate of significant humoral and cellular immune dysfunction. Continued longitudinal study of immune function is critical to understand evolution with age, bone marrow failure, and cancer development. © 2017 Wiley Periodicals, Inc.

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

PubMed

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

1999-04-01

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

Development of electrospun bone-mimetic matrices for bone regenerative applications

NASA Astrophysics Data System (ADS)

Phipps, Matthew Christopher

Although bone has a dramatic capacity for regeneration, certain injuries and procedures present defects that are unable to heal properly, requiring surgical intervention to induce and support osteoregeneration. Our research group has hypothesized that the development of a biodegradable material that mimics the natural composition and architecture of bone extracellular matrix has the potential to provide therapeutic benefit to these patients. Utilizing a process known as electrospinning, our lab has developed a bone-mimetic matrix (BMM) consisting of composite nanofibers of the mechanically sta-ble polymer polycaprolactone (PCL), and the natural bone matrix molecules type-I colla-gen and hydroxyapatite nanocrystals (HA). We herein show that BMMs supported great-er adhesion, proliferation, and integrin activation of mesenchymal stem cells (MSCs), the multipotent bone-progenitor cells within bone marrow and the periosteum, in comparison to electrospun PCL alone. These cellular responses, which are essential early steps in the process of bone regeneration, highlight the benefits of presenting cells with natural bone molecules. Subsequently, evaluation of new bone formation in a rat cortical tibia defect showed that BMMs are highly osteoconductive. However, these studies also revealed the inability of endogenous cells to migrate within electrospun matrices due to the inherently small pore sizes. To address this limitation, which will negatively impact the rate of scaf-fold-to-bone turnover and inhibit vascularization, sacrificial fibers were added to the ma-trix. The removal of these fibers after fabrication resulted in BMMs with larger pores, leading to increased infiltration of MSCs and endogenous bone cells. Lastly, we evaluat-ed the potential of our matrices to stimulate the recruitment of MSCs, a vital step in bone healing, through the sustained delivery of platelet derived growth factor-BB (PDGF-BB). BMMs were found to adsorb and subsequently release greater quantities of PDGF-BB, compared to PCL scaffolds, over an 8-week interval. The released PDGF-BB retained its bioactivity, stimulating MSC chemotaxis in two separate assays. Collectively, these re-sults suggest that electrospun matrices incorporating the bone matrix molecules collagen I and HA, with sacrificial fibers, provide a favorable scaffold for MSC survival and infil-tration as well as the ability to sequester PDGF-BB from solution, leading to sustained local delivery and MSC chemotaxis.

Skeletal unloading inhibits the in vitro proliferation and differentiation of rat osteoprogenitor cells

NASA Technical Reports Server (NTRS)

Kostenuik, P. J.; Halloran, B. P.; Morey-Holton, E. R.; Bikle, D. D.

1997-01-01

Loss of weight bearing in the growing rat decreases bone formation, osteoblast numbers, and bone maturation in unloaded bones. These responses suggest an impairment of osteoblast proliferation and differentiation. To test this assumption, we assessed the effects of skeletal unloading using an in vitro model of osteoprogenitor cell differentiation. Rats were hindlimb elevated for 0 (control), 2, or 5 days, after which their tibial bone marrow stromal cells (BMSCs) were harvested and cultured. Five days of hindlimb elevation led to significant decreases in proliferation, alkaline phosphatase (AP) enzyme activity, and mineralization of BMSC cultures. Differentiation of BMSCs was analyzed by quantitative competitive polymerase chain reaction of cDNA after 10, 15, 20, and 28 days of culture. cDNA pools were analyzed for the expression of c-fos (an index of proliferation), AP (an index of early osteoblast differentiation), and osteocalcin (a marker of late differentiation). BMSCs from 5-day unloaded rats expressed 50% less c-fos, 61% more AP, and 35% less osteocalcin mRNA compared with controls. These data demonstrate that cultured osteoprogenitor cells retain a memory of their in vivo loading history and indicate that skeletal unloading inhibits proliferation and differentiation of osteoprogenitor cells in vitro.

Gender differences in bone turnover in 2-year-old Thoroughbreds.

PubMed

Jackson, B F; Lonnell, C; Verheyen, K; Wood, J L N; Pfeiffert, D U; Price, J S

2003-11-01

Injuries to the skeleton are a major cause of morbidity and mortality in racehorses and age, gender and season have all been shown to influence risk of injury. To use biochemical markers of bone cell activity to establish to whether cellular processes in bone underlie these described effects. Blood samples were collected monthly from 2-year-old horses in race training between November 1998 and September 1999. Mean age at the start of the study was 20 months (range 18-23 months), with no significant difference in average age between colts (n = 84) and fillies (n = 63). Three markers were measured; osteocalcin (OC, bone formation), the carboxyterminal cross-linked telopeptide of type I collagen (ICTP, bone resorption) and the carboxyterminal propeptide of type I collagen (PICP), which is less 'bone-specific' than the other 2 markers. Colts had, on average, 3.62 ng/ml higher OC concentrations (P = 0.044) and 0.68 mg/l higher ICTP concentrations (P = 0.01) than fillies. The effect of gender was not statistically significant for PICP. However, in May, PICP concentrations were on average 157 mg/l higher in fillies than colts. There was no effect of age or season on marker concentrations. This study has shown that there are gender differences in bone turnover markers in 2-year-old Thoroughbreds; however, age, within the limited range studied, did not have a significant effect on bone cell activity. Lower bone marker concentrations may reflect smaller bone size and/or earlier skeletal maturation in fillies. An increase in concentrations of PICP in fillies in spring and early summer may relect an influence of sex hormones on collagen turnover. Gender differences in bone cell activity in 2-year-old colts and fillies may influence bone's adaptive responses to training and risk of injury.

Monitoring tissue inflammation and responses to drug treatments in early stages of mice bone fracture using 50 MHz ultrasound

PubMed Central

Chen, Yen-Chu; Lin, Yi-Hsun; Wang, Shyh-Hau; Lin, Shih-Ping; Shung, K. Kirk; Wu, Chia-Ching

2014-01-01

Bone fracture induces moderate inflammatory responses that are regulated by cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LO) for initiating tissue repair and bone formation. Only a handful of non-invasive techniques focus on monitoring acute inflammation of injured bone currently exists. In the current study, we monitored in vivo inflammation levels during the initial 2 weeks of the inflammatory stage after mouse bone fracture utilizing 50 MHz ultrasound. The acquired ultrasonic images were correlated well with histological examinations. After the bone fracture in the tibia, dynamic changes in the soft tissue at the medial-posterior compartment near the fracture site were monitored by ultrasound on the days of 0, 2, 4, 7, and 14. The corresponding echogenicity increased on the 2nd, 4th, and 7th day, and subsequently declined to basal levels after the 14th day. An increase of cell death was identified by the positive staining of deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and was consistent with ultrasound measurements. The increases of both COX-2 and Leukotriene B4 receptor 1 (BLT1, 5- LO-relative receptor), which are regulators for tissue inflammation, in the immunohistochemistry staining revealed their involvement in bone fracture injury. Monitoring the inflammatory response to various non-steroidal anti-inflammatory drugs (NSAIDs) treatments was investigated by treating injured mice with a daily oral intake of aspirin (Asp), indomethacin (IND), and a selective COX-2 inhibitor (SC-236). The Asp treatment significantly reduced fracture-increased echogenicity (hyperechogenicity, p < 0.05) in ultrasound images as well as inhibited cell death, and expression of COX-2 and BLT1. In contrast, treatment with IND or SC-236 did not reduce the hyperechogenicity, as confirmed by cell death (TUNEL) and expression levels of COX-2 or BLT1. Taken together, the current study reports the feasibility of a noninvasive ultrasound method capable of monitoring post-fracture tissue inflammation that positively correlates with histological findings. Results of this study also suggest that this approach may be further applied to elucidate the underlying mechanisms of inflammatory processes and to develop therapeutic strategies for facilitating fracture healing. PMID:23871514

The Fanconi Anemia Pathway: Repairing the Link Between DNA Damage and Squamous Cell Carcinoma

PubMed Central

Romick-Rosendale, Lindsey E.; Lui, Vivian W. Y.; Grandis, Jennifer R.; Wells, Susanne I.

2013-01-01

Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in one of fifteen genes known to encode FA pathway components. In response to DNA damage, nuclear FA proteins associate into high molecular weight complexes through a cascade of post-translational modifications and physical interactions, followed by the repair of damaged DNA. Hematopoietic cells are particularly sensitive to the loss of these interactions, and bone marrow failure occurs almost universally in FA patients. FA as a disease is further characterized by cancer susceptibility, which highlights the importance of the FA pathway in tumor suppression, and will be the focus of this review. Acute myeloid leukemia is the most common cancer type, often subsequent to bone marrow failure. However, FA patients are also at an extreme risk of squamous cell carcinoma (SCC) of the head and neck and gynecological tract, with an even greater incidence in those individuals who have received a bone marrow transplant and recovered from hematopoietic disease. FA tumor suppression in hematopoietic versus epithelial compartments could be mechanistically similar or distinct. Definition of compartment specific FA activities is now critical to assess the effects of today’s bone marrow failure treatments on tomorrow’s solid tumor development. It is our hope that current therapies can then be optimized to decrease the risk of malignant transformation in both hematopoietic and epithelial cells. Here we review our current understanding of the mechanisms of action of the Fanconi anemia pathway as it contributes to stress responses, DNA repair and squamous cell carcinoma susceptibility. PMID:23333482

Transforming growth factor β induces bone marrow mesenchymal stem cell migration via noncanonical signals and N-cadherin.

PubMed

Dubon, Maria Jose; Yu, Jinyeong; Choi, Sanghyuk; Park, Ki-Sook

2018-01-01

Transforming growth factor-beta (TGF-β) induces the migration and mobilization of bone marrow-derived mesenchymal stem cells (BM-MSCs) to maintain bone homeostasis during bone remodeling and facilitate the repair of peripheral tissues. Although many studies have reported the mechanisms through which TGF-β mediates the migration of various types of cells, including cancer cells, the intrinsic cellular mechanisms underlying cellular migration, and mobilization of BM-MSCs mediated by TGF-β are unclear. In this study, we showed that TGF-β activated noncanonical signaling molecules, such as Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), focal adhesion kinase (FAK), and p38, via TGF-β type I receptor in human BM-MSCs and murine BM-MSC-like ST2 cells. Inhibition of Rac1 by NSC23766 and Src by PP2 resulted in impaired TGF-β-mediated migration. These results suggested that the Smad-independent, noncanonical signals activated by TGF-β were necessary for migration. We also showed that N-cadherin-dependent intercellular interactions were required for TGF-β-mediated migration using functional inhibition of N-cadherin with EDTA treatment and a neutralizing antibody (GC-4 antibody) or siRNA-mediated knockdown of N-cadherin. However, N-cadherin knockdown did not affect the global activation of noncanonical signals in response to TGF-β. Therefore, these results suggested that the migration of BM-MSCs in response to TGF-β was mediated through N-cadherin and noncanonical TGF-β signals. © 2017 Wiley Periodicals, Inc.

Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling.

PubMed

Ying, Xiaozhou; Sun, Liaojun; Chen, Xiaowei; Xu, Huazi; Guo, Xiaoshan; Chen, Hua; Hong, Jianjun; Cheng, Shaowen; Peng, Lei

2013-12-05

Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20 μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs. © 2013 Elsevier B.V. All rights reserved.

Effects of Fenbendazole on Routine Immune Response Parameters of BALB/c Mice

PubMed Central

Cray, Carolyn; Villar, David; Zaias, Julia; Altman, Norman H

2008-01-01

Fenbendazole (FBZ) is an anthelmintic drug widely used to treat and prevent pinworm outbreaks in laboratory rodents. Although data in nonrodent species indicate possible effects of fenbendazole on the bone marrow and lymphocyte proliferation and function, little has been reported regarding possible effects on the rodent immune system. The purpose of the current study was to determine the effects of a therapeutic regimen of FBZ on immune parameters in BALB/c mice. Both 9-wk on–off and 5-wk continuous medicated feed protocols were assessed. No significant differences between normal and FBZ diet treated mice were observed in the following parameters: complete blood count, blood chemistry, quantitation of major T and B cell markers in spleen, quantitation of T cell markers in the thymus, spleen cell proliferation to T and B cell mitogens, bone marrow colony-forming cell assays, skin graft rejection, and primary and secondary humoral immune responses. These data indicate that FBZ treatment does not affect many standard broad measures of immune function. PMID:19049250

Effects of fenbendazole on routine immune response parameters of BALB/c mice.

PubMed

Cray, Carolyn; Villar, David; Zaias, Julia; Altman, Norman H

2008-11-01

Fenbendazole (FBZ) is an anthelmintic drug widely used to treat and prevent pinworm outbreaks in laboratory rodents. Although data in nonrodent species indicate possible effects of fenbendazole on the bone marrow and lymphocyte proliferation and function, little has been reported regarding possible effects on the rodent immune system. The purpose of the current study was to determine the effects of a therapeutic regimen of FBZ on immune parameters in BALB/c mice. Both 9-wk on-off and 5-wk continuous medicated feed protocols were assessed. No significant differences between normal and FBZ diet treated mice were observed in the following parameters: complete blood count, blood chemistry, quantitation of major T and B cell markers in spleen, quantitation of T cell markers in the thymus, spleen cell proliferation to T and B cell mitogens, bone marrow colony-forming cell assays, skin graft rejection, and primary and secondary humoral immune responses. These data indicate that FBZ treatment does not affect many standard broad measures of immune function.

Modeling Vascularized Bone Regeneration Within a Porous Biodegradable CaP Scaffold Loaded with Growth Factors

PubMed Central

Sun, X; Kang, Y; Bao, J; Zhang, Y; Yang, Y; Zhou, X

2013-01-01

Osteogenetic microenvironment is a complex constitution in which extracellular matrix (ECM) molecules, stem cells and growth factors each interact to direct the coordinate regulation of bone tissue development. Importantly, angiogenesis improvement and revascularization are critical for osteogenesis during bone tissue regeneration processes. In this study, we developed a three-dimensional (3D) multi-scale system model to study cell response to growth factors released from a 3D biodegradable porous calcium phosphate (CaP) scaffold. Our model reconstructed the 3D bone regeneration system and examined the effects of pore size and porosity on bone formation and angiogenesis. The results suggested that scaffold porosity played a more dominant role in affecting bone formation and angiogenesis compared with pore size, while the pore size could be controlled to tailor the growth factor release rate and release fraction. Furthermore, a combination of gradient VEGF with BMP2 and Wnt released from the multi-layer scaffold promoted angiogenesis and bone formation more readily than single growth factors. These results demonstrated that the developed model can be potentially applied to predict vascularized bone regeneration with specific scaffold and growth factors. PMID:23566802

Age-related mechanical strength evolution of trabecular bone under fatigue damage for both genders: Fracture risk evaluation.

PubMed

Ben Kahla, Rabeb; Barkaoui, Abdelwahed; Merzouki, Tarek

2018-08-01

Bone tissue is a living composite material, providing mechanical and homeostatic functions, and able to constantly adapt its microstructure to changes in long term loading. This adaptation is conducted by a physiological process, known as "bone remodeling". This latter is manifested by interactions between osteoclasts and osteoblasts, and can be influenced by many local factors, via effects on bone cell differentiation and proliferation. In the current work, age and gender effects on damage rate evolution, throughout life, have been investigated using a mechanobiological finite element modeling. To achieve the aim, a mathematical model has been developed, coupling both cell activities and mechanical behavior of trabecular bone, under cyclic loadings. A series of computational simulations (ABAQUS/UMAT) has been performed on a 3D human proximal femur, allowing to investigate the effects of mechanical and biological parameters on mechanical strength of trabecular bone, in order to evaluate the fracture risk resulting from fatigue damage. The obtained results revealed that mechanical stimulus amplitude affects bone resorption and formation rates, and indicated that age and gender are major factors in bone response to the applied loadings. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sympathetic control of bone mass regulated by osteopontin

PubMed Central

Nagao, Masashi; Feinstein, Timothy N.; Ezura, Yoichi; Hayata, Tadayoshi; Notomi, Takuya; Saita, Yoshitomo; Hanyu, Ryo; Hemmi, Hiroaki; Izu, Yayoi; Takeda, Shu; Wang, Kathryn; Rittling, Susan; Nakamoto, Tetsuya; Kaneko, Kazuo; Kurosawa, Hisashi; Karsenty, Gerard; Denhardt, David T.; Vilardaga, Jean-Pierre; Noda, Masaki

2011-01-01

The sympathetic nervous system suppresses bone mass by mechanisms that remain incompletely elucidated. Using cell-based and murine genetics approaches, we show that this activity of the sympathetic nervous system requires osteopontin (OPN), a cytokine and one of the major members of the noncollagenous extracellular matrix proteins of bone. In this work, we found that the stimulation of the sympathetic tone by isoproterenol increased the level of OPN expression in the plasma and bone and that mice lacking OPN (OPN-KO) suppressed the isoproterenol-induced bone loss by preventing reduced osteoblastic and enhanced osteoclastic activities. In addition, we found that OPN is necessary for changes in the expression of genes related to bone resorption and bone formation that are induced by activation of the sympathetic tone. At the cellular level, we showed that intracellular OPN modulated the capacity of the β2-adrenergic receptor to generate cAMP with a corresponding modulation of cAMP-response element binding (CREB) phosphorylation and associated transcriptional events inside the cell. Our results indicate that OPN plays a critical role in sympathetic tone regulation of bone mass and that this OPN regulation is taking place through modulation of the β2-adrenergic receptor/cAMP signaling system. PMID:21990347

Extended Culture of Bone Marrow with Granulocyte Macrophage-Colony Stimulating Factor Generates Immunosuppressive Cells

PubMed Central

Na, Hye Young; Sohn, Moah; Ryu, Seul Hye; Choi, Wanho; In, Hyunju; Shin, Hyun Soo

2018-01-01

Bone marrow-derived dendritic cells (BM-DCs) are generated from bone marrow (BM) cells cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for a week. In this study we investigated the effect of duration on the BM culture with GM-CSF. Within several months, the cells in the BM culture gradually expressed homogeneous levels of CD11c and major histocompatibility complex II on surface, and they became unable to stimulate allogeneic naïve T cells in mixed lymphocyte reaction (MLR). In addition, when the BM culture were sustained for 32 wk or longer, the BM cells acquired ability to suppress the proliferation of allogeneic T cells in MLR as well as the response of ovalbumin-specific OT-I transgenic T cells in antigen-dependent manner. We found that, except for programmed death-ligand 1, most cell surface molecules were expressed lower in the BM cells cultured with GM-CSF for the extended duration. These results indicate that BM cells in the extended culture with GM-CSF undergo 2 distinct steps of functional change; first, they lose the immunostimulatory capacity; and next, they gain the immunosuppressive ability. PMID:29736292

Parkinson disease-associated LRRK2 G2019S transgene disrupts marrow myelopoiesis and peripheral Th17 response.

PubMed

Park, Jeongho; Lee, Jang-Won; Cooper, Scott C; Broxmeyer, Hal E; Cannon, Jason R; Kim, Chang H

2017-10-01

Parkinson's disease (PD) is a neurodegenerative disease, whereas Crohn's disease is an inflammatory bowel disease. Interestingly, polymorphisms in the LRRK2 gene have been identified as risk factors for both diseases. LRRK2 G2019S is the most prevalent mutation found in PD. To gain insights into the role of the LRRK2 G2019S gene on the development and activation of the immune system in the brain-gut axis, we investigated the effect of LRRK2 G2019S on bone marrow myeloid progenitors and myeloid cell function in the periphery. We used bacterial artificial chromosome transgenic rats harboring the human LRRK2 G2019S gene. LRRK2 G2019S transgene decreased the numbers of monocytic and granulocytic progenitors in the bone marrow. However, the numbers of peripheral, immature myeloid cells with suppressive activity were increased in the gut and blood circulation of LRRK2 G2019S compared with control rats in various acute and chronic inflammatory responses. In inflammatory conditions, Th17 cell activity was suppressed, but tissue-associated phylum Bacteroidetes was abnormally increased in the intestine of LRRK2 G2019S rats. The abnormally expanded myeloid cells because of the LRRK2 G2019S gene were highly suppressive on Th17 cell differentiation. Moreover, we found that inhibition of LRRK2 kinase affects myeloid progenitors and myeloid cell differentiation. Taken together, the results indicate that abnormal LRRK2 activity can alter bone marrow myelopoiesis, peripheral myeloid cell differentiation, and intestinal immune homeostasis. These findings may have ramifications in immune and inflammatory responses in patients with LRRK2 abnormalities. © Society for Leukocyte Biology.

Gene expression markers in circulating tumor cells may predict bone metastasis and response to hormonal treatment in breast cancer

PubMed Central

WANG, HAIYING; MOLINA, JULIAN; JIANG, JOHN; FERBER, MATTHEW; PRUTHI, SANDHYA; JATKOE, TIMOTHY; DERECHO, CARLO; RAJPUROHIT, YASHODA; ZHENG, JIAN; WANG, YIXIN

2013-01-01

Circulating tumor cells (CTCs) have recently attracted attention due to their potential as prognostic and predictive markers for the clinical management of metastatic breast cancer patients. The isolation of CTCs from patients may enable the molecular characterization of these cells, which may help establish a minimally invasive assay for the prediction of metastasis and further optimization of treatment. Molecular markers of proven clinical value may therefore be useful in predicting disease aggressiveness and response to treatment. In our earlier study, we identified a gene signature in breast cancer that appears to be significantly associated with bone metastasis. Among the genes that constitute this signature, trefoil factor 1 (TFF1) was identified as the most differentially expressed gene associated with bone metastasis. In this study, we investigated 25 candidate gene markers in the CTCs of metastatic breast cancer patients with different metastatic sites. The panel of the 25 markers was investigated in 80 baseline samples (first blood draw of CTCs) and 30 follow-up samples. In addition, 40 healthy blood donors (HBDs) were analyzed as controls. The assay was performed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with RNA extracted from CTCs captured by the CellSearch system. Our study indicated that 12 of the genes were uniquely expressed in CTCs and 10 were highly expressed in the CTCs obtained from patients compared to those obtained from HBDs. Among these genes, the expression of keratin 19 was highly correlated with the CTC count. The TFF1 expression in CTCs was a strong predictor of bone metastasis and the patients with a high expression of estrogen receptor β in CTCs exhibited a better response to hormonal treatment. Molecular characterization of these genes in CTCs may provide a better understanding of the mechanism underlying tumor metastasis and identify gene markers in CTCs for predicting disease progression and response to treatment. PMID:24649289

Gene expression markers in circulating tumor cells may predict bone metastasis and response to hormonal treatment in breast cancer.

PubMed

Wang, Haiying; Molina, Julian; Jiang, John; Ferber, Matthew; Pruthi, Sandhya; Jatkoe, Timothy; Derecho, Carlo; Rajpurohit, Yashoda; Zheng, Jian; Wang, Yixin

2013-11-01

Circulating tumor cells (CTCs) have recently attracted attention due to their potential as prognostic and predictive markers for the clinical management of metastatic breast cancer patients. The isolation of CTCs from patients may enable the molecular characterization of these cells, which may help establish a minimally invasive assay for the prediction of metastasis and further optimization of treatment. Molecular markers of proven clinical value may therefore be useful in predicting disease aggressiveness and response to treatment. In our earlier study, we identified a gene signature in breast cancer that appears to be significantly associated with bone metastasis. Among the genes that constitute this signature, trefoil factor 1 (TFF1) was identified as the most differentially expressed gene associated with bone metastasis. In this study, we investigated 25 candidate gene markers in the CTCs of metastatic breast cancer patients with different metastatic sites. The panel of the 25 markers was investigated in 80 baseline samples (first blood draw of CTCs) and 30 follow-up samples. In addition, 40 healthy blood donors (HBDs) were analyzed as controls. The assay was performed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with RNA extracted from CTCs captured by the CellSearch system. Our study indicated that 12 of the genes were uniquely expressed in CTCs and 10 were highly expressed in the CTCs obtained from patients compared to those obtained from HBDs. Among these genes, the expression of keratin 19 was highly correlated with the CTC count. The TFF1 expression in CTCs was a strong predictor of bone metastasis and the patients with a high expression of estrogen receptor β in CTCs exhibited a better response to hormonal treatment. Molecular characterization of these genes in CTCs may provide a better understanding of the mechanism underlying tumor metastasis and identify gene markers in CTCs for predicting disease progression and response to treatment.

Deficiency of bone marrow beta3-integrin enhances non-functional neovascularization.

PubMed

Watson, Alan R; Pitchford, Simon C; Reynolds, Louise E; Direkze, Natalie; Brittan, Mairi; Alison, Malcolm R; Rankin, Sara; Wright, Nicholas A; Hodivala-Dilke, Kairbaan M

2010-03-01

beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.

Antinociceptive effect of intrathecal microencapsulated human pheochromocytoma cell in a rat model of bone cancer pain.

PubMed

Li, Xiao; Li, Guoqi; Wu, Shaoling; Zhang, Baiyu; Wan, Qing; Yu, Ding; Zhou, Ruijun; Ma, Chao

2014-07-08

Human pheochromocytoma cells, which are demonstrated to contain and release met-enkephalin and norepinephrine, may be a promising resource for cell therapy in cancer-induced intractable pain. Intrathecal injection of alginate-poly (l) lysine-alginate (APA) microencapsulated human pheochromocytoma cells leads to antinociceptive effect in a rat model of bone cancer pain, and this effect was blocked by opioid antagonist naloxone and alpha 2-adrenergic antagonist rauwolscine. Neurochemical changes of cerebrospinal fluid are in accordance with the analgesic responses. Taken together, these data support that human pheochromocytoma cell implant-induced antinociception was mediated by met-enkephalin and norepinephrine secreted from the cell implants and acting at spinal receptors. Spinal implantation of microencapsulated human pheochromocytoma cells may provide an alternative approach for the therapy of chronic intractable pain.

Herbal preparation (HemoHIM) enhanced functional maturation of bone marrow-derived dendritic cells mediated toll-like receptor 4.

PubMed

Lee, Sung-Ju; Kim, Jong-Jin; Kang, Kyung-Yun; Hwang, Yun-Ho; Jeong, Gil-Yeon; Jo, Sung-kee; Jung, Uhee; Park, Hae-Ran; Yee, Sung-Tae

2016-02-19

HemoHIM, which is an herbal preparation of three edible herbs (Angelicam gigas Nakai, Cnidium offinale Makino, and Peaonia japonica Miyabe), is known to have various biological and immunological activities, but the modulatory effects of this preparation on dendritic cells (DCs)-mediated immune responses have not been examined previously. DCs are a unique group of white blood cells that initiate primary immune responses by capturing, processing, and presenting antigens to T cells. In the present study, we investigated the effect of HemoHIM on the functional and phenotypic maturation of murine bone marrow-derived dendritic cells (BMDCs) both in vitro and in vivo. The expression of co-stimulatory molecules (CD40, CD80, CD86, MHC I, and MHC II) and the production of cytokines (IL-1β, IL-6, IL-12p70, and TNF-α) were increased by HemoHIM in BMDCs. Furthermore, the antigen-uptake ability of BMDCs was decreased by HemoHIM, and the antigen-presenting ability of HemoHIM-treated mature BMDCs increased TLR4-dependent CD4(+) and CD8(+) T cell responses. Our findings demonstrated that HemoHIM induces TLR4-mediated BMDCs functional and phenotypic maturation through in vivo and in vitro. And our study showed the antigen-presenting ability that HemoHIM-treated mature BMDCs increase CD4(+) and CD8(+) T cell responses by in vitro. These results suggest that HemoHIM has the potential to mediate DC immune responses.

Assessment of Benzene-Induced Hematotoxicity Using a Human-Like Hematopoietic Lineage in NOD/Shi-scid/IL-2Rγnull Mice

PubMed Central

Takahashi, Masayuki; Tsujimura, Noriyuki; Yoshino, Tomoko; Hosokawa, Masahito; Otsuka, Kensuke; Matsunaga, Tadashi; Nakasono, Satoshi

2012-01-01

Despite recent advancements, it is still difficult to evaluate in vivo responses to toxicants in humans. Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγnull (NOG) mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice). Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences. PMID:23226520

Epstein–Barr virus in bone marrow of rheumatoid arthritis patients predicts response to rituximab treatment

PubMed Central

Brisslert, Mikael; Zendjanchi, Kiandoht; Lindh, Magnus; Bokarewa, Maria I.

2010-01-01

Objectives. Viruses may contribute to RA. This prompted us to monitor viral load and response to anti-CD20 therapy in RA patients. Methods. Blood and bone marrow from 35 RA patients were analysed for CMV, EBV, HSV-1, HSV-2, parvovirus B19 and polyomavirus using real-time PCR before and 3 months after rituximab (RTX) treatment and related to the levels of autoantibodies and B-cell depletion. Clinical response to RTX was defined as decrease in the 28-joint disease activity score (DAS-28) >1.3 at 6 months. Results. Before RTX treatment, EBV was identified in 15 out of 35 patients (EBV-positive group), of which 4 expressed parvovirus. Parvovirus was further detected in eight patients (parvo-positive group). Twelve patients were negative for the analysed viruses. Following RTX, EBV was cleared, whereas parvovirus was unaffected. Eighteen patients were responders, of which 12 were EBV positive. The decrease in the DAS-28 was significantly higher in EBV-positive group compared with parvo-positive group (P = 0.002) and virus-negative patients (P = 0.04). Most of EBV-negative patients that responded to RTX (75%) required retreatment within the following 11 months compared with only 8% of responding EBV-positive patients. A decrease of RF, Ig-producing cells and CD19+ B cells was observed following RTX but did not distinguish between viral infections. However, EBV-infected patients had significantly higher levels of Fas-expressing B cells at baseline as compared with EBV-negative groups. Conclusions. EBV and parvovirus genomes are frequently found in bone marrow of RA patients. The presence of EBV genome was associated with a better clinical response to RTX. Thus, presence of EBV genome may predict clinical response to RTX. PMID:20547657

The skeletal cell-derived molecule sclerostin drives bone marrow adipogenesis.

PubMed

Fairfield, Heather; Falank, Carolyne; Harris, Elizabeth; Demambro, Victoria; McDonald, Michelle; Pettitt, Jessica A; Mohanty, Sindhu T; Croucher, Peter; Kramer, Ina; Kneissel, Michaela; Rosen, Clifford J; Reagan, Michaela R

2018-02-01

The bone marrow niche is a dynamic and complex microenvironment that can both regulate, and be regulated by the bone matrix. Within the bone marrow (BM), mesenchymal stromal cell (MSC) precursors reside in a multi-potent state and retain the capacity to differentiate down osteoblastic, adipogenic, or chondrogenic lineages in response to numerous biochemical cues. These signals can be altered in various pathological states including, but not limited to, osteoporotic-induced fracture, systemic adiposity, and the presence of bone-homing cancers. Herein we provide evidence that signals from the bone matrix (osteocytes) determine marrow adiposity by regulating adipogenesis in the bone marrow. Specifically, we found that physiologically relevant levels of Sclerostin (SOST), which is a Wnt-inhibitory molecule secreted from bone matrix-embedded osteocytes, can induce adipogenesis in 3T3-L1 cells, mouse ear- and BM-derived MSCs, and human BM-derived MSCs. We demonstrate that the mechanism of SOST induction of adipogenesis is through inhibition of Wnt signaling in pre-adipocytes. We also demonstrate that a decrease of sclerostin in vivo, via both genetic and pharmaceutical methods, significantly decreases bone marrow adipose tissue (BMAT) formation. Overall, this work demonstrates a direct role for SOST in regulating fate determination of BM-adipocyte progenitors. This provides a novel mechanism for which BMAT is governed by the local bone microenvironment, which may prove relevant in the pathogenesis of certain diseases involving marrow adipose. Importantly, with anti-sclerostin therapy at the forefront of osteoporosis treatment and a greater recognition of the role of BMAT in disease, these data are likely to have important clinical implications. © 2017 Wiley Periodicals, Inc.

Development and characterization of a lung-protective method of bone marrow transplantation in the mouse.

PubMed

Janssen, William J; Muldrow, Alaina; Kearns, Mark T; Barthel, Lea; Henson, Peter M

2010-05-31

Allogeneic bone marrow transplantation is a common method used to study the contribution of myeloid and lymphoid cell populations in murine models of disease. The method requires lethal doses of radiation to ablate the bone marrow. Unintended consequences of radiation include organ injury and inflammatory cell activation. The goal of our study was to determine the degree to which bone marrow transplantation alters lungs and to develop a system to protect the lungs during radiation. C57BL/6 mice were subjected to total body irradiation with 900cGy and then transplanted with bone marrow from green fluorescent protein (GFP) expressing mice. Resultant chimeras exhibited a significant decline in alveolar macrophage numbers within 72h, modest influx of neutrophils in the lungs at 14days, and repopulation of the lungs by alveolar macrophages of bone marrow origin by 28days. Neutrophil influx and alveolar macrophage turnover were prevented when 1cm thick lead shields were used to protect the lungs during radiation, such that 8weeks after transplantation less than 30% of alveolar macrophages were of donor origin. Lung-shielded mice achieved a high level of bone marrow engraftment with greater than 95% of circulating leukocytes expressing GFP. In addition, their response to intratracheal lipopolysaccharide was similar to non-transplanted mice. We describe a model whereby lead shields protect resident cell populations in the lungs from radiation during bone marrow transplantation but permit full bone marrow engraftment. This system may be applicable to other organ systems in which protection from radiation during bone marrow transplantation is desired.

Effects of low level laser therapy (LLLT) on pressured human osteoblasts: A histomorphologic and quantitative study

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.

Ibrutinib enhances IL-17 response by modulating the function of bone marrow derived dendritic cells

PubMed Central

Natarajan, Gayathri; Terrazas, Cesar; Oghumu, Steve; Varikuti, Sanjay; Dubovsky, Jason A; Byrd, John C; Satoskar, Abhay R

2016-01-01

Ibrutinib (PCI-32765) is an irreversible dual Btk/Itk inhibitor shown to be effective in treating several B cell malignancies. However, limited studies have been conducted to study the effect of this drug on myeloid cell function. Hence, we studied the effect of ibrutinib treatment on TLR-4 mediated activation of bone marrow derived dendritic cell culture (DCs). Upon ibrutinib treatment, LPS-treated DCs displayed lower synthesis of TNF-α and nitric oxide (NO) and higher induction of IL-6, TGF-β, IL-10 and IL-18. While ibrutinib dampened MHC-II and CD86 expression on DCs, CD80 expression was upregulated. Further, ibrutinib-treated DCs promoted T cell proliferation and enhanced IL-17 production upon co-culture with nylon wool enriched T cells. Taken together, our results indicate that ibrutinib modulates TLR-4 mediated DC activation to promote an IL-17 response. We describe a novel mode of action for ibrutinib on DCs which should be explored to treat other forms of cancer besides B cell malignancies. PMID:26942065

Ibrutinib enhances IL-17 response by modulating the function of bone marrow derived dendritic cells.

PubMed

Natarajan, Gayathri; Terrazas, Cesar; Oghumu, Steve; Varikuti, Sanjay; Dubovsky, Jason A; Byrd, John C; Satoskar, Abhay R

Ibrutinib (PCI-32765) is an irreversible dual Btk/Itk inhibitor shown to be effective in treating several B cell malignancies. However, limited studies have been conducted to study the effect of this drug on myeloid cell function. Hence, we studied the effect of ibrutinib treatment on TLR-4 mediated activation of bone marrow derived dendritic cell culture (DCs). Upon ibrutinib treatment, LPS-treated DCs displayed lower synthesis of TNF-α and nitric oxide (NO) and higher induction of IL-6, TGF-β, IL-10 and IL-18. While ibrutinib dampened MHC-II and CD86 expression on DCs, CD80 expression was upregulated. Further, ibrutinib-treated DCs promoted T cell proliferation and enhanced IL-17 production upon co-culture with nylon wool enriched T cells. Taken together, our results indicate that ibrutinib modulates TLR-4 mediated DC activation to promote an IL-17 response. We describe a novel mode of action for ibrutinib on DCs which should be explored to treat other forms of cancer besides B cell malignancies.

Intrinsic functional defects of type 2 innate lymphoid cells impair innate allergic inflammation in promyelocytic leukemia zinc finger (PLZF)-deficient mice.

PubMed

Verhoef, Philip A; Constantinides, Michael G; McDonald, Benjamin D; Urban, Joseph F; Sperling, Anne I; Bendelac, Albert

2016-02-01

The transcription factor promyelocytic leukemia zinc finger (PLZF) is transiently expressed during development of type 2 innate lymphoid cells (ILC2s) but is not present at the mature stage. We hypothesized that PLZF-deficient ILC2s have functional defects in the innate allergic response and represent a tool for studying innate immunity in a mouse with a functional adaptive immune response. We determined the consequences of PLZF deficiency on ILC2 function in response to innate and adaptive immune stimuli by using PLZF(-/-) mice and mixed wild-type:PLZF(-/-) bone marrow chimeras. PLZF(-/-) mice, wild-type littermates, or mixed bone marrow chimeras were treated with the protease allergen papain or the cytokines IL-25 and IL-33 or infected with the helminth Nippostrongylus brasiliensis to induce innate type 2 allergic responses. Mice were sensitized with intraperitoneal ovalbumin-alum, followed by intranasal challenge with ovalbumin alone, to induce adaptive TH2 responses. Lungs were analyzed for immune cell subsets, and alveolar lavage fluid was analyzed for ILC2-derived cytokines. In addition, ILC2s were stimulated ex vivo for their capacity to release type 2 cytokines. PLZF-deficient lung ILC2s exhibit a cell-intrinsic defect in the secretion of IL-5 and IL-13 in response to innate stimuli, resulting in defective recruitment of eosinophils and goblet cell hyperplasia. In contrast, the adaptive allergic inflammatory response to ovalbumin and alum was unimpaired. PLZF expression at the innate lymphoid cell precursor stage has a long-range effect on the functional properties of mature ILC2s and highlights the importance of these cells for innate allergic responses in otherwise immunocompetent mice. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. All rights reserved.

Epigenetic regulation of HGF/Met receptor axis is critical for the outgrowth of bone metastasis from breast carcinoma.

PubMed

Bendinelli, Paola; Maroni, Paola; Matteucci, Emanuela; Desiderio, Maria Alfonsina

2017-02-02

Our translational research deals with the influence of microenvironment on the phenotype and colonization of bone metastases from breast carcinoma, and on pre-metastatic niche formation. The aim of the present study was to clarify the origin of hepatocyte growth factor (HGF), ligand of Met receptor, the control of the axis HGF/Met by DNA methylation, and its importance for the nexus supportive cells-metastatic cells and for metastasis outgrowth. In bone metastasis of the 1833-xenograft model, DNA methyltransferase blockade using the chemotherapic drug 5-aza-2'-deoxycytidine (decitabine) strongly reduced the expression of HGF/Met receptor axis and of E-cadherin, with decrease of metastasis wideness and osteolysis, prolonging mice survival. Thus, DNA methylation events acted as commanders of breast carcinoma cells metastatizing to bone influencing the epithelial phenotype. HGF emerged as a bone-marrow stimulus, and the exosomes seemed to furnish HGF to metastatic cells. In fact, decitabine treatment similarly affected some markers of these microvesicles and HGF, indicating that its supply to recipient cells was prevented. Notably, in bone metastasis the hypomethylation of HGF, Met and E-cadherin promoters did not appear responsible for their elevated expression, but we suggest the involvement of hypermethylated regulators and of Wwox oncosuppressor, the latter being affected by decitabine. Wwox expression increased under decitabine strongly localizing in nuclei of bone metastases. We hypothesize a role of Wwox in Met activity since in vitro Wwox overexpression downregulated the level of nuclear-Met protein fragment and Met stability, also under long exposure of 1833 cells to decitabine. HGF enhanced phosphoMet and the activity in nuclei, an effect partially prevented by decitabine. Altogether, the data indicated the importance to target the tumor microenvironment by blocking epigenetic mechanisms, which control critical events for colonization such as HGF/Met axis and Wwox, as therapy of bone metastasis.

Simulated Space Radiation: Murine Skeletal Responses During Recovery and with Mechanical Stimulation

NASA Technical Reports Server (NTRS)

Shirazi-Fard, Yasaman; Zaragoza, Josergio; Schreurs, Ann-Sofie; Truong, Tiffany; Tahimic, Candice; Alwood, Joshua S.; Castillo, Alesha B.; Globus, R. K.

2016-01-01

Simulated space radiation at doses similar to those of solar particle events or a round-trip sojourn to Mars (1-2Gy) may cause skeletal tissue degradation and deplete stem/progenitor cell pools throughout the body. We hypothesized that simulated space radiation (SSR) causes late, time-dependent deficits in bone structure and bone cell function reflected by changes in gene expression in response to anabolic stimuli. We used a unique sequential dual ion exposure (proton and iron) for SSR to investigate time-dependence of responses in gene expression, cell function, and microarchitecture with respect to radiation and an anabolic stimulus of axial loading (AL). Male 16-wk C57BL6/J mice (n=120 total) were exposed to 0Gy (Sham, n=10), 56Fe (2Gy, positive control dose, n=10), or sequential ions for SSR (1Gy 1H/56Fe/1H, n=10) by total body irradiation (IR), and the tissues were harvested 2 or 6 mo. later. Further, to assess the response to anabolic stimuli, we subjected additional Sham-AL (n=15) and SSR-AL (n=15) groups to rest-inserted tibial axial loading (AL) starting at 1 and 5 months post-IR (-9N, 60 cycles/day, 3 days/wk, 4 wks). Exposure to 56Fe caused a significant reduction in cancellous bone volume fraction (BV/TV) compared to Sham (-34%) and SSR (-20%) in the proximal tibia metaphysis at 2-months post-IR; however BV/TV for SSR group was not different than Sham. Both 56Fe and SSR caused significant reduction in trabecular number (Tb.N) compared to Sham (-33% and -16%, respectively). Further, Tb.N for 56Fe (2Gy) was significantly lower than SSR (-21%). Ex vivo culture of marrow cells to assess growth and differentiation of osteoblast lineage cells 6 months post-IR showed that both 56Fe and SSR exposures significantly impaired colony formation compared to Sham (-66% and -54%, respectively), as well as nodule mineralization (-90% and -51%, respectively). Two-way analysis of variance showed that both mechanical loading and radiation reduced BV/TV, mechanical loading reduced trabecular thickness (Tb.Th), and radiation reduced Tb.N, at both time points. To assess acute response to mechanical stimuli, samples were harvested from a subset of Sham-AL (n=5) and SSR-AL (n=5) to measure changes in gene expression levels. Preliminary results indicate that axial loading increased expression of the antioxidant response gene Nfe2l2 and the osteoprogenitor-associated marker Runx2 in the bone marrow cells, and there was an interaction effect between axial loading and radiation at 2-months post-IR. Additional analyses of gene expression levels in the mineralized tissue are in progress. Results indicate that SSR caused persistent impairment of osteoblast colony formation and nodule mineralization 6-mo post-IR. Contrary to our hypothesis, simulated space radiation did not impair the ability of cancellous bone to respond to a mechanical anabolic stimulus, consistent with our previous findings [1]. Hence, compressive loading may be a potential countermeasure against spaceflight-induced bone loss.

Simulated Space Radiation: Murine Skeletal Responses During Recovery and with Mechanical Stimulation

NASA Technical Reports Server (NTRS)

Shirazi-Fard, Yasaman; Zaragoza, Josergio; Schreurs, Ann-Sofie; Truong, Tiffany; Tahimic, Candice; Alwood, Joshua S.; Globus, R. K.

2016-01-01

Simulated space radiation at doses similar to those of solar particle events or a round-trip sojourn to Mars (1-2Gy) may cause skeletal tissue degradation and deplete stem/progenitor cell pools throughout the body. We hypothesized that simulated space radiation (SSR) causes late, time-dependent deficits in bone structure and bone cell function reflected by changes in gene expression in response to anabolic stimuli. We used a unique sequential dual ion exposure (proton and iron) for SSR to investigate time-dependence of responses in gene expression, cell function, and microarchitecture with respect to radiation and an anabolic stimulus of axial loading (AL). Male 16-wk C57BL6/J mice (n=120 total) were exposed to 0Gy (Sham, n=10), 56Fe (2Gy, positive control dose, n=10), or sequential ions for SSR (1Gy 1H/56Fe/1H, n=10) by total body irradiation (IR), and the tissues were harvested 2 or 6 mo. later. Further, to assess the response to anabolic stimuli, we subjected additional Sham-AL (n=15) and SSR-AL (n=15) groups to rest-inserted tibial axial loading (AL) starting at 1 and 5 months post-IR (-9N, 60 cycles/day, 3 days/wk, 4 wks). Exposure to 56Fe caused a significant reduction in cancellous bone volume fraction (BV/TV) compared to Sham (-34%) and SSR (-20%) in the proximal tibia metaphysis at 2-months post-IR; however BV/TV for SSR group was not different than Sham. Both 56Fe and SSR caused significant reduction in trabecular number (Tb.N) compared to Sham (-33% and -16%, respectively). Further, Tb.N for 56Fe (2Gy) was significantly lower than SSR (-21%). Ex vivo culture of marrow cells to assess growth and differentiation of osteoblast lineage cells 6 months post-IR showed that both 56Fe and SSR exposures significantly impaired colony formation compared to Sham (-66% and -54%, respectively), as well as nodule mineralization (-90% and -51%, respectively). Two-way analysis of variance showed that both mechanical loading and radiation reduced BV/TV, mechanical loading reduced trabecular thickness (Tb.Th), and radiation reduced Tb.N, at both time points. To assess acute response to mechanical stimuli, samples were harvested from a subset of Sham-AL (n=5) and SSR-AL (n=5) to measure changes in gene expression levels. Preliminary results indicate that axial loading increased expression of the antioxidant response gene Nfe2l2 and the osteoprogenitor-associated marker Runx2 in the bone marrow cells, and there was an interaction effect between axial loading and radiation at 2-months post-IR. Additional analyses of gene expression levels in the mineralized tissue are in progress. Results indicate that SSR caused persistent impairment of osteoblast colony formation and nodule mineralization 6-mo post-IR. Contrary to our hypothesis, simulated space radiation did not impair the ability of cancellous bone to respond to a mechanical anabolic stimulus, consistent with our previous findings. Hence, compressive loading may be a potential countermeasure against spaceflight-induced bone loss.

Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

PubMed

Chai, Ryan C; Kouspou, Michelle M; Lang, Benjamin J; Nguyen, Chau H; van der Kraan, A Gabrielle J; Vieusseux, Jessica L; Lim, Reece C; Gillespie, Matthew T; Benjamin, Ivor J; Quinn, Julian M W; Price, John T

2014-05-09

Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ Dendritic Cells to the colon in response to Trichuris muris infection

PubMed Central

Bowcutt, Rowann; Bramhall, Michael; Logunova, Larisa; Wilson, Jim; Booth, Cath; Carding, Simon R.; Grencis, Richard; Cruickshank, Sheena

2014-01-01

The ability of the colon to generate an immune response to pathogens, such as the model pathogen Trichuris muris, is a fundamental and critical defense mechanism. Resistance to T.muris infection is associated with the rapid recruitment of dendritic cells (DCs) to the colonic epithelium via epithelial chemokine production. However, the epithelial-pathogen interactions that drive chemokine production are not known. We addressed the role of the cytosolic pattern recognition receptor Nod2. In response to infection, there was a rapid influx of CD103+CD11c+ DCs into the colonic epithelium in wild type (WT) mice whereas this was absent in Nod2−/− animals. In vitro chemotaxis assays and in vivo experiments using bone marrow chimeras of WT mice reconstituted with Nod2−/− bone marrow and infected with T. muris demonstrated that the migratory function of Nod2−/− DCs was normal. Investigation of colonic epithelial cell (CEC) innate responses revealed a significant reduction in epithelial production of the chemokines CCL2 and CCL5 but not CCL20 by Nod2-deficient CEC. Collectively, these data demonstrate the importance of Nod2 in CEC responses to infection and the requirement for functional Nod2 in initiating host epithelial chemokine mediated responses and subsequent DC recruitment and T cell responses following infection. PMID:24448097

Effects of alpha/beta-androstenediol immune regulating hormones on bone remodeling and apoptosis in osteoblasts.

PubMed

Urban, Nicole H; Chamberlin, Brett; Ramage, Samuel; Roberts, Zachary; Loria, Roger M; Beckman, Matthew J

2008-06-01

A large body of evidence suggests that the immune system directly impacts bone physiology. We tested whether immune regulating hormones (IRH), 17beta-androstenediol (beta-AED), 7beta,17beta-androstenetriol (beta-AET) or the 17alpha-androstenediol (alpha-AED), and 7alpha,17beta-androstenetriol (alpha-AET) metabolites could directly influence bone remodeling in vitro using human fetal osteoblasts (FOB-9). The impact on bone remodeling was examined by comparing the ratio of RANKL/OPG gene expression in response to AED and AET compounds. The alpha-AED was found to significantly increase in the ratio of RANKL/OPG gene expression and altering the morphology of RANKL stained FOB-9 cells. Cell viability was assessed using a Live/Dead assay. Again alpha-AED was unique in its ability to reduce the proportion of viable cells, and to induce mild apoptosis of FOB-9 cells. Treatment of FOB-9 cells with WY14643, an activator of PPAR-alpha and -gamma, also significantly elevated the percentage of dead cells. This increase was abolished by co-treatment with GW9962, a specific inhibitor of PPAR-gamma. Analysis of PPAR-gamma mRNA by Quantitative RT-PCR and its activation by DNA binding demonstrated that alpha-AED increased PPAR-gamma activation by 19%, while beta-AED conferred a 37% decrease in PPAR-gamma activation. In conclusion, alpha-AED opposed beta-AED by elevating a bone resorption scenario in osteoblast cells. The increase in RANKL/OPG is modulated by an activation of PPAR-gamma that in turn caused mild apoptosis of FOB-9 cells.

Leukemia and Benzene

PubMed Central

Snyder, Robert

2012-01-01

Excessive exposure to benzene has been known for more than a century to damage the bone marrow resulting in decreases in the numbers of circulating blood cells, and ultimately, aplastic anemia. Of more recent vintage has been the appreciation that an alternative outcome of benzene exposure has been the development of one or more types of leukemia. While many investigators agree that the array of toxic metabolites, generated in the liver or in the bone marrow, can lead to traumatic bone marrow injury, the more subtle mechanisms leading to leukemia have yet to be critically dissected. This problem appears to have more general interest because of the recognition that so-called “second cancer” that results from prior treatment with alkylating agents to yield tumor remissions, often results in a type of leukemia reminiscent of benzene-induced leukemia. Furthermore, there is a growing literature attempting to characterize the fine structure of the marrow and the identification of so called “niches” that house a variety of stem cells and other types of cells. Some of these “niches” may harbor cells capable of initiating leukemias. The control of stem cell differentiation and proliferation via both inter- and intra-cellular signaling will ultimately determine the fate of these transformed stem cells. The ability of these cells to avoid checkpoints that would prevent them from contributing to the leukemogenic response is an additional area for study. Much of the study of benzene-induced bone marrow damage has concentrated on determining which of the benzene metabolites lead to leukemogenesis. The emphasis now should be directed to understanding how benzene metabolites alter bone marrow cell biology. PMID:23066403

Tumor Response After Stereotactic Body Radiation Therapy to Nonspine Bone Metastases: An Evaluation of Response Criteria

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

McDonald, Rachel; Probyn, Linda; Poon, Ian

Purpose: To evaluate the applicability of the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) and University of Texas MD Anderson (MDA) Cancer Center criteria in the setting of stereotactic body radiation therapy (SBRT) to nonspine bone metastases. Methods: Patients who were treated with SBRT to nonspine bone metastases were identified by retrospective chart review. An independent musculoskeletal radiologist evaluated response to treatment using computed tomography (CT) scans. Results: Thirty-three patients were treated to 42 nonspine bone metastases. The most common primary cancer cites were renal cell carcinoma (RCC) (33.3%), lung (24.2%), and prostate (18.2%). Bone metastases were either mainlymore » lytic (57.1%), mainly sclerotic (28.6%), or mixed (14.3%). When lytic and sclerotic lesions were evaluated according to RECIST 1.1, local control (LC) was 83%, 85%, 88%, and 80% for those with CT imaging between months 1 to 3, 4 to 6, 7 to 9, and 10 to 12, respectively. When evaluated by the MDA criteria by density, LC within each time period was slightly greater. Overall LC decreased considerably when evaluated by MDA in terms of size. Conclusions: Consensus definitions of response are required as they have implications on clinical trials and disease management. Without consistent response criteria, outcomes from clinical trials cannot be compared and treatment efficacy remains undetermined.« less

Role of Complement on Broken Surfaces After Trauma.

PubMed

Huber-Lang, Markus; Ignatius, Anita; Brenner, Rolf E

2015-01-01

Activation of both the complement and coagulation cascade after trauma and subsequent local and systemic inflammatory response represent a major scientific and clinical problem. After severe tissue injury and bone fracture, exposure of innate immunity to damaged cells and molecular debris is considered a main trigger of the posttraumatic danger response. However, the effects of cellular fragments (e.g., histones) on complement activation remain enigmatic. Furthermore, direct effects of "broken" bone and cartilage surfaces on the fluid phase response of complement and its interaction with key cells of connective tissues are still unknown. Here, we summarize data suggesting direct and indirect complement activation by extracellular and cellular danger associated molecular patterns. In addition, key complement components and the corresponding receptors (such as C3aR, C5aR) have been detected on "exposed surfaces" of the damaged regions. On a cellular level, multiple effects of complement activation products on osteoblasts, osteoclasts, chondrocytes and mesenchymal stem cells have been found.In conclusion, the complement system may be activated by trauma-altered surfaces and is crucially involved in connective tissue healing and posttraumatic systemic inflammatory response.

Bone Repair Cells for Craniofacial Regeneration

PubMed Central

Pagni, G; Kaigler, D; Rasperini, G; Avila-Ortiz, G; Bartel, R; Giannobile, WV

2012-01-01

Reconstruction of complex craniofacial deformities is a clinical challenge in situations of injury, congenital defects or disease. The use of cell-based therapies represents one of the most advanced methods for enhancing the regenerative response for craniofacial wound healing. Both Somatic and Stem Cells have been adopted in the treatment of complex osseous defects and advances have been made in finding the most adequate scaffold for the delivery of cell therapies in human regenerative medicine. As an example of such approaches for clinical application for craniofacial regeneration, Ixmyelocel-T or bone repair cells are a source of bone marrow derived stem and progenitor cells. They are produced through the use of single pass perfusion bioreactors for CD90+ mesenchymal stem cells and CD14+ monocyte/macrophage progenitor cells. The application of ixmyelocel-T has shown potential in the regeneration of muscular, vascular, nervous and osseous tissue. The purpose of this manuscript is to highlight cell therapies used to repair bony and soft tissue defects in the oral and craniofacial complex. The field at this point remains at an early stage, however this review will provide insights into the progress being made using cell therapies for eventual development into clinical practice. PMID:22433781

NF-κB as a Therapeutic Target in Inflammatory-Associated Bone Diseases.

PubMed

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.

NF-κB as a Therapeutic Target in Inflammatory-Associated Bone Diseases

PubMed Central

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

In the trail of a new bio-sensor for measuring strain in bone: osteoblastic biocompatibility.

PubMed

Carvalho, Lídia; Alberto, Nélia J; Gomes, Pedro S; Nogueira, Rogério N; Pinto, João L; Fernandes, Maria H

2011-06-15

Fibre Bragg Grating (FBG) is an optical sensor recorded within the core of a standard optical fibre, which responds faithfully to strain and temperature. FBG sensors are a promising alternative to other sensing methodologies to assess bone mechanics in vivo. However, response of bone cells/bone tissue to FBGs and its sensing capability in this environment have not been recorded yet. The present study addressed these issues in long-term human osteoblastic cell cultures. Results showed that osteoblastic cells were able to adhere and proliferate over the fibre and, also, the protective polymer coating. RT-PCR analysis showed the expression of Col I, ALP, BMP-2, M-CSF, RANKL and OPG. In addition, cultures presented high ALP activity and the formation of a calcium phosphate mineralized extracellular matrix. Cell behavior over the fibre without and with the coating polymer was similar to that found in cultures grown in standard tissue culture plates (control). In addition to the excellent osteoblastic cytocompatibility, FBGs maintained the physical integrity and functionality, as its sensing capability was not affected through the culture period. Results suggest the possibility of in vivo osseointegration of the optical fibre/FBGs anticipating a variety of applications in bone mechanical dynamics. Copyright © 2011 Elsevier B.V. All rights reserved.

A crucial role for thiol antioxidants in estrogen-deficiency bone loss

PubMed Central

Lean, Jenny M.; Davies, Julie T.; Fuller, Karen; Jagger, Christopher J.; Kirstein, Barrie; Partington, Geoffrey A.; Urry, Zoë L.; Chambers, Timothy J.

2003-01-01

The mechanisms through which estrogen prevents bone loss are uncertain. Elsewhere, estrogen exerts beneficial actions by suppression of reactive oxygen species (ROS). ROS stimulate osteoclasts, the cells that resorb bone. Thus, estrogen might prevent bone loss by enhancing oxidant defenses in bone. We found that glutathione and thioredoxin, the major thiol antioxidants, and glutathione and thioredoxin reductases, the enzymes responsible for maintaining them in a reduced state, fell substantially in rodent bone marrow after ovariectomy and were rapidly normalized by exogenous 17-β estradiol. Moreover, administration of N-acetyl cysteine (NAC) or ascorbate, antioxidants that increase tissue glutathione levels, abolished ovariectomy-induced bone loss, while L-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis, caused substantial bone loss. The 17-β estradiol increased glutathione and glutathione and thioredoxin reductases in osteoclast-like cells in vitro. Furthermore, in vitro NAC prevented osteoclast formation and NF-κB activation. BSO and hydrogen peroxide did the opposite. Expression of TNF-α, a target for NF-κB and a cytokine strongly implicated in estrogen-deficiency bone loss, was suppressed in osteoclasts by 17-β estradiol and NAC. These observations strongly suggest that estrogen deficiency causes bone loss by lowering thiol antioxidants in osteoclasts. This directly sensitizes osteoclasts to osteoclastogenic signals and entrains ROS-enhanced expression of cytokines that promote osteoclastic bone resorption. PMID:12975476

Cell Therapy for Prophylactic Tolerance in Immunoglobulin E-mediated Allergy.

PubMed

Baranyi, Ulrike; Farkas, Andreas M; Hock, Karin; Mahr, Benedikt; Linhart, Birgit; Gattringer, Martina; Focke-Tejkl, Margit; Petersen, Arnd; Wrba, Fritz; Rülicke, Thomas; Valenta, Rudolf; Wekerle, Thomas

2016-05-01

Therapeutic strategies for the prophylaxis of IgE-mediated allergy remain an unmet medical need. Cell therapy is an emerging approach with high potential for preventing and treating immunological diseases. We aimed to develop a cell-based therapy inducing permanent allergen-specific immunological tolerance for preventing IgE-mediated allergy. Wild-type mice were treated with allergen-expressing bone marrow cells under a short course of tolerogenic immunosuppression (mTOR inhibition and costimulation blockade). Bone marrow was retrieved from a novel transgenic mouse ubiquitously expressing the major grass pollen allergen Phl p 5 as a membrane-anchored protein (BALB/c-Tg[Phlp5-GFP], here mPhl p 5). After transplantation recipients were IgE-sensitized at multiple time points with Phl p 5 and control allergen. Mice treated with mPhl p 5 bone marrow did not develop Phl p 5-specific IgE (or other isotypes) despite repeated administration of the allergen, while mounting and maintaining a strong humoral response towards the control allergen. Notably, Phl p 5-specific T cell responses and allergic airway inflammation were also completely prevented. Interestingly allergen-specific B cell tolerance was maintained independent of Treg functions indicating deletional tolerance as underlying mechanism. This proof-of-concept study demonstrates that allergen-specific immunological tolerance preventing occurrence of allergy can be established through a cell-based therapy employing allergen-expressing leukocytes. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A Novel Injectable Magnesium/Calcium Sulfate Hemihydrate Composite Cement for Bone Regeneration

PubMed Central

2015-01-01

Objective. A novel injectable magnesium/calcium sulfate hemihydrate (Mg/CSH) composite with improved properties was reported here. Methods. Composition, setting time, injectability, compressive strength, and bioactivity in simulated body fluid (SBF) of the Mg/CSH composite were evaluated. Furthermore, the cellular responses of canine bone marrow stromal cells (cBMSCs) and bone formation capacity after the implantation of Mg/CSH in tibia defects of canine were investigated. Results. Mg/CSH possessed a prolonged setting time and markedly improved injectability and mechanical property (p < 0.05). Mg/CSH samples showed better degradability than CSH in SBF after 21 days of soaking (p < 0.05). Moreover, the degrees of cell attachment, proliferation, and capability of osteogenic differentiation on the Mg/CSH specimens were higher than those on CSH, without significant cytotoxicity and with the increased proliferation index, ALP activity, and expression levels of integrin β1 and Coll I in cBMSCs (p < 0.05). Mg/CSH enhanced the efficiency of new bone formation at the tibia defect area, including the significantly elevated bone mineral density, bone area fraction, and Coll I expression level (p < 0.05). Conclusions. The results implied that this new injectable bone scaffold exhibited promising prospects for bone repair and had a great potential in bone tissue engineering. PMID:26114102

IL-20 bone diseases involvement and therapeutic target potential.

PubMed

Wang, Hsiao-Hsuan; Hsu, Yu-Hsiang; Chang, Ming-Shi

2018-04-24

Millions of people around the world suffer from bone disorders, likes osteoporosis, rheumatoid arthritis (RA), and cancer-induced osteolysis. In general, the bone remodeling balance is determined by osteoclasts and osteoblasts, respectively responsible for bone resorption and bone formation. Excessive inflammation disturbs the activities of these two kinds of cells, typically resulting in the bone loss. IL-20 is emerging as a potent angiogenic, chemotactic, and proinflammatory cytokine related to several chronic inflammatory disorders likes psoriasis, atherosclerosis, cancer, liver fibrosis, and RA. IL-20 has an important role in the regulation of osteoclastogenesis and osteoblastogenesis and is upregulated in several bone-related diseases. The anti-IL-20 monoclonal antibody treatment has a therapeutic potential in several experimental disease models including ovariectomy-induced osteoporosis, cancer-induced osteolysis, and bone fracture. This review article provides an overview describing the IL-20's biological functions in the common bone disorders and thus providing a novel therapeutic strategy in the future.

Engineered Fibrin Gels for Parallel Stimulation of Mesenchymal Stem Cell Proangiogenic and Osteogenic Potential

PubMed Central

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

Aggregatibacter actinomycetemcomitans, a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis

PubMed Central

Herbert, Bethany A.; Novince, Chad M.; Kirkwood, Keith L.

2015-01-01

Summary Aggregatibacter actinomycetemcomitans is a perio-pathogenic bacteria that has long been associated with localized aggressive periodontitis. The mechanisms of its pathogenicity have been studied in humans and pre-clinical experimental models. Although different serotypes of A. actinomycetemcomitans have differential virulence factor expression, A. actinomycetemcomitans cytolethal distending toxin (CDT), leukotoxin, and lipopolysaccharide (LPS) have been most extensively studied in the context of modulating the host immune response. Following colonization and attachment in the oral cavity, A. actinomycetemcomitans employs CDT, leukotoxin, and LPS to evade host innate defense mechanisms and drive a pathophysiologic inflammatory response. This supra-physiologic immune response state perturbs normal periodontal tissue remodeling/turnover and ultimately has catabolic effects on periodontal tissue homeostasis. In this review, we have divided the host response into two systems: non-hematopoietic and hematopoietic. Non-hematopoietic barriers include epithelium and fibroblasts that initiate the innate immune host response. The hematopoietic system contains lymphoid and myeloid-derived cell lineages that are responsible for expanding the immune response and driving the pathophysiologic inflammatory state in the local periodontal microenvironment. Effector systems and signaling transduction pathways activated and utilized in response to A. actinomycetemcomitans will be discussed to further delineate immune cell mechanisms during A. actinomycetemcomitans infection. Finally, we will discuss the osteo-immunomodulatory effects induced by A. actinomycetemcomitans and dissect the catabolic disruption of balanced osteoclast-osteoblast mediated bone remodeling, which subsequently leads to net alveolar bone loss. PMID:26197893

FoxO4 inhibits atherosclerosis through its function in bone marrow derived cells

PubMed Central

Zhu, Min; Zhang, Qing-Jun; Wang, Lin; Li, Hao; Liu, Zhi-Ping

2011-01-01

Objectives FoxO proteins are transcription factors involved in varieties of cellular processes, including immune cell homeostasis, cytokine production, anti-oxidative stress, and cell proliferation and differentiation. Although these processes are implicated in the development of atherosclerosis, very little is known about the role of FoxO proteins in the context of atherosclerosis. Our objectives were to determine whether and how inactivation of Foxo4, a member of the FoxO family, in vivo promotes atherosclerosis. Methods and Results Apolipoprotein E-deficient (apoE−/−) mice were crossbred with animals lacking Foxo4 (Foxo4−/−). After 10 weeks on a high fat diet (HFD), Foxo4−/−apoE−/− mice showed elevated atherosclerosis and increased amount of macrophages and T cells in the plaque compared to apoE−/− mice. Bone marrow transplantations of chimeric C57B/6 mice reconstituted with either wild-type or Foxo4−/− bone marrows indicate that Foxo4-deficiency in bone marrow derived cells sufficiently promoted atherosclerosis. Foxo4-null macrophages produced elevated inflammatory cytokine IL-6 and levels of reactive oxygen species (ROS) in response to lipopolysaccharides in vitro. Serum levels of IL-6 were upregulated in HFD-fed Foxo4−/−apoE−/− mice compared to those of apoE−/− mice. Conclusions FoxO4 inhibits atherosclerosis through bone marrow derived cells, possibly by inhibition of ROS and inflammatory cytokines that promote monocyte recruitment and/or retention. PMID:22005198

Modulation of Ocular Inflammation by Mesenchymal Stem Cells

DTIC Science & Technology

2017-03-01

mature myeloid cells in 64 host defense and resolution of inflammation, excessive innate immune response can have 65 deleterious effects on tissue...that MSCs can regulate 69 functions of mature innate immune cells , including polarization of inflammatory macrophages 70 into an anti-inflammatory... cells 191 As immune cells are primarily developed in lymphoid organs, single cell suspensions from bone 192 marrow, spleen, and submandibular lymph

IL-18 Contributes to Bone Cancer Pain by Regulating Glia Cells and Neuron Interaction.

PubMed

Liu, Su; Liu, Yue-Peng; Lv, You; Yao, Jun-Li; Yue, Dong-Mei; Zhang, Mao-Yin; Qi, Dun-Yi; Liu, Gong-Jian

2018-02-01

Glial cell hyperactivity has been proposed to be responsible for chronic pain, however, the mechanisms remain unclear. Interleukin (IL)-18, released from glial cells, has been reported to be involved in neuropathic pain. In this study, we investigated the role of IL-18 in bone cancer pain. Bone cancer pain was mimicked by injecting Walker-256 mammary gland carcinoma cells into the intramedullary space of the tibia in rats. Expression and location of IL-18 and the IL-18 receptor were tested. To investigate the contribution of IL-18 signaling to bone cancer pain, IL-18 binding protein and recombinant IL-18 were used. To investigate the mechanisms of glial cells effects, MK801, N-methyl-D-aspartate (NMDA) receptor inhibitor, and Src kinase-specific inhibitor PP1 were used. Tumor cell implantation (TCI) treatment increased expression of IL-18 and IL-18 receptor in spinal cord. The time course of IL-18 upregulation was correlated with TCI-induced pain behaviors. Blocking the IL-18 signaling pathway prevented and reversed bone cancer-related pain behaviors. Meanwhile, blocking IL-18 signaling also suppressed TCI-induced glial cell hyperactivity, as well as activation of GluN2B and subsequent Ca 2+ -dependent signaling. Spinal administration of recombinant IL-18 in naive rat induced significant mechanical allodynia, as well as GluN2B activation. However, intrathecal injection of MK801 failed to suppress recombinant IL-18-induced GluN2B phosphorylation, whereas Src kinase inhibitor PP1 significantly inhibited IL-18-induced GluN2B activation. IL-18-mediated glial-glia and glial-neuron interaction may facilitate bone cancer pain. Blocking IL-18 signaling may effectively prevent and/or suppress bone cancer pain. IL-18 signaling may be a new target for cancer pain therapy. Copyright © 2017 The American Pain Society. Published by Elsevier Inc. All rights reserved.

Establishment of a cell line producing bone morphogenetic protein from a human osteosarcoma.

PubMed

Takaoka, K; Yoshikawa, H; Masuhara, K; Sugamoto, K; Tsuda, T; Aoki, Y; Ono, K; Sakamoto, Y

1989-07-01

A human osteosarcoma cell line was established from a biopsy specimen from a 13-year-old girl. The osteosarcoma tissue was maintained in athymic nude mice (Balb C nu/nu) by serial transplantation for three years. The tumor was excised from a host mouse and digested with collagenase. The isolated cells were cultured by 98 passages in 14 months, and clones of osteosarcoma cells were obtained by limiting dilution. A clone named human osteosarcoma cell 6 (H-OS-6) that showed the osteoblastic phenotypes of productions of bone morphogenetic protein (BMP) and alkaline phosphatase and a response to human parathyroid hormone (h-PTH 1-34) was selected. The morphology of its chromosomes indicated its human origin. This human osteosarcoma cell line is unique in producing BMP under in vitro conditions.

Monomethylarsonous acid (MMA+3) Inhibits IL-7 Signaling in Mouse Pre-B Cells

PubMed Central

Ezeh, Peace C.; Xu, Huan; Lauer, Fredine T.; Liu, Ke Jian; Hudson, Laurie G.; Burchiel, Scott W.

2016-01-01

Our previously published data show that As+3 in vivo and in vitro, at very low concentrations, inhibits lymphoid, but not myeloid stem cell development in mouse bone marrow. We also showed that the As+3 metabolite, monomethylarsonous acid (MMA+3), was responsible for the observed pre-B cell toxicity caused by As+3. Interleukin-7 (IL-7) is the primary growth factor responsible for pre-lymphoid development in mouse and human bone marrow, and Signal Transducer and Activator of Transcription 5 (STAT5) is a transcriptional factor in the IL-7 signaling pathway. We found that MMA+3 inhibited STAT5 phosphorylation at a concentration as low as 50 nM in mouse bone marrow pre-B cells. Inhibition of STAT5 phosphorylation by As+3 occurred only at a concentration of 500 nM. In the IL-7 dependent mouse pre-B 2E8 cell line, we also found selective inhibition of STAT5 phosphorylation by MMA+3, and this inhibition was dependent on effects on JAK3 phosphorylation. IL-7 receptor expression on 2E8 cell surface was also suppressed by 50 nM MMA+3 at 18 h. As further evidence for the inhibition of STAT5, we found that the induction of several genes required in B cell development, cyclin D1, E2A, EBF1, and PAX5, were selectively inhibited by MMA+3. Since 2E8 cells lack the enzymes responsible for the conversion of As+3 to MMA+3 in vitro, the results of these studies suggest that As+3 induced inhibition of pre-B cell formation in vivo is likely dependent on the formation of MMA+3 which in turn inhibits IL-7 signaling at several steps in mouse pre-B cells. PMID:26518055

Ammonium tetrathiomolybdate enhances the antitumor effects of cetuximab via the suppression of osteoclastogenesis in head and neck squamous carcinoma.

PubMed

Morisawa, Ayaka; Okui, Tatsuo; Shimo, Tsuyoshi; Ibaragi, Soichiro; Okusha, Yuka; Ono, Mitsuaki; Nguyen, Thi Thu Ha; Hassan, Nur Mohammad Monsur; Sasaki, Akira

2018-03-01

Head and neck squamous cell carcinoma (HNSCC) poses a significant challenge clinically where one of the mechanisms responsible for the invasion into facial bones occurs via the activation of osteoclasts. Copper has been demonstrated to play a key role in skeletal remodeling. However, the role of copper in cancer-associated bone destruction is thus far unknown. Lysyl oxidase (LOX) is a copper-dependent enzyme that promotes osteoclastogenesis. In the present study, we investigated the effects of copper on HNSCC with bone invasion by the copper chelator, ammonium tetrathiomolybdate (TM) in vitro and in vivo. We demonstrate that TM blocks the proliferation of HNSCC cells, inhibits LOX activation and decreases the expression of the receptor activator of nuclear factor-κB ligand (RANKL) in osteoblasts and osteocytes, subsequently suppressing bone destruction. These findings suggest that copper is a potential target for the treatment of HNSCCs associated with bone destruction.

The effects of simulated hypogravity on murine bone marrow cells

NASA Technical Reports Server (NTRS)

Lawless, Desales

1989-01-01

Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

Role of geometrical cues in bone marrow-derived mesenchymal stem cell survival, growth and osteogenic differentiation.

PubMed

Gupta, Dhanak; Grant, David M; Zakir Hossain, Kazi M; Ahmed, Ifty; Sottile, Virginie

2018-02-01

Mesenchymal stem cells play a vital role in bone formation process by differentiating into osteoblasts, in a tissue that offers not a flat but a discontinuous three-dimensional (3D) topography in vivo. In order to understand how geometry may be affecting mesenchymal stem cells, this study explored the influence of 3D geometry on mesenchymal stem cell-fate by comparing cell growth, viability and osteogenic potential using monolayer (two-dimensional, 2D) with microsphere (3D) culture systems normalised to surface area. The results suggested lower cell viability and reduced cell growth in 3D. Alkaline phosphatase activity was higher in 3D; however, both collagen and mineral deposition appeared significantly lower in 3D, even after osteogenic supplementation. Also, there were signs of patchy mineralisation in 3D with or without osteogenic supplementation as early as day 7. These results suggest that the convex surfaces on microspheres and inter-particulate porosity may have led to variable cell morphology and fate within the 3D culture. This study provides deeper insights into geometrical regulation of mesenchymal stem cell responses applicable for bone tissue engineering.

Scaffolds of hydroxyl apatite nanoparticles disseminated in 1, 6-diisocyanatohexane-extended poly(1, 4-butylene succinate)/poly(methyl methacrylate) for bone tissue engineering.

PubMed

Kaur, Kulwinder; Singh, K J; Anand, Vikas; Bhatia, Gaurav; Kaur, Raminderjit; Kaur, Manpreet; Nim, Lovedeep; Arora, Daljit Singh

2017-02-01

Poly(1, 4-butyl succinate) extended 1, 6-diisocyanatohexane (PBSu-DCH) polymers and Polymethylmethacrylate (PMMA) scaffolds decorated with nano hydroxyl apatite have been prepared and characterized for regeneration of bone in cranio-maxillofacial region. Synthesized scaffolds revealed good response in bone regeneration and excellent cell viability in comparison to commercial available glass plate, which lead to better proliferation of MG-63 cell lines. Additionally, they demonstrate high porosity and excellent water retention ability. Moreover, controlled degradation (in pH=7.4) and sustained drug release in pH (4.5 and 7.4) are advantages of these scaffolds to serve as delivery vehicles for therapeutic drugs. Samples also provide the protection against Escherichia coli and Methicillin Resistant Staphylococcus aureus microorganisms which can be helpful for quick recovery of the patient. In-vitro inflammatory response has been assessed via adsorption of human plasma/serum proteins on the surface of the scaffolds. Results suggest that prepared scaffolds have good bone regeneration ability and provide friendly environment for the cell growth with the additional advantage of protection of the surrounding tissues from microbial infection. With all these features, it is speculated that these scaffolds will have wide utility in the area of tissue engineering and regenerative medicine. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of space relevant radiation exposure on differentiation and mineralization of murine osteoprogenitor cells

NASA Astrophysics Data System (ADS)

Lau, Patrick; Hu, Yueyuan; Hellweg, Christine; Baumstark-Khan, Christa; Reitz, Guenther

Extended exposure to altered gravity conditions like during long-term space flight results in significant bone loss. Exposure to ionizing radiation for cancer therapy causes bone damage and may increase the risk of fractures. Similarly, besides altered gravity conditions, astronauts on exploratory missions beyond low-Earth orbit will be exposed to high-energy heavy ions in addition to proton and photon radiation, although for prolonged periods and at lower doses and dose rates compared with therapy. Space conditions may place astronauts at a greater risk for mission-critical fractures. Until now, little is known about the effects of space radiation on the skeletal system especially on osteoprogenitor cells. Accelerator facilities are used to simulate parts of the radiation environment in space. Heavy ion accelerators therefore could be used to assess radiation risks for astronauts who will be exposed to higher radiation doses e.g. on a Mars mission. The aim of the present study was to determine the biological effects of spaceflight-relevant radiation exposure on the cellular level using murine osteoprogenitor cell lines compared to nonirradiated controls. To gain a deeper understanding of bone cell differenti-ation and mineralization after exposure to heavy ions, we examined gene expression modulation of bone specific transcription factors, osteoblast specific marker genes as well as genes function as coupling factors that link bone resorption to bone formation. We investigated the transcrip-tional modulation of type I collagen (Col I), osteocalcin (Ocn), Transforming growth factor-β1 (TGF-β1), interleukin-6 (IL-6) and the bone specific transcription factor Runx2 (Cbfa1). To gain deeper insight into potential cellular mechanisms involved in cellular response after ex-posure to heavy ions, we investigated gene expression modulations after exposure to energetic carbon ions (35 MeV/u, 73.2 keV/µm), iron ions (1000 MeV/u, 150 keV/µm) and lead ions (29 MeV/u, 9600 keV/µm) versus low LET X-rays. Exposure to X-irradiation dose-dependently increased the mRNA levels of Runx2 (cbfa1) whereas expression values of OCN and TGF-β1 were elevated at later time points. Exposure to heavy ions provoked a more marked effect on bone specific gene expression within the differentiation process. Collectively, our results indi-cate that heavy ions facilitate differentiation more effectively than X-rays as a major response in the progeny of irradiated osteoprogenitor cells. The data presented allow us to suggest that exposure to ionizing radiation interferes with bone formation at the level of cellular differenti-ation. In this regard, further experiments are needed to investigate gene expression patterns in mammalian cells that respond to differentiation after exposure to ionizing radiation.

T-cell chronic lymphocytic leukemia in a double yellow-headed Amazon parrot (Amazona ochrocephala oratrix).

PubMed

Osofsky, Anna; Hawkins, Michelle G; Foreman, Oded; Kent, Michael S; Vernau, William; Lowenstine, Linda J

2011-12-01

An adult, male double yellow-headed Amazon parrot (Amazona ochrocephala oratrix) was diagnosed with chronic lymphocytic leukemia based on results of a complete blood cell count and cytologic examination of a bone marrow aspirate. Treatment with oral chlorambucil was attempted, but no response was evident after 40 days. The bird was euthanatized, and the diagnosis of chronic lymphocytic leukemia was confirmed on gross and microscopic examination of tissues. Neoplastic lymphocytes were found in the bone marrow, liver, kidney, testes, and blood vessels. Based on CD3-positive immunocytochemical and immunohistochemical immunophenotyping, the chronic lymphocytic leukemia was determined to be of T-cell origin.

Quantifying migration and polarization of murine mesenchymal stem cells on different bone substitutes by confocal laser scanning microscopy.

PubMed

Roldán, J C; Chang, E; Kelantan, M; Jazayeri, L; Deisinger, U; Detsch, R; Reichert, T E; Gurtner, G C

2010-12-01

Cell migration is preceded by cell polarization. The aim of the present study was to evaluate the impact of the geometry of different bone substitutes on cell morphology and chemical responses in vitro. Cell polarization and migration were monitored temporally by using confocal laser scanning microscopy (CLSM) to follow green fluorescent protein (GFP)±mesenchymal stem cells (MSCs) on anorganic cancellous bovine bone (Bio-Oss(®)), β-tricalcium phosphate (β-TCP) (chronOS(®)) and highly porous calcium phosphate ceramics (Friedrich-Baur-Research-Institute for Biomaterials, Germany). Differentiation GFP±MSCs was observed using pro-angiogenic and pro-osteogenic biomarkers. At the third day of culture polarized vs. non-polarized cellular sub-populations were clearly established. Biomaterials that showed more than 40% of polarized cells at the 3rd day of culture, subsequently showed an enhanced cell migration compared to biomaterials, where non-polarized cells predominated (p<0.003). This trend continued untill the 7th day of culture (p<0.003). The expression of vascular endothelial growth factor was enhanced in biomaterials where cell polarization predominated at the 7th day of culture (p=0.001). This model opens an interesting approach to understand osteoconductivity at a cellular level. MSCs are promising in bone tissue engineering considering the strong angiogenic effect before differentiation occurs. Copyright © 2010 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Antithetical effects of hemicellulase-treated Agaricus blazei on the maturation of murine bone-marrow-derived dendritic cells

PubMed Central

Kawamura, Masaki; Kasai, Hirotake; He, Limin; Deng, Xuewen; Yamashita, Atsuya; Terunuma, Hiroshi; Horiuchi, Isao; Tanabe, Fuminori; Ito, Masahiko

2005-01-01

We report the effects of hemicellulase-treated Agaricus blazei (ABH) on the maturation of bone-marrow-derived dendritic cells (BMDCs). ABH activated immature BMDCs, inducing up-regulation of surface molecules, such as CD40, CD80 and major histocompatibility complex class I antigens, as well as inducing allogeneic T-cell proliferation and T helper type 1 cell development. However, unlike lipopolysaccharide (LPS), ABH did not stimulate the BMDCs to produce proinflammatory cytokines, such as interleukin-12 (IL-12) p40, tumour necrosis factor-α, or IL-1β. In addition, ABH suppressed LPS-induced DC responses. Pretreatment of DCs with ABH markedly reduced the levels of LPS-induced cytokine secretion, while only slightly decreasing up-regulation of the surface molecules involved in maturation. ABH also had a significant impact on peptidoglycan-induced or CpG oligodeoxynucleotide-induced IL-12p40 production in DCs. The inhibition of LPS-induced responses was not associated with a cytotoxic effect of ABH nor with an anti-inflammatory effect of IL-10. However, ABH decreased NF-κB-induced reporter gene expression in LPS-stimulated J774.1 cells. Interestingly, DCs preincubated with ABH and then stimulated with LPS augmented T helper type 1 responses in culture with allogeneic T cells as compared to LPS-stimulated but non-ABH-pretreated DCs. These observations suggest that ABH regulates DC-mediated responses. PMID:15720441

Genetic models in applied physiology: selected contribution: effects of spaceflight on immunity in the C57BL/6 mouse. I. Immune population distributions

NASA Technical Reports Server (NTRS)

Pecaut, Michael J.; Nelson, Gregory A.; Peters, Luanne L.; Kostenuik, Paul J.; Bateman, Ted A.; Morony, Sean; Stodieck, Louis S.; Lacey, David L.; Simske, Steven J.; Gridley, Daila S.

2003-01-01

There are several aspects of the spaceflight environment that may lead to changes in immunity: mission-related psychological stress, radiation, and changes in gravity. On December 5, 2001, the space shuttle Endeavor launched for a 12-day mission to examine these effects on C57BL/6 mice for the first time. On their return, assays were performed on the spleen, blood, and bone marrow. In response to flight, there were no significant differences in the general circulating leukocyte proportions. In contrast, there was an increase in splenic lymphocyte percentages, with a corresponding decrease in granulocytes. There was an overall shift in splenic lymphocytes away from T cells toward B cells, and a decrease in the CD4-to-CD8 ratios due to a decrease in T helpers. In contrast, there were proportional increases in bone marrow T cells, with decreases in B cells. Although the blast percentage and count were decreased in flight mice, the CD34(+) population was increased. The data were more consistent with a shift in bone marrow populations rather than a response to changes in the periphery. Many of the results are similar to those using other models. Clearly, spaceflight can influence immune parameters ranging from hematopoiesis to mature leukocyte mechanisms.

HLA-B27 Alters the Response to TNFα and Promotes Osteoclastogenesis in Bone Marrow Monocytes from HLA-B27 Transgenic Rats

PubMed Central

Layh-Schmitt, Gerlinde; Yang, Eva Y.; Kwon, Grace; Colbert, Robert A.

2013-01-01

Objective To determine whether HLA-B27 expression alters the response of bone marrow monocytes (BMMo) from HLA-B27/human β2-microglobulin transgenic (B27-Tg) rats to tumor necrosis factor-α (TNFα), and whether this affects cells involved in bone homeostasis. Methods BMMo were treated with receptor activator of NF-κB ligand or TNFα to promote osteoclast formation. Osteoclasts were quantified by counting. Gene expression was measured using quantitative polymerase chain reaction, and protein was detected by enzyme-linked immunosorbent assay, immunoblotting, or immunofluorescence. Effects of endogenously produced cytokines on osteoclast formation were determined with neutralizing antibodies. Results TNFα enhanced osteoclast formation 2.5-fold in HLA-B27-expressing cells compared to either wild type or HLA-B7/human β2-microglobulin expressing monocytes. TNFα induced approximately 4-fold upregulation of HLA-B27, which was associated with accumulation of misfolded heavy chains, binding of the ER chaperone BiP, and activation of an ER stress response, which was not seen with HLA-B7. No differences were seen with RANKL-induced osteoclastogenesis. Enhanced interleukin-1α (IL-1α) production from ER stressed B27-Tg BMMo was found to be necessary and sufficient for enhanced osteoclast formation. However, B27-Tg BMMo also produced more interferon-β (IFNβ), which attenuated the effect of IL-1α on osteoclast formation. Conclusions HLA-B27-induced ER stress alters the response of BMMo from B27-Tg rats to TNFα, which is associated with enhanced production of IL-1α and IFNβ, cytokines that exhibit opposing effects on osteoclast formation. The altered response of cells expressing HLA-B27 to pro-inflammatory cytokines suggests that this MHC class I allele may contribute to the pathogenesis of spondyloarthritis and its unique phenotype through downstream effects involving alterations in bone homeostasis. PMID:23666508

Receptors and effects of gut hormones in three osteoblastic cell lines.

PubMed

Pacheco-Pantoja, Elda L; Ranganath, Lakshminarayan R; Gallagher, James A; Wilson, Peter J M; Fraser, William D

2011-07-29

In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells. mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB]) were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63) showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP), procollagen type 1 amino-terminal propeptides (P1NP), and osteocalcin production. The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor) expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and obestatin. ALP showed higher levels in Saos-2 after GIP, GHR and OB and in TE-85 after GHR. P1NP showed higher levels after GIP and OB in Saos-2. Decreased levels of P1NP were observed in TE-85 and MG-63 after GLP-1, GLP-2 and OB. MG-63 showed opposite responses in osteocalcin levels after GLP-2. These results suggest that osteoblast activity modulation varies according to different development stage under different nutrition related-peptides.

Short exposure of maturing, bone marrow-derived dendritic cells to norepinephrine: impact on kinetics of cytokine production and Th development.

PubMed

Maestroni, Georges J M

2002-08-01

The information gathered by dendritic cells (DC) during the innate immune response to a pathogen is determinant for the type of adaptive response. Here we show that short-term (3 h) exposure of bone marrow-derived DC to norepinephrine (NE), at the beginning of lipopolysaccharide (LPS) or keyhole limpet hemocyanin (KLH) stimulation hampers IL-12 production and increases IL-10 release. The NE effect was mediated by both beta- and alpha2-adrenergic receptors. The capacity of NE-exposed DC to produce IL-12 upon CD40 cross-linking as well as to stimulate allogeneic T-helper (Th) lymphocytes was reduced. Adoptive transfer of NE-exposed DC induced a Th2 slanted response in vivo. Thus, a brief NE exposure of antigen-stimulated DC seems to limit their Th1 polarizing properties. Noteworthy, the ganglionic blocker pentolinium administered in mice before skin sensitization with fluoroscein isothiocyanate (FITC) could increase the Th1-type response in the draining lymph nodes. Our results suggest that the extent of Th differentiation in the response to an antigen might be influenced by the local sympathetic nervous activity in the early phase of dendritic cell stimulation.

The clinical use of regenerative therapy in COPD

PubMed Central

Lipsi, Roberto; Rogliani, Paola; Calzetta, Luigino; Segreti, Andrea; Cazzola, Mario

2014-01-01

Regenerative or stem cell therapy is an emerging field of treatment based on stimulation of endogenous resident stem cells or administration of exogenous stem cells to treat diseases or injury and to replace malfunctioning or damaged tissues. Current evidence suggests that in the lung, these cells may participate in tissue homeostasis and regeneration after injury. Animal and human studies have demonstrated that tissue-specific stem cells and bone marrow-derived cells contribute to lung tissue regeneration and protection, and thus administration of exogenous stem/progenitor cells or humoral factors responsible for the activation of endogenous stem/progenitor cells may be a potent next-generation therapy for chronic obstructive pulmonary disease. The use of bone marrow-derived stem cells could allow repairing and regenerate the damaged tissue present in chronic obstructive pulmonary disease by means of their engraftment into the lung. Another approach could be the stimulation of resident stem cells by means of humoral factors or photobiostimulation. PMID:25548520

Zirconia toughened alumina ceramic foams for potential bone graft applications: fabrication, bioactivation, and cellular responses.

PubMed

He, X; Zhang, Y Z; Mansell, J P; Su, B

2008-07-01

Zirconia toughened alumina (ZTA) has been regarded as the next generation orthopedic graft material due to its excellent mechanical properties and biocompatibility. Porous ZTA ceramics with good interconnectivity can potentially be used as bone grafts for load-bearing applications. In this work, three-dimensional (3D) interconnected porous ZTA ceramics were fabricated using a direct foaming method with egg white protein as binder and foaming agent. The results showed that the porous ZTA ceramics possessed a bimodal pore size distribution. Their mechanical properties were comparable to those of cancellous bone. Due to the bio-inertness of alumina and zirconia ceramics, surface bioactivation of the ZTA foams was carried out in order to improve their bioactivity. A simple NaOH soaking method was employed to change the surface chemistry of ZTA through hydroxylation. Treated samples were tested by conducting osteoblast-like cell culture in vitro. Improvement on cells response was observed and the strength of porous ZTA has not been deteriorated after the NaOH treatment. The porous 'bioactivated' ZTA ceramics produced here could be potentially used as non-degradable bone grafts for load-bearing applications.

Hydrogen gas treatment prolongs replicative lifespan of bone marrow multipotential stromal cells in vitro while preserving differentiation and paracrine potentials.

PubMed

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.

Hydrogen gas treatment prolongs replicative lifespan of bone marrow multipotential stromal cells in vitro while preserving differentiation and paracrine potentials

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

Supplying osteogenesis to dead bone using an osteogenic matrix cell sheet.

PubMed

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.

Osteoblast and osteoclast responses to A/B type carbonate-substituted hydroxyapatite ceramics for bone regeneration.

PubMed

Germaini, Marie-Michèle; Detsch, Rainer; Grünewald, Alina; Magnaudeix, Amandine; Lalloue, Fabrice; Boccaccini, Aldo R; Champion, Eric

2017-06-06

The influence of carbonate substitution (4.4 wt%, mixed A/B type) in hydroxyapatite ceramics for bone remodeling scaffolds was investigated by separately analyzing the response of pre-osteoblasts and osteoclast-like cells. Carbonated hydroxyapatite (CHA) (Ca 9.5 (PO 4 ) 5.5 (CO 3 ) 0.5 (OH)(CO 3 ) 0.25 -CHA), mimicking the chemical composition of natural bone mineral, and pure hydroxyapatite (HA) (Ca 10 (PO 4 ) 6 (OH) 2 -HA) porous ceramics were processed to obtain a similar microstructure and surface physico-chemical properties (grain size, porosity ratio and pore size, surface roughness and zeta potential). The biological behavior was studied using MC3T3-E1 pre-osteoblastic and RAW 264.7 monocyte/macrophage cell lines. Chemical dissolution in the culture media and resorption lacunae produced by osteoclasts occur with both HA and CHA ceramics, but CHA exhibits much higher dissolution and greater bioresorption ability. CHA ceramics promoted a significantly higher level of pre-osteoblast proliferation. Osteoblastic differentiation, assessed by qRT-PCR of RUNX2 and COLIA2, and pre-osteoclastic proliferation and differentiation were not significantly different on CHA or HA ceramics but cell viability and metabolism were significantly greater on CHA ceramics. Thus, the activity of both osteoclast-like and osteoblastic cells was influenced by the carbonate substitution in the apatite structure. Furthermore, CHA showed a particularly interesting balance between biodegradation, by osteoclasts and chemical dissolution, and osteogenesis through osteoblasts' activity, to stimulate bone regeneration. It is hypothesized that this amount of 4.4 wt% carbonate substitution leads to an adapted concentration of calcium in the fluid surrounding the ceramic to stimulate the activity of cells. These results highlight the superior biological behavior of microporous 4.4 wt% A/B CHA ceramics that could beneficially replace the commonly used HA of biphasic calcium phosphates for future applications in bone tissue engineering.

Multilineage response in aplastic anemia patients following long-term administration of filgrastim (recombinant human granulocyte colony stimulating factor).

PubMed

Sonoda, Y; Ohno, Y; Fujii, H; Takahashi, T; Nakayama, S; Haruyama, H; Nasu, K; Shimazaki, C; Hara, H; Kanamaru, A

1993-11-01

The present multicenter study was undertaken to confirm whether filgrastim/recombinant human granulocyte colony stimulating factor (rhG-CSF) could mobilize residual multipotential stem cells by its G0-shortening effect in patients with aplastic anemia (AA) and induce a multilineage response. Twenty-seven patients with acquired severe or moderate AA received long-term administration (2 to 12+ months) of rhG-CSF in doses from 100 to 400 micrograms/body/day by s.c. injection or 250 to 1,500 micrograms/body/day by i.v. infusion. Twenty-six out of the 27 evaluable patients showed a substantial increase in neutrophils associated with a recovery of myeloid precursors in bone marrow within one month of therapy. Interestingly, 10 out of the 27 patients showed a dramatic improvement in severe anemia after two to ten months of therapy. Moreover, severe thrombocytopenia improved after two to four months of therapy in three out of these ten patients accompanied by a significant increase in megakaryocytes in bone marrow. Clonal cultures of bone marrow cells revealed a recovery in myeloid as well as erythroid precursors in most of these ten patients. In two patients who showed a trilineage response, mixed and megakaryocyte colony formations also recovered. These results suggest that long-term administration of rhG-CSF mobilizes myeloid, erythroid, megakaryocyte and multipotential progenitor cells and induces a multilineage response in some patients with AA.

Accelerated Bone Repair After Plasma Laser Corticotomies

PubMed Central

Leucht, Philipp; Lam, Kentson; Kim, Jae-Beom; Mackanos, Mark A.; Simanovskii, Dmitrii M.; Longaker, Michael T.; Contag, Christopher H.; Schwettman, H Alan; Helms, Jill A.

2007-01-01

Objective: To reveal, on a cellular and molecular level, how skeletal regeneration of a corticotomy is enhanced when using laser-plasma mediated ablation compared with conventional mechanical tissue removal. Summary Background Data: Osteotomies are well-known for their most detrimental side effect: thermal damage. This thermal and mechanical trauma to adjacent bone tissue can result in the untoward consequences of cell death and eventually in a delay in healing. Methods: Murine tibial corticotomies were performed using a conventional saw and a Ti:Sapphire plasma-generated laser that removes tissue with minimal thermal damage. Our analyses began 24 hours after injury and proceeded to postsurgical day 6. We investigated aspects of wound repair ranging from vascularization, inflammation, cell proliferation, differentiation, and bone remodeling. Results: Histology of mouse corticotomy sites uncovered a significant difference in the onset of bone healing; whereas laser corticotomies showed abundant bone matrix deposition at postsurgical day 6, saw corticotomies only exhibited undifferentiated tissue. Our analyses uncovered that cutting bone with a saw caused denaturation of the collagen matrix due to thermal effects. This denatured collagen represented an unfavorable scaffold for subsequent osteoblast attachment, which in turn impeded deposition of a new bony matrix. The matrix degradation induced a prolonged inflammatory reaction at the cut edge to create a surface favorable for osteochondroprogenitor cell attachment. Laser corticotomies were absent of collagen denaturation, therefore osteochondroprogenitor cell attachment was enabled shortly after surgery. Conclusion: In summary, these data demonstrate that corticotomies performed with Ti:Sapphire lasers are associated with a reduced initial inflammatory response at the injury site leading to accelerated osteochondroprogenitor cell migration, attachment, differentiation, and eventually matrix deposition. PMID:17592303

The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

PubMed

Ruggiu, Alessandra; Ulivi, Valentina; Sanguineti, Francesca; Cancedda, Ranieri; Descalzi, Fiorella

2013-12-01

Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sustained release of sphingosine 1-phosphate for therapeutic arteriogenesis and bone tissue engineering.

PubMed

Sefcik, Lauren S; Petrie Aronin, Caren E; Wieghaus, Kristen A; Botchwey, Edward A

2008-07-01

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that impacts migration, proliferation, and survival in diverse cell types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. In this study, we investigated the effects of sustained release of S1P on microvascular remodeling and associated bone defect healing in vivo. The murine dorsal skinfold window chamber model was used to evaluate the structural remodeling response of the microvasculature. Our results demonstrated that 1:400 (w/w) loading and subsequent sustained release of S1P from poly(lactic-co-glycolic acid) (PLAGA) significantly enhanced lumenal diameter expansion of arterioles and venules after 3 and 7 days. Incorporation of 5-bromo-2-deoxyuridine (BrdU) at day 7 revealed significant increases in mural cell proliferation in response to S1P delivery. Additionally, three-dimensional (3D) scaffolds loaded with S1P (1:400) were implanted into critical-size rat calvarial defects, and healing of bony defects was assessed by radiograph X-ray, microcomputed tomography (muCT), and histology. Sustained release of S1P significantly increased the formation of new bone after 2 and 6 weeks of healing and histological results suggest increased numbers of blood vessels in the defect site. Taken together, these experiments support the use of S1P delivery for promoting microvessel diameter expansion and improving the healing outcomes of tissue-engineered therapies.

Sustained release of sphingosine 1-phosphate for therapeutic arteriogenesis and bone tissue engineering

PubMed Central

Sefcik, Lauren S.; Petrie Aronin, Caren E.; Wieghaus, Kristen A.

2009-01-01

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid that impacts migration, proliferation, and survival in diverse cells types, including endothelial cells, smooth muscle cells, and osteoblast-like cells. In this study, we investigated the effects of sustained release of S1P on microvascular remodeling and associated bone defect healing in vivo. The murine dorsal skinfold window chamber model was used to evaluate the structural remodeling response of the microvasculature. Our results demonstrated that 1:400 (w/w) loading and subsequent sustained release of S1P from poly(lactic-co-glycolic acid) (PLAGA) significantly enhanced lumenal diameter expansion of arterioles and venules after 3 and 7 days. Incorporation of 5-bromo-2-deoxyuridine (BrdU) at day 7 revealed significant increases in mural cell proliferation in response to S1P delivery. Additionally, three-dimensional (3D) scaffolds loaded with S1P (1:400) were implanted into critical-size rat calvarial defects and healing of bony defects was assessed by radiograph x-ray, microcomputed tomography (μCT), and histology. Sustained release of S1P significantly increased the formation of new bone after 2 and 6 weeks of healing and histological results suggest increased numbers of blood vessels in the defect site. Taken together, these experiments support the use of S1P delivery for promoting microvessel diameter expansion and improving the healing outcomes of tissue-engineered therapies. PMID:18405965

Comparison of the mechanobiological performance of bone tissue scaffolds based on different unit cell geometries.

PubMed

Rodríguez-Montaño, Óscar L; Cortés-Rodríguez, Carlos Julio; Uva, Antonio E; Fiorentino, Michele; Gattullo, Michele; Monno, Giuseppe; Boccaccio, Antonio

2018-07-01

Enhancing the performance of scaffolds for bone regeneration requires a multidisciplinary approach involving competences in the fields of Biology, Medicine and Engineering. A number of studies have been conducted to investigate the influence of scaffolds design parameters on their mechanical and biological response. The possibilities offered by the additive manufacturing techniques to fabricate sophisticated and very complex microgeometries that until few years ago were just a geometrical abstraction, led many researchers to design scaffolds made from different unit cell geometries. The aim of this work is to find, based on mechanobiological criteria and for different load regimes, the optimal geometrical parameters of scaffolds made from beam-based repeating unit cells, namely, truncated cuboctahedron, truncated cube, rhombic dodecahedron and diamond. The performance, -expressed in terms of percentage of the scaffold volume occupied by bone-, of the scaffolds based on these unit cells was compared with that of scaffolds based on other unit cell geometries such as: hexahedron and rhombicuboctahedron. A very intriguing behavior was predicted for the truncated cube unit cell that allows the formation of large amounts of bone for low load values and of very small amounts for the medium-high ones. For high values of load, scaffolds made from hexahedron unit cells were predicted to favor the formation of the largest amounts of bone. In a clinical context where medical solutions become more and more customized, this study offers a support to the surgeon in the choice of the best scaffold to be implanted in a patient-specific anatomic region. Copyright © 2018 Elsevier Ltd. All rights reserved.

Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors

PubMed Central

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

Host response mechanisms in periodontal diseases

PubMed Central

SILVA, Nora; ABUSLEME, Loreto; BRAVO, Denisse; DUTZAN, Nicolás; GARCIA-SESNICH, Jocelyn; VERNAL, Rolando; HERNÁNDEZ, Marcela; GAMONAL, Jorge

2015-01-01

Periodontal diseases usually refer to common inflammatory disorders known as gingivitis and periodontitis, which are caused by a pathogenic microbiota in the subgingival biofilm, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola that trigger innate, inflammatory, and adaptive immune responses. These processes result in the destruction of the tissues surrounding and supporting the teeth, and eventually in tissue, bone and finally, tooth loss. The innate immune response constitutes a homeostatic system, which is the first line of defense, and is able to recognize invading microorganisms as non-self, triggering immune responses to eliminate them. In addition to the innate immunity, adaptive immunity cells and characteristic cytokines have been described as important players in the periodontal disease pathogenesis scenario, with a special attention to CD4+ T-cells (T-helper cells). Interestingly, the T cell-mediated adaptive immunity development is highly dependent on innate immunity-associated antigen presenting cells, which after antigen capture undergo into a maturation process and migrate towards the lymph nodes, where they produce distinct patterns of cytokines that will contribute to the subsequent polarization and activation of specific T CD4+ lymphocytes. Skeletal homeostasis depends on a dynamic balance between the activities of the bone-forming osteoblasts (OBLs) and bone-resorbing osteoclasts (OCLs). This balance is tightly controlled by various regulatory systems, such as the endocrine system, and is influenced by the immune system, an osteoimmunological regulation depending on lymphocyte- and macrophage-derived cytokines. All these cytokines and inflammatory mediators are capable of acting alone or in concert, to stimulate periodontal breakdown and collagen destruction via tissue-derived matrix metalloproteinases, a characterization of the progression of periodontitis as a stage that presents a significantly host immune and inflammatory response to the microbial challenge that determine of susceptibility to develop the destructive/progressive periodontitis under the influence of multiple behavioral, environmental and genetic factors. PMID:26221929

Functional Attachment of Soft Tissues to Bone: Development, Healing, and Tissue Engineering

PubMed Central

Lu, Helen H.; Thomopoulos, Stavros

2014-01-01

Connective tissues such as tendons or ligaments attach to bone across a multitissue interface with spatial gradients in composition, structure, and mechanical properties. These gradients minimize stress concentrations and mediate load transfer between the soft and hard tissues. Given the high incidence of tendon and ligament injuries and the lack of integrative solutions for their repair, interface regeneration remains a significant clinical challenge. This review begins with a description of the developmental processes and the resultant structure-function relationships that translate into the functional grading necessary for stress transfer between soft tissue and bone. It then discusses the interface healing response, with a focus on the influence of mechanical loading and the role of cell-cell interactions. The review continues with a description of current efforts in interface tissue engineering, highlighting key strategies for the regeneration of the soft tissue–to-bone interface, and concludes with a summary of challenges and future directions. PMID:23642244

Role of inflammation in the aging bones.

PubMed

Abdelmagid, Samir M; Barbe, Mary F; Safadi, Fayez F

2015-02-15

Chronic inflammation in aging is characterized by increased inflammatory cytokines, bone loss, decreased adaptation, and defective tissue repair in response to injury. Aging leads to inherent changes in mesenchymal stem cell (MSC) differentiation, resulting in impaired osteoblastogenesis. Also, the pro-inflammatory cytokines increase with aging, leading to enhanced myelopoiesis and osteoclastogenesis. Bone marrow macrophages (BMMs) play pivotal roles in osteoblast differentiation, the maintenance of hematopoietic stem cells (HSCs), and subsequent bone repair. However, during aging, little is known about the role of macrophages in the differentiation and function of MSC and HSC. Aged mammals have higher circulating pro-inflammatory cytokines than young adults, supporting the hypothesis of increased inflammation with aging. This review will aid in the understanding of the potential role(s) of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages in differentiation and function of osteoblasts and osteoclasts in relation to aging. Copyright © 2014 Elsevier Inc. All rights reserved.

MicroRNA-101a regulates microglial morphology and inflammation.

PubMed

Saika, Reiko; Sakuma, Hiroshi; Noto, Daisuke; Yamaguchi, Shuhei; Yamamura, Takashi; Miyake, Sachiko

2017-05-30

Microglia, as well as other tissue-resident macrophages, arise from yolk sac progenitors. Thus, it is likely that the central nervous system environment is critical for the acquisition of a distinct microglial phenotype. Several microRNAs that are enriched in the brain play crucial roles in brain development and may also play a role in the differentiation of microglia. To track the differentiation of hematopoietic cells into microglia, lineage-negative bone marrow cells were co-cultured with astrocytes in the absence or presence of microRNAs or their inhibitors. Microglia-like cells were identified as small, round cells that were immunopositive for CD11b, Iba1, CX3CR1, and triggering receptor expressed on myeloid cells (TREM)-2. Five microRNAs (miR-101a, miR-139-3p, miR-214 * , miR-218, and miR-1186) were identified as modifiers of the differentiation of bone marrow-derived microglia-like cells. Among them, miR-101a facilitated the differentiation of bone marrow cells into microglia-like cells most potently. Small, round cells expressing CD11b, Iba1, CX3CR1, and TREM-2 were predominant in cells treated by miR-101a. miR-101a was abundantly expressed in non-microglial brain cells. Transfection of miR-101a into microglia significantly increased the production of IL-6 in response to LPS. Finally, miR-101a downregulated the expression of MAPK phosphatase-1. miR-101a, which is enriched in the brain, promotes the differentiation of bone marrow cells into microglia-like cells.

Systematical Evaluation of Mechanically Strong 3D Printed Diluted magnesium Doping Wollastonite Scaffolds on Osteogenic Capacity in Rabbit Calvarial Defects

PubMed Central

Sun, Miao; Liu, An; Shao, Huifeng; Yang, Xianyan; Ma, Chiyuan; Yan, Shigui; Liu, Yanming; He, Yong; Gou, Zhongru

2016-01-01

Wollastonite (CaSiO3; CSi) ceramic is a promising bioactive material for bone defect repair due to slightly fast degradation of its porous constructs in vivo. In our previous strategy some key features of CSi ceramic have been significantly improved by dilute magnesium doping for regulating mechanical properties and biodegradation. Here we demonstrate that 6 ~ 14% of Ca substituted by Mg in CSi (CSi-Mgx, x = 6, 10, 14) can enhance the mechanical strength (>40 MPa) but not compromise biological performances of the 3D printed porous scaffolds with open porosity of 60‒63%. The in vitro cell culture tests in vitro indicated that the dilute Mg doping into CSi was beneficial for ALP activity and high expression of osteogenic marker genes of MC3T3-E1 cells in the scaffolds. A good bone tissue regeneration response and elastoplastic response in mechanical strength in vivo were determined after implantation in rabbit calvarial defects for 6‒12 weeks. Particularly, the CSi-Mg10 and CSi-Mg14 scaffolds could enhance new bone regeneration with a significant increase of newly formed bone tissue (18 ~ 22%) compared to the pure CSi (~14%) at 12 weeks post-implantation. It is reasonable to consider that, therefore, such CSi-Mgx scaffolds possessing excellent strength and reasonable degradability are promising for bone reconstruction in thin-wall bone defects. PMID:27658481

C/EBPβ in bone marrow is essential for diet induced inflammation, cholesterol balance, and atherosclerosis.

PubMed

Rahman, Shaikh M; Baquero, Karalee C; Choudhury, Mahua; Janssen, Rachel C; de la Houssaye, Becky A; Sun, Ming; Miyazaki-Anzai, Shinobu; Wang, Shu; Moustaid-Moussa, Naima; Miyazaki, Makoto; Friedman, Jacob E

2016-07-01

Atherosclerosis is both a chronic inflammatory disease and a lipid metabolism disorder. C/EBPβ is well documented for its role in the development of hematopoietic cells and integration of lipid metabolism. However, C/EBPβ's role in atherosclerotic progression has not been examined. We assessed the impact of hematopoietic CEBPβ deletion in ApoE(-/-) mice on hyperlipidemia, inflammatory responses and lesion formation in the aorta. ApoE(-/-) mice were reconstituted with bone marrow cells derived from either WT or C/EBPβ(-/-) mice and placed on low fat or high fat/high cholesterol diet for 11 weeks. Hematopoietic C/EBPβ deletion in ApoE(-/-) mice reduced blood and hepatic lipids and gene expression of hepatic stearoyl CoA desaturase 1 and fatty acid synthase while expression of ATP binding cassette transporter G1, cholesterol 7-alpha-hydroxylase, and liver X receptor alpha genes were significantly increased. ApoE(-/-) mice reconstituted with C/EBPβ(-/-) bone marrow cells also significantly reduced blood cytokine levels and reduced lesion area in aortic sinuses compared with ApoE(-/-) mice reconstituted with WT bone marrow cells. Silencing of C/EBPβ in RAW264.7 macrophage cells prevented oxLDL-mediated foam cell formation and inflammatory cytokine secretion in conditioned medium. C/EBPβ in hematopoietic cells is crucial to regulate diet-induced inflammation, hyperlipidemia and atherosclerosis development. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The role of versican G3 domain in regulating breast cancer cell motility including effects on osteoblast cell growth and differentiation in vitro – evaluation towards understanding breast cancer cell bone metastasis

PubMed Central

2012-01-01

Background Versican is detected in the interstitial tissues at the invasive margins of breast carcinoma, is predictive of relapse, and negatively impacts overall survival rates. The versican G3 domain is important in breast cancer cell growth, migration and bone metastasis. However, mechanistic studies evaluating versican G3 enhanced breast cancer bone metastasis are limited. Methods A versican G3 construct was exogenously expressed in the 66c14 and the MC3T3-E1 cell line. Cells were observed through light microscopy and viability analyzed by Coulter Counter or determined with colorimetric proliferation assays. The Annexin V-FITC apoptosis detection kit was used to detect apoptotic activity. Modified Chemotactic Boyden chamber migration invasion assays were applied to observe tumor migration and invasion to bone stromal cells and MC3T3-E1 cells. Alkaline phosphatase (ALP) staining and ALP ELISA assays were performed to observe ALP activity in MC3T3-E1 cells. Results In the four mouse breast cancer cell lines 67NR, 66c14, 4T07, and 4T1, 4T1 cells expressed higher levels of versican, and showed higher migration and invasion ability to MC3T3-E1 cells and primary bone stromal cells. 4T1 conditioned medium (CM) inhibited MC3T3-E1 cell growth, and even lead to apoptosis. Only 4T1 CM prevented MC3T3-E1 cell differentiation, noted by inhibition of alkaline phosphatase (ALP) activity. We exogenously expressed a versican G3 construct in a cell line that expresses low versican levels (66c14), and observed that the G3-expressing 66c14 cells showed enhanced cell migration and invasion to bone stromal and MC3T3-E1 cells. This observation was prevented by selective EGFR inhibitor AG1478, selective MEK inhibitor PD 98059, and selective AKT inhibitor Triciribine, but not by selective JNK inhibitor SP 600125. Versican G3 enhanced breast cancer cell invasion to bone stromal cells or osteoblast cells appears to occur through enhancing EGFR/ERK or AKT signaling. G3 expressing MC3T3-E1 cells showed inhibited cell growth and cell differentiation when cultured with TGF-β1 (1 ng/ml), and expressed enhanced cell apoptosis when cultured with TNF-α (2 ng/ml). Enhanced EGFR/JNK signaling appears to be responsible for G3 enhanced osteoblast apoptosis and inhibited osteoblast differentiation. Whereas repressed expression of GSK-3β (S9P) contributes to G3 inhibited osteoblast growth. Versican G3 functionality was dependent on its EGF-like motifs. Without the structure of EGF-like repeats, the G3 domain would not confer enhancement of tumor cell migration and invasion to bone with concordant inhibition of osteoblast differentiation and promotion of osteoblast apoptosis. Conclusions Versican enhances breast cancer bone metastasis not only through enhancing tumor cell mobility, invasion, and survival in bone tissues, but also by inhibiting pre-osteoblast cell growth, differentiation, which supply favorable microenvironments for tumor metastasis. PMID:22862967

Expression of CD 68, CD 45 and human leukocyte antigen-DR in central and peripheral giant cell granuloma, giant cell tumor of long bones, and tuberculous granuloma: An immunohistochemical study.

PubMed

Kumar, Anoop; Sherlin, Herald J; Ramani, Pratibha; Natesan, Anuja; Premkumar, Priya

2015-01-01

Multinucleated giant cells (MNCs) form an integral part of numerous bone and soft tissue tumors, tumor-like lesions and are often associated with granulomas of immunological and nonimmunological origin. The presence of various types of giant cells depends on the lesions in which they are present which are difficult to be diagnosed under routine histological techniques. Immunohistochemistry can be used for a better diagnosis and understanding of the origin of various giant cells using various markers of immune response like human leukocyte antigen-DR (HLA-DR) and those expressed on monocytes and macrophages like CD 68 and leukocyte common antigen (LCA). The study group consisted of 10 cases of giant cell tumor (GCT) of long bones, tuberculous granuloma, and giant cell granuloma to evaluate and analyze the expression pattern of LCA, CD 68, and HLA-DR in various giant cell lesions. Strong expression of CD 68 was observed in 80% of the lesions, strong and moderate expression of CD 45 observed in 70% of the lesions among and within the groups. In contrast, HLA-DR demonstrated negative expression in 80% of cases except for tuberculous granuloma where all the 10 cases showed moderate to strong immunoreactivity. CD 68 and CD 45 expression was found in central giant cell granuloma, peripheral giant cell granuloma and GCT, suggesting the origin from mononuclear phagocyte system and considering their clinical behavior of osteoclast type. High expressivity of HLA-DR in tuberculous granulomas which is an essential factor for presentation of the microbial antigen to CD 4 helper cells thus reassuring the fact that they are up-regulated in response to infection.

Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response

PubMed Central

Rui, Yuxiang; Honjo, Tasuku; Chikuma, Shunsuke

2013-01-01

Programmed cell death 1 (PD-1) is an inhibitory coreceptor on immune cells and is essential for self-tolerance because mice genetically lacking PD-1 (PD-1−/−) develop spontaneous autoimmune diseases. PD-1−/− mice are also susceptible to severe experimental autoimmune encephalomyelitis (EAE), characterized by a massive production of effector/memory T cells against myelin autoantigen, the mechanism of which is not fully understood. We found that an increased primary response of PD-1−/− mice to heat-killed mycobacteria (HKMTB), an adjuvant for EAE, contributed to the enhanced production of T-helper 17 (Th17) cells. Splenocytes from HKMTB-immunized, lymphocyte-deficient PD-1−/− recombination activating gene (RAG)2−/− mice were found to drive antigen-specific Th17 cell differentiation more efficiently than splenocytes from HKMTB-immunized PD-1+/+ RAG2−/− mice. This result suggested PD-1’s involvement in the regulation of innate immune responses. Mice reconstituted with PD-1−/− RAG2−/− bone marrow and PD-1+/+ CD4+ T cells developed more severe EAE compared with the ones reconstituted with PD-1+/+ RAG2−/− bone marrow and PD-1+/+ CD4+ T cells. We found that upon recognition of HKMTB, CD11b+ macrophages from PD-1−/− mice produced very high levels of IL-6, which helped promote naive CD4+ T-cell differentiation into IL-17–producing cells. We propose a model in which PD-1 negatively regulates antimycobacterial responses by suppressing innate immune cells, which in turn prevents autoreactive T-cell priming and differentiation to inflammatory effector T cells. PMID:24043779

Strategies to reverse endothelial progenitor cell dysfunction in diabetes.

PubMed

Petrelli, Alessandra; Di Fenza, Raffaele; Carvello, Michele; Gatti, Francesca; Secchi, Antonio; Fiorina, Paolo

2012-01-01

Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial), their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs). Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

LIM mineralization protein-1 potentiates bone morphogenetic protein responsiveness via a novel interaction with Smurf1 resulting in decreased ubiquitination of Smads.

PubMed

Sangadala, Sreedhara; Boden, Scott D; Viggeswarapu, Manjula; Liu, Yunshan; Titus, Louisa

2006-06-23

Development and repair of the skeletal system and other organs is highly dependent on precise regulation of bone morphogenetic proteins (BMPs), their receptors, and their intracellular signaling proteins known as Smads. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, control of cellular responsiveness to BMPs is now a critical area that is poorly understood. We determined that LMP-1, a LIM domain protein capable of inducing de novo bone formation, interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads. In the region of LMP responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and can effectively compete with Smad1 and Smad5 for binding. We have shown that small peptides containing this motif can mimic the ability to block Smurf1 from binding Smads. This novel interaction of LMP-1 with the WW2 domain of Smurf1 to block Smad binding results in increased cellular responsiveness to exogenous BMP and demonstrates a novel regulatory mechanism for the BMP signaling pathway.

The Effect of Gabapentin and Tramadol in Cancer Pain Induced by Glioma Cell in Rat Femur.

PubMed

Corona-Ramos, Janette Nallely; Déciga-Campos, Myrna; Romero-Piña, Mario; Medina, Luis A; Martínez-Racine, Issac; Jaramillo-Morales, Osmar A; García-López, Patricia; López-Muñoz, Francisco Javier

2017-08-01

Preclinical Research The presence of pain as part of the cancer process is variable. Glioblastoma multiform (GBM) can produce bone metastasis, a condition that involves other pathological phenotypes including neuropathic and inflammatory pain. Tramadol and gabapentin are drugs used in the treatment of neuropathic pain. However, there are no studies evaluating their analgesic effects in bone metastasis. We produced a pain model induced by the inoculation of glioma cells (10 5 ) into the rat femur, by perforating the intercodiloid fossa. Painful behavior was evaluated by measuring mechanical allodynia using the Von Frey test while thermal hyperalgesia was assessed in the plantar test. Histopathological features were evaluated and antinociceptive responses were compared using tramadol and gabapentin. The inoculation of cells inside the right femur produced nociceptive behaviors. Tramadol and gabapentin produced an anti-allodynic effect in this condition, but tramadol did not produce an anti-hyperalgesic response. The development of this model will allow us to perform tests to elucidate the pathology of bone metastasis, cancer pain, and in particular the pain produced by glioma. Drug Dev Res 78 : 173-183, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Fabrication and Characterization of Carbon Fiber-Reinforced Nano-Hydroxyapatite/Polyamide46 Biocomposite for Bone Substitute.

PubMed

Deng, Zhennan; Han, Hongjuan; Yang, Jingyuan; Li, Yuanyuan; Du, Shengnan; Ma, Jianfeng

2017-05-24

BACKGROUND Ideal bone repair material should be of good biocompatibility and high bioactivity. Besides, their mechanical properties should be equivalent to those of natural bone. The objective of this study was to fabricate a novel biocomposite suitable for load-bearing bone defect repair. MATERIAL AND METHODS A novel biocomposite composed of carbon fiber, hydroxyapatite and polyamide46 (CF/HA/PA46) was fabricated, and its mechanical performances and preliminary cell responses were evaluated to explore its feasibility for load-bearing bone defect repair. RESULTS The resultant CF/HA/PA46 biocomposite showed a bending strength of 159-223 MPa, a tensile strength of 127-199 MPa and a tensile modulus of 7.7-10.8 GPa, when the CF content was 5-20% (mass fraction) in biocomposite. The MG63 cells, showing an osteogenic phenotype, were well adhered and spread on the surface of the CF/HA/PA46 biocomposite. Moreover, the cells vitality and differentiation on the CF/HA/PA46 biocomposite surface were obviously increased during the culture time and there was no significant difference between the CF/HA/PA46 biocomposite and HA/PA (as control) at all the experimental time (P>0.05). CONCLUSIONS The addition of CF into HA/PA46 composite manifest improved the mechanical performances and showed favorable effects on biocompatibility of MG63 cells. The obtained biocomposite has high potential for bone repair in load-bearing sites.

Fabrication and Characterization of Carbon Fiber-Reinforced Nano-Hydroxyapatite/Polyamide46 Biocomposite for Bone Substitute

PubMed Central

Deng, Zhennan; Han, Hongjuan; Yang, Jingyuan; Li, Yuanyuan; Du, Shengnan; Ma, Jianfeng

2017-01-01

Background Ideal bone repair material should be of good biocompatibility and high bioactivity. Besides, their mechanical properties should be equivalent to those of natural bone. The objective of this study was to fabricate a novel biocomposite suitable for load-bearing bone defect repair. Material/Methods A novel biocomposite composed of carbon fiber, hydroxyapatite and polyamide46 (CF/HA/PA46) was fabricated, and its mechanical performances and preliminary cell responses were evaluated to explore its feasibility for load-bearing bone defect repair. Results The resultant CF/HA/PA46 biocomposite showed a bending strength of 159–223 MPa, a tensile strength of 127–199 MPa and a tensile modulus of 7.7–10.8 GPa, when the CF content was 5–20% (mass fraction) in biocomposite. The MG63 cells, showing an osteogenic phenotype, were well adhered and spread on the surface of the CF/HA/PA46 biocomposite. Moreover, the cells vitality and differentiation on the CF/HA/PA46 biocomposite surface were obviously increased during the culture time and there was no significant difference between the CF/HA/PA46 biocomposite and HA/PA (as control) at all the experimental time (P>0.05). Conclusions The addition of CF into HA/PA46 composite manifest improved the mechanical performances and showed favorable effects on biocompatibility of MG63 cells. The obtained biocomposite has high potential for bone repair in load-bearing sites. PMID:28536416

Bioelectric modulation of wound healing in a 3D in vitro model of tissue-engineered bone.

PubMed

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.

Osteoblast hydraulic conductivity is regulated by calcitonin and parathyroid hormone

NASA Technical Reports Server (NTRS)

Hillsley, M. V.; Frangos, J. A.

1996-01-01

It is our hypothesis that osteoblasts play a major role in regulating bone (re)modeling by regulating interstitial fluid (ISF) flow through individual bone compartments. We hypothesize that osteoblasts of the blood-bone membrane lining the bone surfaces are capable of regulating transosseous fluid flow. This regulatory function of the osteoblasts was tested in vitro by culturing a layer of rat calvarial osteoblasts on porous membranes. Such a layer of osteoblasts subjected to 7.3 mm Hg of hydrostatic pressure posed a significant resistance to fluid flow across the cell layer similar in magnitude to the resistance posed by endothelial monolayers in vitro. The hydraulic conductivity, the volumetric fluid flux per unit pressure drop, of the osteoblast layer was altered in response to certain hormones. Hydraulic conductivity decreased approximately 40% in response to 33 nM parathyroid hormone, while it exhibited biphasic behavior in response to calcitonin: increased 40% in response to 100 nM calcitonin and decreased 40% in response to 1000 nM calcitonin. Further, activation of adenylate cyclase by forskolin dramatically increased the hydraulic conductivity, while elevation of intracellular calcium, [Ca2+]i, by the calcium ionophore A23187 initially decreased the hydraulic conductivity at 5 minutes before increasing conductivity by 30 minutes. These results suggest that cyclic adenosine monophosphate (cAMP) and [Ca2+]i may mediate changes in the osteoblast hydraulic conductivity. The increase in hydraulic conductivity in response to 100 nM calcitonin and the decrease in response to PTH suggest that the stimulatory and inhibitory effects on bone formation of calcitonin and parathyroid hormone, respectively, may be due in part to alterations in bone fluid flow.

Intra-osseous injection of donor mesenchymal stem cell (MSC) into the bone marrow in living donor kidney transplantation; a pilot study.

PubMed

Lee, Hyunah; Park, Jae Berm; Lee, Sanghoon; Baek, Soyoung; Kim, HyunSoo; Kim, Sung Joo

2013-04-11

Mesenchymal stem cells (MSCs) are multi-potent non-hematopoietic progenitor cells possessing an immune-regulatory function, with suppression of proliferation of activated lymphocytes. In this study, adult living donor kidney transplantation (LDKT) recipients were given MSCs derived from the donor bone marrow to evaluate the safety and the feasibility of immunological changes related to the intra-osseous injection of MSC into the bone marrow. MSCs were derived from negative HLA cross-match donors. Donor bone marrow was harvested 5 weeks prior to KT. At the time of transplantation, 1 x 106 cell/kg of donor MSC was directly injected into the bone marrow of the recipient's right iliac bone. Patients' clinical outcomes, presence of mixed chimerism by short tandem repeat polymerase chain reaction, analysis of plasma FoxP3 mRNA and cytokine level, and mixed lymphocyte reaction (MLR) were performed. Seven patients enrolled in this study and received donor MSC injections simultaneously with LDKT. The median age of recipients was 36 years (32 ~ 48). The number of HLA mismatches was 3 or less in 5 and more than 3 in 2. No local complications or adverse events such as hypersensitivity occurred during or after the injection of donor MSC. There was no graft failure, but the biopsy-proven acute rejections were observed in 3 recipients during the follow-up period controlled well with steroid pulse therapy (SPT). The last serum creatinine was a median of 1.23 mg/dL (0.83 ~ 2.07). Mixed chimerism was not detected in the peripheral blood of the recipients at 1 and 8 week of post-transplantation. Donor-specific lymphocyte or T cell proliferation and Treg priming responses were observed in some patients. Plasma level of IL-10, a known mediator of MSC-induced immune suppression, increased in the patients with Treg induction. Donor MSC injection into the iliac bone at the time of KT was feasible and safe. A possible correlation was observed between the induction of inhibitory immune responses and the clinical outcome in the MSC-kidney transplanted patients. Further research will be performed to evaluate the efficacy of MSC injection for the induction of mixed chimerism and subsequent immune tolerance in KT.