rhBMP6 in autologous blood coagulum is a preferred osteoinductive device to rhBMP2 on bovine collagen sponge in the rat ectopic bone formation assay.

Osteoinductive BMPs require a suitable delivery system for treating various pathological conditions of the spine and segmental bone defects. INFUSE, the only commercially available BMP-based osteoinductive device, consisting of rhBMP2 on bovine absorbable collagen sponge (ACS) showed major disadvantages due to serious side effects. A novel osteoinductive device, OSTEOGROW, comprised of rhBMP6 dispersed within autologous blood coagulum (ABC) is a promising therapy for bone regeneration, subjected to several clinical trials for diaphysial bone repair and spinal fusion. In the present study, we have examined the release dynamics showing that the ABC carrier provided a slower, more steady BMP release in comparison to the ACS. Rat subcutaneous assay was employed to evaluate cellular events and the time course of ectopic osteogenesis. The host cellular response to osteoinductive implants was evaluated by flow cytometry, while dynamics of bone formation and maintenance in time were evaluated by histology, immunohistochemistry and micro CT analyses. Flow cytometry revealed that the recruitment of lymphoid cell populations was significantly higher in rhBMP6/ABC implants, while rhBMP2/ACS implants recruited more myeloid populations. Furthermore, rhBMP6/ABC implants more efficiently attracted early and committed progenitor cells. Dynamics of bone formation induced by rhBMP2/ACS was characterized by a delayed endochondral ossification process in comparison to rhBMP6/ABC implants. Besides, rhBMP6/ABC implants induced more ectopic bone volume in all observed time points in comparison to rhBMP2/ACS implants. These results indicate that OSTEOGROW was superior to INFUSE due to ABC's advantages as a carrier and rhBMP6 superior efficacy in inducing bone.

Comparison of synthetic ceramic products formulated with autologous blood coagulum containing rhBMP6 for induction of bone formation.

PURPOSE: Osteogrow, an osteoinductive device containing recombinant human Bone Morphogenetic Protein 6 (rhBMP6) in autologous blood coagulum, is a novel therapeutic solution for bone regeneration. This study aimed to evaluate different commercially available calcium phosphate synthetic ceramic particles as a compression-resistant matrix (CRM) added to Osteogrow implants to enhance their biomechanical properties. METHODS: Osteogrow implants with the addition of Vitoss, ChronOs, BAM, and Dongbo ceramics (Osteogrow-C, where C stands for ceramics) were evaluated in the rodent subcutaneous ectopic bone formation assay. Osteogrow-C device was prepared as follows: rhBMP6 was added to blood, and blood was mixed with ceramics and left to coagulate. Osteogrow-C was implanted subcutaneously in the axillary region of Sprague-Dawley rats and the outcome was analyzed 21 days following implantation using microCT, histology, morphometric analyses, and immunohistochemistry. RESULTS: Osteogrow-C implants with all tested ceramic particles induced the formation of the bone-ceramic structure containing cortical bone, the bone between the particles, and bone at the ceramic surfaces. The amount of newly formed bone was significant in all experimental groups; however, the highest bone volume was measured in Osteogrow-C implants with highly porous Vitoss ceramics. The trabecular number was highest in Osteogrow-C implants with Vitoss and ChronOs ceramics while trabeculae were thicker in implants containing BAM and Dongbo ceramics. The immunological response and inflammation were comparable among ceramic particles evaluated in this study. CONCLUSION: Osteogrow-C bone regenerative device was effective with a broad range of commercially available synthetic ceramics providing a promising therapeutic solution for the regeneration of long bone fracture nonunion, large segmental defects, and spinal fusion surgeries.

Autologous bone graft substitute containing rhBMP6 within autologous blood coagulum and synthetic ceramics of different particle size determines the quantity and structural pattern of bone formed in a rat subcutaneous assay.

Bone morphogenetic proteins (BMPs) are potent osteoinductive agents for bone tissue engineering. In order to define optimal properties of a novel autologous bone graft substitute (ABGS) containing rhBMP6 within the autologous blood coagulum (ABC) and ceramic particles as a compression resistant matrix (CRM), we explored the influence of their amount, chemical composition and particle size on the quantity and quality of bone formation in the rat subcutaneous assay. Tested ceramic particles included tricalcium phosphate (TCP), hydroxyapatite (HA) and biphasic calcium phosphate ceramic (BCP), containing TCP and HA in 80/20 ratio of different particle sizes (small 74-420 µm, medium 500-1700 µm and large 1000-4000 µm). RhBMP6 was either mixed with ABC or lyophilized on CRM prior to use with ABC. The experiments were terminated on day 21 and implants were analysed by microCT, histology and histomorphometry. Addition of CRM to ABGS containing rhBMP6 in ABC significantly increased the amount of newly formed bone and the optimal CRM/ABC ratio was found to be around 100 mg/500 µL. MicroCT analyses revealed that all tested ABGS formulations induced an extensive new bone formation and there were no differences between the two methods of rhBMP6 application as determined by the bone volume. However, the particle size played a significant role in the quantity and quality of newly formed bone. ABGS containing small particles induced new bone forming a dense trabecular network, cortical bone at the rim, bone and bone marrow in apposition to and in between ceramic particles. ABGS containing medium and large particles also resulted in new bone on the surface of particles as well as inside the pores. Histomorphometric analysis revealed that the ceramics particle size correlated with the quality of trabecular pattern of newly formed bone, bone/bone marrow ratio as observed in apposition and between particles, and the ratio between the cortical and trabecular bone. By employing rat subcutaneous implant assay, we showed for the first time that the size of synthetic ceramics particles affected the osteogenesis as defined by both the quantity and quality of ectopic bone.

Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss.

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro --> Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.

Bone morphogenetic protein-7 from serum of pregnant mice is available to the fetus through placental transfer during early stages of development.

BACKGROUND: BMP-7 is an important mediator of metanephric mesenchyme differentiation during kidney development. Gene knockout studies have shown that BMP-7 null mutation mice die shortly after birth due to renal failure, although the induction of metanephric structures has initially occurred (E11-E13). MATERIALS AND METHODS: Iodinated BMP-7 was injected into the tail vein of pregnant mice and its availability to tissues and fetuses was further analyzed by tissue uptake, LM autoradiography and SDS-PAGE electrophoresis. RESULTS: Studies on the distribution of 125I-BMP-7 injected through the tail vein of pregnant mice indicated that 125I-BMP-7 passed across the placenta and localized in developing fetal organs, in particular kidneys, up to day 14 of gestation. At later stages of pregnancy 125I-BMP-7 did not pass the trophoblasts of the placental barrier and did not enter the fetal blood vessels. CONCLUSION: The analysis of the distribution of 125I-BMP-7 from pregnant mice to fetal organs, in particular the kidney, suggests a cross-over of maternal circulating BMP-7 to the fetus through the placental barrier.

Bone morphogenetic protein-7 reduces the severity of colon tissue damage and accelerates the healing of inflammatory bowel disease in rats.

Bone morphogenetic protein-7 (BMP-7) is a growth and differentiation factor and belongs to the TGF-beta superfamily of proteins. Previous studies have shown an abundant expression of BMP-7 in the developing intestine and an association with a perturbed BMP/SMAD downstream signaling leading to a malignant phenotype and inflammation in the gut. In the present study, we have evaluated the effect of systemically administered recombinant human BMP-7 against trinitrobenzenesulfonic (TNBS) acid induced inflammatory bowel disease (IBD) in rats. The TNBS administered rats treated with BMP-7 have developed much less severe form of colitis based on macroscopic and histological scoring when administered 1.5 h before or 24 h after colitis induction. Bioavailability studies in healthy rats have revealed that significant portion (3.6%) of i.v. administered BMP-7 is targeted for BMP-7 receptors in the stomach and ileum, respectively, suggesting its availability to target tissue upon administration. Immunohistochemical and RT-PCR analyses have shown elevated expression of pro-inflammatory (IL-6, TNF-beta, ICAM-1) and pro-fibrogenic (TGF-beta) cytokines, and BMP-7 treatment significantly reduced their expression in the intestine; among which the suppression of IL-6 appeared to be the most important. Taken together, the results of this study suggest that BMP-7 plays an important role in the regulation of anti-inflammatory response in the adult gut tissue.

Characterization of proliferating human skeletal muscle-derived cells in vitro: differential modulation of myoblast markers by TGF-beta2.

Adult human skeletal muscle-derived cells (HuSkMC) propagated in vitro are under investigation as a cell-based therapy for the treatment of myocardial infarction. We have characterized HuSkMC with respect to cell identity and state of differentiation as a prerequisite to their clinical use. Flow cytometric analysis of propagated HuSkMC revealed a population of cells that expressed the myoblast markers CD56 and desmin. The presence of myoblasts in these cultures was further confirmed by their capacity to form myotubes and increase creatine kinase activity when cultured in low serum conditions. The non-myoblast fraction of these propagated cells expressed TE7, a marker associated with the fibroblast phenotype. Spontaneous differentiation of myoblasts occurred during serial propagation of HuSkMC, as judged by myotube formation, thereby reducing the myoblast representative fraction with continued cell expansion. We examined transforming growth factor beta2 (TGF-beta2) for its utility in controlling this spontaneous differentiation of adult human myoblasts in vitro. Propagation of HuSkMC in the presence of 1 ng/ml TGF-beta2 for 5 days decreased desmin expression within the myoblast population and caused a parallel reduction of creatine kinase activity. CD56 expression was unaffected, indicating a differential regulation of these myoblast markers. The reduction in desmin expression and creatine kinase activity was, however, reversible upon the removal of TGF-beta. These data collectively indicate that TGF-beta2 restrained differentiation of adult human skeletal myoblasts during propagation without causing irreversible loss of the myoblast phenotype, demonstrating the potential utility of using TGF-beta2 during cultivation and expansion of HuSkMC intended for therapeutic implantation.

Chondrocytes provide morphogenic signals that selectively induce osteogenic differentiation of mesenchymal stem cells.

During endochondral bone development cartilage formation always precedes that of bone, leading to the hypothesis that chondrocytes provide inductive signals for osteogenesis. To test this hypothesis, C3H10T1/2 mesenchymal stem cells were cocultured in membrane separated trans-well culture chambers with nonhypertrophic chondrocytes, hypertrophic chondrocytes, calvaria osteoblasts, or tendon fibroblasts derived from embryonic chickens to assess if individual cell types would selectively promote osteogenic differentiation. Then, differentiation of C3H10T1/2 mesenchymal stem cells in coculture were compared with that induced by bone morphogenetic protein 7 or osteogenic protein-1 (BMP-7; OP-1) treatment. Osteogenesis, as determined by the expression of Cbfa1 and osteocalcin (OC) messenger RNAs (mRNAs), was induced strongly in C3H10T1/2 cells cocultured with both chondrocyte cell populations but was not induced by coculture with either osteoblasts or skin fibroblasts. Interestingly, treatment of C3H10T1/2 cells with BMP-7 induced both chondrogenesis and osteogenesis, and only osteogenic differentiation was observed in the C3H10T1/2 cells cocultured with chondrocytes. No alterations in the expression of mRNAs for BMP-1 to -8 were observed in the C3H10T1/2 cells under any of the coculture conditions. This shows that the induction of endogenous BMPs by coculture does not regulate osteogenesis in an autocrine manner. These results show that chondrocytes express soluble morphogenetic factors that selectively promote osteogenesis, and this selective effect is not mimicked by an exogenously added BMP.