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.

BMP3 Affects Cortical and Trabecular Long Bone Development in Mice.

Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3-/- mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3-/- mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3-/- mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3-/- mice.

Synthetic ceramic macroporous blocks as a scaffold in ectopic bone formation induced by recombinant human bone morphogenetic protein 6 within autologous blood coagulum in rats.

PURPOSE: We have recently developed an autologous bone graft substitute (ABGS) containing recombinant human bone morphogenetic protein 6 (rhBMP6) in autologous blood coagulum (ABC) that induces new bone formation in vivo. In order to improve biomechanical properties of the implant, compression resistant matrix (CRM) consisting of synthetic ceramics in the form of macroporous cylinders was added to the ABGS and we evaluated the biomechanical properties and the quantity and quality of bone formation following subcutaneous implantation in rats. METHODS: ABGS implants containing rhBMP6 in ABC with cylindrical ceramic blocks were implanted subcutaneously (n = 6 per time point) in the axillary region of Sprague-Dawley rats and removed at specified time points (7, 14, 21, 35, and 50 days). The quantity and quality of newly formed bone were analyzed by microCT, histology, and histomorphometric analyses. Biomechanical properties of ABGS formulations were determined by employing the cut test. RESULTS: MicroCT analyses revealed that ABGS implants induced formation of new bone within ceramic blocks. Histological analysis revealed that on day seven following implantation, the endochondral ossification occupied the peripheral part of implants. On days 14 and 21, newly formed bone was present both around the ceramic block and through the pores inside the block. On both days 35 and 50, cortical bone encircled the ceramic block while inside the block, bone covered the ceramic surface surrounding the pores. Within the osseous circles, there were few trabeculae and bone marrow containing adipocytes. ABGS containing cylindrical ceramic blocks were more rigid and had significantly increased stiffness compared with implants containing ceramic particles as CRM. CONCLUSION: We demonstrated that macroporous ceramic blocks in a form of cylinders are promising CRMs with good handling and enhanced biomechanical properties, supporting bone formation with ABGS containing rhBMP6 within autologous blood coagulum. Hence, ABGS containing ceramic blocks should be tested in preclinical models including diaphyseal segmental defects and non-unions in larger animals.

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.

Bone morphogenetic protein 1.3 inhibition decreases scar formation and supports cardiomyocyte survival after myocardial infarction.

Despite the high prevalence of ischemic heart diseases worldwide, no antibody-based treatment currently exists. Starting from the evidence that a specific isoform of the Bone Morphogenetic Protein 1 (BMP1.3) is particularly elevated in both patients and animal models of myocardial infarction, here we assess whether its inhibition by a specific monoclonal antibody reduces cardiac fibrosis. We find that this treatment reduces collagen deposition and cross-linking, paralleled by enhanced cardiomyocyte survival, both in vivo and in primary cultures of cardiac cells. Mechanistically, we show that the anti-BMP1.3 monoclonal antibody inhibits Transforming Growth Factor ß pathway, thus reducing myofibroblast activation and inducing cardioprotection through BMP5. Collectively, these data support the therapeutic use of anti-BMP1.3 antibodies to prevent cardiomyocyte apoptosis, reduce collagen deposition and preserve cardiac function after ischemia.

Iron overload in aging Bmp6­/­ mice induces exocrine pancreatic injury and fibrosis due to acinar cell loss.

The relationship between hemochromatosis and diabetes has been well established, as excessive iron deposition has been reported to result in impaired function of the endocrine and exocrine pancreas. Therefore, the objective of the present study was to analyze the effects of iron accumulation on the pancreata and glucose homeostasis in a bone morphogenetic protein 6­knockout (Bmp6­/­) mouse model of hemochromatosis. The sera and pancreatic tissues of wild­type (WT) and Bmp6­/­ mice (age, 3 and 10 months) were subjected to biochemical and histological analyses. In addition, 18F­fluorodeoxyglucose biodistribution was evaluated in the liver, muscle, heart, kidney and adipose tissue of both animal groups. The results demonstrated that 3­month­old Bmp6­/­ mice exhibited iron accumulation preferentially in the exocrine pancreas, with no signs of pancreatic injury or fibrosis. No changes were observed in the glucose metabolism, as pancreatic islet diameter, insulin and glucagon secretion, blood glucose levels and glucose uptake in the liver, muscle and adipose tissue remained comparable with those in the WT mice. Aging Bmp6­/­ mice presented with progressive iron deposits in the exocrine pancreas, leading to pancreatic degeneration and injury that was characterized by acinar atrophy, fibrosis and the infiltration of inflammatory cells. However, the aging mice exhibited unaltered blood glucose levels and islet structure, normal insulin secretion and moderately increased α­cell mass compared with those in the age­matched WT mice. Additionally, iron overload and pancreatic damage were not observed in the aging WT mice. These results supported a pathogenic role of iron overload in aging Bmp6­/­ mice leading to iron­induced exocrine pancreatic deficiency, whereas the endocrine pancreas retained normal function.