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Bone tissue engineering seeks to adequately restore functions related to physical and biological properties, aiming at a repair process similar to natural bone. The use of compatible biopolymers, such as bacterial cellulose (BC), as well as having interesting mechanical characteristics, presents a slow in vivo degradation rate, and the ability to be chemically modified. To promote better bioactivity towards BC, we synthesized an innovative BC membrane associated to hydroxyapatite (HA) and anti-bone morphogenetic protein antibody (anti-BMP-2) (BC-HA-anti-BMP-2). We present the physical-chemical, biological and toxicological characterization of BC-HA-anti-BMP-2. Presence of BC and HA components in the membranes was confirmed by SEM-EDS and FTIR assays. No toxic potential was found in MC3T3-E1 cells by cytotoxicity assays (XTT Assay and Clonogenic Survival), genotoxicity (Comet Assay) and mutagenicity (Cytokinesis-blocked micronucleus Test). The in vitro release kinetics of anti-BMP-2 antibodies detected gradually reducing antibody levels, reducing approximately 70% in 7 days and 90% in 14 days. BC-HA-anti-BMP-2 increased SPP1, BGLAP, VEGF, ALPL, RUNX2 and TNFRSF11B expression, genes involved in bone repair and also increased mineralization nodules and phosphatase alcalin (ALP) activity levels. In conclusion, we developed BC-HA-anti-BMP-2 as an innovative and promising biomaterial with interesting physical-chemical and biological properties which may be a good alternative to treatment with commercial BMP-2 protein., Competing Interests: The authors have declared that no competing interests exist.

Recently, we have shown that anti-BMP2 monoclonal antibodies (mAbs) can trap endogenous osteogenic BMP ligands, which can in turn mediate osteodifferentiation of progenitor cells. The effectiveness of this strategy requires the availability of the anti-BMP-2 monoclonal antibodies antigen-binding sites for anti-BMP-2 monoclonal antibodies to bind to the scaffold through a domain that will leave its antigen-binding region exposed and available for binding to an osteogenic ligand. We examined whether antibodies bound to a scaffold by passive adsorption versus through Protein G as a linker will exhibit differences in mediating bone formation. In vitro anti-BMP-2 monoclonal antibodies was immobilized on absorbable collagen sponge (ACS) with Protein G as a linker to bind the antibody through its Fc region and implanted into rat calvarial defects. The biomechanical strength of bone regenerated by absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies immune complex was compared to ACS/anti-BMP-2 monoclonal antibodies or ACS/Protein G/isotype mAb control group. Results demonstrated higher binding of anti-BMP-2 monoclonal antibodies/BMPs to C2C12 cells, when the mAb was initially attached to recombinant Protein G or Protein G-coupled microbeads. After eight weeks, micro-CT and histomorphometric analyses revealed increased bone formation within defects implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies compared with defects implanted with absorbable collagen sponge/anti-BMP-2 monoclonal antibodies (p < 0.05). Confocal laser scanning microscopy (CLSM) confirmed increased BMP-2, -4, and -7 detection in sites implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies in vivo. Biomechanical analysis revealed the regenerated bone in sites with Protein G/anti-BMP-2 monoclonal antibodies had higher mechanical strength in comparison to anti-BMP-2 monoclonal antibodies. The negative control group, Protein G/isotype mAb, did not promote bone regeneration and exhibited significantly lower mechanical properties (p < 0.05). Altogether, our results demonstrated that application of Protein G as a linker to adsorb anti-BMP-2 monoclonal antibodies onto the scaffold was accompanied by increased in vitro binding of the anti-BMP-2 mAb/BMP immune complex to BMP-receptor positive cell, as well as increased volume and strength of de novo bone formation in vivo., (© The Author(s) 2015.)

Endogenous molecular and cellular mediators modulate tissue repair and regeneration. We have recently described antibody mediated osseous regeneration (AMOR) as a novel strategy for bioengineering bone in rat calvarial defect. This entails application of anti-BMP-2 antibodies capable of in vivo capturing of endogenous osteogenic BMPs (BMP-2, BMP-4, and BMP-7). The present study sought to investigate the feasibility of AMOR in other animal models. To that end, we examined the efficacy of a panel of anti-BMP-2 monoclonal antibodies (mAbs) and a polyclonal Ab immobilized on absorbable collagen sponge (ACS) to mediate bone regeneration within rabbit calvarial critical size defects. After 6 weeks, de novo bone formation was demonstrated by micro-CT imaging, histology, and histomorphometric analysis. Only certain anti-BMP-2 mAb clones mediated significant in vivo bone regeneration, suggesting that the epitopes with which anti-BMP-2 mAbs react are critical to AMOR. Increased localization of BMP-2 protein and expression of osteocalcin were observed within defects, suggesting accumulation of endogenous BMP-2 and/or increased de novo expression of BMP-2 protein within sites undergoing bone repair by AMOR. Considering the ultimate objective of translation of this therapeutic strategy in humans, preclinical studies will be necessary to demonstrate the feasibility of AMOR in progressively larger animal models.

Bone engineering strategies often exploit modulation of the extracellular environment, including delivery of cell and growth factors to repair and regenerate damaged tissues. During bone healing, the expression of endogenous bone morphogenetic proteins is an essential component of the healing response. However, in some situations, the inherent reparative capacity available in the local microenvironment is exceeded by the requirements of the defects. We have recently reported on a novel strategy, that exploits the specificity of antibodies to capture and make available endogenous osteogenic growth factors, referred to as "antibody-mediated osseous regeneration" (AMOR). The objective of the present study was to identify some of the cellular and molecular events involved in AMOR in an effort to begin to elucidate the mechanism of AMOR. The rat critical-sized calvarial defect model was used, where anti-bone morphogenetic protein (BMP)-2 monoclonal antibody (mAb), isotype-control mAb, or recombinant human (rh)BMP-2 were immobilized on absorbable collagen calvarial sponge (ACS) by adsorption, and then implanted into calvarial defects. The results demonstrated persistence of implanted mAbs for short term from 1 to 2 weeks after implantation. Increased cell infiltration was found in defects treated with anti-BMP-2 mAb. Examination of proteins on ACS scaffolds retrieved from defect sites demonstration increased levels of BMP-2, BMP-4, and BMP-7 proteins in sites implanted with anti-BMP-2 mAb. Moreover, BMP-2, BMP-4, and BMP-7 gene expression levels were increased in sites implanted with anti-BMP-2 mAb. Micro-computed tomography and histological analysis demonstrated that the bone within calvarial defects was fully regenerated in sites implanted with either anti-BMP-2 mAb or rhBMP-2. However, rhBMP-2-regenerated bone exhibited aberrant histomorphology with dystrophic calcification and invasion of subjacent areas. Altogether, the results revealed evidence for anti-BMP-2 mAbs to form an immune complex with BMP-2, BMP-4, and BMP-7, and bind to cells to mediate osteogenesis bone regeneration in vivo. This approach suggests a significant role for antibodies in regenerative orthopedic medicine.

Bone regeneration often requires harvesting of autologous bone with significant potential morbidity and cost. Recombinant human bone morphogenetic protein (rhBMP)-2 has been approved by the U.S. Food and Drug Administration for specific regenerative indications. However, administration of exogenous growth factors has many drawbacks. The objective of the present proof-of-concept study was to determine whether immobilized anti-BMP-2 antibodies (Abs) could capture endogenous BMP-2 in local sites to mediate osteogenesis, a strategy we refer to as antibody-mediated osseous regeneration (AMOR). We have generated a murine anti-BMP-2 monoclonal antibody library, which was tested along with commercially available Abs in vitro and in vivo for their ability to mediate AMOR. In vitro studies demonstrated that only some anti-BMP-2 Abs tested formed immune complexes with BMP-2, which can bind to BMP cellular receptor, whereas other BMP-2/anti-BMP-2 complexes failed to bind. To investigate whether anti-BMP-2 Abs were able to mediate AMOR in vivo, anti-BMP-2 Abs were immobilized on absorbable collagen sponge (ACS) and surgically placed in rat calvarial defects. Microcomputed tomography analysis of live animals at 2, 4, and 6 weeks demonstrated that some anti-BMP-2 Abs immobilized on ACS mediated significant bone regeneration, whereas other clones did not mediate any bone regeneration. In situ BMP-2 and osteocalcin expression was investigated by immunohistochemistry. Results demonstrated higher BMP-2 and osteocalcin expression in sites with increased bone regeneration. Results provide first evidence for the ability of anti-BMP2 Abs to form an immune complex with endogenous BMP-2 and mediate bone regeneration in vivo, suggesting a promising therapeutic method for tissue engineering.

The concept of using mesenchymal stem cells (MSCs) in bone repair has progressively evolved, and the goal of cell-mediated therapy is to prolong the natural physiological abilities of healing, or substitute them, when these are lacking, failing, or progressing too slowly. The future application of MSCs in human therapies depends on the establishment of preclinical studies with other mammals, such as mouse. Surprisingly, the immunologic properties of murine MSCs remain poorly documented. In the present study, flow cytometry revealed that undifferentiated murine MSCs and osteogenic cells differentiated from MSCs (DOCs) express major histocompatibility complex (MHC) class I (H-2(b)), but not class II (I-A(b)). After exposure to interferon-gamma (IFN-gamma) for 48h, MHC class II and costimulatory molecules (B7-1 and B7-2) on the cell surface showed evident up-regulation. Undifferentiated MSCs and DOCs proved to be poor stimulators of T cell proliferation, eliciting alloreactive lymphocyte proliferative responses as low-allogenic stimulators. Initial results show that the expression of MHC class I, MHC class II, B7-1 and B7-2 was similar on human bone morphogenetic protein 2 (BMP2)-expressing recombinant adenoviral vector (AdBMP2) transduced MSCs (30 MOI) when compared with non-transduced cells. However, AdBMP2 gene transfected MSCs elicited significant stimulatory responses. The findings will be important for studying the in vivo behaviour and the fate of MSCs after grafting in mouse pathology models in bone regeneration.