Antibody-mediated Osseous Regeneration Research Articles

The sialylation profile of IgG determines the efficiency of antibody directed osteogenic differentiation of iMSCs by modulating local immune responses and osteoclastogenesis

Antibody-mediated osseous regeneration (AMOR) has been proved as a promising strategy for osteogenic differentiation of induced pluripotent stem cells derived MSCs (iMSCs). The key characteristic of antibody that determines the AMOR potential is largely unknown. The glycosylation profile of immunoglobulin G (IgG) represents a key checkpoint that determines its effector functions. Herein, we modified the sialylation profile of BMP2 antibodies to investigate the effects of glycosylation on antibody-mediated osteogenic differentiation of iMSCs. We found that over-sialylated BMP2 antibodies stimulated the highest amount of new bone while those non- or low-sialylated led to bone porosity and collapse. The immune response aroused by BMP2 immune complexes (BMP2-ICs) was intensified by desialylation, which contributed to an environment that favored osteoclastogenesis while inhibited osteoblastogenesis. In vitro study further demonstrated that the osteogenic potential of BMP2-ICs was not significantly affected by the degree of sialylation. On the other hand, BMP2-ICs could stimulate osteoclastogenesis by binding FcγRs on preosteoclasts directly, which was significantly intensified by desialylation and attenuated by over-sialylation. Bone defects implanted with alginate microbeads loaded with iMSCs and over-sialylated antibodies showed more bone formation than those sites with non- or low sialylated antibodies. Taken together, our study demonstrated that sialylation profile is one of the traits that decide the AMOR potential of BMP2 antibodies. Enhancement of sialylation may be a promising strategy to optimize antibody for iMSCs application in bone tissue engineering. Statement of significanceAntibody-mediated osseous regeneration (AMOR) is a promising strategy for bone tissue engineering that takes advantage of the specific reactivity of antibodies to sequester endogenous BMP2 and present it to osteoprogenitor cells. We previously demonstrated that BMP2 immune complex can drive iPSCs derived MSCs to osteogenic lineage. In this study, we analyze the effects of glycosylation profile on antibody directed osteogenic differentiation of iMSCs because glycosylation profile represents a key checkpoint that determines the effector functions of antibodies, and it is susceptible to variations in different clones. The results showed that sialylation profile is one of the traits that decides the AMOR potential of BMP2 antibody, and the enhancement of sialylation maybe a promising strategy to optimize antibodies for AMOR.

Antibody-Mediated Osseous Regeneration for Bone Tissue Engineering in Canine Segmental Defects

Among many applications of therapeutic monoclonal antibodies (mAbs), a unique approach for regenerative medicine has entailed antibody-mediated osseous regeneration (AMOR). In an effort to identify a clinically relevant model of craniofacial defect, the present study investigated the efficacy of mAb specific for bone morphogenetic protein- (BMP-) 2 to repair canine segmental mandibular continuity defect model. Accordingly, a 15 mm unilateral segmental defect was created in mandible and fixated with a titanium plate. Anorganic bovine bone mineral with 10% collagen (ABBM-C) was functionalized with 25 μg/mL of either chimeric anti-BMP-2 mAb or isotype-matched mAb (negative control). Recombinant human (rh) BMP-2 served as positive control. Morphometric analyses were performed on computed tomography (CT) and histologic images. Bone densities within healed defect sites at 12 weeks after surgery were 1360.81 ± 10.52 Hounsfield Unit (HU), 1044.27 ± 141.16 HU, and 839.45 ± 179.41 HU, in sites with implanted anti-BMP-2 mAb, rhBMP-2, and isotype mAb groups, respectively. Osteoid bone formation in anti-BMP-2 mAb (42.99% ± 8.67) and rhBMP-2 (48.97% ± 2.96) groups was not significantly different but was higher (p < 0.05) than in sites with isotype control mAb (26.8% ± 5.35). In view of the long-term objective of translational application of AMOR in humans, the results of the present study demonstrated the feasibility of AMOR in a large clinically relevant animal model.

Collagen sponge functionalized with chimeric anti-BMP-2 monoclonal antibody mediates repair of nonunion tibia defects in a nonhuman primate model: An exploratory study.

Recombinant human bone morphogenetic protein (BMP)-2 is an FDA-approved therapy for nonunion tibia fracture, though it has a number of biological and practical disadvantages. Our research group has developed a novel tissue engineering strategy termed antibody-mediated osseous regeneration. This entails application of anti-BMP-2 monoclonal antibodies (mAbs) to capture endogenous BMP's to mediate invivo bone formation. This has been documented in a number of animal models. The present exploratory study sought to investigate the application of antibody-mediated osseous regeneration for repair of nonunion tibia defect in a nonhuman primate model. A 20 mm segmental osteotomy was performed in tibia of 6 Macaca fascicularis and was implanted with absorbable collagen sponge that was functionalized with chimeric anti-BMP-2 or isotype matched control mAb. Cone beam computed tomography (CBCT), histologic and histomorphometric analyses were performed 12 weeks post-operatively. CBCT analyzed by quantitative 3D volumetric analysis revealed that sites implanted with absorbable collagen sponge functionalized with anti-BMP-2 mAb demonstrated numerically higher mineralized tissue (408 ± 127 mm3) compared with sites implanted with isotype matched control mAb (214 ± 81 mm3), though the difference was not statistically significant ( p = 0.09). Histologic and histomorphometric analysis showed de novo bone formation with greater ( p < 0.01) percentage of bone volume in sites implanted with anti-BMP-2 (41.3 ± 4.4%), compared with isotype matched control mAb (14.6 ± 5.6%). Results from the present exploratory study provide evidence for the potential of anti-BMP-2 mAb to mediate repair of a large segmental tibia defects in a nonhuman primate model. Therapeutic antibodies have generally been shown to have great safety and efficacy profile, though their application in tissue engineering has been limited in the past. Following further investigation, anti-BMP-2 mAbs immobilized on appropriate scaffold may have application in repair of large skeletal defects without the need for exogenous growth factors.

Collagen Sponge Functionalized with Chimeric Anti-BMP-2 Monoclonal Antibody Mediates Repair of Critical-Size Mandibular Continuity Defects in a Nonhuman Primate Model.

Antibody-mediated osseous regeneration (AMOR) has been introduced by our research group as a tissue engineering approach to capture of endogenous growth factors through the application of specific monoclonal antibodies (mAbs) immobilized on a scaffold. Specifically, anti-Bone Morphogenetic Protein- (BMP-) 2 mAbs have been demonstrated to be efficacious in mediating bone repair in a number of bone defects. The present study sought to investigate the application of AMOR for repair of mandibular continuity defect in nonhuman primates. Critical-sized mandibular continuity defects were created in Macaca fascicularis locally implanted with absorbable collagen sponges (ACS) functionalized with chimeric anti-BMP-2 mAb or isotype control mAb. 2D and 3D analysis of cone beam computed tomography (CBCT) imaging demonstrated increased bone density and volume observed within mandibular continuity defects implanted with collagen scaffolds functionalized with anti-BMP-2 mAb, compared with isotype-matched control mAb. Both CBCT imaging and histologic examination demonstrated de novo bone formation that was in direct apposition to the margins of the resected bone. It is hypothesized that bone injury may be necessary for AMOR. This is evidenced by de novo bone formation adjacent to resected bone margins, which may be the source of endogenous BMPs captured by anti-BMP-2 mAb, in turn mediating bone repair.

Biomechanical analysis of engineered bone with anti-BMP2 antibody immobilized on different scaffolds.

Recently we have demonstrated the ability of monoclonal antibodies (mAb) specific for bone morphogenetic protein (BMP)-2 immobilized on different scaffolds to mediate bone formation, a process referred to as Antibody Mediated Osseous Regeneration (AMOR). One of the key properties of regenerated bone is its biomechanical strength, in particular in load-bearing areas. This study sought to test the hypothesis that the biomechanical strength of regenerated bone depends of the mode of regeneration, as well as the scaffold used. Four different scaffolds, namely titanium granules (Ti), alginate hydrogel, anorganic bovine bone mineral (ABBM), and absorbable collagen sponge (ACS) were functionalized with anti-BMP-2 or isotype control mAb and implanted into rat critical-size calvarial defects. The morphology, density and strength of the regenerated bone were evaluated after 8 weeks. Results demonstrated that scaffolds functionalized with anti-BMP-2 mAb exhibited varying degrees of bone volume and density. Ti and ABBM achieved the highest bone volume, density, and strength of bone. When anti-BMP-2 mAb was immobilized on Ti or ABBM, the strength of the regenerated bone were 80% and 77% of native bone respectively, compared with 60% of native bone in sites implanted with rh-BMP-2. Control interventions with isotype mAb did not promote considerable bone regeneration and exhibited significantly lower mechanical properties. SEM analysis showed specimens immobilized with anti-BMP-2 mAb formed new bone with organized structure bridging the crack areas. Altogether, the present data demonstrated that the morphological and mechanical properties of bone bioengineered through AMOR could approximate that of native bone, when appropriate scaffolds are used. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1465-1473, 2016.

Effects of the orientation of anti-BMP2 monoclonal antibody immobilized on scaffold in antibody-mediated osseous regeneration.

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.

Application of AMOR in craniofacial rabbit bone bioengineering.

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.

Immobilization of murine anti-BMP-2 monoclonal antibody on various biomaterials for bone tissue engineering.

Biomaterials are widely used as scaffolds for tissue engineering. We have developed a strategy for bone tissue engineering that entails application of immobilized anti-BMP-2 monoclonal antibodies (mAbs) to capture endogenous BMPs in vivo and promote antibody-mediated osseous regeneration (AMOR). The purpose of the current study was to compare the efficacy of immobilization of a specific murine anti-BMP-2 mAb on three different types of biomaterials and to evaluate their suitability as scaffolds for AMOR. Anti-BMP-2 mAb or isotype control mAb was immobilized on titanium (Ti) microbeads, alginate hydrogel, and ACS. The treated biomaterials were surgically implanted in rat critical-sized calvarial defects. After 8 weeks, de novo bone formation was assessed using micro-CT and histomorphometric analyses. Results showed de novo bone regeneration with all three scaffolds with immobilized anti-BMP-2 mAb, but not isotype control mAb. Ti microbeads showed the highest volume of bone regeneration, followed by ACS. Alginate showed the lowest volume of bone. Localization of BMP-2, -4, and -7 antigens was detected on all 3 scaffolds with immobilized anti-BMP-2 mAb implanted in calvarial defects. Altogether, these data suggested a potential mechanism for bone regeneration through entrapment of endogenous BMP-2, -4, and -7 proteins leading to bone formation using different types of scaffolds via AMOR.

Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration

Recent studies have demonstrated the ability of murine anti-BMP-2 monoclonal antibodies (mAb) immobilized on an absorbable collagen sponge (ACS) to mediate de novo bone formation, a process termed antibody-mediated osseous regeneration (AMOR). The objectives of this study were to assess the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating AMOR, as well as to evaluate the suitability of different biomaterials as scaffolds to participate in AMOR. Chimeric anti-BMP-2 mAb was immobilized on 4 biomaterials, namely, titanium microbeads (Ti), alginate hydrogel, macroporous biphasic calcium phosphate (MBCP) and ACS, followed by surgical implantation into rat critical-size calvarial defects. Animals were sacrificed after 8 weeks and the degree of bone fill was assessed using micro-CT and histomorphometry. Results demonstrated local persistence of chimeric anti-BMP-2 mAb up to 8 weeks, as well as significant de novo bone regeneration in sites implanted with chimeric anti-BMP-2 antibody immobilized on each of the 4 scaffolds. Ti and MBCP showed the highest volume of bone regeneration, presumably due to their resistance to compression. Alginate and ACS also mediated de novo bone formation, though significant volumetric shrinkage was noted. In vitro assays demonstrated cross-reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. Immune complex of anti-BMP-2 mAb with BMP-2 induced osteogenic differentiation of C2C12 cells in vitro, involving expression of RUNX2 and phosphorylation of Smad1. The present data demonstrated the ability of chimeric anti-BMP-2 mAb to functionalize different biomaterial with varying characteristics to mediate osteogenesis.

Antibody-Mediated Osseous Regeneration: The Early Events in the Healing Response

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.

Antibody-Mediated Osseous Regeneration: A Novel Strategy for Bioengineering Bone by Immobilized Anti–Bone Morphogenetic Protein-2 Antibodies

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.