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

Abstract

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.

Keywords: Bone morphogenetic protein; Growth factor; Monoclonal antibody; Scaffold; Tissue engineering.

Figure 1

(a) Schematic representation of the flow cytometric assay to study the binding ability of anti-BMP mAb + rhBMP-2 immune complexes to BMP-receptor positive cells. Anti-BMP-2 mAb and rhBMPs were incubated to form immune complexes, and the resultant complexes were followed by incubation of immune complexes incubated with C2C12 cells. The cell-bound immune complexes were detected with a fluorescently- conjugated secondary antibody and flow cytometric analysis. (b) Flow cytometric data examining binding of anti-BMP-2 mAb immune complexes with rhBMP-2, rhBMP-4 and rhBMP-7 to BMP-receptor-positive C2C12 cells. The mean fluorescent intensity (MFI) of flow cytometric analysis showed significant binding between anti-BMP-2 mAb and BMP-2, BMP-4 and BMP-7. Results confirmed the presence of binding interaction between the chimeric anti-BMP-2 and BMP-2, BMP-4 and BMP- 7. **, p<0.01.

Figure 2

(a) Alizarin red staining indicating mineralized nodule formation of cultured C2C12 cells after treatment with either BMP-2 or chimeric anti-BMP-2 mAb after four weeks. (b) Quantitative analysis of the amount of alizarin staining. (c) Western blot analysis showing the effect of chimeric mAb on the levels of expression of regulators of osteogenesis in C2C12 cells. (d) Graphical summary of interactions between chimeric anti-BMP-2 mAb and the receptors (BMPR-I and BMPR-II) that mediate AMOR through the BMP signaling pathway. NS: not significant.

Figure 3

Dose response study of chimeric anti-BMP-2 mAb adsorbed on ACS implanted in rat calvarial defects. Representative 3D reconstruction of micro-CT images (a) and histomicrographs (b) of rat calvarial defects 8 weeks after implantation with different concentrations of chimeric anti-BMP-2 mAb adsorbed on ACS (scale bar = 1 mm). (c) Quantitative analysis of micro-CT data, expressed as % bone fill within calvarial defects. (d) Histomorphometric analysis of Trichrome-stained sections in panel b, expressed as % osteoid bone. Each concentration was compared to isotype mAb as the negative control. Anti-BMP-2 mAb at 25 μg/mL and rhBMP-2 showed the largest amounts of new bone formation. *, Asterisk symbols show the groups that are significantly different (p<0.05).

Figure 4

(a) Representative 3D reconstruction of micro-CT images of rat calvarial defects 8 weeks after implantation with chimeric anti-BMP-2 mAb adsorbed on 4 different scaffold materials. (b) Quantitative analysis of micro-CT data, expressed as % bone fill within calvarial defects. **, p< 0.01.

Figure 5

(a) Histological analysis of rat calvarial bone defects implanted with chimeric anti-BMP-2 mAb immobilized on 4 different types of scaffolds (scale bar = 1 mm for low magnification images and 50 μm for histomicrographs in high magnification). (b) Quantitative histomorphometric analysis performed on H&E-stained sections showing percentage of new bone formation. **, p< 0.01.

Figure 6

Confocal laser scanning microcopy analysis of scaffolds functionalized with anti-BMP-2 mAb retrieved at different time points. Representative CLSM images (a) and quantitative analysis (b, c) of immunofluorescence of 4 different biomaterials with immobilized anti-BMP-2 mAb. Calvarial specimens were retrieved at 1, 14 and 56 days and immunofluorescently labeled with FITC-conjugated goat anti-Human IgG Ab and imaged by CLSM. To control for non-specific labeling, controls were immunolabeled with donkey anti-goat IgG-FITC. (b) Quantitative analysis of fluorescence intensity. (c) Reduction in the fluorescence intensity for each scaffold from day 0 to day 56 after implantation. Three specimens for each group were tested. *p<0.05.