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

Abstract

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.

Keywords: Bone regeneration; Protein G; biomaterials; chimeric anti-BMP2 monoclonal antibodies; tissue engineering.

Figure 1

(a) AMOR: (1) anti-BMP-2 mAb is immobilized on a scaffold. (2) mAb captures endogenous BMP-2 (and other homologous osteogenic BMPs) from the microenvironment. (3) BMP-2 captured by specific mAb binds its cellular receptor on osteoprogenitor cells, promoting their osteogenic differentiation. (b) Schematic representation of the binding of the immune complex (IC) between Protein G/anti-BMP-2 mAb/BMP’s to cellular receptors in vivo.

Figure 2

Investigation of the effect of orientation of anti-mAb on the binding of the immune complex of anti-BMP2/BMP2 to target cells. Flow cytometric analysis of binding of the immune complex between anti-BMP-2 mAb/recombinant protein-G/BMP-2, BMP-4 or BMP-7/BMP-2 cellular receptor on C2C12 cells. Fluorochrome-labeled cells were analyzed by flow cytometer and the mean fluorescence intensity (MFI) of PE was calculated. Controls included cells alone (−) or substitution of chimeric anti-BMP2 mAb with isotype-matched Ab (Iso mAb). The MFI of flowcytometric analysis showed significant binding between PG/chimeric antibody complex and BMP2. **p < 0.01, and ***p < 0.001.

Figure 3

Characterization of the in vitro release profile and binding of chimeric mAb and chimeric mAb Protein G complex-loaded scaffolds. (a) The in vitro release of mAb was calculated by measuring mAb concentrations in solution at various time points. (b) Fluorescence microscopic analysis demonstrating binding of anti-BMP-2 mAb on ACS scaffold detected by FITC-conjugated goat anti-human secondary antibody at different time intervals. *p < 0.05.

Figure 4

(a) Representative 3D reconstruction of micro-CT images of bone volume within rat calvaria. Anti-BMP-2 mAb immobilized on ACS with or without Protein G-coupled microbeads linker implanted within rat calvarial defects. Isotype-matched mAb immobilized on ACS served as the control. (b) Bone volume/total volume (BV/TV) within calvarial defects in each specimen was measured by micro-CT. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

Histological analysis of rat calvarial bone defects implanted withanti-BMP-2 mAb immobilized on ACS with or without Protein G-coupled microbeads linker. Protein G-coupled microbeads (yellow dots) are shown with arrows. Animals were sacrificed at 8 weeks after surgery and calvarial bones were processed for histologic and Histomorphometric analysis. (a) Histomicrographs in low (4×) and high magnification (40×) of H&E stained calvaria. (b) Histomorphometric analysis was performed on H&E stained sections and percentage of new bone formation was quantified. The percentage of osteoid bone coverage was measured within histomicrographs by histomorphometric analysis. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6

Immunofluorescence labeling of tissue specimens harvested from rat calvarial bone defects implanted with anti-BMP-2 mAb immobilized on ACS with or without Protein G linker. Primary antibodies included those with specificity against BMP-2, -4, and -7. (a) Representative immunofluorescent CLSM images demonstrated positive labeling within sites implanted with anti-BMP-2 mAb immobilized on ACS with rec-Protein G linker, as well those sites with anti-BMP-2 mAb immobilized directly on ACS. (b) Analysis of the percentage of positively stained area for anti-BMP-2, -4, and -7 antibodies, showing that G protein-anti-BMP2 mAb complex presented the highest expression of BMP-2, -4, and -7 proteins in comparison to those sites with anti-BMP-2 mAb immobilized directly on ACS, or isotype-matched control mAb, *p < 0.05 and **p < 0.01, respectively.

Figure 7

Results of biomechanical evaluation of the regenerated bone and comparative analysis of strength of the newly formed bone in comparison to native bone. *p < 0.05. NS: not significant.