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.