Immunomodulation of Growth Factor Microenvironment Activates Bone Healing

Objective :The purpose of this study was to characterize the  biological and molecular mechanisms of tethered monoclonal antibodies (mAbs) in the extracellular microenvironment.

Methods : In vivo analyses was performed by immobilizing Anti-BMP-2 mAb , isotype control Ab (25 ug/ml), were adsorbed in each of the scaffolds by incubation at room temperature for 1 hour. We treated the critical size defects of both rat and rabbits with the matrices. . To investigate  protein expression of BMP-2,-4,-7 and presence of abs in early stages ( 1,3,7 days) after surgical implantation, confocal analysis was utilized. After 4 and 6 weeks, bone formation was evaluated morphologically by Micro-CT scan and histology, and molecularly by immunohistochemistry of BMP-2 and osteocalcin. To characterize Ab binding kinetics  in vitro, ElISA was developed. Epitope mapping analysis was studied by protein array.

Results:  Bone Regeneration  revealed by micro-CT and histological analysis  after4 and 6 weeks demonstrated that in both rat and rabbit wounds there was a significant increase  bone formation (p<0.001)  when compared to isotype controls. Confocal analyses of in situ  expression BMPs showed  increase in BMP-2,-4,-7 after 1 to 7 days. In addition, after 4 and6 weeks, BMP-2 and osteocalcin expression was increased in bone regeneration sites only. In vitro  cross reactivity of Abs by western blots was positive for BMP-2,-7. Kinetics Abs and BMPs interaction showed high dissociation constant  (k_D=11nM). Protein array utilizing 12 overlapping peptides of each mature BMPs, showed that  the N-terminus region of BMP-2,-7 was a positive functional region of Ab binding.

Conclusion: This data provide the first molecular elucidation of the interaction of AMOR and BMP-2,-4,-7. These  antibodies modulate the extracellular matrix,  forming a complex  with BMPs in vivo, increasing its local concentration and mediate bone regeneration in vivo, suggesting a novel role of monoclonal antibodies in bioengineering.