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 (kD=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.
Keywords: Antigens-antibodies, Bioengineering, Extracellular matrix molecules, Therapeutics and Wound healing
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