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Uncoupling Of Osteogenic And Osteoclastogenic Differentiation On Nanoparticulate Mineralized Collagen Scaffolds Through Physisorption Of Osteoprotegerin
Dillon Dejam, BS1, Xiaoyan Ren, MD, PhD1, Qi Zhou, DDS, PhD1, Michelle K. Oberoi, BA, BS1, Natalie J. Dahan, BA1, Kelly X. Huang, HSD1, Rachel M. Caprini, BS1, Meiwand Bedar, MD, MSc1, Brendan A.C. Harley, SB, ScD2, Justine C. Lee, MD, PhD1.
1Division of Plastic and Reconstructive Surgery, University of California, Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA, 2Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Purpose Osteoprotegerin (OPG) plays an important role in bone metabolism by functioning as a decoy receptor for receptor activator of nuclear factor kB (RANK) ligand (RANKL) in the RANK/RANKL/OPG axis, resulting in the inhibition of osteoclastogenesis and bone resorption. Our previous work has demonstrated that the introduction of OPG through an adenoviral vector into primary human mesenchymal stem cells (hMSCs) seeded onto nanoparticulate mineralized collagen glycosaminoglycan scaffolds (MC) promotes osteogenesis and potentiates the inhibition of osteoclast resorption activity. However, there are disadvantages to using viral vectors in translational medicine, such as non-integration, immunogenicity, replication competence, and off-target effects. In this work, we investigate an alternative physisorption-based approach to deliver recombinant OPG onto both non-mineralized collagen-glycosaminoglycan scaffolds (CG) and MC both in vitro and in vivo. Methods CG and MC scaffolds were soaked in recombinant OPG, generating S-OPG CG and S-OPG MC, respectively. Western blot, enzyme-linked immunosorbent assays, and co-immunoprecipitation were performed in order to characterize the release of OPG from soaked scaffolds and to evaluate for interactions with RANKL. After seeding and culturing scaffolds with hMSCs, mineralization of scaffolds was evaluated using microtomography. Fluorescent staining of scaffold sections from both osteoclast-only and osteoblast-osteoclast cocultures was performed in order to evaluate for changes in osteoclast differentiation, and pit assays were performed in order to determine the effect of physisorption of OPG on osteoclast resorption activity. Finally, in vivo implantation of OPG-soaked scaffolds was performed in a rabbit cranial defect model. Results Scaffolds soaked with OPG were confirmed to continually release OPG into culture medium, and co-immunoprecipitation confirmed that OPG released from soaked scaffolds was able to bind RANKL. In addition, scaffolds soaked with OPG did not demonstrate any decreases in mineralization or osteogenic differentiation compared to non-soaked scaffolds, and fluorescent staining demonstrated that osteoclasts on soaked scaffolds were poorly differentiated compared to non-soaked scaffolds. Results of pit assays demonstrated that osteoclast resorption activity was significantly inhibited on S-OPG MC. In our in vivo rabbit cranial defect model, both S-OPG CG and S-OPG MC demonstrated increased mineralization compared to non-soaked controls. Conclusions Our results demonstrate that physisorption of OPG onto CG and MC inhibits osteoclast differentiation and resorption activity without affecting osteogenic differentiation or mineralization, and these results are corroborated by in vivo experiments where soaking scaffolds prior to implantation leads to increased mineralization. Thus, the physisorption of OPG onto MC may represent a potential mechanism for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration.

Figure 1. Representative microtomography images used to evaluate mineralization of scaffolds (left). Physisorption of OPG onto scaffolds does not affect mineralization (right).

Figure 2. Scaffolds cultured in osteoblast-osteoclast coculture demonstrate poor osteoclast differentiation on staining (left), as well as decreased osteoclast resorptive activity on S-OPG MC (right).

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