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Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy

Abstract

Structural allograft healing is limited because of a lack of vascularization and remodeling. To study this we developed a mouse model that recapitulates the clinical aspects of live autograft and processed allograft healing. Gene expression analyses showed that there is a substantial decrease in the genes encoding RANKL and VEGF during allograft healing. Loss-of-function studies showed that both factors are required for autograft healing. To determine whether addition of these signals could stimulate allograft vascularization and remodeling, we developed a new approach in which rAAV can be freeze-dried onto the cortical surface without losing infectivity. We show that combination rAAV-RANKL- and rAAV-VEGF-coated allografts show marked remodeling and vascularization, which leads to a new bone collar around the graft. In conclusion, we find that RANKL and VEGF are necessary and sufficient for efficient autograft remodeling and can be transferred using rAAV to revitalize structural allografts.

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Figure 1: The mouse femoral allograft model.
Figure 2: Altered Tnfsf11 and Vegfa gene expression during allograft healing.
Figure 3: Systemic and local loss of either RANKL or VEGF results in defective autograft healing.
Figure 4: Transduction efficiency of rAAV-β-gal following freeze-drying onto allografts and implants in vitro and in vivo.
Figure 5: Revitalization of processed allografts via rAAV mediated-RANKL and VEGF gene transfer.
Figure 6: rAAV-mediated gene transfer of RANKL and VEGF induces cortical bone resorption, vascularization and remodeling in processed allografts in vivo.

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Acknowledgements

We thank: H. Burchardt (Musculoskeletal Transplant Foundation) for advice with this research. J. Huard (University of Pittsburgh) for providing us with the sFlt-1 cDNA, W. Min (Yale University) for providing us with the Vegfa cDNA, Amgen Inc. for providing us with the OPG and Tnfsf11 cDNA and the RANK:Fc. We also thank C. Hock and D. Reynolds for assistance with the serum ELISA studies, B, Fan, L. Gehan and B. Stroyer for assistance with the histology, H. Awad for assistance with manuscript preparation, and R. Guldberg and A. Lin for μCT analyses. This work was supported by research grants from the Orthopedic Research and Education Foundation, the Musculoskeletal Transplant Foundation, US National Institutes of Health grants AR51469, AR48149, AR48681, AR43510, ES011854 and HL066973, and unrestricted research grants from DePuy, J&J Inc.

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Correspondence to Edward M Schwarz.

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J. Rabinowitz and R.J. Samulski are founders of Asklepios BioPharmaceutical, Inc. P.T. Rubery, R.J. O'Keefe and E.M. Schwarz are founding members of LAGeT, Inc.

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Ito, H., Koefoed, M., Tiyapatanaputi, P. et al. Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy. Nat Med 11, 291–297 (2005). https://doi.org/10.1038/nm1190

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