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Inducible regulation of Runx2-stimulated osteogenesis

Abstract

Ex vivo gene therapy is a promising approach to orthopedic regenerative medicine. These strategies typically focus on the constitutive overexpression of osteogenic factors to induce osteoblastic differentiation and matrix mineralization. However, the unregulated production of osteoinductive molecules has also resulted in abnormal bone formation and tumorigenesis. To address these limitations, this work describes a retroviral system to deliver the Runx2 osteoblastic transcription factor under control of the tetracycline-inducible (tet-off) promoter in primary skeletal myoblasts. Runx2 expression was tightly regulated by anhydrotetracyline (aTc) concentration in cell culture media. Osteoblastic gene expression, alkaline phosphatase activity, and matrix mineralization were also controlled by aTc in a dose-dependent manner. Additionally, osteoblastic differentiation was temporally regulated by adding and removing aTc from the culture media. Engineered cells were seeded onto collagen scaffolds and implanted intramuscularly in the hind limbs of syngeneic mice. In vivo mineralization by these constructs was regulated by supplementing the drinking water with aTc, as demonstrated by micro-computed tomography and histological analyses. Collectively, these results present a novel system for regulating osteoblastic differentiation of a clinically relevant autologous cell source. This system is significant to developing controlled and effective orthopedic gene therapy strategies and studying the regulation of osteoblastic differentiation.

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Acknowledgements

Collagen scaffolds were generously donated by the Kensey Nash Corporation. We thank TJ Murphy and Grace K Pavlath for generously providing retroviral vectors and primary skeletal myoblasts, respectively. We also thank Jennifer E Phillips and Benjamin A Byers for helpful discussions and Angela S Lin, Tracey L Couse and Kellie L Burns for technical assistance with micro-CT, histology and animal surgeries, respectively. This research was funded by the NIH (R01-EB003364), the Georgia Tech/Emory Center NSF Engineering Research Center on Tissue Engineering (EEC-9731643), the Emory-Georgia Tech Biomedical Technology Research Center, and a Medtronic Foundation Fellowship and NIH Biotechnology Training Grant (T32-GM08433).

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Correspondence to A J García.

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Gersbach, C., Le Doux, J., Guldberg, R. et al. Inducible regulation of Runx2-stimulated osteogenesis. Gene Ther 13, 873–882 (2006). https://doi.org/10.1038/sj.gt.3302725

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