Novel Approach to Regeneration of Bone: Using Focused Ultrasound for the Spatiotemporal Patterning of Angiogenic and Osteogenic Factors.

摘要

This project is developing an innovative method for regenerating bone that incorporates both temporal and spacial control of angiogenic and osteogenic genes, thereby allowing the patterning of vasculature and bone formation. The approach uses high intensity focused ultrasound (HIFU) and heat shock/ligand-dependent gene switches. Focused ultrasound generates localized hyperthermia to activate the heat shock response in a spatially restricted fashion, while ligand dependency improves stringency of expression. Substantial progress was made in all 3 task areas during year 1. 1. Generate adenoviral vectors for use in primary cells. Plasmid-based and retrovirus-based systems were developed for HIFU/heat shock inducible expression of VEGF165 and BMP2. 2. Establish stringency of therapeutic gene expression in transduced BMSCs. The plasmid expression systems developed under Task 1 were transfected into C3H10T1/2 mesenchymal cells. Tight control of luciferase, VEGF165 and BMP2 expression was demonstrated after heat-shock. 3. Determine the doses of HIFU required for inducing gene expression in vitro and in vivo. Optimal conditions for HIFU-induction were identified using a luciferase reporter gene stably integrated into C3H10T1/2 cells embedded in fibrin scaffolds. Using this approach, precisely patterned gene expression was achieved in vitro. HIFU- inducible VEGF and BMP2 expression were demonstrated. Lastly, heat shock induction of VEGF expression was demonstrated in vivo and shown to induce a robust subcutaneous angiogenic response.