789. Regulated VEGF Expression Modulates Bone Formation Elicited by Muscle Stem Cells Expressing BMP4 (2006)

Our prior study demonstrated synergy between angiogenic and osteogenic factors in bone formation, indicating that the former is very important for endochondral ossification. We also found that excessive VEGF in relation to BMP4 impairs bone healing. Here, we hypothesized that the timing of VEGF induction will affect bone formation mediated by muscle-derived stem cells (MDSCs) expressing BMP4 (B4 cells). We constructed a self-inactivating tet-on retroviral vector expressing VEGF under the control of a tetracycline-inducible promoter. ELISA was used to confirm inducible VEGF expression by the transduced MDSCs (TVE cells). TVE cells were exposed to different doses of doxycycline (Dox; 0, 1, and 2 μg/mL), and the amount of VEGF in conditioned medium was measured over 2, 4, or 6 days. TVE cells stimulated with 2 μg/mL of Dox for 6 days secreted 4 times less VEGF than did MDSCs transduced with a conventional retroviral vector (VE cells). Based on these findings, a normalized number of B4 and TVE cells (B4+TVE) was seeded onto Gelfoam scaffolds and implanted into the right thigh muscles of normal mice. Control mice received scaffolds containing B4 and VE cells (B4+VE). Left thigh muscles of all mice received scaffolds containing B4 cells only (B4). The 25 animals then were divided into 5 groups depending on whether or not they received Dox- supplemented drinking water and when Dox was administered after surgery. Mice in group 1 (control) and group 2 received no Dox. Dox administration began on day 0 after surgery in group 3, on day 3 in group 4, and on day 7 in group 5 and continued until euthanization. Ectopic bone formation at the implantation sites was monitored radiographically and histologically at 3 and 6 weeks postsurgery. Exorbitant ossification was detected at B4+VE implantation sites in control mice. Superior bone formation was evident in Dox-treated animals at B4+TVE implantation sites compared to B4 implantation sites any time VEGF expression was induced by Dox administration. Radiomorphometry showed larger bone area at B4+TVE implantation sites in Dox-treated mice than in nontreated mice. There was a significant increase in bone area and density after 6 weeks when Dox was administered at times other than immediately after surgery. At the end of the experiment we isolated bony nodules from the muscles of remaining mice and measured their dry weight. Bone from B4+TVE implantation sites of Dox-treated animals weighted more than bone from contralateral B4 implantation sites, but the increase was significant only when VEGF expression began on day 3. VEGF induction time had no significant effect on the ectopic bone weight in Dox-treated mice. Our study shows that controlled VEGF expression is achievable with the inducible tet-on retroviral vector developed in our laboratory. After induction with Dox TVE cells secreted enough VEGF to enhance heterotopic bone formation mediated by B4 cells. We were able to modulate bone formation by altering the start time of VEGF expression. Our study has important implications for the development of better bone tissue engineering strategies based on optimizing the timing of VEGF supplementation.