Plastic Surgery Research Council
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Presenter: Akishige Hokugo, DDS, PhD
Co-Authors: Li A; Buck A; Nguyen P; Li J; Jarrahy R

Background: Autologous bone grafting is the gold standard for bone reconstruction and creates significant donor site morbidity. Regenerative medicine can circumvent this through constructs that combine stem cells, growth factors, and scaffolds. Human mesenchymal stem cells (hMSCs) and vascular endothelial growth factor (VEGF) are both promising in this context, the former due to their pluripotent capacity and the latter due to its osteogenic and chemotactic activity. In this study, we combine hMSCs and VEGF to develop a smart scaffold for use in bone tissue engineering.

Methods: MSCs were transfected with human VEGF-A and blasticidin-red-fluorescent-protein (RFP) via lentivirus vectors. Expression of RFP in the hMSCs confirmed successful transfection. Levels of VEGF were measured in conditioned media taken from transfected and non-transfected hMSCs through enzyme-linked immunosorbent assay (ELISA). The chemotactic activity of VEGF-transfected cells was evaluated via a trans-well assay: conditioned media was collected from transfected and non-transfected hMSC cultures. MSCs were placed in the upper chamber and conditioned media from both cell types filled the lower chambers. After 3 hours, cells that migrated through the semipermeable membranes separating upper and lower chambers were counted with calcein-AM using a fluorescent plate reader.

Results: Transfection of RFP occurred at nearly 100%, as evidenced by red fluorescence of transfected hMSCs. Non-transfected hMSCs did not express red fluorescence. Levels of VEGF secreted by transfected hMSCs were significantly higher than levels secreted by non-transfected hMSCs. Migration through semipermeable membranes was significanty greater in chambers filled with medium conditioned by VEGF-transfected cells.

Conclusions: Human MSCs can be transfected with VEGF gene, and the expressed VEGF chemoattracts other hMSCs in vitro. The incorporation of VEGF into bone substitutes may attract pluripotent cells to the site of reconstruction. We are currently examining the chemotactic and regenerative capacity of VEGF-transfected hMSCs in animal models.

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