The Use of Chemotactic “Smart Scaffold ” in Healing Craniofacial Defects: Experimental Studies

Background: The gold standard for bone reconstruction involves autologous bone grafting, which creates significant donor site morbidity. Regenerative medicine approaches to these clinical problems aim to obviate the need for autologous bone grafting through the use of bioengineered constructs that combine stem cells, growth factors, and biocompatible vehicles. Human mesenchymal stem cells (hMSCs) and vascular endothelial growth factor (VEGF) [1,2] have both shown promise for use in this context, the former due to their pluripotent capacity and the latter due to its osteogenic and chemotactic activity. In this study we harness the regenerative potential of these cells and this growth factor to develop a “Smart Scaffold” for use in bone tissue engineering.

Methods: MSCs were transfected with human VEGF-A and 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. For the chemotactic in vivo study, VEGF-transfected hMSCs cells were seeded on apatite-coated PLGA scaffold to prepare the “Smart Scaffold”. The scaffold was then implanted in dorsal subcutaneous pocket and cranial defect of immunocompromised animal. HMSCs tagged with a dye (DiR) were injected intravenously in the postoperative period to evaluate the chemotactic and osteogenic capabilities (N=5).

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. In the result of in vivo chemotactic examination, DiR-tagged hMSCs were accumulated in the chemotactic scaffold. Successful bone regeneration was shown in the defect treated with “Smart Scaffold”.

Conclusions: These observations suggest that incorporation of VEGF may play a vital role in the design of clinically relevant bone graft substitutes, or chemotactic scaffolds attracting pluripotent cells to the site of reconstruction.

References:

1, Street, J., Bao, M., deGuzman, L., et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proceedings of the National Academy of Sciences of the United States of America. 2002;99:9656-9661.

2, Fiedler, J., Leucht, F., Waltenberger, J., et al. VEGF-A and PlGF-1 stimulate chemotactic migration of human mesenchymal progenitor cells. Biochemical and biophysical research communications. 2005;334:561-568.