Expression of vascular endothelial growth factor protein in mRNA-transfected human periodontal ligament cells

Abstract

The complete regeneration of periodontal tissues following current periodontal therapy remain challenging and unpredictable. Nucleoside-modified messenger RNA (mRNA) technology can be a promising novel platform in regenerative medicine. The aims of this study were to evaluate whether pseudouridine modified mRNA encoding vascular endothelial growth factor (VEGF) could induce VEGF production in human periodontal ligament cells (PDLCs) and this translated protein function by promoting in vivo blood vessel formation using chorioallantoic membrane (CAM) assay. Isolated PDLCs from healthy periodontal tissue were transfected with modified mRNA encoding VEGF (VEGF mRNA) complexed with a transfecting agent, Lipofectamine 2000 (L2000) and L2000 alone (control). Supernatants collected at 24 hours (h) after transfection were evaluated for protein production by ELISA and cell viability by Alamar Blue assay. The supernatants of the VEGF mRNA-L2000, L2000 (L2000 control), and DPBS (negative control) were applied on filter papers, individually placed these grafts on to the CAM surface through the window on day 8 of embryonic development (E8) and incubated for another three days. Angiogenesis assessment, counting number of blood vessels convergence to the grafts, was carried out by photographed with stereomicroscopic on E8 and E11. The result showed that PDLCs, transfected with mRNA encoding VEGF, produced high level of VEGF protein than controls at 24 h (p < 0.001). The transfection of mRNA encoding VEGF showed negligible effect on PDLC viability. When supernatants were applied in CAM assay, translated protein VEGF protein was able to significantly induce blood vessel formation (p < 0.001). In conclusion, modified mRNA encoding VEGF promoted VEGF production and had angiogenic properties, increased blood vessel formation in the CAM. Thus, this mRNA platform technology may allow future application as a novel therapeutic platform for periodontal regeneration.