In Vitro Cell Proliferation, Osteogenic Differentiation, and In Vivo Angiogenesis Induced by Modified mRNA Encoding Vascular Endothelial Growth Factor-A.

Abstract

Aims: This study aimed to determine whether pseudouridine-modified mRNA encoding vascular endothelial growth factor (VEGF) -A induces VEGF production in human periodontal ligament cells (PDLCs), and to evaluate its effects on PDLC proliferation, osteogenic differentiation, and in vivo angiogenesis.

Methods: PDLCs derived from healthy periodontal tissues were transfected with pseudouridine-modified VEGF-A mRNA using Lipofectamine 2000 (L2000) or treated with L2000 alone. VEGF secretion was quantified at 24-72 h posttransfection. Cell viability and immune activation were also assessed. Supernatants collected at 48 h were used to evaluate PDLC proliferation using the AlamarBlue assay. Osteogenic differentiation was assessed by Alizarin Red S staining and the expression of alkaline phosphatase gene (ALPL), RUNX2, and osteocalcin (OCN). Angiogenic activity was evaluated using the chorioallantoic membrane (CAM) assay in fertilized White Leghorn chicken eggs (n = 7 per group). On embryonic day 8, filter papers loaded with supernatants from L2000 + mRNA, L2000 groups, Dulbecco's phosphate-buffered saline, or recombinant VEGF (rhVEGF) were placed on the CAM. After a 3-day incubation, newly formed blood vessels were imaged and quantified.

Results: PDLCs transfected with pseudouridine-modified VEGF-A mRNA produced significantly higher VEGF levels than the control groups for up to 72 h (p < 0.05), without affecting cell viability or inducing innate immune activation. VEGF-containing supernatants promoted PDLC proliferation. Direct VEGF mRNA transfection enhanced osteogenic differentiation and upregulated expression of osteogenic markers ALPL, RUNX2, and OCN. In the CAM assay, abundant microvascular networks with a characteristic spoked-wheel pattern were observed in both the VEGF mRNA and rhVEGF groups. Furthermore, the VEGF mRNA group showed a significantly greater increase in blood vessel formation than the control groups (p < 0.01).

Conclusion: Pseudouridine-modified VEGF-A mRNA effectively induced VEGF production, which subsequently enhanced PDLC proliferation, osteogenesis, and in vivo angiogenesis. These findings support the potential of pseudouridine-modified VEGF-A mRNA as a promising therapeutic approach for periodontal regeneration.