In Vitro Cell Proliferation, Osteogenic Differentiation, and In Vivo Angiogenesis Induced by Modified mRNA Encoding...

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.

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.

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).

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.