Priming integrin alpha 5 promotes the osteogenic differentiation of human periodontal ligament stem cells due to cytoskeleton and cell cycle changes.

To seek a potential target for periodontal tissue regeneration, this study aimed to explore the role of Integrin alpha 5 (ITGA5) in human periodontal ligament stem cells (PDLSCs). Transwell assay, Cell Counting Kit 8 (CCK8) assay, cell cycle assay, alkaline phosphatase (ALP) activity, alizarin red staining, and western blot were used to investigate the effects of ITGA5 on PDLSC migration, proliferation and osteogenic differentiation. The in vivo effect was investigated by nude mice subcutaneous transplantation with cell and hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) complex. The involved mechanism was explored by the iTRAQ proteomic technique and validated by western blot and immunofluorescence. We found that ITGA5forced expression enhanced the proliferation, migration, and osteogenic capacity of PDLSCs, while inhibited ITGA5 expression had the opposite effects. The phosphorylation of focal adhesion kinase (FAK), phosphatidylinositide 3-kinases/protein kinase B (PI3K/AKT), and mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinases 1 and 2 (MEK1/2/ERK1/2) were crucial in this process. Forced expression of ITGA5 in PDLSCs increased osteoid and PDL-like tissue formation in vivo. Proteomic and bioinformatic analysis revealed that cytoskeleton and cell cycle changes were involved. Keratin, type II cytoskeletal 6B (KRT6B) and desmin (DES) may distinguish this process and serve as new markers of PDLSC differentiation. SIGNIFICANCE: Periodontitis is highly prevalent and can impair PDL and teeth functioning. One of the most promising therapies to periodontitis therapies is PDL regeneration by utilizing PDLSCs. While many obstacles remain to be resolved, the regulation of PDLSC osteogenic differentiation is a main concern. The present study demonstrated the potential clinical value of an ITGA5 priming peptide, which may be utilized in PDL tissue repair and regeneration. The mechanism elucidated in this study would help to fuel its application.