Rapamycin ameliorates senescence of periodontal ligament stem cells and promotes their osteogenesis via the PI3K/AKT pathway

Periodontal ligament stem cells (PDLSCs) have been regarded as ideal candidates for tissue regeneration due to their excellent self-renewal and multipotent differentiation ability. Rapamycin (RAPA) is reported to play an important role in the regulation of biological properties of stem cells and a variety of physiological processes. This study investigates whether RAPA could ameliorate the senescence and accelerate the osteogenic differentiation of PDLSCs, particularly the regenerative potential in a rat calvarial bone defect model, and the underlying mechanisms involved. β-galactosidase staining, quantitative real-time polymerase chain reaction, and western blot analysis were performed to assess the effects of RAPA on senescent PDLSCs. The osteogenic differentiation ability of PDLSCs was detected by alkaline phosphatase staining and activity, Alizarin Red S staining, and gene and protein levels of osteogenesis-related markers. The underlying signaling pathways were investigated via RNA transcriptome sequencing analysis and WB tests. Calvarial bone defects in rat were treated with PDLSCs pre-incubated with or without RAPA and/or H₂O2. The results showed that RAPA could enhance the osteogenic potentials of PDLSCs via PI3K/AKT signaling pathway, and reversed H₂O₂-induced senescence and osteogenic differentiation inhibition of PDLSCs. Moreover, calvarial defects transplanted with RAPA-treated PDLSCs showed significantly greater new bone formation compared with other groups, and also improved the H₂O₂-induced impairment of bone formation, whether by micro-computed tomography examination or by histological analysis. Collectively, RAPA was capable of promoting osteogenic differentiation of PDLSCs via PI3K/AKT signaling pathway in vitro, facilitating calvarial bone regeneration and reversing H₂O₂-induced impairment of osteogenic differentiation and cell senescence in PDLSCs.