Abstract: Objective: To investigate the changes in stemness characteristics of human periodontal ligament stem cells (hPDLSCs) under hypoxia environment and their effects on osteogenic differentiation in vitro and in vivo. Methods: hPDLSCs were cultured under normoxia and hypoxia environment, and their proliferative ability was studied by CCK-8 and colony forming unit experiments. The senescence was detected by β-galactosidase staining. The expression of stemnessrelated genes sex determining region of Y-box protein 2 (SOX2), octamer binding transcription factor 4 (OCT4) and NANOG was studied by qRT-PCR and Western blot experiments. The supernatant of hPDLSCs under two culture environments was collected and used as the conditioned medium to configure the osteogenic induction medium, then osteogenic induction of hPDLSCs was performed under normoxia. The alkaline phosphatase (ALP) staining, alizarin red staining and the expression of osteogenicrelated genes collagen type Ⅰ(COL-1), osteopontin (OPN), osteocalcin (OCN) and runt related transcription factor 2 (RUNX2) detected by qRT-PCR assay were used to study the ability of hPDLSCs to promote osteogenic differentiation in vitro. Cell membrane sheets of hPDLSCs pretreated in two culture environments were implanted into the critical bone defects of rats skulls, tissue samples were collected after 8 weeks, and microcomputed tomography (micro-CT), HE staining, Masson staining, immunohistochemical staining and fluorescent dyes labeling new bone experiment were used to study the ability of hPDLSCs to promote osteogenic differentiation in vivo. Results: Under hypoxia conditions, the proliferation ability of hPDLSCs was increased (P＜0.05), the level of senescence was decreased (P＜0.05), and the expressions of SOX2, OCT4, and NANOG genes and proteins were all enhanced (P＜0.05). Using hPDLSCs conditioned medium cultured in hypoxia to promote hPDLSCs osteogenic differentiation in vitro, the ALP activity increased, calcium nodule formation increased (P＜0.05), and the expressions of COL-1, OPN, OCN and RUNX2 were increased (P＜0.05). After implanting the hypoxia cultured hPDLSCs cell sheets into the bone defects, new bone formation increased, the area of fluorescent emitted by new bone was larger and the intensity was stronger. Bone volume fraction of rat skull ［bone volume (BV)/tissue volume (TV)］ was significantly increased (P＜0.05), and a large number of new bone tissues at the edge of the bone defect with surrounding osteocytes and a large number of collagen fibers were observed by HE and Masson staining. Immunohistochemical results showed increased expression of OPN and OCN proteins (P＜0.05). Conclusion: Under hypoxia environment, the stemness of hPDLSCs is enhanced, and hypoxia pretreated hPDLSCs promote osteogenic differentiation of cells in vitro and in vivo.

Key words: Human periodontal ligament stem cells, Hypoxia, Stemness, Bone regeneration