Semaphorin 3A attenuates the hypoxia suppression of osteogenesis in periodontal ligament stem cells

Objective and background: The occurrence and development of periodontitis are closely related to hypoxia of the periodontal microenvironment. Periodontal ligament stem cells (PDLSCs) are considered to have potential to regenerate periodontal tissues. Semaphorin 3A (Sema3A) plays an essential role in promoting osteogenesis. However, the effect of Sema3A on osteogenesis of PDLSCs under hypoxia remains unclear. The aim of this study was to investigate the effect of Sema3A on osteogenesis of PDLSCs under hypoxia.

Methods: Isolated PDLSCs were identified using flow cytometry. Adipogenic differentiation potential was identified by oil red O staining. Osteogenesis was measured using Alizarin Red S staining and ALP staining. Intracellular hypoxia was induced using cobalt chloride (CoCl₂ ). The expression level of hypoxia-inducible factor-1α (HIF-1α) was detected via ELISA. Expression of osteogenic markers and Sema3A was analyzed using western blot and Real-Time PCR.

Results: The proliferation and osteogenesis of PDLSCs were markedly inhibited with increased concentrations of CoCl₂ . Under the treatment with a low concentration of CoCl₂ , expression of related osteogenic markers and Sema3A decreased in a time-dependent manner. ARS and ALP staining results also showed that osteogenic calcification decreased under hypoxia. Apigenin, an inhibitor of HIF-1α, effectively up-regulated expression of Sema3A and osteogenic markers with CoCl₂ treatment. Moreover, exogenous Sema3A significantly increased the expression of osteogenesis-related markers and mineralization of PDLSCs according to ALP and ARS staining with CoCl₂ treatment.

Conclusions: Hypoxia markedly inhibited osteogenesis of PDLSCs. Sema3A explicitly attenuated the hypoxia suppression of osteogenesis in PDLSCs.