Physiologic levels of endogenous hydrogen sulfide maintain the proliferation and differentiation capacity of periodontal ligament stem cells

Background: Many invading oral bacteria are known to produce considerable amounts of hydrogen sulfide (H₂S). The toxic activity of exogenous H₂S in periodontal tissue has been demonstrated, but the role of endogenous H₂S in the physiologic function of periodontal tissue remains poorly understood. The purpose of the present study is to investigate the biologic functions of H₂S in the proliferation and differentiation of human periodontal ligament stem cells (PDLSCs). Methods: PDLSCs were isolated from periodontal ligament tissues of periodontally healthy volunteers or patients with periodontitis. Immunocytochemical staining, flow cytometry, and Western blot analysis were used to examine the expression of H₂S-synthesizing enzymes cystathionine-b-synthase (CBS) and cystathionine-γ-lyase (CSE). The proliferation capacity of PDLSCs was determined by cell counting kit-8 assay, carboxyfluorescein succinimidyl ester analysis, and 5-ethynyl- 29-deoxyuridine assay. The osteogenic potential of PDLSCs was tested using alkaline phosphatase staining, Alizarin Red staining, and in vivo transplantation experiments. Oil Red O staining was used to analyze adipogenic ability. Results: The results show that human PDLSCs express both CBS and CSE and produce H₂S. Blocking the generation of endogenous H₂S with CBS inhibitor hydroxylamine significantly attenuated PDLSC proliferation and reduced the osteogenic and adipogenic differentiation capacity of PDLSCs. In contrast, CSE inhibitor DL-propargylglycine had no effect on PDLSC function. Exogenous H₂S could inhibit the production of endogenous H₂S and impair PDLSC function in a dose-dependent manner. Conclusion: Physiologic levels of endogenous H₂Smaintain the proliferation and differentiation capacity of PDLSCs, and CBS may be the main source of endogenous H₂S in PDLSCs.