OBJECTIVEThis study aimed to investigate the role and mechanism of neuropeptide substance P (SP) in ST2 cell (bone mesenchymal stem cells of mice) osteogenic differentiation to provide a basis for the treatment of temporomandibular joint osteoarthritis.

METHODSThird-generation ST2 cells were cultured with different concentrations of SP (0, 10⁻¹⁰, 10⁻⁸, 10⁻⁶, and 10⁻⁵ mol·L⁻¹). After 24, 48, and 72 h, cell proliferation was detected by CCK-8. The ST2 cells were cultured with 10⁻⁶ mol·L⁻¹ SP for 1, 3, 5, and 7 days. Subsequently, the expression of alkaline phosphatase (ALP), collagen typeⅠ(CollaⅠ), and osteocalcin (OCN) in the culture supernatant was tested by enzyme-linked immunosorbent assay (ELISA). ALP activity was detected by immunofluorescence staining. The ST2 cells were cultured with SP, Noggin (inhibitor of the bone morphogenetic protein signaling pathway), SP+Noggin, and 2% fetal bovine serum, respectively. Finally, the expression of ALP, CollaⅠ, and OCN in the culture supernatant was tested by ELISA.

RESULTSCCK-8 showed that the effect of cell proliferation was most obvious when the SP concentration was 10⁻⁶ mol·L⁻¹ (P<0.01). The ELISA results demonstrated that ALP expression significantly increased at day 5 compared with that in the control group (P<0.01), whereas the expression of CollaⅠand OCN significantly increased at day 7 (P<0.05). Immunofluorescence results showed that ALP activity was strongest at day 5. The expression of ALP, CollaⅠ, and OCN decreased after Noggin addition (P<0.05).

CONCLUSIONSSP can promote the proliferation and osteogenic differentiation of ST2 cells, and the bone morphogenetic protein signaling pathway may be involved in this process.