Melatonin differentially modulates chondro-osteogenic lineage commitment and suppresses proinflammatory mediator production in equine mesenchymal stem cells.

OBJECTIVE: To evaluate the effects of melatonin on chondrogenic and osteogenic differentiation, chondro-osteogenic transdifferentiation, and inflammatory responses in equine mesenchymal stem cells (eMSCs) in vitro. METHODS: This in vitro experimental study was conducted from October 2023 through June 2024. The eMSCs were cultured under chondrogenic, osteogenic, and chondro-osteogenic transdifferentiation conditions with or without melatonin and exposed to proinflammatory cytokines (IL-1β and tumor necrosis factor-α [TNF-α]). Expression of chondrogenic and osteogenic markers was measured by quantitative reverse-transcription PCR. Concentrations of IL-1β, TNF-α, IL-6, and IL-8 in culture supernatants were quantified by multiplex immunoassay. RESULTS: In chondrogenic cultures, melatonin was associated with increased collagen type II (COLII) and sex-determining region Y-box transcription factor 9 (SOX9) expression and was associated with higher COLII-to-collagen type I (COLI) and SOX9-to-runt-related transcription factor 2 (RUNX2) ratios, consistent with preservation of chondrogenic phenotype. During chondro-osteogenic transdifferentiation, melatonin increased RUNX2 and ALP expression and decreased COLII:COLI and SOX9:RUNX2 ratios, suggesting promotion of an early osteogenic or hypertrophic shift. In contrast, in osteogenic cultures, melatonin decreased RUNX2, ALP, and COLI expression, indicating context-dependent effects on osteogenic differentiation. Across culture conditions, melatonin significantly reduced IL-1β, TNF-α, IL-6, and IL-8 concentrations in culture supernatants. CONCLUSIONS: Melatonin differentially modulated lineage behavior in eMSCs, preserving chondrogenic characteristics in some settings, promoting early osteogenic transition during transdifferentiation, and suppressing proinflammatory mediator production. CLINICAL RELEVANCE: These findings support further investigation of melatonin as a potential adjunct for modulating proinflammatory responses during equine cartilage repair and other inflammatory musculoskeletal disorders.