Spheroid culture of human periodontal ligament stem cells on agarose enhances stemness and osteogenic differentiation potential.

OBJECTIVE: To develop an agarose (AG)-based spheroid culture system to enhance the stemness and osteogenic potential of human periodontal ligament stem cells (hPDLSCs), addressing limitations of current periodontal tissue engineering approaches. METHODS: hPDLSCs were isolated from extracted premolars and cultured as spheroids in AG-coated 96-well plates. Spheroid formation was optimized by varying cell seeding densities and culture durations. Monolayer-cultured hPDLSCs served as controls. Stemness was evaluated using RT-qPCR and immunofluorescence detection of pluripotency markers. Cell migratory capacity was assessed by scratch assays. Osteogenic differentiation was induced for 10-21 days, and calcium deposition was quantified by Alizarin Red S staining. RESULTS: Spheroid-derived hPDLSCs showed significantly higher expression of stem cell markers (OCT4 and NANOG) and greater migratory capacity compared to monolayer-cultured cells. Upon osteogenic induction, spheroid-derived hPDLSCs exhibited upregulated expression of osteogenesis-related genes (BSP and OPN) and formed more extensive calcium nodules than their monolayer counterparts. CONCLUSION: The AG-based spheroid culture system effectively maintains hPDLSC stemness and enhances their osteogenic differentiation potential. This scalable and cost-effective platform provides high-quality seed cells for periodontal regeneration and holds promise for improving clinical outcomes in periodontal therapy.