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

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. 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. 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. 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.