Zhang Qinyao, Huang Shuwei, Wu Jiumei, Liu Peng, Sun Jun, Xie Xing, Ge Mengjun, Li Chuanmei, Zhang Mengyue, Lei Lei
Stem Cell and Regenerative Medicine Research Group, Xiangya School of Stomatology Central South University Changsha 410000, Hunan, China.
College of Materials Science and Engineering, Hunan University Changsha 410082, Hunan, China.
Am J Transl Res. 2025 Jul 15;17(7):5257-5270. doi: 10.62347/PPEI1766. eCollection 2025.
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.
开发一种基于琼脂糖（AG）的球体培养系统，以增强人牙周膜干细胞（hPDLSCs）的干性和成骨潜能，解决当前牙周组织工程方法的局限性。
从拔除的前磨牙中分离出hPDLSCs，并在AG包被的96孔板中培养成球体。通过改变细胞接种密度和培养时间来优化球体形成。单层培养的hPDLSCs作为对照。使用RT-qPCR和多能性标志物的免疫荧光检测来评估干性。通过划痕试验评估细胞迁移能力。诱导成骨分化10 - 21天，并用茜素红S染色定量钙沉积。
与单层培养的细胞相比，球体来源的hPDLSCs显示出干细胞标志物（OCT4和NANOG）的表达显著更高，且迁移能力更强。在成骨诱导后，球体来源的hPDLSCs表现出成骨相关基因（BSP和OPN）的表达上调，并且比单层培养的细胞形成更广泛的钙结节。
基于AG的球体培养系统有效地维持了hPDLSCs的干性并增强了它们的成骨分化潜能。这个可扩展且具有成本效益的平台为牙周再生提供了高质量的种子细胞，并有望改善牙周治疗的临床结果。
