2023, Vol. 27 ›› Issue (6): 846-852

Three-dimensional culture affects morphology, activity and osteogenic differentiation of human periodontal ligament stem cells

Li Xinyue1, Li Xiheng1, Mao Tianjiao2, Tang Liang1, Li Jiang1, 2   

Abstract: BACKGROUND: Periodontal ligament stem cells, as a member of odontogenic mesenchymal stem cells, are the research hotspot of periodontal bone tissue engineering in recent years. Due to the insufficient number of stem cells and the influence of microenvironment, the regeneration ability of periodontal tissue often cannot meet the needs of treatment. In recent years, flaky cultured cell sheets and three-dimensional cultured cell spheres, as substitutes for single cell suspension injection, can effectively improve the problems of insufficient quantity and microenvironment, and become the research trend of tissue engineering. However, there are few studies on the formation of periodontal ligament stem cell spheres by three-dimensional culture technology.
OBJECTIVE: To explore the effects of three-dimensional culture technology on the morphology, activity and osteogenic differentiation of human periodontal ligament stem cells.
METHODS: Human periodontal ligament stem cells were cultured by modified enzyme digestion method, and then identified. The morphology of cell spheres was detected by cytoskeleton and nuclear fluorescence labeling. The activity of cell spheres was detected by Live/Dead staining kit. The effects of three-dimensional culture on osteogenic differentiation of human periodontal ligament stem cells were analyzed by alkaline phosphatase activity and osteogenic related gene expression.
RESULTS AND CONCLUSION: (1) The morphology and activity of human periodontal ligament stem cell spheroid cells changed in a time-dependent manner. With the increase of time, the diameter of cell sphere became smaller and the number of dead cells in the core of sphere increased. (2) Compared with two-dimensional culture, three-dimensional culture of human periodontal ligament stem cells increased alkaline phosphatase activity at 3 days of osteogenic induction and the expression of osteogenic related genes and β-catenin at 3, 4, and 7 days of osteogenic induction, suggesting that three-dimensional culture technology can promote the osteogenic differentiation of human periodontal ligament stem cells, which may be related to Wnt/ β-catenin pathway.
Key words: three-dimensional culture technology, periodontal ligament stem cell, osteogenic differentiation, tissue engineering, Wnt/β-catenin signaling pathway