| Objective:Human periodontal ligament stem cells(h PDLSC)have become a research hotspot in periodontal bone tissue engineering in recent years.At present,h PDLSC is mainly based on the two-dimensional(2D)culture technology,and there are few related studies on the formation of h PDLSC cell spheroids using the three-dimensional(3D)culture technology.As a substitute for single cells,3D cultured cell spheroids can effectively improve the problems of insufficient number and microenvironment,and become a research trend in tissue engineering.Therefore,the research purpose of this experiment is to construct a three-dimensional culture condition by making agarose microplates,to explore the change law of h PDLSC morphology and activity with culture time under 3D culture,and to explore the 3D cell spheroid growth conditions by comparing with 2D culture.Effects on osteogenic differentiation of h PDLSCs.Methods:1.The hPDLSCs were isolated and cultured from the human periodontal ligament by the modified enzyme digestion method,and the cultured cells were identified by flow cytometry after labeling with cell surface proteins CD45,CD73,CD34,CD90 and CD105 fluorescent antibodies;Osteogenic and adipogenic induction experiments combined with staining techniques were used to detect the osteogenic and adipogenic differentiation potential of cultured cells.2.Agarose was used to cover the mold,and the microporous pattern with uniform size was successfully obtained on the surface of agarose to form agarose microporous culture plate.After h PDLSC were laid in,they settled within 5 hours and gathered themselves to form a pile of cells.After 24 hours,the shape of cell sphere was stable,and the stem cell sphere gradually narrowed with the growth of culture time.3.After 3,7 and 10 days of osteogenic induction of h PDLSC spheres,the morphological changes of the cell body at different time points were detected by fluorescent labeling of the cytoskeleton(filamentous protein of cell membrane)and fluorescent labeling of the nucleus.4.After osteogenic induction of h PDLSC spheres for 3,7 and 10 days,the activity changes of cell bodies at different time points were detected by Live/ Dead staining kit.5.After 3 days of osteogenic induction,compared with the 2D culture group,the ALP activity of the 3D culture group was significantly enhanced;after 7 days of osteogenic induction,the ALP activity of the 3D culture group was weakened compared with the 2D culture group.6.Detect the gene expression levels of h PDLSCs at 3 d,4 d and 7 d after osteogenic induction under 2D and 3D culture conditions by RT-q PCR,and analyze the effect of 3D culture technology on h PDLSC osteogenic differentiation compared with2 D culture.In addition,the gene expression level of ?-catenin was also detected.Results:1.The hPDLSC cells were successfully isolated and cultured from the human periodontal ligament by the modified enzyme digestion method.Flow cytometry results showed that the proportions of cell surface antigens were: CD45(0.01%),CD34(0.15%),CD105(99.92%),CD73(99.23%)and CD90(99.1%),confirming that the cells were of mesenchymal origin.After 3 weeks of osteogenic induction and adipogenic induction,the cultured cells were stained to prove that they had multi-directional differentiation potential in vitro.2.The agarose is covered on the rear surface of the mold to obtain a uniform pattern of micropores,forming an agarose microwell culture plate.After the h PDLSC was plated,it settled within 5 h and self-aggregated to form a bunch of cells;after 24 h,the shape of cell spheroids began to stabilize;24h-7d was stable.Then the stem cell spheroids gradually shrunk as the culture time increased.3.After the cell spheroids were stained with skeleton,the membrane filamentous actin was stained in green,and the nucleus was stained in blue.Cell spheroid cytoskeleton staining changed within 7 d.Compared with 3 d staining,the 10 d staining results showed that the microspheres were compact,the green staining part of the cell spheroids increased,and the cortical actin network became more and more narrow.4.The results of Live/Dead staining in cell spheroid culture for 3,7 and 10 days showed that as the culture time became longer,the number of dead cells in the core of the spheroid gradually increased.5.After 3d of osteogenesis induction,the ALP activity of the 3D culture group was significantly enhanced compared with that of the 2D culture group,and the results of the bone induction for 7 days showed that the ALP activity of the 3D culture group was reduced compared with that of the 2D culture group.6.RT-qPCR results showed that: after 3 d osteogenic induction,the expressions of ALP and OCN in the 3D culture group were significantly increased compared with 2D culture;The expressions of Runx2 and OCN were significantly increased;after 7 days of osteogenic induction,only the expression of OCN was significantly increased in the 3D culture group compared with the 2D culture.In addition,the results showed that the expression of ?-catenin was significantly increased at 3,4,and 7 days after osteogenic induction,with a time-dependent effect.Conclusion:The h PDLSC spheroids were successfully cultured in agarose microplates,and the morphology of the cell spheroids changed in a time-dependent manner.With the increase of culture time,the diameter of cell spheroids became smaller and more compact,and the number of dead cells inside the spheroids increased;compared with the 2D culture group,the ALP activity in the first 3 days and the expression of some osteogenic differentiation-related genes in the 3D culture group increased significantly.High,suggesting that 3D culture technology can promote h PDLSC osteogenic differentiation.After 3D culture,the transcript level of ?-catenin increased,suggesting that the spheroid changes may be related to the Wnt/?-catenin pathway.The results established the possibility of h PDLSC spheroids as adult stem cells in periodontal tissue,and provided a new technical means for the field of stem cell research. |
PDF Full Text Request