| Objects:The aim of this study is to explore the biological effects and the associated signaling pathway of culturing late passage periodontal ligament cells on early passge periodontal ligament cells derived extracellular matrix(ECM).We want to obtain a large amount of seeding cells with high expression level of stemness by using tissue –specific ECM as a template.Furthermore,cells expanded on this material were used to fabricate cell sheets in vitro.Periodontal tissue regeneration was examined after in vivo implantation.All of them were provided experimental and therologic supporting for future clinical application of periodontal ligament cells.Materials and methods:1.To culture hPDLCs with tissue-explant method.Cultured cells at passage 2 were obtained for keratin staining and vimentin staining to make the source identification.Cells were continuously cultured for 14 days at a low density for fabricated cell monolayer.After decellularization,the micro-structure of ECM was investigated by phase-inverted microscope and scaning electron microscope(SEM).The main structure proteins of ECM,Collagen-I and Fibronectin,was examined using immunofluorescence.2.The proliferation effect of ECM on reseeding cells was assessed by blood plate count method.MAPK siganaling pathway underlying cell proliferation was explored by using western-blot.Flow cytometry were used for examined the change in cell cycle and reactive oxygen species generation.In the meantime,cell cycle related marker genes were also investigated by real-time PCR.3.Cells were cultured for 7 days on different plates,with or without ?-catenin inhibitor XAV-939,then the expression of stemness associated marker genes Nanog,Sox-2,Oct-4 were exmined by real-time PCR.4.Cells were cultured for 7 days in osteogenic medium with or without ?-catenin inhibitor XAV-939,JNK inhibitor SP600125.The osteogenic differentiation of PDLCs cultured onto plastic or ECM was quantitatively investigated by measuring alizarin red-S(ARS)staining,the alkaline phosphatase(ALP)staining,and osteogenic marker gene and protein expression.5.Cells expanded on different template were used to fabricate cell sheet in vitro.Western-blot and Real-time PCR were used to examinate the gene expression and protein expression of ECM protein.Periodontal regeneration in vivo were investigated using.H&E staining.Results:1.After 14 days continuous culturing,the cells present cell monolayer morphology.After decellularization,there was no cell-like structure under invertion microscope.Both low and high magnification SEM showed that the scaffolds present a cross network of fibers whichis more clear than fresh cell sheets.We also found that the structural network of two main ECM proteins Collagen-I and Fibronectin were not destroyed.Besides,cell nuclei were rarely examined.2.hPDLCs grown on ECM exhibited a tendency towards directional migration along the ECM fibril.In contrast,cells expanded on plastic grew randomly.Compared with the plastic group,ECM promotes the proliferation of reseeding cells.The phosphorylation of MAPK were increased along with culturing time.Cells expanded on ECM present a low rate at G0/G1 phase(p<0.01)but a higher cell rate at both S phase(p<0.01)and G2/M phase(p>0.05).Consistently,the m RNA expression of CDK4 and Cyclin D1 was significantly increased in the ECM group(p<0.001).In the meantime,the intracellular ROS were decreased after expanding on ECM.3.ECM promotes the expression of pluriopotent related marker,Nanog,Sox-2,Oct-4.These genes also increase significantly after ?-catenin XAV-939 application.These promotion effects were enchanced largely in the ECM group.Consistently,the protein expression of active ?-catenin and the pluripotent associated marker Nanog was synchronously upregulated by ECM expansion,and this increment was enhanced by XAV-939 treatment.4.Real-time PCR data showed that Run X-2 m RNA level was significantly higher in ECM group than that in plastic group(p<0.05).After XAV-939 treatment,Run X-2gene expression was decreased in both ECM and plastic groups.However,JNK inhibitor treatment led to the downregulation of Run X-2 m RNA level in ECM expanded cells(p<0.05)but not in plastic expanded ones(p>0.05).The enhanced osteogenic potential in ECM group was further confirmed by ALP and ARS staining.Western blot data indicated that,the increased active ?-catenin levels indeed downregulated Run X-2 protein expression in both ECM and plastic groups.Meanwhile,after JNK protein was inhibited,the Run X-2 protein level was decreased in ECM group but not in plastic group.5.Cell sheets formed by hPDLCs derived from ECM expansion appeared easier to frizzle and thicker than the control.Except the protein expression of laminin,the levels of these indexes were all significantly higher in the cell sheets derived from hPDLCs expanded by ECM,than the control(p<0.05 for Col-I and Laminin m RNA;p<0.01 for Fibronectin m RNA).After 8 week transplantation,cell sheets of the two groups both regenerated periodontal tissue-like structures.H&E staining showed that,periodontal ligament formed by ECM group lined more regularly and appeared thicker than the control group.Moreover,there were some fibers seemed to be embedded in the cementum-like structures,especially in the ECM group.Conclusions:1.Tissue-expanded method could successfully obtain periodontal ligament cells,which is derived from the mesoderm.We successfully fabricate tissue specific ECM with an intact structure by using appropriate decellularization method.2.ECM could decrease the intracellular ROS of reseeding cells,which may promote cell cycle transition and MAPK activation,followed by cell proliferation.3.ECM could promote the stemness of reseeding cells via increasing the expression of active ?-catenin.ECM promotes the osteogenic differentiation of reseeding cells via inhibiting the expression of active?-catenin and increasing the expression of p-JNK expression.4.Cell sheet formed by cells expanded on ECM present more dense and higher periodontal tissue regeneration. |
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