Effect On Osteogenetic Ability Of P/Aged-PDLSCs By Co-cultured With Epithelial Cell Rests Of Malassez

PDLSCs are one of the best candidates for periodontal regeneration.Whatever,the extracellular microenvironment is known to affect the proliferation and differentiation of stem cells.Therefore,the multidifferentiation capacity of PDLSCs would be impaired by adverse microenvironment which would affect periodontal regeneration,especially the decreased osteogensis capacity.Consider this,we would concentrate on the manner how to optimize the decresed multidifferentiation capacity of PDLSCs as the result of microenvironment.As priviously demonstrated,the osteogensis capacity of PDLSCs could be enhanced by co-cultured with HERS.However, the soure of HERS is limited which exisit only in puberty cells. The fate of HERS cells is, eventually, to form the epithelial rests of Malassez(ERM),distributing in the mature periodontal ligament tissue,which is always been overlooked.Given that the way of co-culture could better iminate the microenvironment in vivo,we establish a co-culture system of ERM and P/Aged-PDLSCs.Our hypothesis is that ERM cells play pivotal roles in cementum formation through the interplay with PDLSCs from inflamentary or aging tissues,which can provide a better scheme for periodontal regeneration. The study includes three parts: 1.Cell isolation,culture and identificationObjective:to obtain PDLSCs from different source and culture human ERM.Methods:H/P/Aged-PDLSCs were obtained from health people,patients diagnosed with periodontitis and aged but health donors.After purification,we observed the condition of H/P/Aged-PDLSCs by using staining of toluidine blue,identified phenotype characteristics of H/P/Aged-PDLSCs by FCM, and identified multidifferentiation capacity by using osteogenenic and adipogenic induction experiment.ERM were isolated and purified from PDL mesenchymal cell using differential digestion method.To identify it,we use Immunofluorescence staining and RT-PCR.Results:We obtained H/P/Aged-PDLSCs successfully,and the three cells all highly expressed CD29,CD90,CD105 and negatively expressed CD45. Compared with controls,the osteogenic induction experiment shows that ALP staining darker and more mineralized nodules appearing in the experimental part.The adipogenic induction experiment shows that more lipid cluster forming compared with controls.For ERM,cell immunofluorescence staining shows that CK14 positively expressed,at the same time,RT-PCR highly expresssed sepecific marker of epithelial cell.Conclusion:H/P/Aged-PDLSCs are all MSCs which have mutidifferentiation capacity and ERM is epithelial cell which grow well. 2.Effect of ERM on the osteogensis capacity of P/Aged-PDLSCsObjective:to confirm the effect of micro-environment and study the effect of ERM on the osteogensis capacity of P/Aged-PDLSCs.Methods:Firstly,we detect the osteogensis capacity of H/P/Aged-PDLSCs from gene and protein lever respectively to confirm that the effect of microenvironment.Built the co-culture system of ERM/PPDLSCs and ERM/Aged-PDLSCs.In addition,in the co-culture system we contrast the osteogensis capacity of the P/Aged-PDLSCs in the lower champer of transwell by using the techonology of RT-PCR and western blot.At the same time,alkaline phosphatase(ALP) staining and alizarin red S staining intuitively detect the changes of osteogensis capacity of P/Aged-PDLSCs.Result:ALP,Runx-2 gene and protein lever down-expressed in the P/Aged-PDLSCs compared with HPDLSCS.After co-cultured with ERM,up-regulation of ALP,Runx-2 gene and protein lever.In addition,afer co-cultured with ERM,ALP staining darker and appearing more mineralized nodules.Conclusion:Adversed microenvironment could impared the osteogensis capacity of PDLSCs.ERM could recover the decreased osteogensis capacity of P/Aged-PDLSCs. 3.Preliminary research on the mechanism of effect of ERM on the osteogensis capacity of P/Aged-PDLSCsObjective:to sudy the mechanism of effect of ERM on the osteogensis capacity of P/Aged-PDLSCs initialy.Methods:By western blot,we detect the total-β-catenin,active-β-catenin,GSK-3β and P-GSK-3β which are the special marker of wnt signaling pathway in co-culture and monoculture system.Result:the level of total-β-catenin,GSK-3β was essentially unchanged in both parts,while,the protein expression of active-β-catenin and P-GSK-3β in the co-culture was lower than monoculture system.Conclusion:Wnt signaling pathway play the role in affecting osteogenensis capacity of P/Aged-PDLSCs by co-cultured with ERM.In conclusion,ERM,as the descendants of Hertwig’s epithelial root sheath are useful which could optimize the decreased osteogensis capacity of P/Aged-PDLSCs at some extent.Wnt signaling pathway play the role during the course.Whatever,future studies should further explore the specific mechanism.