Feasibility Study On Differentiation Of Human Dental Pulp Stem Cells Into Corneal Epithelial Cells

ObjectiveTry to induce human dental pulp stem cells(DPSCs)differentiate into corneal epithelial-like cells by simulating corneal epithelial cell growth microenvironment and observes their ability to differentiate into corneal epithelial-like cells.Biocompatibility of DPSCs with acellular porcine small intestinal intestine submucosa(SIS)scaffolds was investigated.Feasibility of DPSCs as seed cells in construction of tissue engineered corneal epithelium was also discussed.MethodsCells were isolated and cultured by tissue block digestion.Flow cytometry was used to identify cell surface markers.The conditioned medium(CM)was mixed with a basic medium(BM)to prepare a medium in a proportion of 10%,20%,30%,40%,60%,90%,100%CM for culturing DPSCs.Detection of cell proliferation activity was detected by cck-8 assay.DPSCs were induced in medium with 30%,60%,and 90%CM.DPSCs cultured with BM was set as control group.Immunofluorescence assay was used to detect corneal epithelial cell markers cytokeratin 3(CK3)and cytokeratin 12.Preparation of aseptic acellular SIS and its extract.The DPSCs were cultured in the acellular SIS extract.DPSCs was cultured in a vessel containing SIS and detection of cell proliferation activity was detected by cck-8 assay.DPSCs were inoculated on the surface of acellular SIS,after a certain time of culture,HE staining was used to observe the growth of DPSCs on the surface of acellular SIS.ResultsAfter 4 days of culture,spindle-shaped cells crawled out of the pulp tissue.After 7 days of culture,the cells grew as colonies.On the 9th day of culture,the cells grew radially.At 14 days,there was a dense spiral growth.On the second day of subculture,the cell density could reach 80%,and cells morphology presents a typical long-shuttle.After 7 passages,the cell morphology was stable,the cytoplasm was full and uniform,and cells maintain a strong proliferative capacity.The cells expressed the mesenchymal stem cell surface marker CD90(99.26%),CD 105(98.69%),and hardly expressed the hematopoietic stem cell surface marker CD34(1.19%),CD45(0.99%).cells in the 30%,60%,90%CM group did not express CK3 at 3 days of induction,cells in the 60%CM group and the 90%CM group began to express CK12;CK3 and CK12 were expressed in the 30%,60%,90%CM group at 7 days;At 11 and 14 days,Cells continued to express CK3 and CK12 in the 30%,60%,and 90%CM groups.At 3,7,11 and 14 days,no expression of CK3 and CK12 was observed in the BM group.The acellular SIS extract and acellular SIS had no significant effect on the proliferation of DPSCs,and there was no significant difference compared with the control group(P>0.05).HE staining showed that the cells could grow on the surface of acellular SIS after DPSCs seeding.Conclusion1.DPSCs can differentiate into corneal epithelial-like cells induced under conditioned medium;2.DPSCs have some biocompatibility with acellular SIS;3.It is feasible to use DPSCs as seed cells for the construction of tissue engineered corneal epithelium.