Rat Oral Mucosa Epithelial Cells Induce Bone Marrow Mesenchymal Stem Cells Differentiate To Epithelial Cells In Vitro

Patients whose vagina are deficiency due to all sorts of reasons needvaginoplasty. Traditional operations always bring out troubles such assacrificing normal tissue, leaving scar, immunological rejection etc. Tissueengineering technology makes up the insufficiency in the form and function ofartificial vagina formatted by traditional surgery, provides the ideal tissuelining for vaginal reconstruction, and the reconstructed vagina has ideal depthand width. The seed cells is the most important element for tissue constructionby engineering. Acquiring enough seed cells that maintaining specificbiological activity can guarantee the success of the tissue construction. Atpresent, the seed cells for tissue engineering vagina mostly adopt from selfhomologous cells, but there are defects such as the limited source, cell aging,difficulty in amplification and the new trauma caused by drawing materials.Therefore, bone marrow mesenchymal stem cells (BMSCs) differentiated toepithelial cells is the key steps in vaginal tissue engineering application. Thisstudy was desigened to use oral mucosa epithelial cells inducing BMSCsdifferentiatiate to epithelial cells in vitro, which may lay the foundation ofBMSCs as seed cells used in vaginal tissue engineering.Part one: Primary Culture of Rat Oral Mucosal Epithelial Cells in vitroObjective: To study the culture method of rat oral mucosal epithelial cellsin vitro, in order to be used in inducing BMSCs differentiate to epithelial cells.Methods: The oral mucosal tissues of rats used enzymatic digestion in0.6U/ml Dispase Ⅱ respectively dissolving in PBS and D-KSFM, anddigested with0.6U/ml and1.2U/ml Dispase Ⅱdissolving in D-KSFM. Wetaked notes the separation results grade of epithelium at each group and theviable count and observed the attached conditions under the inverted phasecontrast microscope when replaced medium for the first time. Trypsin digested 10min twice and20min once respectively. The cells were cultured usingKBM-GOLD medium and KBM-GOLD medium containing6%FBS. Theviable count of each group were recorded and the cells morphology underinverted phase contrast microscope and ultrastructure under the scanningelectron microscopy was observed. The cells were identified withanti-pan-cytokeratin (AE1/AE3) antibody by immunohistochemistry stain andimmunofluorescence.Results: The score of the separation results after enzymatic digestion with0.6U/ml Dispase Ⅱ dissolving in PBS was basically the same compared toD-KSFM with no statistical difference (P=1.0>0.05). The number of viablecount was less than the group of D-KSFM, but the two groups had nostatistical difference (P>0.05). Attached cells were more in the group ofD-KSFM when replaced medium for the first time. After digestion with1.2U/ml Dispase Ⅱ, the epithelium were more likely to be separated completely,and the viable count was higher than0.6U/ml Dispase Ⅱwith statisticalsignificance (P=0.025<0.05; P=0.036<0.05). Trypsin digestion10mintwice could obtain higher viable count than20min once, but the differencehas no statistical significance (P=0.388). The attached cell observed underinverted microscope after trypsin digestion10min twice was more than20min when replaced medium for the first time. The cells cultured inKBM-GOLD medium adhered in48h, increased slightly between3-6days,became confluent gradually after7-8days, and showed the appearances of“paving stone”. The cells grew faster in KBM-GOLD medium containing6%FBS, reached85%confluency after4-6days, but mixed with the elongatedspindle fibroblasts, and morphology of epithelial cell has changed. Theepithelial cells were polygon-like or irregular globular and showed typicalappearances of “paving stone” after confluence under inversion microscope.Microvilli ridge were observed on cells surface with scanning electronmicroscope. The anti-pan-cytokeratin (AE1/AE3) by immunohistochemistrystain was positive. Immunofluoresence displayed that the cytoplasm was red.Conclusion: The oral mucosal tissues of rats digest with1.2U/ml Dispase Ⅱdissolving in D-KSFM and with trypsin10min twice in succession,then culture in the KBM-GOLD medium can obtain more rat oral mucosalepithelial cells demonstrating epithelial characterization.Part two: Inducing Rat Bone Marrow Mesenchymal Stem CellsDifferentiate to Epithelial Cells in vitroObjective: To study the culture method of inducing BMSCs differentiateto epithelial cells by rat oral mucosal epithelial cells in vitro, and to providethe basis for using BMSCs as the seed cells in vaginal tissue engineering.Methods: Epithelial cells was obtained after the rat oral mucosal tissuesdigested in DispaseⅡand trypsin in succession, and then seeded in Transwellinsert, the bone marrow mesenchymal stem cells on third to fifth passageseeded in six-well plate with the density of3×10~3/cm~2(co-cultured for14days) and5×10~3/cm~2(co-cultured for7days). After adhered, BMSCs wereco-cultured with rat oral mucosal epithelial cells which has cultured2days inTranswell insert supplied by KBM-GOLD medium containing3%FBS, andchanged the insert which planned to co-cultured for14days in the8th day.The changes of morphology were observed under inverted microscope everyday, and the expression of pan-cytokeratin (AE1/AE3) which is the specificmarker of epithelial cells was identified by immunohistochemistry andwestern blot on7th and14th day respectively.Results: In preliminary stage, the induced cells showed a spindle type,uniform size and no obvious protuberance. On the7th day, a few BMSCschanged into round or oval, short villous protuberance appeared around,nuclear presented as round or oval, cytoplasm was rich, and thepan-cytokeratin (AE1/AE3) by immunohistochemistry stain was positivewhere claybank particles emerged in cytoplasm. On the14th day, round oroval cells and the expression of AE1/AE3increased significantly, thearrangement of BMSCs changed into irregular or clumped together. Westernblot results showed that the induced cells had AE1/AE3expression, and thelevel increased significantly on the14th day.Conclusion: When BMSCs are co-cultured indirectly with oral mucosal epithelial cells in vitro, KBM-GOLD medium containing3%FBS couldinduce BMSCs differentiate to epithelioid cells, it is expected to substitute forvaginal epithelial cells and applied in the tissue engineering construction ofvagina.