Tissue Engineering Vagina With Vaginal Epithelial Cells And Porcine Acellular Dermal Matrix(padm)-fibrin Gel In Mouse

Part One: Culture and Identification of Mouse Vaginal Epithelial Cells invitroObjective: To find out a suitable method to isolate, identify, and culturemouse vaginal epithelial cells for engineering of vaginal tissue.Methods:(1) Enzyme digestion methods: The vaginal tissues fromeight-week female mice were digested by dispase. The epithelial layer wasisolated and digested by trypsine before the cell suspension and cultured inK-SFM.(2) Observation and identification: The cell morphology, time ofattachment and proliferation characteristics were observed by inversionmicroscope. The growth curve was drawn by MTT method. The epithelialcells were identified by AE1/AE3immunohistochemistry stain. Theultrastructure was oberserved by scanning electron microscope.Results:(1) The epithelial cells became small globular after digestion,and they attached with typical polygonal epithelial morphology after24～48hcultured in K-SFM. The proliferation was notable within3～5d and coloniesprogressively shaped. The cells reached80%confluency within7~10days andshowed typical appearances of "paving stone".(2) The expression ofAE1/AE3staining of the cultured cells was positive, and purity reached98%.(3) Microvilli ridges were observed on cells' surface with scanning electronmicroscope.Conclusion: Vaginal epithelial cells obtained by enzyme digestionmethod displayed fast attachment, short growth time, and high success rate.The large numbers of epithelial cells can be rapidly obtained for engineeringof vaginal tissue.Part Two: Biocompatibility of the Mouse Vaginal Epithelial Cells and Porcine Acellular Dermal Matrix(PADM)-Fibrin gelObjective: To investigate the feasibility of engineering a vagina usingvaginal epithelial cells, fibrin gel and porcine acellular dermal matrix(PADM).Methods:(1) Intermixing fibrinogen, thrombin, aprotinin and calciumchloride in different order and concentration to finding out the optimalproportion.(2) Mouse vaginal epithelial cells were suspended in fibrin gel,and cell morphology and proliferation characteristics were observed byinversion microscope.(3) Cultured vaginal epithelial cells were suspended infibrin and grafted together with PADM. Biologic attachment and growth ofcultured mice vaginal epithelial cells were observed daily. The cell-seededscaffolds were subcutaneously implanted into mice after cultured48hours invitro. The animals were sacrificed at2,4,8and12weeks after implantation.The tissue-engineered constructs were prepared for HE and Van Gieson (VG)staining to evaluate the status of various layer growth. Epithelial broadspectrum of monoclonal antibodies of AE1／AE3was used to identify theexistence of epithelial cells.Results:(1) Epithelial cells were suspended uniformly in fibrin gel andgrew in three-dimensional space.(2) There was no significant immunologicalrejection.(3) Two weeks after transplantation, only a few vaginal epithelialcells were observed under the light microscopy. Within4weeks2-3layerepitheliums were observed. Within8-12weeks epithelization was intensified.AE1／AE3staining was consistent with HE staining. The reducedperformance of the grafts and cellular ingrowth were observed in VG staining.Conclusion: Cell seeded polymer scaffold was able to form vascularizedvaginal tissue in vivo with phenotypic properties similar to those of normalvaginal tissue.Part Three: Comparison of Vaginal Epithelial Cells Seeded and UnseededPorcine Acellular Dermal Matrix for Vagina ReconstructionObjective: To compare the effect of vagina reconstruction with vaginalepithelial cells seeded and unseeded porcine acellular dermal matrix (PADM)in an animal model． Methods: Twenty-four female Kun Ming mice were randomly dividedinto group A and group B. Vagina excision and vagina reconstruction withvaginal epithelial cells unseeded and seeded PADM were performed in groupA and group B, correspondingly. At week2,4,8and12after implantation, theanimals were sacrificed and the neovaginas were prepared forhematoxylin-eosin (HE) staining, Van Gieson (VG) staining and epithelialbroad spectrum of monoclona1antibodies of AE1/AE3immunohistochemicalstaining to evaluate the status of various layer growth of vagina．Theultrastructure of neovagina was observed by transmission electron microscopeat week12after implantation．Results: In group A, only2-3layers of epitheliums were observed underthe light microscopy at week2after implantation. At week4, epithelization inmucosa of neovaginal canal was intensified to4-5layers, but the epithelialcells were disarranged. Within8-12weeks, the differences between theneovagina and the native vagina were harldy noted. In group B, the neovaginahad almost the same structure as the native vagina after2weeks. Within4-12weeks the same results could be observed. In both groups the structure ofPADM were became loose as time went by. At week12, degradation of PADMcould be observed in group A, while the structure of PADM still remainedcomplete. Within4-12weeks, smooth muscle cells and fibroblasts wereobserved in both groups．In group B, fibroblasts, nerve fibre and capillarycould be observed under transmission electron microscope.Conclusion: Vagina reconstruction with vaginal epithelial cells seededand unseeded PADM were feasible. Vagina reconstruction with unseededPADM was a simple method, but the epithelization taked more time comparedwith cells seeded PADM method which was more complex.