A Study Of Adipose-dervied Stem Cells Combined With Acellular Tissue Matrix For Vaginal Reconstruction In A Rat Model

Part one:Preparation and test of porcine SIS,UBM and AVM.Objective: To explore an effective method for preparation of porcine SIS,UBM and AVM and to compare the biological mechanical properties and growth factor content.Methods: Porcine SIS,UBM and AVM were prepared by the methods of physical,chemical and enzymatic methods,using HE staining,DAPI staining and DNA content and fragment size to assess the effectiveness of the acellular method.Tensile tests and ELISA methods were used to compare the biomechanical properties and contents of growth factors respectively.Results: By the means of physical,chemical and enzymatic methods,we successfully prepared the acellular matrix materials——SIS,SISk,UBM,UBMk,AVM and AVMk.There were some differences in the tissue structure,biomechanical properties and the contents of growth factors.5% peroxyacetic acid increased the porosity of the material,reduced biomechanical properties of the material,and the growth factor content of the material was greatly reduced.Conclusion: Applying the method of physical,chemical combined with enzymatic methods can successfully prepare six kinds of acellular matrix materials-SIS,SISk,UBM,UBMk AVM and AVMk.Part two:Isolation,culture and characterization of rat adipose-derived stem cells in vitro and the biocompatibilities of acellular matrix materials.Objective: To establish a stable protocol for isolation and culture of adipose-derived stem cells in vitro,assess the biocompatibilities of six kinds of acellular matrix materials ——SIS,SISk,UBM,UBMk,AVM and AVMk.Methods: The adipose tissue was obtained from bilateral groins of the ratand was digested with 0.1% I-collagenase.The phenotypes of ASCs were confirmed by flow cytometry analysis.Adipogenic differentiation and osteogenic differentiation were used to confirm its property of pluripotency.Direct contact MTT quantitative detection method and laser scanning confocal microscope observing the cell morphology were combined to assess the in vitro cytotoxicity of six kinds of scaffold materials.Six kinds of scaffold materials were transplanted subcutaneous to observe the biocompatibility in vivo.Results: We successfully isolated rat inguinal ASCs by enzyme digestion.The surface markers of the isolated cells were consistent with ASCs,and they had adipogenic and osteogenic differentiation capacity.ASCs grew well on six kinds of scaffolds,cells on perforated materials grew better than on not perforated materials,SISk,AVMk and AVM can better promote cell growth.Subcutaneous transplantation test also showed that all the six kinds of scaffolds have good biocompatibility.Conclusion: The enzyme digestion method can successfully isolate and culture rat inguinal ASCs.SIS,SISk,UBM,UBMk,AVM and AVMk had good biocompatibility,among which SISk,AVMk and AVM were optimal.Part three: The comparison of different acellular matrix materials in the reconstruction of rat vaginaObjective: To construct the model of partial vaginal resection of rat.Verify the possibility of SIS,SISk,UBM,UBMk,AVM and AVMk on the construction of tissue engineered vagina,and compare of them.Methods: The whole uterus and part of the vagina were removed to construct the model of partial excision of the rat vagina.SIS,SISk,UBM,UBMk,AVM and AVMk were used to reconstruct tissue engineered vigina,and the gross morphology observation,general micromorphological observation and immunohistochemistry observation of vaginal were used for morphological assessment.Tissue bath method was used to evaluate the new vaginal function.Results: SIS,SISk,UBM,UBMk,AVM and AVMk can successfully construct rat tissue engineered vigina.Drilling scaffolds were better than non perforated scaffolds.The SISk group,UBM group,UBMk group,AVM group and AVMk group had better effect on the contraction and relaxation to the drugs than the SIS group.AVMk group was superior to other groups in terms of the morphology and the function.Conclusion: SIS,SISk,UBM,UBMk,AVM and AVMk six kinds of acellular matrix materials can be successfully constructed rat tissue engineered vagina.The structure and function of the tissue engineered vagina constructed by AVMk was the best.Part four: ASCs combined with AVMk reconstruction of rat vaginaObjective: To construct the model of vaginal resection of rat.Verify the ASCs compositing AVMk to build rat tissue engineered vagina is better than pure AVMk application.Methods: The whole vagina was removed to construct the model of total excision of the rat vagina.AVMk compositing ASCs or not reconstructed rat tissue engineered vagina.The gross morphology observation and general micromorphological observation were used for morphological assessment.Immunofluorescence was used to assess the proliferation differentiation of ASCs.Then tissue bath method was used to evaluate the new vaginal function.Results: AVMk compositing ASCs or not all can successfully reconstructe rat tissue engineered vagina.ASCs could differentiate into muscle cells,fibroblasts,nerve cells and estrogen receptor expression cells,and may differentiate into endothelial cells to promote tissue regeneration.Tissue engineered vagina reconstructed with AVMk and ASCs was superior to AVMk not only in morphology but also in function.Conclusion: Through direct differentiation ASCs can promote the reconstruction of the tissue engineered vagina.