Study On The Regulation Of Tissue Repair Function By Artificial Extracellular Matrix And Its Application In Pelvic Floor Reconstruction Mes

Objective:To establish an immortalized human vaginal wall fibroblasts(hVWFs)cell line.To establish a rat model of vaginal wall pelvic organ prolapse(POP).Based on the cell line and rat model,we sought to evaluate the effectiveness of artificial extracellular matrix(ECM)modified pelvic floor reconstruction mesh on reducing the complications of mesh exposure.Methods:The hTERT gene was overexpressed in primary hVWFs by lentivirus,cells were cultured continuously,and cell senescence was assessed by Senescence-associatedβ-galactosidase(SA-β-gal)staining.CCK-8 assay,EdU assay,and wound healing assay were used to evaluate the proliferation and migration of primary and immortalized cells.Real-time quantitative PCR(qPCR)and Western blot(WB)were used to detect the expression of key genes in immortalized cells during continuous passage.Karyotype stability was determined by chromosome karyotype analysis,and tumorigenesis assay in nude mice was performed to evaluate the tumorigenicity of immortalized cells in vivo.Female SD rats at 9-10 weeks were ovariectomized and vaginal balloon dilation was performed 2 weeks later.Masson staining was performed at 2,4,and 12 weeks after dilation.Immunohistochemical staining was used to evaluate the matrix metalloproteinase(MMPs)and tissue inhibitors of metalloproteins(TIMPs),muscle fibers,and collagen.The expression of collagen was evaluated by Sirius red staining,and the difference of protein expression was confirmed by WB assay.Biomechanical changes in tissues were assessed using uniaxial tension tests.The coaxial electrospinning technique was used to prepare the nano-spinning with polylactic acid(PLA)inner core and gelatin shell,and then the nano-fiber film was prepared as an artificial extracellular matrix to improve the polypropylene mesh.The modified mesh was implanted into the vaginal wall of the model rats and evaluated at 4 and 12 weeks postoperatively.The tissue morphology was evaluated by HE staining,the expression levels of muscle fibers,MMPs,and TIMPs were evaluated by immunohistochemistry and WB assay.The expression levels of collagen were evaluated by Sirius red staining and WB assay.The software GraphPad was used for statistical analysis of data.Results:Compared with the primary cells,the senescence rate of hVWFs overexpressing hTERT gene was significantly lower(P=0.014).In the process of continuous passage,immortalized hVWFs cells maintained stable proliferation and migration,key genes maintained stable expression at mRNA level and protein level,chromosome karyotype remained stable after overexpression of hTERT gene,and immortalized hVWFs cells did not have tumorigenesis in vivo.Compared with the control group,the rats undergoing balloon dilation after ovariectomy showed epithelial atrophy,muscle fiber disorder,significantly decreased collagen expression,increased MMPs expression,and significantly decreased TIMPs expression,basically consistent with the histopathological characteristics of POP.In terms of biomechanics,the stiffness of the tissue increased significantly under both low and high deformation,which was consistent with the biomechanical characteristics of POP tissue.The modified mesh could significantly promote the proliferation of fibroblasts in vitro(P=0.012).After implantation of the modified mesh into the vaginal wall of rats,no mesh exposure occurred compared with the normal polypropylene mesh.The modified mesh maintained a relatively fixed histological position without intruding into muscle layer or breaking through epidermis.The collagen content in the vaginal wall of rats with modified mesh was significantly higher,and the COL1/COL3 ratio was lower,indicating better tissue elasticity.MMPs expression was lower and TIMPs expression was higher in the modified mesh group,indicating that collagen catabolism was inhibited.The expression of TGF-β1 and the phosphorylation levels of Smad3,p38 and ERK1/2 were also significantly increased in the modified mesh implantation tissues(P<0.05).Conclusion:We successfully immortalized hVWFs cells by overexpressing hTERT gene in primary hVWFs.It is a feasible method to construct POP model of rat vaginal wall by vaginal wall balloon dilation after ovariectomy.The use of artificial ECM coated polypropylene mesh can increase biocompatibility and reduce mesh complications.The ECM coated polypropylene mesh could potentially be clinically used in the future.