Tissue Engineering Study Of Mesenchymal Stem Cell-based Bioengineered Patch To Promote Tissue Repair Ability Of Rhesus Monkey Vaginal Wall

Introduction and hypothesisPelvic organ prolapse(POP),a kind of herniation of pelvic organs into or through the vagina,is a major gynecological condition that can significantly affect the physical and mental health of women.Transvaginal polypropylene meshes are widely used in prolapse surgery to reduce the anatomic recurrence rate;however,the risk of severe mesh complications,including exposure or erosion,limits their intensive usage worldwide.The FDA had halted the usage of surgical mesh intended for transvaginal repair of POP in 2019,creating an emergency requirement for developing new therapies.Strategies that embody the principles of tissue engineering and regenerative medicine have gained considerable traction in the past decade.Due to the capability of regenerating injured tissues and inhibiting foreign body reactions,mesenchymal stem cells(MSCs)have attracted widespread interest as an effective tool for POP repair.Small intestine submucosa(SIS)graft,a kind of extracellular matrix-derived graft,plays an important role in regulating cell proliferation,differentiation and migration through intricate relationships among cellular and acellular tissue components and further provides support for the weak pelvic floor.Thus,there is urgent need to combine the mechanical and biochemical properties of bioscaffolds with the therapeutic capabilities of MSCs to maximize the regenerative capability in treating POP.In the Part one section of this study,we seeded HUMSCs on the SIS graft and produced a bioengineered graft.We investigated the secretome changes and differentiation ability of HUMSCs after culture on SIS grafts for 5 days in vitro.Methods1.HUMSCs were isolated from the umbilical cord and were identified by the flow cytometric analysis;2.The viability and proliferation of HUMSCs was assessed by the CellTiter-Blue viability assay.The appropriate culture time for in vivo implantation was chosen.The morphology of HUMSCs seeded on SIS grafts was evaluated by scanning electron microscopy;3.The differentiation potential of HUMSCs into smooth muscle cells/fibroblasts was evaluated by the external growth factor stimulation;4.Comparison of the complete transcriptome profile of HUMSCs cultured on the TCP and SIS graft was revealed by RNA-Seq and several key genes associated with extracellular matrix secretion and reorganization and some key growth factors were confirmed by q-PCR.Results1.HUMSCs were successfully isolated and expressed the surface markers of MSCs by flow-cytometric analysis;2.The isolated HUMSCs were seeded on the SIS graft to obtain the bioengineered graft,HUMSC can proliferate well on the SIS graft,and maintain a high proliferation rate and an appropriate density on the 5th day,which provides the basis for implantation in vivo.As shown by the scanning electron microscope,HUMSCs secret the extracellular matrix and filled the cracks of the SIS grafts;3.HUMSCs successfully differentiated to smooth muscle cells and fibroblasts,and expressed corresponding markers with external growth factor stimulation.4.The complete transcriptomic profile of HUMSCs revealed that HUMSCs cultured on grafts produced significantly higher levels of proangiogenic cytokines than cells cultured in tissue culture plates(p<0.05).The GO enrichment analysis indicated that the up-regulated genes were mostly concentrated on the pathways related to extracellular matrix reorganization,indicating that the HUMSCs seeded on the SIS graft experienced the process of extracellular matrix remodeling.ConclusionsHUMSCs proliferated well and the density was appropriate after 5 days of culture.In addition,we demonstrated that the differentiation potential of HUMSCs was maintained with external growth factor stimulation.The complete transcriptomic profile of HUMSCs revealed that HUMSCs cultured on grafts produced significantly higher levels of proangiogenic cytokines than cells cultured on TCPs.HypothesisIn the Part two section of this study,we confirmed the functional relevance of these bioengineered grafts in biocompatibility,tissue regeneration potential and mechanical properties by implanting them into the vaginas of ovariectomized(OVX)rhesus monkeys via sacrocolpopexy,to simulate the clinical operation to the great extent and provide experimental data for the bioengineered grafts in treating POP.MethodsEstablish an ovariectomized model of rhesus monkey,which is divided into 3 groups:sham group,SIS group and SIS+HUMSCs group.A simple hysterectomy,the implantation of SIS graft or bioengineered graft by sacrocolpopexy was performed respectively.The full-length vaginal tissue was taken for analysis 3 months after the surgery;1.Masson staining was performed to analyze the changes of the structure and thickness of each layer of the vaginal wall;Sirius red staining was performed to compare the type?/? collagen,and EVG staining was used to evaluate the elastic fibers of the lamina propria;2.a-SMA immunohistochemistry was performed to compare the diameter of the muscle bundle(miniferet);3.vWF immunofluorescence was performed to evaluate the angiogenesis of the vaginal tissue;4.The expression analysis of several genes associated with extracellular matrix content and reorganization and several key growth factors were performed by q-PCR;5.Biomechanical testing was conducted to evaluate the mechanical properties of the vaginal tissue.The above indicators were used to evaluate the effect of bioengineered graft on impaired vaginal tissue.Results1.Compared with the sham and SIS group,increased type I collagen were observed in SIS+HUMSCs group(Sham group:p=0.048;SIS group:p=0.027).Decreased type III collagen(p=0.014)and increased elastic fiber(p=0.042)were only observed in SIS+HUMSCs group compared with the sham group;2.We found that the thicker smooth muscle layer(Sham group:/p=0.02;SIS group:p=0.036)and larger muscle bundle formation(Sham group:p=0.001;SIS group:p=0.01)in the vaginal tissue of the SIS+HUMSCs group compared with the sham and SIS group;3.Promising angiogenesis were found 3 months after the implantation in SIS+HUMSCs group(Sham group:p=0.001;SIS group:p=0.005).4.The mRNA level of actin in the SIS+HUMSCs group was significantly higher than other groups(Sham group:p=0.007;SIS group:p=0.008),and the expression of angiogenic factor(VEGF)was higher than the sham group(p=0.016).5.Improved elastic modulus were observed in SIS+HUMSCs group compared with the sham group(p=0.031).ConclusionsThree months after implantation of the bioengineered grafts into ovariectomized(OVX)rhesus monkeys via sacrocolpopexy,extracellular matrix reorganization,large muscle bundle formation,angiogenesis,and mechanical properties of the vagina were enhanced.To our knowledge,this is the first demonstration of the utility of stem cell-based bioengineered grafts for repairing damaged vaginal tissue in rhesus monkeys.These results elucidate a new approach for treating POP,providing the clinical translational value.