| Vaginal loss can be caused by a variety of congenital and acquired factors.At present,a variety of clinical methods for the treatment of vaginal loss,such as parietal pressure,rectovesical acupoint + lining and intestinal replacement of vagina,not only have the malpractice of "treating injury with injury",but also it is difficult to achieve the perfect substitution of structure and function.In recent years,with the continuous development of tissue engineering technology,a variety of tissue engineering materials,represented by small intestinal submucosal matrix(SIS)and acellular dermal matrix(ADM),have been successfully applied to the repair of vaginal defects.Therefore,vaginoplasty based on biomaterials has attracted more and more attention.IThe vagina is mainly composed of four layers: epithelial layer,lamina propria,muscular layer,and adventitia.The epithelium provides a physical immune barrier,maintains the acidic environment of the vagina,and plays a significant role in lubrication.The muscular layer plays an important role in supporting pelvic organs,and it is also related to women’s sexual pleasure.The vagina in the physiological state has a multi-layer tissue structure,so we hope to find suitable biomaterials to make the anatomical structure and function of the reconstructed vagina closer to the physiological state,so as to avoid contracture,stenosis and fibrosis of the reconstructed tissue.The Double-sided biomembrane(DBM)developed and produced by Wuqi Biomedical Technology(Jiangsu)Co.,Ltd.The product is designed to have a unique double-layer structure of smooth layer and rough layer,the smooth layer is not only conducive to the migration and proliferation of epithelial cells,but also can exercise an effective barrier function to inhibit adhesion and scar tissue formation;Porous layer is a three-dimensional skeleton structure composed of collagen fibers,which can promote the anchoring and proliferation of smooth muscle cells and vascular endothelial cells.Collagen can stop bleeding and promote wound healing.Based on the synergistic effect of the two structures,collagen matrix biofilm can induce rapid epithelialization,prevent adhesion,and induce muscle regeneration and angiogenesis.Moreover,the product is a clippable stitchable patch that can be cut to the desired shape according to the location and size of the expected repair implant.In vaginal reconstruction research,the establishment of animal models is still a challenge.At present,the research on the application of biomaterials in vaginal reconstruction is mainly focused on small animal models such as mice,rats and rabbits.However,these models have many limitations for preclinical applications.Minipigs are becoming the preferred large animal model for tissue engineering and regenerative medicine because of their similarities in genetics,reproductive tract anatomy,physiology,and pathophysiology with humans.However,successful research in large animal models requires more specialized knowledge and skills,higher procurement and veterinary care costs,which limit large-scale studies.In this study,a fullthickness(4cm)vaginal defect model was established in minipigs,and DBM was used for vaginal reconstruction.At the same time,SIS material was used as a control to evaluate the effect of DBM regeneration.When the graft is implanted,insufficient angiogenesis is often associated with adverse clinical outcomes.In the early stage of implantation,the inability to establish effective microcirculation is an important factor restricting the treatment of soft tissue injury by tissue engineering,especially in large tissue defects.When biomaterials are implanted,the cell / material interface is a complex and dynamic microenvironment,in which biomaterials can determine the fate of cells through their inherent properties(such as hardness,surface structure,pore structure,etc.).For example,some experimental results show that when the surface roughness of biomaterials increases,the adhesion and proliferation of vascular endothelial cells will be enhanced accordingly,and the high porosity of materials can also promote vascular endothelial cells to a certain extent.these effects are superimposed to improve the vascularization of tissue grafts.Mechanical signals in extracellular microenvironment can be mediated by YAP/TAZ to regulate cell morphology,play a key role in cell growth and differentiation,and ultimately determine the fate of cells.Therefore,HIPPO signal pathway is an important part of vascular remodeling.By detecting the expression of YAP/TAZ and other proteins in neovagina,we preliminarily discussed the mechanism of vascular remodeling.Part One Characterization of double-sided biomembrane and small intestinal submucosaObjective: Evaluation of surface morphology,composition and mechanical properties of biomaterials.Methods:1.The composition and acellular condition of DBM and SIS were evaluated by HE,Masson and Sirius red staining,and the surface morphology was observed by SEM.2.Testing the mechanical properties of DBM and SIS by Instron3366 electronic material tension load tester.3.Detection of hydrophilicity of DBM by water contact angle test.3.Mass spectrometric analysis was performed to determine the protein composition and contents of DBM.Results:1.Through HE staining and DAPI staining analysis of DBM and SIS,the results showed that there were no obvious cell and nucleic acid residues and no obvious immunogenicity in DBM and SIS.In addition,the smooth layer of DBM was thinner,while the loose layer was thicker,but there was no obvious delamination structure in SIS.DBM and SIS were stained with Masson and Sirius red staining results were analyzed.It was found that there were lots of elastic and collagen fibers in DBM and SIS,and they were mainly composed of type I and III.Surface structure of DBM and SIS was analyzed by SEM.The results indicate that DBM has a smooth inner surface,the outer surface is rough,and a large number of fibrous tissues are interlaced with each other,while there is no obvious difference between the inner and outer surfaces of SIS.2.The maximum tensile strength and elastic modulus of DBM tested by Instron 3366 electronic material tensile load tester are 0.82±0.08 Mpa and4.53±0.09 Mpa.3.The water contact angle of the smooth side was 88.95±3.13°,while the rough side was 74.05±3.52°.4.Protein mass spectrometric analysis identified that DBM has a variety of structural components of extracellular matrix(ECM),such as collagen(I,III,IV,VI,XII and XXI),proteoglycans(PGs),fibronectin(FN),fibrillin-1(FBN1)and so on.Conclusions:1.The results of structure and composition analysis of DBM and SIS materials show that both materials have no obvious immunogenicity and contain a lot of elastic fibers and collagen,which can effectively support the tissue regeneration and repair.2.The analysis of the mechanical properties shows that DBM is closer to the mechanical properties of normal vaginal tissue.3.The water contact angle shows that DBM has high hydrophilicity,which is beneficial for cell adhesion.4.Protein mass spectrometric analysis of DBM shows that it is a kind of natural ECM and its tissue composition is beneficial to the regeneration and reconstruction of vagina.Part Two Construction of a large animal model of vaginal loss and reconstruct vagina.Objective: Construction of a large animal model of vaginal loss by laparotomy,and the neovagina was reconstructed by DBM and SIS.Methods: Animals were classified into 3 groups: DBM(n=12),SIS(n=10)and sham(n=6).Part of the vaginal tissue(4cm)of minipigs was removed by laparotomy and replaced by appropriate size of DBM or SIS,while the sham group only underwent laparotomy without removal of vaginal tissue.Serum samples were taken at 1,2,3,4,8,12 weeks after surgery to detect TNF-α and IL-1β.The histocompatibility of DBM and SIS was evaluated.After 4 and 12 weeks,half of the animals were sacrificed,and the reconstruction were observed,the stenosis and necrosis were observed.Results:1.All the miniature pigs survived to the end of the experiment,with good appetite and mental state,normal defecation and urination,slight redness and swelling of the incision,and no obvious infection,fistula,rejection and other phenomena,indicating successful establishment of a large animal model of vaginal loss.2.The level of TNF-α in DBM group increased significantly in 1 week,reached a peak in 3 weeks,then decreased gradually and returned to normal after 8 weeks.The level of IL-1β was also increased in the first week,and reached its peak in 4 weeks,and then decreased gradually.The changes of TNF-α and IL1-β in SIS group were similar to those in DBM group.3.In DBM group,the vaginal tissue at 4 weeks after reconstruction was pink mucosa covering the whole vagina,and the mucosal surface was smooth and moist.The muscular layer was thin and tough without obvious elasticity.After 12 weeks,longitudinal folds were distributed on the surface of vaginal mucosa,which was similar to that of normal vagina,and the muscular layer was significantly thickened and elastic.In SIS group,the vaginal tissue at 4weeks after reconstruction also showed that the pink mucosa covered the vagina,the mucous membrane was smooth,the muscular layer was thin and inelastic,and 12 weeks after reconstruction,the vaginal mucosal surface was still smooth with no obvious wrinkles,and the muscular layer was slightly thickened and elastic.In both groups,the neovaginal tissue adhered to the surrounding tissue(rectum,bladder and surrounding connective tissue),and the vaginal epithelium had slight contracture,but there was no stenosis and atresia.Conclusions:1.We successfully established the model of vaginal loss in miniature pigs by laparotomy.2.DBM and SIS do not cause strong immune response,have low immunogenicity,and are safe to use.3.After 4 weeks,the formation of neovagina could be seen in both DBM group and SIS group.At 12 weeks after operation,the new muscle layer in DBM and SIS groups was thickened and had certain elasticity.Part Three Evaluation of the effect of vaginal tissue reconstruction with DBM and SIS and a preliminary study on the mechanism of vascular reconstructionObjective: The histology,epithelial morphology and specific protein expression of the reconstructed vagina were evaluated by HE,Masson staining,scanning electron microscope and immunohistochemical staining.The landmark proteins in the neovagina were quantitatively analyzed by Western blot,the synthesis of glycogen in the newborn vagina was evaluated by PAS staining,and the contraction function of the newborn vagina at 12 weeks after operation was evaluated by tissue bath method.The expression of key proteins of Hippo pathway in neovagina of DBM group was detected by Western blot.Methods:1.The morphological characteristics of reconstructed vaginal tissue in each group were evaluated by HE staining and Masson staining.The surface structure of neovaginal tissue was observed by scanning electron microscope,and the arrangement of epithelial cells in each group was evaluated.Immunohistochemical staining of CK14,α-actin,HSP47,CD31,PGP9.5 and PCNA was performed to evaluate the expression of specific protein molecules in each tissue layer and proliferating cell nuclear antigen(PCNA)in reconstructed blood vessels.Western blot was used to determine the expression level of these molecular proteins and the expression of key proteins in HIPPO pathway.2.PAS staining was used to assess the ability of the neoepithelial cells of the two groups to synthesize glycogen.3.Tissue bath test was used to evaluate whether the vaginal tissue of the two groups reconstructed for 12 weeks had contractile function.Results:1.HE staining showed that in the 4-week DBM reconstruction group,the newborn vagina had formed a complete epithelium,about 3-4 layers,and the lamina propria could be seen under the epidermis,which was loose connective tissue,rich neovascularization,and a large number of lymphocytes,neutrophils and other inflammatory cells.12 weeks after operation,the epithelium further thickened and was stratified squamous epithelium.In the lamina propria,the lumen of the artery is regular and round,and the wall has a complete muscular layer,while the shape of the venule is irregular and contains few lymphocytes and neutrophils.The reconstruction results of SIS group were similar to those of DBM group.2.Masson staining showed that the epithelium of normal vaginal tissue was red,indicating that the cytoplasm was rich,the lamina propria was blue,and the collagen fibers were rich,and the smooth muscle layer was also red,which was a regular,large bundle of smooth muscle tissue.After 4 weeks of reconstruction,smooth muscle formation was observed in DBM group,but muscle bundles were thinner and arranged irregularly,and larger muscle bundles were observed in 12-week reconstruction group,similar to normal vaginal tissue.The formation of smooth muscle layer in 4-week SIS reconstruction group was similar to that in DBM reconstruction 4-week group,but the smooth muscle bundles in 12-week reconstruction group were still small and disorderly.3.The morphology and arrangement of epithelial cells could be observed by scanning electron microscope.the results showed that the normal vaginal epithelial cells were round and regular,and there seemed to be granular secretion on the surface of normal vaginal epithelial cells with small vesicles.The neovaginal epithelial cells in the 4-week DBM reconstruction group were flat or irregular and arranged disorderly,while in the 12-week group,mucosal folds,full epithelial cells and regular arrangement were observed,and there was no significant difference between the neovaginal epithelial cells and the normal vaginal epithelial cells.The reconstruction results of SIS group were similar to those of DBM group.4.Immunohistochemical staining showed that vaginal tissues reconstructed for 4 weeks in DBM group could express markers of various layers of vaginal tissues,such as CK14,α-actin,HSP47,CD31,and PGP9.5.After 12 weeks of operation,the area shown by each marker increased,which was not significantly different from that of normal vaginal tissue.Similar results were observed in SIS group reconstruction of vaginal tissue.5.The positive area of PCNA in the 4-week DBM reconstruction group was significantly higher than that in the normal group,decreased in the12-week reconstruction group,but still higher than that in the normal group.6.The results of Western blot showed that compared with the normal vaginal tissue,the expression of CK14 in the 4-week DBM reconstruction group was significantly lower than that in the normal group,but there was no significant difference between the 12-week reconstruction group and the normal group,while the expression of CK14 in the vaginal tissue increased gradually in the 4-week and 12-week SIS reconstruction group,and the expression in the 12-week reconstruction group was significantly higher than that in the normal group.The expression of α-actin in vaginal tissue of 4-week and 12-week DBM reconstruction showed an increasing trend,but there was no significant difference compared with normal group.The expression trend of α-actin in vaginal tissue of SIS reconstruction was the same as that of DBM group,but the expression of α-actin in 12-week reconstruction group was significantly higher than that of normal group.Compared with the normal group,the expression of CD31 and HSP47 in vaginal tissue of 4 weeks and 12 weeks after DBM reconstruction increased at first and then decreased,and the expression of CD31 and HSP47 in vaginal tissue of SIS group increased significantly at 4 weeks and 12 weeks after reconstruction,which was significantly higher than that in normal group.After 4 weeks of DBM reconstruction,the expression of PGP9.5 in vaginal tissue was significantly lower than that in the normal group,but it increased significantly after 12 weeks of reconstruction,and there was no significant difference between the normal group and the normal group.The expression trend of PGP9.5 in SIS group was consistent with that in DBM group.7.There was no obvious positive area of PAS staining in the vaginal tissue of the two groups after 4 weeks of reconstruction,while the positive areas of purplish red could be observed in the vagina of the two groups after12 weeks of reconstruction,indicating that there was a large amount of glycogen synthesis.8.The results of tissue bath experiment showed that the contraction induced by norepinephrine and carbachol were observed in both groups at 12 weeks after reconstruction,which could be antagonized by phentolamine and atropine,respectively.9.The expression of Yap1/TAZ/TEAD1/Cyclin D1 in 4-week DBM reconstruction group and 12-week reconstruction group increased at first and then decreased,and the expression in 4-week reconstruction group was significantly higher than that in normal group,with statistical difference,which was consistent with the trend of CD31 expression.Conclusions:1.The morphology of vaginal tissue reconstructed by DBM and SIS was similar to that of normal vagina,but thicker smooth muscle bundles could be formed in 12-week DBM reconstruction group.In the early stage of reconstruction,both groups had epithelialization and smooth muscle layer reconstruction,and a large number of vascular endothelial cells and fibroblasts proliferated;in the later stage of reconstruction,the epithelial layer and smooth muscle layer were further repaired and remodeled,and the nerve recovery was the slowest.2.After 12 weeks of reconstruction,the epithelial cells of neovaginal tissues in both groups could synthesize glycogen,and the neovaginal tissues in both groups showed contractile response to norepinephrine and carbachol.3.Yap1/TAZ pathway promotes the proliferation of vascular endothelial cells and the early reconstruction of microcirculation. |
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