Experimental Study Of Bone Mesenchymal Stem Cells-urinary Bladder Matrix-polypropylene Composite Mesh

Pelvic organ prolapse (POP) is a common gynecological disease in middle-andold-aged women, which can severely affect the patients’ quality of life and cause hugepsychological burden. Effective treatment is in urgent need in clinical setting.Reconstructive pelvic surgery is a common and effective approach for the treatment of POP.The traditional POP repair surgery is performed on the fragile pelvic floor tissues, such asthe fasciae, connective tissues or ligaments, which have already been injured or containabnormal collagen; thus, it is unable to repair the defect completely and leads to a highrecurrence rate. Therefore more safety and stable alternative materials are needed toimprove the operation.In recent years, the advances in the technology of tissue engineering enable theengineered tissue to be used as the alternative materials to support the fragile or injuredpelvic floor, thus to efficiently reconstruct its anatomy and restore its function.Polypropylene mesh (PM), a widely used synthetic mesh in pelvic floor reconstruction, canresult in a series of adverse reactions, such as infection,erosion,contracture,rejection,chronic pain, dyspareunia, etc., seriously affecting the qualityof life. ECM has a good tissue compatibility, which can play a immunomodulatoryrole after implantation in vivo in promoting Th1(immune rejection) to Th2(immune tolerance) and M1(pro-inflammatory and cytotoxic) to M2(anti-inflammatory and tissue repair) transition. One kind of ECM is the decellularizedporcine urinary bladder matrix (UBM), which is easily available and has low productioncost. UBM is one of the hot areas of biomaterials research and its main shortcomingsinclude fast degradation after implantation. As bone mesenchymal stem cells (BMSCs), onetype of pluripotent stem cells, are abundant and have multi-differentiation potential. It is acommonly used seed cell in tissue engineering, and can differentiate into functional cells in certain biological microenvironment involved in the repair of a variety of tissue injuries.Meanwhile, BMSCs exhibited significant anti-inflammatory and immunosuppressiveeffects in vivo and in vitro.In this research, we prepared porcine UBM with a combinationof physical and chemical methods. We succeeded in isolating and culturing the BMSCs ofrats using whole bone marrow attachment culture method, and inoculated the BMSCs tourinary bladder matrix-polypropylene scaffold (UBM-PM) based on the principle of tissueengineering, to build the bone mesenchymal stem cells-urinary bladdermatrix-polypropylene composite mesh (BMSCs-UBM-PM). Meanwhile, by focusing on thechanges in histopathology、inflammation and immune tolerance after the implantation ofBMSCs-UBM-PM underneath the abdominal skin, we discuss the feasibility of usingBMSCs-UBM-PM as the alternative material to repair and reconstruct the pelvic floor.Materials and Methods:1. The preparation of UBMWe decellularized the urinary bladder of the pigs by washing the stains, stirring andenzyme digestion to prepare the samples of UBM. The scanning electron microscope wasused to observe the morphology of freeze-dried UBM. After paraffin sectioning, HE andDAPI staining was performed to observe the effect of decellularization. Alkali hydrolysiswas used to assay the content of hydroxyproline in UBM samples, and the method ofmolecular biology was adopted to determine the concentration of DNA in UBM samples.2. The isolation and identification of BMSCs of rates, and research on the growth andproliferation of BMSCs on UBMWe used whole bone marrow attachment culture method to isolate and culture BMSCs,and adenovirus (with the fragments of GFP gene) to infect BMSCs of SD rats. We adoptedflow cytometry to examine expressions of CD29, CD44, CD31and CD45cell surfacefactors; and adipogenic, osteogenic and chondrogenic induction kits to detect thedifferentiation of BMSCs into adipocytes, osteoblasts and chondrocytes. CCK-8cellproliferation assay and scanning electron microscope were used to observe growth andproliferation of BMSCs on UBM.3. BMSCs-UBM-PM in ratsSD rats were randomly divided into4groups, each containing20rats. Based ondifferent pre-implanted materials, the4groups were designated as BMSCs-UBM-PM group (PM was inserted between two layers of UBM loaded with BMSCs), UBM-PMgroup (PM was inserted between two layers of UBM not loaded with BMSCs), PMgroup (only PM was used) and sham-operated group. The rats were sacrificed in thefirst, second, fourth and sixth week respectively after the operation. Samples weretaken to receive HE, Masson, immunohistochemical, and immunofluorescent staining,and mechanical test.Results1. We did not find obvious residuals of cells after the urinary bladder wasdecellularized, but observed the matrix in the homogeneous state and network-likestructural change in collagen fiber. The freeze-dried UBM was hard, had high-tensilestrength, and reduced extensibility. When viewed under a scanning electronmicroscope, the collagen fibers were neatly arranged, coarse-grained, and cord-like.Average hydroxyproline content in cellular UBM is2.45μg/mg and afterdecellularization, average hydroxyproline content in UBM is2.34μg/mg, thedifference was not statistically significan（tP＞0.05）. The average DNA concentrationin cellular UBM is443.71μg/ml and decreased to4.32μg/ml in UBM afterdecellularization, the difference was statistically significant（P＜0.05）.2. The whole bone marrow attachment culture method was used to isolate and culturethe BMSCs of SD rats, the part of BMSCs adhered to the wall at24h,the BMSCs adhered tothe wall began to devide and proliferate at48h,and grown in the shape of spindle orpolygonal.Three generations of BMSCs were observed,which almost remained unchangedmorphologically. CD29, CD44, CD31and CD45positive expression rates of BMSCs were98.74%,95.43%,2.03%and1.14%, respectively. After adipogenic, osteogenic andchondrogenic induction, BMSCs were positive for Oil Red O, alizarin red and alcian bluestaining. Adenovirus (with the fragments of GFP gene) can successfully infect BMSCs.TheCCK-8cell proliferation experiments showed a good growth condition of BMSCs on UBMand a strong proliferation capacity.Under the scanning electron microscope, we observedthat the BMSCs growed on UBM in the shape of fusiform or polygon.3. The BMSCs-UBM-PM group had a low degree of inflammatory cell infiltration, anda large number of collagen fibers and new blood vessels. Immunofluorescence showed thatBMSCs can survive in vivo for at least6weeks, and part of them can differentiate into smooth muscle cells. Immunohistochemical results suggest that CXCR3(Th1cellsurface marker) positive ratio in BMSCs-UBM-PM of each week was lower thanother groups, while CXCR3positive ratio in PM of each week was higher than othergroups,CXCR3positive ratio in UBM-PM of each week is between these two. CCR4(Th2cell surface marker) positive rates in both BMSCs-UBM-PM and UBM-PM atthe second week reach to the highest level, and CCR4positive rate in PM is always ata low level.Mechanical test showed that the maximum load (kN) of the BMSCs-UBM-PMgroup was0.31±0.08, the highest tension (mm/mm) was0.62±0.19, and Young’s moduluswas43.36±11.83, all of which were significantly higher than the results of other groups.Conclusions1. The UBM prepared by a combination of physical and chemical methods has agood histocompatibility, loss of collagen content was less,and is convenient to storeafter sterilization and freeze-drying. Therefore, it is an ideal biological scaffoldmaterial that can be used in tissue engineering, and pelvic floor repair andreplacement.2. The BMSCs of SD rats isolated and cultured by the whole bone marrow adherencemethod have a high cellular purity, a strong proliferation capacity and a good viability invitro; thus, can be used as seed cells in tissue engineering.3. Compared with conventional polypropylene mesh, BMSCs-UBM-polypropylene composite mesh which has a good biocompatibility induced a mildinflammation and transplantation tolerance after implantation in rats, and hadhigh-tensile strenth,so the composite mesh can be used as an ideal candidate materialin repairing and reconstructing of the pelvic floor tissues.