Experimental Study On Muscle-derived Stem Cell Transplantation Combined With Injectable Fibrin Glue In Rat Models Of Female Stress Urinary Incontinence

Objective To explore the effect and mechanism of Muscle-Derived Stem Cells transplantation combined with injectable fibrin glue in rat models of female stress urinary incontinence in order to provide a new cytotherapy method.Methods 1.The rat MDSCs were isolated and cultivated by modified preplating process. The expression of cell surface antigens was tested by flow cytometry , immunofluorescence and immunohistochemical method respectively. The P3-MDSCs were cultivated by differentiation medium. The myotubes were identified by the expression of MyHC via immunofluorescence method. 2.The P3-MDSCs were seeded on sterile coverslips in 6-well plates with a density of 1×105/ml to culture inside three-dimensional condition and on surface of fibrin glue respectively. The morphology of MDSCs was observated by scanning electron microscope at 1st and 7th day of cultivation. The P3-MDSCs were seeded in 96-well plates with a density of 5×103/well and then devided into three groups: A group (culture inside three-dimensional condition of fibrin glue), B group (culture on surface of fibrin glue), C group (culture on surface of 96-well plate). The cell growth in different culture conditions was observed by a inverted microscope. The MTS method was used to test OD value of each group to assess the cell proliferation . 3. The P1-MDSCs were infected by pGC-FU-GFP-LV, a lentiviral vector with green fluorescent protein (GFP), at optimal MOI and then passaged. The GFP-positive cells were quantified by FACS. Pudendal nerve-transected(PNT) rats were utilized as stress urinary incontinence (SUI) models. Seventy five animals were divided into five groups (n = 15): Sham (S), PNT+FG-injection(F),vehic control (D), PNT+MDSCs-injection (M), and PNT+MDSCs plus FG-injection (FM); And they were then split into 1,2 and 4 weeks subgroups. GFP-labeling MDSCs with a density of 1×106/rat injections were performed into the proximal urethra of the models. One,two and four weeks after injection , the urodynamics was used to assess the urethral rhabdosphincter function. The surviving cells were tracked and their differentiation into myotubes was evaluated by immunofluorescent staining after transplantation at 1 and 4 weeks. Expression of IGF-1, VEGF in the proximal urethra was assay by Real-time quantitative PCR and immunostaining methods after 1,2 and 4 weeks injection. At 4 weeks after MDSCs transplantation,the pathological changes were assessed by HE and Masson staining and angiogenesis was assessed by detecting the expression of Factor VIII-related Antigen in the proximal urethra of the models .Results 1.The rat MDSCs by primary cultivation were adhered after 72 hours.They reached 70% confluence after 14 to 16 days of primary cultivation and then could generated. The P1 and P3 proliferated quickly while the P7 proliferated slowely. The flow cytometry showed the results of the cell surface antigen CD34 being weak positive(21.15%) and CD45 being negative (0.85%). The expression of Sca-1 was strong positive and the expression rate of desmin was high. The myotubes were strongly expressed MyHC by immunofluorescence method. 2.After about 4-days incubation period, the cells in group A proliferated into a logarithmic growth phase and then into a plateau phase after 8-days growth. After about 3-days incubation period, the cells in group B and C proliferated into a logarithmic growth phase and then into a plateau phase after 7-days growth. The doubling time in group A was longer than that of group B and group C but there was no significant difference among them(P>0.05). The doubling time of group B and C was similar . There was no significant difference between the two groups(P>0.05).3.The P1-MDSCs almost expressed GFP after being infected 96 hours with pGC-FU-GFP-LV at MOI 20. More than 91.74% of successively passaged P3 MDSCs were GFP-positive. In vivo, the cellular survival of the combine-injection group(FM) increased as compared to the cell injection group(M) after 1 week of transplantation (P﹤0.05). It can be seen that the MDSCs integrated into the host muscle tissue in group FM and group M . The MDSCs were differentiated into myotubes partly when detected the expression of MyHC in both group. The expression of MyHC in group FM was more than that of group M.The Abdominal Leak Point Pressure( ALPP) and maximal bladder capacity significantly increased in group FM and M at different time point of postimplantation when compared with the control group D (P<0.01) but there were no difference to group S. The ALPP and maximal bladder capacity of the FM group were slightly higher than those of the M group but there were no difference between them. At 1 week and 2 weeks after transplantation, expression of IGF-1 and VEGF in the proximal urethra of the FM group was higher than that of the vehic group(P﹤0.01). The expression of these growth factors in the M group was also higher than that of the vehic group at 1 week(P﹤0.05).The expression of VEGF was higer in the FM group than that of the M group at 1 week postimpltation(P﹤0.05).After four weeks of injection, it was shown an increased microvessel density in the proximal urethra of the FM group as compared to other groups (P﹤0.01). The histology examination indicated that an increased muscle/collagen ratio was observed in group FM as compared to the other group(P﹤0.01)but not the sham group(P﹤0.01).Conclusions The modified preplating method is simple and easy to isolate and cultivate MDSCs and can get high purification population of MDSCs. Fibrin glue exhibited a good biocompatibility with rat muscle-derived stem cells. The present study indicates that fibrin glue may be used as a tissue-engineering scaffold to improve transplanted muscle-derived stem cell survival and differentiatation in the injected area , improve expression of VEGF and IGF-1, induce neovasculature formation and potentialy enhance the effect of transplanted cells on urethral rhabdosphincter function in SUI rat models.