Experimental Study Of The Vascularization In Tissue Engeering Bladder

Since the endogenous protein secreated by transinfected cells showed high biological activity with little immunologic rejection and side effects, which were effectively binding to surface receptor, we tried to explore better methods for the vascularization in tissue engeering bladder with transinfected cells. The VEGF165 cDNA was cloned into the eukaryotic expression vector pcDNA3.1(-). The reconstructed vector pcDNA3.1(-)/VEGF165 was transinfected into rat bladder muscular cells by electroporation. The VEGF expression in rat bladder muscular cells was determined by RT-PCR. The biological activity of the VEGF in the supernant of the transinfected cell cultures were tested by MTT method. The expression of VEGF in the transinfected bladder muscular cells of rat was improved. The proliferation of the endothelial cell was stimulated by the supernant of the transinfected cell cultures.To understand the role of vascularization induced by transinfected bladder muscular cells in vivo, the rat's bladders were augumentated with scaffold of small intestinal submucosa (SIS) implanted with transinfected bladder muscular cell of rat for 7 days. At the same time, another two groups of rats augumentated with SIS without transinfected cell or partial bladder mucosa and smooth muscle destroyed were used as controls. Samples were taken at regular intervals, and were detected with histological and immunohistochemistry staining against CD34 and VEGFR2 expression. In the control group without SIS transplantation, the urinary bladder transitional epithelium was observed at 1 week after operation. Neogenesis mucosa covered the destroyed area at 2 weeks. The obvious inflammatory cell infiltration and some urinary bladder transitional epithelium were observed at 1 week in the both two SIS transplanted groups (only SIS and SIS with transinfected cells). At 2 weeks after transplantation, intact epithelium was observed with no significant difference between two groups. But the numbers of neogenesis capillary and positive VEGFR2 expression cells were significantly more in the group of SIS with transinfected cells than those in only SIS group(P<0.05). At 4 weeks, the number of neogenesis capillary was significantly more than those at 2 weeks (P<0.05), but there was no significant difference between the only SIS group and SIS with transinfected cells group. Few smooth muscle fibres were observed in both groups at 4 weeks, while at 6 weeks more could be found. At 10 weeks, smooth muscle bundles were observed and there were no significant difference between the two groups of the number of neogenesis capillary and positive VEGFR2 expression cells. To understand the release of exogenous growth factors from small intestinal submuscosa (SIS) in bladder regeneration, the release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from SIS in vitro were evaluated by ELISA and MTT method. The defected bladder walls of rats in experimental group were repaired by porcine small intestinal submuscosa. Partial bladder mucosa and smooth muscle of the rats in control groups were destroyed. At regular intervals, the VEGF and bFGF expression were observed by histological and immunohistochemical methods. The concentration of VEGF and bFGF released in vitro from SIS in PBS solution were 121.8±2.683ng/L and 93.8±3.033ng/L respectively, and showed proliferation of vascular endothelial cell. In the SIS framework, the capillary and smooth muscle were observed followed histological evaluation. The weak expression of VEGF and bFGF in both experimental and control groups were found in the first week. Since the second week the VEGF and bFGF expression in experimental group began to increase with a peak in the 6th week, and began to decrease after 8 weeks. In the control group, the weak VEGF and bFGF expression were shown during the observation. SIS functions as a carrier for exogenous growth factors release in rat bladder regeneration. So it still needs further research on how to use the characteristics of SIS better to keep sustained growth factors level in bladder reconstruction.