| Urethral defect is one of the most common diseases in urology, which is caused by congenital disease such hypospadias and epispadias, or caused by urethral injuries and infection, for most of time, urethral fibrosis and stricture occur after the injuries, which compromise the urinary and reproductive function of the urethra. Short urethral stricture can be cured by urethral dilation, or urethrotomy under direct invision or the urethrectomy of the fibrosis segment and anastomosis of the resident urethra. However, long segment urethral stricture need graft substitute for urethroplasty. Currently, the mostly used substitute grafts are lingual mucosa, oral mucosa, and penile skin. However, some complications accompany the donor site, such as limitation of mouth opening, oral infection, ulceration, numb and pain. In addition, stricture may recur sometimes after urethroplasty with those grafts, for those patients, the second urethoplasty may lack of the adequate donor graft resources. So seeking substantial graft resources is imperative.On the basis of the achievements in last 2 decades, tissue engineered graft may provide a new alternative for urethral reconstruction. However, Chapple et al argued that further research must be conducted to find more suitable material for tissue engineered urethral replacement before transition from bench to bedside. The mostly used materials include chemical synthesis material, natural accellular matrix, natural biological matrix extraction and the mixed compound of the matrix. Small intestinal submucosa (SIS) is attributed to natural accellular matrix, which has natural histocompatibility with cells, including collagen, GAG and growth factor, which can promote cell growth. However, the density structure of the SIS makes it difficult for penetration of the nutrients and the exchange of the metabolic materials, so those two disadvantages limited its use. In addition, the resident heterogenous nuclear component also interfere its further usage. The ideal tissue engineered material should have 3 dimensional porous structures and the least retained nuclear component. We fabricated the PAA modified SIS for tissue engineered urethral reconstruction in our study. Our study is divided into 3 sections as below:Section 1 Fabrication of PAA modified small intestinal submucosaObjective:to fabricate PAA modified small intestinal submucosa (SIS), and to evaluate its morphological mechanical characteristicsMethods:take fresh pig small intestinal, removal of mucosa and seromuscular layer, leave it in distilled water, then subjected to 5%PAA in modified group, while it was subjected to PBS in unmodified group, then left in 1% Triton X-100. Lastly, disinfection with 75% ethanol, stored in distilled water for use. Sampling for HE stain, MASSON stain, and scanning electron microscopy. Porosity was calculated by density bottle. Sampling of the material was taken for mechanical analysis, including strain, stress and Young Module. The data were analyzed for comparison between two groups.Results:Non-PAA treated SIS has dense structure and some nuclear component retained. The pore size is 1.75±0.32μm, the porosity was 18.5±2.6%. the pore size in PAA treated SIS is 5.16±1.83μm, the porosity was 66.8±3.9%(P<0.01).the maximum strain in non PAA and PAA SIS was 16.3±1.6% and 15.0±1.2%, respectively, the maximum stress was 35.1±7.2 Mpa和24.3±5.6 Mpa, respectively, Young module was 150.4±39.2Mpa and 118.5±28.4Mpa, respectively.Conclusion:Compared to non-PAA SIS, PAA modified SIS has 3D porous structure, almost scarcity of nuclear component retained, but still maintained mechanical characteristics.Section 2:Fabrication of tissue engineered bladder urothelial cells-PAA modified SIS compoundObjective:To fabricate tissue engineered bladder urothelial cells-PAA modified SIS compound, and to evaluate its morphology and histocompatibility.Method:A sampling of bladder tissue (0.6×0.6 cm) from adult New Zealand white rabbits, washed in PBS。Then it was digested in pronase E including DMEM overnight, then urothelial cells were scraped from the mucosal side slightly. The urothelial cells were cultured in ECM including 5% FBS and 1% streptomycin-penicilin. Cells were subcultured using 0.05% trypsin. The passage 3 cells were identified using AE1/AE3. The histocompatibility of the SIS and PAA modified SIS were examined using MTT assay to calculate the OD value of the cell in both groups. Cells were transplant onto the SIS and PAA modified SIS scaffold and cultured for 2 weeks, sampling for HE stain and scanning electron microscopy (SEM). The expression of Uroplakin was examined by RT-PCR and Western-blot.Results:Urothelial cells grow well in both SIS and PAA modified SIS extracts, and have better trend than simple ECM though not significant(p=0.35). Single layer of urothelial cells formed on SIS scaffold, however,2-3 layers of structure developed on the PAA modified SIS scaffold. Under SEM, cells were polygonal, flat and stretched with pseudopodia and attached tightly to the SIS scaffold. Cells on 5% PAA modified PAA SIS scaffold were more densely populated, and confused into 3 dimensional structure, the cells were round, ellipse and cube-like. The expression of Uroplakin was higher in PAA SIS-UC than in non PAA SIS-UC.Conclusions:Live bladder urothelial cells were isolated and expanded by combination of enzyme digestion and mechanical scrape. The cells expanded positive expressed AE1/AE3. Both SIS and PAA modified SIS have good cell compatibility. In addition, PAA modified SIS promoted cell growth and form multi-layered structure. The PAA modified SIS promoted the expression of Uroplakin in UC.Section 3 Tissue engineered urothelial cells-PAA modified SIS compound for onlay urethroplasty in a rabbit modelObjective:To investigate the efficacy of cell seeded PAA modified SIS compound in the onlay urethroplasy in a rabbit modelMethods:18 healthy adult New Zealand white rabbits were divided into 3 groups, cell-seeded PAA modified SIS (n=6), cell-seeded non PAA modified SIS (n=6) and PAA modified SIS (n=6). When cell seeded SIS compound were prepared, after the anesthesia of the rabbits, the urethra was separated and the mucosa exposed, a mucosa of 1.5cm×0.8cm2 defect was created, repaired by a scaffold of 1.7×1 cm2. Antibiotics were used to prevent infection, and the urethral tube was washed. Urethrography was performed 6 months after the operation. In addition, gross and micro pathology including HE/MASSON and immunohistochemistry analysis were conducted also.Results:All rabbits in cell-seeded 5% PAA modified SIS survived with wide caliber, and the regenerated urethra seems normal-like. Multi-layered transitional cells, smooth muscle cells and vessel cells were seen using H&E and MASSON stain. By comparison, fistulae and ulceration were seen in cell-seeded non PAA group. Unorganized and thin epithelium and chronic imflammatory cells were also seen in the group. Smooth muscle was also less than cell-seed PAA SIS arm. Urethral stricture was accompanied by rabbits in simple PAA modified SIS group. The regenerated mucosa seemed pale, stiff and shrinkage-like. The pathological exam indicated extensive fibrosis formation and scarce of vessel and smooth muscle. The expression of epithelium and smooth muscle in cell-seeded PAA modified SIS was higher than cell-seeded non PAA SIS (P<0.05), the vessel and smooth muscle expression were higher than PAA SIS arm (P<0.05).Conclusions:Bladder urothelial cells-seeded SIS compound provided an ideal alternative to urethroplasty. |
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