双层蚕丝支架复合脂肪干细胞构建的组织工程膀胱补片用于膀胱修复重建的效果

The study on the tissue engineered bladder patch constructed with the double-layer silk scaffold and adipose-derived stem cells for bladder repair and reconstruction

目的探讨双层蚕丝支架复合脂肪干细胞(ADSCs)构建的组织工程膀胱补片在膀胱修复重建中的效果。方法2020年5月至2021年3月利用家蚕蚕茧获得丝素蛋白(SF)水溶液,制备由丝素蛋白膜和丝素蛋白海绵组成的双层蚕丝支架。分离培养大鼠ADSCs,并对ADSCs表面标志物(CD29、CD90、CD45、CD106)进行流式细胞鉴定。将ADSCs种植在双层蚕丝支架上构建组织工程膀胱补片。将36只雄性SD大鼠按随机数字表法分为组织工程膀胱补片组(SF-ADSCs组,15只)、双层蚕丝支架组(SF组,15只)、对照组(6只)。SF-ADSCs组和SF组分别将组织工程膀胱补片和双层蚕丝支架包埋于大鼠的大网膜,以促进移植物血管化。包埋7 d后,两组分别处死3只大鼠,行HE和免疫荧光染色检查评估血管化情况。SF-ADSCs组(12只)和SF组(12只)于膀胱顶部切除超过50%的膀胱组织,保留三角区和输尿管开口,分别采用大网膜包埋后的组织工程膀胱补片和双层蚕丝支架修补膀胱缺损;对照组在充分暴露膀胱组织后随即关闭切口。术后4周,SF-ADSCs组和SF组分别随机取6只大鼠,观察膀胱组织的大体形态,行膀胱造影检查观察膀胱壁形态,移植物取材后行HE和Masson's 3色染色、免疫荧光染色检查观察膀胱壁组织再生情况。术后12周,3组均行尿动力学检查,均行上述检查比较膀胱组织形态学、组织再生情况的差异。结果流式细胞实验结果显示,分离的细胞阳性表达CD29和CD90,未见CD45和CD106显著表达。大体观察和扫描电镜结果证实制备的双层蚕丝支架不仅具备利于细胞种植的孔隙结构,还具备良好的韧性利于手术缝合。大网膜包裹后,SF-ADSCs组血管样结构的数量[(43.50±2.66)个]和面积占比[(0.73±0.03)%]明显高于SF组[(24.50±3.51)个,(0.55±0.05)%],差异均有统计学意义(P<0.05)。膀胱修补术后4周,SF-ADSCs组和SF组移植物组织学染色可见大量双层蚕丝支架降解碎片;术后12周形态学检测结果显示,SF-ADSCs组移植物膀胱形态均一,与正常膀胱组织形态相近,免疫荧光染色结果表明SF-ADSCs组中可见连续的尿路上皮层、大量的平滑肌组织、血管结构和再生的神经元。尿动力学检查结果显示,SF-ADSCs组膀胱最大容积[(0.74±0.03)ml]和顺应性[(16.68±0.44)μl/cmH2O]明显优于SF组[(0.47±0.05)ml、(14.89±0.37)μl/cmH2O],但低于对照组[(1.12±0.08 ml)、(19.34±0.45)μl/cm H2O],差异均有统计学意义(P<0.05)。结论双层蚕丝支架和ADSCs构建的组织工程膀胱补片,可促进膀胱组织形态学修复、膀胱壁结构再生和膀胱生理功能的恢复。

Objective To investigate the effect of tissue engineered bladder patch constructed by double-layer silk scaffold and adipose-derived stem cells(ADSCs)in the repair and reconstruction of bladder.Methods This study was conducted from May 2020 to March 2021.The silk fibroin(SF)aqueous solution was obtained from silkworm cocoons,and a double-layer silk scaffold composed of silk fibroin film and silk fibroin sponge was further prepared.The rat ADSCs were isolated,cultured,and the ADSCs surface markers(CD29,CD90,CD45,CD106)were identified by flow cytometry.The ADSCs were planted on a double-layer silk scaffold to construct a tissue-engineered bladder patch.Thirty-six male SD rats were randomly divided into three groups:tissue engineered bladder patch group(SF-ADSCs group,n=15),double-layer silk scaffold group(SF group,n=15),control group(n=6).The tissue engineered bladder patch(SF-ADSCs group)and double-layer silk scaffold(SF group)were wrapped on the omentum to promote vascularization.The vascularization was evaluated by HE and immunofluorescence staining.The wrapped tissue engineered bladder patch and double-layer silk scaffold were used to repair the defective bladder.In the control group(six rats),the incision was closed immediately after the bladder tissue fully exposed.At 4 weeks and 12 weeks after operation,the general morphology of bladder tissue and cystography were performed to evaluate the recovery of bladder morphology.After the graft was harvested,HE and Masson's trichrome staining and immunofluorescence staining were used to observe the regeneration of bladder wall tissue.Urodynamics was used to assess the recovery of bladder function at 12 weeks after operation.Results The flow cytometry results confirmed that the isolated cells positively expressed CD29 and CD90,and there was no significant expression of CD45 and CD106.Gross observation and scanning electron microscope confirmed that the preparation of double-layer silk scaffold not only had a pore structure that was conducive to cell planting,but also had good toughness and was facilitated to surgical suture.The number(43.50±2.66)and area(0.73±0.03)%of vascular-like structures in the SF-ADSCs group after the omentum encapsulation was significantly higher than that in the SF group[(24.50±3.51),(0.55±0.05)%],and the difference was statistically significant(P<0.05).At 4 weeks after bladder repair,the histological staining of the grafts in the SF-ADSCs and SF groups showed a large number of degraded fragments of double-layer silk scaffold.At 12 weeks,the morphology of the graft in the SF-ADSCs group showed uniform bladder morphology,which was similar to that of normal bladder tissue.Immunofluorescence staining showed that the continuous urothelial layer,abundant smooth muscle tissue,vascular structure and regenerated neurons could be observed in the SF-ADSCs group.Urodynamic test showed that the bladder maximum volume(0.74±0.03)ml and compliance(16.68±0.44)μl/cm H2O in the SF-ADSCs group,which were better than that in the SF group[(0.47±0.05)ml,(14.89±0.37)μl/cm H2O],but lower than that in the control group[(1.12±0.08)ml,(19.34±0.45)μl/cm H2O],and the difference was statistically significant(P<0.05).Conclusions The tissue engineered bladder patch constructed with double-layer silk scaffolds and ADSCs could promote the morphological repair of bladder tissue,the regeneration of bladder wall structure and the recovery of bladder physiological function.