脂肪干细胞与膨体聚四氟乙烯管体外构建组织工程化尿液流出道行犬尿流改道

Construction of tissue-engineered urine outflow tract using adipose stem cells and expanded polytetrafluoroethylene for canine urinary diversion

背景:利用组织工程技术在体外构建形成具有良好抗尿液渗漏性能的组织工程管,能够显著降低各类围术期并发症的发生率。目的:应用脂肪干细胞与膨体聚四氟乙烯管在体外构建组织工程化尿液流出道,尝试用于犬尿流改道手术,评估其可行性。方法:实验组取4只雌性比格犬,分离培养脂肪干细胞,在体外向尿路上皮细胞定向诱导分化14 d,将诱导分化后的细胞种植于膨体聚四氟乙烯工程管内表面,构成组织工程尿流改道装置;切除犬膀胱组织(仅保留三角区1.0 cm×1.0 cm组织),将组织工程尿流改道装置两端分别与三角区、腹壁造口吻合,完成尿流改道手术,术后1,2,4,8周取材。对照组2只犬采用膨体聚四氟乙烯工程管完成尿流改道手术,术后8周取材。动物实验获武汉大学人民医院实验动物伦理委员会批准,许可证号为WDRM动(福)第20180810号。结果与结论:(1)两组植入的组织工程尿流改道装置均与腹壁或肠壁有不同程度的粘连,实验组2只犬存在轻度肾积水,无尿外渗、感染等严重并发症;对照组1只犬形成严重的尿瘘,术后7周死亡,另1只组织工程流出道被结石包裹,长满结石;(2)苏木精-伊红染色显示,实验组流出道内腔在术后1周后开始出现连续生长的薄层上皮细胞,细胞间连接较松散;术后2周细胞间连接开始变得致密,上皮细胞数量增多;术后4周开始细胞出现堆积、增厚,逐步变为多层结构;术后8周上皮层已经覆盖整个管腔,并可见新生血管生成;(3)结果表明,脂肪干细胞向尿路上皮细胞定向诱导分化后与膨体聚四氟乙烯共同构建组织工程尿液流出道,在比格犬体内进行尿流改道是可行的,流出道内腔可形成具有紧密连接的尿路上皮层,具备引流尿液、防止尿外渗的功能。

BACKGROUND:Tissue engineering technology was used to construct and form tissue engineering,tubes which can significantly reduce the incidence of various perioperative complications with good anti-urine leakage performance in vitro.OBJECTIVE:To evaluate the feasibility of using adipose stem cells and expanded polytetrafluoroethylene to construct tissue-engineered urine outflow tract in vitro for canine urinary diversion surgery.METHODS:In the experimental group,adipose stem cells were isolated and cultured from four female beagles,and then differentiated into urothelial cells in vitro for 14 days.The differentiated cells were implanted on the inner surface of expanded polytetrafluoroethylene tubes to form a tissue-engineered urinary diversion device.Bladders were removed from dogs(only 1.0 cm×1.0 cm of trigonometric area was retained),and both ends of the tissue engineered urinary diversion device were anastomosed with trigonometric area and abdominal wall respectively to complete the urinary diversion operation.The samples were collected at 1,2,4 and 8 weeks postoperatively.The control group(two dogs)underwent urinary diversion with expanded polytetrafluoroethylene engineered tube,and samples were collected 8 weeks after operation.The animal experiment was approved by laboratory animal ethics committee of Renmin Hospital of Wuhan University(approval No.WDRM(Fu)20180810).RESULTS AND CONCLUSION:(1)There were different degrees of adhesion between the two groups of implanted tissue-engineered urinary diversion devices and the abdominal wall or intestinal wall.Two dogs of the experimental group had mild hydronephrosis,and no serious complications such as urinary exosmosis or infection.One dog in the control group developed severe urinary fistula and died 7 weeks after operation.The tissue-engineered urinary diversion device of the other dog was covered with stones.(2)Hematoxylin-eosin staining showed that thin epithelial cells with loose intercellular connections began to grow continuously in the outflow tract lumen of the experimental group 1 week after operation.At 2 weeks postoperatively,the intercellular connections became dense and the number of epithelial cells increased.Four weeks after operation,the cells began to accumulate and thicken,and gradually changed into a multilayer structure.The entire lumen was covered by the epidermis,and neovascularization was observed 8 weeks after surgery.(3)The results showed that it was feasible to construct tissue-engineered urine outflow tract together with expanded polytetrafluoroethylene after adipose stem cells were differentiated into urothelial cells for urinary diversion in beagles.The inner cavity of outflow tract could form a tightly connected urothelial cortex with the function of draining urine and preventing urinary extravasation.