BIOENGINEERING OF BLADDER TISSUES

从胎儿发育开始，膀胱就暴露在各种可能的伤害中。除先天性异常外，患者还可能患有其他疾病，如癌症、创伤、感染、炎症、医源性损伤或其他可能导致膀胱损伤或丧失的疾病，最终需要更换或修复膀胱。目前，胃肠段是膀胱置换或修复的常用组织。然而，当胃肠道组织与尿路接触时，可能会出现多种并发症，如感染、代谢紊乱、尿石穿孔、粘液生成增加和恶性肿瘤。尽管在100多年前首次提出用肠来替代膀胱组织，但它仍然是今天的黄金标准，尽管它存在已知的相关问题，并且由于缺乏更好的替代品。很明显，尿路上皮与尿路上皮的吻合术在功能上更可取。然而，用于重建的自体泌尿相关组织数量有限，通常排除了这种选择。目前迫切需要组织来替代膀胱丢失和功能缺陷的组织。使用选择性细胞移植的生物工程膀胱组织可能提供一种创造功能新颖的泌尿生殖系统组织的方法。在我们的实验室中，我们所遵循的生物工程组织方法包括最终使用自体细胞，从而避免排斥，其中从宿主获得组织活检，细胞在体外分离和扩增，重新附着在可生物降解的基质上，并植入同一宿主。这一应用是基于我们过去9年的工作中得到的几个经过验证的概念，这些概念导致了大部分生物工程膀胱组织的发展，用于增强和次全替换。然而，在需要全膀胱置换的患者从这项技术中获益之前，需要进行进一步的研究，其中需要进行全膀胱切除术。这并不需要像实验证明的那样，部分更换膀胱组织，而是完全更换膀胱并重新植入输尿管。因此，本提案的总体目标是在实验模型中使用生物工程组织实现完全膀胱替代，从而避免目前使用的胃肠道部分相关的问题。与其他类型的由胃肠道段制成的原位膀胱一样，预计由生物工程组织制成的新膀胱大部分可以通过间歇导管排空，但这些应该能够提供低压力和高顺应性的天然自体组织储存库。本提案的具体目的：具体目的1：确定输尿管在组织工程膀胱中长期再植的可行性。在这些实验中，我们将测试输尿管可以植入工程膀胱组织的假设，输尿管不会阻塞，输尿管不会反流。具体目标2：确定使用组织工程技术实现完全膀胱置换的可行性。在这些实验中，我们将验证以下假设：生物工程膀胱组织可以与天然尿道吻合；使用生物工程膀胱组织进行完全膀胱置换是可行的；输尿管置换在全膀胱置换模型中是可行的，没有输尿管梗阻或膀胱输尿管反流；生物工程膀胱可以以间歇性的方式有效排空。具体目标3：确定用于次全置换和完全置换的膀胱组织的短期和长期表型和功能特征。在这个目标中，我们将尝试确定生物工程膀胱组织的表型和功能特征，随着时间的推移，与对照组相比。

The bladder is exposed to a variety of possible injuries from the time the fetus develops. Aside from congenital abnormalities, individuals may also suffer from other disorders such as cancer, trauma, infection, inflammation, iatrogenic injuries r other conditions which may lead to painful bladder damage or loss, requiring eventual bladder replacement or repair. Currently, gastrointestinal segments are commonly used as tissues for bladder replacement or repair. However, when gastrointestinal tissues are in contact with the urinary tract, multiple complications may ensure, such as infection, metabolic disturbances, urolithiasis perforation, increased mucous production, and malignancy. Even though the use of bowel for bladder tissue replacement was first proposed over 100 years ago, it still remains the gold standard today, despite its known associated problems and due to a lack of a better alternative. It is evident that urothelial to urothelial anastomoses are preferable functionally. However, the limited amount of autologous urologic associated tissues fro reconstruction generally precludes this option. There is a critical need for tissues to replace lost and functionally- deficient bladder tissues. Bioengineering bladder tissues using selective cell transplantation may provide a means to create functional new genitourinary tissues. The approach which we have followed to bioengineer tissues in our laboratory involves the eventual use of autologous cells, thus avoid rejection, wherein a biopsy of tissue is obtained from the host, the cells are dissociated and expanded in vitro, reattached to a biodegradable matrix, and implanted into the same host. The proposed application is based on several proven concepts derived from our previous work, performed over the last 9 years, which have lead to the development of large portions of bioengineered bladder tissue for augmentation and subtotal replacement. However, before patients who require complete bladder replacement can benefit from this technology, further studies need to be performed wherein total bladder cystectomies are performed. This would not entail the partial replacement of bladder tissue, as had been demonstrated experimentally, but rather, complete bladder replacement with ureteral reimplantation. Therefore, the overall goal of this proposal is to achieve complete bladder replacement using bioengineered tissues in an experimental model, thus avoiding the problems associated with the currently used gastrointestinal segments. Like other types of orthotopic bladders created with gastrointestinal segments, it is expected that the neobladders created with bioengineered tissues could empty mostly by intermittent catheterization, but these should be able to provide low pressure and high compliance reservoirs with native autologous tissues. The specific aims of this proposal: Specific Aim 1: To determine the feasibility of reimplanting ureters long term in tissue engineered bladders. In these experiments we will test the hypotheses that ureters can be hyper-implanted into engineered bladder tissue, that the ureters will not obstruct, and the ureters will not reflux. Specific Aim 2: To determine the feasibility of attaining complete bladder replacement using tissue engineering techniques. In these experiments we will test to the hypothesis that bioengineered bladder tissues can be anastomosed to native urethras, that complete bladder replacement is feasible using bioengineered bladder tissues that ureteral replacement in a model of total bladder replacement is feasible, without ureteral obstruction or vesicoureteral reflux, and that bioengineered bladders can be emptied efficiently in an intermittent fashion. Specific Aim 3: To determine the short term and long term phenotypic and functional characteristics of bladder tissues engineered for subtotal and complete replacement. In this aim we will attempt to determine the phenotypic and functional characteristics, over time, of the bioengineered bladder tissues, as compared to controls.