BIOENGINEERING OF BLADDER TISSUES

从胎儿发育开始，膀胱就暴露在各种可能的损伤中。除了先天性异常之外，个体还可能患有其他病症，例如癌症、创伤、感染、炎症、医源性损伤或可能导致疼痛的膀胱损伤或丧失的其他病症，最终需要膀胱置换或修复。目前，胃肠道段通常用作膀胱置换或修复的组织。然而，当胃肠道组织与泌尿道接触时，可能会出现多种并发症，如感染、代谢紊乱、尿石穿孔、粘液产生增加和恶性肿瘤。尽管在100多年前首次提出使用肠进行膀胱组织置换，但尽管其已知的相关问题并且由于缺乏更好的替代方案，但它仍然是当今的黄金标准。很明显，尿路上皮对尿路上皮的功能是优选的。然而，用于重建的自体泌尿相关组织的数量有限，通常排除了这种选择。迫切需要组织来替代丧失的和功能缺陷的膀胱组织。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性新的泌尿生殖系统组织的方法。我们在实验室中遵循的生物工程组织的方法涉及最终使用自体细胞，从而避免排斥，其中从宿主获得组织活检，细胞在体外解离和扩增，重新附着到可生物降解的基质上，并植入同一宿主中。拟议的应用程序是基于几个证明的概念，来自我们以前的工作，在过去的9年中进行，这导致了大部分生物工程膀胱组织的发展，以增强和次全置换。然而，在需要完全膀胱置换的患者可以从这项技术中受益之前，需要进行进一步的研究，其中进行全膀胱切除术。这不需要像实验所证明的那样部分替换膀胱组织，而是完全替换膀胱并输尿管再植。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置换，从而避免与当前使用的胃肠道段相关的问题。与用胃肠道段创建的其他类型的原位膀胱一样，预期用生物工程组织创建的新膀胱可以主要通过间歇性导管插入术排空，但是这些应该能够用天然自体组织提供低压和高顺应性储液器。本研究的具体目的：具体目的1：确定组织工程膀胱长期输尿管再植的可行性。在这些实验中，我们将测试输尿管可以超植入工程膀胱组织，输尿管不会阻塞，输尿管不会回流的假设。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，我们将测试以下假设：生物工程化膀胱组织可以与天然膀胱结合，使用生物工程化膀胱组织进行完全膀胱置换是可行的，在完全膀胱置换模型中进行输尿管置换是可行的，没有输尿管阻塞或膀胱输尿管反流，并且生物工程化膀胱可以以间歇方式有效排空。具体目标3：目的：探讨膀胱组织工程化移植的近期和远期表型及功能特征。在这个目标中，我们将试图确定随着时间的推移，与对照相比，生物工程膀胱组织的表型和功能特征。

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.