Intrasurgical tissue engineering of autologous grafts using irreversible electroporation for bladder reconstruction

项目概要 膀胱重建（BR）对于恢复神经源性膀胱患者的泌尿功能至关重要， 先天性疾病，以及膀胱或盆腔恶性肿瘤手术治疗的后遗症。目前的 标准护理是在 BR 期间使用患者自身的肠道组织（回肠段；IS）作为移植材料 创建新膀胱或尿流改道。虽然 IS 移植物不具有免疫原性并且易于获得，但 移植物中肠细胞的手术持续性阻碍了膀胱壁的再生，导致结石 形成、代谢性酸中毒和继发性癌症的风险。我们的目标是开发新技术 通过使用不可逆细胞成分敲低来进行 IS 的术中组织工程 电穿孔（IRE）并确定功能性膀胱壁再生的基本因素。 IRE 用于 通过超短电脉冲诱导细胞死亡来消融患者的肿瘤。我们提出的战略构建 我们的初步数据显示 (i) IRE 可以击倒 IS 移植物中的肠细胞，帮助重新增殖 在 BR 大鼠模型中使用尿路上皮，（ii）用于局灶性敲除的新脉冲应用策略的可行性 粘膜或去细胞化，同时保留 IS 中的脉管系统和 ECM，以及 (iii) 表型变化 尿路上皮化后 IRE 治疗的 IS，在生理条件下的假手术对照中未观察到 膀胱充盈和排尿。在具体目标 1 中，我们将定义移植物灌注对膀胱壁的影响 通过执行 IS 的脉管系统保护 IRE 进行再生。在具体目标 2 中，阐明 IS 的作用 通过使用 IRE 敲低粘膜来治疗 BR 后并发症。在具体目标 3 中，调查的作用 IRE 治疗的 IS 中膀胱功能发育的机械转导。术中创建灌注， 组织相容性移植物（目标 1）和粘膜局部脱细胞，同时保留下层 IS（目标 2）是使用 IRE 进行体内击倒组织工程的第一个例子。该研究和 应用机械传导原理增强 IS 移植物泌尿屏障功能的发育 （目标 3）之前没有描述过。从拟议的研究中获得的知识将产生一个简单的外科手术 技术（mt-IRE或vs-IRE），结合了已经成熟的技术（IRE）和嫁接技术（与IS） 在临床中，能够快速转化，使接受 BR 的患者立即受益。最终，我们 预计我们的工作将推进以患者为主体的功能化移植物体内生产的概念 生物材料来源和生物反应器，应用于涉及其他管状结构的重建手术 食道、气管或大血管等器官。

PROJECT SUMMARY Bladder reconstruction (BR) is essential to restore urinary function in patients with neurogenic bladder, congenital disorders, and as sequalae to the surgical treatment of bladder or pelvic malignancies. The current standard of care is to use the patient's own intestinal tissue (ileal segment; IS) as graft material during BR to create a neobladder or urinary diversion. While IS grafts are non-immunogenic and readily available, post- surgical persistence of intestinal cells in the graft impedes regeneration into bladder wall, resulting in stone formation, metabolic acidosis and risk of secondary cancers. Our objective is to develop new technology for intrasurgical tissue engineering of the IS by knockdown of cellular components using irreversible electroporation (IRE) and identify factors fundamental for regeneration of functional bladder wall. IRE is used in patients for tumor ablation by inducing cell death with ultrashort electric pulses. Our proposed strategy builds upon our preliminary data showing (i) IRE can knockdown intestinal cells in an IS graft, aiding repopulation with urothelium in a rat model of BR, (ii) feasibility of new pulse application strategies for the focal knockdown of mucosa, or decellularization while preserving vasculature and ECM in the IS, and (iii) phenotypic changes in IRE treated IS following urothelialization, that were not observed in sham controls under physiologic conditions of bladder filling and voiding. In specific aim 1, we will Define the impact of graft perfusion on bladder wall regeneration by performing vasculature sparing IRE of the IS. In specific aim 2, Elucidate the role of IS mucosa in post-BR complications by knockdown with IRE. In specific aim 3, Investigate the role of mechanotransduction in bladder function development in IRE treated IS. Intrasurgical creation of a perfused, histocompatible graft (Aim 1) and focal decellularization of the mucosa while sparing the underlying layers in the IS (Aim 2) are the first examples of in vivo knockdown tissue engineering using IRE. The study and application of mechanotransduction principles to augment urinary barrier function development in IS grafts (Aim 3) is previously undescribed. Knowledge gained from proposed research will yield a simple intrasurgical technique (mt-IRE or vs-IRE) that combines technology (IRE) and grafting technique (with IS) that are already in the clinic, enabling rapid translation for the immediate benefit of patients undergoing BR. Eventually, we anticipate our work to advance the concept of in vivo production of functionalized grafts using the patient as the source of biomaterial and the bioreactor, with application to reconstructive surgery involving other tubular organs such as the esophagus, trachea or large blood vessels.