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

项目总结 膀胱重建术(BR)是恢复神经源性膀胱患者排尿功能的关键， 先天性疾病，以及手术治疗膀胱癌或盆腔恶性肿瘤的后遗症。海流 标准护理是在BR期间使用患者自己的肠道组织(回肠段；IS)作为移植物材料 创造一个新的膀胱或尿流改道。虽然IS移植物是非免疫原性的，很容易获得，但术后 移植物中肠细胞在外科手术中的滞留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 通过使用不可逆性击倒细胞成分进行IS的外科内组织工程 电穿孔(IRE)和确定功能性膀胱壁再生的基本因素。IRE用于 超短电脉冲诱导细胞死亡进行肿瘤消融的患者。我们提出的战略建立在 根据我们的初步数据显示：(I)IRE可以击倒移植物中的肠道细胞，帮助重新繁殖。 在BR大鼠模型中使用尿路上皮，(II)新的脉冲应用策略对局灶性击倒的可行性 在保留IS中的血管系统和细胞外基质的同时，粘膜的脱细胞或去细胞，以及(Iii) IRE治疗是在尿路结石之后进行的，而假手术对照组在生理条件下没有观察到这种情况 膀胱充盈和排尿。在特定的目标1中，我们将确定移植物灌流对膀胱壁的影响。 通过对IS进行血管重建来进行再生。在具体目标2中，阐明IS的作用 用IRE击倒BR术后黏膜并发症。在具体目标3中，调查 机械转导在IRE治疗后膀胱功能发育中的作用。在外科手术中创造了一个灌流的， 组织相容移植物(目标1)和局灶性脱细胞粘膜，同时保留下层 IS(AIM 2)是使用IRE的体内击倒组织工程的第一个例子。该研究和 应用机械转导原理增强IS移植物的尿屏障功能 (Aim 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手术内 将已有技术(IRE)和嫁接技术(具有IS)相结合的技术(MT-IRE或VS-IRE) 在临床上，实现快速翻译，直接造福于接受BR手术的患者。最终，我们 期待我们的工作，以病人为研究对象，推进体内生产功能化移植物的概念 生物材料的来源和生物反应器，以及在涉及其他管子的重建手术中的应用 器官，如食道、气管或大血管。

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.