Functional Tissue Engineering for Stress Incontinence

描述(申请人提供)：利用我们实验室开发的技术，我们建议对基于肌肉干细胞的组织工程治疗压力性尿失禁(SUI)进行系统的体外和体内生理和生物力学分析。Sui是一个重大的医学问题，影响着大约2500万美国女性1。尽管SUI的发病率很高，但利用组织工程技术进行的以治疗为导向的研究很少。利用这些技术，我们建议开发一种真正的生理学吊衣，而不是来自合成或身体组织，而是一种工程化的、功能性的干细胞肌肉支架，可以植入修复受损的尿道括约肌。假设：1)肌源性干细胞(MDSC)在体外提高了小肠粘膜下层(SIS)支架的力学性能。2)MDSC/SIS悬带可改善SUI大鼠模型的可控性。本研究的主要目的包括：1)建立MDSC-SIS双轴力学性能改变的机制：a)建立将MDSC种植到SIS的可行性；b)建立MDSC在SIS上的长期持久性；以及c)通过不同时间点的综合力学测试，评估单独的SIS和MDSC/SIS的顺应性。2)对MDSC/SIS的收缩特性进行评价：a)将不同浓度的MDSC接种于SIS上，培养7天，检测MDSC/SIS的肌肉和神经诱发收缩特性；b)采用2a的最佳接种密度，在体外不同时间点培养MDSC/SIS，测试肌肉和神经诱发的收缩特性；c)采用2a的最佳种植密度，在体内和不同时间点测试MDSC/SIS的肌肉和神经诱导的收缩特性。3)评估模拟分娩创伤后MDSC/SIS与SIS悬带置入的功能结果：a)评估SIS或MDSC/SIS悬带在连续膀胱测压中对排尿的影响；b)使用可靠灵敏的垂直倾斜膀胱内压力钳方法评估SIS或MDSC/SIS对漏点压力(LPP)的影响；c)评估植入的SIS和MDSC/SIS悬带的免疫组织学；以及d)重新测试LPP测试后MDSC/SIS悬带的力学性能及肌肉和神经诱发的收缩特性。我们计划通过功能组织工程学对缺陷的尿括约肌进行再造，从而显著提高SUI的治疗水平。我们想强调的是，我们的研究完全符合联邦关于胚胎干细胞研究的指导方针。这些干细胞不是从胚胎(动物或人类)或胚胎干细胞的细胞系中获得的。

DESCRIPTION (provided by applicant): Utilizing techniques developed in our laboratory, we propose to mount a systematic in vitro and in vivo physiological and biomechanical analysis of muscle stem cell based tissue engineering treatment of stress urinary incontinence (SUI). SUI is a significant medical problem affecting approximately 25 million American women 1. Despite the high prevalence of SUI, there is very little treatment-oriented research utilizing tissue engineering techniques. Using such techniques, we propose to develop a truly physiologic sling, not from synthetic or cadaveric tissue, but rather an engineered, functional stem cell muscle scaffold that can be implanted to repair a damaged urethral sphincter.Hypothesis: 1) Muscle Derived Stem Cells (MDSC) improve the mechanical properties of small intestinal submucosa (SIS) scaffold in vitro. 2) MDSC/SIS suburethral sling improves continence in an in vivo rat model of SUI.The Key Aims of this grant include: 1) Establish the mechanisms underlying the alterations in biaxial mechanical properties of MDSC-seeded SIS: a) Establish the feasibility of seeding MDSC to SIS; b) Establish the long-term persistence of MDSC on SIS; and c) Evaluate the compliance properties of SIS alone and MDSC/SIS in vitro and in vivo with comprehensive mechanical testing at different time points. 2) Evaluate the contractile properties of MDSC/SIS: a) Test muscle and neurally evoked contractile properties of MDSC/SIS based on seeding different densities of MDSC onto SIS and culturing for 7 days; b) Using the best seeding density from 2a, test muscle and neurally evoked contractility properties of MDSC/SIS, culture in vitro at different time points; and c) Using the best seeding density from 2a, test muscle and neurally evoked contractility properties of MDSC/SIS cultured in vivo and at different time points. 3) Assess the functional outcome of MDSC/SIS versus SIS suburethral sling placement following simulated birth trauma: a) Assess the effect of SIS or MDSC/SIS suburethral slings on micturition during continuous cystometry; b) Assess the effect of SIS or MDSC/SIS on leak point pressure (LPP) using the reliable and sensitive vertical tilt intravesical pressure clamp method; c) Evaluate the immunohistology of implanted SIS and MDSC/SIS suburethral slings; and d) Retest the mechanical properties as well as muscle and neurally evoked contractile properties of MDSC/SIS suburethral slings following LPP testing for correlative purposes. By reengineering the deficient urinary sphincter through functional tissue engineering, we plan to significantly improve the treatment of SUI. We want to strongly emphasize that our research is in complete compliance with the federal guidelines on embryonic stem cell research. These stem cells have not been obtained from embryos (animal or human) or cell lines of embryonic stem cells.