Implantation of physiologically functional bioengineered innervated IAS construct

生理功能生物工程神经支配 IAS 构建体的植入

• 批准号: 8316630
• 负责人: KHALIL N BITAR
• 金额: $ 1.36万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8316630
• 关键词: Implantation; physiologically; functional; bioengineered; innervated

DESCRIPTION (provided by applicant): Challenge Area (11) Regenerative Medicine: 11-DK-101: Promote regeneration and repair in the digestive system, liver, pancreas, hematology, kidneys and urological system. Fecal incontinence is a condition with ramifications that extend well beyond the physical manifestations. Many individuals find themselves withdrawing from their social lives and attempting to hide the problem from their families, friends, and even their doctors. The shame, embarrassment, and stigma associated with these conditions pose significant barriers to seeking professional treatment, resulting in many persons who suffer from these conditions without help. As baby boomers approach their sixties, the incidence and public health burden of incontinence are likely to increase. The burdens of fecal incontinence fall into economic and non- economic categories, and each is complex. Individuals who are incontinent may experience anxiety about "accidents," depression, social isolation, and social exclusion. The management of incontinence itself is burdensome. Incontinence requires greater amounts of informal and formal care giving. This was emphasized in the NIH State-of-the-Science Conference in December 2007 [1]. We need to provide solution to enhance the quality of life for individuals with fecal incontinence. Novel hypothesis suggests the effective utilization of knowledge about smooth muscle cells, organs, sphincters and possible replacement of defective IAS sphincters with implantation of bioengineered IAS sphincters are reasonable expectations. This grant proposal offers an effective approach to tackle fecal incontinence. We were able to successfully implant bioengineered rings that were constructed from IAS smooth muscle cells and neuronal precursor cells. In essence we have developed intrinsically innervated IAS constructs in culture that could be used for implantation. The objective of this grant proposal is to implant bioengineered functional IAS constructed from either human or mouse IAS smooth muscle cells with intrinsic innervations connected to extrinsic neural network. These bioengineered IAS constructs will have bioengineered intrinsic neuronal circuitry and will be connected to extrinsic neural network from the animal. Our preliminary data indicates that: (A) in culture, mouse IAS smooth muscle cells co- cultured with Immortomouse Fetal Enteric Neurons (IM-FEN), formed a tight ring around a central post. Peripheral to the ring, the neuronal cells were observed to elongate, branch and form networks around the tight IAS ring. The innervated constructs: 1) contracted and generated sustained force in response to acetylcholine and PdBU; and 2) relaxed in response to VIP and Electrical Filed Stimulation (EFS). (B) Same results were obtained from constructs bioengineered using human IAS smooth muscle cells co-cultured with IM-FEN cells. (C) The innervated bioengineered mouse IAS constructs were successfully implanted under the skin of a strain matched mouse. Rings became vascularized and survived in the animals without any signs of rejection for up to 27 days. (D) Similarly, the innervated bioengineered human IAS constructs were successfully implanted under the skin of an immuno-deficient mouse. Rings became vascularized and survived in the animals without any signs of rejection. (E) Upon harvesting, the vascularized innervated rings maintained their physiological characteristics observed prior to implantation. Our physiological studies confirm that the rings maintain their functional properties are able to develop basal tone and response to contractile and relaxant neurotransmitters as well as EFS. Our preliminary results confirm the proof of concept that bioengineered rings are vascularized upon implantation. The animals tolerate implantation without signs of rejection. This is the first demonstration of physiologically functional bioengineered innervated smooth muscle constructs. These constructs upon implantation were tolerated by recipient animal and became vascularized. These findings represent a substantial advance in GI tissue replacement and transplantation. Based on this, the specific aims of the proposal are: 1. Develop protocols for the culture of ENS progenitor cells and their differentiation into enteric neural and glial cells. 2. Bioengineer a 3-D physiologically functional model of the IAS produced in culture from smooth muscle cells isolated from the IAS of either human or mouse and mouse ENS progenitor cells. 3. Implant an innervated physiologically functional 3-D IAS tissue into a mouse. This is an innovative approach that could result in implantation of bioengineered IAS from autologous cells. This has potential to provide enhanced quality of life to persons with Fecal Incontinence. Furthermore it would have positive implications for research focusing on people suffering from urinary incontinence. PUBLIC HEALTH RELEVANCE: This grant proposal represents an innovative approach that could result in implantation of Bioengineered Internal Anal Sphincter (IAS) from autologous cells. This Bioengineered IAS would have intrinsic neural circuitry derived from embryonic neural progenitor cells. This has potential to provide enhanced quality of life to persons with fecal incontinence. Furthermore it would have positive implication for people suffering from urinary incontinence.

描述（由申请人提供）：挑战区域（11）再生医学：11-DK-101：促进消化系统，肝脏，胰腺，血液学，肾脏和泌尿科系统的再生和修复。粪便尿失禁是一种疾病，其影响远远超出了物理表现。许多人发现自己正在退出社交生活，并试图向家人，朋友甚至医生掩盖问题。与这些条件相关的耻辱，尴尬和污名构成了寻求专业治疗的重大障碍，导致许多人在没有帮助的情况下遭受这些条件。随着婴儿潮一代的六十多岁，尿失禁的发生率和公共卫生负担可能会增加。粪便失禁的负担属于经济和非经济类别，每个类别都很复杂。失火的人可能会对“事故”，抑郁，社会隔离和社会排斥感到焦虑。失禁本身的管理很繁重。尿失禁需要更多的非正式和正式护理。在2007年12月的NIH最先进的会议上强调了这一点[1]。我们需要提供解决方案，以增强粪便失禁的个体的生活质量。新的假设表明，在植入生物工程的IAS括约肌的情况下，有效利用了有关平滑肌细胞，器官，括约肌以及可能替代有缺陷的IAS括约肌的知识，这是合理的期望。该赠款提案提供了解决粪便尿失禁的有效方法。我们能够成功植入由IAS平滑肌细胞和神经元前体细胞构建的生物工程环。从本质上讲，我们已经开发了可以用于植入的文化中内在神经支配的IAS构建体。该赠款建议的目的是植入由人或小鼠IAS平滑肌细胞构建的生物工程功能IAS，其内在神经与外部神经网络有关。这些生物工程的IAS构建体将具有生物工程的固有神经元电路，并将与动物的外部神经网络连接。我们的初步数据表明：（a）在培养中，小鼠IAS平滑肌细胞与不朽的胎儿肠神经元（IM-FEN）共同培养，在中央柱子周围形成了一个紧密的环。观察到环上的周围，观察到神经元细胞在紧密的IAS环周围拉长，分支和形成网络。神经支配的构建体：1）响应于乙酰胆碱和PDBU的收缩和产生的持续力； 2）响应VIP和电归档刺激（EFS）而放松。 （b）使用与IM富含细胞共培养的人IAS平滑肌细胞进行生物工程的构建体获得了相同的结果。 （c）神经工程的小鼠IAS构建体成功地植入了与小鼠相匹配的皮肤下。戒指变成了血管化并在动物中生存，没有任何拒绝迹象长达27天。 （d）类似地，支配的生物工程的人IAS构建体成功地植入了免疫缺陷小鼠的皮肤下。环成为血管化并在动物中幸存下来，没有任何拒绝迹象。 （e）收获后，血管化神经环保持其在植入前观察到的生理特征。我们的生理研究证实，环保持其功能性能能够发展基础语调，并对收缩和松弛神经递质以及EFS产生反应。我们的初步结果证实了生物工程环在植入后被血管的概念证明。动物耐受植入而没有排斥迹象。这是生理功能性生物工程支配平滑肌构建体的首次演示。这些植入后的结构是通过受体动物耐受的，并变得血管化。这些发现代表了胃肠道组织的替代和移植方面的重大进展。基于此，该提案的具体目的是：1。为ENS祖细胞的培养及其分化为肠神经和神经胶质细胞的培养方案。 2。生物工程师是由从人或小鼠和小鼠ENS祖细胞分离的平滑肌细胞中产生的IAS的3-D生理功能模型。 3。植入一个神经的生理功能性3-D IAS组织中。这是一种创新的方法，可以导致自体细胞中生物工程的IAS植入。这有可能为粪便失禁的人提供增强的生活质量。此外，这将对关注患有尿失禁的人的研究具有积极的影响。 公共卫生相关性：该赠款提案代表了一种创新的方法，可能导致自体细胞中生物工程的内部肛门括约肌（IAS）植入。该生物工程的IAS将具有源自胚胎神经祖细胞的固有神经回路。这有可能为粪便失禁的人提供增强的生活质量。此外，这将对患有尿失禁的人产生积极的影响。