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: 7942997
• 负责人: KHALIL N BITAR
• 金额: $ 48.24万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 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 2011-06-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7942997
• 关键词: 3-Dimensional; Accidents; Acetylcholine; Address; Adult; Affect; Age; Animals; Anogenital region; Anus; Anxiety; Applications Grants; Area; Autologous; Biological Neural Networks; Biomedical Engineering; Categories; Cells; Characteristics; Child; Circular layer of muscularis propria of anal canal; Clinical; Clothing; Coculture Techniques; Complex; Contracts; Data; Defect; Devices; Disease; Distress; Drops; Economics; Elderly; Electric Stimulation; Embryo; Enteral; Exclusion; Family; Fecal Impaction; Fecal Incontinence; Female; Fibrosis; Friends; Functional Gastrointestinal Disorders; Harvest; Hematology; High Prevalence; Human; Implant; Incidence; Incontinence; Individual; Infection; Institutionalization; Kidney; Knowledge; Life; Liver; Mechanics; Mental Depression; Modality; Modeling; Mus; Muscle; Muscle Contraction; National Institute of Diabetes and Digestive and Kidney Diseases; Natural History; Natural regeneration; Nature; Neuroglia; Neurons; Neurotransmitters; Organ; Pancreas; Patient Care; Patients; Peripheral; Persons; Physiological; Premature aging syndrome; Prevalence; Prevention; Property; Protocols documentation; Public Health; Quality of life; Regenerative Medicine; Reporting; Research; Risk Factors; Science; Severities; Shame; Skeletal Muscle; Skin; Smooth Muscle; Smooth Muscle Myocytes; Social isolation; Soil; Solutions; Sphincter; Stem cells; Stigmata; Structure of thyroid parafollicular cell; Surgical Flaps; Surveys; Symptoms; System; Time; Tissues; Transplantation; United States National Institutes of Health; Urinary Incontinence; base; caregiving; enema administration; expectation; experience; falls; fetal; gastrointestinal system; high school; human old age (65+); implantation; improved; innovation; nerve stem cell; nerve supply; neural circuit; neuronal circuitry; novel; physical conditioning; precursor cell; pressure; prevent; psychologic; public health relevance; relating to nervous system; repaired; response; sex; social; social stigma; symposium; urologic

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（申请人提供）：挑战领域（11）再生医学：11-DK-101：促进消化系统、肝脏、胰腺、血液、肾脏和泌尿系统的再生和修复。大便失禁是一种其后果远远超出身体表现的病症。许多人发现自己退出了社交生活，并试图向家人、朋友甚至医生隐瞒这个问题。与这些病症相关的耻辱、尴尬和耻辱对寻求专业治疗构成了重大障碍，导致许多人在没有帮助的情况下遭受这些病症的困扰。随着婴儿潮一代年近六十，失禁的发生率和公共健康负担可能会增加。大便失禁的负担分为经济和非经济类别，每种类别都很复杂。失禁的人可能会因“事故”而感到焦虑、抑郁、社会孤立和社会排斥。失禁的治疗本身就是一个负担。失禁需要更多的非正式和正式护理。 2007 年 12 月的 NIH 科学状况会议强调了这一点 [1]。我们需要提供解决方案来提高大便失禁患者的生活质量。新的假设表明，有效利用有关平滑肌细胞、器官、括约肌的知识以及通过植入生物工程 IAS 括约肌可能替代有缺陷的 IAS 括约肌是合理的预期。该拨款提案提供了解决大便失禁的有效方法。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。本质上，我们已经在培养物中开发了可用于植入的内在神经支配的 IAS 结构。该拨款提案的目的是植入由人类或小鼠 IAS 平滑肌细胞构建的生物工程功能性 IAS，其内部神经支配与外部神经网络相连。这些生物工程 IAS 构建体将具有生物工程内在神经元电路，并将连接到动物的外在神经网络。我们的初步数据表明：(A) 在培养中，小鼠 IAS 平滑肌细胞与永生鼠胎儿肠神经元 (IM-FEN) 共培养，在中心柱周围形成紧密的环。在环的外围，观察到神经元细胞在紧密的 IAS 环周围伸长、分支并形成网络。受神经支配的结构：1）响应乙酰胆碱和 PdBU 收缩并产生持续的力； 2) 对 VIP 和电场刺激 (EFS) 做出放松反应。 (B) 使用与 IM-FEN 细胞共培养的人 IAS 平滑肌细胞进行生物工程构建，获得了相同的结果。 (C) 神经支配的生物工程小鼠 IAS 构建体成功植入品系匹配小鼠的皮下。环变得血管化，并在动物体内存活长达 27 天，没有任何排斥迹象。 (D) 同样，受神经支配的生物工程人类 IAS 构建体成功植入免疫缺陷小鼠的皮下。环变得血管化并在动物体内存活下来，没有任何排斥的迹象。 (E) 收获后，血管化神经支配环保持了植入前观察到的生理特征。我们的生理学研究证实，环保持其功能特性，能够产生基础张力以及对收缩和松弛神经递质以及 EFS 的反应。我们的初步结果证实了生物工程环在植入后血管化的概念证明。动物能够耐受植入而没有排斥迹象。这是生理功能生物工程神经支配平滑肌结构的首次演示。这些构建体在植入后被受体动物耐受并变得血管化。这些发现代表了胃肠道组织替代和移植的重大进步。基于此，该提案的具体目标是： 1. 制定 ENS 祖细胞培养及其分化为肠神经细胞和神经胶质细胞的方案。 2. 通过生物工程设计 IAS 的 3-D 生理功能模型，该模型是在从人或小鼠的 IAS 和小鼠 ENS 祖细胞的 IAS 中分离出的平滑肌细胞培养物中产生的。 3. 将受神经支配的生理功能 3-D IAS 组织植入小鼠体内。这是一种创新方法，可以实现自体细胞生物工程 IAS 的植入。这有可能提高大便失禁患者的生活质量。此外，它还将对针对尿失禁患者的研究产生积极影响。 公共健康相关性：这项拨款提案代表了一种创新方法，可以实现利用自体细胞植入生物工程肛门内括约肌 (IAS)。这种生物工程 IAS 将具有源自胚胎神经祖细胞的内在神经回路。这有可能提高大便失禁患者的生活质量。此外，它对患有尿失禁的人也有积极的影响。