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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
NIH Challenge Grants and Partnerships Program (RC1)
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Special Emphasis Panel (ZRG1-DKUS-A (58))
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Carrington, Jill L
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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Gilmont, Robert R; Raghavan, Shreya; Somara, Sita et al. (2014) Bioengineering of physiologically functional intrinsically innervated human internal anal sphincter constructs. Tissue Eng Part A 20:1603-11
Raghavan, Shreya; Miyasaka, Eiichi A; Gilmont, Robert R et al. (2014) Perianal implantation of bioengineered human internal anal sphincter constructs intrinsically innervated with human neural progenitor cells. Surgery 155:668-74
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