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Neuroglial differentiation and neo-innervation in extracellular matrix-based tissue engineered innervated smooth muscle sheets

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Excitatory and inhibitory motor neurons of the myenteric plexus in the enteric nervous system regulate and coordinate smooth muscle motility. Enteric neurons and glia of the myenteric plexus are organized into ganglia, that are surrounded by an extracellular matrix composed primarily of collagen IV, laminin and heparan sulfate. Disruption of ganglionic integrity, loss or damage of all or specific subsets of enteric neurons occur in several pathological states resulting in intestinal dysmotility and reduced quality of life. Neural-crest derived enteric neuronal progenitor cells persist in the adult mammalian gut, and contribute to the plasticity observed in the adult enteric nervous system. Neural stem cell transplantation is an emerging therapeutic paradigm aimed at repopulating enteric ganglia and restoring intestinal motility. However, transplantation of neural stem cells within the gut has demonstrated poor long-term phenotype maintenance and stability. We hypothesized that extracellular matrix (ECM) microenvironments could be used to direct the differentiation of enteric neuronal progenitor cells in vitro, prior to transplantation. The ECM plays an enormous role developmentally, in the migration of neural crest cells into the gastrulating mesoderm, and subsequent differentiation into neuroglial phenotypes appropriate for the intestinal microenvironment. We evaluated the potential of ECM microenvironments in modulating enteric neuroglial differentiation in 2D and 3D culture. We isolated enteric neuronal progenitor cells in sufficient numbers from small intestinal biopsies of adult rabbits. When presented with various ECM culture substrata, we observed that glial differentiation was inhibited in surfaces promoting neuronal differentiation. A pre-ponderance of differentiated glia was obtained in substrata that contained no ECM components. Neuronal differentiation was enhanced in the presence of collagen IV, with the emergence of branched neurons. Neurites on laminin coated substrata were the longest, averaging ~400µm. Heparan sulfate substrata promoted organization and preliminary neuronal networking. A tissue engineering model of innervated intestinal smooth muscle sheets was utilized to facilitate neuronal differentiation in viscoelastic hydrogels. Uniaxially aligned smooth muscle monolayers were obtained using substrate microtopography. ECM hydrogels were used to encapsulate enteric neuronal progenitor cells, generating innervated intestinal smooth muscle sheets. By varying ECM hydrogel composition, proportions of differentiated neurons were altered in the tissue engineered sheets. Sheets manufactured with collagen I and laminin had enriched populations of excitatory cholinergic neurons with minimal inhibitory neurons. Sheets manufactured with collagen IV had enriched populations of inhibitory nitrergic neurons. Cholinergic and nitrergic neuronal mediated contraction/relaxation matched their expression levels, when assessed using organ bath studies and electrical field stimulation. A therapeutic application for tissue engineered innervated sheets was evaluated as the potential to neo-innervate explant cultures of colon. Benzalkonium chloride treatments were used to denervate explant colons. Tissue engineered sheets were manufactured with enriched populations of nitrergic neurons, and maintained in co-culture with the recipient denervated colons. Neo-innervation was visualized following co-culture using immunohistochemistry and immunoblot expression for neuronal marker βIII Tubulin. Neuronal physiology was also improved following co-culture, indicating functional neo-innervation.
Enteric Nervous System
Gastrointestinal tract
Neural stem cells
Regenerative Medicine
Tissue Engineering
Raghavan, Shreya (author)
Bitar, Khalil N (committee chair)
Van Dyke, Mark E (committee member)
Rajagopalan, Padmavathy (committee member)
Goldstein, Aaron S (committee member)
Christ, George J (committee member)
2014-09-10T08:35:13Z (accessioned)
2016-09-10T08:30:09Z (available)
2014 (issued)
Biomedical Engineering (discipline)
2016-09-10 (terms)
http://hdl.handle.net/10339/39383 (uri)
en (iso)
Wake Forest University
Neuroglial differentiation and neo-innervation in extracellular matrix-based tissue engineered innervated smooth muscle sheets

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