166 Successful Implantation of Intrinsically Innervated Physiologically Functional Bioengineered Human Internal Anal Sphincter (IAS)

Abstract

Background: We have bioengineered intrinsically innervated IAS tissue by co-culturing human IAS circular smooth muscle and immorto mouse fetal enteric neurons (IM-FENs) Objective: We provide proof of concept that intrinsically innervated bioengineered human IAS rings implanted in RAG1-/mice preserve physiological functionality of constituent myogenic and neuronal components. Methods: Intrinsically innervated human IAS rings were bioengineered by embedding human IAS smooth muscle cells and de-differentiated IM-FENs on a collagen/laminin matrix. IM-FENs were allowed to differentiate at 39C for 12 days. Bioengineered rings were implanted for 25 days under the skin of RAG1-/mice. Results: Histology of harvested post-implant IAS revealed neovascularization. Real-time force acquisition from these constructs revealed that physiological responses of post-implant innervated IAS were similar to control non-implanted innervated IAS rings: (1)Spontaneous basal tone generated was tetrodotoxin (TTX)-insensitive and hence purely myogenic; (2)Vasoactive Intestinal Peptide(VIP) caused a relaxation of basal tone upto 300μN. VIPinduced relaxation in the presence of TTX was only 20% of the original, indicating dual involvement of neuronal and myogenic component in VIP-induced relaxation;(3)Electrical Field Stimulation(10Hz,0.3ms) caused relaxation of basal tone upto 153μN. EFS response was abolished in presence of TTX indicating selective stimulation of neuron mediated relaxation pathway; (4)Acetylcholine(Ach) response was dose-dependent and elicited a rapidrising, sustained contraction of 477±77μN. Contraction was attenuated by 90% in the presence of TTX, implying synergistic involvement of both myogenic and neuronal components in cholinergic contraction; (5)30mM KCl elicited a rapid rising spike contraction, indicating preservation of physiologically relevant ion channels. Summary: Implanted intrinsically innervated IAS maintained: 1-Spontaneous generation of myogenic basal tone, a constitutive property of IAS. 2Neurotransmitter-mediated contraction and relaxation, and their TTX sensitivity displaying distinct contributions of viable intrinsic innervation (IM-FEN) and constituent myogenic component (human IAS smooth muscle).Conclusion: Intrinsically innervated bioengineered human IAS tissue preserved the integrity and physiological functionality of both myogenic and neuronal components, upon implantation into RAG1-/mice. This is the first report of implantation of intrinsically innervated human IAS bioengineered construct, with vast potential to create functional autologous graft tissue to repair degenerated IAS. Supported by NIHRO1DK071614 and 1RC1DK087151