Successful implantation of physiologically functional bioengineered mouse internal anal sphincter

Abstract

We have previously developed bioengineered three-dimensional internal anal sphincter (IAS) rings from circular smooth muscle cells isolated from rabbit and human IAS. We provide proof of concept that bioengineered mouse IAS rings are neovascularized upon implantation into mice of the same strain and maintain concentric smooth muscle alignment, phenotype, and IAS functionality. Rings were bioengineered by using smooth muscle cells from the IAS of C57BL/6J mice. Bioengineered mouse IAS rings were implanted subcutaneously on the dorsum of C57BL/6J mice along with a microosmotic pump delivering fibroblast growth factor-2. The mice remained healthy during the period of implantation, showing no external signs of rejection. Mice were killed 28 days postsurgery and implanted IAS rings were harvested. IAS rings showed muscle attachment, neovascularization, healthy color, and no external signs of infection or inflammation. Assessment of force generation on harvested IAS rings showed the following: 1) spontaneous basal tone was generated in the absence of external stimulation; 2) basal tone was relaxed by vasoactive intestinal peptide, nitric oxide donor, and nifedipine; 3) acetylcholine and phorbol dibutyrate elicited rapid-rising, dose-dependent, sustained contractions repeatedly over 30 min without signs of muscle fatigue; and 4) magnitudes of potassium chloride-induced contractions were 100% of peak maximal agonist-induced contractions. Our preliminary results confirm the proof of concept that bioengineered rings are neovascularized upon implantation. Harvested rings maintain smooth muscle alignment and phenotype. Our physiological studies confirm that implanted rings maintain 1) overall IAS physiology and develop basal tone, 2) integrity of membrane ionic characteristics, and 3) integrity of membrane associated intracellular signaling transduction pathways for contraction and relaxation by responding to cholinergic, nitrergic, and VIP-ergic stimulation. IAS smooth muscle tissue could thus be bioengineered for the purpose of implantation to serve as a potential graft therapy for dysfunctional internal anal sphincter in fecal incontinence.

Cell and organ bioengineering technology as applied to gastrointestinal diseases

“…One successful approach to promoting angiogenesis is to deliver fibroblast growth factor to bioengineered small intestine segments or smooth muscle constructs forming the anal sphincter 121 122. Platelet-derived growth factor, an angiogenic growth factor, has also been used successfully to maintain survival of bioengineered IAS constructs in mice 123…”

Bioengineering of Colo-Rectal Tissue

“…Raghavan and colleagues were able to successfully bioengineer a ringshaped SMC construct, that, once subcutaneously implanted remained viable until harvest at 28 days. The muscle-ring further maintained its alignment, phenotype as well as some physiological properties of SMCs (Raghavan et al, 2010b). Very recently, the same research group has co-cocultured SMCs originating from human internal anal sphincter with immortomouse fetal enteric neurons, which they eventually implanted in mice (Raghavan et al, 2011).…”

Advances in Neo-Innervation of the Gut

“…Implantation of this bioengineered construct was performed in mice; it showed no signs of rejection or inflammation, and remained viable for at least 28 days. More recently, implanted constructs with human intestinal SMCs and neurons were shown to be viable following transplantation into an immunecompromised mouse [63]. The implanted constructs became vascularized by host blood vessels.…”