Bioengineering of the digestive tract: approaching the clinic.

The field of regenerative medicine is developing technologies that, in the near future, will offer alternative approaches to either cure diseases affecting the gastrointestinal tract or slow their progression by leveraging the intrinsic ability of our tissues and organs to repair after damage. This article will succinctly illustrate the three technologies that are closer to clinical translation-namely, human intestinal organoids, sphincter bioengineering and decellularization, whereby the cellular compartment of a given segment of the digestive tract is removed to obtain a scaffold consisting of the extracellular matrix. The latter will be used as a template for the regeneration of a functional organ, whereby the newly generated cellular compartment will be obtained from the patient's own cells. Although clinical application of this technology is approaching, product development challenges are being tackled to warrant safety and efficacy.

Bioengineering functional human sphincteric and non-sphincteric gastrointestinal smooth muscle constructs.

Digestion and motility of luminal content through the gastrointestinal (GI) tract are achieved by cooperation between distinct cell types. Much of the 3 dimensional (3D) in vitro modeling used to study the GI physiology and disease focus solely on epithelial cells and not smooth muscle cells (SMCs). SMCs of the gut function either to propel and mix luminal contents (phasic; non-sphincteric) or to act as barriers to prevent the movement of luminal materials (tonic; sphincteric). Motility disorders including pyloric stenosis and chronic intestinal pseudoobstruction (CIPO) affect sphincteric and non-sphincteric SMCs, respectively. Bioengineering offers a useful tool to develop functional GI tissue mimics that possess similar characteristics to native tissue. The objective of this study was to bioengineer 3D human pyloric sphincter and small intestinal (SI) constructs in vitro that recapitulate the contractile phenotypes of sphincteric and non-sphincteric human GI SMCs. Bioengineered 3D human pylorus and circular SI SMC constructs were developed and displayed a contractile phenotype. Constructs composed of human pylorus SMCs displayed tonic SMC characteristics, including generation of basal tone, at higher levels than SI SMC constructs which is similar to what is seen in native tissue. Both constructs contracted in response to potassium chloride (KCl) and acetylcholine (ACh) and relaxed in response to vasoactive intestinal peptide (VIP). These studies provide the first bioengineered human pylorus constructs that maintain a sphincteric phenotype. These bioengineered constructs provide appropriate models to study motility disorders of the gut or replacement tissues for various GI organs.

Tissue engineering in the gut: developments in neuromusculature.

The complexity of the gastrointestinal (GI) tract lies in its anatomy as well as in its physiology. Several different cell types populate the GI tract, adding to the complexity of cell sourcing for regenerative medicine. Each cell layer has a specialized function in mediating digestion, absorption, secretion, motility, and excretion. Tissue engineering and regenerative medicine aim to regenerate the specific layers mimicking architecture and recapitulating function. Gastrointestinal motility is the underlying program that mediates the diverse functions of the intestines, as an organ. Hence, the first logical step in GI regenerative medicine is the reconstruction of the tubular smooth musculature along with the drivers of their input, the enteric nervous system. Recent advances in the field of GI tissue engineering have focused on the use of scaffolding biomaterials in combination with cells and bioactive factors. The ability to innervate the bioengineered muscle is a critical step to ensure proper functionality. Finally, in vivo studies are essential to evaluate implant integration with host tissue, survival, and functionality. In this review, we focus on the tubular structure of the GI tract, tools for innervation, and, finally, evaluation of in vivo strategies for GI replacements.

Treatment of fecal incontinence: state of the science summary for the National Institute of Diabetes and Digestive and Kidney Diseases workshop.

This is the second of a two-part summary of a National Institutes of Health conference on fecal incontinence (FI) that summarizes current treatments and identifies research priorities. Conservative medical management consisting of patient education, fiber supplements or antidiarrheals, behavioral techniques such as scheduled toileting, and pelvic floor exercises restores continence in up to 25% of patients. Biofeedback, often recommended as first-line treatment after conservative management fails, produces satisfaction with treatment in up to 76% and continence in 55%; however, outcomes depend on the skill of the therapist, and some trials are less favorable. Electrical stimulation of the anal mucosa is ineffective, but continuous electrical pulsing of sacral nerves produces a ≥50% reduction in FI frequency in a median 73% of patients. Tibial nerve electrical stimulation with needle electrodes is promising but remains unproven. Sphincteroplasty produces short-term clinical improvement in a median 67%, but 5-year outcomes are poor. Injecting an inert bulking agent around the anal canal led to ≥50% reductions of FI in up to 53% of patients. Colostomy is used as a last resort because of adverse effects on quality of life. Several new devices are under investigation but not yet approved. FI researchers identify the following priorities for future research: (1) trials comparing the effectiveness, safety, and cost of current therapies; (2) studies addressing barriers to consulting for care; and (3) translational research on regenerative medicine. Unmet patient needs include FI in special populations (e.g., neurological disorders and nursing home residents) and improvements in behavioral treatments.

Stem cell therapy for GI neuromuscular disorders.

The enteric nervous system is the intrinsic innervation of the gut. Several neuromuscular disorders affect the neurons and glia of the enteric nervous system adversely, resulting in disruptions in gastrointestinal motility and function. Pharmacological interventions to remedy gastrointestinal function do not address the underlying cause of dysmotility arising from lost, absent, or damaged enteric neuroglial circuitry. Cell-based therapies have gained traction in the past decade, following the discovery of several adult stem cell niches in the human body. Adult neural stem cells can be isolated from the postnatal and adult intestine using minimally invasive biopsies. These stem cells retain the ability to differentiate into several functional classes of enteric neurons and enteric glia. Upon identification of these cells, several groups have also established that transplantation of these cells into aganglionic or dysganglionic intestine rescues gastrointestinal motility and function. This chapter highlights key studies performed in the field of stem cell transplantation therapies that are targeted towards the remedy of gastrointestinal motility and function.

Cell and organ bioengineering technology as applied to gastrointestinal diseases.

This review illustrates promising regenerative medicine technologies that are being developed for the treatment of gastrointestinal diseases. The main strategies under validation to bioengineer or regenerate liver, pancreas, or parts of the digestive tract are twofold: engineering of progenitor cells and seeding of cells on supporting scaffold material. In the first case, stem cells are initially expanded under standard tissue culture conditions. Thereafter, these cells may either be delivered directly to the tissue or organ of interest, or they may be loaded onto a synthetic or natural three-dimensional scaffold that is capable of enhancing cell viability and function. The new construct harbouring the cells usually undergoes a maturation phase within a bioreactor. Within the bioreactor, cells are conditioned to adopt a phenotype similar to that displayed in the native organ. The specific nature of the scaffold within the bioreactor is critical for the development of this high-function phenotype. Efforts to bioengineer or regenerate gastrointestinal tract, liver and pancreas have yielded promising results and have demonstrated the immense potential of regenerative medicine. However, a myriad of technical hurdles must be overcome before transplantable, engineered organs become a reality.

Distraction-induced intestinal enterogenesis: preservation of intestinal function and lengthening after reimplantation into normal jejunum.

BACKGROUND: Significant bowel lengthening can occur in an isolated intestinal segment with the use of linearly directed distractive forces, resulting in increased surface area and epithelial cell proliferation. We hypothesized that reimplantation of this lengthened intestine into normal jejunum would preserve this gain in intestinal length and function similar to normal jejunum. METHODS: An intestinal lengthening device was inserted into isolated jejunal segments in pigs, and fully expanded over 8 days. Lengthened segments were then reimplanted into normal intestinal continuity. Pigs were studied after another 28 days. Function was assessed by motility, mucosal enzyme activity, barrier function, and intestinal ion transport. RESULTS: Lengthened segments were significantly longer than control segments and had nearly 2-fold greater surface area. Bowel lengthening was maintained 4 weeks after reimplantation. Motility after reimplantation was similar to nonoperated pigs. Barrier function, mucosal disaccharidase levels, and electrophysiologic measures declined immediately after lengthening but returned to nearly normal levels 28 days after reimplantation. CONCLUSION: Bowel lengthening results in a transient decline in mucosal absorptive function and smooth muscle contractility. However, function approaches that of normal bowel after reimplantation into enteric flow. These data may support the use of this technique as a potential new option for the treatment of patients with short bowel syndrome.

Regenerative medicine as applied to general surgery.

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.

Successful implantation of bioengineered, intrinsically innervated, human internal anal sphincter.

BACKGROUND & AIMS: To restore fecal continence, the weakened pressure of the internal anal sphincter (IAS) must be increased. We bioengineered intrinsically innervated human IAS to emulate sphincteric physiology in vitro. METHODS: We cocultured human IAS circular smooth muscle with immortomouse fetal enteric neurons. We investigated the ability of bioengineered innervated human IAS, implanted in RAG1-/- mice, to undergo neovascularization and preserve the physiology of the constituent myogenic and neuronal components. RESULTS: The implanted IAS was neovascularized in vivo; numerous blood vessels were observed with no signs of inflammation or infection. Real-time force acquisition from implanted and preimplant IAS showed distinct characteristics of IAS physiology. Features included the development of spontaneous myogenic basal tone; relaxation of 100% of basal tone in response to inhibitory neurotransmitter vasoactive intestinal peptide (VIP) and direct electrical field stimulation of the intrinsic innervation; inhibition of nitrergic and VIPergic electrical field-induced relaxation (by antagonizing nitric oxide synthesis or receptor interaction); contraction in response to cholinergic stimulation with acetylcholine; and intact electromechanical coupling (evidenced by direct response to potassium chloride). Implanted, intrinsically innervated bioengineered human IAS tissue preserved the integrity and physiology of myogenic and neuronal components. CONCLUSIONS: Intrinsically innervated human IAS bioengineered tissue can be successfully implanted in mice. This approach might be used to treat patients with fecal incontinence.

Real-time dynamic movement of caveolin-1 during smooth muscle contraction of human colon and aged rat colon transfected with caveolin-1 cDNA.

Caveolin-1 (cav-1) plays a key role in PKC-α and RhoA signaling pathways during acetylcholine (ACh)-induced contraction of colonic smooth muscle cells (CSMC). Aged rat CSMC showed sluggish contractility, concomitant with reduced expression of cav-1 with an associated reduction in activation of PKC-α and RhoA signaling pathway. Real-time monitoring of live human CSMC transfected with yellow fluorescent protein-tagged wild-type caveolin 1 cDNA (YFP-wt-cav-1) cDNA in the present study suggests that cav-1 cycles within and along the membrane in a synchronized, highly organized cytoskeletal path. These studies provide, for the first time, the advantages of real-time monitoring of the dynamic movement of caveolin in living cells. Rapid movement of cav-1 in response to ACh suggests its dynamic role in CSMC contraction. Human CSMC transfected with YFP-ΔTFT-cav-1 dominant negative cDNA show fluorescence in the cytosol of the CSMC and no movement of fluorescent cav-1 in response to ACh mimicking the response shown by aged rat CSMC. Transfection of CSMC from aged rat with YFP-wt-cav-1 cDNA restored the physiological contractile response to ACh as well as the dynamic movement of cav-1 along the organized cytoskeletal path observed in normal adult CSMC. To study the force generation by CSMC, three-dimensional colonic rings were bioengineered. Colonic bioengineered rings from aged CSMC showed reduced force generation compared with colonic bioengineered rings from adult CSMC. Colonic bioengineered rings from aged CSMC transfected with wt-cav-1 cDNA showed force generation similar to colonic bioengineered rings from adult rat CSMC. The data suggest that contraction in CSMC is dependent on cav-1 reorganization dynamics, which restores the physiological contractile response in aged CSMC. We hypothesize that dynamic movement of cav-1 is essential for physiological contractile response of colonic smooth muscle.

In vivo growth of a bioengineered internal anal sphincter: comparison of growth factors for optimization of growth and survival.

PURPOSE: Our laboratory has developed and implanted a novel bioengineered internal anal sphincter (IAS) to treat anal incontinence. Fibroblast growth factor-2 (FGF-2) has been used in mice; however, the optimal growth factor for successful IAS implantation is unclear. This study compares several growth factors in order to optimize IAS viability and functionality. METHODS: Bioengineered IAS rings were implanted subcutaneously into the dorsum of wildtype C57Bl/6 mice, with an osmotic pump dispensing FGF-2, vascular endothelial growth factor (VEGF), or platelet-derived growth factor (PDGF) (n = 4 per group). Control mice received IAS implants but no growth factor. The IAS was harvested approximately 25 days post-implantation. Tissue was subjected to physiologic testing, then histologically analyzed. Muscle phenotype was confirmed by immunofluorescence. RESULTS: All implants supplemented with growth factors maintained smooth muscle phenotype. Histological scores, blood vessel density and muscle fiber thickness were all markedly better with growth factors. Neovascularization was comparable between the three growth factors. Basal tonic force of the constructs was highest with VEGF or PDGF. CONCLUSION: All growth factors demonstrated excellent performance. As our ultimate goal is clinical implantation, our strong results with PDGF, a drug approved for use in the United States and the European Union, pave the way for translating bioengineered IAS implantation to the clinical realm.

p70 Ribosomal S6 kinase is required for airway smooth muscle cell size enlargement but not increased contractile protein expression.

We examined the contribution of p70 ribosomal S6 kinase (p70S6K) to airway smooth muscle hypertrophy, a structural change found in asthma. In human airway smooth muscle cells, transforming growth factor (TGF)-beta, endothelin-1, and cardiotrophin-1 each induced phosphorylation of p70S6K and ribosomal protein S6 while increasing cell size, total protein synthesis, and relative protein abundance of alpha-smooth muscle actin and SM22. Transfection of myocytes with siRNA against either p70S6K or S6, or infection with retrovirus encoding a kinase-dead p70S6K, reduced cell size and protein synthesis but had no effect on contractile protein expression per mg total protein. Infection with a retrovirus encoding a constitutively active, rapamycin-resistant (RR) p70S6K increased cell size but not contractile protein expression. siRNA against S6 decreased cell size in myocytes expressing RR p70S6K. Finally, TGF-beta treatment, but not RR p70S6K expression, increased KCl-induced fractional shortening. Together, these data suggest that p70S6K activation is both required and sufficient for airway smooth muscle cell size enlargement but not contractile protein expression. Further, ribosomal protein S6 is required for p70S6K-mediated cell enlargement. Finally, we have shown for the first time in a functional cell system that p70S6K-mediated myocyte enlargement alone, without preferential contractile protein expression, is insufficient for increased cell shortening.

Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle.

Small heat shock proteins HSP27 and HSP20 have been implicated in regulation of contraction and relaxation in smooth muscle. Activation of PKC-α promotes contraction by phosphorylation of HSP27 whereas activation of PKA promotes relaxation by phosphorylation of HSP20 in colonic smooth muscle cells (CSMC). We propose that the balance between the phosphorylation states of HSP27 and HSP20 represents a molecular signaling switch for contraction and relaxation. This molecular signaling switch acts downstream on a molecular mechanical switch [tropomyosin (TM)] regulating thin-filament dynamics. We have examined the role of phosphorylation state(s) of HSP20 on HSP27-mediated thin-filament regulation in CSMC. CSMC were transfected with different HSP20 phosphomutants. These transfections had no effect on the integrity of actin cytoskeleton. Cells transfected with 16D-HSP20 (phosphomimic) exhibited inhibition of acetylcholine (ACh)-induced contraction whereas cells transfected with 16A-HSP20 (nonphosphorylatable) had no effect on ACh-induced contraction. CSMC transfected with 16D-HSP20 cDNA showed significant decreases in 1) phosphorylation of HSP27 (ser78); 2) phosphorylation of PKC-α (ser657); 3) phosphorylation of TM and CaD (ser789); 4) ACh-induced phosphorylation of myosin light chain; 5) ACh-induced association of TM with HSP27; and 6) ACh-induced dissociation of TM from caldesmon (CaD). We thus propose the crucial physiological relevance of molecular signaling switch (phosphorylation state of HSP27 and HSP20), which dictates 1) the phosphorylation states of TM and CaD and 2) their dissociations from each other.

Successful implantation of physiologically functional bioengineered mouse internal anal sphincter.

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.

Bioengineered internal anal sphincter derived from isolated human internal anal sphincter smooth muscle cells.

BACKGROUND & AIMS: The internal anal sphincter (IAS) is a specialized circular smooth muscle that maintains rectoanal continence. In vitro models are needed to study the pathophysiology of human IAS disorders. We bioengineered sphincteric rings from human IAS smooth muscle cells (SMC) and investigated their response to cholinergic stimulation as well as investigated whether protein kinase C (PKC) and Rho kinase signaling pathways remain functional. METHODS: 3-Dimensional bioengineered ring (3DBR) model of the human IAS was constructed from isolated human IAS SMC obtained from surgery. Contractile properties and force generation in response to acetylcholine, PKC inhibitor calphostin-C, Rho/ROCK inhibitor Y-27632, permeable Rho/ROCK inhibitor c3-exoenzyme, and PKC activator PdBU was measured. RESULTS: The human IAS 3DBR has the essential characteristics of physiologically functional IAS; it generated a spontaneous myogenic basal tone, and the constructs were able to relax in response to relaxants and contract in response to contractile agents. The constructs generated dose-dependent force in response to acetylcholine. Basal tone was significantly reduced by calphostin-C but not with Y-27632. Acetylcholine-induced force generation was also significantly reduced by calphostin-C but not with Y-27632. PdBU generated force that was equal in magnitude to acetylcholine. Thus, calphostin-C inhibited PdBU-induced force generation, whereas Y-27632 and c3 exoenzyme did not. CONCLUSIONS: These data indicate that basal tone and acetylcholine-induced force generation depend on signaling through the PKC pathway in human IAS; PKC-mediated force generation is independent of the Rho/ROCK pathway. This human IAS 3DBR model can be used to study the pathophysiology associated with IAS malfunctions.

Functional restoration of ex vivo model of pylorus: Co-injection of neural progenitor cells and interstitial cells of Cajal.

Transplantation of neural stem cells is a promising approach in treatment of intestinal dysfunctionality. The interstitial cells of Cajal (ICCs) are also critical in conditions such as pyloric dysfunctionality and gastroparesis. The objective of this study was to replenish neurons and ICCs in a dysfunctional pylorus as cell-based therapy to restore functionality. ICCs and enteric neural progenitor cells (NPCs) were isolated from rat duodenum and transduced with fluorescent proteins. Rat pylorus was harvested, and an ex-vivo neuromuscular dysfunctional model was developed by selective ablation of neurons and ICCs via chemical treatments. Cellular repopulation and restoration of motility were assessed by immunohistochemistry, qPCR, and functional analysis after delivery of fluorescently tagged cells. Chemical treatment of pylorus resulted in significant depletion of ICCs (67%, P = .0024; n = 3) and neural cells (83%, P = .0012; n = 3). Delivered ICCs and NPCs survived and integrated with host muscle layers. Co-injection of ICCs with NPCs exhibited 34.4% (P = .0004; n = 3) and 61.0% (P = .0003; n = 3) upregulation of ANO1 and ßIII tubulin, respectively. This regeneration resulted in the restoration of agonist-induced excitatory contraction (82%) and neuron evoked relaxation (83%). The functional studies with specific neuronal nitric oxide (NO) synthase blocker confirmed that restoration of relaxation was NO mediated and neuronally derived. The simultaneous delivery of ICCs observed 35.7% higher neuronal differentiation and functional restoration compared with injection of NPCs alone. Injected NPCs and ICCs integrated into the dysfunctional ex vivo pylorus tissues and restored neuromuscular functionality. The co-transplantation of NPCs and ICCs can be used to treat neurodegenerative disorders of the pylorus.

Role of thin-filament regulatory proteins in relaxation of colonic smooth muscle contraction.

Coordinated regulation of smooth muscle contraction and relaxation is required for colonic motility. Contraction is associated with phosphorylation of myosin light chain (MLC(20)) and interaction of actin with myosin. Thin-filament regulation of actomyosin interaction is modulated by two actin-binding regulatory proteins: tropomyosin (TM) and caldesmon (CaD). TM and CaD are known to play crucial role in actomyosin interaction promoting contraction. Contraction is associated with phosphorylation of the small heat shock protein HSP27, concomitant with the phosphorylation of TM and CaD. Phosphorylation of HSP27 is attributed as being the prime modulator of thin-filament regulation of contraction. Preincubation of colonic smooth muscle cells (CSMC) with the relaxant neurotransmitter vasoactive intestinal peptide (VIP) showed inhibition in phosphorylation of HSP27 (ser78). Attenuation of HSP27 phosphorylation can result in modulation of thin-filament-mediated regulation of contraction leading to relaxation; thus the role of thin-filament regulatory proteins in a relaxation milieu was investigated. Preincubation of CSMC with VIP exhibited a decrease in phosphorylation of TM and CaD. Furthermore, CSMC preincubated with VIP showed a reduced association of TM with HSP27 and with phospho-HSP27 (ser78) whereas there was reduced dissociation of TM from CaD and from phospho-CaD. We thus propose that, in addition to alteration in phosphorylation of MLC(20), relaxation is associated with alterations in thin-filament-mediated regulation that results in termination of contraction.

Bioengineering and regeneration of gastrointestinal tissue: where are we now and what comes next?

INTRODUCTION: The field of tissue engineering and regenerative medicine has been applied to the gastrointestinal (GI) tract for a couple decades. Several achievements have been accomplished that provide promising tools for treating diseases of the GI tract. AREAS COVERED: The work described in this review covers the traditional aspect of using cells and scaffolds to replace parts of the tract. Several studies investigated different types of biomaterials and different types of cells. A more recent approach involved the use of gut-derived organoid units that can differentiate into all gut cell layers. The most recent approach introduced the use of organ-on-a-chip concept to understand the physiology and pathophysiology of the GI system. EXPERT OPINION: The different approaches tackle the diseases of the GI tract from different perspectives. While all these different approaches provide a promising and encouraging future for this field, the translational aspect is yet to be studied.

BioSphincters to treat Fecal Incontinence in Nonhuman Primates.

Loss of anorectal resting pressure due to internal anal sphincter (IAS) dysfunctionality causes uncontrolled fecal soiling and leads to passive fecal incontinence (FI). The study is focused on immediate and long-term safety and potential efficacy of bioengineered IAS BioSphincters to treat passive FI in a clinically relevant large animal model of passive FI. Passive FI was successfully developed in Non-Human Primates (NHPs) model. The implantation of autologous intrinsically innervated functional constructs resolved the fecal soiling, restored the resting pressure and Recto Anal Inhibitory Reflex (RAIR) within 1-month. These results were sustained with time, and efficacy was preserved up to 12-months. The histological studies validated manometric results with the regeneration of a well-organized neuro-muscular population in IAS. The control groups (non-treated and sham) remained affected by poor anal hygiene, lower resting pressure, and reduced RAIR throughout the study. The pathological assessment of implants, blood, and the vital organs confirmed biocompatibility without any adverse effect after implantation. This regenerative approach of implanting intrinsically innervated IAS BioSphincters has the potential to offer a better quality of life to the patients suffering from FI.

VIP induces PKA-mediated rapid and sustained phosphorylation of HSP20.

The small molecular weight heat shock protein HSP20 has been proposed to regulate smooth muscle relaxation in a manner dependent on its phosphorylated state. We present the first evidence of HSP20 phosphorylation in response to a naturally occurring neurotransmitter. HSP20 was rapidly phosphorylated in colonic circular smooth muscle cells exposed to the physiologically relevant relaxant neuropeptide, Vasoactive Intestinal Peptide (VIP). HSP20 phosphorylation was significantly and substantially increased by 30s following VIP treatment and remained elevated for 30 min. VIP-induced HSP20 phosphorylation was dose dependent. Both basal and VIP-induced HSP20 phosphorylations were solely mediated by Protein Kinase A. Maximal phosphorylation of HSP20 was induced by the same VIP concentration range which induces maximal relaxation. Increased phosphorylation of HSP20 occurred in both cytosolic and particulate cell fractions. Our findings represent evidence for neurogenic modulation of the cyclic molecular regulation of relaxation required for peristalsis via a VIP-PKA-HSP20 pathway.