Tissue engineering for the treatment of short bowel syndrome in children.

Short bowel syndrome is a major cause of morbidity and mortality in children. Despite decades of experience in the management of short bowel syndrome, current therapy is primarily supportive. Definitive treatment often requires intestinal transplantation, which is associated with significant morbidity and mortality. In order to develop novel approaches to the treatment of short bowel syndrome, we and others have focused on the development of an artificial intestine, by placing intestinal stem cells on a bioscaffold that has an absorptive surface resembling native intestine, and taking advantage of neovascularization to develop a blood supply. This review will explore recent advances in biomaterials, vascularization, and progress toward development of a functional epithelium and mesenchymal niche, highlighting both success and ongoing challenges in the field.

Ancylostoma ceylanicum infective third-stage larvae are activated by co-culture with HT-29-MTX intestinal epithelial cells.

BACKGROUND: Human hookworm larvae arrest development until they enter an appropriate host. This makes it difficult to access the larvae for studying larval development or host-parasite interactions. While there are in vivo and in vitro animal models of human hookworm infection, there is currently no human, in vitro model. While animal models have provided much insight into hookworm biology, there are limitations to how closely this can replicate human infection. Therefore, we have developed a human, in vitro model of the initial phase of hookworm infection using intestinal epithelial cell culture. RESULTS: Co-culture of the human hookworm Ancylostoma ceylanicum with the mucus-secreting, human intestinal epithelial cell line HT-29-MTX resulted in activation of infective third-stage larvae, as measured by resumption of feeding. Larvae were maximally activated by direct contact with fully differentiated HT-29-MTX intestinal epithelial cells. HT-29-MTX cells treated with A. ceylanicum larvae showed differential gene expression of several immunity-related genes. CONCLUSIONS: Co-culture with HT-29-MTX can be used to activate A. ceylanicum larvae. This provides an opportunity to study the interaction of activated larvae with the human intestinal epithelium.

Microscale Bioreactors for in situ characterization of GI epithelial cell physiology.

The development of in vitro artificial small intestines that realistically mimic in vivo systems will enable vast improvement of our understanding of the human gut and its impact on human health. Synthetic in vitro models can control specific parameters, including (but not limited to) cell types, fluid flow, nutrient profiles and gaseous exchange. They are also "open" systems, enabling access to chemical and physiological information. In this work, we demonstrate the importance of gut surface topography and fluid flow dynamics which are shown to impact epithelial cell growth, proliferation and intestinal cell function. We have constructed a small intestinal bioreactor using 3-D printing and polymeric scaffolds that mimic the 3-D topography of the intestine and its fluid flow. Our results indicate that TEER measurements, which are typically high in static 2-D Transwell apparatuses, is lower in the presence of liquid sheer and 3-D topography compared to a flat scaffold and static conditions. There was also increased cell proliferation and discovered localized regions of elevated apoptosis, specifically at the tips of the villi, where there is highest sheer. Similarly, glucose was actively transported (as opposed to passive) and at higher rates under flow.

Electrochemical reverse engineering: A systems-level tool to probe the redox-based molecular communication of biology.

The intestine is the site of digestion and forms a critical interface between the host and the outside world. This interface is composed of host epithelium and a complex microbiota which is "connected" through an extensive web of chemical and biological interactions that determine the balance between health and disease for the host. This biology and the associated chemical dialogues occur within a context of a steep oxygen gradient that provides the driving force for a variety of reduction and oxidation (redox) reactions. While some redox couples (e.g., catecholics) can spontaneously exchange electrons, many others are kinetically "insulated" (e.g., biothiols) allowing the biology to set and control their redox states far from equilibrium. It is well known that within cells, such non-equilibrated redox couples are poised to transfer electrons to perform reactions essential to immune defense (e.g., transfer from NADH to O2 for reactive oxygen species, ROS, generation) and protection from such oxidative stresses (e.g., glutathione-based reduction of ROS). More recently, it has been recognized that some of these redox-active species (e.g., H2O2) cross membranes and diffuse into the extracellular environment including lumen to transmit redox information that is received by atomically-specific receptors (e.g., cysteine-based sulfur switches) that regulate biological functions. Thus, redox has emerged as an important modality in the chemical signaling that occurs in the intestine and there have been emerging efforts to develop the experimental tools needed to probe this modality. We suggest that electrochemistry provides a unique tool to experimentally probe redox interactions at a systems level. Importantly, electrochemistry offers the potential to enlist the extensive theories established in signal processing in an effort to "reverse engineer" the molecular communication occurring in this complex biological system. Here, we review our efforts to develop this electrochemical tool for in vitro redox-probing.

Generation of an artificial intestine for the management of short bowel syndrome.

PURPOSE OF REVIEW: This article discusses the current state of the art in artificial intestine generation in the treatment of short bowel syndrome. RECENT FINDINGS: Short bowel syndrome defines the condition in which patients lack sufficient intestinal length to allow for adequate absorption of nutrition and fluids, and thus need parenteral support. Advances toward the development of an artificial intestine have improved dramatically since the first attempts in the 1980s, and the last decade has seen significant advances in understanding the intestinal stem cell niche, the growth of complex primary intestinal stem cells in culture, and fabrication of the biomaterials that can support the growth and differentiation of these stem cells. There has also been recent progress in understanding the role of the microbiota and the immune cells on the growth of intestinal cultures on scaffolds in animal models. Despite recent progress, there is much work to be done before the development of a functional artificial intestine for short bowel syndrome is successfully achieved. SUMMARY: Continued concerted efforts by cell biologists, bioengineers, and clinician-scientists will be required for the development of an artificial intestine as a clinical treatment modality for short bowel syndrome.

Development of a synthetic receptor protein for sensing inflammatory mediators interferon-γ and tumor necrosis factor-α.

Intestinal inflammation has been implicated in a number of diseases, including diabetes, Crohn's disease, and irritable bowel syndrome. Important components of inflammation are interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), which are elevated both on the luminal and submucosal sides of the intestinal epithelial barrier in several diseases. Here, we developed a novel Escherichia coli based detection system for IFN-γ and TNF-α comprised of a chimeric protein and a simple signal transduction construct, which could be deployed on the luminal side of the intestine. OmpA of E. coli was engineered to detect IFN-γ or TNF-α through the replacement of extracellular loops with peptide fragments from OprF of P. aeruginosa. OmpA/OprF chimeras were developed, capable of binding IFN-γ or TNF-α. The specific peptide fragments that bind IFN-γ were identified. IFN-γ or TNF-α binding the OmpA/OprF chimera induced the pspA promoter, driving ß-galactosidase production. The OmpA/OprF chimera had a detection limit of 300 pM for IFN-γ and 150 pM for TNF-α. This work will further the development of bacteria based therapeutics for the treatment of inflammatory diseases of the gut.

Intestinal stem cell growth and differentiation on a tubular scaffold with evaluation in small and large animals.

AIMS: To investigate the growth and differentiation of intestinal stem cells on a novel tubular scaffold in vitro and in vivo. MATERIALS & METHODS: Intestinal progenitor cells from mice or humans were cultured with myofibroblasts, macrophages and/or bacteria, and evaluated in mice via omental implantation. Mucosal regeneration was evaluated in dogs after rectal mucosectomy followed by scaffold implantation. RESULTS: Intestinal progenitor cells differentiated into crypt-villi structures on the scaffold. Differentiation and scaffold coverage was enhanced by coculture with myofibroblasts, macrophages and probiotic bacteria, while the implanted scaffolds enhanced mucosal regeneration in the dog rectum. CONCLUSION: Intestinal stem cell growth and differentiation on a novel tubular scaffold is enhanced through addition of cellular and microbial components, as validated in mice and dogs.

Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes.

The inactive full-length form of GLP-1(1-37) stimulates conversion of both rat and human intestinal epithelial cells into insulin-secreting cells. We investigated whether oral administration of human commensal bacteria engineered to secrete GLP-1(1-37) could ameliorate hyperglycemia in a rat model of diabetes by reprogramming intestinal cells into glucose-responsive insulin-secreting cells. Diabetic rats were fed daily with human lactobacilli engineered to secrete GLP-1(1-37). Diabetic rats fed GLP-1-secreting bacteria showed significant increases in insulin levels and, additionally, were significantly more glucose tolerant than those fed the parent bacterial strain. These rats developed insulin-producing cells within the upper intestine in numbers sufficient to replace ∼25-33% of the insulin capacity of nondiabetic healthy rats. Intestinal tissues in rats with reprogrammed cells expressed MafA, PDX-1, and FoxA2. HNF-6 expression was observed only in crypt epithelia expressing insulin and not in epithelia located higher on the villous axis. Staining for other cell markers in rats treated with GLP-1(1-37)-secreting bacteria suggested that normal function was not inhibited by the close physical proximity of reprogrammed cells. These results provide evidence of the potential for a safe and effective nonabsorbed oral treatment for diabetes and support the concept of engineered commensal bacterial signaling to mediate enteric cell function in vivo.

Rapid and repeatable redox cycling of an insoluble dietary antioxidant: electrochemical analysis.

There are many unresolved questions concerning the health benefits of dietary antioxidants due in part to the complexity of the materials and mechanisms of action. We applied a new electrochemical method and report new observations for one of the richest sources of dietary antioxidants. We observed that the insoluble fraction of clove is redox-active and can be rapidly and repeatedly switched between oxidized and reduced states. Also, the radical scavenging antioxidant properties of insoluble clove are largely independent of this reversible redox activity, which is similar to observations made with the natural phenolic melanin. In contrast to melanin, insoluble clove was observed to have little pro-oxidant activity (as measured by H2O2 generation) irrelevant to whether it was poised in an oxidized or reduced state. These results suggest that dietary antioxidants, even when insoluble and nonabsorbed, can undergo important redox interactions in the intestinal tract.

3-D intestinal scaffolds for evaluating the therapeutic potential of probiotics.

Biomimetic in vitro intestinal models are becoming useful tools for studying host-microbial interactions. In the past, these models have typically been limited to simple cultures on 2-D scaffolds or Transwell inserts, but it is widely understood that epithelial cells cultured in 3-D environments exhibit different phenotypes that are more reflective of native tissue, and that different microbial species will preferentially adhere to select locations along the intestinal villi. We used a synthetic 3-D tissue scaffold with villous features that could support the coculture of epithelial cell types with select bacterial populations. Our end goal was to establish microbial niches along the crypt-villus axis in order to mimic the natural microenvironment of the small intestine, which could potentially provide new insights into microbe-induced intestinal disorders, as well as enabling targeted probiotic therapies. We recreated the surface topography of the small intestine by fabricating a biodegradable and biocompatible villous scaffold using poly lactic-glycolic acid to enable the culture of Caco-2 with differentiation along the crypt-villus axis in a similar manner to native intestines. This was then used as a platform to mimic the adhesion and invasion profiles of both Salmonella and Pseudomonas, and assess the therapeutic potential of Lactobacillus and commensal Escherichia coli in a 3-D setting. We found that, in a 3-D environment, Lactobacillus is more successful at displacing pathogens, whereas Nissle is more effective at inhibiting pathogen adhesion.

Synthetic small intestinal scaffolds for improved studies of intestinal differentiation.

In vitro intestinal models can provide new insights into small intestinal function, including cellular growth and proliferation mechanisms, drug absorption capabilities, and host-microbial interactions. These models are typically formed with cells cultured on 2D scaffolds or transwell inserts, but it is widely understood that epithelial cells cultured in 3D environments exhibit different phenotypes that are more reflective of native tissue. Our focus was to develop a porous, synthetic 3D tissue scaffold with villous features that could support the culture of epithelial cell types to mimic the natural microenvironment of the small intestine. We demonstrated that our scaffold could support the co-culture of Caco-2 cells with a mucus-producing cell line, HT29-MTX, as well as small intestinal crypts from mice for extended periods. By recreating the surface topography with accurately sized intestinal villi, we enable cellular differentiation along the villous axis in a similar manner to native intestines. In addition, we show that the biochemical microenvironments of the intestine can be further simulated via a combination of apical and basolateral feeding of intestinal cell types cultured on the 3D models.

On chip porous polymer membranes for integration of gastrointestinal tract epithelium with microfluidic 'body-on-a-chip' devices.

We describe a novel fabrication method that creates microporous, polymeric membranes that are either flat or contain controllable 3-dimensional shapes that, when populated with Caco-2 cells, mimic key aspects of the intestinal epithelium such as intestinal villi and tight junctions. The developed membranes can be integrated with microfluidic, multi-organ cell culture systems, providing access to both sides, apical and basolateral, of the 3D epithelial cell culture. Partial exposure of photoresist (SU-8) spun on silicon substrates creates flat membranes with micrometer-sized pores (0.5-4.0 µm) that--supported by posts--span across 50 µm deep microfluidic chambers that are 8 mm wide and 10 long. To create three-dimensional shapes the membranes were air dried over silicon pillars with aspect ratios of up to 4:1. Space that provides access to the underside of the shaped membranes can be created by isotropically etching the sacrificial silicon pillars with xenon difluoride. Depending on the size of the supporting posts and the pore sizes the overall porosity of the membranes ranged from 4.4 % to 25.3 %. The microfabricated membranes can be used for integrating barrier tissues such as the gastrointestinal tract epithelium, the lung epithelium, or other barrier tissues with multi-organ "body-on-a-chip" devices.

Microscale 3-D hydrogel scaffold for biomimetic gastrointestinal (GI) tract model.

Here we describe a simple and efficient method for fabricating natural and synthetic hydrogels into 3-D geometries with high aspect ratio and curvature. Fabricating soft hydrogels into such shapes using conventional techniques has been extremely difficult. Combination of laser ablation and sacrificial molding technique using calcium alginate minimizes the stress associated with separating the mold from the hydrogel structure, and therefore allows fabrication of complex structures without damaging them. As a demonstration of this technique, we have fabricated a microscale collagen structure mimicking the actual density and size of human intestinal villi. Colon carcinoma cell line, Caco-2 cells, was seeded onto the structure and cultured for 3 weeks until the whole structure was covered, forming finger-like structures mimicking the intestinal villi covered with epithelial cells. This method will enable construction of in vitro tissue models with physiologically realistic geometries at microscale resolutions.

Secretion of insulinotropic proteins by commensal bacteria: rewiring the gut to treat diabetes.

Here, we show that commensal bacteria can stimulate intestinal epithelial cells to secrete insulin in response to glucose. Commensal strains were engineered to secrete the insulinotropic proteins GLP-1 and PDX-1. Epithelia stimulated by engineered strains and glucose secreted up to 1 ng ml(-1) of insulin with no significant background secretion.

Fluorescently labeled liposomes for monitoring cholera toxin binding to epithelial cells.

Vibrio cholerae, the causative agent for cholera, expresses a toxin required for virulence consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of pathogenic V. cholerae and binds to the G(M1) ganglioside on the surface of epithelial cells. To study V. cholerae virulence (CTB expression) in the presence of human epithelia, we devised an inexpensive, simple, and rapid method for quantifying CTB bound on epithelial surfaces in microtiter plates. G(M1) ganglioside was incorporated into the lipid bilayer of liposomes both encapsulating the fluorescent dye sulforhodamine B (SRB) and with SRB tagged to lipids in the bilayer (BEGs). In addition, G(M1)-embedded liposomes encapsulating SRB only (EGs) and with SRB in their bilayers only (BGs) were synthesized. The three types of liposomes were compared with respect to their efficacy for both visualizing and quantifying CTB attached to the surface of Caco-2 cells. The BEGs were the most effective overall, providing both visualization under a fluorescence microscope and quantification after lysis in a microtiter plate reader. A limit of detection corresponding to 0.2 8microg/ml applied CTB was attained for the on-cell assay using the microtiter plate reader approach, whereas as low as 2 microg/ml applied CTB could be observed under the fluorescence microscope.

Interrupting Vibrio cholerae infection of human epithelial cells with engineered commensal bacterial signaling.

Vibrio cholerae El Tor serotypes are largely responsible for outbreaks of cholera in the developing world. The infection cycle for some strains of V. cholerae is coordinated, at least in part, through quorum sensing. That is, the expression of virulence genes depends on the concentration of V. cholerae autoinducers cholera autoinducer 1 (CAI-1) and autoinducer 2 (AI-2). High concentrations of CAI-1 and AI-2 have been shown previously to inhibit virulence gene expression. We have demonstrated here that a commensal bacterium, E. coli Nissle 1917 (Nissle), can be engineered to express CAI-1 (Nissle expresses AI-2 natively) and effectively interrupt V. cholerae virulence. We engineered Nissle to express CAI-1 under control of the lac promoter, and demonstrated inhibition of V. cholerae expression of cholera toxin (CT, as indicated by presence of the CT subunit B (CTB)) and of the toxin co-regulated pilus (TCP, as indicated by the relative transcript of TCP subunit A (TCPA)) in both monocultures of V. cholerae and co-cultures with epithelial cells, Nissle, and V. cholerae. In the model system of Caco-2 epithelia incubated with V. cholerae, we demonstrated that co-cultures with Nissle expressing CAI-1 activity reduced CTB binding to Caco-2 cells by 63% over co-cultures with wild-type Nissle. Further, cultures with Nissle expressing CAI-1 had significantly lower TCPA transcription than controls with wild-type Nissle. These results represent a significant step towards a prophylactic method for combating enteric disease through engineered quorum signaling within a commensal bacterial strain.

Engineering eukaryotic signal transduction with RNAi: enhancing Drosophila S2 cell growth and recombinant protein synthesis via silencing of TSC1.

RNAi has been useful in the study of biochemical pathways, but has not been widely used as a tool in metabolic engineering. The work described here makes use of double-stranded RNA (dsRNA) for the post-transcriptional gene silencing of TSC1 in Drosophila S2 cells. TSC1 downregulates the insulin-mediated signal transduction pathway, and serves as a metabolic control to guard against cellular overproliferation and tumorogenesis in both flies and mammals. By silencing TSC1 with in vitro-synthesized dsRNA, we have created a tunable and specific metabolic "throttle" that, like insulin, apparently increases the specific growth rate of S2 cells in a dose-dependent manner. This "throttle," augments the benefits of insulin addition while apparently avoiding deleterious and pleiotropic effects which can lead to lysis. During the period wherein dsRNA was active, cell growth rate was increased by 11% by the addition of 15 microg/mL dsTSC1 and by over 20% by the addition of 30 microg/mL dsTSC1. Additionally, synthesis of recombinant green fluorescent protein (GFP) was increased nearly 50% in a stable S2 cell line inducibly expressing GFP. Accordingly, we have "tuned" a normally tumorogenic pathway in animals into an advantage for both growth and recombinant product synthesis in cell culture. Potential applications for improving eukaryotic cell culture are anticipated.

luxS-dependent gene regulation in Escherichia coli K-12 revealed by genomic expression profiling.

The bacterial quorum-sensing autoinducer 2 (AI-2) has received intense interest because the gene for its synthase, luxS, is common among a large number of bacterial species. We have identified luxS-controlled genes in Escherichia coli under two different growth conditions using DNA microarrays. Twenty-three genes were affected by luxS deletion in the presence of glucose, and 63 genes were influenced by luxS deletion in the absence of glucose. Minimal overlap among these gene sets suggests the role of luxS is condition dependent. Under the latter condition, the metE gene, the lsrACDBFG operon, and the flanking genes of the lsr operon (lsrR, lsrK, tam, and yneE) were among the most significantly induced genes by luxS. The E. coli lsr operon includes an additional gene, tam, encoding an S-adenosyl-l-methionine-dependent methyltransferase. Also, lsrR and lsrK belong to the same operon, lsrRK, which is positively regulated by the cyclic AMP receptor protein and negatively regulated by LsrR. lsrK is additionally transcribed by a promoter between lsrR and lsrK. Deletion of luxS was also shown to affect genes involved in methionine biosynthesis, methyl transfer reactions, iron uptake, and utilization of carbon. It was surprising, however, that so few genes were affected by luxS deletion in this E. coli K-12 strain under these conditions. Most of the highly induced genes are related to AI-2 production and transport. These data are consistent with the function of LuxS as an important metabolic enzyme but appear not to support the role of AI-2 as a true signal molecule for E. coli W3110 under the investigated conditions.