Current understanding of the interplay between extracellular matrix remodelling and gut permeability in health and disease.

The intestinal wall represents an interactive network regulated by the intestinal epithelium, extracellular matrix (ECM) and mesenchymal compartment. Under healthy physiological conditions, the epithelium undergoes constant renewal and forms an integral and selective barrier. Following damage, the healthy epithelium is restored via a series of signalling pathways that result in remodelling of the scaffolding tissue through finely-regulated proteolysis of the ECM by proteases such as matrix metalloproteinases (MMPs). However, chronic inflammation of the gastrointestinal tract, as occurs in Inflammatory Bowel Disease (IBD), is associated with prolonged disruption of the epithelial barrier and persistent damage to the intestinal mucosa. Increased barrier permeability exhibits distinctive signatures of inflammatory, immunological and ECM components, accompanied by increased ECM proteolytic activity. This narrative review aims to bring together the current knowledge of the interplay between gut barrier, immune and ECM features in health and disease, discussing the role of barrier permeability as a discriminant between homoeostasis and IBD.

An in vitro model to study immune activation, epithelial disruption and stromal remodelling in inflammatory bowel disease and fistulising Crohn's disease.

At present, preclinical models of inflammatory bowel disease (IBD) are insufficient, limiting translation between research and new therapeutics. This is especially true for fistulising Crohn's disease (CD), as the severe lack of relevant models hinders research progression. To address this, we present in vitro human IBD mucosal models that recapitulate multiple pathological hallmarks of IBD simultaneously in one model system - immune cell infiltration, stromal remodelling and epithelial disruption. Stimulation of models induces epithelial aberrations common in IBD tissue including altered morphology, microvilli abnormalities, claudin gene expression changes and increased permeability. Inflammatory biomarkers are also significantly increased including cytokines and chemokines integral to IBD pathogenesis. Evidence of extracellular matrix remodelling, including upregulated matrix-metalloproteinases and altered basement membrane components, suggests the models simulate pathological stromal remodelling events that closely resemble fistulising CD. Importantly, MMP-9 is the most abundant MMP and mimics the unique localisation observed in IBD tissue. The inflamed models were subsequently used to elucidate the involvement of TNF-α and IFN- γ in intestinal stromal remodelling, in which TNF-α but not IFN- γ induced MMP upregulation, specifically of MMP-3 and MMP-9. Collectively, our results demonstrate the potential of the IBD models for use in preclinical research in IBD, particularly for fistulising CD.

Applying Stirred Perfusion to 3D Tissue Equivalents to Mimic the Dynamic In Vivo Microenvironment.

Complex three-dimensional (3D) tissue equivalents have been widely developed with applications with a multitude of organs and tissues. While these systems lead to significant improvements over conventional two-dimensional culture, the static conditions of the surrounding medium still present a limitation to the physiological relevance of these models. Medium perfusion and convective mixing can be introduced to these models through a variety of techniques using equipment such as pumps and rockers. These systems can easily become very complex or suffer from limited control over the fluid flow properties. We have developed a bioreactor enabling controlled perfusion of 3D tissue equivalents utilizing a magnetic stirrer-based system, allowing for scalability and ease of use. This system has demonstrated potential applications in a range of tissues such as the liver, intestine, and skin, with many other potential applications yet to be tested. Our solution provides users with a low cost and easy to use alternative to complex bioreactor systems while still providing high levels of control over fluid flow and structural properties of the tissue constructs.

Use of Porous Polystyrene Scaffolds to Bioengineer Human Epithelial Tissues In Vitro.

In vitro epithelial models are valuable tools for both academic and industrial laboratories to investigate tissue physiology and disease. Epithelial tissues comprise the surface epithelium, basement membrane, and underlying supporting stromal cells. There are various types of epithelial tissue and they have a diverse and intricate architecture in vivo, which cannot be successfully recapitulated using two-dimensional (2D) cell culture. Tissue engineering strategies can be applied to bioengineer the organized, multilayered, and multicellular structure of epithelial tissues in vitro. Alvetex® is a porous, polystyrene scaffold that enables fibroblasts to synthesize a complex network of endogenous, humanized extracellular matrix proteins. This creates a physiologically relevant three-dimensional (3D) subepithelial microenvironment, enriched with mechanical and chemical cues, which supports the organization and differentiation of epithelial cells. Such technology has been used to bioengineer different epithelial architectures in vitro, including the simple, columnar structure of the intestine and the stratified, squamous, and keratinized structure of skin. Epithelial tissue models provide a useful platform for fundamental and translational research, with multifaceted applications including disease modeling, drug discovery, and product development.

Bioengineering Novel in vitro Co-culture Models That Represent the Human Intestinal Mucosa With Improved Caco-2 Structure and Barrier Function.

The Caco-2 monolayer is the most widely used in vitro model of the human intestinal mucosa to study absorption. However, models lack communication from other cells present in the native intestine, such as signals from fibroblasts in the lamina propria. In this study, we have investigated the effects of fibroblasts upon the Caco-2 epithelium through two mechanisms: indirect signaling from fibroblasts and direct contact with fibroblasts. Culture of Caco-2 cells with paracrine signals from fibroblasts, through the use of conditioned media, did not induce a significant change in epithelial cell morphology or function. To examine the effects of direct contact between the epithelium and fibroblasts, we developed novel, humanized three-dimensional (3D) co-culture models whereby Caco-2 cells are grown on the surface of a subepithelial-like tissue construct containing intestinal or dermal fibroblasts. In our models, we observed endogenous extracellular matrix production from the fibroblasts that provides support to the above epithelium. The Caco-2 epithelium displayed morphological changes in 3D co-culture including enhanced polarization and the formation of a basement membrane-like attachment to the underlying stromal compartment. An important structural alteration was the significantly straightened lateral membrane that closely mimics the structure of the in vivo intestinal mucosa. This enhanced lateral membrane phenotype, in correlation with an reduction in TEER to levels more similar to the human intestine, is thought to be responsible for the increased paracellular permeability observed in 3D co-cultures. Our results demonstrate that direct contact between epithelial and mesenchymal cells results in an enhanced epithelial barrier. The in vitro models described herein have the potential to be used for studying intestinal epithelial-fibroblast interactions and could provide more accurate tools for drug permeability studies.