Perspectives on the extracellular matrix in inflammatory bowel disease and bowel decellularization protocols

Abstract

The extracellular matrix (ECM) is a non-cellular three-dimensional structure present in all tissues that is essential for the intestinal maintenance, function and structure, as well as for providing physical support for tissue integrity and elasticity. ECM enables the regulation of various processes involved in tissue homeostasis, being vital for healing, growth, migration and cell differentiation. Structurally, ECM is composed of water, polysaccharides and proteins, such as collagen fibers and proteoglycans, which are specifically arranged for each tissue. In pathological scenarios, such as inflammatory bowel disease (IBD), the deposition and remodeling of the ECM can be altered in relation to the homeostatic composition. IBD, such as Ulcerative colitis and Crohn’s disease, can be differentiated according to ECM alterations, such as circulating levels of collagen, laminin and vimentin neoepitopes. In this context, ECM presents particularities in both physiological and pathological processes, however, exploring methods of tissue decellularization is emerging as a promising frontier for new therapeutic interventions and clinical protocols, promoting the development of new approaches to intestinal diseases.

Key Words: Extracellular matrix; Inflammatory bowel disease; Decellularization protocols; Regenerative medicine; Intestine

Core Tip: Regenerative medicine provides a promising perspective in the treatment of inflammatory bowel diseases (IBD) by targeting tissue repair. The development of decellularization techniques to produce scaffolds that mimic the native environment of the intestine is crucial to the advances in this field. This article addresses how research focused on the extracellular matrix in IBD tissues and studies ways to improve regenerative therapies that represent fundamental steps towards furthering the efficacy and safety of IBD treatments.

INTRODUCTION

The extracellular matrix (ECM) plays a fundamental role in intestinal maintenance, function and structure[1]. It has a non-cellular three-dimensional conformation and is present in all tissues, with each tissue having a unique composition. Structurally, the ECM is composed of water, polysaccharides and proteins as well as collagen fibers and proteoglycans[2].

The ECM has several functions, including providing physical support for cell integrity, tissue elasticity and remodeling to control tissue homeostasis[3-5]. The molecular interaction between ECM and cells is complex and reciprocal, allowing the regulation of various processes involved in tissue homeostasis and in the wound healing process, as well as taking part in cell growth, migration, differentiation and in the production of neo-ECM[6]. Despite this, it is often neglected in relation to both its physiological functions and complications. Regarding injury and disease processes, matrix deposition and remodeling can be strongly altered according to the homeostasis composition. The pathological formation of the matrix may be associated with changes in the expression and activation of enzymes along with other factors that alter the matrix[7].

Inflammatory bowel diseases (IBD) are conditions characterized by an inadequate immune response, presenting chronic inflammation with symptomatic moments and clinical remission. The most prevalent forms of IBD are Crohn’s disease (CD) and Ulcerative colitis (UC)[8]. UC affects the superficial mucosa, starting in the rectum and being limited to the colon, while CD causes transmural inflammation and can affect any region of the gastrointestinal tract[9].

Regenerative medicine seeks to develop approaches to replace or regenerate tissues and organs that have lost their functional effectiveness, as well as to treat tissue injuries. Scaffolds are therefore responsible for providing a support structure for cells, growth factors, signaling molecules and offering structural, biochemical and biomechanical properties capable of guiding as well as regulating cell behavior and tissue development[10]. This brief review proposes tissue bioengineering as a relevant technique in regenerative medicine for IBD, considering the aspects of the ECM in tissues presenting IBD as well as different protocols used to obtain a scaffold in order to properly remove cells, DNA and cell contents.

ECM AND COMPONENTS

ECM is recognized by its role as a support for delicate cells of the body, as well as for providing mechanical properties for each function[11]. In addition, it supplies the chemical and mechanical signals necessary for the regulation of cell proliferation, migration, survival and differentiation, crucial to maintain homeostasis and proper tissue function[12,13]. The ECM can be classified into two types, according to its components and structure: The basement membrane and the interstitial matrix surrounding the cells, and the pericellular matrix, which is in close contact with the cells[14]. The interstitial matrix is located in a thin layer below the basement membrane, consisting of four main components: Collagen IV, laminin, nidogen and perlecan[15]. The matrisome, proteins associated with the matrix, is made up of the main ECM constituents (collagens, glycoproteins, proteoglycans and polysaccharides), associated regulators and secreted factors. It is produced by unique combinations of resident cells found in different tissues[16].

Studies indicate that the mesenchyme is responsible for tissue differentiation, the matrisome being dynamically maintained and associated with various responses, such as cell growth and differentiation, through direct interactions between ligands and receptors. It is also responsible for the biomechanical properties, being able to sequester and regulate the availability of cytokines along with growth factors. Embryonic precursor cells grown on complex ECM structures from decellularized tissues are capable of differentiating into epithelia of the same type of tissue from which the structure was derived[16].

Fibroblasts are fundamental in parenchymal organs and are responsible for providing structure, regulation, survival, differentiation as well as migration of cells. Thus, fibroblasts support the architecture of the mucosal crypt, the remodeling of the ECM and the efficiency of the immune system in the intestine[17-19]. In general terms, ECM components, such as collagens, represent the most abundant family of proteins found in the ECM. Collagens are proteins that form a triple helix of three polypeptide chains, forming supramolecular structures in the ECM that vary in size, function and distribution throughout the tissues[20].

The elastic fibre is a specialized structure of connective tissue and its main component is elastin, a structural glycoprotein. However, different proportions of elastin and microfibrils mediate different functions adaptable to each local tissue demand. Mature elastic fibers are responsible for the reversible distension of many elastic tissues and ligaments, being synthesized by fibroblasts, chondrocytes and hydrolyzed by elastases[21]. ECM regulators such as family members are classified into subgroups of mainly collagenases, stromelysins, matrilysins, membrane-type metalloproteinases and gelatinases[22].

Proteoglycans are among the structural and functional biomacromolecules of the tissue and are made up of a protein core covalently linked to one or more glycosaminoglycan chains of either the same type or different. In addition, glycosaminoglycans are long, negatively charged heteropolysaccharides containing disaccharides comprising: N-acetylated hexosamines (N-acetyl-D-galactosamine or N-acetyl-D-glucosamine) and D-/L-hexuronic acid (D-glycuronic acid or L-iduronic acid). Six types of glycosaminoglycans are recognized: The chondroitin sulphate/dermatan sulphate galactosaminoglycans, and the heparan sulphate, heparin, keratan sulphate and hyaluronic acid glycosaminoglycans[23].

Metalloproteinases are zinc-dependent[24-27] composed of 24 families grouped by domain structure and substrate preference, being able to degrade matrix components such as cytokines, chemokines, adhesion molecules, growth factors and their receptors, which is why they are important for the homeostasis processes of ECM remodeling. They also contribute to angiogenesis, cell migration, tissue repair and inflammation[24]. The family of metalloproteinases have common structural and functional elements, with substrate specificity and affinity, being divided into different classes such as collagenases [matrix metalloproteinase (MMP)-1, -8 and -13], gelatinases (MMP-2 and -9), stromelysins (MMP-3, -10, -11 and -19), matrilysins (MMP-7 and -26), membrane-type MMPs (MT-MMPs), macrophage elastase (MMP-12), among others[24].

In normal tissues, the expression of collagenases is low and they are upregulated in inflammation processes linked to tissue destruction. Since they are potentially dangerous to tissues, their activity and inhibition are highly regulated by tissue inhibitors of metalloproteinases (TIMPs). MMPs are synthesized and secreted rapidly, and may be stored in granules of inflammatory cells with release upon stimulation[24]. MMPs are controlled by three different pathways: Regulation of transcription, inhibition by specific inhibitors or proenzyme activation[26].

ECM COMPONENTS IN THE GASTROINTESTINAL TRACT

Fibroblasts are present in the intestinal compartment and are responsible for producing ECM components, while myofibroblasts are fibroblasts with smooth muscle cell properties, such as the expression of alpha-smooth muscle actin, which plays a role in smooth muscle contraction[28]. They may be present and support the architecture of mucosal crypts, ECM remodeling and immune fitness. They provide support to the architecture of the crypts in the colon by creating discrete anatomical zones that maintain epithelial stem cell niches in defined areas, while differentiating epithelial cells and inhibiting cell proliferation in others[29]. Fibroblasts are recognized as the main source of MMPs in the intestine, furthermore, interleukin (IL)-21 is part of the regulation of intestinal fibroblasts synthesis and the production of MMPs increases as a result of the cells’ cytokine production[30].

MMPs and TIMPs (MMPs tissue inhibitors) are expressed by different cell types in all layers of the intestine and are important in the remodeling process of the ECM in the intestine[31,32]. In normal tissue, epithelial cells express mainly MMP-1, -3, -7, -10 and -12, while mononuclear cells express MMP-2, -3, -9 and TIMP-1. Polymorphonuclear cells express only MMP-9 and fibroblasts just express MMP-2[33].

Type IV collagen, described as a basement membrane collagen, performs the structural function of the intestine and is the most abundant basement membrane collagen. The α1, α2, α5, and α6 chains of collagen IV are the most abundant in the intestine, while the α3 and α4 chains present in the basement membrane of the intestinal epithelium are only found on the surface of the mucosa[34,35]. Type IV collagen is found at the interface between the basement membrane and the interstitial matrix, separating the two layers. It binds to various proteins in the ECM, the basement membrane and the interstitial matrix, such as type IV collagen, perlecan, decorin and fibronectin[36].

In one study, the expression of collagen chains was examined in both human and animal small intestines[37]. The classic α1 and α2 (IV) chains were found to be individually distributed in the basement membrane throughout the epithelium, as well as in cellular elements present in the fetal and adult small intestine’s lamina propria, and α5 was found in the mucosa of the fetal small intestine. However, the α3 and α4 (IV) chains were not identified in the intestine, which is consistent with the restricted distribution of these chains in tissues[34].

ECM IN IBD

Cells are not able to grow in solutions and need to be anchored to a solid matrix. This way, cells can bind to receptors such as integrins, which are responsible for the cells’ perception of the ECM rigidity and thus alter the intracellular state, hence verifying that the ECM is capable of altering the behavior of cells, including differentiation[28]. During chronic inflammation, the excessive degradation and inadequate deposition of ECM by proteases makes it impossible to restore the matrix, a process that is called fibrosis, and may be responsible for the development of cancer. Therefore, ECM has a great influence on cancer development and ECM remodeling is fundamental to the development of cancer as a consequence of chronic inflammation[38-42].

IBD are chronic and relapsing diseases. In CD, inflammation can occur throughout the gastrointestinal tract in all layers of the intestine, but is most present in the ileum and colon[43]. In UC, there is a greater severity of inflammation and pathological alterations in the mucosa and submucosa. However, it is possible to observe that in intestinal diseases, an increased remodeling of the ECM is common due to the excessive and prolonged inflammatory response, interfering in the structure and functioning of the intestinal tissue’s ECM[44].

Fibrosis is a pathophysiological mechanism that promotes the deposition of connective tissue in the ECM as a consequence of injury[45], and is a serious complication in the intestine[46] (Figure 1). Seen mainly in CD, intestinal fibrosis occurs through the activation of fibroblasts. Excessive deposition of ECM impairs the gastrointestinal motility and can lead to intestinal stenosis and obstruction, which is a cause of morbidity and mortality in IBD. Collagen deposited in intestinal tissue can cause chronic hypoxia and stimulates neoangiogenesis through the positive regulation of the vascular endothelial growth factor, allowing more fibrosis to be deposited, which cannot be reversed by controlling inflammation[45].

Figure 1 Summary of changes in the extracellular matrix in inflammatory bowel disease. There is an increase in collagen fibres influenced by the action of metalloproteinases, contributing to the development of fibrosis. There is a reduction in elastic fibres and a change in glycosaminoglycans, affecting the elasticity and hydration of the tissue. ECM: Extracellular matrix; MMP: Matrix metalloproteinase; GAG’s: Glycosaminoglycans.

In general, mucosal inflammation influences MMP gene variation and deregulation promotes the development of fibrosis. The deposition of collagen fibres can also be observed, especially type I and type III, due to the high activity of fibroblasts and myofibroblasts. Biglican proteoglycan, which is involved in the assembly of collagen fibres, is also abnormally degraded and contributes to the fibrotic process. In addition, an accumulation of ECM fragments can be observed, which are released during the remodelling process and cause deterioration of intestinal function in IBD patients[47].

Furthermore, it is possible to observe that, during inflammatory processes, alterations in the composition of the ECM lead to a greater affinity for leukocyte binding and a greater retention of immune cells in the inflammation, despite the possibility of the matrix metalloproteinases production by immune cells and the consequent ECM degradation, however, the deregulated activity and inhibition of metalloproteinases by their inhibitors can lead to serious complications such as fistulas and fibrosis[24].

In IBD, it is possible to observe regulatory molecular mechanisms in the maintenance of the ECM integrity, such as hyaluronan, a non-sulfated branched glycosaminoglycan that participates in wound healing processes, in the migration and proliferation of cells and in the modulation of the inflammatory process, which means that there is a glycosaminoglycan increase during IBD[25]. Overexpression of tenascin-C, a protein that mediates the inflammatory process, is also observed in IBDs. Studies with tenascin-C knockout animals have shown that the absence of this protein can trigger an anti-inflammatory and tissue regenerative process[48]. Tenascin-C has already been described in other studies demonstrating its expression in the adult intestine, which is one of the few organs in which tenascin remains at maturity[37,49], as well as being present in IBD such as UC[49]. Other alterations in the ECM may be correlated with the circulating levels of collagen, laminin and vimentin neoepitopes, which correlate with IBD subtypes and may act as a differentiation mechanism between UC and CD[7].

Treatment with dextran sulfate sodium, an inducer of IBD, causes an increase in the levels of collagen I, integrins and focal adhesion kinases which may impair intestinal repair after treatment with dextran sulfate sodium. It is suggested that Yes-associated protein (YAP) is essential for the detection of ECM properties during the regenerative process, since focal adhesion kinases inhibition is responsible for the YAP decrease. It can be seen that ECM detection is fundamental for modulating the intestinal regeneration response[28].

The main proteases in intestinal tissue responsible for remodeling the matrix are neutrophil elastases and meprins. The expression of periostin is higher when compared to normal tissue, and it may be deposited in the stromal component in IBD as well as in tumors, in addition, YAP/TAZ has also been shown to be significantly increased in colorectal tumors and weakly increased in inflammatory diseases[50].

In CD, the inflamed mucosa presents a lower gene expression of MMP-2, MMP-9[51] and MMP-3[24] than in UC. In another publication, there was an overexpression of MMP-1, -3, -9, and -12 in UC when compared to CD[52]. Some studies have indicated the role in the induction of MMPs in IBD, based on this, IL-17A and IL-17F may be associated with an increased secretion of MMP-1 and -3 in the subepithelial myofibroblasts, it may also increase the actions of IL-1 and β and tumor necrosis factor-α on MMPs being mediated by mitogen-activated protein kinases[27].

TISSUE BIOENGINEERING

When a tissue suffers an injury, it can be said that the ECM is also damaged, which impairs the functional support of the tissue repair process and results in the formation of scar tissue[53], which can become an aggravating factor for an effective treatment. In this context, tissue bioengineering has been seen as an alternative to free patients from these exhausting treatments, offering new perspectives for tissue repair[54].

A challenge in this process is to obtain an ECM with suitable physicochemical properties and the complex biological characteristics native to ECM for functional tissue production. These necessary physicochemical properties consist of surface structure, pore size, mechanical properties, biocompatibility, biodegradability and cell adhesion[6]. The main product of the decellularization process is the ECM, so it must act as the physical structure in which the cells are integrated, as well as regulate processes such as growth, differentiation, migration and morphogenesis[12]. In order to decellularize tissues, there are enzymatic, chemical and/or physical processes for removing cells from the ECM[2]. The complete removal of DNA, the preservation of the tissue’s specific mechanical properties, along with the maintenance of the structural and functional proteins of the ECM are of fundamental importance for the process effectiveness. In this process, a large number of cells and immunogenic molecules must be removed, while proteins such as collagen, elastin, fibronectin and macromolecules such as proteoglycans and glycosaminoglycans must be maintained[2,55].

In the bioengineering of ECM biomaterials for the gastrointestinal tract, there is the challenge of mimicking the mucosal and muscular layers, as well as restoring motor functions so that proper gastrointestinal peristalsis occurs. It is therefore necessary to try to modulate chronic inflammation and stimulate the deposition of neo-ECM in the gastrointestinal tract[6]. Furthermore, in order to produce collagen, the cells need to have sufficient adhesion to the ECM for cell proliferation. Thus, a biomaterial that resembles the structure and functions of ECM is required[56].

Biomaterials produced from decellularized ECM are used in different reconstructive surgical applications and are increasingly applied in regenerative medicine strategies[57]. These biomaterials are capable of supporting specialized cell types, promoting a regenerative process and providing a microenvironment similar to the target tissue. For example, some biomaterials such as the submucosa of the small intestine and the matrix of the urinary bladder have been approved by the Food and Drug Administration for the manufacture of regenerative biomaterials with evidence in skin, muscle and gastrointestinal tissues[6].

Scaffolds are classified as either natural or synthetic. Natural scaffolds include natural polymers such as collagen, silk fibroin and hyaluronic acid, while synthetic scaffolds include synthetic polymers such as polyglycolic acid, polylactic acid, polylactic acid-co-glycolic acid and polycaprolactone. The natural ECM is an ideal biomaterial for tissue scaffolding. ECM provides support for the cells structure, regulates cell growth, proliferation and differentiation through the action of bioactive substances, growth factors and cytokines. Hence, natural ECM is composed of a variety of complex components such as proteins and glycosaminoglycans with a highly complex spatial structure and proportion of components, making therefore difficult to reconstitute with the same composition using conventional physicochemical methods[58]. Thus, decellularization is an effective method for the preparation of biomimetic ECM structures, and studies have shown that decellularized ECM scaffolds can remodel various tissues, such as for the regeneration of the heart, valves, blood vessels, skin, nerves, lungs, cornea, esophagus, as well as of the submucosa of the small intestine and various other types of tissues[58].

TECHNIQUES FOR DECELLULARIZING INTESTINAL TISSUE

There are different agents that act to aid the decellularization of each tissue, and it is necessary to investigate various factors such as cellularity, density and lipid content in order to choose the best agent. Ionic detergents, such as sodium dodecyl sulfate (SDS), solubilize cell membranes and dissociate proteins from the ECM, being highly effective at removing cells, although they may damage the ECM. Non-ionic detergents, such as Triton X-100, are less damaging to the ECM and can remove cell debris from thicker tissues; however, it may be necessary to combine detergents for better results[57]. In general, other agents take part in the decellularization process, such as deionized water, which is capable of causing hyposmotic shock in the cells[2].

Studies published by Kim et al[59] adapted decellularization techniques in the small intestine and stomachs of pigs based on the type (non-ionic or ionic) and time of treatment. In a first optimized protocol, they used Triton X-100, a non-ionic detergent, and observed the removal of cellular components along with the preserved components of the ECM. In a second protocol using sodium deoxycholate, an ionic detergent, the effective complete removal of cellular components was verified, however, it was not possible to preserve the structure of the ECM due to the glycosaminoglycans decrease. However, they observed that ionic detergents may be responsible for damaging the ECM and impairing the bioactivity and properties of ECM hydrogels[59].

On the other hand, other studies have indicated that the decellularization process used sodium deoxycholate in the distal neck of mice allowed the tissue to become opaque and translucent and completely removed the DNA, so there was no cell growth after the tissue was immersed in cell culture medium. However, the nature of the decellularization agents such as deionized water, sodium deoxycholate and DNAse used in the protocol were ideal for removing the cells as well as for preserving the ECM structure[2,60].

Another publication defining the protocol with 1% (v/v) Triton X-100 with 0.1% (v/v) ammonium hydroxide overnight in rat ileum showed that decellularization was successful, given the minimal amount of DNA present after the perfusion decellularization process, the tissue was translucent, as well as preserved villous structures on the luminal side of the tissue[61]. Another study in the small intestine of rats also using 1% (v/v) Triton X-100 with 0.1% (v/v) ammonium hydroxide in dH₂O for 24 hours followed by immersion in peracetic acid to sterilize the tissue found translucent tissue, preservation of the mesentery and macroscopic visualization and permeability of the vascular channels. There was almost total removal of cells and 97% removal of DNA after the decellularization process[62].

Decellularization by serial perfusion using SDS, triton X-100 and deionized water reduced the total DNA content per unit length of original small intestine to less than 3%, immunofluorescence confirmed the presence of major ECM proteins such as fibronectin, laminin and collagen I, also maintaining about 40% of the intestinal collagen, while reducing sulfated glycosaminoglycans and elastin[63]. Another study used 0.5% SDS in human fetal intestines for 12 hours in which it was possible to observe the preservation of the ECM structure and removal of cells, with no degradation of collagen and no nuclear content observed after decellularization[64].

In order to define a suitable protocol for the decellularization of submucosal tissue from the small intestine of goats, Singh et al[65] used four different types of protocols: In the first protocol, they used the ionic detergent 0.05% SDS and the non-ionic detergent 0.1% Triton-X 100, while the second used a degreaser (methanol/chloroform, 1:1 v/v), enzymatic digestion with 0.05% trypsin/0.05% EDTA and was incubated with 0.5% SDS in a 0.9% sodium chloride solution. The third protocol used 1% SDS, then was incubated in 1% triton-X 100 solution, however, the last one used incubation with a 1.0 M KI solution, followed by treatment with 0.1% TX-100. Although all the protocols decellularized the caprine SIS, the DP4 method stood out for preserving the ECM, presenting a greater viability and cell proliferation compared to the other methods[65]. Another publication used 2% SDS for 24 hours to decellularize the small intestine of goats. This way, they were able to observe a rapid and effective decellularization, as well as biocompatibility and cytocompatibility[54]. Despite the various techniques described (Table 1), it can be said that there are major challenges for the application of regenerative medicine procedures in clinical treatments, as well as the source of cells, meaning that further in-depth studies on the subject are needed[66].

Table 1 A summary of some of the agents used in the decellularisation process, including the type of agent, concentrations applied, treatment times, characteristics and effects on the tissue, and the preservation of the extracellular matrix.

Agent	Type	Concentration	Time	Characteristics and effects	Preservation of the ECM
Sodium dodecyl sulfate	Ionic detergent	0.05%-2%	12-24 hours	Highly effective at removing cells, solubilises cell membranes; can degrade ECM (especially glycosaminoglycans)	May degrade ECM, including collagen and glycosaminoglycans
Triton X-100	Non-ionic detergent	0.1%-1% (v/v)	Variable (e.g., 24 hours)	Less damaging to the ECM, effective in removing cell debris in thick tissues	Good preservation of the ECM structure
Ammonium hydroxide	Alkaline	0.1% (v/v)	Overnight	Used in combination with Triton X-100, aids decellularisation	Preserves villous structures and ECM
Deionized water	Osmotic agent	N/A	Variable	Causes hyposmotic shock in cells, contributing to cell removal	Helps remove cellular content
Sodium deoxycholate	Ionic detergent	Variable (e.g., 0.05%)	Variable	Removes cells effectively, but can compromise the MEC	May degrade glycosaminoglycans, impacting the ECM
Methanol/chloroform	Degreaser	1:1 (v/v)	Variable	Used to remove lipids before decellularisation	Helps remove cellular content
Trypsin/EDTA	Digestive enzymes	0.05%/0.05%	Variable	Used for cellular digestion, in combination with detergents	Helps remove cellular content
KI (potassium iodine)	Additional agent	1.0 M	Variable	Used in combination with Triton X-100 for decellularisation	Helps remove cellular content
Peracetic acid	Sterilising	Variable	Variable	Used to sterilise tissue after decellularisation	Preserves the structure of the mesentery and vascular channels
DNAse	Enzyme	Variable	Variable	Removes residual DNA after decellularisation	No direct effect on ECM, focuses on DNA removal

ECM: Extracellular matrix; N/A: Not applicable.

FUTURE PROSPECTS FOR TISSUE BIOENGINEERING IN IBD

In general, regenerative medicine and tissue bioengineering aim to develop functional organs and overcome immunological and transplantation limits. Methods such as decellularisation and recellularisation have been seen as effective in traditional organ transplantation, which integrates a cell-free framework (ECM)[67]. Many studies have elucidated the behaviour of cells in a two-dimensional model based on cell culture on a flat surface with the interaction between the cells and the substrate, which are effective for an initial study. While the three-dimensional model favours the simulation of various cell behaviours such as migration, proliferation, differentiation and morphogenesis, and the relationship between the cells and the ECM[68].

Future research into three-dimensional intestinal models aims to develop in vitro models with a greater capacity to reproduce a more immunocompetent physiological environment, further investigating microbial interactions, immunological responses, pathogenicity mechanisms and cellular alterations mediated by dysfunctions in microbial composition. Examples of immunocompetent three-dimensional intestinal models using organ-on-chip technology were compared with organoid and two-dimensional models, in which the model showed greater cell viability with greater expression of E-cadherin and zonula occludens-1, although non-biological supports were used to construct three-dimensional models, such as polystyrene membranes[69].

Decellularisation and recellularisation methods can present difficulties based on the microarchitecture of the ECM during re-endothelialisation, and differences in ECM components such as glycosaminoglycans, elastin and fibronectin can affect the development of the endothelium. It is therefore essential to develop and apply suitable methods using physical, chemical and enzymatic techniques. Finally, although the use of bioreactors has helped in this regard, there are still immunological and inadequate flow challenges. Modifications to the surface used heparin molecules can be improved according to regulatory requirements. In addition, other studies using mesenchymal stem cells and exosomes have been promising, but require further investigation[67].

Other relevant studies about intestinal fibrosis, a consequence of the chronic inflammation of IBD, point to new methodologies for decellularisation in the human duodenum, obtaining three-dimensional scaffolds of intestinal ECM. These techniques preserve essential tissue properties and maintain three-dimensional architecture that surpass traditional two-dimensional culture models and animal models. Recellularisation using primary intestinal myofibroblasts showed superior efficacy while preserving differentiation characteristics. In this way, these scaffolds can present an improvement in relation to IBD research and develop new therapies and targeting to optimise decellularisation and recellularisation techniques with a view to expanding clinical applicability[70].

However, decellularised ECM has excellent functionality as a biomaterial, although its application in the construction of organoids is limited. Based on this, studies have advanced in the search to combine decellularised ECM with other biomaterials to form hydrogels that optimise cell behaviour. It is therefore essential to understand the composition of decellularised ECM and to customise it for different organoids. Finally, it is necessary to adapt these models for clinical use and investigate them in different diseases to improve diagnosis and treatment[71].

CONCLUSION

In summary, IBD requires innovative approaches to improve the treatment of this disease. Regenerative medicine has emerged as a promising proposal for restoring intestinal function through the repair and regeneration of damaged tissues. To this end, the development of decellularization techniques to obtain scaffolds that mimic the native environment of the intestine is essential. Research regarding the ECM alterations caused by IBD is therefore crucial, as are studies to advance the development of more effective and safer regenerative therapies for IBD.