Profile of matrix-entrapped extracellular vesicles of microenvironmental and infiltrating cell origin in decellularized colorectal cancer and adjacent mucosa.

Cellular elements that infiltrate and surround tumours and pre-metastatic tissues have a prominent role in tumour invasion and growth. The extracellular vesicles specifically entrapped and stored within the extracellular matrix (ECM-EVs) may reflect the different populations of the tumour microenvironment and their change during tumour progression. However, their profile is at present unknown. To elucidate this aspect, we isolated and characterized EVs from decellularized surgical specimens of colorectal cancer and adjacent colon mucosa and analyzed their surface marker profile. ECM-EVs in tumours and surrounding mucosa mainly expressed markers of lymphocytes, natural killer cells, antigen-presenting cells, and platelets, as well as epithelial cells, representing a multicellular microenvironment. No difference in surface marker expression was observed between tumour and mucosa ECM-EVs in stage II-III tumours. At variance, in the colon mucosa adjacent to stage IV carcinomas, ECM-EV profile showed a significantly increased level of immune, epithelial and platelet markers in comparison to the matrix of the corresponding tumour. The increase of EVs from immune cells and platelets was not observed in the mucosa adjacent to low-stage tumours. In addition, CD25, a T-lymphocyte marker, resulted specifically overexpressed by ECM-EVs from stage IV carcinomas, possibly correlated with the pro-tolerogenic environment found in the corresponding tumour tissue. These results outline the tissue microenvironmental profile of EVs in colorectal carcinoma-derived ECM and unveil a profound change in the healthy mucosa adjacent to high-stage tumours.

Decellularized tissues as platforms for digestive system cancer models.

The extracellular matrix (ECM) is a multifunctional network of macromolecules that regulate various cellular functions and physically support the tissues. Besides physiological conditions, the ECM also changes during pathological conditions such as cancer. As tumor cells proliferate, notable changes occur in the quantity and makeup of the surrounding ECM. Therefore, the role of this noncellular component of tissues in studies of tumor microenvironments should be considered. So far, many attempts have been made to create 2-dimensional (2D) or 3-dimensional (3D) models that can replicate the intricate connections within the tumor microenvironment. Decellularized tissues are proper scaffolds that imitate the complex nature of native ECM. This review aims to summarize 3D models of digestive system cancers based on decellularized ECMs. These ECM-based scaffolds will enable us to study the interactive communication between cells and their surrounding environment which brings new potential for a better understanding of the pathophysiology of cancer.

Scaffold-based 3D cell culture models in cancer research.

Three-dimensional (3D) cell cultures have emerged as valuable tools in cancer research, offering significant advantages over traditional two-dimensional (2D) cell culture systems. In 3D cell cultures, cancer cells are grown in an environment that more closely mimics the 3D architecture and complexity of in vivo tumors. This approach has revolutionized cancer research by providing a more accurate representation of the tumor microenvironment (TME) and enabling the study of tumor behavior and response to therapies in a more physiologically relevant context. One of the key benefits of 3D cell culture in cancer research is the ability to recapitulate the complex interactions between cancer cells and their surrounding stroma. Tumors consist not only of cancer cells but also various other cell types, including stromal cells, immune cells, and blood vessels. These models bridge traditional 2D cell cultures and animal models, offering a cost-effective, scalable, and ethical alternative for preclinical research. As the field advances, 3D cell cultures are poised to play a pivotal role in understanding cancer biology and accelerating the development of effective anticancer therapies. This review article highlights the key advantages of 3D cell cultures, progress in the most common scaffold-based culturing techniques, pertinent literature on their applications in cancer research, and the ongoing challenges.

Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment.

The extracellular matrix (ECM) is a pericellular network of proteins and other molecules that provides mechanical support to organs and tissues. ECM biophysical properties such as topography, elasticity and porosity strongly influence cell proliferation, differentiation and migration. The cell's perception of the biophysical microenvironment (mechanosensing) leads to altered gene expression or contractility status (mechanotransduction). Mechanosensing and mechanotransduction have profound implications in both tissue homeostasis and cancer. Many solid tumours are surrounded by a dense and aberrant ECM that disturbs normal cell functions and makes certain areas of the tumour inaccessible to therapeutic drugs. Understanding the cell-ECM interplay may therefore lead to novel and more effective therapies. Controllable and reproducible cell culturing systems mimicking the ECM enable detailed investigation of mechanosensing and mechanotransduction pathways. Here, we discuss ECM biomimetic systems. Mainly focusing on collagen, we compare and contrast structural and molecular complexity as well as biophysical properties of simple 2D substrates, 3D fibrillar collagen gels, cell-derived matrices and complex decellularized organs. Finally, we emphasize how the integration of advanced methodologies and computational methods with collagen-based biomimetics will improve the design of novel therapies aimed at targeting the biophysical and mechanical features of the tumour ECM to increase therapy efficacy.

[Conjunctival reconstruction-State of the art of regenerative treatment forms beyond the limbus]. / Bindehautrekonstruktion ­ Status quo regenerativer Therapieformen jenseits des Limbus.

BACKGROUND: The demands on conjunctival replacement tissues are high: they need to be elastic, clinically compatible, surgically feasible and support goblet cell growth. OBJECTIVE: This article provides an overview of currently applied conjunctival replacement tissues and those under investigation. METHOD: Current publications on clinically applied conjunctival replacement tissues and substrates which are the subject of scientific research and those already tested in animal models are presented and discussed. RESULTS: Replacement tissues in clinical use are autologous and allogenic conjunctiva, nasal and oral mucous membranes, amniotic membrane and decellularized tissues. Autologous conjunctiva shows good results but is not suitable for large defects due to limited availability. In these cases autologous nasal and oral mucous membranes can be used; however, success is limited in cases of autoimmune diseases. Amniotic membranes are frequently applied clinically but goblet cell growth is limited. Different decellularized tissues are used clinically and goblet cell growth was found in vivo. Robust comparative studies are not yet available. Biological matrices such as fibrin, collagen, elastin, gelatin or hyaluronate and synthetic tissues from the group of polyesters are being investigated in the laboratory and in animal models. These studies show good epithelialization and goblet cell growth in vivo. CONCLUSION: Transplantation of conjunctiva, nasal and oral mucous membranes and amniotic membranes show satisfactory clinical results but exhibit individual weaknesses. Further studies in animal models and clinical settings are required to further evaluate the benefits of other matrices, such as cell-free tissues or other biological and synthetic matrices.

Extracellular Matrix Stiffness and Composition Regulate the Myofibroblast Differentiation of Vaginal Fibroblasts.

Fibroblast to myofibroblast differentiation is a key feature of wound-healing in soft tissues, including the vagina. Vaginal fibroblasts maintain the integrity of the vaginal wall tissues, essential to keep pelvic organs in place and avoid pelvic organ prolapse (POP). The micro-environment of vaginal tissues in POP patients is stiffer and has different extracellular matrix (ECM) composition than healthy vaginal tissues. In this study, we employed a series of matrices with known stiffnesses, as well as vaginal ECMs, in combination with vaginal fibroblasts from POP and healthy tissues to investigate how matrix stiffness and composition regulate myofibroblast differentiation in vaginal fibroblasts. Stiffness was positively correlated to production of α-smooth muscle actin (α-SMA). Vaginal ECMs induced myofibroblast differentiation as both α-SMA and collagen gene expressions were increased. This differentiation was more pronounced in cells seeded on POP-ECMs that were stiffer than those derived from healthy tissues and had higher collagen and elastin protein content. We showed that stiffness and ECM content regulate vaginal myofibroblast differentiation. We provide preliminary evidence that vaginal fibroblasts might recognize POP-ECMs as scar tissues that need to be remodeled. This is fundamentally important for tissue repair, and provides a rational basis for POP disease modelling and therapeutic innovations in vaginal reconstruction.

Deconstructing, Replicating, and Engineering Tissue Microenvironment for Stem Cell Differentiation.

One of the most crucial components of regenerative medicine is the controlled differentiation of embryonic or adult stem cells into the desired cell lineage. Although most of the reported protocols of stem cell differentiation involve the use of soluble growth factors, it is increasingly evident that stem cells also undergo differentiation when cultured in the appropriate microenvironment. When cultured in decellularized tissues, for instance, stem cells can recapitulate the morphogenesis and functional specialization of differentiated cell types with speed and efficiency that often surpass the traditional growth factor-driven protocols. This suggests that the tissue microenvironment (TME) provides stem cells with a holistic "instructive niche" that harbors signals for cellular reprogramming. The translation of this into medical applications requires the decoding of these signals, but this has been hampered by the complexity of TME. This problem is often addressed by a reductionist approach, in which cells are exposed to substrates decorated with simple, empirically designed geometries, textures, and chemical compositions ("bottom-up" approach). Although these studies are invaluable in revealing the basic principles of mechanotransduction mechanisms, their physiological relevance is often uncertain. This review examines the recent progress of an alternative, "top-down" approach, in which the TME is treated as a holistic biological entity. This approach is made possible by recent advances in systems biology and fabrication technologies that enable the isolation, characterization, and reconstitution of TME. It is hoped that these new techniques will elucidate the nature of niche signals so that they can be extracted, replicated, and controlled. This review summarizes these emerging techniques and how the data they generated are changing our view on TME. Impact statement This review summarizes the current state of art of the understanding of instructive niche in the field of tissue microenvironment. Not only did we survey the use of different biochemical preparations as stimuli of stem cell differentiation and summarize the recent effort in dissecting the biochemical composition of these preparations, through the application of extracellular matrix (ECM) arrays and proteomics, but we also introduce the use of open-source, high-content immunohistochemistry projects in contributing to the understanding of tissue-specific composition of ECM. We believe this review would be highly useful for our peer researching in the same field. "Mr. Tulkinghorn is always the same… so oddly out of place and yet so perfectly at home." -Charles Dickens, Bleak House.

Spare Parts from Discarded Materials: Fetal Annexes in Regenerative Medicine.

One of the main aims in regenerative medicine is to find stem cells that are easy to obtain and are safe and efficient in either an autologous or allogenic host when transplanted. This review provides an overview of the potential use of the fetal annexes in regenerative medicine: we described the formation of the annexes, their immunological features, the new advances in the phenotypical characterization of fetal annexes-derived stem cells, the progressions obtained in the analysis of both their differentiative potential and their secretoma, and finally, the potential use of decellularized fetal membranes. Normally discarded as medical waste, the umbilical cord and perinatal tissue not only represent a rich source of stem cells but can also be used as a scaffold for regenerative medicine, providing a suitable environment for the growth and differentiation of stem cells.

Application of injectable hydrogels for cardiac stem cell therapy and tissue engineering.

Cardiovascular diseases are responsible for approximately one-third of deaths around the world. Among cardiovascular diseases, the largest single cause of death is ischemic heart disease. Ischemic heart disease typically manifests as progressive constriction of the coronary arteries, which obstructs blood flow to the heart and can ultimately lead to myocardial infarction. This adversely affects the structure and function of the heart. Conventional treatments lack the ability to treat the myocardium lost during an acute myocardial infarction. Stem cell therapy offers an excellent solution for myocardial regeneration. Stem cell sources such as adult stem cells, embryonic and induced pluripotent stem cells have been the focal point of research in cardiac tissue engineering. However, cell survival and engraftment post-transplantation are major limitations that must be addressed prior to widespread use of this technology. Recently, biomaterials have been introduced as 3D vehicles to facilitate stem cell transplantation into infarct sites. This has shown significant promise with improved cell survival after transplantation. In this review, we discuss the various injectable hydrogels that have been tried in cardiac tissue engineering. Exploring and optimizing these cell-material interactions will guide cardiac tissue engineering towards developing stem cell based functional 3D constructs for cardiac regeneration.

3-D physiomimetic extracellular matrix hydrogels provide a supportive microenvironment for rodent and human islet culture.

Organ-on-a-chip platforms serve as cost-efficient testbeds for screening pharmaceutical agents, mimicking natural physiology, and studying disease. In the field of diabetes, the development of an islet-on-a-chip platform would have broad implications in understanding disease pathology and discovering potential therapies. Islet microphysiological systems are limited, however, by their poor cell survival and function in culture. A key factor that has been implicated in this decline is the disruption of islet-matrix interactions following isolation. Herein, we sought to recapitulate the in vivo peri-islet niche using decellularized extracellular matrix (ECM) hydrogels. Sourcing from porcine bladder, lung, and pancreas tissues, 3-D ECM hydrogels were generated, characterized, and validated using both rodent and human pancreatic islets. Optimized decellularization protocols resulted in hydrogels with distinctive viscoelastic properties that correlated to their matrix composition. The in situ 3-D encapsulation of human or rat islets within ECM hydrogels resulted in improved functional stability over standard culture conditions. Islet composition and morphology were also altered, with enhanced retention of islet-resident endothelial cells and the formation of cord-like structures or sprouts emerging from the islet spheroid. These supportive 3-D physiomimetic ECM hydrogels can be leveraged within microfluidic platforms for the long-term culture of islets.

Tissue-Engineered Neo-Urinary Conduit from Decellularized Trachea.

Decellularized tissues have been increasingly popular for constructing scaffolds for tissue engineering applications due to their beneficial biological compositions and mechanical properties. It is therefore natural to consider decellularized trachea for construction of tissue-engineered trachea, as well as other tubular organs. A Neo-Urinary Conduit (NUC) is such a tubular organ that works as a passage for urine removal in bladder cancer patients who need a urinary diversion after their diseased bladder is removed. In this study, we report our findings on the feasibility of using a decellularized trachea for NUC applications. As a NUC scaffold, decellularized trachea provides benefits of having not only naturally occurring biological components but also having sufficient mechanical properties and structural integrity. We, therefore, decellularized rabbit trachea, evaluated its mechanical performance, and investigated its ability to support in vitro growth of human smooth muscle cells (hSMCs) and human urothelial cells (hUCs). The decellularized trachea had appropriate biomechanical properties with ultimate tensile strength of ∼0.34 MPa in longitudinal direction and ∼1.0 MPa in circumferential direction and resisted a radial burst pressure of >155 mm Hg. Cell morphology study by scanning electron microscopy further showed that hUCs grown on decellularized trachea adopted a typical flatten and interconnected network structure in the lumen of the scaffold, while they formed a round spherical shape and did not spread on the outer surfaces. SMCs, on the other hand, spread well throughout the scaffold. The gene expression analysis by real time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence studies further confirmed scaffold's ability to support long-term growth of hSMCs. Since uroepithelium has been shown to regenerate itself over time in vivo, these findings suggest that it is possible to construct a NUC from decellularized trachea without any preseeding of UCs. In future, we plan to translate decellularized trachea in a preclinical animal model and evaluate its biological performance.

Heart Valve Replacements with Regenerative Capacity.

The incidence of severe valvular dysfunctions (e.g., stenosis and insufficiency) is increasing, leading to over 300,000 valves implanted worldwide yearly. Clinically used heart valve replacements lack the capacity to grow, inherently requiring repetitive and high-risk surgical interventions during childhood. The aim of this review is to present how different tissue engineering strategies can overcome these limitations, providing innovative valve replacements that proved to be able to integrate and remodel in pre-clinical experiments and to have promising results in clinical studies. Upon description of the different types of heart valve tissue engineering (e.g., in vitro, in situ, in vivo, and the pre-seeding approach) we focus on the clinical translation of this technology. In particular, we will deepen the many technical, clinical, and regulatory aspects that need to be solved to endure the clinical adaptation and the commercialization of these promising regenerative valves.