Development of a physiomimetic model of acute respiratory distress syndrome by using ECM hydrogels and organ-on-a-chip devices

Acute Respiratory Distress Syndrome is one of the more common fatal complications in COVID-19, characterized by a highly aberrant inflammatory response. Pre-clinical models to study the effect of cell therapy and anti-inflammatory treatments have not comprehensively reproduced the disease due to its high complexity. This work presents a novel physiomimetic in vitro model for Acute Respiratory Distress Syndrome using lung extracellular matrix-derived hydrogels and organ-on-a-chip devices. Monolayres of primary alveolar epithelial cells were cultured on top of decellullarized lung hydrogels containing primary lung mesenchymal stromal cells. Then, cyclic stretch was applied to mimic breathing, and an inflammatory response was induced by using a bacteriotoxin hit. Having simulated the inflamed breathing lung environment, we assessed the effect of an anti-inflammatory drug (i.e., dexamethasone) by studying the secretion of the most relevant inflammatory cytokines. To better identify key players in our model, the impact of the individual factors (cyclic stretch, decellularized lung hydrogel scaffold, and the presence of mesenchymal stromal cells) was studied separately. Results showed that developed model presented a more reduced inflammatory response than traditional models, which is in line with what is expected from the response commonly observed in patients. Further, from the individual analysis of the different stimuli, it was observed that the use of extracellular matrix hydrogels obtained from decellularized lungs had the most significant impact on the change of the inflammatory response. The developed model then opens the door for further in vitro studies with a better-adjusted response to the inflammatory hit and more robust results in the test of different drugs or cell therapy

hLMSC Secretome Affects Macrophage Activity Differentially Depending on Lung-Mimetic Environments

Mesenchymal stromal cell (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate the activity of macrophages. Despite recent advances, there is scarce information regarding changes of the secretome content attributed to physiomimetic cultures and, especially, how secretome content influence on macrophage activity for therapy. hLMSCs from human donors were cultured on devices developed in house that enabled lung-mimetic strain. hLMSC secretome was analyzed for typical cytokines, chemokines and growth factors. RNA was analyzed for the gene expression of CTGF and CYR61. Human monocytes were differentiated to macrophages and assessed for their phagocytic capacity and for M1/M2 subtypes by the analysis of typical cell surface markers in the presence of hLMSC secretome. CTGF and CYR61 displayed a marked reduction when cultured in lung-derived hydrogels (L-Hydrogels). The secretome showed that lung-derived scaffolds had a distinct secretion while there was a large overlap between L-Hydrogel and the conventionally (2D) cultured samples. Additionally, secretome from L-Scaffold showed an HGF increase, while IL-6 and TNF-α decreased in lung-mimetic environments. Similarly, phagocytosis decreased in a lung-mimetic environment. L-Scaffold showed a decrease of M1 population while stretch upregulated M2b subpopulations. In summary, mechanical features of the lung ECM and stretch orchestrate anti-inflammatory and immunosuppressive outcomes of hLMSCs

Innovative three-dimensional models for understanding mechanisms underlying lung diseases: powerful tools for translational research

Chronic lung diseases result from alteration and/or destruction of lung tissue, inevitably causing decreased breathing capacity and quality of life for patients. While animal models have paved the way for our understanding of pathobiology and the development of therapeutic strategies for disease management, their translational capacity is limited. There is, therefore, a well-recognised need for innovative in vitro models to reflect chronic lung diseases, which will facilitate mechanism investigation and the advancement of new treatment strategies. In the last decades, lungs have been modelled in healthy and diseased conditions using precision-cut lung slices, organoids, extracellular matrix-derived hydrogels and lung-on-chip systems. These three-dimensional models together provide a wide spectrum of applicability and mimicry of the lung microenvironment. While each system has its own limitations, their advantages over traditional two-dimensional culture systems, or even over animal models, increases the value of in vitro models. Generating new and advanced models with increased translational capacity will not only benefit our understanding of the pathobiology of lung diseases but should also shorten the timelines required for discovery and generation of new therapeutics. This article summarises and provides an outline of the European Respiratory Society research seminar "Innovative 3D models for understanding mechanisms underlying lung diseases: powerful tools for translational research", held in Lisbon, Portugal, in April 2022. Current in vitro models developed for recapitulating healthy and diseased lungs are outlined and discussed with respect to the challenges associated with them, efforts to develop best practices for model generation, characterisation and utilisation of models and state-of-the-art translational potential. </p

The evaluation of thermoresponsive biomaterials for chondrocyte culture

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Biomaterials for in vitro models in lung research

In this chapter we introduce biomaterials science to a nonexpert reader in order to provide new perspectives for the development of in vitro three-dimensional models that are relevant in lung research. Specifically, we provide a comprehensive description of key concepts in the fields of biomaterials science and lung research to explain how merging them can give rise to new models of lung biology and disease that prevent overuse of animal models while making it possible to do tests in human lung cells and tissue. Along with the different examples of lung models reviewed here, we answer questions that can help a researcher in the lung field to understand the studies: what biomaterial, why this biomaterial, and how it has been used. Moreover, we discuss important findings and breakthroughs that have been made in the lung field with the use of biomaterials, and we comment on the advantages and disadvantages of these approaches

Oxidized Dextran as Crosslinker for Chitosan Cryogel Scaffolds and Formation of Polyelectrolyte Complexes between Chitosan and Gelatin.

Macroporous scaffolds composed of chitosan and using oxidized dextran as a crosslinker are produced through cryogelation. Introducing gelatin as a third component into the structure results in the formation of mesopores in the pore walls, which are not seen if gelatin is excluded. The mesoporous structure is explained by the formation of polyelectrolyte complexes between chitosan and gelatin before crosslinking takes place. The scaffolds exhibit highly elastic properties withstanding compressions up to 60%. The in vitro biocompatibility of the cryogels is evaluated using fibroblasts from a mouse cell line (L929) and it is seen that the cells adhere and proliferate on the scaffolds. The mesoporous structure seems to have a positive effect on proliferation

Model visualization : from micro to macro

Because of increasing demand, rapid development of in vitro and in vivo models to be used to study lung regeneration and lung repair has occurred during the last years. Even if imaging has always been an important tool in diagnosing disease and validating models, the current disease models, including three-dimensional (3D) lung models, put a higher demand on advanced imaging techniques. Moreover, choosing the most relevant technique for a specific question is not a trivial task, and the rapid development of new techniques has not made this task easier. Therefore the aim of this chapter is to provide an overview of different advanced imaging techniques that can be used to evaluate and validate 3D lung models, to provide a discussion on the current state of the art, and to list the pros and cons of the available techniques

Evaluation of macroporous blood and plasma scaffolds for skeletal muscle tissue engineering

The field of tissue engineering has a growing need for suitable scaffold materials to become attractive as a clinical therapy. To use a completely autologous construct to repair a damaged or diseased tissue is an appealing thought. As a model system, two types of scaffolds were prepared from biological fluids: blood and plasma. The prepared scaffolds formed a macroporous structure with elastic mechanical properties that were further evaluated with myoblast cell line (C2C12) cultivation and transplantation into mouse skeletal muscle. The cells were found to attach, proliferate, and migrate through all the different scaffolds. Moreover, the cells underwent myogenic differentiation, showing typical cell morphology aligned in a parallel fashion. An increased level of myogenin mRNA was found with the time of culture. Furthermore, myogenic markers MyoD1, desmin, myogenin and myosin, as well as beta-dystroglycan and the laminin alpha 2 chain, were found to be expressed. In vivo data indicated that the scaffolds degraded and were replaced with regenerated muscle fibres. We conclude that the two types of macroporous scaffolds based on blood or plasma have potential in the field of skeletal muscle tissue engineering

Porous protein-based scaffolds prepared through freezing as potential scaffolds for tissue engineering.

Successful tissue engineering with the aid of a polymer scaffold offers the possibility to produce a larger construct and to mould the shape after the defect. We investigated the use of cryogelation to form protein-based scaffolds through different types of formation mechanisms; enzymatic crosslinking, chemical crosslinking, and non-covalent interactions. Casein was found to best suited for enzymatic crosslinking, gelatin for chemical crosslinking, and ovalbumin for non-covalent interactions. Fibroblasts and myoblasts were used to evaluate the cryogels for tissue engineering purposes. The stability of the cryogels over time in culture differed depending on formation mechanism. Casein cryogels showed best potential to be used in skeletal tissue engineering, whereas gelatin cryogels would be more suitable for compliable soft tissues even though it also seemed to support a myogenic phenotype. Ovalbumin cryogels would be better suited for elastic tissues with faster regeneration properties due to its faster degradation time. Overall, the cryogelation technique offers a fast, cheap and reproducible way of creating porous scaffolds from proteins without the use of toxic compounds

Laminin α2 Chain-Deficiency is Associated with microRNA Deregulation in Skeletal Muscle and Plasma.

microRNAs (miRNAs) are widespread regulators of gene expression, but little is known of their potential roles in congenital muscular dystrophy type 1A (MDC1A). MDC1A is a severe form of muscular dystrophy caused by mutations in the gene encoding laminin α2 chain. To gain insight into the pathophysiological roles of miRNAs associated with MDC1A pathology, laminin α2 chain-deficient mice were evaluated by quantitative PCR. We demonstrate that expression of muscle-specific miR-1, miR-133a, and miR-206 is deregulated in laminin α2 chain-deficient muscle. Furthermore, expression of miR-223 and miR-21, associated with immune cell infiltration and fibrosis, respectively, is altered. Finally, we show that plasma levels of muscle-specific miRNAs are markedly elevated in laminin α2 chain-deficient mice and partially normalized in response to proteasome inhibition therapy. Altogether, our data suggest important roles for miRNAs in MDC1A pathology and we propose plasma levels of muscle-specific miRNAs as promising biomarkers for the progression of MDC1A