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

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

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

Converging pathways in pulmonary fibrosis and Covid-19 - The fibrotic link to disease severity : Common molecular pathways in Covid-19 and pulmonary fibrosis

As Covid-19 affects millions of people worldwide, the global health care will encounter an increasing burden of the aftermaths of the disease. Evidence shows that up to a fifth of the patients develop fibrotic tissue in the lung. The SARS outbreak in the early 2000 resulted in chronic pulmonary fibrosis in a subset (around 4%) of the patients, and correlated to reduced lung function and forced expiratory volume (FEV). The similarities between corona virus infections causing SARS and Covid-19 are striking, except that the novel coronavirus, SARS-CoV-2, has proven to have an even higher communicability. This would translate into a large number of patients seeking care for clinical signs of pulmonary fibrosis, given that the Covid-19 pandemic has up till now (Sept 2020) affected around 30 million people. The SARS-CoV-2 is dependent on binding to the angiotensin converting enzyme 2 (ACE2), which is part of the renin-angiotensin system (RAS). Downregulation of ACE2 upon virus binding disturbs downstream activities of RAS resulting in increased inflammation and development of fibrosis. The poor prognosis and risk of developing pulmonary fibrosis are therefore associated with the increased expression of ACE2 in risk groups, such as obesity, heart disorders and aging, conferring plenty of binding opportunity for the virus and subsequently the internalization of ACE2, thus devoiding the enzyme from acting counter-inflammatory and antifibrotic. Identifying pathways that are associated with Covid-19 severity that result in pulmonary fibrosis may enable early diagnosis and individualized treatment for these patients to prevent or reduce irreversible fibrotic damage to the lung

Glycosaminoglycans: a link between development and regeneration in the lung : a link between development and regeneration in the lung

What can we learn from embryogenesis to increase our understanding of how regeneration of damaged adult lung tissue could be induced in serious lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and asthma? The local tissue niche determines events in both embryogenesis and repair of the adult lung. Important constituents of the niche are extracellular matrix (ECM) molecules including proteoglycans and glycosaminoglycans (GAG). GAGs, strategically located in the pericellular and extracellular space, bind developmentally active growth factors and morphogenes such as fibroblast growth factors (FGF), transforming growth factor- (TGF-) and bone morphogenetic proteins (BMPs) aside from cytokines. These interactions affect activities in many cells, including stem cells, important in development and tissue regeneration. Moreover, it is becoming clear that the "inherent code", such as sulfation of disaccharides of GAGs is a strong determinant of cellular outcome. Sulfation pattern, deacetylations and epimerizations of GAG chains function as tuning forks in gradient formation of morphogens, growth factors and cytokines. Learning to tune these fine instruments, i.e. interactions between growth factors (GF), chemokines and cytokines with the specific disaccharide code of GAGs in the adult lung, could become the key to unlock inherent regenerative forces to override pathological remodeling. This review aims to give an overview of the role GAGs play during development and similar events in regenerative efforts in the adult lung