185 An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition INTRODUCTION Extracellular matrix (ECM) is the structural component of every tissue, formed by a complex network of proteins, glycosaminoglycans and proteoglycans [1]. The highly tissue-specific nature of the ECM is dictated by the presence of a defined grouping of matrisome elements, incorporating demarcated ratios of ECM proteins [2]. These distinctions also result in different mechanical properties of the ECM, depending on the origin of the tissue [3]. Next to being structural support for the cells, the ECM provides biochemical and biomechanical cues to cells in vivo [4]. As such, it has proven challenging to mimic and incorporate the ECM structure and mechanics into (in vitro) studies regarding the structure and function of the ECM in health and disease. In fibrotic lung diseases, not only is the ECM composition altered but also its mechanical properties, resulting in higher stiffness and decreases in stress relaxation [5, 6]. All the changes that are evident within a fibrotic ECM have been revealed to instruct cells and influence their responses to contribute to the progression of fibrosis, as reported and reviewed elsewhere [7-13]. To investigate the mechanical properties of (fibrotic) ECM in vitro, the ECM is often mimicked using hydrogels. ECM-derived hydrogels, which have been introduced to the field in the last decade, are a promising alternative to other types of hydrogels such as collagen, gelatine, or hyaluronic acid [14]. ECM hydrogels which are developed from native decellularized tissue, retain most of the native ECM composition and, in general, resemble the mechanical properties of the parent tissue [6]. The most common method to produce hydrogels from ECM is to digest decellularized ECM powder with porcine pepsin at low pH with constant agitation [14]. Our recent study illustrated the preparation of ECM-derived hydrogels from human decellularized lung ECM, and established that the mechanical properties of the diseased (fibrotic) lung ECM-derived hydrogels resembled the mechanics of the decellularized fibrotic lung ECM [6]. Fibrotic lung ECM (both in native and hydrogel form) showed decreased viscoelastic stress relaxation compared to control lung ECM [6]. The stiffness of fibrotic lung tissue was ~10 times higher than its hydrogel counterpart, possibly due to the absence of chemical crosslinks and lung-resident cells in the ECM hydrogel. Previous studies showed that the composition of fibrotic lung ECM is different to that seen in control lung due to dysregulation of the ECM degradation/deposition processes resulting in an aberrant ECM [15]. To investigate the separate influences on the cells of the altered mechanical properties or ECM composition in the fibrotic microenvironment, advanced and innovative in vitro models are needed. Recently, altering the mechanical properties of methacrylate or thiol functionalized ECMderived hydrogels using click-chemistry has been shown [16, 17]. Given that these 8
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