Abstract
The mechanical properties of the cellular microenvironment play a crucial role in modulating cell function, and many pathophysiological processes are accompanied by variations in extracellular matrix (ECM) stiffness. Lysyl oxidase (LOx) is one of the enzymes involved in several ECM-stiffening processes. Here, we engineered poly(ethylene glycol) (PEG)-based hydrogels with controlled mechanical properties in the range typical of soft tissues. These hydrogels were functionalized featuring free primary amines, which allows an additional chemical LOx-responsive behavior with increase in crosslinks and hydrogel elastic modulus, mimicking biological ECM-stiffening mechanisms. Hydrogels with elastic moduli in the range of 0.5–4 kPa were obtained after a first photopolymerization step. The increase in elastic modulus of the functionalized and enzyme-responsive hydrogels was also characterized after the second-step enzymatic reaction, recording an increase in hydrogel stiffness up to 0.5 kPa after incubation with LOx. Finally, hydrogel precursors containing HepG2 (bioinks) were used to form three-dimensional (3D) in vitro models to mimic hepatic tissue and test PEG-based hydrogel biocompatibility. Hepatic functional markers were measured up to 7 days of culture, suggesting further use of such 3D models to study cell mechanobiology and response to dynamic variation of hydrogels stiffness. The results show that the functionalized hydrogels presented in this work match the mechanical properties of soft tissues, allow dynamic variations of hydrogel stiffness, and can be used to mimic changes in the microenvironment properties of soft tissues typical of inflammation and pathological changes at early stages (e.g., fibrosis, cancer).
Highlights
Many in vitro models have been developed to mechanistically investigate biological processes
A number of studies have shown the influence of substrate elasticity on biological processes, for example, by culturing cells on a variety of natural and synthetic hydrogels mimicking the native stiffness of different biological tissues (Engler et al, 2006; Mattei et al, 2015, 2017; Olivares-Navarrete et al, 2017)
While engineering a biomaterial to model biological tissues, is the control over dynamic variation of the mechanical properties: several biological processes in vivo involve a constant remodeling of the surrounding extracellular matrix (ECM) changing physicochemical properties to match the values critical for tissue development and function (Bonnans et al, 2014; Manou et al, 2019)
Summary
Many in vitro models have been developed to mechanistically investigate biological processes. Poly(ethylene glycol)based hydrogels were engineered to have a first gelation step using visible light to photochemically crosslink the monomer mixtures, and a second on-demand step (i.e., enzymatic reaction) after incubation with enzymes to form additional crosslinks and further increase hydrogel elastic modulus to mimic in vivo processes. The initial elastic modulus of PEG hydrogels was tuned through the monofunctional/difunctional monomer ratio to match the values of a healthy human liver and being in the range of 0.1–5 kPa. The additional curing step was designed to mimic the fibrotic/stiffening events reported in liver injury: we selected LOx, an enzyme that is upregulated early in liver injury (Desmoulière et al, 1997; Mesarwi et al, 2015) and contributes to ECM stiffening during liver fibrosis (Perepelyuk et al, 2013; Liu et al, 2016), to crosslink further PEG hydrogels on demand, increase the elastic modulus and match early and late events of hepatic tissue fibrosis
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