The elastic modulus of the extracellular matrix is a dynamic property that changes during various biological processes, such as disease progression or wound healing. Most cell culture platforms, however, have traditionally exhibited static properties, making it necessary to replate cells to study the effects of different elastic moduli on cell phenotype. Recently, much progress has been made in the development of substrates with mechanisms for either increasing or decreasing stiffness in situ, but there are fewer examples of substrates that can both stiffen and soften, which may be important for simulating the effects of repeated ECM injury and resolution. In the work presented here, poly(ethylene glycol)-based hydrogels reversibly stiffen and soften with multiple light stimuli via photoisomerization of an azobenzene-containing crosslinker. Upon irradiation with cytocompatible doses of 365 nm light (10 mW/cm2, 5 min), isomerization to the azobenzene cis configuration leads to a softening of the hydrogel up to 100-200 Pa (shear storage modulus, G’). This change in gel properties is maintained over a timescale of several hours due to the long half-life of the cis isomer. The initial modulus of the gel can be recovered upon irradiation with similar doses of visible light. With applications in mechanobiology in mind, cytocompatibility with a mechanoresponsive primary cell type is demonstrated. Porcine aortic valvular interstitial cells were encapsulated in the developed hydrogels and shown to exhibit high levels of survival, as well as a spread morphology. The developed hydrogels enable a route to the noninvasive control of substrate modulus independent of changes in the chemical composition or network connectivity, allowing for investigations of the effect of dynamic matrix stiffness on adhered cell behavior.
Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis. To better understand and quantify how microenvironmental cues influence VIC phenotype and myofibroblast activation, we compared expression profiles of VICs cultured on poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. In general, VICs cultured in hydrogel matrices had lower levels of activation (<10%), similar to levels seen in healthy valve tissue, while VICs cultured on TCPS were ~75% activated myofibroblasts. VICs cultured on TCPS also exhibited a higher magnitude of perturbations in gene expression than soft hydrogel cultures when compared to the native phenotype. Using peptide-modified PEG gels, VICs were seeded on (2D), as well as encapsulated in (3D), matrices of the same composition and modulus. Despite similar levels of activation, VICs cultured in 2D had distinct variations in transcriptional profiles compared to those in 3D hydrogels. Genes related to cell structure and motility were particularly affected by the dimensionality of the culture platform, with higher expression levels in 2D than in 3D. These results indicate that dimensionality may play a significant role in dictating cell phenotype (e.g., through differences in polarity, diffusion of soluble signals), and emphasize the importance of using multiple metrics when characterizing cell phenotype.
In their native extracellular microenvironment, cells respond to a complex array of biochemical and mechanical cues that can vary in both time and space. High-throughput methods that allow characterization of cell-laden matrices are valuable tools to screen through many combinations of variables, ultimately helping to evolve and test hypotheses related to cell-ECM signaling. Here, we developed a platform for high-throughput encapsulation of cells in peptide-functionalized poly(ethylene glycol) hydrogels. Hydrogels were synthesized using a thiol-ene, photoclick reaction, which allowed the cell matrix environment to be modified in real time. Matrix signals were dynamically altered by in situ tethering of RGDS (0-1.5 mM), a fibronectin-derived adhesive peptide that induced more elongation than RLD or IKVAV, and/or by increasing the matrix modulus (1 to 6 kPa). This method was demonstrated with aortic valvular interstitial cells (VICs), a population of cells responsible for the pathological fibrosis and matrix remodeling that leads to aortic stenosis. VIC response to cell-matrix interactions was characterized by quantifying cell morphology and the fraction of cells exhibiting α-smooth muscle actin (αSMA) stress fibers, a hallmark of the myofibroblast phenotype. VICs elongated in response to RGDS addition, with a dramatic change in morphology within 24 h. Myofibroblast activation was also dependent on RGDS addition, with VICs exhibiting high activation (16-24%) in 1 kPa gels with RGDS. Response to RGDS was path-dependent, with the amount of time exposed to the adhesive ligand important in determining VIC morphology and activation. Although VIC aspect ratios were dependent on the amount of time spent in a stiff vs soft gel, low levels of VIC activation (≤4%) were observed in any gels cultured in higher modulus (6 kPa vs 1 kPa) microenvironments.
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