Dynamic stiffening of poly(ethylene glycol)-based hydrogels to direct valvular interstitial cell phenotype in a three-dimensional environment

Abstract

Valvular interstitial cells (VICs) are active regulators of valve homeostasis and disease, responsible for secreting and remodeling the valve tissue matrix. As a result of VIC activity, the valve modulus can substantially change during development, injury and repair, and disease progression. While two-dimensional biomaterial substrates have been used to study mechanosensing and its influence on VIC phenotype, less is known about how these cells respond to matrix modulus in a three-dimensional environment. Here, we synthesized MMP-degradable poly(ethylene glycol) (PEG) hydrogels with elastic moduli ranging from 0.24 kPa to 12 kPa and observed that cell morphology was constrained in stiffer gels. To vary gel stiffness without substantially changing cell morphology, cell-laden hydrogels were cultured in the 0.24 kPa gels for 3 days to allow VIC spreading, and then stiffened in situ via a second, photoinitiated thiol-ene polymerization such that the gel modulus increased from 0.24 kPa to 1.2 kPa or 13 kPa. VICs encapsulated within soft gels exhibited αSMA stress fibers (∼ 40%), a hallmark of the myofibroblast phenotype. Interestingly, in stiffened gels, VICs became deactivated to a quiescent fibroblast phenotype, suggesting that matrix stiffness directs VIC phenotype independent of morphology, but in a manner that depends on the dimensionality of the culture platform. Collectively, these studies present a versatile method for dynamic stiffening of hydrogels and demonstrate the significant effects of matrix modulus on VIC myofibroblast properties in three-dimensional environments.

Keywords: ECM; Elasticity; Heart valve; Hydrogel; Three-dimensional cell culture; Valvular interstitial cells.

Figure 1

Schematic of VIC encapsulation within PEG-based hydrogels. PEGnb average molecular weight and concentration of PEGnb and MMP-degradable crosslinking peptide were varied as shown in the table to achieve a range of moduli. VICs were encapsulated at a density of 10 million cells per mL of gel. Insets (50 μm) show Live/Dead staining at day 0 and day 2.

Figure 2

A. Live/Dead staining of VICs 48 hours after encapsulation within hydrogels. VICs within 0.24 kPa hydrogels exhibit an elongated morphology and high cell viability, while VICs within 4 kPa and 12 kPa retained a rounded morphology and experienced greater cell death. Scale bar = 100 μm. B. Quantification of Live/Dead staining shows decreased cell viability with increasing modulus. C. Increasing modulus correlates with decreasing αSMA mRNA relative to the L30 internal standard as determined by qRT-PCR. * = p<0.05.

Figure 3

Schematic of stiffening the cell-laden hydrogels. 8-arm PEG-thiol, 8-arm PEGnb, and LAP were swollen into the cell-laden gels for 12 minutes. Gels were then re-polymerized using UV light to increase the gel modulus. Final stiffened modulus was varied by changing the concentration of PEG-thiol and PEGnb while maintaining a stoichiometric reaction.

Figure 4

A. Live/Dead staining of VICs encapsulated within soft and stiffened hydrogels 48 hours after stiffening. VICs in all conditions exhibited an elongated morphology and high cell viability. Scale bar = 100 μm. B. Quantification of Live/Dead staining shows high cell viability across the range of moduli. C. Quantification of cell aspect ratio. High modulus standard encapsulations result in greatly reduced cell aspect ratio, while aspect ratio in stiffened gels remains approximately the same as in the softest condition. * = p<0.05.

Figure 5

A. Immunostaining for αSMA (green), f-actin (red) and nuclei (blue) with two representative images per condition. In soft gels, many cells exhibit αSMA stress fibers. In “to medium” gels, there are fewer cells positive for stress fibers, but diffuse αSMA is common. In “to stiff” gels, very little αSMA is present in either form. Scale bars = 100 μm. B. Fraction of activated VICs as defined by the presence of αSMA stress fibers was quantified. Activation decreased with increasing final modulus. C. A non-gelling formulation of the stiffening solution was used as a control to show that the UV light, radical intermediates, and other possible strains of the stiffening process were not responsible for the reduction in activation. * = p<0.05.

Figure 6

qRT-PCR comparing genes of interest in soft (white), to medium (striped), and to stiff (solid) conditions. All genes were normalized to the L30 housekeeping gene. Myofibroblast markers αSMA and CTGF decreased with increasing modulus. The fibroblast marker S100A4 increased with increasing modulus. MMP was higher in the stiffened gels than in the soft gel. Col1A1 was unchanged in the examined conditions. * = p < 0.05.