Mesenchymal Stromal Cell Chondrogenic Differentiation Induced by Continuous Stiffness Gradient in Photocrosslinkable Hydrogels

Abstract

Chondrogenic differentiation of stem and progenitor cells is dependent on the biophysical properties of the surrounding matrix. Current biomaterials-based approaches for chondrogenesis are limited to discrete platforms, slowing our ability to interrogate the role of mechanical cues such as substrate stiffness and other signals. Thus, novel platforms must incorporate a range of biophysical properties within a single construct to effectively assess changes in cell response. We encapsulated human mesenchymal stromal cells (MSCs) within biodegradable, photocurable oxidized, and methacrylated alginate (OMA). Cell-laden hydrogels were crosslinked when exposed to light through a grayscale photomask to form substrates with a continuous stiffness gradient. We also tested the influence of the adhesive ligand Arg-Gly-Asp (RGD) on chondrogenic differentiation. Compared to unmodified gels possessing uniform biophysical properties, RGD-modified OMA hydrogels with the same modulus promoted chondrogenic differentiation of MSCs as evidenced by gene expression, matrix deposition, and histological analysis. MSCs entrapped in OMA hydrogels exhibiting a biologically relevant stiffness gradient (2–13 kPa over 8 mm) demonstrated increased chondrogenic differentiation with increases in stiffness. MSC chondrogenic differentiation was dependent upon the ability to mechanosense the modulus of the surrounding matrix, confirmed by the addition of Latrunculin A (LatA), a soluble inhibitor of actin polymerization. These findings validate a methodology for customizing hydrogel platforms for chondrogenic differentiation and identifying the interplay of key variables to instruct cell function.