Mind the Viscous Modulus: The Mechanotransductive Response to the Viscous Nature of Isoelastic Matrices Regulates Stem Cell Chondrogenesis

Abstract

The design of hydrogels as mimetics of tissues’ matrices typically disregards the viscous nature of native tissues and focuses only on their elastic properties. In the case of stem cell chondrogenesis, this has led to contradictory results, likely due to unreported changes in the matrices’ viscous modulus. Here, by employing isoelastic matrices with Young's modulus of ≈12 kPa, variations in viscous properties alone (i.e., loss tangent between 0.1 and 0.25) are demonstrated to be sufficient to drive efficient growth factor‐free chondrogenesis of human mesenchymal stem cells, both in 2D and 3D cultures. The increase of the viscous component of RGD‐functionalized polyacrylamide or polyethylene glycol maleimide hydrogels promotes a phenotype with reduced adhesion, alters mechanosensitive signaling, and boosts cell–cell contacts. In turn, this upregulates the chondrogenic transcription factor SOX9 and supports neocartilage formation, demonstrating that the mechanotransductive response to the viscous nature of the matrix can be harnessed to direct cell fate.

Matrix viscoelasticity controls epithelial cell mechanobiology through dimensionality

“…Various strategies have been proposed to independently control the stiffness and viscoelasticity of PAAm hydrogels, including integrating viscous pre-polymerized linear acrylamide into an elastic network (14, 40), adjusting the initiator and activator concentrations (20), or modifying the Aam-to-Bis ratio (16). We recently refined the latter approach to generate isoelastic matrices with diverse dissipative profiles (18), where viscoelasticity is enhanced by increasing the concentration of monomer while concurrently decreasing the concentration of crosslinker ( Fig. 1b ).…”

“…1b ). In this scenario, the network’s elasticity is governed by the monomer concentration, while the low concentration of crosslinker favours physical entanglements between long and loosely bound polyacrylamide chains, providing viscoelastic characteristics to the material (16, 18). By maintaining the Bis percentage at ∼0.01 % and increasing the AAm percentage from 15% to 35%, we produced viscoelastic PAAm hydrogels with initial E akin to that of elastic hydrogels ( Figs.…”

“…Stress relaxation measurements were conducted using a fiber-optic based nanoindentation device (Chiaro, Optics 11 Life) mounted on top of an inverted optical microscope (Axiovert 200M, Zeiss), adapting a previously described approach (18). The instrument operated in closed-loop mode (Indentation mode), maintaining a constant indentation depth, δ, over time to characterise the local stress relaxation response of the material.…”

“…Concurrently, the load signal was recorded. The acquired data were analysed using a previously described open source Jupyter notebook available on GitHub (18). At least two hydrogels per conditions were tested, with over 100 points per hydrogel, spaced at least twice the contact radius () from the previous one to ensure different points were tested.…”

“…While exploration of relevant relaxation times in vivo is still at its early stages (9, 12), the role of viscoelasticity in influencing cell responses in vitro has gained appreciation over the past decade. Notably, research has shown that matrix energy dissipation plays a regulatory role in fundamental processes such as cell spreading (13–15), differentiation (14, 16–18) and more recently cell migration (19, 20) and cancer progression (12). In the body, cell migration plays a crucial role in diverse processes, including development, wound healing and cancer metastasis (21).…”