(395a) In Situ Control of Hydrogel Modulus with Light to Direct Cell Phenotype

The structure and chemistry of material substrates are known to influence cellular functions such as migration [1], cytoskeletal organization [2], and differentiation [3]. Of particular interest recently is how the elasticity of a material, as measured by modulus, affects cell phenotype. To study this, researchers have developed hydrogels with different discrete moduli or with gradients in modulus, where modulus has been shown to influence the differentiation pathway of mesenchymal stem cells [3], the fibroblast-to-myofibroblast transition [4], and smooth muscle cell migration [1]. While great advances have been made in the fabrication of these hydrogels, the properties of these gel systems typically are fixed upon gel formation, limiting the ability to study how in situ changes in modulus influence dynamic cellular processes such as differentiation. Here, we have developed a culture platform that allows manipulation of the gel properties in situ with light and used it to study the effect of dynamic changes in modulus on the fibroblast-to-myofibroblast transition. This transition is important for understanding wound healing and tissue fibrosis. This specific culture platform is a photodegradable hydrogel with an entrapped adhesion protein, fibronectin, that promotes cell attachment and is formed by copolymerization of a photodegradable PEG-based diacrylate crosslinking monomer [5] with a monoacrylate PEG monomer. The modulus of the hydrogel surface is subsequently tuned with irradiation, degrading the photolabile crosslinks and decreasing the gel modulus. The gel degradation under cytocompatible conditions was characterized in bulk with rheometry (365 nm at 10 mW/cm² for up to 5 min). Gradients of modulus on the gel surface were created with photolithography over 9 mm and characterized with atomic force microscopy, where a range of surface moduli that span collagenous bone to muscle was created. Subsequently, this system was used to explore how modulus affects the phenotype of fibroblasts, specifically valvular interstitial cells (VICs). Moduli that promote or suppress VIC myofibroblastic differentiation were identified with gradient moduli gels by immunostaining for alpha smooth muscle actin, a conclusive indicator of myofibroblastic differentiation. VICs were subsequently cultured on myofibroblast promoting substrates and irradiated, decreasing the gel modulus in situ and de-differentiating the VICs from myofibroblasts to fibroblasts. This 2D dynamic cell culture platform offers promise for exploring how real-time changes in gel modulus may influence dynamic cellular processes such as differentiation and migration.

References

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[2] S. R. Peyton, C. B. Raub, V. P. Keschrumrus, A. J. Putnam, Biomaterials 2006, 27, 4881.

[3] A. J. Engler, S. Sen, H. L. Sweeney, D. E. Discher, Cell 2006, 126, 677.

[4] B. Hinz, J. Invest. Dermatol. 2007, 127, 526.

[5] A. M. Kloxin, A. M. Kasko, C. N. Salinas, K. S. Anseth, Science 2009, 324, 59.