To develop and optimize new scaffold materials for tissue engineering applications, it is important to understand how changes to the scaffold affect the cells that will interact with that scaffold. In this study, we used a hyaluronic acid- (HA-) based hydrogel as a synthetic extracellular matrix, containing modified HA (CMHA-S), modified gelatin (Gtn-S), and a crosslinker (PEGda). By varying the concentrations of these components, we were able to change the gelation time, enzymatic degradation, and compressive modulus of the hydrogel. These changes also affected fibroblast spreading within the hydrogels and differentially affected the proliferation and metabolic activity of fibroblasts and mesenchymal stem cells (MSCs). In particular, PEGda concentration had the greatest influence on gelation time, compressive modulus, and cell spreading. MSCs appeared to require a longer period of adjustment to the new microenvironment of the hydrogels than fibroblasts. Fibroblasts were able to proliferate in all formulations over the course of two weeks, but MSCs did not. Metabolic activity changed for each cell type during the two weeks depending on the formulation. These results highlight the importance of determining the effect of matrix composition changes on a particular cell type of interest in order to optimize the formulation for a given application. 1. Introduction Tissue engineering continues to grow as a field, and with it the number of potential scaffolds also grows. Among the plethora of synthetic and natural scaffolds available are synthetic extracellular matrices (ECMs), scaffolds that are generally a mixture of multiple components and are meant to mimic various functions of the natural ECM. These functions include both structural support and cellular signaling, thereby influencing cell shape, fate, and metabolism. Thus, the goal of optimizing a synthetic ECM should be to direct cell function toward recapitulating a natural ECM, and therefore a natural tissue. Hyaluronic acid (HA) is one component that has been used in synthetic ECMs due to the benefits that HA provides to the body, including water homeostasis, joint lubrication, and its role during development and wound healing processes [1–7]. Crosslinked hydrogels based on HA remain a promising tool for a wide range of applications [8]. For example, HA-based hydrogels have been used for skin and corneal wound healing, postsurgical adhesion prevention, and as scaffolds for tissue engineering and in vitro 3D cell culture applications [9–13]. There are many methods available for crosslinking HA to
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