In tissue regeneration, the goal is to regenerate tissue similar to what was damaged or missing while preventing fibrotic scarring, which may lead to decreased mechanical strength and dissimilar tissue characteristics compared to native tissue. We believe collagen orientation plays a critical role in wound contraction and scarring and that it is modulated by myofibroblasts. We used macrophage conditioned medium to simulate complex events that can influence the fibroblast phenotype during the wound healing process. In addition to examining the effect of macrophage phenotype on fibroblasts, we inhibited focal adhesion kinase (FAK), Rho-associated protein kinase (ROCK), and myosin II for fibroblasts cultured on both tissue culture plastic and methacrylated gellan gum to understand how different pathways and materials influence fibroblast responses. Collagen orientation, α-SMA expression, focal adhesion area, and cell migration were altered by inhibition of FAK, ROCK, or myosin II and macrophage phenotype, along with the substrate. An increase in either focal adhesion area or α-smooth muscle actin (α-SMA) expression correlated with an aligned collagen orientation. Gellan gum hydrogel upregulated α-SMA expression in ROCK inhibited conditioned media and downregulated the FAK area in FAK and ROCK inhibited conditioned media. Myosin II had no impact on the α-SMA expression on the substrate compared to coverslip except for M2 conditioned medium. Gellan gum hydrogel significantly increased cell migration under FAK and Myosin II mediated conditioned media and unconditioned media. Collectively, our study examined how macrophage phenotype influences fibroblast response, which would be beneficial in controlling scar tissue formation.