Gellan gum-gelatin viscoelastic hydrogels as scaffolds to promote fibroblast differentiation

Fabricating hydrogel scaffolds that are both bioreactive toward fibroblasts while still mechanically compatible with surrounding tissue is a major challenge in tissue engineering. This is because the outcome of scaffold implantation is largely determined by fibroblasts differentiating toward myofibroblasts, which is characterized by the expression of α-smooth muscle actin (α-SMA). Previous studies promoted fibroblasts differentiation by increasing scaffold substrate stiffness. However, the stiffness of scaffold has to be compatible with surrounding tissue, as mismatched stiffness may cause initial hyperplasia and inappropriate endothelial layer development. Therefore, we adjusted the hydrogel chemical component, and thus viscoelasticity to affect the mechano-signaling of fibroblasts and promote fibroblasts differentiation. Elastic gellan gum and viscoelastic gelatin were hybridized at different ratios to fabricate hydrogel scaffold with varied stress-relaxation. Vitronectin (VN) was used to further regulate the interaction between fibroblasts and the substrate. Fibroblast differentiation, characterized by α-SMA area per cell, increased from~3000-4000 μm²/cell on less viscoelastic gels to ~5000 μm²/cell on the most viscoelastic gel. Fibroblasts seeded on hydrogels had a slower migration rate on more viscoelastic hydrogels (slowest at 38 ± 14 μm/h) compared to the migration speed on less viscoelastic hydrogels (74 ± 20 μm/h). VN slowed the migration speed on all hydrogels. The organization of collagen deposited by fibroblasts cultured on the hydrogels was characterized by second harmonic generation (SHG), which showed that collagen was more organized (parallel) on more viscoelastic hydrogels. In summary, we provided a novel strategy to fabricate hydrogel scaffolds that can promote fibroblasts differentiation while keeping the stiffness compatible with blood vessels. The most viscoelastic hydrogel studied here meets these requirements best.