The stiffness of a crosslinked hyaluronan hydrogel affects its chondro-induction activity on hADSCs

Matrix stiffness plays an important role in stem cell differentiation. This study reports the synthesis of methacrylated hyaluronan (MeHA) with different degrees of methacrylation, ranging from 15 to 140% per disaccharide unit, which corresponds to a matrix stiffness ranging from 1.5 to 8 KPa. The swelling ratio was inversely proportional to the matrix stiffness, but the water content remained constant at >97% of the hydrogel mass. A fibril-like surface morphology and larger pore size were observed in lyophilized MeHA hydrogel with a lower stiffness. The matrix stiffness also affected the degradability of the MeHA hydrogel, where softer MeHA hydrogels (MeHA₁₅ and MeHA₃₀) were completely degraded within 6 days and a stiffer MeHA hydrogel (MeHA₁₄₀) was able to retain ∼25% of its initial mass after 30 days. Subsequently, the crosslinked MeHA hydrogel was used as a scaffold to encapsulate human adipose-derived stem cells (hADSCs). The embedded cells remained viable and expressed ∼11-fold higher levels of aggrecan and 42-fold higher levels of collagen type II in MeHA₁₄₀ compared with ADSCs cultured in HA-coated wells. In addition, cells grown in MeHA₁₄₀ exhibited the highest rates of glycosaminoglycan and collagen type II synthesis of ∼5 ng/DNA and 0.4 ng/DNA, respectively. Immunofluorescence staining showed an increase of collagen type II synthesis in MeHA₆₅, MeHA₈₅ and MeHA₁₄₀. This study showed that the matrix stiffness of a hydrogel can be modulated by the degree of methacrylation, thus affecting the efficacy of chondrogenesis in hADSCs.