Biopolymer hydrogels have become an important group of biomaterials in experimental and clinical use. However, unlike metallic or mineral materials, they are quite sensitive to sterilization. The aim of this study was to compare the effects of gamma irradiation and supercritical carbon dioxide (scCO2) treatment on the physicochemical properties of different hyaluronan (HA)- and/or gelatin (GEL)-based hydrogels and the cellular response of human bone marrow-derived mesenchymal stem cells (hBMSC). Hydrogels were photo-polymerized from methacrylated HA, methacrylated GEL, or a mixture of GEL/HA. The composition and sterilization methods altered the dissolution behavior of the biopolymeric hydrogels. There were no significant differences in methacrylated GEL release but increased methacrylated HA degradation of gamma-irradiated samples. Pore size/form remained unchanged, while gamma irradiation decreased the elastic modulus from about 29 kPa to 19 kPa compared to aseptic samples. HBMSC proliferated and increased alkaline phosphatase activity (ALP) particularly in aseptic and gamma-irradiated methacrylated GEL/HA hydrogels alike, while scCO2treatment had a negative effect on both proliferation and osteogenic differentiation. Thus, gamma-irradiated methacrylated GEL/HA hydrogels are a promising base for multi-component bone substitute materials.