Bone-Like Tissue Formation by Three-Dimensional Culture of MG63 Osteosarcoma Cells in Gelatin Hydrogels Using Calcium-Enriched Medium

The aim of this study was to investigate the effect of Ca²⁺ concentration in culture medium on the promotion of osteogenesis by MG63 osteoblast-like cells and to prepare bone-like tissues by supplying Ca²⁺-enriched medium to MG63 cells immobilized in three-dimensional gelatin hydrogels. Human osteosarcoma MG63 cells were cultured on tissue culture dish under various Ca²⁺ concentrations to evaluate the effect of Ca²⁺ concentration on calcium deposition. When Ca²⁺ concentration was 8 mM, the maximum calcium deposition was obtained at day 28. Then MG63 cells were entrapped in gelatin hydrogels cross-linked by transglutaminase and cultured for 28 days, either in a standard culture medium or in medium containing 8 mM Ca²⁺. Effects of Ca²⁺-enriched medium on osteoblastic phenotype of MG63 cells in gelatin hydrogels were analyzed in terms of cell number, calcium deposition content, and alkaline phosphatase (ALP) activity. The characteristics of calcified gelatin hydrogels were evaluated by x-ray diffraction (XRD), histological analysis, and scanning electron microscopy (SEM). After 28 days of culture, no significant difference in cell numbers was found between the different culture conditions. However, calcium content of gelatin hydrogels with cells cultured in Ca²⁺-enriched media was significantly higher than that of hydrogels with cells cultured in standard Ca²⁺ concentration medium. After 14 days of culture, ALP activity of cells cultured in Ca²⁺-enriched media was down-regulated compared with that of cells cultured in standard Ca²⁺ concentration media. XRD analysis indicated the formation of hydroxyapatite in gelatin hydrogels cultured in the Ca²⁺-enriched media at day 14, and the XRD pattern of the composite at day 21 was almost similar to that of mouse tibia. Moreover, histological analysis and SEM analysis revealed that cross-sections of hydrogels cultured in Ca²⁺-enriched media had an organic/mineral layer structure analogous to that of mouse tibia.