The Impact of Gamma-linolenic Acid on Osteogenic Differentiation in Monolayer and Spheroid Cultures

This study investigated the effects of gamma-linolenic acid (GLA) on osteogenic differentiation of bone marrow stem cells (BMSCs) in monolayer and 3D spheroid cultures.BMSCs were isolated from the femurs and tibias of 6-week-old male C57BL/6 mice and cultured in 2D monolayer and 3D spheroid conditions.Cells at various concentrations were cultured to form spheroids, and their morphology was analyzed using phase contrast microscopy over time.After 7 days of osteogenic induction, extracellular matrix mineralization was assessed using Alizarin Red staining.Significantly greater mineralization was observed in spheroid cultures than in monolayer cultures, as indicated by strong positive staining and higher absorbance values (P < 0.01).Live/dead assays performed after 48 hours of culture showed no significant difference in cell viability between GLAtreated and control groups.Quantitative real-time PCR analysis demonstrated that the expression levels of key osteogenic markers (alkaline phosphatase, ALP; osteocalcin, OCN; osteopontin, OPN; osterix, OSX; and runt-related transcription factor 2, RUNX2) were significantly higher in 3D spheroid cultures than in monolayer cultures.Additionally, expression of these markers was stimulated by GLA treatment.These findings suggest that 3D spheroid cultures offer a more supportive environment for the osteogenic differentiation of BMSCs compared to monolayer cultures.Furthermore, GLA markedly enhances this differentiation, highlighting its potential as a therapeutic agent in bone tissue engineering and regenerative medicine.