Three-Dimensional Hydrogel Culture Reveals Novel Differentiation Potential of Human Bone Marrow-Derived Stem Cells

Objectives: Traditional 2D cell cultures on flat surfaces fail to replicate 3D environments, affecting cellular morphology and function. Various 3D techniques (e.g., spheroids, organoids, organs-on-chips, 3D bioprinting) have been used for disease modelling and drug testing, but their application in hard tissues remains challenging. This study aimed to develop a biocompatible 3D culture method for bone tissue organoids using human bone marrow-derived stem cells (hBMSCs) and hydrogels. Methods: hBMSCs were isolated from human jawbone marrow. The control group was cultured under 2D conditions, whereas the experimental group was cultured in a 3D hydrogel environment. In vitro analyses, including flow cytometry and RNA sequencing, were performed. Quantitative data were statistically analysed at a 0.05 level of significance. Results: hBMSCs cultured in 3D hydrogel conditions indicated enhanced reproducibility, increased cell viability, and significant osteogenic differentiation. Genes such as MMP-13, LPL, and SP7 showed substantially higher expression in 3D cultures, with protein-level confirmation by Western blot. These findings suggest that 3D culture more effectively supports the natural growth and differentiation of hBMSCs. Conclusions: Culturing hBMSCs in a 3D environment more closely mimics in vivo conditions, thus promoting the expression and activity of critical proteins involved in hBMSC differentiation.