Dexamethasone (dex) is an anti-inflammatory steroid drug widely used in medicine, but long-term use can contribute to osteoporosis. Interestingly, in laboratory research, dex is also commonly used to help human bone marrow mesenchymal stromal cells (hBMSCs) develop into bone-forming cells, together with another compound called β-glycerophosphate (β-GP). However, dex can trigger mixed biological responses: while it promotes some early steps of bone formation, it may also activate pathways linked to fat cell development and reduce the expression of certain genes important for healthy bone maturation. The aim of this study was to improve current laboratory methods for bone differentiation by reducing or refining the amount of dex used.
To identify the most suitable conditions, we first tested different cell culture surfaces (polystyrene, polystyrene coated with collagen, polystyrene coated with calcium phosphate and a combination of both), several dex concentrations (0–10 nM), and β-GP concentrations (0–5 mM) using hBMSCs from three donors. In total, 128 experimental combinations were evaluated. Lower/intermediate dex concentrations (0.312/0.625 nM) combined with moderate β-GP levels (1.25/2.5 mM) produced the most balanced results, supporting bone-related activity while limiting potentially unwanted effects.
Based on these findings, we carried out a second, more focused study using the selected conditions, alongside positive and negative controls, across all tested surfaces. We then analyzed gene expression, alkaline phosphatase (ALP) staining, and proteins related to mineral formation to further evaluate bone differentiation.
After 7 days, increasing dex concentrations promoted early signs of bone commitment, but also showed signals associated with fat-related pathways. At later stages (days 14 and 21), the highest dex concentration (10 nM) reduced the expression of genes linked to mature bone formation, while intermediate conditions maintained a more stable and balanced profile. ALP expression and staining were slightly higher in the 10 nM dex condition, although results varied between donors. Additional analyses of mineralization and osteonectin, a protein linked to mineral deposits, are currently ongoing.
Overall, our results show that dexamethasone has dose-dependent effects during bone differentiation of hBMSCs. High dex levels promoted early osteogenic activity but also increased unwanted adipogenic signalling and reduced markers associated with later bone maturation. In contrast, lower/intermediate dex concentrations combined with moderate β-GP levels produced a more balanced osteogenic response, suggesting that reducing dex supplementation could improve the stability and quality of bone differentiation protocols.