Abstract
BACKGROUND: The ability to guide stem cells toward the bone-forming lineage is a fundamental aspect of regenerative medicine. Conventional protocols for osteogenic differentiation of mesenchymal stem cells typically rely on continuous exposure to the synthetic steroid dexamethasone, in combination with other supplements such as ascorbate and glycerophosphate. Although dexamethasone effectively induces bone-related gene expression and matrix mineralization, prolonged or excessive exposure can lead to adverse catabolic and cytotoxic effects. Therefore, there is a need to develop improved culture conditions that retain efficacy while enhancing physiological relevance, safety, and reproducibility. This study investigated whether 1-week dexamethasone exposure and supplementing the medium with vitamin D3 and the synthetic vitamin K analogue K3 could enhance osteogenic differentiation of bone marrow-derived mesenchymal stem cells. METHODS: Stem cells were cultured for 28 days in four types of osteogenic media differing in the duration of dexamethasone exposure and the presence of vitamins D3 and K3. Osteogenic differentiation was evaluated using histochemical staining for mineralized matrix, quantification of calcium deposition, analysis of alkaline phosphatase activity, fluorescence microscopy of cellular morphology and mitochondrial organization, and gene expression of osteogenic markers including RUNX2, alkaline phosphatase, osteocalcin, and bone morphogenetic protein 2. RESULTS: A short exposure to dexamethasone alone was insufficient to support robust osteogenic progression, confirming the importance of sustained stimulation. In contrast, the combination of vitamins D3 and K3 produced a clear synergistic effect, characterized by stronger mineralization, increased alkaline phosphatase activity, and enhanced expression of key osteogenic genes. CONCLUSIONS: This study demonstrates that the combined use of vitamins D3 and K3, together with optimized dexamethasone exposure, provides a simple and biologically compatible strategy to improve the osteogenic differentiation of mesenchymal stem cells. This approach offers a physiologically relevant, safe, and cost-effective modification of standard osteogenic protocols and may contribute to the development of more efficient cell-based systems for bone tissue engineering and regenerative medicine.