Ultrastructural changes during osteogenic differentiation in mesenchymal stromal cells cultured in alginate hydrogel

Background: Osteogenic differentiation of mesenchymal stem cells has been extensively investigated with regards to different aspects, including the analysis of cell intracellular and extracellular proteome, cell gene expression pattern, and morphology. During the osteogenic differentiation, osteoblasts produce and release specific proteins, like osteocalcin and osteopontin. Simultaneously, cells produce the extracellular matrix (ECM) that resembles the bone ECM, with high quantity of calcium and phosphorus. We focused on the ultrastructural changes occurring during the osteogenic differentiation of MSC cultured in alginate hydrogel. Results: The analysis revealed that during the osteogenic differentiation the most of cells become dead, and these dead cells contain large quantities of calcium and deposition is strictly connected with the cellular death and small membrane vesicles released by cells. Cell organelles were not present within differentiated cells, while in cells from non-osteogenic group the cellular ultrastructure was proper, with single nuclei, endoplasmic reticulum and numerous mitochondria. Conclusion: The ECM synthesis and deposition during the osteogenic differentiation of MSC involves cellular programmed death. The small membrane vesicles become the mineralization sites of formed bone ECM.