Modelli di organoidi da cellule staminali di polpa dentale (DPSCs)

In recent decades, two-dimensional (2D) cell cultures have been the experimental model of choice in biological and biomedical research. This simple, inexpensive and versatile system is easily reproducible and has enabled us to understand mechanisms and identify pathways. However, 2D models have significant limitations, including unnatural morphology, a lack of extracellular matrix and tissue architecture, and reduced complexity, which makes it difficult to fully understand cellular behaviour. For this reason, interest has increasingly shifted towards 3D cell models — systems in which cells organise themselves into complex structures, such as organoids. However, organoids themselves have significant drawbacks, such as higher cost and difficulty in standardisation. Currently, there is no unified protocol for generating organoids. The primary aim of this study has been to generate organoids from dental pulp stem cells (DPSCs) to create a useful model for translational studies. These stem/mesenchymal cells are of interest due to their high regenerative potential and ease of recovery. The cells were subjected to conditions that allowed osteogenic differentiation using a specific differentiation medium. Cell viability was assessed using the CCK-8 assay and trypan blue staining, while osteogenic morphology and mineralisation were evaluated via Alizarin Red analysis and Raman microspectroscopy. Finally, immunofluorescence analysis was used to evaluate the expression of odonto/osteogenic markers such as RUNX and OSX. Alizarin Red analysis revealed a greater presence of calcium phosphate deposits compared to the control groups (CTR), a finding that was confirmed by Raman spectral profiles attributable to a hydroxyapatite-based biomaterial. Immunofluorescence analysis also revealed increased expression of odonto/osteogenic markers (RUNX and OSX), alongside reduced expression of stemness markers. In summary, the organoids derived from dental pulp stem cells (DPSCs) exhibited highly effective osteogenic differentiation. This was evidenced by the organised deposition of a mineralised extracellular matrix rich in hydroxyapatite, which is the primary component of native bone and dentin tissue, as revealed by colorimetric and spectral analyses. This phenotypic maturation was accompanied by the coordinated activation of specific markers of mature cell lines. These results suggest that dental pulp organoids could be used as a valuable model for studying mineralization and a potential platform for drug screening and regenerative medicine applications.