Dental Pulp Stem Cell-Derived Organoids: Advancing the Development of 3D Structures

Highlights: What are the main findings? The main aim of this article is to propose a validated and standardized protocol for characterising and conducting advanced structural analyses of dental pulp-like organoids using Raman microspectroscopy. What is the implication of the main findings? These findings demonstrate improved reproducibility and reliability of these models and could have significant implications for their use in preclinical studies and drug screening. Furthermore, enabling Raman microspectroscopy to investigate the structural organisation of 3D dental pulp organoids paves the way for future clinical applications in the critical fields of bone tissue engineering and regenerative medicine. Two-dimensional cell cultures are crucial research tools, and they have been widely used, although they are not completely representative of biological processes in vivo due to the lack of tissue architecture and complexity. Recent advances in organoid technology have addressed these limitations and are revolutionizing the tools available for in vitro culture. Although there are no unified protocols for generating organoids, they can be obtained with various techniques, leading to cell aggregation by promoting cell adhesion. This work aims to generate and characterise organoid models of dental pulp from dental pulp stem cells (DPSCs), a type of mesenchymal stem/stromal cells known for their high regenerative potential and ease of accessibility, to establish a model for translational studies. The organoids were subjected to osteogenic differentiation conditions. Cell viability was evaluated using a CCK-8 assay, while osteogenic morphology and mineralization were confirmed by Alizarin red analysis, Raman microspectroscopy, and by immunofluorescence for the lineage markers expression. The Alizarin red analysis indicated a higher presence of calcium phosphate deposits in the differentiated organoids than in the control group (CTR). These results were confirmed by spectral profiles obtained using Raman microspectroscopy, which were 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 conclusion, the organoids appeared to have successfully differentiated into an osteogenic lineage, forming a mineralized matrix containing hydroxyapatite and showing increased expression of relevant lineage markers.