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.

Background/Objectives: Bone metastases are a common complication of breast cancer. In our previous study, we reported that extracellular vesicles released by osteotropic human (MDA-MB-231) and murine (4T1) breast cancer cells disrupt bone homeostasis by enhancing osteoclast differentiation and impairing osteoblast function. Based on these findings, we investigated whether microRNAs contained within tumour-derived EVs could mediate these bone-altering effects. Methods: MDA-MB-231- and 4T1-EVs were tagged with the RNA-specific fluorophore SYTORNA and employed to treat mouse primary bone marrow macrophages (BMMs) and osteoblasts (OBs). We also performed RNAseq on MDA-MB-231- and 4T1-EVs to assess their miRNAs content. Finally, we evaluated the effect of selected miRNA-mimics on OBs, BMMs and HUVEC cells. Results: Fluorescence microscopy demonstrated EV-RNAs shuttling to recipient cells, while RNA sequencing on MDA-MB-231- and 4T1-EVs revealed that, of the top 20 expressed miRNAs, 10 were common. Among them, we first focused on the following four: miR-26a-5p, miR-24-3p, miR-29a-3p, and miR-29b-3p, which were linked to bone biology. We confirmed their presence in MDA-MB-231-/4T1-EVs by qPCR. Then, we evaluated their EV-mediated shuttling to BMMs and OBs using affinity tags. Among all the conditions tested, miR-29a and miR-29b were the best-shuttled miRNAs, with efficiency between 50–100% in both OBs and BMMs, both for MDA-MB-231- and 4T1-EVs. Finally, to test whether miR-29a and miR-29b could have a functional role in bone cells, OBs were transfected with miR-29a and 29b-mimics, discovering that this treatment reduced collagen1α1 and 1α2 mRNA as well as the OBs’ mineralisation ability, while the same miRNA mimics were found to have no effect on osteoclastogenesis or on in vitro angiogenesis. Conclusions: MDA-MB-231- and 4T1-EVs shuttle miRNAs to bone cells, which likely contributes to OBs’ activity impairment.