## 4 Discussion
In the present work, MC3T3-E1 spheroids were uniform and stable from day 4. The aggregates present a regular and compact surface when observed by SEM, remaining intact in the course of 28 days, as revealed by histological observation, even though no cell viability assessment had been performed yet for such a long culture time. They also presented small vesicles sprouting from the cell membrane, with typical dimensions of matrix vesicles. It is possible to observe a morphological modification in the spheroid cells cultured for 28 days in an osteogenic medium. This morphological modification and high numbers of filopodia can be due to thedifferentiation of osteoblasts, which when involved with the mineralized matrix, become osteocytes, which have dendritic characteristics [27]. Usually, MC3T3 cells in a bidimensional, monolaminar culture show mineralization only when cultivated in the presence of the induction medium and, in most studies, evidence of mineralization begins after 14 days of cultivation [28; 29]. The proposed MC3T3 spheroids, on the other hand, presented mineralized matrix formation in 21 days of cultivation even without treatment with induction medium, while aggregates that were treated with osteogenic medium presented a strong deposition of calcium already on the 7 th day of culture. It is possible that the close cell-cell and cell-interactions promoted by 3D models improve cell signaling and increase the local concentration of self-secreted induction factors, such as growth factors, stimulating osteoblasts to differentiate and produce mineralized matrix regardless of medium supplementation [30; 31]. This is a feature that may increase the similarity of the model with bone microtissue during medical materials testing. We tested this 3D model with different colorimetric cytotoxicity assays. Among those, MTT is a metabolic method and, therefore, both the number of viable cells and interferences cell metabolism can influence the result. However, the testing with the MC3T3 model did not identify a relationship between the number of cells on an aggregate unexposed to toxicants and the resulting Optical Density at 540 nm, indicating the lack of sensitivity of the regular protocol of MTT for this model. In contrast to our results, the literature presents different studies using MTT with 3D models, that have an apparent success investigating drug cytotoxicity using human colorectal cancer cells spheroids [6], scaffold evaluation using human osteosarcoma MG63 cells [32] and dental biomaterials biocompatibility with mesenchymal stem-cell spheroids [33]. However, other authors suggest that colorimetric assays using the reduction in tetrazolium are not applicable to three-dimensional models and micro-tissues with the collagen matrix, since the dense matrix can affect the absorption and kinetic diffusion of the dye, which impacts OD readings [34; 35]. The high production of junction proteins such as N-cadherins and E-cadherins during the development of the spheroids, which do not occur in the two-dimensional model, can be another source of interference [36]. It is interesting to note that the MTT and XTT have negative and positive charges, respectively [37] that maybe interacts with collagen charges that can be more negative or more positive depending on the pH [38]. It is possible that the formazan resulting from the tetrazolium conversion by cells would not be able to leave the inner layers of the spheroid due to the dense collagen matrix Type I collagen production that increases with time in osteoblasts and osteoblast-like cells [39]. Indeed, the sections of the spheroids after staining seem to corroborate this hypothesis as it is possible to observe that the tetrazolium is transformed to formazan even in the inner layers, and even after the extraction step with an organic solvent, the aggregates remained stained.