## 4 Discussion
Other protocol adaptations, such as increased incubation and extraction times, were not effective in this study, indicating that further strategies might be tested, such as the use of other solvents, in order to enable the adequate use of this method with bone cell spheroids. Although XTT is a soluble version of the tetrazolium test, therefore not requiring an extraction step, it presented similar results as the MTT for the 3D model. Analyzing the histological sections, it is possible to observe that the dye left the cells of the outer layers, but remained trapped inside the spheroid, which was strongly stained. A similar result was observed for the third colorimetric assay tested, using the Neutral Red dye, which also failed to leave the inner layers of the aggregates during extraction, as revealed by the histological analysis. It is important to note that although the XTT, MTT, and NR tests did not perform adequately for the spectrophotometric analysis, the formazan labeling is still present after the cryosections, indicating the viability of the cells throughout the spheroid. Thus, those reagents/dyes can still be used in the 3D model as a qualitative histochemical analysis of cell viability, including the assessment of the penetration of the effects of a given toxicant or material, reinforcing the usefulness of 3D models for histological approaches. Many authors employ the enzymatic LDH assay to determine cell viability of 3D models [40; 41]. In the present study, LDH was the assay with best performance in the detection of different cell densities inside the MC3T3 spheroids. The test was sensible to differentiate different densities and showed a dose-response when the spheroids were exposed to latex, resulting in an IC\({}_{50}\) estimated at 175 mg/mL. Curiously, a previous study employing monolaminar bidimensional MC3T3-E1 culture was able to identify a much higher sensitivity of these cells to latex samples of the same origin as this study, with an IC\({}_{50}\) value approximately ten times lower (16 mg/mL) [42]. This expected pattern of lower sensitivity of 3D models to toxicants is often reported in the literature and is most probably related to the strong cell-cell and cell--CM connections that alter the drug/toxic penetration [43], in a manner that is more relatable to the in vivo response to these molecules. An important observation was made regarding the response of the LDH test to aggregates with different times of cultivation, as the optical density observed for spheroids at 4 days was higher than that observed for those on the 7 th day. In this study, we were not able to experimentally identify if this reduced signal could be attributed to a smaller proportion of cells at day seven, as the mineralized spheroids at this experiment could not be disaggregated for cell counting with trypan blue. On the other hand, it is also possible that the spheroids become more difficult to be quantified over time, most probably, due to the mineralization and strengthening of the matrix. This hypothesis remains untested and should be assessed by further studies but is a possibility of great relevance for researchers that intend to investigate cell density during studies of the osteogenic potential of drugs and materials, that usually employ longer experimental times, demanding further refinement of the available quantification methods. In summary, the results of the present study describe a reproductive model, with characteristics more similar to the bone tissue in vivo, presenting mineralized matrix production even without induction, but that demands the development of particular methodologies for cytotoxicity assessments, with special issues related to colorimetric assays. Methods that are very dependent on dye extraction may not be suitable for quantitative analysis of the three-dimensional bone model but can be used qualitatively on histological sections.