## 4 Discussion  
In the present work, we developed and characterized a preosteoblast cell spheroid model, aiming to provide a simple, low-cost, easy reproduction tool to achieve one further step towards the simulation of bone microtissue, and improve the predictivity of cytotoxicity assessments of bone-substitute biomaterials and pharmaceuticals related to bone treatment. For this, we verified the applicability of the model for common cytotoxicity tests employed in the scientific literature and recommended in international standards (ISO10993-5:2009), including the colorimetric assays MTT, XTT, and NR, as well as the enzymatic LDH method. Our results indicate that the methods and parameters used for the 2D models are not directly applicable to the wide use of this type of 3D models, and that adaptations must be developed for these systems to achieve their full potential. The choice of the cell type used in an in vitro biological assessment is an important feature that must balance its advantages and limitations. While primary cells have the benefit of a closer response to that observed in vivo, since they retain most characteristics of the tissue of origin, obtaining these cells is usually difficult, and limited by ethical approval and availability of donors. A limited number of passages and high variability among donors are other interfering factors that restrict the use of primary cells in several studies [15]. Immortalized cell lines, on the other hand, have the accumulation of mutations and metabolic alterations as their main disadvantages, but have important advantages in their commercial availability, uniformity, unlimited growth, and being well-characterized in the scientific literature, therefore, contributing to achieving standardized and reproducible tests [15]. In this context, MC3T3-E1 is a well-established non-transformed cell line derived from mouse calvaria bone, often used in materials research and osteogenesis studies, accumulating a large amount of data in the literature [16]. While its animal origin requires interspecies extrapolation for human hazard purposes [17], MC3T3 cells have close similarities to human osteoblast behavior and phenotype [17; 18]. Regardless of the cell origin, the characterization of a 3D model is an important step to enable its safe and reproducible use in different experimental scenarios. In the present study, the proposed MC3T3-E1 spheroids presented a pattern of reduction in diameter over time. This phenomenon has already been observed in other works with spheroids of other cell types [19; 20; 21; 22]. Possible explanations for this phenomenon may reside in differences in Extracellular Matrix (ECM) secretion and reorganization [19], or an increase in interdigital connections between the cells, increasing the expression of tight junctional proteins such as E-cadherin [22]. Another possibility is cell death in the spheroid core, due to the lack of oxygen and nutrients in their central areas. It is interesting to observe that the MC3T3 spheroids were stabilized on an average diameter a little under 400 \(\upmu\)m considering that oxygen and nutrient diffusion rarely exceeds 200 \(\upmu\)m, which is the maximum distance between a cell and the nearest capillary [23]. Furthermore, the initial aggregated presented apoptotic cells in the core, while the histological evaluation did not evidence the presence of a necrotic core. In conjunction, this characterization points out important cytotoxicity assessments with this model to ensure the use of spheroids with similar time of formation and diameter to generate comparable results. The time for the complete formation of a 3D model varies according to the cell type. Some studies indicate that the best time for spheroid use is around day 7, as described for human mesenchymal stem cell spheroids [24] and primary human hepatocyte spheroids [25]. Other works show that the first day of the formation may be already adequate for aggregates composed of tumor cells, such as glioma spheroids [26].