University of São Paulo Sao Paulo, Sao Paulo, Brazil
Abstract: It is well known that dental pulp stem cells (DPSCs) cultured in monolayers commit to the osteogenic lineage after 7 days under differentiation stimulus. This is defined by the peak of runx2 gene expression. When cultured in a three-dimensional conformation (3D), like a spheroid, this time could be different, furthermore, the spheroid diameter also seemed to influence this process due to the different metabolic zones inside. Our research group is studying the factors that could influence DPSCs commitment towards osteoblasts, including extracellular matrix, secretome from other cells, like macrophages and endothelial cells. Thus, the aim of the present study was to establish the period of osteogenic commitment of DPSC spheroids in different sizes in comparison to the monolayer. We cultured dental pulp stem cell monolayers (5.000 cells/cm2) and two sizes of spheroids, 100 µm (1.000 cells - fabricated in 2% agarose micromolds) and 500 µm (55.000 cells - fabricated in low adherent u-shape 96 wells). We then induced osteogenic differentiation on these cells, collected total RNA and performed histology at days 7 and 14, after spheroid formation. As a result, we observed that in monolayers and 100 µm spheroid, the peak of runx2 expression occurred on day 7, with accentuated differentiation and mineralization on day 14, shown by other RT-qPCR markers, like collagen-1, and alizarin red staining. The 500 µm spheroid, on the other hand, presented a later runx2 peak. At least on day 7, the 500 µm showed a β-catenin peak, usually seen before the runx2 peak, indicating a tendency towards differentiation. We conclude from this that the size of the spheroid is a determining factor for the period of cell osteogenic commitment and that the smaller size (100 µm) carries out the process more quickly, resembling the process in monolayer and in vivo. The delay in the largest size may be due to a difficulty in diffusing factors to all cell layers of the spheroid, or cell death, or secretome. With this, we suggest that the DPSCs spheroid smaller size (100 µm) has a similar osteogenic commitment than the monolayer.
