Effect of different osteogenic media and saline solutions on the osteogenesis protocol using MC3T3-E1 subclones

Adequate laboratory protocols may improve the study of bone tissue and its metabolism. Thus, the use of effective techniques for staining bone cells in vitroand evaluating their function is significant. The cell line used for this purpose was MC3T3-E1, which contains preosteoblasts with mineralization potential. Several osteogenic media are used in the culture of these cells, and a variety of saline solutions are used for washing cultures in mineralization staining protocols and in situ alkaline phosphatase detection. Thus, the objective of the present study was to evaluate the effects of different osteogenic media in the culture of MC3T3-E1 cells, subclones 4 and 14, in addition to washing with Dulbecco’s phosphate saline solution (DPBS) and Hanks’ balanced salt solution (HBSS) in an alizarin red staining assay and situ alkaline phosphatase labeling via the Fast Red method. The cells were seeded at a density of 1x104 cells/well for 7 and 10 days for the Fast Red assay and 12, 14, and 17 days for the staining of mineralization nodules. The data were statistically analyzed and significance was set for p < 0.05. Data obtained showed that the presence of dexamethasone significantly enhanced ALP detection in subclone 14 osteoblastic cells after 7 and 10 days as well as in subclone 4 cells washed with DPBS after 7 and 10 days when compared to control. Washing with Hanks’ solution significantly increased the quantification of ALP at 10 days and of mineralized nodules in 4 subclone cells after 17 days. Moreover, alizarin red staining improved, resulting in a more intense red color, in the group that was washed with Hanks’ solution for both subclones in all experimental periods. Thus, it is suggested that washing with Hanks’ salt solution is better for in vitro staining of calcium nodules when using the alizarin red method.