Avaliação in vitro da capacidade de diferenciação osteogênica de células-tronco de dentes decíduos esfoliados humanos cultivadas em grânulos de hidroxiapatita e beta-tricálcio fosfato.

Abstract

Substantial limitations present in the use of autologous bone graft point to the need to establish concrete alternatives in the context of bone reconstructive surgery. The bioceramic of biphasic calcium phosphate (BCP), in its various proportions of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), has been launched as a peremptory possibility for this demand. In this context, the present study aimed to investigate the in vitro osteogenic response of stem cells from human exfoliated deciduous teeth (SHED) in association with BCP granules with different proportions of HA and β-TCP. SHED were cultured for 1, 7, 14 and 21 days and divided into eight groups according to the HA/β-TCP proportions of the BCP granules and the culture media employed: MC80-20 (SHED + HA/β-TCP 80/20 in conventional medium - CM); MC60-40 (SHED + HA/β-TCP 60/40 in CM); MC20-80 (SHED + HA/β-TCP 20/80 in CM); MO80-20 (SHED + HA/β-TCP 80/20 in osteogenic medium - OM); MO60-40 (SHED + HA/β-TCP 60/40 in OM); MO20-80 (SHED + HA/β-TCP 20/80 in OM); C- (SHED in CM) and C+ (SHED in OM). The viability and proliferation of SHED were assessed using MTT assay. The quantitative and qualitative analysis of the deposition of calcium ions in the mineralized matrix of SHED was carried out using the colorimetric assay with alizarin red.Gene expression for osteogenic differentiation markers, OCN and RUNX2, was evaluated by real time RT-PCR. The viability and proliferation of SHED were not affected by the presence of the granules, except on day 1, whose results were lower than in the other periods. None of the proportions of HA/β-TCP of the granules had a greater influence on the mineral deposition process, when compared to the other employed ones. In addition, the osteogenic medium, by itself, induced mineralization similar to that generated in association with granules after the 14th day. As for the osteogenic differentiation markers, some groups containing granules expressed OCN lately, on the 21st day, with no statistical difference between those who did (p> 0.05). In contrast, the expression for RUNX2 started early on the first day, in all groups, with no statistical difference between them (p>0.05), followed by a decrease on days 7 and 14 and a subsequent increase on the 21st day, in some groups. In addition, on the 21st day, the C+ group showed greater expression of RUNX2 than the other groups. Taken together and considering the limitations of the study, we concluded that BCPs were able to stimulate osteogenic differentiation in SHED, with no difference between the three proportions used.