Dose- and time-dependent effects of taxifolin on viability and mineralization markers of osteoblast-like cells

Abstract

The current study evaluated the effects of taxifolin treatments on the viability of osteoblast-like cells, and on the expression of early mineralization markers, as part of the ongoing search for new endodontic materials able to induce periapical healing without causing cytotoxicity. Saos-2 osteoblast-like cells were exposed to different concentrations of taxifolin (5 and 10 µM), applied as pretreatments either for 24h and 72h, or continuously throughout the experimental protocol. Cell viability using the methylthiazole tetrazolium (MTT) assay, alkaline phosphatase activity using thymolphthalein monophosphate assays, deposition of mineralized nodules using alizarin red staining, and expression of ALP and COL-1 by qPCR were determined after 6 and 13 days of treatment. The data were analyzed statistically (p<0.05). Taxifolin was not cytotoxic in the concentrations tested. Pretreatments with taxifolin for 24h and 72h at 10 µM stimulated ALP activity, and increased mineralized nodule deposition by Saos-2 cells. Continuous treatment with taxifolin was not effective in stimulating ALP activity and mineralization. ALP and COL-1 gene expression increased with taxifolin pretreatments, since the highest mRNA levels were observed after 72h of pretreatment with taxifolin at 10 µM on day 13. In conclusion, taxifolin was cytocompatible, and induced mineralization markers when applied for short periods in osteoblast-like cell culture.

Cytotoxicity and Biomineralization Potential of Flavonoids Incorporated into PNVCL Hydrogels

“…Cells were seeded (T2) and evaluated for cytotoxicity at 48 h (T4), 8 days (T5) and 14 days (T6); for ALP at 8 days (T5); and for MN deposition at 14 days (T6) ( Figure 1 B). All assays were performed in triplicate in three independent experiments [ 36 ].…”

“…The cell viability was evaluated using resazurin colorimetric assays at 48 h, 8 days and 14 days after treatments, according to previous studies [ 34 , 35 , 36 , 37 ]. Briefly, cells were seeded into 96-well plates (5 × 10 3 cells/well) and incubated for 48 h. For flavonoid screening, culture medium was aspirated and cells were treated with AMP, ISO and RUT at 100, 50 and 25 µM for 48 h under standard cell culture conditions.…”

“…After all these periods, cells were washed with sodium phosphate buffer (PBS, 10 mM, pH 6.8) and stained with resazurin (#7017, Sigma Aldrich) for 4 h. Absorbance values were read at each time point at 570 and 600 nm in a spectrophotometer (Biotek, Winooski, VT, USA). These values were converted into percentages of cell viability considering DMEM medium as 100% of growth [ 34 , 35 , 36 , 37 ].…”

“…MDPC-23 at 3 × 10 2 cells/well in 48-wells plates were treated with flavonoids (from 25 to 100 μM) or the PNVCL extracts (diluted at 1/16 and 1/32) for 48 h with subsequent osteogenic DMEM changing until completing 8 days. Total protein (TP) quantification and alkaline phosphatase activity (ALP) assays were conducted according to previous studies [ 35 , 36 , 37 ]. Briefly, cells were washed after aspiration of cell culture, and 0.1% sodium lauryl sulfate (Sigma-Aldrich) was added to each well for 10 min to lyse cells.…”

“…ALP activity was calculated based on a standard curve with known enzyme concentrations. The final ALP data (percentages in relation to the osteogenic DMEM control) were divided by the values of total protein (percentages in relation to the osteogenic DMEM control) to normalize the ALP results [ 35 , 36 , 37 ].…”