Quantifying Osteogenic Gene Expression

The genetic expression of human alkaline phosphatase (ALP), collagen type 1 (Col1A1), dentin sialophosphoprotein (DSPP), osteonectin (ON), runt-related transcription factor 2 (RUNX2), and bone sialoprotein progenitor (BSP) was determined by quantitative polymerase chain reaction (qPCR) from cells treated with discs of the different material eluates after 14 days (n = 3) as described previously [16 (link)]. Briefly, cells were harvested and total RNA was isolated (Purelink RNA Mini Kit, Invitrogen, Thermo Fisher Scientific). Following that, cDNA synthesis was performed (iScript RT Supermix, Bio-Rad, Hercules, CA, USA). The 2^‑ΔΔCT method was used to calculate the relative gene expression values obtained by qPCR analysis for each gene compared to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression. Cells cultured with basal growth media were used as negative control, and cells treated with a commercial differentiation media (StemMACS OsteoDiff Media, Miltenyi Biotec, Bergisch Gladbach, Germany) acted as the positive control. Each experimental condition was carried out in triplicate for each sample and analyzed in three independent experiments.