(4) Gene expression and real-time polymerase chain reaction (PCR) analysis

The expression of osteogenic genes, including OCN, HSPA5, bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), collagen type I (col1a1), transcription factor Sp7 (Osterix), and Runx2, in C3H10 cells cultured on various specimens was analyzed by real-time PCR assay. After culturing for 7 days, total RNA was extracted using Trizol reagent from cells. The concentration of RNA was measured by a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). The primers used are shown in Table 1.

Primer sequences of osteogenic genes

F, forward; R, reverse; BMP2, bone morphogenetic protein 2; ALP, alkaline phosphate; col1a1, collagen type I; OCN; osteocalcin.