2.4.3. Gene Expressions

To assess the osteogenic differentiation effects of different substrates, gene expressions of osteogenic differentiation markers, that is, osteocalcin (OCN) and osteopontin (OPN), were investigated by real-time PCR. Cells were seeded at 1 × 105 cells/mL on Ti, PDGF/Hepa/Ti, BMP/Hepa/Ti, and PDGF/BMP/Hepa/Ti, and cultured for 7 or 21 days (*n* = 5). RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA). 1 μg of total RNA was reverse transcribed into cDNA using AccuPower RT PreMix (Bioneer, Daejeon, Korea), according to the manufacturer's protocol. Primer sequences of target genes were as follows: OCN (F) 5 -TTG GTG CAC ACC TAG CAG AC-3 , (R) 5 -ACC TTA TTG CCC TCC TGC TT-3 ; and OPN (F) 5 -GAG GGC TTG GTT GTC AGC-3 , (R) 5 -CAA TTC TCA TGG TAG TGA GTT TTC C-3 . PCR amplification and detection were performed using an ABI7300 Real-Time Thermal Cycler (Applied Biosystems, Foster, CA, USA).

#### *2.5. In Vivo Animal Study*
