RNeasy kit (Qiagen, Valencia, CA) was used to extract total RNA from scaffolds at 0, 3, and 14 days of culture. Gene sequences for 18S, Runx2, OPN, OPG, and receptor activator of RANKL were obtained from the National Center for Biotechnology Information gene database, and primers were designed (table S1). qRT-PCR was performed on the Opticon Continuous Fluorescence System (Bio-Rad Laboratories Inc., Hercules, CA) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen). Cycle conditions were as follows: reverse transcription at 50°C (30 min); activation of HotStarTaq DNA polymerase/inactivation of reverse transcriptase at 95°C (15 min); and 45 cycles of 94°, 58°, and 72°C for 15, 30, and 45 s, respectively. Results were analyzed and presented as representative graphs of triplicate experiments.

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