Total protein lysates were prepared from scaffolds at 0, 3, 14, 28, 42, and 56 days of culture by incubating scaffolds morselized using scissors in 3× SDS reducing sample buffer. Lysates were then incubated at 95°C for 5 min and centrifuged in 0.2 μm of Spin-X filters (Corning Costar, Corning, NY) at 14,000 rpm for 5 min. Protein concentration was measured, and equal amounts were subjected to 4 to 20% SDS–polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). Western blot analysis was carried out with antibodies against OPG, RANKL, p-Smad1/5, total Smad5, p-ERK1/2, total ERK1/2, and β-actin, followed by 1:4000 dilutions of HRP-conjugated immunoglobulin G antibodies (Bio-Rad, Hercules, CA) and an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL). For the detection of p-Smad1/5 and total Smad5, 10 μg of lysate was loaded per lane. For the detection of OPG, RANKL, p-ERK1/2, total ERK1/2, and β-actin, 20 μg of lysate was loaded per lane. All antibodies were obtained from Cell Signaling Technology (Danvers, MA), with the exception of RANKL, OPG, and β-actin antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). Imaging analysis was carried out using ImageJ (NIH, Bethesda, MD). The RANKL/OPG-relative protein ratios were calculated by quantifying the densitometry of all RANKL and OPG normalized to actin using ImageJ (NIH, Bethesda, MA).

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