The inhibitory effects of rGPC1, rGPC3 and rGPC1 + 3 released from proposed TNT-based cranial implants on BMP2 signaling in murine C2C12 cells. The FITC-BSA-loaded and non-loaded TNT/Ti discs acted as positive and negative controls, respectively. Cells were co-transfected with a BMP-responsive luciferase reporter, a control luciferase vector for inducing transfection and then subsequently treated with 100 ng/ml of BMP2 and 100 ng/ml of (A) control recombinant glypicans (day 0, added from the stock solution), (B) released proteins on day 1, (C) released proteins on day 8 and (D) released proteins on day 15. Luciferase expression was assayed 24 h post-treatment. The statistical significant difference compared to BMP2 treatment is denoted by * (P ≤ 0.01 for day 0, P ≤ 0.05 for day 1 and P ≤ 0.001 for day 8). Basal luciferase activity was measured for each reading to eliminate background attenuation generated from endogenous BMPs and glypicans, if any. Error bars represent mean values ± SD of the 3 technical replicates and RLU indicates relative light unit.