Effects of the Hedgehog antagonist cyclopamine (CLP) on osteoblast differentiation and the expression of Hedgehog receptors in osteoblasts grown on polished Ti (Ti-Control) and Ti with nanotopography (Ti-Nano). The gene expression of the osteoblastic markers runt-related transcription factor 2 (Runx2), osteopontin (Opn), and alkaline phosphatase activity (Alp) on day 10 (A), RUNX2 protein expression on day 10 (B), ALP activity on day 7 (C), extracellular matrix mineralization on day 17 (D), gene expression of the Hedgehog receptors zinc finger 1, 2 and 3 transcription factors (Gli1, Gli2, and Gli3) on day 10 (E) and GLI1 protein expression on day 10 (F) in osteoblasts cultured on Ti-Control and Ti-Nano with either vehicle or CLP 10 nM. The original diameter of the Ti discs presented in C and D is 13 mm. The data of gene expression (n = 4), protein expression (n = 3), ALP activity (n = 5), and extracellular matrix mineralization (n = 5) are presented as the mean ± SD, and * indicates statistically significant differences (p ≤ 0.05).