Mbs2533600

Osteoblasts separated from rat calvariae were cultured in group a: osteoblast culture medium alone as mentioned in 4.8; group b: osteoblast culture medium with C59; group c: half of osteoclast culture medium as mentioned in 4.5 (conditioned medium) and half of osteoblast culture medium with 50 mg/mL ascorbic acid (A1968, Sigma Aldrich, USA) and 2 mM b-glycerophosphate (G9422, Sigma Aldrich, USA) (bone formation medium); group d: half of conditioned medium with C59 and half of bone formation medium; group e: half of 3 nM calcitonin-treated conditioned medium and half of bone formation medium; group f: half of 3 nM calcitonin-treated conditioned medium with C59 and half of bone formation medium. For the ALP stain and ALP activity assay, each group was cultured for 7 days. For the alizarin red stain, each group was cultured for 18 days. Before osteoblast were cultured, the concentration of Wnt10b in each group of medium were detected by ELISA (MBS2533600, MyBioSource, San Diego, USA).

The concentrations in sera of Wnt10b and the bone resorption marker TRAP5b (tartrate-resistant acid phosphatase from 5b) were measured by ELISA (Wnt 10b (MBS2533600, MyBioSource, San Diego, CA, USA), TRAP5b (SB-TR102, Immunodiagnosticsystems, East Boldon, UK), and resorption marker CTX-1 (C-telopeptide of type I collagen) and formation markers osteocalcin and P1NP (type 1 procollagen amino-terminal—propeptide) were measured EIA (AC-06F1, AC-12F1, and AC-33F1 Immunodiagnosticsystems, UK).