Levels of alkaline phosphatase and osteocalcin in human osteoarthritis subchondral bone osteoblasts. Alkaline phosphatase activity (a) and osteocalcin level (b) were determined after treatment with chondroitin sulfate (CS) (200 μg/mL), glucosamine sulfate (GS) (50 or 200 μg/mL), or both (200 μg/mL each) in the absence or presence of vitamin D3 at 50 nM. Alkaline phosphatase activity (a) was determined in the cell lysate by substrate hydrolysis using p-nitrophenylphosphate, whereas osteocalcin level (b) was determined in the culture media by using a specific enzyme immunoassay. Data are from eight independent experiments. Statistical significance was assessed by paired Student t test. P value indicates the statistical difference between control (C, basal conditions) and vitamin D3-treated specimens.