OB functions of matrix mineralization, procollagen-α1 expression, and matrix metalloproteinase (MMP) activity. (A, B) Mineralization in vitro by normal human OBs. Cells were cultured 20 days in the presence of 10 mM β-glycerophosphate and stimulated with vehicle (control) or with 0.5 and 1 mg MSU for 12 days before alizarin red staining (ARS). (A) Representative ARS staining in vehicle- or MSU-stimulated OBs. (B) Quantification of ARS, values are shown as mean ± SEM from three different donors. (C) Expression of procollagen-α1 by OBs stimulated with MSU. Cells were incubated with vehicle or with 1 mg MSU for 12, 24, and 48 hours. Gene expression was evaluated with QRT-PCR. Results were GAPDH-normalized and expressed as procollagen-α1 expression by MSU-stimulated OBs relative to unstimulated cells (fold increase). (D) Evaluation of generic MMP activity. Confluent OBs were incubated with vehicle (Control) or with 0.5 mg MSU for 24 hours, and MMP activity in the supernatant was evaluated according to the manufacturer’s protocol. MMP activity in MSU-stimulated cells was calculated as the ratio of MSU-stimulated cells to vehicle-treated cells (control referred to as 100%). Statistics: (B) and (C) were analyzed by using one-way ANOVA followed by Tukey multiple-comparison test (n = 4 different donors); means without a common letter differ: P < 0.05. (D) Absorbance values were compared by using paired two-tailed t tests. **P = 0.002 (n = 5 different donors).