Bzb induces matrix mineralization and calcium secretion by MSCs. C3H10T1/2 (A,B), MSC from normal donors (C,D), and MM patients (E,F) were cultured at 6-well plates for 24 hours at an initial density of 3 × 105/cm2. The cells were cultured in the absence of (A,C,E) or presence of 2 nM Bzb (B,D,F) for 21 days. Matrix mineralization was assessed by von Kossa staining. Stained cultures were photographed at 100× magnification. C3H10T1/2 cells (G) and MSCs from an MM patient (H) were cultured in the absence or presence of Bzb at indicated concentrations for 21 days using BMP-2 (200 ng/mL) or OB differentiation media (BDM) as the positive controls. Calcium staining was assessed by ARS as described. Results are mean plus or minus SD (n = 3). *P < .01, **P < .01, relative to control. An Olympus IMT2 inverted research microscope (Olympus) using an Olympus 40×/0.65 P was used. The fluorescent images were acquired using a SPOT camera (Diagnostic Instruments) Model: 2.21 and were processed with an Advanced STOP version 4.7 software (Diagnostic Instruments) and Adobe Photoshop CS2 version 9.02 software (Adobe Systems).
