Figure 3

A. Osteoblast Activation. A: Mineralization Assay. Left; pre-osteoblasts were seeded evenly to confluence and treated for 6 days with 8 ng/ml rhBMP-2 plus varying concentrations of the rhPln.D1. Dark stain represents dose-dependent mineralization as a result of alizarin red staining. Right; Stained mineralization in the wells was quantified by densitometry. Data represent fold increase beyond the level of mineralization seen with low level BMP-2 alone. Star represents statistical difference between the mineralizing effects of both 2 and 4 μg/ml Pln.D1 conditions with either 0 or 1 μg/ml Pln.D1 (p = 0.02). Data represent replicates in three separate experiments. B. Alkaline Phosphatase Assay. Activation of pre-osteoblasts was determined by AP activity released by pre-osteoblasts into serum-free medium after 5 days incubation. Conditions compared BMP-2 dilutions immobilized either on plastic (BMP-2) or onto rhPln.D1-coated wells (rhPln.D1/BMP-2). Equal levels of BMP-2 in wells was confirmed by ELISA (not shown). K_act for each condition was determined from dilution series equilibrium point representing 50% maximum activity release (inset).