Journal: Bone

Article Title: Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures

doi: 10.1016/j.bone.2013.01.020

Figure Lengend Snippet: MC3T3-E1 osteoblasts were cultured in osteogenic media and treated with either 10 μM PolyP5 or PolyP65 for the indicated 2-day intervals. After 12 days of culture, mineral was stained in the plates (A) by the von Kossa method (black), or (B) the cell/matrix layer was embedded in LR White acrylic resin, sectioned perpendicular to the culture dish plane, and sections were stained with the von Kossa method followed by counterstaining with toluidine blue. By both methods, less mineral is seen after PolyP treatment starting at days 4–6 when the matrix starts to mineralize; earlier treatments when matrix is being first secreted and assembled had little effect. (C) Direct binding of PolyPs to hydroxyapatite (HAP) was determined by incubating the indicated concentrations of synthetic HAP beads in either 0.5 mM PolyP5 or PolyP65 solutions, and quantifying the remaining concentration of PolyP in the supernatant (by acid hydrolysis and phosphate quantification). Increasing amounts of HAP clearly leads to increasing depletion of PolyPs from solution. Data are presented as mean values ± SE. *p<0.05, **p<0.01, ***p<0.001 Student's t-test relative to 0 mg/ml HAP. (D) When 20 μg PolyP are bound to 2 mg HAP, hydrolysis of the polyphosphates by TNAP is less than that of polyphosphate alone. Adsorption of polyphosphate onto HAP was verified by supernatant acid hydrolysis followed by phosphate quantification. Data are presented as mean values ± SE. **p<0.01, ***p<0.001 Student's t-test relative to free PolyP.

Article Snippet: Hydroxyapatite binding assay A 20 mg/ml stock hydroxyapatite bead (5 μm average diameter, Berkeley Advanced Biomaterials, Berkeley, CA, USA) slurry was generated by washing the beads as received from the supplier three times in Tris-buffered saline, pH 7.0 (TBS), and then reconstituting in TBS.

Techniques: Cell Culture, Staining, Binding Assay, Concentration Assay, Adsorption