Figure 7.

Colorimetric determination of alkaline phosphatase expression and mineralized matrix synthesis. (A) Aliquots of individual scaffold homogenate were diluted to a known concentration and analyzed for alkaline phosphatase expression. Optical density measurements were determined at 415 nm. Quantities of alkaline phosphatase were normalized by dividing the measured quantity by the total number of cells present on the scaffold from which the homogenate was prepared. (B) To quantify the amount of ALZ that reacts with mineralized matrix, we adapted a technique that solubilizes the red matrix precipitate with cetyl pyridinium chloride to yield a purple solution suitable for optical density measurements at 562 nm. The concentration of Ca²⁺ equivalents in scaffold homogenate was determined by using a CaCl₂ standard. For each time point, molar equivalent quantities were normalized for the average number of cells per scaffold as determined by companion fluorometric DNA analysis.