An optimized method for the chemiluminescent detection of alkaline phosphatase levels during osteodifferentiation by bone morphogenetic protein 2

Differentiation of osteoprogenitor cells into osteoblasts is a pivotal step during the normal development and repair of bone. Upregulation of endogeno us cellular alkaline phosphatase activity (AP) is a commonly used intracell ular marker for the assessment of osteoprogenitor cell differentiation into the osteoblastic phenotype. Current methods for assaying AP involve colori metric detection of the enzyme's activity using the synthetic substrate p-n itrophenol phosphate. In this paper, we explored an alternative method of d etecting AP using the chemiluminescent substrate disodium 3-(4-methoxyspiro (1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1(3,7)]decan)-4-yl) phenyl ph osphate (CSPD) for enhanced AP sensitivity and a more simplified assay. Usi ng calf intestinal alkaline phosphatase as a standardizing enzyme, we deter mined that the chemiluminescent detection system was four orders of magnitu de more sensitive than the standard colorimetric method of detection. Moreo ver, the chemiluminescent assay was faster and marked ly simpler to perform . To maximize the utility of this assay system, two osteoprogenitor cell li nes were compared for their ability to generate alkaline phosphatases in vi tro when exposed to recombinant human bone morphogenetic protein (rhBMP-2). The W20-17 cell line was substantially more sensitive to rhBMP-2 than the C3H10T1/2 cell line, where each cell line produced detectable increases in AP after exposure to rhBMP-2 levels of 5 and 25 ng/ml, respectively. The ex perimental design for AP responsiveness to rhBMP-2 was further optimized fo r chemiluminescent detection with the W20-17 cell line by comparing the eff ects of reporter cell seeding density and the day of assay. In summary, the data presented in this paper demonstrate a faster, simpler, and more sensi tive chemiluminescent method to monitor changes in AP levels during osteodi fferentiation. I. Cell. Biochem. 80:532-537, 2001. (C) 2001 Wiley-Liss, Inc .