Assessment of bisphosphonate treated-osteoblast behaviors by conventional assays and a simple digital image analysis

The main objective of this study was to analyze the changes in osteoblast behaviors by two different methods, a simple digital image analysis method and conventional assays. Osteoblast progenitor cells and osteoblasts were treated with alendronate (ALN; a nitrogen-containing bisphosphonate). Osteoblast behaviors such as the uptake of ALN, cell proliferation, cell differentiation, and mineralized nodule formation were examined. Quantitative assessments were conducted using a publically available ImageJ software along with conventional methods. Furthermore, ImageJ method and conventional assay for mineralized nodule formation were performed simultaneously and were compared in order to demonstrate the reliability of ImageJ analysis. Osteoblast precursors and osteoblasts responded to ALN treatments. The software could identify various colors and allowed for the quantification of staining intensity and area coverage. Both image analysis and conventional assays detected the changes in cell behaviors between treated and untreated samples. For alizarin red S assay, the staining intensity calculated by ImageJ analysis was comparable to the absorbance measured by conventional assay. These findings showed that digital image analysis along with conventional assays could be used for quantitative assessment to evaluate osteoblast alteration by drug treatment. Image analysis method is practical and might be useful for other applications in the field of biology and medical sciences. It could also be employed in a combination with the conventional assays to strengthen the data.