Abstract
Novel bone substitute materials need to be evaluated in terms of their osteogenic differentiation capacity and possible unwanted cytotoxic effects in order to identify promising candidates for the therapy of bone defects. The activity of alkaline phosphatase (ALP) is frequently quantified as an osteogenic marker, while various colorimetric assays, like MTT assay, are used to monitor cell viability. In addition, the DNA or protein content of the samples needs to be quantified for normalization purposes. As this approach is time consuming and often requires the analysis of multiple samples, we aimed to simplify this process and established a protocol for the combined fluorescence-based quantification of ALP activity and cell viability within one single measurement. We demonstrate that the fluorogenic substrate 4-methylumbelliferone-phosphate (4-MUP) and the commonly used para-nitrophenylphosphate (p-NPP) produce comparable and highly correlating results. We further show that fluorescein–diacetate (FDA) can be used to quantify both cell viability and cell number without interfering with the quantification of ALP activity. The measurement of additional normalization parameters is, therefore, unnecessary. Therefore, the presented assay allows for a time-efficient, simple and reliable analysis of both ALP activity and cell viability from one sample and might facilitate experiments evaluating the osteogenic differentiation of osteoblast precursor cells.
Highlights
Many bone substitute materials, e.g., bioactive glasses, are known to promote osteogenic differentiation in osteoblast precursor cells like human mesenchymal stromal cells, making them therapeutic options for the repair of bone defects [1]
As the absolute alkaline phosphatase (ALP) activity of a sample lacks explanatory power, values usually need to be normalized to the total number of cells or correlating values in a second step
As the individual measurement of alkaline phosphatase activity, cell viability and normalization parameters can be very time consuming, especially when multiple materials and cell types need to be investigated, we aimed to simplify these measurements by a combined fluorescence-based approach
Summary
E.g., bioactive glasses, are known to promote osteogenic differentiation in osteoblast precursor cells like human mesenchymal stromal cells, making them therapeutic options for the repair of bone defects [1]. Besides the quantification of extracellular calcium deposits or the gene expression analysis of osteogenic marker genes, alkaline phosphatase (ALP) activity functions as an approved marker for osteogenic differentiation of in vitro cultured osteoblast precursor cells [2,3]. ALP converts the transparent p-NPP into the yellow para-nitrophenol (p-NP). This color change can be quantified by photometry. As the absolute ALP activity of a sample lacks explanatory power, values usually need to be normalized to the total number of cells or correlating values in a second step. Comparing the ALP activity to the total protein or dsDNA concentration of the investigated sample are approved ways to give reasonable context to the obtained data [6,7]
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