Abstract
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Objectives 18F-labeled sodium fluorine was the first widely used agent for skeletal scintigraphy in the 1960s. 18F-NaF exchanges rapidly for hydroxylgroups of the hydroxyapatite covalently binding to the surface of new bone, forming fluoroapatite.[1;3] Osteogenic differentiation of human mesenchymal stem cells (hMSC) is a standard procedure in todays tissue engineering. Analyzing the osteogenic potential (=hydroxyapatite) of MSCs in vitro is of major interest in order to maximize bone formation in vitro/vivo.[2] Within this abstract we present a new non-destructive method to quantify the amount of newly formed hydroxyapatite by hMSCs using 18F-NaF.
Methods hMSCs (n=6) were seeded in 3,5cm diameter flat bottom petri dishes (15.000 cells/cm2). Osteogenic induction was established with DMEM low glucose + 10%FCS + 1%P/S + 10mM beta-glycerol phosphate + 173µM ascorbic acid 2-phosphate + 100nM dexamethasone. As a control the same media without the osteogenic supplements was used. Cultures were kept at 37°C, 5% CO2, mediachange every 2 days. After 21 days 10 MBq 18F-NaF in 1ml 0,9%NaCl was added to each dish. After 2 hours of incubation, dishes were washed and and the amount of MBq bound to each dish was measured in an activitimeter. The dishes were placed under a µ-PET for imaging. We validated our method by alizarin red staining followed by spectral quantification for hydroxyapatite content.
Results The 18F-activity in the osteogenic inducted group showed a statistically significant increase of the activity compared to the according control; students t-test p=0,012. The mean activity in the osteogenic group was 0,61MBq, compared to 0,04 MBq in the control group. The alizarin red stain showed equal results. The two methods showed a high significant correlation (Pearson correlation coefficient 0,88; p=0,001).
Conclusions Measurement of 18F-NaF uptake is a sensitive method to proof and quantify the osteogenic potential of hMSCs.