Real-time assessment of the osteogenic potential of hmscs by 99mtc-hdp-labelling during an ongoing cell culture

The novel, highly-sensitive and non-destructive method for the quantification of the osteogenic potential of bone marrow mesenchymal stem cells (BM-MSCs), by the evaluation of its hydroxyapatite (HA), in vitro is 99mTc-HDP-Labelling. 99mTc-HDP (tracer) binds rapidly to HA and this uptake can be visualized and quantified. This study was performed to evaluate if this method is suitable to perform a real-time assessment during an ongoing cell culture and if the radioactive tracer may influence the cells and their ability to differentiate. BM-MSCs (n=3) were cultivated in 35mm-dishes. Groups 1 and 3 received DMEM-LG based osteogenic media while Groups 2 und 4 were non-osteogenic controls. Groups 1 and 2 (multi-labelling) were incubated with 5 MBq 99mTc-HDP for 30min on day 7 (d7) and the bound activity was measured using an activimeter. Subsequently the cell-culture was continued and again labelled with 99mTc-HDP on day 14 and 21 (d14, d21). Groups 3 and 4 (single labelling), cultivation of the respective triplicates, ended on day 7, 14 and 21 (d7, d14, d21) followed by 99mTc-HDP-Labelling. Statistical analysis using one-factor ANOVA (p<0.05). Absolute tracer uptake increased steadily in both osteogenic groups: 1 (d7: 0.315; d14: 1.093; d21: 3.283 MBq) and 3 (d7: 0.208; d14: 0.822; d: 212.437 MBq) and was significantly higher than in the corresponding non-osteogenic control-group (Group 2 and 4) at all timepoints. (p<0.001). No significant negative effect of the radioactive tracer could be revealed in group 1 (multi radioactive labelling on d7, d14, d21) compared to Group 3 (singe labelling). The 99mTc-Uptake of groups 2 and 4 was not significantly different at any time. Our data show that the repeated exposition to 99mTc-HDP has no negative influence on the osteogenic differentiation potential of BM-MSCs. Therefore, the method is capable of determining the amount of HA during an ongoing cell culture.