Article
Quantification of newly produced hydroxyapatite in monolayer cultures of osteogenically induced hMSCs using the three different radiopharmaceuticals 99mTc-MDP, 99mTc-DPD and 99mTc-HDP
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Authors
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T. Großner
- Unfallchirurgie der Chirurgischen Klinik Campus Großhadern, LMU München, München, Germany
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E. Volkmer
- Chirurgische Klinik - Innenstadt, LMU München, Experimentelle Chirurgie und Regenerative Medizin, München, Germany
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M. Saller
- Chirurgische Klinik - Innenstadt, LMU München, Experimentelle Chirurgie und Regenerative Medizin, München, Germany
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B. Cherian Kallukalam
- Chirurgische Klinik - Innenstadt, LMU München, Experimentelle Chirurgie und Regenerative Medizin, München, Germany
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F.J. Gildehaus
- Klinik und Poliklinik für Nuklearmedizin, LMU München, Germany
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M. Schieker
- Chirurgische Klinik - Innenstadt, LMU München, Experimentelle Chirurgie und Regenerative Medizin, München, Germany
| Published: | October 21, 2010 |
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Outline
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Objective: Osteogenic induction of human mesenchymal stem cells is a well-estblished method used in tissue engineering for experimental orthopaedic research. Proof of osteogenic differentiation is commonly made by Alizarin red and von Kossa staining. Recently 99mTc-MDP, a radiolabelled diphosphonate, that binds to newly-forming hydroxyapatite, was successfully employed to quantify the degree of mineralization of osteoblasts and osteogenically induced hMSCs in monolayer and 3D-culture. In nuclear medicine three different 99mTc bone agents are routinely used. To make this promising method widely usable it is important to show that this method works equally well with all three tracers. The objective of the present study was to determine whether the three different radiopharmaceuticals 99mTc-MDP, 99mTc-DPD and 99mTc-HDP perform comparably well in terms of quantification of the degree of osteogenic differentiation of hMSCs.
Methods: hMSCs from three human donors (P6) were seeded in triplicates in 35mm diameter petri dishes at a density of 30.000 cells/cm2 and induced towards the osteogenic lineage by the addition of osteogenic media containing DMEM high glucose with 10% FBS and 1% penicillin and streptomycin, 4mM L-Glutamine, 100nM Dexamethasone, 10 mM ß-glycerolphosphate and 50µM L-ascorbic acid 2-phosphate (OSM=osteogenic medium). As a control cell-seeded dishes were grown in Alpha-MEM with 10% FBS and 1% penicillin and streptomycin (=CNTRL). After 21 days of culture one dish of each donor and group was incubated with 1ml NaCl 0,9% containing either 5,5 MBq 99mTc-MDP, 5,5 MBq 99mTc-DPD, or 5,5 MBq 99mTc-HDP. After 2 hours of incubation the dishes were washed 3 times in 0.,9% NaCl to remove the unbound tracer and then placed under a double-headed gamma camera (Siemens ECAM). Counts were acquired for 3 minutes. To analyze the acquired gamma counts, Siemens software (Siemens ICON) was used.
Results and conclusions: Student’s-t-test revealed a highly significant effect of osteogenic medium when compared to controls on the total gamma counts of p<0,001 when assessed by 99mTc-MDP (Median: OSM 69.135 vs. CNTRL 1818 counts/180 sec). The two other tracers 99mTc-DPD and 99mTc-HDP performed equally well in picking up the degree of osteogenic differentiation (p<0,001). A non-parametric Test (Mann-Whitney-Test) also showed a two sided significant difference between the osteogenic and the control groups of p=0,05.
Within the presented work we are able to measure the amount of newly deposited hydroxyapatite without destroying the cell layer using three kinds of Technetium-based tracers. All Tracers used work equally well when assessing the extent of osteogenic differentiation. We believe that this method is superior in quantifying osteogenic differentiation as it is highly accurate and sensitive, and the cell cultures remain viable and thus available for further experiments. Additional studies are now warranted to determine the sensitivity of the method.
