Abstract
Summary Statement
One of the most challenging problems in osteogenic 3D-tissue engineering is, to quantify the amount of new hydroxylapatite deposition. 18F-NaF-Labeling is a new, high-sensitive method to proof and quantify the osteogenic potential of hMSCs in an in vitro 3D-model.
Introduction
18F-labeled sodium fluorine was the first widely used agent for skeletal scintigraphy in the 1960s. 18F-NaF is rapidly exchanged for hydroxylgroups of the hydroxylapatite, covalently binding to the surface of new bone, which results in the formation of fluoroapatite. Three dimensional scaffolds are used to favor osteogenic differentiation of precursor cells. Cell-loaded scaffolds are investigated for their healing potential of critical size bone defects. Assessing the osteogenic potential of MSCs in 3D-in vitro cultures is of major interest in tissue engineering in order to maximise bone formation in vitro and in vivo.
One of the most challenging problems is, to quantify directly the amount of new hydroxylapatite deposition without destroying the evaluated cell-loaded scaffold. Within this abstract, we present a novel, non-destructive, high-sensitive method to quantify the amount of local hydroxylapatite deposition in 3D-cultures using 18F-NaF.
Material & Methods
hMSCs (n=5) were seeded in duplicates on highly porous collagen I/III scaffolds (circular, 8 × 2mm, 550.000 cells/scaffold). Osteogenic differentiation of the MSCs was established with DMEM low glucose + 10% FCS + 1% P/S + 10mM beta-glycerol phosphate + 173µM ascorbicacid-2-phosphate + 100nM dexamethasone. As control the same media without osteogenic supplements was used. Cultures were kept at 37°C and 5% CO2, media was changed every 2 days. After 21 days 40 MBq 18F-NaF in 1ml 0,9%NaCl was added to each scaffold, placed in a 2ml tube. After 2 hours of incubation, scaffolds were washed in aqua dest. (3x). To measure the bound activity, two methods were used. First the scaffolds were placed in an activity-meter (dose-calibrator) to assess the amount of bound radioactive tracer in MBq. Then, all scaffolds were placed in a µ-PET-scanner also for quantification of the bound activity (Bq/ml) and for 3D-imaging. To quantify the absolute calcium content, the 8mm scaffolds were lysed and analyzed by inductively coupled plasma massspectrometry (ICP-MS). In advance a DNA-Pico-Green assay was performed and all results were normalised to the DNA-amount of each scaffold.
Qualitative proof for the presence of hydroxylapatite was made by alizarin red staining as well as by scanning electron microscopy followed by energy dispersive X-ray-spectroscopy (SEM/EDX) of the histology sections (6µm) from the paraffin embedded duplicate scaffolds.
Results
The 18F activity evaluated by µ-PET scan and the results from the activity meter showed a statistically significant tracer uptake in the osteogenic induced group when compared to controls; Mann-Whitney-U Test: p=0,008 (µ-PET), p=0,008 (activity-meter). Spearman-Correlation analysis showed a statistical significant high correlation for the two methods (correlation-coefficient 0,712; p = 0,019).
Results from ICP-MS revealed significant amounts of calcium within the osteogenic group samples while within the control group was no calcium detection at all. The alizarin red stained slides also showed a positive stain for the osteogenic group, within the control group there was positive stain. SEM/EDX confirmed the presence of calcium and phosphorus for the osteogenic group.
Conclusion
Measurement of 18F-NaF uptake is a high-sensitive method to proof and quantify the osteogenic potential of hMSCs in a collagen scaffold based three dimensional tissue engineering model.
