ESCUELA DE POSGRADO 4.2 COINVESTIGADORES:

between conditions +βGP and -βGP in both control and BMP-stimulated conditions indicated that the staining was able to distinguish between mineralized and unmineralized nodules and thus indicates specificity. Like Alizarin Red S, BoneTag displayed low background staining in unmineralized nodules, while OsteoSense staining seemed to have a higher background and could not distinguish between + and - βGP without BMP-6 stimulation. This indicates that BoneTag has a higher specificity to the bone mineral compared to OsteoSense.

Bisphosphonates are compounds that bind with high affinity to bone and are used in the clinic for treatment of osteoporosis because of their ability to inhibit bone resorption by osteoclasts [19]. Because of their high affinity, bisphosphonates have a long half-life when bound to a mineralized matrix [19, 20]. OsteoSense is based on the bisphosphonate pamidronate [12], and care must be taken when using this probe in bisphosphonate research, since competition between the administered bisphosphonate and OsteoSense staining may occur. This potential problem is not expected when using BoneTag, which is a tetracylin-based probe [11]. In addition, it is important to note that in our experience OsteoSense is stable for a limited period of time (up to 8 weeks), even when stored at -80°C (data not shown), while BoneTag storage could be extended to over a year without loss of specificity.

Alizarin Red S is the standard staining for mineralized matrix in bone research. It can be dissolved and quantified by spectrophotometry. There are several advantages of using BoneTag and OsteoSense compared to Alizarin Red S staining:

1) They are added in the culture medium prior to fixation of the cells, and require no additional washing or staining procedures.

2) Both BoneTag and OsteoSense are available with fluorescence at 800nm and 680nm. The long wavelength of these probes makes them ideal for use with mineralized matrix as this greatly reduces autofluorescence.

3) These probes are universal in use for in vivo and in vitro bone research as they can also be used for in vivo imaging of bone formation. They are commercially available as quality controlled validated batches, reducing variability.

4) They can be quantified with great sensitivity using the dedicated Odyssey near-infrared imaging system.

5) Because small amounts of dye are needed for staining, the dyes are very cost-effective; up to 12,000 samples can be stained with a single vial of 24 pmol.

Other options for fluorescent detection of in vitro mineralization may be xylenol orange or calcein blue. Wang et al. [21] have for example used these substances to evaluate mineralized nodule formation in living osteoblastic cultures. However, this method was used to count the number of positive nodules, and not to quantify mineralization. Furthermore, these fluorescent dyes emit blue or red light, which is sensitive to autofluorescence of the mineralized matrix.

In conclusion, BoneTag and OsteoSense are good substitutes with many advantages over Alizarin Red S staining. The results of both fluorescent compounds provide good correlation with Alizarin Red S staining in a range of different experiments stimulating and inhibiting differentiation and mineralization. BoneTag is more specific in conditions of low mineralization and can be stored for extended periods of time. The long emission wavelength in the near-infrared range reduces autofluorescence during detection. For both compounds numerous possibilities exist for combination with other fluorescent markers at different wavelengths, such as DNA stains. This opens up many possibilities for quantitative and high-throughput experiments in bone research.

Acknowledgments

This work was supported by grants from NL Agency/IOP Genomics (IGE07001A), the Netherlands Institute for Regenerative Medicine (NIRM, FES0908) and the Dutch Arthritis Foundation (Reumafonds LLP-13). The authors acknowledge prof. S. Vukicevic for generously providing BMP-6 and dr. A.B. Chan for critically reading the manuscript.

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Supplementary figure 1. Mineralized nodule in a differentiated KS483 culture stained with BoneTag (LI-COR) and photographed by a Nuance 2.8 camera mounted on a Leica DM IRE2 fluorescence microscope (Leica). Magnification 400x. A) Brightfield image, B) Fluorescence image with BoneTag in green (800 nm), C) Composite of brightfield and fluorescence images. Fluorescence overlaps the mineralized area.

Chapter

General discussion