Quantifying Bone Remodeling Dynamics

The rationales, protocols, and quantifications of bone remodeling imaging were detailed in our work published previously^{6 (link)}. In brief, two calcium-binding dyes were administered sequentially. The first calcium-binding dye (Dye 1, Calcein Blue, Sigma, 30 mg/kg) was administered intraperitoneally 48 h before imaging to label and track the change of bone fronts over the course of 2 days, which approximately represents one cycle of bone resorption. Calcein Blue was chosen to be spectrally compatible with Rhod-5N and AF488. Its effect was transient (Fig. 2d) and did not alter the serum calcium on the day of imaging as verified by the Arsenazo assay. The second calcium-binding dye to label all the bone fronts (Alizarin Red, 40 mg/kg) was injected on the day of imaging but after the acquisition of the Rhod5N/AF488 data so it does not interfere with the calcium measurement. As Dye 1 would be eroded if bone resorption has occurred, the Dye 1 to Dye 2 ratio in a single BM cavity (the concave endosteum) indicates the stage of bone remodelling during the 48 h period. We then defined bone cavities as (i) deposition type (D-type; dye 1:dye 2 > 75%); (ii) resorption type (R-type; dye 1:dye 2 < 25%), and (iii) mixed type (M-type; dye 1:dye 2 between 25 and 75%. For quantifying fractions of cavity types, 3D maps of calvaria were acquired and analyzed.