Fig. 6
LIMB approach reveals kinetics of vascular remodeling during bone healing and homeostasis on time scales from hours to months. C57/B6J mice received the LIMB implant and were injected intravenously with Qdots (red) prior to each LIMB imaging session to label the vasculature. Vessels were three-dimensionally imaged at increasing time resolution over the course of a weeks b days and c several hours. In line with our previous observations, we noted prominent changes in the vasculature, which continued over the whole monitoring time period, even after homeostasis is reached (n = 5 mice, two independent experiments, scale bar = 50 µm). Small vessels continuously appear and disappear, larger vessels change their position and shape. The trace of such a larger vessel is displayed at all time points as a line in c. Blood vessels which can be used as landmarks are labeled by arrowheads and those that completely disappear within days are labeled by asterisks. d Overlap of the 3D projections of blood vessels in a mouse 35 days post-surgery (+0 h, green) and 24 h later (+24 h, red). A differential image between the two 3D images was generated. Blood vessel volume change was calculated by dividing the fraction of the volume difference between +24 h and 0 h (cyan areas in the middle panel indicate positive values, i.e., appearance of blood vessels; yellow areas indicate negative values, i.e., disappearance of blood vessels) by the total volume of the blood vessel at +24 h (delineated by white lines in the left panel) to obtain a normalized parameter of vessel volume change. The normalized volume changes (right panel) are dependent on the blood vessel diameter, with small vessels remodeling more rapidly than large vessels (n = 6 mice, scale bar = 100 µm). e Similar to the observations in the deep femoral marrow, repeated imaging of blood vessels in calvarial bone and bone marrow also showed remodeling of the vasculature (n = 3 mice, two independent experiments). Scale bar = 100 µm. Error bars represent s.d. values. Statistical analysis in d was performed using an ANOVA test (*p < 0.05; **p < 0.01; ***p < 0.001)
