Fig. 3: Blood flow in sprouting and regenerating bone microvessels.

a Intravital multiphoton imaging of sprouting Flk1-GFP⁺ (green) microvasculature near the SHG⁺ (blue) calvarial bone edge in Flk1-GFP⁺ reporter mice at PLD7. Intravenously injected TexasRed-dextran (red) visualizes the vascular lumen including circulating blood cells as dark round cells. White arrowheads indicate extended filopodia of Flk1-GFP⁺ tip cells. Yellow arrowheads indicate luminal spaces of extended vascular sprouts. White arrows indicate perfused vessel segments. Data in a-c are from n = 3 biologically independent experiments. b Flow map of RBC velocities in sprouting microvasculature shown in a Flow velocities were grouped by flow rate. Flk1-GFP⁺ endothelial lining is shown in light green. Perfused luminal spaces are shown in light red. Non-perfused luminal spaces (grey) are often found in vascular sprouts where flow is stationary. c Multiphoton images and histograms (right) of temporal variations of RBC flow velocities in a given vessel of the sprouting microvasculature shown in a. Vessel segments (v1-v4) are indicated in the flow map in b Data are presented as mean values ± SD. n = 7 over 3 biologically independent experiments. d, e Blood flow velocities and vessel diameters in individual segments of the regenerating microvasculature at PLD9 (d) and PLD21 (e) according to previously defined flow velocities for different BM vessel types: arterial vessels (av), post-arterial vessels (pv), intermediate capillaries (ic) and sinusoidal capillaries (ssc)⁴¹. Data are presented as mean values ± SD, n = 3 (av), n = −8 (pav), n = 3 or 6 (ic), n = 6 or 9 (ssc) from 3 biologically independent experiments. f Multiphoton microscopy showing a SHG⁺ growing bone edge at PLD21 with multiple GFP⁺ microvessels in close proximity. Histograms with blood flow velocities and vessel diameters are shown. Data are presented as mean values ± SD. n = 4 over 3 biologically independent experiments g Multiphoton images and histograms (right) of temporal variation in RCB flow velocities in the indicated microvessels near the growing SHG⁺ calvarial bone. Data are presented as mean values ± SD and show temporal variation in blood flow rate in the indicated vessel. n = 6 over 3 biologically independent experiments. Source data for Fig. 3c, d, e, f, g are provided as a Source Data file.