Fig. 7.

LIMB and immunofluorescence analysis indicate possible mechanisms of vascular morphological changes deep in the femoral bone marrow, during regeneration, and in steady-state homeostasis. a Immunofluorescence analysis shows that type H vessels, characterized by CD31^hiEmcn^hi-expressing endothelial cells, are induced and present around the implant at day 3 after LIMB implantation. Their presence may vary individually but normalizes within 28 days post-surgery. Sinusoidal and type H vessel morphology adjacent to the wc is irregular in the first week and completely reorganizes to an appearance comparable to vessels found at endosteal areas distant from the injury site (n = 3 mice). bm bone marrow, cb cortical bone. Scale bar = 500 µm (left panels). b Immunofluorescence analysis after EdU pulse-chase experiments indicates similar EdU-uptake in the bone marrow of LIMB-implanted femurs and contralateral bones. Proliferating endothelial cells were rarely present at late time points after implantation. This result also supports the conclusion that 28 days after LIMB implantation both the bone and the bone marrow reach homeostasis (n = 3 mice in each cohort). c 3D fluorescence image (300 × 300 × 66 µm³, left and right panel) acquired by LIMB 26 days post-surgery, in a paGFP mouse with the vasculature labeled by Qdots. Photoactivation was performed within a volume of 100 × 100 × 9 µm³ in the center of the image. The fluorescence image was acquired 2 h post activation. Scale bar = 50 µm. The middle panel shows time-lapse 3D images of the inset from the left panel, indicating that paGFP fluorescent cells outside the initial photoactivation volume are present 3 h after photoactivation and that they fluctuate in number and position within the tissue. Passive displacement of the relatively immobile stromal and vascular compartments by continuous proliferation and movement of hematopoietic cells is a possible mechanism of tissue and vascular re-localization during homeostasis (see Supplementary Movies 10, 11)