Three-Dimensional Characterization of the Vascular Bed in Bone Metastasis of the Rat by Microcomputed Tomography (MicroCT)

Abstract

BackgroundAngiogenesis contributes to proliferation and metastatic dissemination of cancer cells. Anatomy of blood vessels in tumors has been characterized with 2D techniques (histology or angiography). They are not fully representative of the trajectories of vessels throughout the tissues and are not adapted to analyze changes occurring inside the bone marrow cavities.Methodology/Principal FindingsWe have characterized the vasculature of bone metastases in 3D at different times of evolution of the disease. Metastases were induced in the femur of Wistar rats by a local injection of Walker 256/B cells. Microfil®, (a silicone-based polymer) was injected at euthanasia in the aorta 12, 19 and 26 days after injection of tumor cells. Undecalcified bones (containing the radio opaque vascular casts) were analyzed by microCT, and a first 3D model was reconstructed. Bones were then decalcified and reanalyzed by microCT; a second model (comprising only the vessels) was obtained and overimposed on the former, thus providing a clear visualization of vessel trajectories in the invaded metaphysic allowing quantitative evaluation of the vascular volume and vessel diameter. Histological analysis of the marrow was possible on the decalcified specimens. Walker 256/B cells induced a marked osteolysis with cortical perforations. The metaphysis of invaded bones became progressively hypervascular. New vessels replaced the major central medullar artery coming from the diaphyseal shaft. They sprouted from the periosteum and extended into the metastatic area. The newly formed vessels were irregular in diameter, tortuous with a disorganized architecture. A quantitative analysis of vascular volume indicated that neoangiogenesis increased with the development of the tumor with the appearance of vessels with a larger diameter.ConclusionThis new method evidenced the tumor angiogenesis in 3D at different development times of the metastasis growth. Bone and the vascular bed can be identified by a double reconstruction and allowed a quantitative evaluation of angiogenesis upon time.

Assessment of bone vascularization and its role in bone remodeling

“…Indeed, most of the publications using Microfil opacification of bone marrow vessels show few ball-shaped vessels and discontinuous vascular networks. 59,60 In cortical bone, the vessels were reproducibly filled with barium in rats 61 ( Figure 2d-f) but not in mice 34 ( Figure 2b), probably because of inadequate rheology/output of the barium solution for infusion of mouse cortical vessels. In line with our results, Schneider et al 62 quantified the cortical canal network before and after barium infusion and found that the volume of the vascular network was much smaller than that of the cortical porosity.…”

Subtraction micro-computed tomography of angiogenesis and osteogenesis during bone repair using synchrotron radiation with a novel contrast agent

“…High-resolution X-ray micro-computed tomography (mCT) combined with a vascular casting technique has been used for three-dimensional (3D) imaging of bone microvasculature in rodents. [14][15][16][17][18] In this technique, barium sulfate-gelatin or lead chromate-loaded silicon has been used as a vascular contrast-casting agent, and bone was often decalcified for extracting microvessels as the reconstructed absorption values in microvessels and bone do not differ much. Decalcification is disadvantageous for investigating the involvement of angiogenesis in bone repair.…”

Quantitative analysis of bone and soft tissue by micro-computed tomography: applications to ex vivo and in vivo studies

“…Assessment of the differences in vessel volume, number and thickness allows for the distinction of tumour vasculature from that of healthy tissue. 58,59 In addition, studies have shown that osteolytic lesions result in increases in trabecular spacing and decreases in BMD and trabecular number owing to the destruction of the bone tissue. [60][61][62][63] It has recently been shown that micro-CT is superior to radiography in detecting osteolytic lesions.…”