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

Abstract

Quantitative three-dimensional (3D) imaging of angiogenesis during bone repair remains an experimental challenge. We developed a novel contrast agent containing 0.07-to 0.1-mm particles of zirconium dioxide (ZrCA) and established subtraction mCT using synchrotron radiation (sSRCT) for quantitative imaging of angiogenesis and bone repair. This method was applied to a rat model of tibial bone repair 3 days (DAY3; n ¼ 2), 5 days (DAY5; n ¼ 8), or 10 days (DAY10; n ¼ 8) after drill-hole injury. Using the same drill-hole defect model, its potential use was illustrated by comparison of bone repair between hindlimbs subjected to mechanical unloading (n ¼ 6) and normal weight bearing (n ¼ 6) for 10 days. Following vascular casting with ZrCA, the defect site was scanned with 17.9-and 18.1-keV X-rays. In the latter, image contrast between ZrCA-filled vasculature and bone was enhanced owing to the sharp absorption jump of zirconium dioxide at 18.0 keV (k-edge). The two scan data sets were reconstructed with 2.74-mm voxel resolution, registered by mutual information, and digitally subtracted to extract the contrast-enhanced vascular image. K 2 HPO 4 phantom solutions were scanned at 17.9 keV for quantitative evaluation of bone mineral. Angiogenesis had already started, but new bone formation was not found on DAY3. New bone emerged near the defect boundary on DAY5 and took the form of trabecular-like structure invaded by microvessels on DAY10. Vascular and bone volume fractions, blood vessel and bone thicknesses, and mineralization were higher on DAY10 than on DAY5. All these parameters were found to be decreased after 10 days of hindlimb unloading, indicating the possible involvement of angiogenesis in bone repair impairment caused by reduced mechanical stimuli. In conclusion, the combined technique of sSRCT and ZrCA vascular casting is suitable for quantitative 3D imaging of angiogenesis and its surrounding bone regeneration. This method will be useful for better understanding the linkage between angiogenesis and bone repair.

In Vivo Hypobaric Hypoxia Performed During the Remodeling Process Accelerates Bone Healing in Mice

“…Concerning the effect of hind-limb unloading on BM levels of VEGF and HGH, to our knowledge, hardly any related data have been published to date. In a drilled-hole bone-defect model, rats subjected to 10 days of hind-limb unloading exhibited fewer developed blood vessels than control rats [38], hence the claim that low production of VEGF is correlated to the impairment of blood vessel development in the context of hind-limb unloading during bone healing process in rodents, thus accounting for our results with regard to decreased VEGF level in our tail-suspended mice.…”

Stimulating angiogenesis mitigates the unloading-induced reduction in osteogenesis in early-stage bone repair in rats

“…In contrast, the viscosity of the novel contrast agent (ZrCA) is similar to the apparent blood viscosity in microvasculature (Matsumoto et al. ). Thus, using ZrCA for vascular casting and taking advantage of its K‐absorption edge, we were able to determine the angiogenic vascular structure as radiointensity‐enhanced regions while minimizing uneven vascular filling artefacts.…”

“…The detailed methods for SRS μ CT vascular and bone imaging and vascular casting with ZrCA were described previously (Matsumoto et al. ).…”

“…Impaired angiogenesis is likely involved in the underlying mechanism resulting in the decreased osteogenic potential during skeletal unloading conditions (Sakuma-Zenke et al 2005;Vandamme et al 2012;Matsumoto et al 2013). The reduced expressions of HIF-1a and several key angiogenic factors in unloaded skeletal muscles have also been reported (Wagatsuma 2008;McCabe et al 2013).…”

“…Using a contrast agent containing zirconium dioxide particles (ZrCA) for vascular casting and taking advantage of its absorption discontinuity, three‐dimensional images of angiogenic vessels and newly formed bone were acquired by synchrotron radiation‐based subtraction microcomputed tomography (SRS μ CT), a method that was established in a previous study from our group (Matsumoto et al. ).…”