Quantification of vascular density through in vivo micro-computed
tomography
Laura Nebuloni, Gisela A. Kuhn and Ralph Müller
Institute for Biomechanics, ETH Zürich, Switzerland
Introduction
Methods
Results and
Discussion
One of the aims of regenerative medicine is the complete
integration of a biological substitute within the
surrounding tissues. A physiological process that
participates in tissue integration is angiogenesis, the
formation of new blood vessels from pre-existing vascular
networks. To verify the success of tissue engineering
solutions, current strategies rely on the assessment of the
ingrowth of newly formed micro-vessels [1]. To date, no in
vivo imaging technique provides all the requirements
needed to image angiogenesis in a 3D fashion. For this
purpose, a new method based on in vivo micro-computed
tomography (micro-CT) was developed.
A contrast agent containing alkaline metal nanoparticles
(ExiTronTM nano 12000, Miltenyi Biotec, Berlin, Germany)
was injected intravenously in C57BL/6 female mice. Preand post-contrast images were acquired with in vivo
micro-CT (vivaCT 40, Scanco Medical AG, Brüttisellen,
Switzerland) at the muscles of the lower hind limb (figure
1a). The contrast agent concentration in blood was
obtained from the jugular veins (figure 1b). Vascular
density maps were calculated from pre- and post-contrast
images and used to quantify the micro-vascular bed
(figure 1c). Dynamic low resolution imaging (70 µm
voxel-size) was applied to monitor the time behavior of
the contrast agent in blood and muscle. A high resolution
setup (17.5 µm voxel-size) was used to assess
reproducibility, sensitivity, accuracy and robustness of the
method. Vascular corrosion casts were produced at
experiments end term according to the protocol reported
in [2]. Ultra-high resolution images of casts (1.4 µm voxelsize) were acquired with micro-CT (µCT 50, Scanco
Medical AG, Brüttisellen, Switzerland) at the same region
scanned in vivo. Morphometric analysis was performed
on the segmented vascular network.
The results proved that the contrast medium is safe and it
is homogeneously distributed in the arterial-venous
system. Dynamic micro-CT imaging showed that the
agent has a stable concentration in plasma for over 30
minutes and does not diffuse outside the vascular
compartment (figure 2a); moreover, it is completely
excreted within 24 hours after injection (figure 2b). In the
reproducibility study, the vascular volume per tissue
volume (VV/TV) was quantified from vascular density
maps for repeated measurements (n=7, 5 scans each).
The Precision Errors (PEs) for VV/TV and pre- and postcontrast absorption values were calculated in muscle and
blood according to [3]. The results revealed a PE of
10.54% for VV/TV and the highest variations were found
in blood due to movement artifacts in the jugular region. A
sensitivity analysis was conducted by varying the injected
quantity of contrast (0%, 50% and 100% of the standard
volume). The average absorptions calculated from full
and half dose scans presented significant differences
between them in both tissues (p<0.005). In addition, the
corresponding vascular densities were not influenced by
the contrast dose; this was considered a proof of
robustness of the technique. Preliminary accuracy results
showed a good correlation with morphometric parameters
calculated from ultra-high resolution micro-CT of vascular
corrosion casts. A visual representation of the vessel
thickness (V.Th) is shown in figure 3.
Conclusions
and Outlook
Our results proved that vascular density maps revealed
useful in the assessment of the micro-vascular network
and show potential in quantifying angiogenesis.
References
[1] Laschke MW, Tissue Eng, 2006. 12(8):p.2093-104.
[2] Heinzer S, Neuroimage, 2006. 32(2):p.626-36.
[3] Gluer CC, Osteoporos Int, 1995. 5(4):p.262-70.
Acknowledge
The authors acknowledge funding from the European 7th
Framework Programme (FP7-NMP-2008-214402).
a)
b)
c)
Figure 1: Pre- and post-contrast micro-CT images acquired at the muscle tissues of mouse
lower hind limb (a) and at the region of the jugular veins (b). Pre- and post-contrast scans
are used to calculate vascular density maps (c). Bone and bone marrow are excluded from
the evaluation (shown in white).
a)
b)
Figure 2: Short- (a) and long-term (b) kinetics of contrast agent in different tissues (primary
axis: blood; secondary axis: muscle). In a), notice the contrast stability in blood for over 30
min and the absence of extravascular leakage in muscle.
Figure 3: Ultra-high resolution micro-CT of vascular corrosion cast of mouse lower hind
limb. The images were acquired with a resolution of 1.4 µm. The vascular network is colored
by vessel thickness (V.Th).