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Page 2 of 13
2 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
Introduction
The scans of mouse bones can be performed on the Skyscan 1072, 1172, 1076 micro-CT scanners. This report sets out a methodology for morphometric analysis of trabecular and cortical murine bone. Results are
presented for four mouse femur samples, for both trabecular and cortical bone morphometry.
Method
Scan and reconstruction parameters
The mouse bones were removed from alcohol storage and dried superficially on paper tissue, before being wrapped in plastic “cling-film”
or in parafilm, to prevent drying during scanning (and associated movement artefacts). Each plastic-wrapped bone was placed in a plastic / polystyrene foam tube which was mounted vertically in the Skyscan 1172
/ 1072, or horizontally in the 1076/1176 scanner sample chamber, for micro-CT imaging.
The suggested parameters of the scan in the 1172 (10/11 Mpix model) are as follows:
X-ray voltage 50 kV
X-ray current 200 µA.
Filter 0.5 mm aluminium
Image pixel size 4-5 µm
Camera resolution setting Medium or low (2000 or 1000 pixel field width)
Tomographic rotation 180/360
Rotation step 0.3-0.5
Frame averaging 1-2 (depending on required scan time)
Scan duration Medium res. 20-40 min.; low res. 6-10
min.
The suggested parameters of the scan in the 1172 (1.3 Mpix model) are
as follows:
X-ray voltage 50 kV
X-ray current 200 µA.
Filter 0.5 mm aluminium
Image pixel size 4-5 µm
Camera resolution setting High (1280 pixel field width)
Tomographic rotation 180/360
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3 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
Rotation step 0.3-0.5
Frame averaging 1-2
Scan duration 30-50 minutes
The suggested parameters of the scan in the 1072 are as follows:
X-ray voltage 50 kV
X-ray current 200 µA.
Filter 0.5 mm aluminium
Image pixel size 4-5 µm
Camera resolution setting n/a (1024 pixel field width)
Tomographic rotation 180
Rotation step 0.45
Frame averaging 1-2
Scan duration 45-80 minutes
The suggested parameters of the scan in the 1076/1176 (not in vivo, extracted bone) are as follows:
X-ray voltage 50 kV
X-ray current 200 µA.
Filter 0.5 mm aluminium
Image pixel size 8.9 µm
Camera resolution setting High (4000 pixel field width)
Tomographic rotation 180/360
Rotation step 0.3-0.5
Frame averaging 1
Scan duration 20-50 minutes
The suggested parameters of the scan in the 1076/1176 (in vivo, living
mouse under anaesthesia) are as follows:
X-ray voltage 50 kV
X-ray current 200 µA.
Filter 0.5 mm aluminium
Image pixel size 8.9 µm
Camera resolution setting High (4000 pixel field width)
Tomographic rotation 180
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4 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
Rotation step 0.8-0.9
Frame averaging 1
Scan duration 5-9 minutes
Reconstruction parameters suggested (same for all scanners):
Smoothing 0-1
Beam hardening 33%
Ring reduction 5
Post-alignment use value calculated by Nrecon
Histogram limits min 0; max – at right end of high density “tail”; refer to image to get good visible
contrast
Reconstruction was carried out employing a modified Feldkampi algorithm using the Skyscan Nreconii software which facilitates network distributed
reconstruction carried out on four pcs running simultaneously. Time for reconstruction of on scan dataset is usually much less than the scan
duration.
Region of interest selection for trabecular and cortical
bone
Both trabecular (metaphyseal) and cortical (metaphyseal-diaphyseal) were selected with reference to the growth plate. A crossectional slice was
selected as a growth plate reference slice, in the following way. Moving slice-by-slice toward the growth plate from the metaphysis/diaphysis, a point is reached where a clear “bridge” of low density cartilage
(chondrocyte seam) becomes established from one corner of the crossection to another. This bridge is established by the disappearance of
the last band of fine primary spongiosal bone interrupting the chondrocyte seam (see figure 1). This landmark allows a reference level to be defined
for the growth plate: trabecular and cortical volumes of interest are then defined relative to this reference level. (Note that the “bridge” can be at different locations in the growth plate crossection in difference bone
scans, depending on the orientation of the scanned bone.) Such a reference slice can be identified in all micro-CT scans of the proximal tibia
and distal femur.
Note that while in the femur there are four circular “islands” delineated by chondrocyte seams in crossections through the upper growth plate, in the
tibia there are only two such islands; none-the-less, the criterion for a growth plate reference described here can be reliably applied both for the
femur and tibia. The only possible exception is in very old mice or rats
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5 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
where the growth plate is inactive and very little or no chondrocyte seams are present. In such cases, a similar landmark can be found involving
connection of the bony structures of the vestigial growth plate.
Figure 1. Moving slice-by-slice toward the growth plate from the metaphysis/diaphysis, a point is reached where a clear “bridge” of low
density cartilage (chondrocyte seam) becomes established from one corner of the crossection to another, as shown by the arrows. The two images above show the establishment of this bridge with the
disappearance of the last band of fine primary spongiosal bone interrupting the chondrocyte seam. This landmark allows a reference level
to be defined for the growth plate: trabecular and cortical volumes of interest are then defined relative to this reference level. (Note that the “bridge” can be at different locations in the growth plate crossection.)
The regions of interest (ROI) were selected with reference to a growth
plate reference slice, selected as described above. Trabecular and cortical regions were defined as positions along the long axis of the femur relative to the growth plate reference. The trabecular region commenced about
0.215 mm (50 image slices) from the growth plate level in the direction of the metaphysis, and extended from this position for a further 1.72 mm
(450 image slices). The vertical extent of the trabecular and cortical ROIs is indicated in figure 2.
Note that for the trabecular ROI the value of the number of slices of the
“offset” (interval from growth plate reference level to start of ROI, here 50 slices) and the “height” (here 450 slices) can be altered to allow for
different bone geometries in mice of different strain, age and gender. The offset needs to be sufficient so that the trabecular ROI begins in a region of mature secondary trabecular bone, without a significant presence of the
fine-structured growth-plate-associated primary spongiosa in the crossections.
Within the trabecular region, separation of the trabecular from cortical bone was done using a freehand drawing tool for delineating of complex
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6 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
regions of interest (figure 3). The boundaries of the selected trabecular ROI ran parallel and close to the endocortical boundary – but excluding
peripheral vestiges of the growth plate and associated primary spongiosa, as shown in fig. 3. In Skyscan CT-analyser the volume of interest (VOI)
automatically interpolates or morphs the edited ROI shapes (such as freehand drawn shapes) between edited levels. The frequency of edited levels depends on how rapidly the bone crossection shape changes along
the bone axis (in the z direction) and becomes less as you move from the growth plate in the direction of the diaphysis (bone shaft).
The cortical region commenced about 2.15 mm (500 image slices) from the growth plate level in the direction of the metaphysis, and extended from this position for a further 0.43 mm (100 image slices). This probably
represents a diaphyseal site - there were relatively few remaining thin trabecular structures, compared to the much higher relative volume and
number density of trabecular structures at the metaphysis close to the growth plate. The trabecular and cortical bone ROIs were delineated as shown in figure 3.
Note again that for the cortical ROI the value of the number of slices of the “offset” (interval from growth plate reference level to start of ROI,
here 500 slices) and the “height” (here 100 slices) can be altered to allow for different bone geometries in mice of different strain, age and gender.
Figure 2. The vertical extent of the trabecular and cortical ROIs, defined with reference to a standard growth plate reference level (see text).
Growth plate (GP) reference level
Trab ROI: 0.215 – 1.94 mm from GP (50–450 slices); 1.72 mm section height (400 slices)
Cort ROI: 2.15 – 2.58 mm from GP (500–600 slices); 0.43 mm section height (100 slices)
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7 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
Figure 3. Trabecular and cortical ROIs delineated by the Skyscan CT-
analyser software ROI tool, by freehand drawing.
Morphometric analysis
3D and 2D morphometric parameters were calculated for the trabecular
and cortical selected ROIs. Single grey threshold values were applied for the trabecular and cortical ROIs respectively (68 and 120). (The lower density threshold for trabecular bone allows segmentation of the finer
structures; the thicker cortical bone can have higher threshold to represent thickness and porosity more accurately.) The signal to noise
ratios of the reconstructed images were high so that further image processing steps on the binarised images such as despeckle were not likely to significantly improve measurement precision. 3D parameters
were based on analysis of a Marching Cubesiii type model with a rendered surface. Calculation all of 2D areas and perimeters was based on the
Prattiv algorithm. Morphometric parameters measured by CT-analyser have been validated on both virtual objects and aluminium foil and wire phantomsv. Structure thickness in 3D was calculated using the local
thickness or “sphere-fitting” methodvi, and structure model index (an indicator of the relative prevalence of plates and rods) was derived
according to the method of Hildebrand and Ruegseggervii. Degree of anisotropy was calculated by the mean intercept methodviii.
3D Model construction
Rendered 3D models were constructed for 3D viewing of trabecular and cortical analysed regions. Model construction was by the “Double time
cubes” methodix, a modification of the Marching cubes method3.
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8 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
Results
Trabecular bone
Table 1 lists examples of measured morphometric parameters for
trabecular bone, within the volumes of interest referenced to the growth plate, as described above. Of the four examples A-D, samples A and B
have a lower trabecular percent volume (BV/TV), of 4-5%, while C and D have about double this percent volume, at 9-11%. Most of this increase in trabecular BV/TV is due to an increased spatial density of trabecular
structures, as indicated by trabecular number (Tb.N) being twice as high in C and D compared to A and B. By contrast the trabecular thickness
(Tb.Th) is not much different between the four samples.
Images of 3d rendered models of the four samples (the analysed trabecular volumes of interest) are shown in figure 4.
Cortical bone
The morphometric parameters measured for four cortical bone samples are listed in table 2. Samples C and D have greater cortical thickness and
crossectional area; however both periosteal and endosteal perimeter was greater in samples A and B. This implies that the cortical diaphysis was
both wider and thinner in A and B. The wider bone shape in A and B compensates for the reduced cortical thickness and area in terms of cortical strength, as indicated by rotational moment of inertia, which is
about the same in all four samples.
Images of 3d rendered models of the four samples (the analysed cortical
volumes of interest) are shown in figure 5.
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9 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
Table 1. Trabecular morphometric parameters measured by micro-CT in mouse distal femur samples A-D.
Distal femur
Parameter Parameter symbolx, unit
Mouse A Mouse B Mouse C Mouse D
Total
(tissue) volume
TV (mm3) 3.1385 3.1942 2.3077 2.4298
Bone volume BV (mm3) 0.1249 0.1500 0.2510 0.2249
Percent bone volume
BV/TV (%) 3.9786 4.6965 10.8751 9.2577
Bone surface BS (mm2) 11.3041 12.4453 17.1408 16.8528
Bone surface to volume ratio
BS/BV (mm-1) 90.5295 82.9592 68.2993 74.9197
Bone surface density
BS/TV (mm-1) 3.6018 3.8962 7.4276 6.9358
Trabecular thickness
Tb.Th (mm) 0.0475 0.0505 0.0553 0.0514
Trabecular separation
Tb.Sp (mm) 0.3413 0.3507 0.2754 0.2842
Trabecular number
Tb.N (mm-1) 0.8380 0.9293 1.9678 1.8013
Trabecular pattern
factor
Tb.Pf (mm-1) 33.5844 30.8598 18.1766 18.4714
Structure
model index
SMI 2.4090 2.3749 1.7773 1.7549
Euler
connectivity
E.Con 138.0 160.5 204.0 194.0
Euler
connectivity density
E.Con.D
(mm-3) 43.9700 50.2473 88.3997 79.8420
Degree of anisotropy
DA 1.7354 1.2388 1.8672 1.7124
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10 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
Table 2. Cortical morphometric parameters measured by micro-CT in mouse distal femur samples A-D.
Distal femur
Parameter Parameter symbol9, unit
Mouse A Mouse B Mouse C Mouse D
Cortical
thickness (3D)
Ct.Th (mm) 0.1921 0.1921 0.2154 0.2230
Cortical
crossectional thickness
(2D)
Ct.Cs.Th
(mm)
0.1683 0.1724 0.1964 0.2008
Cortical
periosteal perimeter
Ct.Pe.Pm
(mm)
5.1556 5.2004 4.9922 4.9303
Cortical endosteal perimeter
Ct.En.Pm (mm)
4.0622 3.9723 3.8091 3.6415
Cortical crossectional
area
Ct.Ar (mm2) 0.7754 0.7905 0.8643 8.5717
Polar
moment of inertia
MMI(p)
(mm4)
0.3586 0.3613 0.3540 0.3392
Eccentricity Ecc 0.7662 0.7542 0.7713 0.7591
Cortical
porosity
Ct.Po (%) 0.0846 0.0352 0.0709 0.0538
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11 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
(a) (b)
(c) (d)
Figure 4. 3D rendered models from the trabecular ROIs from mouse femur samples A-D
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12 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
(a) (b)
(c) (d)
Figure 5. 3D rendered models of the cortical ROIs from the distal femurs
of samples A-D.
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13 Bruker-MicroCT method note: Long mouse bone micro-CT analysis
i Feldkamp LA, Davis LC, Kress JW (1984) Practical cone-beam algorithm. J. Opt. Soc. Am. 1 (6):612-619.
ii Xuan Liu, Alexander Sasov (2005) Cluster reconstruction strategies for microCT and nanoCT scanners. In: Proceedings of the Fully Three-Dimensional Image Reconstruction Meeting in Radiology and Nuclear
Medicine, July 6-9, 2005, Fort Douglas/Olympic Village, Salt Lake City, Utah, USA.
iii Lorensen WE, Cline HE (1987) Marching cubes: a high resolution 3d surface construction
algorithm. Computer graphics 21 (4): 163-169.
iv Pratt WK. Digital image processing, 2nd ed. New York: Wiley; 1991. Chapter 16.
v Salmon PL, Buelens E, Sasov AY (2003) Performance of In Vivo Micro-CT Analysis of Mouse Lumbar Vertebral and Knee Trabecular Bone
Architecture. J. Bone Miner. Res.18 (suppl. 2) S256.
vi Hildebrand T and Ruegsegger P (1997) A new method for the model independent assessment of thickness in three dimensional images. J.
Microsc.185: 67-75.
vii Hildebrand T and Ruegsegger P (1997) Quantification of bone
microarchitecture with the structure model index. Comp. Meth. Biomech. Biomed. Eng. 1: 15-23.
viii Harrigan T P and Mann R W (1984) Characterisation of microstructural anisotropy in orthotropic materials using a second rank tensor. J. Mater. Sci. 19: 761-767.
ix Bouvier Dennis J (2004) Double-time cubes: a fast 3d surface construction algorithm for volume visualisation. University of Arkansas,
313 Engineering Hall, Fayetteville, AR72701, USA.
x Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR (1987) Bone Histomorphometry: standardization of
nomenclature, symbols and units. J. Bone Miner. Res. 2 (6): 595-610.
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