Micro-CT analysis of mouse long bones (proximal tibia, distal femur)

Transcription

1 Micro-CT analysis of mouse long bones (proximal tibia, distal femur) Application note Page 1 of 13

2 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 X-ray current Filter 50 kv 200 µa. Image pixel size 4-5 µm Camera resolution setting Tomographic rotation Rotation step Frame averaging 0.5 mm aluminium Medium or low (2000 or 1000 pixel field width) 180 / (depending on required scan time) Scan duration Medium res min.; low res min. The suggested parameters of the scan in the 1172 (1.3 Mpix model) are as follows: X-ray voltage X-ray current Filter 50 kv 200 µa. Image pixel size 4-5 µm Camera resolution setting Tomographic rotation 0.5 mm aluminium High (1280 pixel field width) 180 /360 Page 2 of 13

3 3 Bruker-MicroCT method note: Long mouse bone micro-ct analysis Rotation step Frame averaging 1-2 Scan duration 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 minutes The suggested parameters of the scan in the 1076/1176 (not in vivo, extracted bone) are as follows: X-ray voltage X-ray current Filter 50 kv 200 µa. Image pixel size 8.9 µm Camera resolution setting Tomographic rotation Rotation step Frame averaging 1 Scan duration 0.5 mm aluminium High (4000 pixel field width) 180 / minutes The suggested parameters of the scan in the 1076/1176 (in vivo, living mouse under anaesthesia) are as follows: X-ray voltage X-ray current Filter 50 kv 200 µa. Image pixel size 8.9 µm Camera resolution setting Tomographic rotation 0.5 mm aluminium High (4000 pixel field width) 180 Page 3 of 13

4 4 Bruker-MicroCT method note: Long mouse bone micro-ct analysis Rotation step 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 Histogram limits use value calculated by Nrecon 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 Feldkamp i algorithm using the Skyscan Nrecon ii 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 Page 4 of 13

5 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 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 Page 5 of 13

6 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. Cort ROI: mm from GP ( slices); 0.43 mm section height (100 slices) Trab ROI: mm from GP ( slices); 1.72 mm section height (400 slices) Growth plate (GP) reference level Figure 2. The vertical extent of the trabecular and cortical ROIs, defined with reference to a standard growth plate reference level (see text). Page 6 of 13

7 7 Bruker-MicroCT method note: Long mouse bone micro-ct analysis Figure 3. Trabecular and cortical ROIs delineated by the Skyscan CTanalyser 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 Cubes iii type model with a rendered surface. Calculation all of 2D areas and perimeters was based on the Pratt iv algorithm. Morphometric parameters measured by CT-analyser have been validated on both virtual objects and aluminium foil and wire phantoms v. Structure thickness in 3D was calculated using the local thickness or sphere-fitting method vi, and structure model index (an indicator of the relative prevalence of plates and rods) was derived according to the method of Hildebrand and Ruegsegger vii. Degree of anisotropy was calculated by the mean intercept method viii. 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 method ix, a modification of the Marching cubes method 3. Page 7 of 13

8 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. Page 8 of 13

9 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. Parameter Parameter symbol x, unit Distal femur Mouse A Mouse B Mouse C Mouse D Total (tissue) volume TV (mm 3 ) Bone volume BV (mm 3 ) Percent bone volume BV/TV (%) Bone surface BS (mm 2 ) Bone surface to volume ratio Bone surface density Trabecular thickness Trabecular separation Trabecular number Trabecular pattern factor Structure model index Euler connectivity Euler connectivity density Degree of anisotropy BS/BV (mm -1 ) BS/TV (mm -1 ) Tb.Th (mm) Tb.Sp (mm) Tb.N (mm -1 ) Tb.Pf (mm -1 ) SMI E.Con E.Con.D (mm -3 ) DA Page 9 of 13

10 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. Parameter Parameter symbol 9, unit Distal femur Mouse A Mouse B Mouse C Mouse D Cortical thickness (3D) Cortical crossectional thickness (2D) Cortical periosteal perimeter Cortical endosteal perimeter Cortical crossectional area Polar moment of inertia Ct.Th (mm) Ct.Cs.Th (mm) Ct.Pe.Pm (mm) Ct.En.Pm (mm) Ct.Ar (mm 2 ) MMI(p) (mm 4 ) Eccentricity Ecc Cortical porosity Ct.Po (%) Page 10 of 13

11 11 Bruker-MicroCT method note: Long mouse bone micro-ct analysis (a) (b) (c) Figure 4. 3D rendered models from the trabecular ROIs from mouse femur samples A-D (d) Page 11 of 13

12 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. Page 12 of 13

13 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): 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): iv Pratt WK. Digital image processing, 2nd ed. New York: Wiley; 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: vii Hildebrand T and Ruegsegger P (1997) Quantification of bone microarchitecture with the structure model index. Comp. Meth. Biomech. Biomed. Eng. 1: 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: 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): Page 13 of 13