Thoroughbreds are particularly susceptible to joint diseases,1 which result in substantial wastage in the horse racing industry.1–3 Repetitive impact of the carpus during high-speed galloping generates extremely high stresses in the dorsal and medial regions of its opposing joint surfaces.4–7 This, in turn, can lead to localized degeneration of the cartilage and bone4,7,8 and may initiate osteoarthritis with associated subchondral bone sclerosis and necrosis and thus an increased risk of osteochondral fracture.9,10
Although musculoskeletal tissues respond readily to intensive exercise through adaptive changes that can increase their physiologic threshold of safe function,11,12 it remains unclear what might constitute a beneficial versus a potentially harmful program of exercise for an immature equine athlete.13–15 Thus, to increase the resistance to joint injury and degeneration, a series of studies12,16–23 have been conducted investigating the effects of early exercise during the phase of active development in juvenile horses, which were subjected to moderate conditioning exercise from some weeks after birth to 18 months of age and compared with controls.
The purpose of the study reported here was to investigate histomorphometric changes in the cartilage and subchondral bone of the third carpal bone associated with conditioning exercise in young Thoroughbreds. We hypothesized that the histomorphometric variables of the radial facet of the third carpal bones would be significantly different between exercised and control groups.
Materials and Methods
Sample—The 18 left and right carpal bones of 9 Thoroughbreds were used from a group of 12 horses that had been allocated to either spontaneous exercise at pasture only (control group; n = 6) or additional conditioning exercise beginning shortly after birth (additional conditioning exercise group; 6).17 Carpal bones of 3 of these horses were randomly chosen and used in establishing the required experimental procedures; of the remaining 9 horses, 5 were in the additional conditioning exercise group and 4 were in the control group. These 12 horses had been euthanized at 18 months of age, and the tissues were also used in other studies,17–22 including the evaluation of the same third carpal bones of the present study. The study and its procedures were approved by the Massey University Animal Ethics Committee.
Exercise program—For approximately 18 months, beginning at a mean age of 3 weeks, both groups of horses were kept on pasture, but horses in the additional conditioning exercise group were additionally exercised (1,030 m/d on 5 d/wk) on an oval track under controlled conditions described elsewhere.17 During phase 1A (from 3 weeks of age to weaning [approx 120 days]), the mean target speed for exercise was 5.4 m/s; in phase 1B (from weaning to first sprint [approx 100 days]), the mean target speed was 7.5 m/s; and in phase 1C (approx 300 days), the mean target speed was 9.6 m/s, with a brief 129-m sprint at 12.5 m/s. During the 18-month exercise period, prolonged lameness was not detected in any of the 12 horses, whereas during brief periods, mild and moderate effusions in the midcarpal joints were found in an earlier study17 of the larger cohort of horses, of which the 12 in the present study were part.
Sample collection and processing—From each of the 18 third carpal bones, osteochondral slabs (approx 20 × 10 × 5 mm) were sawn immediately adjacent to the location of the slab cut for a study of cartilage swelling (Figure 1).16 The slabs were divided into sites 1 and 2 (exposed to high but intermittent loading) and sites 3 and 4 (exposed to moderate and more constant loading).4,7,24 As a control, site 0 was chosen from the nonloaded region of the joint surface adjacent to site 1, at the extreme edge of the dorsal lip of the radial facet. The slabs were fixed in 10% formaldehyde solution for 1 to 3 days, decalcified for up to 7 days in 10% formic acid solution buffered with sodium formate,25,26 rinsed in running water for 1 hour, and mounted with a formulationa of water-soluble glycols and resins on a sledge microtome for cryosectioning. From each slab, 30-μm-thick osteochondral serial sections (n = 10 to 30) were obtained in the sagittal plane, spanning a distance of approximately 3 mm. Five sections were randomly selected for histologic examination and photographic (bright-field microscopy)b imaging.
Illustrations and photograph of the forelimb and third carpal bone of a horse. Left panel—Diagram of the skeletal structures of the forelimb. The third carpal bone is indicated in red; the arrow indicates the orientation reference between A and B. A—Diagram of the third carpal bone indicating the location of histomorphometric examination sites 0 to 4 (asterisk) immediately adjacent to sampling sites previously used to obtain matrix swelling data.16 Osteochondral lesions are frequently reported in the dorsal region of the facet (red oval). B—Photograph of a portion of the third carpal bone indicating sites 0 to 4 of a typical sagittally sectioned osteochondral slab.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Illustrations and photograph of the forelimb and third carpal bone of a horse. Left panel—Diagram of the skeletal structures of the forelimb. The third carpal bone is indicated in red; the arrow indicates the orientation reference between A and B. A—Diagram of the third carpal bone indicating the location of histomorphometric examination sites 0 to 4 (asterisk) immediately adjacent to sampling sites previously used to obtain matrix swelling data.16 Osteochondral lesions are frequently reported in the dorsal region of the facet (red oval). B—Photograph of a portion of the third carpal bone indicating sites 0 to 4 of a typical sagittally sectioned osteochondral slab.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Illustrations and photograph of the forelimb and third carpal bone of a horse. Left panel—Diagram of the skeletal structures of the forelimb. The third carpal bone is indicated in red; the arrow indicates the orientation reference between A and B. A—Diagram of the third carpal bone indicating the location of histomorphometric examination sites 0 to 4 (asterisk) immediately adjacent to sampling sites previously used to obtain matrix swelling data.16 Osteochondral lesions are frequently reported in the dorsal region of the facet (red oval). B—Photograph of a portion of the third carpal bone indicating sites 0 to 4 of a typical sagittally sectioned osteochondral slab.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
The selected sections were washed with water at room temperature (approx 22°C), wet-mounted under a coverslip on a glass slide for examination at 20× magnification (unstained), and digitally photographed.c If the section contained focally enlarged calcified cartilage as described,19 the affected section was not included in the study.
Histomorphometric assessment—Each high-resolution composite image of the osteochondral section was overlaid with four 3,000 × 3,000-μm square grids covering the matrix beneath the en face sites 1 to 4 (Figures 2 and 3). Within the grid, a pen-tablet digitizerd with image editing softwaree was used to trace by hand the relevant tissue boundaries, which were then digitally colored (blue for hyaline cartilage, green for calcified cartilage, and red for vascular channels). Because vascular channels are thought to participate in remodeling of the osteochondral junction via advancement up to and into the calcified cartilage,27–30 they were counted only if they made contact with the osteochondral junction or were within approximately 50 μm of it. The area of any vascular channel intruding into the calcified cartilage was also included.
Illustration of an osteochondral slab of the third carpal bone of a horse indicating the histomorphometric variables examined in sections obtained as indicated in Figure 1. Vascular channels are labeled with numerals 1 to 5. CCang = Collagen matrix orientation in calcified cartilage. CCt = Calcified cartilage thickness. HCang = Collagen matrix orientation in hyaline cartilage. HCt = Hyaline cartilage thickness. Rug = Osteochondral junction rugosity, defined as length of osteochondral junction divided by the grid unit length (3,000 μm). Vca = Vascular channel area. VCang = Mean orientation of vascular channels. VCn = Number of vascular channels.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Illustration of an osteochondral slab of the third carpal bone of a horse indicating the histomorphometric variables examined in sections obtained as indicated in Figure 1. Vascular channels are labeled with numerals 1 to 5. CCang = Collagen matrix orientation in calcified cartilage. CCt = Calcified cartilage thickness. HCang = Collagen matrix orientation in hyaline cartilage. HCt = Hyaline cartilage thickness. Rug = Osteochondral junction rugosity, defined as length of osteochondral junction divided by the grid unit length (3,000 μm). Vca = Vascular channel area. VCang = Mean orientation of vascular channels. VCn = Number of vascular channels.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Illustration of an osteochondral slab of the third carpal bone of a horse indicating the histomorphometric variables examined in sections obtained as indicated in Figure 1. Vascular channels are labeled with numerals 1 to 5. CCang = Collagen matrix orientation in calcified cartilage. CCt = Calcified cartilage thickness. HCang = Collagen matrix orientation in hyaline cartilage. HCt = Hyaline cartilage thickness. Rug = Osteochondral junction rugosity, defined as length of osteochondral junction divided by the grid unit length (3,000 μm). Vca = Vascular channel area. VCang = Mean orientation of vascular channels. VCn = Number of vascular channels.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Photomicrographs of unstained sections of the third carpal bone of a horse. A—Composite photomicrograph of a dorsal-to-palmar sagittal section of the proximal aspect of the third carpal bone indicating the location of sites 0 to 4, which were selected for histomorphometric analysis; site 0 was not analyzed. The boundary of the radial carpal bone (Cr), indicated by the shaded area, indicates the approximate region of intercarpal contact. Bar = 1,000 μm. B—Colored histomorphometric image of sites 1 to 4 (from panel A; grid size, 3,000 × 3,000 μm). Notice that there are visible differences in vascularity (red) from sites 1 to 4. Bar = 500 μm. C—Representative photomicrographs of sites 0 to 4 indicating preferential orientations of collagen matrices in the hyaline cartilage and calcified cartilage layer (red arrows). The dotted green line indicates the tidemark dividing the hyaline cartilage from the calcified cartilage layer. Notice that there are visible differences in the orientations between the sites. Grid width = 3,000 μm. Bar = 500 μm.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Photomicrographs of unstained sections of the third carpal bone of a horse. A—Composite photomicrograph of a dorsal-to-palmar sagittal section of the proximal aspect of the third carpal bone indicating the location of sites 0 to 4, which were selected for histomorphometric analysis; site 0 was not analyzed. The boundary of the radial carpal bone (Cr), indicated by the shaded area, indicates the approximate region of intercarpal contact. Bar = 1,000 μm. B—Colored histomorphometric image of sites 1 to 4 (from panel A; grid size, 3,000 × 3,000 μm). Notice that there are visible differences in vascularity (red) from sites 1 to 4. Bar = 500 μm. C—Representative photomicrographs of sites 0 to 4 indicating preferential orientations of collagen matrices in the hyaline cartilage and calcified cartilage layer (red arrows). The dotted green line indicates the tidemark dividing the hyaline cartilage from the calcified cartilage layer. Notice that there are visible differences in the orientations between the sites. Grid width = 3,000 μm. Bar = 500 μm.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Photomicrographs of unstained sections of the third carpal bone of a horse. A—Composite photomicrograph of a dorsal-to-palmar sagittal section of the proximal aspect of the third carpal bone indicating the location of sites 0 to 4, which were selected for histomorphometric analysis; site 0 was not analyzed. The boundary of the radial carpal bone (Cr), indicated by the shaded area, indicates the approximate region of intercarpal contact. Bar = 1,000 μm. B—Colored histomorphometric image of sites 1 to 4 (from panel A; grid size, 3,000 × 3,000 μm). Notice that there are visible differences in vascularity (red) from sites 1 to 4. Bar = 500 μm. C—Representative photomicrographs of sites 0 to 4 indicating preferential orientations of collagen matrices in the hyaline cartilage and calcified cartilage layer (red arrows). The dotted green line indicates the tidemark dividing the hyaline cartilage from the calcified cartilage layer. Notice that there are visible differences in the orientations between the sites. Grid width = 3,000 μm. Bar = 500 μm.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Each grid square was analyzed with image analysis softwaref to quantify the tissue variables, namely thickness of hyaline cartilage and calcified cartilage and area and number of vascular channels. A previous study31 revealed that the alignment of the chondrocytes provides a clear indication of the overall fibrillar alignment. By use of this method, the general fibril orientations in the hyaline cartilage and calcified cartilage were measured from a reference line drawn perpendicular to the articular surface by determining the mean of 10 measurements. The orientation of the vascular channels was calculated by determining the mean of all those previously selected. The rugosity of the osteochondral junction was measured by dividing the length of the osteochondral junction by the grid length (3,000 μm).
Statistical analysis—Five osteochondral sections per bone from 18 bones (left and right) of 9 horses resulted in 90 osteochondral sections. This, in turn, generated 360 data sets (ie, 90 sections × 4 sites), and each data set consisted of the 8 histomorphometric variables (hyaline cartilage thickness, calcified cartilage thickness, vascular channel area, number of vascular channels, fibril orientations in the hyaline cartilage, fibril orientations in the calcified cartilage, orientation of the vascular channels, and osteochondral junction rugosity). Thirty-five data sets of the 360 histologic slices from sites 1 or 2 affected with calcified cartilage abnormality19 (abnormally thickened calcified cartilage and disrupted osteochondral junction) were excluded from the assessment. This exclusion did not affect the overall significance (P = 0.53) of the treatment effect as verified by use of an exact χ2 significance test.
The remaining 325 data sets were analyzed with a statistical software packagef via both multivariate ANOVA for a broad assessment of the exercise effects and univariate mixed model analyses (ANOVA) on each of the histomorphometric variables for variable-specific treatment effects. The model included nested random effects of horse and site, and dependence of the left and right leg was also factored into the models. Values of P < 0.05 were considered significant.
Ten correlation coefficients (R) between each pair of the 5 nonangular histomorphometric variables (ie, hyaline cartilage thickness, calcified cartilage thickness, vascular channel area, number of vascular channels, and osteochondral junction rugosity) were calculated and used to construct correlation matrices for each group. Ten thousand permutation tests incorporating Fisher Z transformationg were performed to determine whether there was any evidence of structural difference between the matrices.
Results
An overall exercise effect encompassing all the histomorphometric variables was not detectable (P = 0.396; Figure 4). When each variable was considered individually, there were measurable differences in the vascular channel area between the groups across all sites 1 to 4; the mean values of the additional conditioning exercise group's vascular channel area were consistently (35% to 57%) higher than those of the control group, but this difference was not significant (P = 0.057) because of large variance in the data. The 7 other variables had neither differences in their mean values nor significant detectable exercise effects.
Mean ± SD values of 8 histomorphometric variables in the 4 sites indicated in Figure 1 in horses of the additional conditioning exercise group (squares, dashed lines) and the control group (diamonds, solid lines). *Notice that only the Vca was markedly different between the groups. See Figure 2 for key.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Mean ± SD values of 8 histomorphometric variables in the 4 sites indicated in Figure 1 in horses of the additional conditioning exercise group (squares, dashed lines) and the control group (diamonds, solid lines). *Notice that only the Vca was markedly different between the groups. See Figure 2 for key.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Mean ± SD values of 8 histomorphometric variables in the 4 sites indicated in Figure 1 in horses of the additional conditioning exercise group (squares, dashed lines) and the control group (diamonds, solid lines). *Notice that only the Vca was markedly different between the groups. See Figure 2 for key.
Citation: American Journal of Veterinary Research 74, 4; 10.2460/ajvr.74.4.542
Both groups had strong site-specific findings in all measured histomorphometric variables regardless of the exercise treatment (P < 0.001). Although the largest vascular channel area occurred at site 1, this decreased progressively toward the palmar aspect. All the other nonangular variables (hyaline cartilage thickness, calcified cartilage thickness, number of vascular channels, and osteochondral junction rugosity) had maximums at sites 2 or 3 and minimums at either site 1 or 4. The variables fibril orientations in the hyaline cartilage, fibril orientations in the calcified cartilage, and orientation of the vascular channels all had a consistent increase from site 1 to 4 (range, −40° to 20°), with neutral orientations (ie, perpendicular to the articular surface) prevailing in sites 2 and 3.
The Pearson correlation coefficients (R) for the additional conditioning exercise group and control group indicated that most of the additional conditioning exercise group R values were larger than those of the control group by up to 0.5 (Table 1). However, when tested for the difference between within-group correlation matrix structures by use of the permutation tests, no significant (P = 0.063) difference was detected between the groups.
Correlation matrices and groupwise differences (ΔR) of pairs of histomorphometric variables in horses in exercised and control groups.
| Exercised | Control | ||||
|---|---|---|---|---|---|
| Variable | R | P value | R | P value | ΔR |
| HCt-CCt | 0.59 | < 0.001 | 0.08 | 0.326 | 0.50* |
| HCt-VCa | 0.17 | 0.015 | 0.07 | 0.411 | 0.10 |
| HCt-VCn | 0.34 | < 0.001 | 0.23 | 0.007 | 0.11 |
| HCt-Rug | 0.20 | 0.004 | 0.38 | < 0.001 | −0.18 |
| CCt-VCa | 0.36 | < 0.001 | 0.27 | 0.001 | 0.09 |
| CCt-VCn | 0.42 | < 0.001 | 0.34 | < 0.001 | 0.08 |
| CCt-Rug | 0.30 | < 0.001 | 0.33 | < 0.001 | −0.03 |
| VCn-Rug | 0.42 | < 0.001 | 0.35 | < 0.001 | 0.08 |
Only those pairs with values of P < 0.05 are listed.
Notice that the correlation coefficient of HCt-CCt in the exercised group is much higher than that for the control (ΔR = 0.50) suggesting that there was a measurable exercise effect in terms of the HCt-CCt relationship.
CCang = Collagen matrix orientation in calcified cartilage. CCt = Calcified cartilage thickness. HCang = Collagen matrix orientation in hyaline cartilage. HCt = Hyaline cartilage thickness. Rug = Osteochondral junction rugosity, defined as length of osteochondral junction divided by the grid unit length (3,000 μm). VCa = Vascular channel area. VCang = Mean orientation of vascular channels. VCn = Number of vascular channels.
Discussion
This study measured and compared 8 histomorphometric variables relating to the hyaline and calcified cartilage and the vascular channels in the radial facet of the third carpal bones of the additional conditioning exercise group and the control group. Although the study failed to detect a significant exercise effect, the mean vascular channel area of the additional conditioning exercise group was larger by 35% to 57% in all measured sites, compared with that of the control group. Furthermore, vascular channel area of both groups had the highest values at site 1 (ie, the most dorsal region of radial facet in the third carpal bone), whereas the remaining variables had highest values at site 2 or 3 (ie, midpoint of radial facet).
Vascular channels are the fine osseous cavities (diameter, 10 to 30 μm) formed by ongoing osteoclastic resorption as a part of bone remodeling via the activation-resorption-formation sequence.32 Although the function of the vascular channels is not fully understood, they are thought to be important features in bone, associated with nutritional and signal pathways into the overlying hyaline cartilage33–35 as well as with cartilage mineralization and tidemark advancement.36,37
The detailed quantification of vascular channel area enlargement performed in this study is, to the authors' knowledge, the first of its kind. The findings are supported by others who have also reported positive associations between the vascular channel area and exercise in the femoral head38 and tibial diaphysis39,40 of mice. Thus, greater vascular channel area in the additional conditioning exercise group and in the highly to intermittently loaded site 1 of the third carpal bone4,20–22,41,42 in both groups (additional conditioning exercise group and control group) does suggest that subchondral vascular channel enlargement could be an adaptive response of the subchondral bone to increased stress. However, the mechanism of vascular channel enlargement is not understood well; it could be driven by the need to repair microfractures associated with stress43–46 or by the increased blood flow (hyperemia) from the cyclic loading of the bone.16,47
Although vascular channel enlargement is likely to be a normal adaptive feature in the subchondral bone, the pronounced enlargement observed at site 1 is of potential concern in that such a structural change could compromise the mechanical strength of the third carpal bone's subchondral bone. An increase in both vascular channel area and vascular channel density has been reported to weaken human cortical bone by reducing its fracture toughness48 and fatigue strength,49 respectively. Similarly, the greater vascular channel enlargements in the exercised group in the present study could reduce the bone's structure integrity, thus lowering the strength of the third carpal bone in a repetitive stress environment. This may explain why authors of the present study have also observed osteochondral abnormalities characterized by calcified cartilage thickening and a disruption of the cement line, together with a variety of other unusual structural features in the dorsal region of the third carpal and the opposing radial carpal bone.19 Thus, the observation of vascular channel enlargement suggests that further investigation is required to establish with greater statistical certainty that it does have a true association with exercise. This is especially so given that dorsal site 1 is the region often associated with osteochondral fracture, subchondral bone collapse, and osteoarthritis.10
All 8 histomorphometric variables in both groups had strong site-specific characteristics. A similar site-specific pattern in the swelling behavior of the hyaline cartilage was found in another study16 of the same bones, suggesting that there is an important influence of the pattern of loading across the third carpal bone surface irrespective of the exercise treatment.
With regard to hyaline cartilage thickness and calcified cartilage thickness, their site specificities are thought to arise from localized adaptations that increase joint congruency,50–52 this in turn being driven by the contact stresses generated by weight bearing and joint movement.53–55 Although other studies that used equine tissues have also found an overall positive correlation between hyaline cartilage thickness and added exercise in the metacarpal condyle,18 carpus,14,56 and tarsus,57 an increase in hyaline cartilage thickness in the additional conditioning exercise group tissues was not detected. It is difficult to provide a definitive answer as to why that we did not observe any measurable increase in the hyaline cartilage thickness in the additional conditioning exercise group in sites 1 through 4, in contrast to reports14,18,56,57 of a positive relationship between cartilage thickness and exercise. It is possible that the intensity of the loading stimulus from the conditioning exercise may not have been great enough to induce changes in the hyaline cartilage thickness in the region that was measured. The small sample size used in the present study may also have been a contributing factor. However, in a previous study,16 a significant difference was not found in cartilage swelling behavior between the exercised and control groups, and this is consistent with the absence of a positive relationship between hyaline cartilage thickness and exercise, as in the present study.
Osteochondral junction rugosity appears to arise from the advancing vascular channels; the irregular cement line associated with this advance is thought to facilitate extra anchorage between the cartilage and subchondral bone.58–60 Although other studies have found the degree of interdigitation to be positively correlated both with age60 and stress on the bone,61 the present study did not find any significant variation among the 4 sites where the values ranged from 1.8 to 1.9. However, the pattern of variation resembled that of the number of vascular channels, with the correlation coefficients between osteochondral junction rugosity and number of vascular channels being 0.42 and 0.35 for the additional conditioning exercise group and control group, respectively. These values suggest that there could be a positive correlation between osteochondral junction rugosity and the number of vascular channels.
Collagen fibril orientation and organization are important factors influencing the mechanical function of articular cartilage.62,63 In the present study, semiquantitative measurement of collagen fibril orientation in the deep zone of the hyaline cartilage and calcified cartilage indicated that the collagen fibrils have strong site-specific angular deviations from the primary radial direction (ie, perpendicular to the articular surface; Figure 3). Such deviations may be an adaptive response to accommodate localized loading during the growth phase. We suggest that the collagen fibrils in the dorsal and palmar aspects had undergone lateral spreading because of a combination of axial compression and local shear forces. With the deep anchorage of the collagen fibrils, this may result in an overall fibrillar deviation away from the radial direction. As the joint matures, the deep zone matrix, in its laterally sheared state, is then progressively calcified while embedding the deviated fibrils, as is indicated by the chondrocyte flow lines. Interestingly, the extreme dorsal edge (ie, site 0), which has little or no contact stress from the opposite radial carpal bone, did not have any strong collagen directionality, compared with sites 1 and 4.
Even the orientation of the vascular channels followed closely the site-specific patterns in fibril orientations in the hyaline cartilage and calcified cartilage. Their angular morphology might be a consequence of a developmental adaptation influenced by the loading pattern across the joint surface during the horse's early growth phase. Interestingly, the results appear similar, if not identical, to the changes observed (via different methodologies) in the cartilage of the proximal phalanx of these same horses at the same age.64
This study used histomorphometry to reveal that vascular channel morphology was influenced by both early exercise treatment and regional stress patterns occurring in the radial facet of the third carpal bone. Results suggested that the mild exercise imposed during the developmental period appeared to be associated with an increase in the vascular channel area beneath the calcified cartilage layer in the dorsal aspect of the bone, although the difference from controls was not significant. The potential for such changes to compromise the strength and fracture toughness of the third carpal bone is of particular concern in view of its known vulnerability to osteochondral fracture and related joint disease.
Tissue-Tek OCT Compound, Sakura Finetek USA Inc, Torrance, Calif.
Nikon AZ100, Nikon Instruments Inc, Melville, NY.
PTGui, version 9, New House Internet Services BV, Rotterdam, The Netherlands.
Model Graphire CTE-630, Wacom Co Ltd, Saitama, Japan.
Adobe Photoshop version CS, Adobe Systems Inc, San Jose, Calif.
Image J, version 1.39d, National Institutes of Health, Bethesda, Md.
PROC MIXED, SAS, version 9.1, SAS Institute Inc, Cary, NC.
R, version 2.10.1, R Foundation for Statistical Computing, Vienna, Austria.
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