Editorial Type:
Bone, Joint, and Cartilage

Structural characterization of subchondral bone in the distal aspect of third metacarpal bones from Thoroughbred racehorses via micro–computed tomography

Luis M. Rubio-Martínez Comparative Orthopedics Research Laboratory, Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON NIG 2W1, Canada

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 DVM, PhD, DVSc
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Antonio M. Cruz Comparative Orthopedics Research Laboratory, Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON NIG 2W1, Canada

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Karen Gordon Department of Biological Engineering, School of Engineering, University of Guelph, Guelph, ON NIG 2W1, Canada

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Mark B. Hurtig Comparative Orthopedics Research Laboratory, Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON NIG 2W1, Canada

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DOI:
https://doi.org/10.2460/ajvr.69.11.1413
Volume/Issue:
Volume 69: Issue 11
Online Publication Date:
01 Nov 2008
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Abstract

Objective—To characterize the microstructure of subchondral bone (SCB) plate and trabecular bone (TBB) of the distopalmar aspect of the condyles of third metacarpal bones (MC3s) from Thoroughbred racehorses at 2 different stages of SCB disease via micro–computed tomography (CT).

Sample Population—12 pairs of MC3s from Thoroughbred racehorses euthanized for various reasons.

Procedures—MC3s were collected from horses with mild (n = 6) or severe (6) SCB disease, as determined via micro-CT. Cubic (6 × 6 × 6-cm) specimens of SCB plate and TBB were cut from the palmar aspect of condyles and sagittal ridges and examined with 3-dimensional micro-CT. For each specimen, apparent bone mineral density (aBMD), true BMD (tBMD), bone volume fraction (BVF), trabecular thickness (TBT), trabecular separation (TBS), and connectivity (CN) were calculated.

Results—Condyles had higher aBMD, tBMD, BVF, and TBT and lower TBS than did the sagittal ridge. In bone specimens with mild SCB changes, SCB plate had higher aBMD, TBT, and CN and lower TBS than did TBB. In bone specimens with severe SCB disease, TBB had higher aBMD and TMD and lower TBS than it did in bone specimens with mild disease, and values were similar to those for SCB plate in bone specimens with severe disease.

Conclusions and Clinical Relevance—The microstructure of SCB of the distopalmar aspect of metacarpal condyles of horses varied according to the severity of changes identified via micro-CT. With mild SCB disease, sclerosis existed in the SCB plate of the condyles; with severe disease, sclerosis also invaded condylar TBB.

Abstract

Objective—To characterize the microstructure of subchondral bone (SCB) plate and trabecular bone (TBB) of the distopalmar aspect of the condyles of third metacarpal bones (MC3s) from Thoroughbred racehorses at 2 different stages of SCB disease via micro–computed tomography (CT).

Sample Population—12 pairs of MC3s from Thoroughbred racehorses euthanized for various reasons.

Procedures—MC3s were collected from horses with mild (n = 6) or severe (6) SCB disease, as determined via micro-CT. Cubic (6 × 6 × 6-cm) specimens of SCB plate and TBB were cut from the palmar aspect of condyles and sagittal ridges and examined with 3-dimensional micro-CT. For each specimen, apparent bone mineral density (aBMD), true BMD (tBMD), bone volume fraction (BVF), trabecular thickness (TBT), trabecular separation (TBS), and connectivity (CN) were calculated.

Results—Condyles had higher aBMD, tBMD, BVF, and TBT and lower TBS than did the sagittal ridge. In bone specimens with mild SCB changes, SCB plate had higher aBMD, TBT, and CN and lower TBS than did TBB. In bone specimens with severe SCB disease, TBB had higher aBMD and TMD and lower TBS than it did in bone specimens with mild disease, and values were similar to those for SCB plate in bone specimens with severe disease.

Conclusions and Clinical Relevance—The microstructure of SCB of the distopalmar aspect of metacarpal condyles of horses varied according to the severity of changes identified via micro-CT. With mild SCB disease, sclerosis existed in the SCB plate of the condyles; with severe disease, sclerosis also invaded condylar TBB.

In racing Thoroughbreds, the metacarpophalangeal joint sustains high loads and, consequently, represents one of the most common sites of overload arthrosis and catastrophic joint failure.1–3 As an adaptive response to this mechanical loading during exercise, the subchondral bone of the distal condyles of the MC3 undergoes an increase in BMD and bone volume fraction, which is interpreted radiographically as sclerosis.2,4–7 It is hypothesized that this adaptive response characterized by bone resorption and deposition can be uncoupled, leading to excessive subchondral mineralization, interference with the blood supply to the subchondral bone, and eventual subchondral bone necrosis, microdamage, and failure, a condition referred to as overload arthrosis.2,8,9

An intrinsic characteristic of the adaptive response of subchondral bone within the condyles of MC3 is that it appears to follow a consistent anatomic pattern6 that is exaggerated in racing versus other horses.1 In this response, the subchondral bone located at the condyles becomes denser (higher BMD and bone volume fraction) across the distopalmar aspect than at the sagittal ridge.4,6 This pattern is explained by the articulation of the distopalmar aspect of the condyles with the proximal sesamoid bones, which support the mechanical load and create stress over the condyles,10 leading to an increase in BMD and bone volume fraction of condylar subchondral bone.5,7 The sagittal ridge contacts the intersesamoidean ligament, which transfers less stress onto the opposing surface, resulting in little or no sclerosis of sagittal subchondral bone.1,10

Because the mechanical properties of bone tissue are strongly related to BMD,11 it has been hypothesized that the variations of BMD across the condyles represent gradients in the mechanical properties of bone tissue. As a result, repetitive loading of bone during exercise would hypothetically cause shear stress and strain to concentrate at the interfaces between those regions of different BMDs,1,6 which correspond with the condylar grooves,1,6 the typical location for condylar fractures in racing Thoroughbreds.3,12 In fact, fatigue-related failure of subchondral bone at this location precedes the development of condylar fractures.1,13,14 This fatigue damage could trigger subsequent bone remodelling and cause formation of resorption spaces that may exacerbate a stress-riser effect (accumulation of stress at that location) that predisposes affected horses to formation and propagation of condylar fractures.5,13,14

However, trabecular microarchitecture is also an important determinant of bone strength.15–18 A wide range of methods has been used to evaluate the microstructure of bone. Traditional histomorphometry19 and scanning electron microscopy20 are destructive, are laborious, and do not provide direct information regarding the 3-D structure.19 Clinical CT is a nondestructive 3-D imaging technique that is more sensitive than conventional radiography7; however, it does not provide accurate information of the bone microarchitecture. Micro-CT provides nondestructive assessment and analysis of 3-D bone architecture and mineral density, allowing quantification of focal osteopenia and loss of connectivity within the trabecular bone network.17,21,22 In another study23 at our laboratory, micro-CT was used to characterize morphologic changes that developed in subchondral bone of the metacarpal condyles of Thoroughbred racehorses. Results of that study led to the hypothesis that there are certain stages of the progressive subchondral bone disease in metacarpal condyles, and these stages allow the staging of bone lesions according to a hypothetic timeline of pathologic changes. Because of the structural differences between the subchondral bone plate and deeper trabecular bone and because the earliest signs of failure of subchondral bone appear to develop at the calcified cartilage layer and subchondral bone plate,1,2,13,20 we decided to evaluate trabecular bone and the subchondral bone plate separately.

The purpose of the study reported here was to characterize the microstructure of subchondral bone (subchondral bone plate and trabecular bone) of the distopalmar aspect of the condyles of MC3s in Thoroughbred racehorses by use of micro-CT. In addition, we sought to evaluate the differences in microstructure between 2 different stages of subchondral bone disease as determined via micro-CT morphology. We hypothesized that structural properties of subchondral bone of MC3s would differ between the condyles and the sagittal ridge and between the subchondral bone plate and trabecular bone. We also hypothesized that different morphologic appearances of subchondral bone evaluated via micro-CT would indicate different structural characteristics of the bone tissue.

Materials and Methods

Animals—One hundred twelve Thoroughbred racehorses underwent postmortem examination at the Ontario Veterinary College of the University of Guelph during 2004 and 2005. These horses comprised all Thoroughbreds that died 60 days before or after a race during 2 racing meets at 2 racetracks in Ontario as part of the Ontario Racing Commission Death Registry Program.24 Median age at the time of death was 4 years (range, 2 to 8 years). Both MC3s were collected from all horses and dissected free of tissues.

Sample population—The condylar region of each MC3 was examined by use of micro-CTa (Figure 1). The micro-CT images of the epiphysis of the distal aspect of the MC3 bone were acquired at 45-μm isotropic voxel resolution, corrected for fan-beam projection distortion, and reconstructed into a 3-D format, then imported into analysis software.b At the distopalmar aspect of the condyles (corresponding to the contact area with the proximal sesamoid bone during the stance phase of the gallop), ≥ 1 slice was obtained at the area in which most bone changes were detected in the reconstructed 3-D scout image along a proximodorsal-palmarodistal plane (representing the loading direction of the condylar subchondral bone against the proximal sesamoid bone). Two investigators (AMC and MBH) who were unaware of the identities of the horses independently categorized both condyles of each image according to degree of subchondral bone disease by use of an established scoring system that ranged from 0 (normal) to 8 (condylar fracture).23 When several slices from each condyle were analyzed, the highest score of bone disease assigned to the 2-D image (slice) with the most severely diseased condyle was considered the condyle score. Then each horse was categorized according to the maximal score assigned to the 4 condyles of both MC3s of the horse. Once the acquisition of micro-CT scans was complete, all MC3s were wrapped in gauze soaked in physiologic saline (0.9% NaCl) solution, placed inside 2 plastic bags, and stored at −20°C until analyzed.

Figure 1—
Figure 1—

Reconstructed 3-D scout micro-CT image of the distal epiphysis of an MC3 (left) from a Thoroughbred racehorse with subchondral bone disease. Letters X, Y, and Z represent each of the 3-D planes. In the right column, 2-D images along each of the 3 planes are represented (transverse [top], dorsal [center], sagittal [bottom]). Micro-CT analysis software allowed rotation of the planes, which permitted selection of the slice that best represented the most severe changes detected in the distopalmar aspect of the condyles.

Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1413

From the initial sample of 112 horses, the MC3s of 12 horses (24 MC3s in total) were selected and classified into 2 groups of 6 pairs of bones each, according to the category of subchondral bone disease assigned to the bone. Group 1 (mild) consisted of bones with sclerosis and mild focal or coalescing radiolucent areas in the distopalmar subchondral bone of MC3 without involvement of the articular surface (categories 0, 1, and 2). The characteristics were considered representative of incipient maladaptive remodelling (Figure 2). Group 2 (severe) consisted of bones with more severe multifocal radiolucent areas traversing the subchondral bone plate or affecting the articular surface (categories 5 to 7). These characteristics were considered representative of end-stage maladaptive remodelling (Figure 3). Bones with condylar fractures (score = 8) were not included in the study.

Figure 2—
Figure 2—

Micro-CT image of the MC3 of a Thoroughbred racehorse with mild subchondral bone disease, showing sclerosis (ellipses) and mild focal or coalescing radiolucent areas (arrows) in the distopalmar subchondral bone of the MC3 without involvement of the articular surface. These characteristics were considered representative of incipient maladaptive remodelling. +Y represents location of one of the 3-D planes.

Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1413

Figure 3—
Figure 3—

Micro-CT image of the MC3 of a Thoroughbred racehorse with severe subchondral bone disease, showing more severe multifocal radiolucent areas (arrows) traversing the subchondral bone plate or affecting the articular surface (ellipse). These characteristics were considered representative of end-stage maladaptive remodelling. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1413

Specimen collection—Selected MC3s were thawed at room temperature (18° to 21°C) for 16 to 20 hours. The remaining soft tissues were removed from the bones, and the distal condylar portion of each MC3 was separated from the bone diaphysis with a transverse cut performed at the level of the physeal scar by use of a commercial band saw.c The articular cartilage was then removed from the distopalmar articular condylar surface with a blade, and the condylar portion was mounted on a low-speed diamond saw.d Two parallel diamond bladese separated by 6 mm via washers were used. An initial 6-mm-thick slice of bone was cut in a proximodorsal direction perpendicular to the distopalmar articular surface of the metacarpal condyles, corresponding to the area of contact with the proximal sesamoid bones during the stance phase of the gallop25 and representing the site at which degenerative lesions develop2 (Figure 4). Measurements were obtained and locations for the 3 ROIs were determined on the slice. These 3 ROIs were defined at the sagittal ridge and the midpoint of each condyle. At each ROI, 2 cubes of bone approximately 6 × 6 × 6 mm were collected and measured by use of a calibrated digital caliper.f Final cubes were machined from the initial slice with the same blades. The bone specimens and the blades were maintained under continuous irrigation with saline solution during the whole machining process. The whole process of sample machining was performed at controlled laboratory temperature and humidity conditions.

Figure 4—
Figure 4—

Line drawing of specimen collection from an MC3. A—Lateral view of the distal end of the MC3 showing the distopalmardorsoproximal direction along which the samples were collected (this direction simulates the loading direction against the proximal sesamoid bones). B—Dorsal view of the distal end of the MC3 indicating the location of the samples. Do = Dorsal. Pa = Palmar. P = Proximal (trabecular bone). D = Distal (subchondral bone plate). M = Medial. S = Sagittal. L = Lateral. X = Width of medial condyle. Y = Width of lateral condyle.

Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1413

The dimensions of the cubes of bone were chosen on the basis of the estimated thickness of the subchondral bone plate at this location. The subchondral bone plate has been defined as the bone layer separating the calcified cartilage from the marrow spaces.9,26 In our other study,27 the bone layer had an approximate thickness of 5 to 6 mm at the palmar aspect of the condyles. Bone specimens from distal portions were considered to mostly include the calcified cartilage layer and the subchondral bone plate, whereas specimens from proximal portions included mostly trabecular bone.

Acquisition of micro-CT images— The 6 cubes of subchondral bone collected from each MC3 were wrapped together in gauze soaked in saline solution and sealed with plastic film. Care was taken to ensure maintenance of the correct anatomic position and orientation of the cubes within the micro-CT gantry. The package of 6 cubes from each metacarpal bone was positioned above a calibration phantom and introduced into the gantry of the micro-CT scanner.a Images were obtained at 80 kV and 450 μA, with a 45-μm isotropic voxel resolution. After image acquisition, scan data were corrected and CT values were calibrated into HU by use of the calibration phantom.g The calibration phantom included a bubble filled with water, a piece of bovine hydroxyapatite, and a defect filled with air so that HU values for water, bone, and air, respectively, could be determined. After calibration, fullresolution reconstruction of the 6 cubes of subchondral bone from each MC3 was performed by use of userdefined 3-D ROIs.b The same investigator (LRM), who was unaware of specimen group allocation, performed this procedure for all specimens.

Bone analysis—Each cube of subchondral bone was analyzed with micro-CT advanced bone analysis software.b For each cube, to discriminate bone from nonbone voxels within each ROI for measurement of bone mineral values, the software determined the optimal threshold value on the basis of a method described elsewhere.27 This application performed a virtual biopsy and ashing of bone to determine bone mineral content nondestructively and provided the equivalent mass of hydroxyapatite in milligrams. Apparent BMD (mg/cm3) was calculated on the basis of volume of the total cube (ie, all the voxels that formed the cube), including marrow cavities and vascular channels. True BMD (m/cm3) was calculated considering only the fraction of the bone cube that included bone tissue (ie, only those voxels occupied by bone tissue). Bone volume fraction represented the proportion of the total volume of the cube of bone that was occupied by bone tissue.

Trabecular thickness (mm) and trabecular separation (mm) were calculated by fitting maximal spheres to the trabecular structure, including bone and marrow regions.28 Connectivity density (number of trabecular connections/mm3) was calculated, on the basis of the Euler number,29 as the number of connections within the structure per unit of volume.

Statistical analysis—Data were assessed for normality, and a logarithmic transformation was used for nonnormally distributed data. A factorial design within a partial randomized block ANOVA was used to evaluate differences between front limbs, among specimen locations, or between disease classifications with respect to values of apparent BMD, true BMD, bone volume fraction, trabecular thickness, trabecular separation, and connectivity. A horse from which each pair of MC3s was obtained was designated as a block, and factors investigated included disease group (mild vs severe subchondral bone disease), side (left vs right forelimb), ROI (medial condyle, lateral condyle, or sagittal ridge), and layer of bone (subchondral bone plate vs trabecular bone). When appropriate, paired comparisons were performed with a Tukey test. Values of P ≤ 0.05 were considered significant for all analyses.

Results

The 12 horses included in the study had a median age of 4 years (range, 2 to 7 years). These horses died or were euthanized for carpal fracture (n = 3 horses), cranial trauma and fracture (2), proximal sesamoid bone fracture (2), humeral fracture (1), tibial fracture (1), sudden collapse (1), suspensory ligament rupture (1), and acute severe pulmonary hemorrhage.

Micro-CT images of 6 specimens/horse were acquired (Figures 5 and 6). Subchondral bone specimens from left and right forelimbs were not significantly different structurally. When considering ROI (medial condyle, sagittal ridge, and lateral condyle) overall, condylar samples had a significantly (P < 0.001) higher values for apparent BMD, true BMD, bone volume fraction, and trabecular thickness and lower values for trabecular separation (P ≤ 0.009) than did specimens from the sagittal ridge (Table 1). When layers were compared overall, subchondral bone plate specimens had higher BMD (P = 0.020), higher trabecular thickness (P = 0.002), lower trabecular separation (P = 0.002), and higher connectivity values (P < 0.001) than did trabecular bone cubes (Table 2). Differences with respect to true BMD and bone volume fraction were not significant.

Table 1—

Mean ± pooled SEM values of apparent BMD (aBMD), true BMD (tBMD), and bone volume fraction (BVF) and mean (95% confidence interval) values for tra-becular thickness (TBT) and trabecular separation (TBS) in 3 ROIs of MC3 specimens obtained from 12 horses with mild or severe subchondral bone disease, as determined via micro-CT analysis.
VariableROI
Lateral condyleMedial condyleSagittal ridge
aBMD (mg/cm3)869.750 ± 18.689a873.080 ± 18.627a607.670 ± 18.627b
tBMD (mg/cm3)957.660 ± 18.038a960.040 ± 18.001a730.13 ± 18.000b
BVF (ratio)0.697 ± 0.009a0.710 ± 0.008a0.601 ± 0.009b
TBT (mm)0.408a (0.369 – 0.451)0.415a (0.376 – 0.458)0.219b (0.198 – 0.241)
TBS (mm)0.141a (0.134 – 0.141)0.134a (0.127 – 0.142)0.154b (0.146 – 0.163)

Within each row, values with different superscript letters are significantly (P≤ 0.05) different.

Values for connectivity are not included because they were not significantly different among ROIs.

Table 2—

Mean ± pooled SEM values of aBMD and connectivity (CN) and mean values (confidence intervals) for TBT and TBS in trabecular bone versus subchondral bone plate of MC3 specimens from 12 horses with mild or severe subchondral bone dis-ease, as determined via micro-CT analysis.
VariableLayer of boneP value
Trabecular boneSubchondral bone plate
aBMD (mg/cm3)771.230 ± 17.921795.760 ± 17.8930.02
TBT (mm)0.313 (0.286 – 0.342)0.355 (0.325 – 0.389)0.002
TBS (mm)0.150 (0.143 – 0.157)0.137 (0.130 – 0.144)< 0.001
CN (1/mm3)8.337 ± 0.98719.215 ± 0.987< 0.001

Values for tBMD and bone volume fraction are not included because they were not significantly different between layers.

See Table 1 for remainder of key.

Figure 5—
Figure 5—

Micro-CT images of 6 subchondral bone specimens obtained from a horse that was classified as having mild subchondral bone disease. Figure represents a 2-D image across the 6 specimens along the proximodorsal-distopalmar plane. Order of specimens from left to right and top to bottom is as follows: trabecular bone of the medial condyle, sagittal ridge, and lateral condyle and subchondral bone plate of the medial condyle, sagittal ridge, and lateral condyle.

Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1413

Figure 6—
Figure 6—

Micro-CT image of 6 subchondral bone specimens obtained from a horse that was classified as having severe subchondral bone disease. Figure represents a 2-D image across the 6 specimens along the proximodorsal-distopalmar plane. Order of specimens from left to right and top to bottom is as follows: trabecular bone of the lateral condyle, sagittal ridge, and medial condyle and subchondral bone plate of the lateral condyle, sagittal ridge, and medial condyle.

Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1413

Within the group classified as having mild subchondral bone disease, subchondral bone plate specimens had higher apparent BMD (P < 0.001), closer trabeculae (P = 0.003), and higher connectivity values (P < 0.001) than did trabecular bone specimens (Table 3). However, no differences were evident between both layers in MC3s classified as having severe subchondral bone disease. Trabecular bone specimens in the severe group had higher values for apparent BMD (P = 0.03) and lower values for trabecular separation (P = 0.02) than did similarly located cubes of bone from the mild group. Connectivity values were also higher in trabecular bone cubes in the severe group, but the difference with the mild group was not significant (P = 0.08).

Table 3—

Significant (P≤ 0.05) ANOVA results for the interaction between degree of subchondral bone disease (mild vs severe) and layer of bone (trabecular bone vs subchondral bone plate) and its effects on mean ± pooled SEM values of aBMD, tBMD, and CN and mean values and confidence intervals for TBS in MC3 specimens from 12 horses with mild or severe subchondral bone disease, as determined via micro-CT analysis.
VariableLayer of boneSeverity of subchondral bone diseaseANOVA P value
MildSevere
aBMD (mg/cm3)Trabecular bone731.880 ± 25.304a810.590 ± 25.384b,c< 0.001
Subchondral bone plate794.550 ± 25.304b796.980 ± 25.304a,b,c
 tBMD (mg/cm3)Trabecular bone855.120 ± 24.825b912.980 ± 24.874b0.001
Subchondral bone plate878.510 ± 24.825b883.830 ± 24.825b
TBS (mm)Trabecular bone0.160a (0.150 – 0.172)0.140b (0.130 – 0.149)0.02
Subchondral bone plate0.138b (0.129 – 0.148)0.136b (0.126 – 0.146)
CN (1/mm3)Trabecular bone6.194 ± 1.396b,c10.480 ± 1.396b,c0.01
Subchondral bone plate19.482 ± 1.396a,d18.949 ± 1.369a,d

Within each variable, different superscript letters indicate significant differences.

Values for true bone volume fraction, tBMD, and TBT are not included because they were not significantly different

See Tables 1 and 2 for remainder of key.

The interaction between group and ROI was only significant (P = 0.003) for bone volume fraction. Within both groups, condylar bone specimens had significantly higher bone volume fractions than did sagittal bone specimens. Between groups, sagittal bone specimens in the severe group had significantly (P = 0.02) higher bone volume fractions (mean ± SD, 0.62 ± 0.01) than sagittal samples in the mild group (0.57 ± 0.012).

The interaction of group, layer, and ROI had a significant effect for the variables true BMD, trabecular thickness, and connectivity (Table 4). Subchondral bone specimens obtained from the condyles had significantly higher true BMD than specimens obtained from the sagittal ridge in both layers of trabecular bone and subchondral bone plate. Condylar specimens also had higher trabecular thickness than did sagittal samples in both layers in both groups of subchondral bone disease. Within the medial condyle, groups were different when both layers were compared: in the mild group, subchondral bone plate had thicker trabeculae than did trabecular bone (P < 0.001); however, this difference was not significant in the severe group (P = 0.15). Subchondral bone plate specimens had consistently higher values for connectivity than trabecular bone specimens at every ROI in both groups, except for the lateral condyle in the severe group. At that location, connectivity values in trabecular bone were significantly (P = 0.007) higher than those at the same location in the mild group. This value was also significantly (P = 0.01) higher than that for trabecular bone from the sagittal ridge in the severe group.

Table 4—

Results* from ANOVA assessments of the effect of the interaction of degree of subchondral bone disease, layer of bone, and ROI (lateral condyle [L], sagittal ridge [S], or medial condyle [M]) on mean ± pooled SEM values of aBMD, tBMD, and CN and mean values and confidence intervals for TBT in MC3 specimens from 12 horses with mild to severe subchondral bone disease, as determined via micro-CT analysis.
VariableLayerClassification of subchondral bone diseaseP value
MildSevere
LSMLSM
aBMD (mg/cm3)Trabecular bone810.44 ± 29.237567.34 ± 29.237817.86 ± 29.237905.39 ± 29.853643.57 ± 29.237882.81 ± 29.2370.09
Subchondral bone plate897.29 ± 29.237578.71 ± 29.237907.65 ± 29.237865.87 ± 29.237641.06 ± 29.237884.0 ± 29.237
tBMD (mg/cm3)Trabecular bone922.45 ± 27.262a716.400 ± 2.262b926.52 ± 27.262a995.93 ± 27.663a764.88 ± 27.262b978.13 ± 27.262a0.03
Subchondral bone plate967.13 ± 27.262a691.57 ± 27.262b976.82 ± 27.262a945.12 ± 27.262a747.68 ± 27.262b958.68 ± 27.262a
TBT (mm)Trabecular bone0.374a (0.316 – 0.443)0.216b (0.182 – 0.256)0.351a,A (0.296 – 0.416)0.367a (0.310 – 0.435)0.239b (0.202 – 0.284)0.376a (0.317 – 0.446)0.004
Subchondral bone plate0.443a (0.373 – 0.525)0.212b (0.179 – 0.251)0.520a,A (0.439 – 0.616)0.457a (0.381 – 0.548)0.209b (0.176 – 0.247)0.434a (0.366 – 0.514)
CN (mm)Trabecular bone5.324 ± 1.926†C7.832 ± 1.926D5.426 ± 1.926E14.710 ± 1.926†a7.185 ± 1.926b,G9.545 ± 1.926H0.006
Subchondral21.363 ± 1.926C19.452 ± 1.926D17.630 ± 1.926E16.438 ± 1.92619.677 ± 1.926G20.730 ± 1.926H

Interactions with values of P≥ 0.10 are not included.

For each variable, difference between disease classifications (mild vs severe) for that variable at that specific location (ROI and layer) is significant (P≤ 0.05). Unless specified with a dagger (†), different superscript capital letters do not indicate significant differences between groups at a specific layer.

Within each classification of subchondral bone disease (mild or severe) and each layer of bone, different superscript lowercase letters indicate significant (P≤ 0.05) differences between different ROIs (M, S, or L) at that specific classification and layer.

Within each disease classification and each ROI, identical uppercase superscript letters indicate significant (P ≤ 0.05) differences between the 2 layers for that specific variable at that specific disease classification and ROI. When no letters are included, no significant differences between layers were detected.

See Tables 1 and 2 for remainder of key.

Discussion

Our results indicated that the microstructure of subchondral bone in the distopalmar aspect of MC3s of a select population of Thoroughbred racehorses (from 2 Ontario racetracks and that were euthanized for diverse reasons) had a specific anatomically related architecture that was related to the severity of the subchondral bone changes detected via micro-CT. In general, without distinguishing between degree of subchondral bone disease or layer of bone (trabecular bone vs subchondral bone plate), the subchondral bone located at the condyles was consistently denser than that at the sagittal ridge. This higher density was indicated by higher quantity of bone tissue per unit of total specimen volume (higher apparent BMD and bone volume fraction) and different quality of bone tissue (more mineralized as suggested by higher true BMD). Architecturally, the trabeculae at the condyles were also thicker (almost double, on average) and closer to each other than those at the sagittal ridge. These findings agree with those of another study6 that revealed that subchondral bone of the condyles of MC3 was consistently denser than that of the sagittal ridge by use of radiography, clinical CT, and microradiographic stereology. This phenomenon likely represents the result of the adaptive response of bone tissue to loading during exercise.5,30 In our study, specimens of condylar subchondral bone were obtained from the location of the condyles that is loaded against the proximal sesamoid bones, which support the mechanical load during the stance phase of the gallop.2,10 On the contrary, the axial portion of the sagittal ridge remains relatively unloaded.10,25 This variation in subchondral bone properties according to loading history is consistent with the results of another study31 performed at our laboratory with similar methodology. Values for apparent BMD and bone volume fraction were highest for the subchondral bone located at the load-bearing aspects of the femoral condyles in horses, probably as a result of functional biomechanical demands.31

We also found that structural characteristics differed between subchondral bone plate and trabecular bone and that micro-CT changes detected in subchondral bone of the distal end of MC3s were indicative of changes in the microstructural characteristics of both layers. In bones from horses with mild subchondral bone disease, the subchondral bone plate typically had higher apparent BMD and thicker and closer trabeculae that were also more connected, compared with respective measurements in trabecular bone. However, when severe signs of subchondral bone disease were detected via micro-CT, the trabeculae in the trabecular bone specimans had increased apparent BMD and were closer, reaching similar values to the specimens of subchondral bone plate. This architectural pattern likely represents the result of the additional adaptive response of bone tissue to increased loading.

The adaptive response of the palmar aspect of the MC3 condyles has also been examined.5 Investigators found that increases in bone volume fraction and apparent BMD can be caused by deposition of new bone upon existing lamellar surfaces without prior resorption of older bone, such that the new bone composed of mixed woven and lamellar bone tissue with a lower degree of mineralization than that of previously existing (older) trabeculae occupies the former marrow spaces.5 They suggested that the increase in bone tissue could be underestimated by some radiographic imaging modalities. In the present study, we detected a significant increase in apparent BMD and a significant decrease in trabecular separation in trabecular bone with severe versus mild bone disease, which supports the ability of micro-CT to detect the new, less mineralized bone tissue formed.

On the other hand, aged bone tissue is more mineralized and is indicative of the loading history of the bone tissue.30 Racing histories were not available for the horses in our study, but all of the horses were active in flat racing. Horses in the severe group were selected for having more severe changes identified via micro-CT, which were likely indicative of a more advanced stage of maladaptation of the subchondral bone to exercise.20,23 It would be expected that these horses had aged bone tissue that was likely more mineralized and thus had a higher true BMD than did horses in the mild group. However, we did not detect an increase in mineralization in the condylar subchondral bone plate. Specimens of condylar subchondral bone plate might have already reached a maximum true BMD, from which an additional increase does not develop but subsequent subchondral bone failure does.20 Resorptive lesions are characteristic of advanced stages of subchondral bone disease20,32,33 and were also detected in bone specimens from horses with severe subchondral bone disease in our study. A counterbalance of coexistent increased true BMD and focal resorption33 as well as bone tissue collapse and compaction in progressing subchondral bone disease20 might have interfered with the ability of micro-CT to detect differences in bone structure in bone specimens from horses with mild bone disease. In our study, although results of paired comparisons of bone specimens collected from the same location between groups were not significant, a significant interaction effect was detected for true BMD, trabecular separation, and connectivity. If mean condylar values for true BMD are considered, then values for trabecular bone increased, whereas values for subchondral bone plate slightly decreased. In lateral condyles, true BMD appeared to be higher in trabecular bone than in subchondral bone plate, although that difference was not significant (P = 0.09). The lack of significance for this comparison may have been attributable to the limited sample size. As well, horses were classified as having severe or mild subchondral bone disease on the basis of the maximal score assigned to any particular metacarpal condyle. Some horses may have had high scores in some condyles and low scores in others. In that situation, some condyles classified as mildly diseased would have been included in the severe group, therefore decreasing the ability to detect differences between groups. We opted to consider the horse as a unit to ensure that the same loading history (exercise) existed for forelimbs and metacarpophalangeal joints.

The subchondral bone plate is defined as the bone layer separating the calcified cartilage from the marrow spaces.9,26 The thickness of the subchondral bone plate has been evaluated in some regions in bones of horses34 but not at the distopalmar aspect of the MC3 condyles. Degree of thickness can vary by type of joint and is related to history of local biomechanical load. The subchondral bone plate is thicker in regions of bone with high load than regions with low load,35–37 and it is thicker in flat-racing horses than those of horses in other disciplines.38 The thickness of subchondral bone plate has traditionally been determined by histomorphometry.39 In our study, we used visual assessment of micro-CT images.23 Micro-CT is a precise technique for morphometric characterization of bone tissue and is highly correlated with histomorphometry.22,40–42 We considered that the quality of the micro-CT images was sufficient to provide adequate differentiation of the bone layer separating cartilage from marrow spaces in trabecular bone. We also acknowledged a certain degree of variability in the thickness of subchondral bone among horses, which may have led to variability in the ratio of subchondral bone plate to trabecular bone in bone specimens from the distopalmar aspect of MC3s among different horses in the study. One study20 revealed that the earliest signs of subchondral bone failure develop within 3 mm of the calcified cartilage layer, which warrants the evaluation of this layer separately from the overlying trabecular bone.

We did not detect differences between left and right forelimbs, but we detected some structural differences in the values for subchondral bone between medial and lateral condyles. In bone specimens from horses with mild bone disease, the subchondral bone plate of medial condyles had thicker trabeculae, particularly when compared with the immediately proximally located trabecular bone. However, in bone specimens from horses with severe bone disease, trabecular thickness decreased to values similar to those of trabecular bone. This finding may indicate that, as subchondral bone disease progresses, the subchondral bone plate weakens, particularly in the medial condyle of MC3, and that, as others33 have proposed, the medial condyle is a predominant site for osteoarthritic lesions of the metacarpophalangeal joint. Mean values of apparent and true BMD in the medial condyle varied accordingly, but the differences between the means were not significant.

Also interesting was the significant difference in connectivity within the trabecular bone of lateral condyles between horses classified as having mild versus severe subchondral bone disease. The trabecular bone of this region in the severe group was the only trabecular bone that had values of connectivity similar to those for the subchondral bone plate. Although differences were not significant, the value for true BMD of trabecular bone in lateral condyle was somewhat higher (P = 0.09) than that in the overlying subchondral bone plate in bone specimens from horses with severe subchondral bone disease and was somewhat higher (P = 0.10) than that of similarly located trabecular bone in the mild group. In another study,6 a more intense and extended region of increased bone density in lateral versus medial condyles was identified. The investigators suggested that the difference between anatomic locations could be the result of higher stress on lateral versus medial condyles because of the smaller size of the lateral condyle and higher slope of its articular surface.43

Our results supported and expanded the findings of other researchers6,7 in that our study revealed that across the palmar aspect of the metacarpal condyles exists a steep gradient in density of subchondral bone (apparent BMD, true BMD, and bone volume fraction) and in structural characteristics (trabecular thickness and separation). This gradient may contribute to the pathogenesis of condylar fractures through concentration of shear forces at the area of the condylar groove. Indeed, we have described that, in horses with micro-CT findings indicative of severe subchondral bone disease, sclerosis invaded the trabecular bone in the condyles and, to a lesser extent, the trabecular bone in the sagittal ridge. Consequently, the density and structural gradient expanded in a proximal direction. We believe this pattern is indicative of an increase in loading history of the trabecular bone,30 in accordance with in vitro findings.44 As the sclerosis of subchondral bone plate progressed, it would have become brittle, and shock absorption would have been transferred to the trabecular bone, which in turn would have undergone an adaptive response in an attempt to counteract the increased stress, leading to denser, thicker, and closer trabeculae.

This study had a number of limitations. The number of horses in each group was small, and there was no control over factors such as age, previous work history, or degenerative lesions on the overlying articular cartilage; therefore, correlations between these factors and structural characteristics were limited. In another study at our laboratory, associations between disease of the metacarpophalangeal joint or cumulative work index and subchondral bone abnormalities investigated via micro-CT examination were detected in horses with late-stage disease.23 Horses were selected on the basis of micro-CT images that were believed to represent various stages of subchondral bone disease in metacarpophalangeal joints of Thoroughbred racehorses,23 which agrees with other investigators.20 Because histologic analysis of the bone specimens used was not performed, the micro-CT images and categories could not be compared with actual pathologic lesions. No definitive categorization of the physiologic (adaptive) or pathologic (maladaptive) nature of some of the micro-CT changes can be made at this point. However, results of micro-CT imaging reportedly have excellent agreement with results of histopathologic evaluation in other animal species.40,42 As well, a strong correlation between results of micro-CT analysis with bovine hydroxyapatite and results of bone mineral analysis by ashing techniques has been identified.45

Our sample of horses was selected from 2 racetracks in Ontario. Specific factors related to these particular horse populations and environmental factors at those locations may limit extrapolation of our results. However, epidemiologic characteristics of catastrophic musculoskeletal injuries that occur at these racetracks appear similar to those of other North American racetracks.24 As mentioned before, the possibility that some horses classified as having severe subchondral bone disease had low initial scores in specific condyles might have reduced the likelihood of detecting significant differences between bone specimens from horses with severe versus mild bone disease. Horses with naturally developing disease were used for the study, which is desirable when investigating a chronic performance-related process such as that affecting the metacarpophalangeal joint in Thoroughbred racehorses.

Potential errors related to the methodology used in the study reported here should also be considered, although special attention was maintained to minimize them. Bone specimens were carefully handled and protected from drying by keeping them wrapped in gauze that was soaked in saline solution. Care was taken to maintain the specimens under constant irrigation with saline solution during sample machining. Thin diamond blades were used at low speed to decrease the risk of specimen damage during the machining process. Some variation in the orientation of the blades during machining was likely to have occurred, although the process was carefully supervised by a single investigator to decrease that likelihood.

The imaging protocol used for micro-CT was selected on the basis of other studies performed at our laboratory, and a resolution of 45 μm was considered to be thinner than the bone trabeculae of the subchondral bone at the distal aspect of MC3. However, this choice of imaging resolution meant that details < 45 μm were not detected in our study. Subchondral bone failure at the distal aspect of MC3 originates as fractal arrays or branching microcracks that measure in the nanomillimeter range.14 Although initial changes < 45 μm related to bone failure were not distinguished in this study, characterization of such changes was not our objective.

To the authors' knowledge, this is the first study in which micro-CT was used for structural evaluation of subchondral bone in metacarpal condyles of horses. On the basis of our results, we concluded that the microstructure of subchondral bone in the distopalmar aspect of MC3 followed an anatomic gradient; bone tissue was consistently denser and had thicker and closer trabeculae in the condyles versus the sagittal ridge. Denser bone tissue with thicker and closer trabeculae was also evident in the subchondral bone plate versus trabecular bone in horses with micro-CT changes indicative of mild subchondral bone disease; however, when micro-CT changes were severe, the structural characteristics of trabecular bone tended to resemble those of the subchondral bone plate, especially at the condyles. Application of micro-CT provided detailed information on subchondral bone in metacarpal condyles. Different micro-CT images of the subchondral bone revealed different structural characteristics of that bone. The information provided by our study will be useful for further understanding and characterization of the adaptation and maladaptation of subchondral bone to exercise. This could serve as a base for development of new diagnostic tools that facilitate early diagnosis of subchondral bone disease so that appropriate modifications of training and racing regimens can be performed with the objective of decreasing the incidence of its undesirable consequences.

At this stage, additional studies should include validation of our micro-CT technique by use of a gold standard such as histopathologic evaluation and bone mineral analysis of equine bone as well as investigation of the mechanical properties of the subchondral bone of MC3. Associations need to be evaluated among micro-CT findings and clinical findings such as history of lameness or racing, results from other diagnostic imaging modalities, and evidence of articular cartilage lesions and condylar fractures. Investigation of the clinical significance of the micro-CT images could be also performed by evaluating clinical signs at time of euthanasia or by completing prospective studies with similar diagnostic modalities available for in vivo application.

ABBREVIATIONS

2-D

2-dimensional

3-D

3-dimensional

BMD

Bone mineral density

CT

Computed tomography

HU

Hounsfield units

MC3

Third metacarpal bone

ROI

Region of interest
a.

eXplore Locus micro–computed tomography scanner, GE Medical Systems, London, ON, Canada.

b.

MicroView ABA, version 2.1.2, GE Healthcare, London, ON, Canada.

c.

Band saw, model B1012, Craftex, Mississauga, ON, Canada.

d.

Diamond saw, model 800-900, LECO Corp, St Joseph, Mich.

e.

Diamond wheel, M4D220-N100M99-1/8, Company, Worcester, Mass.

f.

Electronic digital calliper, Marathon Watch Co, Richmond Hill, ON, Canada.

g.

SB3, Gammex RMI, Middleton, Wis.

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Contributor Notes

Supported by the Ontario Horse Racing Industry Association and the Ontario Ministry of Agriculture, Food and Rural Affairs, Canada. The Spanish Ministry of Education and Science provided stipend funding for Dr. Rubio-Martínez during the study.

Presented at the 34th Annual Veterinary Orthopedic Society Conference, Sun Valley, Idaho, March 2007; the 16th Annual Scientific Meeting of the European College of Veterinary Surgeons, Dublin, June 2007; and the American College of Veterinary Surgeons Symposium, Chicago, October 2007.

The authors thank Jon Huska and Dr. Dean Inglis for technical assistance and Gabrielle Monteith for assistance with statistical analysis.

Address correspondence to Dr. Rubio-Martínez.
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
Publication Date:
01 Nov 2008
  • Figure 1—
    View in gallery
    Figure 1—

    Reconstructed 3-D scout micro-CT image of the distal epiphysis of an MC3 (left) from a Thoroughbred racehorse with subchondral bone disease. Letters X, Y, and Z represent each of the 3-D planes. In the right column, 2-D images along each of the 3 planes are represented (transverse [top], dorsal [center], sagittal [bottom]). Micro-CT analysis software allowed rotation of the planes, which permitted selection of the slice that best represented the most severe changes detected in the distopalmar aspect of the condyles.

  • Figure 2—
    View in gallery
    Figure 2—

    Micro-CT image of the MC3 of a Thoroughbred racehorse with mild subchondral bone disease, showing sclerosis (ellipses) and mild focal or coalescing radiolucent areas (arrows) in the distopalmar subchondral bone of the MC3 without involvement of the articular surface. These characteristics were considered representative of incipient maladaptive remodelling. +Y represents location of one of the 3-D planes.

  • Figure 3—
    View in gallery
    Figure 3—

    Micro-CT image of the MC3 of a Thoroughbred racehorse with severe subchondral bone disease, showing more severe multifocal radiolucent areas (arrows) traversing the subchondral bone plate or affecting the articular surface (ellipse). These characteristics were considered representative of end-stage maladaptive remodelling. See Figure 2 for remainder of key.

  • Figure 4—
    View in gallery
    Figure 4—

    Line drawing of specimen collection from an MC3. A—Lateral view of the distal end of the MC3 showing the distopalmardorsoproximal direction along which the samples were collected (this direction simulates the loading direction against the proximal sesamoid bones). B—Dorsal view of the distal end of the MC3 indicating the location of the samples. Do = Dorsal. Pa = Palmar. P = Proximal (trabecular bone). D = Distal (subchondral bone plate). M = Medial. S = Sagittal. L = Lateral. X = Width of medial condyle. Y = Width of lateral condyle.

  • Figure 5—
    View in gallery
    Figure 5—

    Micro-CT images of 6 subchondral bone specimens obtained from a horse that was classified as having mild subchondral bone disease. Figure represents a 2-D image across the 6 specimens along the proximodorsal-distopalmar plane. Order of specimens from left to right and top to bottom is as follows: trabecular bone of the medial condyle, sagittal ridge, and lateral condyle and subchondral bone plate of the medial condyle, sagittal ridge, and lateral condyle.

  • Figure 6—
    View in gallery
    Figure 6—

    Micro-CT image of 6 subchondral bone specimens obtained from a horse that was classified as having severe subchondral bone disease. Figure represents a 2-D image across the 6 specimens along the proximodorsal-distopalmar plane. Order of specimens from left to right and top to bottom is as follows: trabecular bone of the lateral condyle, sagittal ridge, and medial condyle and subchondral bone plate of the lateral condyle, sagittal ridge, and medial condyle.

Figure 1—

Reconstructed 3-D scout micro-CT image of the distal epiphysis of an MC3 (left) from a Thoroughbred racehorse with subchondral bone disease. Letters X, Y, and Z represent each of the 3-D planes. In the right column, 2-D images along each of the 3 planes are represented (transverse [top], dorsal [center], sagittal [bottom]). Micro-CT analysis software allowed rotation of the planes, which permitted selection of the slice that best represented the most severe changes detected in the distopalmar aspect of the condyles.
Figure 2—

Micro-CT image of the MC3 of a Thoroughbred racehorse with mild subchondral bone disease, showing sclerosis (ellipses) and mild focal or coalescing radiolucent areas (arrows) in the distopalmar subchondral bone of the MC3 without involvement of the articular surface. These characteristics were considered representative of incipient maladaptive remodelling. +Y represents location of one of the 3-D planes.
Figure 3—

Micro-CT image of the MC3 of a Thoroughbred racehorse with severe subchondral bone disease, showing more severe multifocal radiolucent areas (arrows) traversing the subchondral bone plate or affecting the articular surface (ellipse). These characteristics were considered representative of end-stage maladaptive remodelling. See Figure 2 for remainder of key.
Figure 4—

Line drawing of specimen collection from an MC3. A—Lateral view of the distal end of the MC3 showing the distopalmardorsoproximal direction along which the samples were collected (this direction simulates the loading direction against the proximal sesamoid bones). B—Dorsal view of the distal end of the MC3 indicating the location of the samples. Do = Dorsal. Pa = Palmar. P = Proximal (trabecular bone). D = Distal (subchondral bone plate). M = Medial. S = Sagittal. L = Lateral. X = Width of medial condyle. Y = Width of lateral condyle.
Figure 5—

Micro-CT images of 6 subchondral bone specimens obtained from a horse that was classified as having mild subchondral bone disease. Figure represents a 2-D image across the 6 specimens along the proximodorsal-distopalmar plane. Order of specimens from left to right and top to bottom is as follows: trabecular bone of the medial condyle, sagittal ridge, and lateral condyle and subchondral bone plate of the medial condyle, sagittal ridge, and lateral condyle.
Figure 6—

Micro-CT image of 6 subchondral bone specimens obtained from a horse that was classified as having severe subchondral bone disease. Figure represents a 2-D image across the 6 specimens along the proximodorsal-distopalmar plane. Order of specimens from left to right and top to bottom is as follows: trabecular bone of the lateral condyle, sagittal ridge, and medial condyle and subchondral bone plate of the lateral condyle, sagittal ridge, and medial condyle.
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