During the last decade, researchers have investigated the relationship between exercise history and changes in the structure of subchondral bone in metacarpophalangeal joints in Thoroughbred racehorses. They found that the palmar aspect of the metacarpal condyles undergoes severe adaptive changes because it sustains high stress during flat racing.1–3 A consistent pattern of this adaptive response has been described as a marked gradient in radiographic opacity across the width of the condylar area, on the palmar aspect. The subchondral bone located at the condyles becomes denser than that at the sagittal ridge.4,5
Because bone density is highly related to the mechanical properties of cancellous bone,6 this gradient of radiographic opacity is believed to result in a gradient in the mechanical properties across the width of the MC3 condyles. The hypothesis is that the gradient causes shear stress and strain accumulation at the interface of regions of different elastic moduli,4,7 leading to increased fatigue damage at that location. Subsequent repair processes with formation of resorptive spaces weaken this location, acting as stress risers (stress accumulation) and predisposing affected horses to condylar fracture. The interfaces of radiographic opacity correspond with the condylar grooves in which condylar fractures typically develop.3 Microcracks and resorption lacunae have been detected in the subchondral bone of the condylar grooves of nonfractured MC3s from racing horses and on the fractured surface of MC3s with fractured condyles.7–9 These findings are evidence of fatigue failure of the subchondral bone at the fracture site, preceding development of fracture, and support the hypothesis that underlying structural deficiencies at specific locations predispose horses to condylar fractures.10
Although bone density is highly related to the mechanical properties of bone tissue, bone architecture is also a determinant factor.11,12 We have described the microstructure of subchondral bone in the palmar aspect of MC3 condyles in Thoroughbred racehorses by use of micro-CT,13 which is a technique that provides nondestructive assessment and microanalysis of the 3-dimensional architecture of trabecular bone.14 Our results were supportive of the hypothesis that condylar fractures are the end result of fatigue damage secondary to a gradient in mechanical properties of subchondral bone in the condylar region, as suggested from the gradient in bone density and bone microstructure at that location. We determined that the gradient was maintained and even extended proximad when severe changes indicative of subchondral bone disease were evident.13 However, to our knowledge, the mechanical properties of subchondral bone in the distopalmar aspect of MC3 condyles have not been investigated.
The objectives of the study reported here were to evaluate the mechanical properties of subchondral bone in the distopalmar aspect of MC3 condyles in Thoroughbred racehorses by compressive mechanical testing; measure the differences in the mechanical properties of subchondral bone between 2 different stages of subchondral bone disease, as determined via micro-CT; and determine whether there is an association between microstructural characteristics and mechanical properties of subchondral bone at this location. Specifically, we hypothesized that mechanical properties of subchondral bone in the distopalmar aspect of MC3 condyles would vary depending on anatomic location and, as such, that properties would differ between the condyles and the sagittal ridge and between subchondral bone plate and trabecular bone. In addition, mechanical properties of the subchondral bone at this location would be related to differences in the morphologic appearance of the subchondral bone, as assessed via micro-CT. Furthermore, values for structural and mechanical properties of the subchondral bone at this location would be correlated.
Material and Methods
Sample population—One hundred twelve Thoroughbred racehorses underwent postmortem examination at the Ontario Veterinary College of the University of Guelph during 2004 and 2005.15 Pairs of MC3s were collected from all horses and dissected free of tissues. The condylar region of each MC3 was examined by use of micro-CT.a The micro-CT images of the epiphysis of the distal aspect of each MC3 bone were acquired and reconstructed on a 3-dimensional format by use of an imaging program.b A dorsofrontal section was obtained from the subchondral bone located at the distopalmar aspect of the condyles in an area representative of subchondral bone changes.13 The obtained images were visually evaluated and classified according to category of bony changes on a scale from 0 (normal) to 8 (condylar fracture). Then, each horse was categorized according to the maximal score assigned to the 4 condyles of both MC3s. 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.
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). These characteristics were considered representative of incipient maladaptive remodeling. 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 endstage maladaptive remodelling. Bones with condylar fractures (score = 8) were not included in the study.
Specimen definition—At each MC3, 3 ROIs were defined at the sagittal ridge and the midpoint of each condyle. These ROIs were located at the distopalmar articular surface, corresponding to the contact area of the proximal sesamoid bones during the stance phase of the gallop,16 which also corresponds to the region at which degenerative lesions develop.3 At each ROI, samples were collected in a distopalmar-proximodorsal direction from the articular surface, simulating the direction of the compressive force applied by the proximal sesamoid bones during maximal extension of the metacarpophalangeal joint during racing. From the MC3s selected, 2 cubic specimens were collected at each ROI after physically removing the overlying articular cartilage manually with a blade. The dimensions of the bone specimens were determined on the basis of the estimated thickness of the subchondral bone plate at each location.13 Specimens from the distal aspect of the bone were considered to include mostly the calcified cartilage layer and the subchondral bone plate, whereas specimens from the proximal aspect of the bone included mostly trabecular bone.
Specimen collection—The selected MC3s were thawed at room temperature (18° to 21°C) for 6 to 20 hours, and 2 cubes of bone, each measuring approximately 6 × 6 × 6 mm, were cut from each of the 3 ROIs (sagittal ridge and the midpoint of each condyle) as described in detail elsewhere,13 yielding 6 specimens/MC3. Once the specimens had been obtained, the dimensions of each were recorded by use of a digital micrometer,c and the transverse cross-sectional area (perpendicular to the dorsofrontal plane along which blocks were machined) of each specimen was calculated. The whole process of specimen machining was performed in conditions of controlled temperature and humidity. The location from which each of the 6 bone specimens was obtained was written on the dorsodistal surface.
Acquisition of micro-CT images—The 6 samples collected from each MC3 were wrapped together in gauzed soaked in saline solution, sealed with plastic film, and imaged with micro-CT.13 Structural characteristics measured included apparent BMD (mg/cm3), true BMD (mg/cm3), bone volume fraction (vol:vol), trabecular thickness (mm), trabecular separation (mm), and connectivity (1/mm3).13 After image acquisition, samples that were wrapped in gauze soaked in saline solution were stored at −20°C until mechanical analyses were performed.
Mechanical testing—Bone specimens were thawed at room temperature (18 to 21°C) before the mechanical testing. Custom-made blocks were used as the lower testing platen (Figure 1). Blocks consisted of a 3 × 3 × 3-cm stainless steel cube and a 2-mm-thick, 3 × 3-cm stainless steel lamina. The lamina was attached to the cube on its upper surface via 4 screws (1 screw at each corner). This lamina had a 7-mm-diameter full-thickness perforation in the center, in which bone specimens were potted by use of a rapidly curing methyl methacrylate copolymer.d The 2 components of the copolymer were mixed according to the manufacturer's instructions, and the mixture was used to fill the central circular perforation. A bone cube was then potted in the mixture by applying digital pressure and maintaining the lateral sides of the cube as vertical as possible. Excess mixture was removed so that it was even with the upper surface of the platform. A minimum of 7 minutes was allowed for the mixture to cure to ensure that polymerization was complete and that a consistent firmness had been achieved. During that time, the bone specimens were maintained covered in gauze soaked with saline solution to prevent dehydration.
Photograph of a custom-made block used as the lower testing platen in the compressive mechanical testing of cubic specimens of subchondral bone (center) obtained from condyles of MC3s from Thoroughbred racehorses. Blocks consisted of a 3 × 3 × 3-cm stainless steel cube and an identical cross section of stainless steel 2-mm-thick lamina. The lamina was attached to the cube on its upper surface via 4 screws (1 screw at each corner).
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1423
Photograph of a custom-made block used as the lower testing platen in the compressive mechanical testing of cubic specimens of subchondral bone (center) obtained from condyles of MC3s from Thoroughbred racehorses. Blocks consisted of a 3 × 3 × 3-cm stainless steel cube and an identical cross section of stainless steel 2-mm-thick lamina. The lamina was attached to the cube on its upper surface via 4 screws (1 screw at each corner).
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1423
Photograph of a custom-made block used as the lower testing platen in the compressive mechanical testing of cubic specimens of subchondral bone (center) obtained from condyles of MC3s from Thoroughbred racehorses. Blocks consisted of a 3 × 3 × 3-cm stainless steel cube and an identical cross section of stainless steel 2-mm-thick lamina. The lamina was attached to the cube on its upper surface via 4 screws (1 screw at each corner).
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1423
Compressive testing was performed by use of a testing device.e The custom-made block was secured to the lower platform of the device to prevent lateral displacement of the block. The upper platform incorporated spherical seating that allowed accommodation to the obliqueness of the upper surface of the bone specimen (Figure 2). All samples sustained an initial preload (mean ± SD, 49.13 ± 4.32 N) and tested in compression (rate, 1% [0.06 mm/s]) until a final strain of 50% was achieved. Time, load, and interplaten displacement data were registered every 0.1 seconds. Load and displacement values at each time were converted into stress and strain values, respectively, by use of initial values for cross-sectional area and height of each specimen. A stress-strain curve was constructed for each specimen by use of commercially available softwaref (Figure 3).
Schematic (A) and photographic (B) illustrations of the compressive mechanical testing process used to evaluate the mechanical properties of specimens of subchondral bone obtained from condyles of MC3s from Thoroughbred racehorses. In panel A, thick arrows indicate the compressive force that a bone specimen was subjected to. The spherical setting (*) permits adaptation of the upper platform to the obliquity of the upper surface of the sample (as can be appreciated in panel B). Thin arrows indicate the acrylic in which specimens were embedded. In panel B, a bone specimen is potted on the customized platform and undergoing in situ compressive mechanical testing. Notice the obliquity of the upper platform.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1423
Schematic (A) and photographic (B) illustrations of the compressive mechanical testing process used to evaluate the mechanical properties of specimens of subchondral bone obtained from condyles of MC3s from Thoroughbred racehorses. In panel A, thick arrows indicate the compressive force that a bone specimen was subjected to. The spherical setting (*) permits adaptation of the upper platform to the obliquity of the upper surface of the sample (as can be appreciated in panel B). Thin arrows indicate the acrylic in which specimens were embedded. In panel B, a bone specimen is potted on the customized platform and undergoing in situ compressive mechanical testing. Notice the obliquity of the upper platform.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1423
Schematic (A) and photographic (B) illustrations of the compressive mechanical testing process used to evaluate the mechanical properties of specimens of subchondral bone obtained from condyles of MC3s from Thoroughbred racehorses. In panel A, thick arrows indicate the compressive force that a bone specimen was subjected to. The spherical setting (*) permits adaptation of the upper platform to the obliquity of the upper surface of the sample (as can be appreciated in panel B). Thin arrows indicate the acrylic in which specimens were embedded. In panel B, a bone specimen is potted on the customized platform and undergoing in situ compressive mechanical testing. Notice the obliquity of the upper platform.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1423
Representative stress-strain curve for a specimen of subchondral bone obtained from the distopalmar aspect of an MC3 of a Thoroughbred racehorse. The yield point was calculated following the 0.02% strain offset criterion. An offset line originating from 0.02% strain and with a slope equal to the elastic modulus for the specimen is included. The intersection (arrow) of the offset line with the stress-strain curve was defined as the yield point. Yield stress and yield strain were defined at the yield point. Energy to failure (shaded area) was calculated as the area under the curve up to the yield point.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1423
Representative stress-strain curve for a specimen of subchondral bone obtained from the distopalmar aspect of an MC3 of a Thoroughbred racehorse. The yield point was calculated following the 0.02% strain offset criterion. An offset line originating from 0.02% strain and with a slope equal to the elastic modulus for the specimen is included. The intersection (arrow) of the offset line with the stress-strain curve was defined as the yield point. Yield stress and yield strain were defined at the yield point. Energy to failure (shaded area) was calculated as the area under the curve up to the yield point.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1423
Representative stress-strain curve for a specimen of subchondral bone obtained from the distopalmar aspect of an MC3 of a Thoroughbred racehorse. The yield point was calculated following the 0.02% strain offset criterion. An offset line originating from 0.02% strain and with a slope equal to the elastic modulus for the specimen is included. The intersection (arrow) of the offset line with the stress-strain curve was defined as the yield point. Yield stress and yield strain were defined at the yield point. Energy to failure (shaded area) was calculated as the area under the curve up to the yield point.
Citation: American Journal of Veterinary Research 69, 11; 10.2460/ajvr.69.11.1423
From each stress-strain curve, elastic modulus and energy to failure (toughness) were derived. Elastic modulus was calculated as the slope of the linear portion of the curve. For this calculation, 2 points that were within and toward the limits of the linear portion of the curve were visually selected. Linear regression analysis was performed to include all data points on the stress-strain curve that were within the 2 selected points. The R2 values calculated for these regression analyses were all ≥ 0.967. The yield point was calculated following the 0.02% strain offset criterion.17,18 An offset line originating from 0.02% strain offset was created with a slope equal to the elastic modulus for the specimen. The intersection of the offset line with the stress-strain curve was defined as the yield point. Yield stress and yield strain were defined at the yield point, which was used to define bone failure. To calculate energy to failure, the portion of the stress-strain curve from 0 to the yield point was first selected and fit to a fourth-order polynomial equation; all equations had an R2 ≥ 0.998. Energy to failure was subsequently calculated by integrating the equation to the yield strain value.
Statistical analysis—Data were assessed for normality of distribution, and those that were nonnormally distributed were logarithmically transformed. A factorial design within a partial randomized block ANOVA was used to evaluate differences in mechanical characteristics of bone (elastic modulus, yield stress, yield strain, and energy to failure) among various factors. Horse from which both MC3s were obtained was designated as a block, and factors investigated included disease classification (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 adjusted with a Tukey test. Correlations were assessed between structural variables (apparent BMD, true BMD, bone volume fraction, trabecular thickness, trabecular separation, and connectivity) and mechanical variables (elastic modulus, yield stress, yield strain, and energy to failure). Values of P ≤ 0.05 were considered significant for all analyses.
Results
Five bone specimens were destroyed during mechanical testing because of technical errors. Consequently, mechanical testing data from 3 specimens from the mild disease group and 2 specimens from the severe disease group were excluded from statistical analyses.
Analyses of variance—Specimens of subchondral bone from the MC3s of right versus left forelimbs were not significantly different with respect to mechanical properties. When considering the factor ROI (lateral condyle, sagittal ridge, and medial condyle), no differences were detected between specimens obtained from the lateral and medial condyles. However, specimens obtained from the condyles had significantly (P < 0.001) higher values for elastic modulus, yield stress, and energy to failure (toughness) than did specimens obtained from the sagittal ridge (Table 1). When values for layers of bone were compared, specimens of subchondral bone plate had significantly lower (P < 0.001) values for elastic modulus and yield stress and significantly higher (P < 0.001) values for yield strain than did specimens of trabecular bone (Table 2).
Mean ± pooled SEM mechanical properties of bone specimens from the distopalmar aspect of MC3s obtained from 12 horses with mild or severe subchondral bone disease, according to ROI.
| Mechanical property | ROI | ||
|---|---|---|---|
| Lateral condyle | Medial condyle | Sagittal ridge | |
| Elastic modulus (MPa) | 2,904.7 ± 112.8a | 3,212.7 ± 111.4a | 1,706.3 ± 113.4b |
| Yield stress (MPa) | 113.3 ± 3.115a | 116.2 ± 3.105a | 65.75 ± 3.136b |
| Energy to failure (MJ/m3) | 3.165 ± 0.149a | 2.95 ± 0.147a | 1.979 ± 0.149b |
Within each variable, values with different superscripts are significantly (P≤ 0.05) different.
Values for yield strain are not provided because they were not significantly different among ROIs.
Mean ± pooled SEM elastic modulus and yield stress and mean (95% CD yield strain of bone specimens from 2 layers of the distopalmar aspect of MC3s obtained from 12 horses with mild or severe subchondral bone disease.
| Outcome | Layer of bone | P value | |
|---|---|---|---|
| Trabecular bone | Subchondral bone plate | ||
| Elastic modulus (MPa) | 3,053.1 ± 90.59 | 2,162.8 ± 93.16 | < 0.001 |
| Yield stress (MPa) | 104.6 ± 2.82 | 92.26 ± 2.865 | < 0.001 |
| Yield strain (%) | 3.984 (3.757 – 4.225) | 4.929 (4.645 – 5.23) | < 0.001 |
Values for energy to failure are not provided because they were not significantly different among ROIs.
Analysis of the effects of the interaction between disease classification and layer of bone on the various mechanical properties yielded some significant results (Table 3). Within specimens from subchondral bone with micro-CT indications of mild disease, values for yield stress were similar between subchondral bone plate and trabecular bone; however, within specimens from MC3s with severe subchondral bone disease, values for yield stress were higher (P < 0.001) in trabecular bone than in subchondral bone plate. A significant effect was also detected for energy to failure, but post hoc comparisons yielded no significant differences between disease classifications or layers. Results for elastic modulus were not significant (P = 0.10).
Results of ANOVA 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 mechanical properties in MC3 specimens from 12 horses with mild or severe subchondral bone disease.
| Outcome | Layer of bone | Seventy of subchondral bone disease | P value | |
|---|---|---|---|---|
| Mild | Severe | |||
| Yield stress (MPa) | Trabecular bone | 99.1 ± 4.003a,b,c | 110.1 ± 3.974c | < 0.001 |
| Subchondral bone plate | 97.57 ± 4.046a,b,c | 86.99 ± 4.058b | ||
| Energy to failure (MJ/m3) | Trabecular bone | 2.539 ± 0.189a | 2.8239 ± 0.185a | 0.04 |
| Subchondral bone plate | 2.855 ± 0.190a | 2.5725 ± 0.189a | ||
Within each variable, values with different superscripts indicate significant (P≤ 0.05) differences.
Results for elastic modulus and yield strain are not included because they were not significant.
The interaction between disease classification and ROI (lateral condyle, medial condyle, and sagittal ridge) was significant for yield stress (Table 4). Yield stress values for condylar specimens were significantly (P < 0.001) higher than those for sagittal ridge specimens.
Results of ANOVA for the interaction between degree of subchondral bone disease and ROI and its effects on mean ± pooled SEM yield stress in MC3 specimens from 12 horses with mild or severe subchondral bone disease.
| ROI | Mild | Severe |
|---|---|---|
| Lateral condyle | 115.5 ± 4.42a | 111.1 ± 4.477a |
| Medial condyle | 118.4 ± 4.362a | 114.1 ± 4.42a |
| Sagittal ridge | 61.1 ± 4.507b | 70.39 ± 4.362b |
Different superscripts indicate significant (P ≤ 0.05) differences among values.
Results for elastic modulus, yield strain, and energy to failure were not significant.
Interactions of disease classification, layer of bone, and ROI had significant effects on values of several mechanical properties (Table 5). Decreases in yield stress and energy to failure were detected in condylar subchondral bone plate of specimens from MC3s classified with severe subchondral bone disease, and the opposite was true for condylar trabecular bone. Condylar subchondral bone plate and trabecular bone had similar values for yield stress in MC3s classified as mildly diseased; however, in MC3s classified as severely diseased, condylar trabecular bone had significantly (P ≤ 0.009) higher values for yield stress than did condylar subchondral bone plates. At the region of the sagittal ridge, trabecular bone had higher values for yield stress than did subchondral bone plates in both disease classifications (P ≤ 0.04). Regarding energy to failure, in MC3s classified with mild disease, the subchondral bone plate of both condyles was higher than the trabecular bone (though only significant for the medial condyle [P = 0.017]). However, in specimens from MC3s with severe subchondral bone disease, differences between both layers were not significant, and mean values for energy to failure in condylar trabecular bone were similar or higher (nonsignificantly) than mean values for condylar subchondral bone plate.
Results of ANOVAs for the effect of the interaction among classification of subchondral bone disease, layer of bone, and ROI on mean ± SD values for yield stress and energy to failure and mean (95% CD values for yield strain in MC3 specimens from 12 horses with subchondral bone disease.
| Outcome | Layer | Classification of subchondral bone disease | P value | |||||
|---|---|---|---|---|---|---|---|---|
| Mild | Severe | |||||||
| L | S | M | L | S | M | |||
| Yield stress (MPa) | TBB | 113.2 ± 5.548a | 70.86 ± 5.35*,b | 113.2 ± 5.359a | 122.4 ± 5.359†a | 82.36 ± 5.359‡,b | 125.6 ± 5.359§,a | 0.02 |
| SCBP | 117.8 ± 5.359a | 51.34 ± 5.821*,b | 123.5 ± 5.359¶,a | 99.92 ± 5.727†,a | 58.43 5.359‡,b | 102.5 ± 5.548§,¶,a | ||
| Yield strain (%) | TBB | 3.818 (3.300 – 4.418) | 4.163 (3.620 – 4.787) | 3.593 (3.144 – 4.100) | 4.159 (3.640 – 4.752) | 4.136 (3.620 – 4.726) | 4.069 (3.561 – 4.649) | 0.07 |
| SCBP | 5.242 (4.588 – 5.990) | 4.923 (4.225 – 5.721) | 4.959 (4.341 – 5.666) | 5.243 (4.559 – 6.029) | 4.713 (4.125 – 5.385) | 4.525 (3.935 – 5.204) | ||
| Energy to failure (MJ/m3) | TBB | 2.857 ± 0.282a | 2.22 ± 0.261a | 2.539 ± 0.261∥,a | 3.237 ± 0.261a | 2.174 ± 0.261b | 3.061 ± 0.262a | 0.02 |
| SCBP | 3.335 ± 0.261a | 1.681 ± 0.287a | 3.550 ± 0.261∥,a | 3.229 ± 0.272a | 1.840 ± 0.261b | 2.648 ± 0.272a,b | ||
Value for yield stress in specimens with mild disease from the sagittal ridge (S) is significantly (P = 0.039) different between layers of bone (TBB and SCBP).
Value foryield stress in specimens with severe disease from the lateral condyle (L) is significantly (P = 0.009) different between layers of bone (TBB and SCBP).
Value for yield stress in specimens with severe disease from the sagittal ridge is significantly (P = 0.005) different between layers of bone (TBB and SCBP).
Value for yield stress in specimens with severe disease from the medial condyle (M) is significantly (P = 0.001) different between layers of bone (TBB and SCBP).
Value for energy to failure in specimens with mild disease from the medial condyle is significantly (P = 0.017) different between layers of bone.
Value for yield stress in specimens of SCBP from medial condyles is significantly (P≤ 0.05) different between classifications of disease.
TBB = Trabecular bone. SCBP = Subchondral bone plate.
For each variable, within each disease classification and each layer, different superscript lowercase letters indicate significant (P ≤ 0.05) differences between different ROIs.
Results for elastic modulus are not included because they were not significant.
Correlation analyses—Significant overall correlations (multivariate ANOVA) were detected between the structural factors apparent BMD, true BMD, and bone volume fraction and the mechanical factors elastic modulus, yield stress, and energy to failure (Table 6). Yield stress was also significantly correlated with trabecular thickness and trabecular separation. A weak and insignificant correlation was evident between connectivity and energy to failure (r = 0.170; P = 0.097) and between elastic modulus and trabecular separation (r = −0.172; P = 0.094). No significant overall correlations were detected for yield strain or connectivity. At specific ROIs, various significant correlations between structural and mechanical variables were detected via calculation of Pearson correlation coefficient (Tables 7 and 8). The strongest correlations detected involved yield stress.
Results of multivariate ANOVAs indicating overall correlations between structural and mechanical properties of subchondral bone specimens from MC3s of 12 Thoroughbred racehorses with subchondral bone disease.
| Outcome | Mechanical property | ||
|---|---|---|---|
| Elastic modulus | Yield stress | Energy to failure | |
| Apparent BMD | 0.457 | 0.767 | 0.377 |
| (< 0.001) | (< 0.001) | (< 0.001) | |
| True BMD | 0.439 | 0.747 | 0.398 |
| (< 0.001) | (< 0.001) | (< 0.001) | |
| Bone volume fraction | 0.235 | 0.501 | 0.204 |
| (0.02) | (< 0.001) | (0.05) | |
| Trabecular thickness | 0.071 | 0.237 | 0.098 |
| (0.49) | (0.02) | (0.34) | |
| Trabecular separation | −0.172 | −0.215 | 0.105 |
| (0.09) | (0.04) | (0.31) | |
| Connectivity | −0.039 | 0.067 | 0.17 |
| (0.71) | (0.52) | (0.10) |
Values in parentheses are P values.
Results for yield strain are not included because they were not significant.
Pearson correlation coefficients for significant (P< 0.05) correlations between the structural and mechanical properties of subchondral bone specimens from MC3s of 12Thoroughbred racehorses with evidence of mild bone disease, according to location of bone specimen.
| Structural property | Elastic modulus in SCBP | Yield stress | Energy to failure | ||
|---|---|---|---|---|---|
| TBB | SCBP | TBB | SCBP | ||
| Lateral condyle | |||||
| Apparent BMD | NS | 0.843 (0.001) | NS | NS | NS |
| True BMD | NS | 0.751 (0.007) | NS | 0.619 (0.04) | NS |
| Sagittal ridge | |||||
| Apparent BMD | 0.663 (0.04) | 0.925 (< 0.001) | 0.977 (< 0.001) | NS | 0.807 (0.005) |
| True BMD | 0.678 (0.03) | 0.914 (< 0.001) | 0.97 (< 0.001) | NS | 0.803 (0.005) |
| Bone volume fraction | NS | 0.904 (< 0.001) | 0.815 (0.004) | NS | 0.677 (0.03) |
| Trabecular thickness | NS | 0.886 (0.001) | 0.647 (0.04) | NS | NS |
| Trabecular separation | −0.674 (0.03) | −0.856 (< 0.001) | −0.68 (0.03) | NS | NS |
| Medial condyle | |||||
| Apparent BMD | NS | 0.658 (0.02) | 0.576 (0.05) | NS | NS |
| True BMD | NS | 0.608 (0.04) | NS | NS | NS |
| Connectivity | NS | 0.681 (0.02) | NS | 0.565 (0.06) | 0.637 (0.03) |
Values in parentheses are P values. NS = Not significant.
See Table 5 for remainder of key.
Pearson correlation coefficients for significant (P ≤ 0.05) correlations between the structural and mechanical properties of subchondral bone specimens from MC3s of 12Thoroughbred racehorses with evidence of severe bone disease, according to location of bone specimen.
| Structural property | Elastic modulus | Yield stress | Yield strain in SCBP | Energy to failure in SCBP | ||
|---|---|---|---|---|---|---|
| TBB | SCBP | TBB | SCBP | |||
| Lateral condyle | ||||||
| Apparent BMD | 0.818 (0.001) | NS | 0.775 (0.003) | NS | −0.607 (0.04) | NS |
| True BMD | 0.075 (0.005) | NS | 0.637 (0.03) | NS | −0.599 (0.04) | NS |
| Bone volume fraction | NS | NS | 0.749 (0.005) | NS | NS | NS |
| Trabecular thickness | NS | NS | −0.756 (0.004) | NS | NS | NS |
| Trabecular separation | −0.580 (0.048) | NS | NS | NS | NS | NS |
| Sagittal ridge | ||||||
| Apparent BMD | NS | NS | NS | 0.778 (0.003) | NS | 0.592 (0.04) |
| True BMD | NS | NS | NS | 0.694 (0.01) | NS | 0.570 (0.05) |
| Bone volume fraction | NS | NS | 0.612 (0.034) | 0.731 (0.007) | NS | NS |
| Trabecular thickness | NS | NS | 0.595 (0.041) | NS | NS | |
| Medial condyle | ||||||
| Apparent BMD | NS | NS | 0.808 (0.001) | NS | NS | NS |
| True BMD | NS | NS | 0.612 (0.03) | NS | NS | NS |
| Bone volume fraction | NS | NS | 0.860 (< 0.001) | NS | −0.602 (0.05) | NS |
| Trabecular thickness | NS | NS | 0.669 (0.02) | NS | NS | NS |
| Trabecular separation | NS | NS | −0.862 (< 0.001) | NS | NS | NS |
| Connectivity | NS | NS | NS | NS | NS | |
See Tables 5 and 7 for remainder of key.
Discussion
Our results indicated that the mechanical properties of subchondral bone at the palmar aspect of the condyles of MC3 varied according to anatomic location and severity of subchondral bone disease, as determined via micro-CT. In MC3s with micro-CT evidence of severe subchondral bone disease, the trabecular bone was stronger, whereas subchondral bone plate was weaker, compared with MC3s with evidence of mild disease; this pattern was particularly evident at the condyles, in which subchondral bone was stronger than it was at the sagittal ridge. These results paralleled those of our study13 on structural characteristics, supporting the hypothesis that a gradient of values for mechanical properties exists across the distopalmar aspect of MC3 condyles in Thoroughbred racehorses. The correlations detected between the microstructural and mechanical properties indicated the potential predictive value of micro-CT for the evaluation of mechanical characteristics of subchondral bone at this location.
Regardless of disease classification, condylar subchondral bone (both subchondral bone plate and trabecular bone) had higher values for elastic modulus and was stronger (higher yield stress) and tougher (higher energy to failure), compared with subchondral bone at the sagittal ridge. This variation by location agrees with the pattern for structural characteristics: subchondral bone in condyles had higher BMD (apparent and true BMD), thicker trabeculae, and closer trabeculae than did subchondral bone in the sagittal ridge.13 These findings support the theory that different mechanical properties of subchondral bone might create stress risers at the interfaces (condylar groove area), which can be involved in the pathogenesis of condylar fractures.4 Unlike specimens from MC3s with evidence of mild subchondral bone disease, when specimens from MC3s with severe disease were evaluated, subchondral bone plate specimens were significantly weaker (lower yield stress) than trabecular bone, but the gradient across the condylar area was still maintained.
Energy to failure (or toughness) is an indicator of the energy that a bone tissue sample is capable of absorbing before the structure is not able to withstand the forces and fails (ie, cracks, collapses, and fractures); a tough bone will be more resistant to fracture.19 We did not detect definitive deteriorations in the structural properties (ie, a significant decrease in bone density, bone volume fraction, or trabecular thickness) of subchondral bone plate in specimens from MC3s classified as severely diseased.13 However, study limitations may have reduced the ability to detect those deteriorations.13 The mechanical findings of the present study support our suggestion13 that the adaptive response of subchondral bone plate in MC3s classified with mild subchondral bone disease could be close to its maximal capacity or even already overwhelmed and further exceeded in bones classified with severe disease. We believe that the severe changes detected via micro-CT were indicative of advanced stages of subchondral bone disease, when bone collapse and failure were already developing,20 leading to deterioration of the mechanical properties. In those circumstances, the subchondral bone plate decreases its capacity for shock absorption21 and the load is then transmitted to the trabecular bone. This trabecular bone undergoes an adaptive response, acquiring a modified microstructure13 that is mechanically superior by increasing values for yield stress and energy to failure.
Interestingly, we found that trabecular bone had significantly higher values for elastic modulus than did subchondral bone plate, regardless of disease status, even though the structure of the trabecular bone differed significantly between disease classification groups.13 We know that although both layers of bone have a highly organized anisotropic structure with trabeculae running in the sagittal plane, the structural anisotropy is significantly more pronounced in trabecular bone versus subchondral bone plate.5 In subchondral bone plate, the anisotropy tends to be masked by any increase in the bone volume fraction (evident as sclerosis).5,13 This is further evidence that both layers are composed of different bone tissue. In our study, the condylar trabecular bone was more similar to cancellous bone than the condylar subchondral bone plate. It appears that the more anisotropic, lamellar trabecular bone with fewer mediolateral connections has a higher elastic modulus, compared with the masked, anisotropic subchondral bone plate.5 On the other hand, we know that the earliest signs of subchondral bone failure develop at the layers of calcified cartilage and subchondral bone plate20 and that they develop as fractal arrays or branching microcracks starting at the nanometer scale,9 which is lower than the 45-μm resolution used in the present study. Therefore, some bone failure may have already existed in specimens of subchondral bone plate from MC3s with mild subchondral bone disease.
In specimens from MC3s classified with severe versus mild subchondral bone disease, trabecular thickness was much lower in the subchondral bone plate of medial versus lateral condyles,13 which may explain the significantly lower values for yield stress in specimens with severe versus mild disease. In this location, differences in mean energy to failure between mildly and severely diseased bone were much greater than they were in lateral condyles. That is, in medial condyles, the mean value for energy to failure for mildly diseased specimens was 1 MJ/mm3 (significantly higher than energy to failure in the immediately proximal trabecular bone), and that for severely diseased specimens was 0.4 MJ/mm3 (lower, but not significantly different, than energy to failure in the immediately proximal trabecular bone). This supports other findings22 that the medial condyle is more severely affected during arthritic degeneration than is the lateral condyle. We did not find any mechanical consequence of the higher values of connectivity in trabecular bone of lateral versus medial condyles that were detected in our other study.13
Significant correlations were detected between structural and mechanical properties of subchondral bone in the distopalmar aspect of MC3 condyles. These findings warrant further investigation and the development of new diagnostic imaging tools that could assist in identifying horses at risk of bone failure. Some overall correlations were detected between trabecular structural values; however, the strongest overall correlations were with BMD and bone volume fraction. This indicates that in the present study, although the microarchitectural characteristics played a role in determining the mechanical properties of subchondral bone in the distopalmar aspect of MC3,6,11,12 apparent BMD, true BMD, and bone volume fraction were better determinants overall. However, when correlations were assessed separately for each anatomic location, architectural characteristics and mechanical properties were correlated in specimens of trabecular bone in all ROIs and in the subchondral bone plate in the sagittal ridge. Other than the structural differences,5,13 results of mechanical testing provided further evidence that both layers were composed of different bone tissue and that trabecular bone in both condyles and trabecular bone and subchondral bone plate in the sagittal ridge had properties similar to cancellous bone. It is in cancellous bone where the architectural organization plays an important role in determining the mechanical properties of bone tissue.23 Yield strain did not correlate well with any structural variable, and this finding is consistent with other studies in which equine24 and human bone material24–26 were evaluated.
To our knowledge, no studies have been specifically designed to investigate the mechanical properties of subchondral bone of metacarpophalangeal joints in horses. Other researchers27 evaluated trabecular bone from the equine third carpal bone, which is also subject to repetitive high compressive loading and represents another typical location for subchondral bone disease in Thoroughbred racehorses.2 They also detected a characteristic regional pattern of SCB stiffness in healthy horses, which was not maintained in third carpal bone specimens with subchondral bone disease.27
Surprisingly, we did not detect any significant correlations between mechanical and structural properties for specimens of the subchondral bone plate from condyles. The reason for this is not clear. It is possible that accumulation of fatigue damage in subchondral bone plate in severely diseased bone, and perhaps even already present in mildly diseased bone, limited our ability to detect such correlations. Reduced sample sizes and heterogeneity and variability of specimen characteristics at each location may also have played a role. Although processes of machining and potting were carefully controlled, some variability may have occurred between specimens, particularly in specimens of subchondral bone plate that did not have distal flat surfaces. In humans, alterations in the trabecular pattern of subchondral bone have been described in osteoarthritic joints,28 with subsequent changes in the mechanical properties of the subchondral bone. Because the calcified cartilage layer tends to cleave in the sagittal plane and the marrow spaces in subchondral bone plates are oriented sagittally,5 we machined the bone specimens along the sagittal plane. Additional stereologic and anisotropic evaluation of the trabeculae at these locations may provide more information.
Caution is urged when considering the absolute values reported here. There is tremendous variation in the literature regarding material properties and anisotropy of bone,29 which are greatly dependent on the anatomic position, in vivo loading history, and functionality of bone. Our results were consistent with those reported for MC3 cancellous bone in horses,30 but higher than those reported for cancellous bone in other animal species (elastic modulus, < 1,000 Mpa; strength, < 50 MPa),29,31 including the subchondral bone of femoral condyles in horses.32 In our study, the specimens of subchondral bone plate partially contained bone tissue with a density similar to that of cortical bone, and the extreme loading conditions that subchondral bone at the palmar aspect of the condyles sustains differ from those sustained by cancellous bone at other locations, such as vertebrae. In fact, our results for mechanical properties approximate some of those reported for cortical bone samples.24,31
Our compression testing methodology simulated the compressive force that condylar subchondral bone sustains when the load is transmitted against the opposing proximal sesamoid bones.4,33,34 The results of this study are indicative of relative static values, which represent the maximal mechanical characteristics that the bone tissue specimens can withstand prior to fracture under a loading destructive test and at specific test conditions.35 Condylar fractures and fetlock subchondral bone failure are believed to be the result of a chronic repetitive insult of the subchondral bone and not the result of 1 traumatic event. Therefore, the methodology that we used did not resemble the pathophysiology of the subchondral bone failure and condylar fractures in racing Thoroughbreds. It revealed, however, the mechanical properties that the subchondral bone at that location possessed as a result of the adaptive or pathological process caused by repeated loading during racing and training.
Scraping of the articular cartilage may have caused some minor defects on the calcified cartilage layer; however, scraping was considered to be less harmful than removal of cartilage by chemical methods. We have been consistently using this methodology at our laboratory, and we believe it has minimal undesirable physical consequences. Interaction of the specimen with the compression platens influences the results of mechanical testing. The distal surface of the subchondral bone plate specimens matched the contour of the articular surface, which may have caused uneven distribution of load and stress and, consequently, underestimation of the mechanical results.36,37 This phenomenon was particularly important to avoid at the sagittal ridge. To lessen the effect, the specimens were embedded in acrylic copolymer. The distal surface of the specimen was then adhered to the lower platen and restricted to lateral expansion, which could have led to overestimation of some mechanical properties.36,38,39 On the other hand, the trabeculae of the upper surface of the specimen were unsupported and likely sustained higher strain, which may have artificially decreased the magnitude of some mechanical values.38,40 To validate comparisons, all samples were treated with identical methodology.
Our results indicated that micro-CT can be used for evaluation of morphology and microarchitecture and for estimation of material properties of subchondral bone in the palmarodistal aspect of MC3s in horses. This method can also be used to assess cortical samples of the equine MC3s24 and cortical samples from rat bones.41 Micro-CT is superior to other 2-dimensional techniques such as x-ray absorptiometry.42 Clinical CT can be used to provide good predictions of some mechanical properties of cancellous bone from pigs,43 but the inclusion of micro-CT measurements improves predictions of values for trabecular bone specimens from pigs43 and humans.44,45 Although the clinical application of micro-CT is limited, this technique offers great potential for further investigation and characterization of pathophysiologic processes such as subchondral bone disease in horses.
The number of specimens evaluated was limited, and there was no control over several factors such as age, previous work history, or degenerative lesions on the overlying articular cartilage; therefore, statistical analyses were limited. The classification of bones as mildly or severely diseased was based on micro-CT changes in the subchondral bone that we believed indicate the various stages of subchondral bone failure.20,46 Bones were classified according to the highest score assigned to a condyle; therefore, condyles of some bones classified as severely diseased had low micro-CT scores. No definitive categorization of the physiologic (adaptive) or pathologic (maladaptive) nature of some of the micro-CT changes can be made at this stage because we did not perform histologic evaluation of bone specimens; however, excellent agreement between results of micro-CT and histologic evaluations exists in other species.47,48
Results of the study reported here indicated that a gradient in mechanical properties exists across the width of the palmar aspect of MC3 condyles in Thoroughbred racehorses, with a steep gradient at the region of the condylar groove. This gradient was detected in bone specimens from MC3s of horses with mild subchondral bone disease and appeared to further extend in a proximal direction in specimens from horses with severe subchondral bone disease because a gradient was also detected in the trabecular bone located proximal to the subchondral bone plate. These findings give support to the supposition that strain accumulates at the condylar grooves, which may be responsible for development of condylar fractures. The mechanical properties of subchondral bone from the distal aspect of MC3s can be predicted to some extent by use of micro-CT. Additional studies with larger numbers of horses are needed to better understand the progression of subchondral bone disease and the predictive value of micro-CT. Additional studies are also necessary to develop new imaging techniques that can help us to diagnose and predict horses at risk and to develop adequate training and racing protocols to decrease the development of such catastrophic injuries in Thoroughbred racehorses.
ABBREVIATIONS
| BMD | Bone mineral density |
| CI | Confidence interval |
| CT | Computed tomography |
| MC3 | Third metacarpal bone |
| ROI | Region of interest |
eXplore Locus micro-CT scanner, GE Medical Systems, London, ON, Canada.
MicroView ABA, version 2.1.2, GE Healthcare, London, ON, Canada.
Electronic digital calliper, Marathon Watch Co, Richmond Hill, ON, Canada.
Technovit 6091 Easy, Heraeus Kulzer GmbH, Wehrheim, Germany.
Instron Materials Testing model 8872, Instron Inc, Burlington, ON, Canada.
Excel 2002, Microsoft Corp, Redmond, Wash.
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