Osteoarthritis is the most prevalent sequela of developmental and acquired joint disease. Although studies have focused on many aspects of osteoarthritis, the specific pathogenesis is not fully understood. Osteoarthritis involves an inflammatory process that is potentiated by specific cytokines and enzymes and affects both bone and cartilage.1–3 Ultimately, the inflammatory process results in a degenerative sequence that includes malacia, fibrillation and clefting, and erosion of the articular cartilage; thickening of the subchondral bone; and outgrowth of osteophytes, leading to signs of pain and suboptimal joint function.4 Mechanical changes in the joint can result in osteoarthritis5; results of several studies6–10 indicate that induction of joint instability via cruciate ligament transection in dogs results in progressive articular cartilage degeneration. Blunt impact trauma can lead to cartilage thickening or chondrocyte proliferation and articular cartilage degeneration.5,11 Thickening of the cartilage and the subchondral bone also develops in osteoarthritis. Whereas thickening of the AC is a characteristic of early stages of osteoarthritis, thinning of the AC is often associated with later or end-stage joint disease.12,13 A thick subchondral plate has traditionally been associated with end-stage disease; however, there is evidence that subchondral bone thinning may be associated with aging and osteoarthritis.14 A quantitative comparison of AC, ZCC, and subchondral bone plate thickness among joints considered normal, joints with early osteoarthritis, and joints with end-stage osteoarthritis has not been performed to our knowledge. The purpose of the study reported here was to quantify and compare the microscopic changes in the AC, ZCC, and subchondral bone plate in femoral heads from clinically normal dogs and dogs with moderate and severe osteoarthritis. We hypothesized that there would be a difference in thickness in the AC, ZCC, and subchondral bone plate in femoral heads from clinically normal dogs, compared with dogs with osteoarthritis. Our goal was to gain further insight into the interactions among the AC, ZCC, and subchondral bone plate and their role in the progression of osteoarthritis.
Materials and Methods
Bone and cartilage specimen preparation—Femoral heads were collected from 54 dogs and allocated into 3 categories (normal, moderate osteoarthritis, and severe osteoarthritis). Samples considered normal (n = 16) were collected from dogs undergoing femoral head and neck excision in an unrelated study. Clinically normal dogs did not have signs of hip joint discomfort, radiographic evidence of osteoarthritic changes, or any macroscopic changes in the articular cartilage surface and histologically were graded as normal by use of a modified Mankin scale (scores ≥ 16 of a possible 17 points).13,15 Clinically normal dogs weighed from 27 to 37 kg and ranged in age from 1.5 to 4 years old. The remaining samples were collected from clinical cases of osteoarthritis believed to be secondary to hip dysplasia and treated with total hip arthroplasty or femoral head and neck excision. Few dogs had obvious subluxation and moderate osteoarthritis, whereas other dogs had severe osteoarthritis with or without obvious subluxation. Samples from dogs with osteoarthritis were determined to be moderate or severe on the basis of clinical signs in dogs; subjective preoperative radiographic findings of the extended hip joint (by use of ventrodorsal radiographic views) and pelvis, such as degree of osteophytosis and degree of thickening in the femoral neck; macroscopic findings; and, ultimately, histologic grade according to a modified Mankin scale.12,16 Moderate osteoarthritis was defined as having a middle-range score for each of the categories of cartilage structure, cellularity, intensity of staining, and calcified zone integrity and a total score ≤ 11 of a possible 17 points; severe osteoarthritis was defined as having a low-range score for each of the categories of cartilage structure, cellularity, intensity of staining, and calcified zone integrity, and a total score ≤ 8 of a possible 17 points.12,13,15–17 Weight and age of dogs from which femoral heads with moderate osteoarthritis (n = 24) were obtained ranged from 12 to 39 kg and 6 months to 7 years, respectively. Weight and age of dogs from which femoral heads with severe osteoarthritis (n = 14) were obtained ranged from 25 to 55 kg and 9 months to 10 years, respectively. Samples of normal and severe osteoarthritis had been used in studies16,17 evaluating the AC via a modified Mankin scale and trabecular bone cancellous architecture in the femoral head via histomorphometry. Samples of moderate osteoarthritis had been used in a study15 evaluating histomorphometric changes in trabecular bone at the epiphyseal and metaphyseal portion of the femoral head. The study reported here focused on changes in thickness of the AC, ZCC, and subchondral bone plate, whereas other studies focused on trabecular bone patterns.
After surgical removal, femoral heads and necks were placed in neutral-buffered 10% formalin for fixation. Femoral heads were cut in either a coronal or transverse plane as indicated: normal coronal (n = 9), normal transverse (7), moderate osteoarthritis coronal (8), moderate osteoarthritis transverse (16), severe osteoarthritis coronal (8), and severe osteoarthritis transverse (6; Figures 1 and 2). Samples were grouped for analysis according to their modified Mankin scores and not according to their radiographic or gross grade. Three equally spaced 2-mm sections were cut from normal femoral heads and those with severe osteoarthritis with a precision saw.a One to 3 samples in corresponding planes were cut from femoral heads with moderate osteoarthritis. Coronal samples were designated cranial, middle, and caudal, whereas transverse samples were designated dorsal, middle, and ventral. Samples were decalcified in citric-buffered formic acid. After paraffin embedding, 6-μm sections were obtained and stained with safranin O stain and fast green counterstain. Each section was divided into 4 approximately equal regions, and the orientations of each section were recorded. All abnormal (osteoarthritis) samples were obtained from dogs with clinical disease.
Illustration of coronal sectioning of the left femoral head from a clinically normal dog. A—Proximal to distal view. B— Medial to lateral view. C—Cranial to caudal view of specimen slice showing regions (1 to 4) and measurement points (1 to 12).
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Illustration of coronal sectioning of the left femoral head from a clinically normal dog. A—Proximal to distal view. B— Medial to lateral view. C—Cranial to caudal view of specimen slice showing regions (1 to 4) and measurement points (1 to 12).
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Illustration of coronal sectioning of the left femoral head from a clinically normal dog. A—Proximal to distal view. B— Medial to lateral view. C—Cranial to caudal view of specimen slice showing regions (1 to 4) and measurement points (1 to 12).
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Illustration of transverse sectioning of the left femoral head from a clinically normal dog. A—Proximal to distal view. B— Cranial to caudal view. C—Proximal to distal view of specimen slice showing regions (1 to 4) and measurement points (1 to 12).
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Illustration of transverse sectioning of the left femoral head from a clinically normal dog. A—Proximal to distal view. B— Cranial to caudal view. C—Proximal to distal view of specimen slice showing regions (1 to 4) and measurement points (1 to 12).
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Illustration of transverse sectioning of the left femoral head from a clinically normal dog. A—Proximal to distal view. B— Cranial to caudal view. C—Proximal to distal view of specimen slice showing regions (1 to 4) and measurement points (1 to 12).
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Histomorphometric analysis of AC—Each specimen was viewed on a stereoscopic microscope,b digitally capturedc at 6.6X magnification, and analyzed by use of computer software.d The AC thickness was recorded at 12 successive locations. Measurements that fell in the region of the fovea (attachment site for the ligament of the head of the femur) were not used in the analysis because of the lack of definition between the subchondral bone plate, ZCC, and AC.
Histomorphometric analysis of ZCC—Specimens from 36 dogs were chosen for analysis of the ZCC. Regions 1 and 3 were analyzed because of the variability of the integrity detected in the ZCC in regions 2 (fovea region) and 4 (junction of cartilage and bone). Twelve specimens from each group, 6 in the coronal plane and 6 in the transverse plane, were analyzed. The ZCC was viewed with light microscopy,e digitally capturedc at 40X magnification, and analyzed.d Two thickness measurements of the ZCC were recorded from each magnified field of view.
Histomorphometric analysis of the subchondral bone plate—Images of the subchondral bone plate were captured by use of both 6.6X magnification (all specimens) and 40X magnification (zones 1 and 3 of 36 specimens).b,c,e The thickness of the subchondral bone plate was analyzedd with 40X magnification.
Definition of terms—A section is 1 slice in the coronal or transverse plane (cranial, middle, caudal, dorsal, middle, or ventral). A measurement point was defined as 1 of 12 successive locations chosen for measurement at approximately equal distance from the femoral head. Region was defined as 1 of 4 quadrants from the femoral head. Region 1 contained measurement points 1, 2, and 3; region 2 contained measurement points 4, 5, and 6; region 3 contained measurement points 7, 8, and 9; and region 4 contained measurement points 10, 11, and 12 (Figures 1 and 2).
Statistical analysis—An ANOVA, using a splitplot model, was used to evaluate the effect of the following parameters: slice orientation (coronal and transverse), slice location (cranial, middle, and caudal for coronal sections; dorsal, samples considered normal in the coronal plane in regions 1, 2, and 4 and in the transverse plane in all regions (Figure 3). A significant increase in thickness was observed in samples with severe osteoarthritis, compared with the normal samples in the coronal plane in region 1 and in the transverse plane in regions 1 and 4. A significant increase in thickness was observed in normal samples, compared with samples with severe osteoarthritis in region 3 of the middle section in both the coronal and transverse planes. Samples with moderate osteoarthritis were significantly thicker than samples with severe osteoarthritis in the coronal plane; in regions 1, 2, and 4 of the caudal sections; and in the transverse plane in most regions of most sections.
Comparisons of mean AC thickness in femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) osteoarthritis (OA) at each specific slice and region in the coronal (A) and transverse (B) planes. a-cFor each cluster of bars, different letters indicate significant (P < 0.05) differences among groups. Error bars represent SE. N/S = Not significant.
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Comparisons of mean AC thickness in femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) osteoarthritis (OA) at each specific slice and region in the coronal (A) and transverse (B) planes. a-cFor each cluster of bars, different letters indicate significant (P < 0.05) differences among groups. Error bars represent SE. N/S = Not significant.
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Comparisons of mean AC thickness in femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) osteoarthritis (OA) at each specific slice and region in the coronal (A) and transverse (B) planes. a-cFor each cluster of bars, different letters indicate significant (P < 0.05) differences among groups. Error bars represent SE. N/S = Not significant.
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
The effect of slice location was significant when AC thickness was compared among groups. In samples with moderate osteoarthritis in the coronal plane, the AC was thicker in the caudal sections, compared with the cranial and middle sections. In all groups in the transverse plane, the AC was thicker in the dorsal sections, compared with the middle and ventral sections.
There was a significant (P < 0.001) difference between mean ZCC thickness for samples with moderate and severe osteoarthritis, compared with normal samples. Mean ZCC thickness was significantly (P < 0.001) greater in samples with moderate (0.22 ± 0.009 mm) and severe osteoarthritis (0.21 ± 0.008 mm), compared with samples considered normal (0.16 ± 0.003 mm).
A significant increase in the ZCC thickness was observed in samples with moderate and severe osteoarthritis, compared with normal samples in the coronal and transverse planes in regions 1 and 3 of many sections (Figure 4). Slice orientation had a significant effect on thickness of the ZCC. In normal samples in the coronal plane, the ZCC in the cranial sections was significantly thicker than the middle and caudal sections, and in the transverse plane, the ZCC of the dorsal and ventral sections was significantly thicker than the middle section. In samples with moderate osteoarthritis in the transverse plane, the ZCC was significantly thicker in the dorsal sections, compared with the middle and ventral sections. In samples with severe osteoarthritis in the coronal plane, the ZCC in the caudal sections was significantly thicker, compared with the cranial and middle sections.
Comparisons of mean ZCC thickness in femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) osteoarthritis at each specific slice in regions 1 and 3 in the coronal (A) and transverse (B) planes. SeeFigure 3 for key.
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Comparisons of mean ZCC thickness in femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) osteoarthritis at each specific slice in regions 1 and 3 in the coronal (A) and transverse (B) planes. SeeFigure 3 for key.
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Comparisons of mean ZCC thickness in femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) osteoarthritis at each specific slice in regions 1 and 3 in the coronal (A) and transverse (B) planes. SeeFigure 3 for key.
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
There was a significant (P < 0.001) difference in mean subchondral bone plate thickness among all groups. The mean thickness of the subchondral bone middle, and ventral for transverse sections), region (1 to 4), measurement point (1 to 12), and group (normal, moderate osteoarthritis, or severe osteoarthritis) on each variable (thickness of AC, ZCC, and subchondral bone plate). A 1-way ANOVA was used to evaluate the effect of the region and the measurement point on each variable. When results of the ANOVA indicated significant differences within any level of the analysis, a Duncan multiple range test was performed. Values of P < 0.05 were considered significant. Results were compared with the normal group unless noted. All data in the results section are reported as mean ± SE.
Results
There was a significant (P < 0.001) difference in mean AC thickness among all groups. Thickness of the AC was significantly (P < 0.001) greater in samples with moderate (1.17 ± 0.03 mm) and severe (0.91 ± 0.04 mm) osteoarthritis, compared with samples considered normal (0.67 ± 0.01 mm; Figure 3). Thickness of the AC in samples with moderate osteoarthritis was significantly (P < 0.001) greater than that in samples with severe osteoarthritis.
A significant increase in thickness was observed in samples with moderate osteoarthritis, compared with plate in samples with severe osteoarthritis (0.67 ± 0.031 mm) was significantly (P < 0.001) greater than that in samples with moderate osteoarthritis (0.36 ± 0.023 mm) and normal samples (0.51 ± 0.019 mm). A significant decrease in the subchondral bone plate thickness was observed in samples with moderate and severe osteoarthritis in the coronal plane in region 1, compared with that same region in normal samples, whereas a significant increase in the subchondral bone plate thickness was observed in samples with severe osteoarthritis in region 3, compared with all sections in normal samples.
In the transverse plane in region 1, the subchondral bone plate thickness was significantly decreased in the middle section of samples with moderate and severe osteoarthritis, compared with normal samples. In region 3, the subchondral bone plate thickness in samples with severe osteoarthritis was greater than that in normal samples and samples with moderate osteoarthritis in only the ventral section (Figure 5).
Comparison of mean subchondral bone plate thickness in femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) OA at each specific slice and region in the coronal (A) and transverse (B) planes. SeeFigure 3 for key.
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Comparison of mean subchondral bone plate thickness in femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) OA at each specific slice and region in the coronal (A) and transverse (B) planes. SeeFigure 3 for key.
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
Comparison of mean subchondral bone plate thickness in femoral heads from clinically normal dogs (n = 16) and dogs with moderate (24) or severe (14) OA at each specific slice and region in the coronal (A) and transverse (B) planes. SeeFigure 3 for key.
Citation: American Journal of Veterinary Research 67, 10; 10.2460/ajvr.67.10.1719
There was a significant effect of slice location on subchondral bone plate thickness. In normal samples in the coronal plane, the caudal sections were significantly thicker than the cranial and middle sections, and in the transverse plane, the dorsal and middle sections were significantly thicker than the ventral sections. No significant effect of slice location on subchondral plate thickness was detected in samples with osteoarthritis.
Discussion
In other studies,12,13,16 health of the AC has been graded according to a scale that characterizes cartilage structure, cellularity, matrix staining, and calcified cartilage integrity but not cartilage thickness. It is generally accepted that the AC becomes thinner in the presence of progressive osteoarthritis, gradually wearing away to expose the subchondral tissues.4,17–19 One of the initial stages of osteoarthritis is swelling of the AC, which results in fibrillation, clefting, and erosion of the matrix.4,12,13,19 It is feasible that this swelling and subsequent increased thickness of the AC is maintained throughout the process of osteoarthritis and only at the end stage of the disease does the cartilage wear away to reveal subchondral tissues. In the study reported here, the AC of femoral heads with osteoarthritis remained thicker in most regions than that in femoral heads considered normal. This thickness was maintained despite major pathologic lesions in the AC detected in another study.17 In our study, the proximomedial region of the femoral head in samples with severe osteoarthritis had thinner AC, which is consistent with findings in 1 study17 in which the greatest cartilage degeneration was detected in dogs with severe osteoarthritis of the femoral head. The proximomedial region also bears most of the forces acting on the femoral head, as reported in other studies.20,21 Normal wear of this area would also contribute to thinning of the AC. Even in the face of increased thickness of the AC, it is unlikely that load transmission through the AC would be normal. The pathologic changes in the AC would be of sufficient magnitude to influence mechanical load transmission.5
Changes in the ZCC, including vascular invasion and advancement into the noncalcified AC, have been associated with osteoarthritis.5,18,22,23
An increase in thickness of the ZCC was detected in samples with moderate and severe osteoarthritis in several areas but most notably in the cranial and proximolateral aspects of the femoral head. Thickening of the calcified cartilage was not accompanied by a concomitant thinning of the AC, as may be anticipated with calcified cartilage advancement. Thickening of the calcified cartilage would invariably develop by calcification of the overlying AC. The only way that the AC could maintain or increase its thickness would be to swell or add matrix at its surface. In the presence of subluxation and accelerated wear associated with hip dysplasia, it is unlikely that cartilage could be added at the surface in any substantial amount.
Thickening of the subchondral bone plate has been observed in osteoarthritis of the femoral heads of dogs.15,17 It has been loosely associated with changes in the subchondral trabecular bone and is thought to be an adaptive response caused by changes in loading of the joint subsequent to cartilage degeneration.15 Changes in the subchondral bone plate detected in our study corresponded to the area in which changes in trabecular bone alignment were detected in another study17 and the area of decreased thickness of the AC in our study. This supports the notion that the subchondral bone plate increases in thickness in association with a thinning of the AC. The concomitant changes in the trabecular bone of the femoral head and neck are further evidence of the adaptive response associated with the loss of AC.15,17 Thickening of the subchondral bone plate in response to alterations in load transmission may be a gradual change and may not develop until severe or end-stage osteoarthritis.
As with any microscopic quantitative study, there was some ambiguity in the samples regarding consistency in locating regions. This was especially true in samples with osteoarthritis in which peripheral osteophytes may extend the area of the femoral head. An attempt was made to standardize this by dividing each femoral head into 12 measurement points with 3 consecutive points making up a region. Peripheral points of measurement (points 1 and 12) were offset by the adjacent points in the region, and a mean and SE were recorded for each region. In our study, weight and age of dogs were not considered in the data analysis. Age of dogs may have had an effect on the severity of osteoarthritis, although all cartilage was graded as normal or as having moderate or severe osteoarthritis by use of a modified Mankin scale.17 However, it is possible that dogs of various ages may have had different responses to osteoarthritis in the calcified zone and subchondral bone plate. Causes of osteoarthritis in samples in the moderate and severe groups were not known, although in all dogs, hip dysplasia was strongly suspected, as determined by clinical history and radiographic findings. Results of 1 study24 indicate that dogs with osteoarthritis have altered load distribution to the femoral head. In dogs with luxation or subluxation of the hip joint, an abnormally high shear force is generated in the proximomedial region of the femoral head.24 Animals with luxation or subluxation of the hip joint would be predisposed to cartilage wear in the proximomedial region of the femoral head because of the increased forces present in that area. Although we did not take into account the severity of subluxation, all abnormal samples (samples with moderate and severe osteoarthritis) were obtained from dogs that underwent surgical treatment for hip dysplasia and secondary degenerative changes of the hip joint.
We were not able to distinguish a cause and effect association between thickening of the subchondral structures and thinning and loss of the overlying AC. We believe that this was caused by persistent swelling of the AC and variability in response of the calcified cartilage and subchondral bone plate. Perhaps greater numbers of samples would be necessary to detect a more global response in the femoral head. Regardless, we were able to detect changes in AC thickness, which were in agreement with results of other studies15,17 in which pathologic lesions of the AC and histomorphometric changes of trabecular bone were recorded. These changes were associated with changes in the subchondral bone plate, and this is consistent with the theory of adaptation in response to altered load distribution.
ABBREVIATIONS
| AC | Articular cartilage |
| ZCC | Zone of calcified cartilage |
Model 11-2480 Isomet, Buehler Ltd, Lake Bluff, Ill.
Model SMZ-10, Nikon Instruments Inc, Melville, NY
Model DXC-390, Sony Corp, Tokyo, Japan
Image J, National Institutes of Health, Bethesda, Md. Available at: rsb.info.nih.gov/ij/. Accessed Jun 15, 2002
Model Eclipse 6600, Nikon Instruments Inc, Melville, NY.
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