Cranial cruciate ligament disease, which is clinically characterized by partial or complete rCCL, is a daunting orthopedic problem, with an estimated annual economic burden exceeding $1.3 billion for US pet owners.1 Despite medical and surgical interventions, dysfunction of the CCL commonly leads to initiation and progression of stifle joint osteoarthritis, which is a leading cause of disability in dogs. The pathogenesis of CCLD is not fully understood2; however, studies3,4 have revealed that most of the diagnosed ruptures of the CCL occur in ligaments with chronic degenerative changes. Various factors, including genetics, aging, body weight and body composition, developmental influences, stifle joint anatomy, and joint inflammation, have been implicated as contributors to CCL degeneration.2,3,5
Tensile strength of the CCL decreases with age, particularly in dogs weighing > 15 kg.3 Mechanical weakening of the CCL in aging dogs is associated with histologic changes of degeneration, including decreased ligamentocyte cellularity, chondroid metaplasia of ligamentocytes, mucoid-to-cartilaginous changes of the extracellular matrix, and disruption of the arrangement of collagen bundles.3–5 Cranial cruciate ligaments with degenerative changes are mechanically weak,3 which suggests that rCCL occurs in most dogs during normal loading conditions without supraphysiologic trauma.2,3,5,6
Vascular disturbance resulting in decreased vascular density with hypoxia has been mentioned as a potential contributing factor in CCL degeneration in dogs.3,7–9 However, investigators of 1 study8 reported that histomorphometric analysis revealed an increase in vascular density in the core region of rCCLs of dogs. The increase in vascular density in CCLs may have been associated with an attempted reparative response later during the course of the disease process. On the basis of these competing hypotheses and contradictory results, the role of the vascularity of the CCL core region in CCL degeneration and CCLD has not been clearly delineated and warrants further study.
Synovial inflammation has been associated with risk of rCCL.5,10,11 Hyperplastic changes and lymphoplasmacytic inflammation are often observed in synovium obtained from stifle joints with CCLD.12–15 Importantly, synovitis has been detected in stifle joints with an intact CCL that subsequently developed CCLD when assessed in dogs undergoing surgical intervention for CCLD in the contralateral stifle joint.10,11 Although histologic changes in synovium related to CCLD have been studied,10–15 the relationships between spontaneous CCL degeneration and synovitis are unclear.
Because of the economic and quality-of-life issues associated with CCLD, novel approaches for prevention and treatment of this pervasive problem are critically required and currently represent an unmet need in veterinary medicine. Such new approaches can be successful only if they effectively address mechanisms of disease. Therefore, the objective of the study reported here was to investigate the relationship between histologic degeneration of CCLs and severity of synovitis and ligament vascularity. Our hypothesis was that the degree of histologic degeneration in the CCL core region would be significantly related to the severity of synovitis and the vascularity of the CCL core region.
Materials and Methods
Sample
Specimens of CCL and synovium were obtained from intact stifle joints of juvenile and adult dogs during necropsy at the University of Missouri Veterinary Medical Diagnostic Laboratory as part of routine gross and histologic examinations. Exclusion criteria included gross evidence of partial or complete rupture of the CCL, meniscal injury, or gross evidence of osteoarthritis including cartilage defects, osteophytosis, or eburnation. The CCL specimens were collected by sharp dissection with a No. 11 scalpel blade; specimens included the entire ligament from origin to insertion. Synovium specimens were obtained by sharp dissection from the fat pad and suprapatellar to tibial insertion area of the medial side of the stifle joint.
Specimens of CCL and synovium also were obtained from stifle joints of dogs with partial or complete rCCL. Specimens were collected during surgical intervention performed with client consent to treat clinical dysfunction. The CCL specimens were collected by surgical resection and included all remaining ligament from origin to insertion. Synovium specimens were obtained by surgical biopsy from the fat pad or the medial side of the stifle joint (or both).
Specimens were routinely processed for histologic examination and stained with H&E stain. All tissues were assessed by a board-certified veterinary pathologist who was unaware of demographic, pathological, and clinical data. Tissues were assessed for degenerative changes of the CCL core region by use of a modified Bonar score16 that excluded assessment of vascularity. Variables comprising the modified Bonar score were cell morphology, extracellular matrix ground substance, and collagen arrangement, which were used to determine a score (scale, 0 to 9); vascularity of the CCL core region was excluded from the modified Bonar score because it was evaluated separately. Changes of the synovium indicative of synovitis were assessed by use of the Osteoarthritis Research Society International synovitis system (scale, 0 to 18).17
Unstained paraffin-embedded tissue sections (thickness, 5 μm) were prepared, and horseradish peroxidase-based immunohistochemical staining (CD31 antibodya; dilution, 1:40) for endothelial cells was performed to enable identification of vessels in the CCL core region. Vascularity was scored on a scale of 0 to 3 (0 = inconspicuous blood vessels, 1 = an occasional cluster of vessels [< 1 cluster of vessels/10 hpf], 2 = 1 or 2 clusters of vessels/10 hpf, and 3 = > 2 clusters of vessels/10 hpf).16 Phosphotungstic acid hematoxylin stain was used to specifically identify fibrin when vascular necrosis with fibrin thrombi were suspected on the basis of examination of H&E-stained CCL sections.
Statistical analysis
Statistical analyses were performed by use of a computer software program.b Data for each group (juveniles, adults, and dogs with rCCL) were combined, and mean ± SD was determined. To assess differences among groups but considering nonindependence of the samples (samples were obtained from > 1 stifle joint of some dogs), a linear model with a random subject effect was used to assess differences. This same structure was used to assess relationships between variables, adjusting for group and subject effects. When significant differences among groups were detected, post hoc analyses with the Sidak method were used to adjust for multiple comparisons. Significance was considered at values (2 sided) of P < 0.05.
Results
Tissue samples of CCL and synovium were obtained from 15 intact stifle joints of 12 juvenile dogs, 25 intact stifle joints of 22 adult dogs, and 19 stifle joints of 19 dogs with rCCL (Table 1). Juvenile dogs (mean ± SD age, 3.3 ± 1.6 months; range, 5 days to 5 months) comprised 8 females and 4 males consisting of 1 each of Australian Shepherd, Beagle, Bloodhound, English Bulldog, French Bulldog, Great Dane, Mastiff, Rottweiler, Saint Bernard, and Welsh Corgi; there were 2 mixed-breed dogs. Adult dogs (mean age, 9.1 ± 4.0 years; range, 2 to 16 years) comprised 12 females (11 spayed and 1 sexually intact) and 10 males (7 castrated and 3 sexually intact) consisting of 2 American Bulldogs, 2 Boston Terriers, and 1 each of Basset Hound, Chow Chow, Coonhound, Doberman Pinscher, French Bulldog, German Shepherd Dog, German Shorthaired Pointer, Golden Retriever, Great Dane, Great Pyrenees, Labrador Retriever, Miniature Schnauzer, Pomeranian, Rottweiler, Shar-Pei, and Welsh Corgi; there were 2 mixed-breed dogs. Dogs with rCCL (mean age, 8.1 ± 2.4 years; range, 3 to 12 years) comprised 8 females (7 spayed and 1 sexually intact) and 11 males (6 castrated and 5 sexually intact) consisting of 2 German Shepherd Dogs, 2 Toy Poodles, and 1 each of Boxer, French Bulldog, Golden Retriever, Labrador Retriever, Maltese, Rat Terrier, Rottweiler, Shiba, Siberian Husky, and Welsh Corgi; there were 5 mixed-breed dogs. Mean body weight was 9.7 ± 10.1 kg (range, 0.3 to 33.0 kg) for juvenile dogs, 24.6 ± 16.2 kg (range, 4.2 to 59.4 kg) for adult dogs, and 22.6 ± 14.9 kg (range, 1.5 to 52.0 kg) for dogs with rCCL. There were no significant differences in age (P = 0.313) or body weight (P = 0.672) between adult dogs and dogs with rCCL.
Characteristics of juvenile dogs with an intact CCL (15 joints in 12 dogs), adult dogs with an intact CCL (25 joints in 22 dogs), and dogs with rCCL (19 joints in 19 dogs).
| Group | Sex | Side of stifle joint | Age | Body weight (kg) | Synovitis score | Modified Bonar score | Vascularity score |
|---|---|---|---|---|---|---|---|
| Juvenile | F-8, M-4 | L-11, R-4 | 3.3 ± 1.6 mo (5 d to 5 mo) | 9.7 ± 10.1 (0.3–33.0) | 2.80 ± 0.66 (0–6) | 1.13 ± 0.55 (0–5) | 3 (NA) |
| Adult* | F-1, FS-11, M-3, MC-7 | L-21,R-4 | 9.1 ± 4.0 y (2 to 16 y) | 24.6 ± 16.2 (4.2–59.4) | 3.70 ± 0.81 (0–11) | 4.85 ± 0.44 (1–7) | 1.53 ± 0.27 (0–3) |
| rCCL† | F-1, FS-7, M-5, MC-6 | L-10, R-9 | 8.1 ± 2.4 y (3 to 12 y) | 22.6 ± 14.9 (1.5–52.0 kg) | 5.84 ± 0.68 (1–11) | 5.69 ± 0.49 (1–9) | 0.78 ± 0.23 (0–3) |
Values reported for sex and side of the stifle joint are counts and for other variables are mean ± SD (range). Synovitis score was on a scale of 0 to 18. Modified Bonar score was on a scale of 0 to 9. Vascularity score was on a scale of 0 to 3.
Includes 4 dogs with hematoidin deposits and 4 dogs with evidence of vascular necrosis.
Includes 2 dogs with hematoidin deposits.
F = Female, sexually intact. FS = Female, spayed. L = Left. M = Male, sexually intact. MC = Male, castrated. NA = Not applicable. R = Right.
Degenerative changes of the CCL were characterized by ligamentocytes with conspicuous cytoplasm and round nuclei, chondroid changes of ligamentocytes, mucoid or cartilaginous changes of the extracellular matrix ground substance, loss of demarcation of collagen bundles, separation of collagen bundles, and disorganization and loss of collagen bundles (Figure 1). These findings were similar to those reported for degeneration of the anterior cruciate ligament in humans and CCL degeneration in dogs.3,4,18,19 Modified Bonar scores were analyzed by use of a random effects model, which revealed that the mean ± SD values for dogs with rCCL (5.69 ± 0.49) and adult dogs (4.85 ± 0.44) were significantly (P = 0.003 and 0.006, respectively) higher than the mean for the juvenile dogs (1.13 ± 0.55); there was not a significant (P = 0.576) difference between means for dogs with rCCL and adult dogs.
Photomicrographs of sections of the CCL from representative juvenile (A through C) and adult (D through F) dogs with an intact CCL and dogs with rCCL (G through I). For the specimens from the juvenile dogs, there are ligamentocytes with elongated spindle-shaped nuclei, no mucoid changes in the extracellular matrix ground substance, and tightly, cohesively arranged dense collagen. Blood vessels are readily evident in the photomicrographs and insets. For the specimens from the adult dogs, there are degenerative changes characterized by ligamentocytes with conspicuous cytoplasm and round nuclei; ligamentocytes with chondroid changes; extracellular matrix ground substance with mucoid to cartilaginous changes; loss of demarcation of collagen bundles; and separation, disorganization, and loss of collagen bundles. Blood vessels are readily evident in panel D but indiscernible in panels E and F. For the specimens from dogs with rCCL, the degenerative changes are extremely similar to those seen for the CCLs of the adult dogs. There are no discernible blood vessels in panels G, H, or I. H&E stain for the photomicrographs and horseradish peroxidase-based CD31 immunohistochemical stain for the insets; bar in photomicrographs = 50 μm in A and 100 μm in B through I, and bar in insets = 50 μm in A and C, 100 μm in B and D, and 200 μm in E through I.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.152
Multiple clusters of capillaries were readily observed in the CCL core region in all specimens from juvenile dogs, whereas blood vessels were indiscernible in many specimens of adult dogs and dogs with rCCL (Figure 1; Table 1). Every juvenile CCL had a vascularity score of 3 (ie, > 2 clusters of vessels/10 hpf), whereas mean ± SD vascularity scores were significantly lower for the adult dogs (1.53 ± 0.27; range, 0 to 3; P = 0.019) and dogs with rCCL (0.78 ± 0.23; range, 0 to 3; P = 0.002). Vascularity scores did not differ significantly (P = 0.218) between adult dogs and dogs with rCCL. In addition to the typical degenerative changes that were evident, hematoidin deposits in the CCL core region were detected in 2 specimens from dogs with rCCL and 4 specimens from adult dogs (Figure 2). Vascular necrosis in the CCL core region, which was characterized by denuded endothelium with homogeneously pale eosinophilic vessel walls, was detected in 4 CCL specimens from adult dogs; 1 of these specimens also contained hematoidin deposits. Fibrin thrombi in necrotic vessels were suspected in 2 specimens from adult dogs, as determined by evaluation of tissues stained with phosphotungstic acid hematoxylin stain. Neither vascular necrosis nor fibrin thrombi were detected in specimens from juvenile dogs or dogs with rCCL.
Photomicrographs of representative CCLs obtained from adult dogs with an intact CCL (A, B, D, E, and F) and a dog with rCCL (C). Notice the bright yellow crystalline deposits consistent with hematoidin (A through C). Vascular necrosis characterized by denuded endothelium with homogeneously pale eosinophilic vessel walls (arrow) is evident (B, D, E, and F). Fibrin thrombi were suspected on the basis of examination of the photomicrographs and was supported by blue staining with phosphotungstic acid hematoxylin stain (inset of panels D and E). H&E stain for photomicrographs and phosphotungstic acid hematoxylin stain for insets; bar = 50 μm for photomicrographs and insets.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.152
Synovial hyperplasia characterized by stacked plump synoviocytes was commonly seen in CCLs from intact stifle joints regardless of the dog's age, whereas lymphoplasmacytic infiltrates with or without lymphoid aggregates were often detected in CCL specimens obtained from adult dogs regardless of their clinical status (Figure 3). Mean synovitis score for dogs with rCCL (5.84 ± 0.68) was higher than, but not significantly (P = 0.054) different from, the score for juvenile dogs (2.80 ± 0.66). Mean synovitis score for adult dogs (3.70 ± 0.81) was not significantly different from the mean values for the other 2 groups.
Photomicrographs of representative synovium obtained from a juvenile (A) and adult (B) dog with an intact CCL and a dog with rCCL (C). Synovial hyperplasia characterized by > 2 cell layers of plump synoviocytes is evident in the specimen from the juvenile dog. Synovial hyperplasia with frond-like projections, stacked plump synoviocytes, and lymphoplasmacytic infiltrates with or without lymphoid aggregates are evident in the specimens from the adult dog with an intact stifle joint and the dog with rCCL. H&E stain; bar = 100 μm.
Citation: American Journal of Veterinary Research 80, 2; 10.2460/ajvr.80.2.152
Analysis with a random effects model between the juvenile and adult dogs and between the adult dogs and dogs with rCCL was used to identify variables that were significantly related with CCL degeneration and rupture of the CCL. There was a significant (P = 0.018) negative relationship between modified Bonar scores and vascularity scores for the juvenile and adult dogs; however, comparison between the modified Bonar scores and synovitis scores did not reveal a significant (P = 0.087) relationship.
Analysis with a random effects model between the adult dogs and dogs with rCCL revealed that there was a significant (P = 0.037) positive relationship between the modified Bonar scores and body weight, whereas there was not a significant relationship between the modified Bonar scores and vascularity scores or between the modified Bonar scores and synovitis scores. However, on the basis of results from comparisons between the juvenile and adult dogs, these relationships may have been influenced by aging or other natural processes. After controlling for age of the dogs, there was a significant (P = 0.043) negative relationship between the modified Bonar scores and vascularity scores, although the relationship between the modified Bonar scores and synovitis scores was not significant (P = 0.211).
Discussion
Results of the study reported here supported a portion of our overall hypothesis. Histologic degeneration in the CCL was related to a decrease in vascularity in the CCL core region; however, no significant relationship was detected between histologic degeneration and severity of synovitis. Degeneration of the CCL in adult dogs was also related to a higher body weight. Considered together, the data suggested that decreased vascularity in the CCL core region during aging in at-risk dogs could have been a key underlying mechanism for spontaneous degeneration of the ligament and that CCL degeneration among adult dogs was influenced by body weight, whereas synovitis appeared to have a lesser role in initiating CCL degeneration in dogs.
Although histologic assessment of CCL vascularity is subjective,3 the grading criteria of vascularity in the original Bonar system is straightforward and repeatable and can provide a practical overview of histologic vascularity. Results of the present study reaffirmed that the CCL core region in juvenile dogs was a vascular-rich area and that the core region developed a paucity of vascularity in adult dogs, often with inconspicuous blood vessels, regardless of breed or size. Vascularity is reduced in the central core relative to other regions of the CCL.3,7,20 It has been suggested that biomechanical stress produced by twisting of the 2 functional components (bands) of the CCL reduces blood flow to the central region, which is the location where most ruptures occur.3,21 Thus, inadequate blood supply has been implicated as a potential contributing cause of CCL degeneration and rupture in dogs.3,7,8 However, contrary to this hypothesis, an increase in vascular density in the core region of rCCLs of dogs was detected in 1 study.8 Because only rCCLs were examined in that study,8 it is unknown whether the increased vascular density was contributory to CCLD or a manifestation of the condition.
Responses of the human anterior cruciate ligament after rupture have been characterized22 and include inflammation followed by epiligamentous regeneration, revascularization of the ligament remnant, and remodeling and maturation of the anterior cruciate ligament. Investigators of 1 study16 reported that neovascularization is not a primary change in asymptomatic tendinosis in humans; rather, it develops after other histopathologic changes, including alterations of tenocyte morphology and extracellular matrix ground substance and arrangement of collagen.16 Considered together with the results of the present study, the increased vascular density in rCCL specimens reported previously likely was a reflection of a reparative response rather than a causative contributor to CCL degeneration and rupture. Differences in results among studies may be attributable to timing of the assessment. The elapsed time between initiation of CCLD, ligament rapture, and surgery cannot be determined for the study reported here or a previous study8; however, we speculate that the elapsed time may have been shorter in the present study on the basis of the primary care settings at which surgical biopsy specimens were obtained. In addition, the inclusion of juvenile and adult control groups in the present study provided cross-sectional data regarding temporal progression of ligament changes in the absence of CCLD or before rCCL.
The present study indicated that decreased vascularity in the CCL core region was significantly related with CCL degeneration characterized by cellular morphological changes of ligamentocytes, mucoid to cartilaginous changes of extracellular matrix ground substance, disorganization of collagen bundles, and separation and loss of collagen bundles. These findings suggested that decreased vascularity and the associated inadequate blood supply in the CCL core region may have been an important change that could have directly contributed to CCL degeneration and risk for CCLD.
Hematoidin deposits were detected in 2 rCCL specimens as well as in the core region of CCLs of at least 4 adult dogs with intact CCLs in the present study. Hematoidin is a hematogenous pigment derived from erythrocytes and is deposited in tissues at sites of hemorrhage.23 Although the underlying pathophysiology of a paucity of vascularity in the CCL core region of adult dogs cannot be determined from the present study, the hematoidin deposits and vascular necrosis with fibrin thrombi suspected in some intact CCLs of adult dogs provided evidence of a hemorrhagic incident and vascular disturbance in the CCL core region for at least a subset of at-risk dogs. We cannot determine whether these findings were associated with age-related physiologic vascular involution or vascular disorders or with local trauma. Nevertheless, these degenerative changes associated with a paucity of vascularity in the CCL core region microstructure would certainly make ligaments susceptible to disruption and dysfunction even from physiologic loading. This hypothesis fits the clinical picture well because CCLD in dogs is typically a chronic degenerative process with a culminating event that prompts recognition and diagnosis.2 It also supports the current assumptions that genetic and epigenetic influences contribute to the development of CCLD in dogs.2,3,5,6
Although rupture of the CCL subsequent to synovitis has been confirmed in dogs undergoing surgical intervention for CCLD in the contralateral stifle joint,10,11 severity of synovitis was not an important contributing factor associated with CCL degeneration in the present study. Although mild to moderate synovitis was often observed in specimens obtained from intact stifle joints in the study reported here, we suspect that these changes were related to factors apart from the initiation of CCLD because of a lack of a significant relationship with degenerative changes in the ligament. Synovitis has been found to be an early feature of CCLD and has been implicated as a contributing factor for CCLD10–15; however, a causal relationship between synovitis and CCL degeneration has not been definitively proven. Although it is possible that chronic synovitis could play an initiating role in CCLD in a subset of canine patients, synovial inflammatory changes detected in stifle joints with CCLD could be a manifestation of the disease processes rather than a contributing factor, as has been suggested for some studies.2,13,14,24
Limitations of the present study included the population of dogs included in the affected and control groups, the inability to assess material properties of CCLs included in the study, and the cross-sectional and subjective nature of the assessments. However, the results reaffirmed that the core region of the CCL has a paucity of vascularity in adult dogs and indicated that decreased vascularity was significantly related to degeneration in the CCL core region. This evidence suggested that a poor blood supply to the CCL core region with resultant hypoxia could be an important contributor to CCL degeneration and subsequent CCLD in dogs, whereas synovitis was not intimately related to initiation of CCL degeneration. Results of a similar histopathologic investigation of specific breeds known to have a high incidence of CCLD (eg, Labrador Retrievers and Rottweilers)25,26 or to have lower odds of developing CCLD (eg, Greyhounds)25,26 could be used to extend the findings reported here and solidify the association between CCL degeneration and vascularity. Although investigators of 1 study27 found a higher prevalence of fibrocartilaginous changes in CCLs of adult Greyhounds (7/8 dogs) than in adult Labrador Retrievers (3/8 dogs), degenerative changes (when detected) were milder in the Greyhounds than in the Labrador Retrievers. However, degenerative changes in these 2 breeds were not investigated with respect to CCL vascularity, hemorrhagic changes (ie, hematoidin), or vasculopathy in that study.27 Further delineation of the mechanisms for decreased vascularity in the CCL core region may shed light on development of novel preventative and therapeutic strategies for CCLD in dogs.
Acknowledgments
The authors thank the Veterinary Medical Diagnostic Laboratory at the University of Missouri and Idexx Japan for contributing histology slides.
ABBREVIATIONS
| CCL | Cranial cruciate ligament |
| CCLD | Cranial cruciate ligament disease |
| rCCL | Cranial cruciate ligament rupture |
References
1. Wilke VL, Robinson DA, Evans RB, et al. Estimate of the annual economic impact of treatment of cranial cruciate ligament injury in dogs in the United States. J Am Vet Med Assoc 2005;227:1604–1607.
2. Cook JL. Cranial cruciate ligament disease in dogs: biology versus biomechanics. Vet Surg 2010;39:270–277.
3. Vasseur PB, Pool RR, Arnoczky SP, et al. Correlative biomechanical and histologic study of the cranial cruciate ligament in dogs. Am J Vet Res 1985;46:1842–1854.
4. Hayashi K, Frank JD, Dubinsky C, et al. Histologic changes in ruptured canine cranial cruciate ligament. Vet Surg 2003;32:269–277.
5. Hayashi K, Manley PA, Muir P. Cranial cruciate ligament pathophysiology in dogs with cruciate disease: a review. J Am Anim Hosp Assoc 2004;40:385–390.
6. de Rooster H, de Bruin T, van Bree H. Morphologic and functional features of the canine cruciate ligaments. Vet Surg 2006;35:769–780.
7. Tirgari M. The surgical significance of the blood supply of the canine stifle joint. J Small Anim Pract 1978;19:451–462.
8. Hayashi K, Bhandal J, Rodriguez CO Jr, et al. Vascular distribution in ruptured canine cranial cruciate ligament. Vet Surg 2011;40:198–203.
9. Hayashi K, Bhandal J, Kim SY, et al. Immunohistochemical and histomorphometric evaluation of vascular distribution in intact canine cranial cruciate ligament. Vet Surg 2011;40:192–197.
10. Muir P, Schwartz Z, Malek S, et al. Contralateral cruciate survival in dogs with unilateral non-contact cranial cruciate ligament rupture. PLoS One 2011;6:e25331.
11. Bleedorn JA, Greuel EN, Manley PA, et al. Synovitis in dogs with stable stifle joints and incipient cranial cruciate ligament rupture: a cross-sectional study. Vet Surg 2011;40:531–543.
12. Galloway RH, Lester SJ. Histopathological evaluation of canine stifle joint synovial membrane collected at the time of repair of cranial cruciate ligament rupture. J Am Anim Hosp Assoc 1995;31:289–294.
13. Kuroki K, Stoker AM, Sims HJ, et al. Expression of Toll-like receptors 2 and 4 in stifle joint synovial tissues of dogs with or without osteoarthritis. Am J Vet Res 2010;71:750–754.
14. Erne JB, Goring RL, Kennedy FA, et al. Prevalence of lymphoplasmacytic synovitis in dogs with naturally occurring cranial cruciate ligament rupture. J Am Vet Med Assoc 2009;235:386–390.
15. Lemburg AK, Meyer-Lindenberg A, Hewicker-Trautwein M. Immunohistochemical characterization of inflammatory cell populations and adhesion molecule expression in synovial membranes from dogs with spontaneous cranial cruciate ligament rupture. Vet Immunol Immunopathol 2004;97:231–240.
16. Cook JL, Feller JA, Bonar SF, et al. Abnormal tenocyte morphology is more prevalent than collagen disruption in asymptomatic athletes’ patellar tendons. J Orthop Res 2004;22:334–338.
17. Cook JL, Kuroki K, Visco D, et al. The OARSI histopathology initiative—recommendations for histological assessments of osteoarthritis in the dog. Osteoarthritis Cartilage 2010;18(suppl 3):S66–S79.
18. Hasegawa A, Otsuki S, Pauli C, et al. Anterior cruciate ligament changes in the human knee joint in aging and osteoarthritis. Arthritis Rheum 2012;64:696–704.
19. Hasegawa A, Nakahara H, Kinoshita M, et al. Cellular and extracellular matrix changes in anterior cruciate ligaments during human knee aging and osteoarthritis. Arthritis Res Ther 2013;15:R29.
20. Arnoczky SP, Rubin RM, Marshall JL. Microvasculature of the cruciate ligaments and its response to injury. An experimental study in dogs. J Bone Joint Surg Am 1979;61:1221–1229.
21. Alm A, Ekstrom H, Stromberg B. Tensile strength of the anterior cruciate ligament in the dog. Acta Chir Scand Suppl 1974;445:15–23.
22. Murray MM, Martin SD, Martin TL, et al. Histological changes in the human anterior cruciate ligament after rupture. J Bone Joint Surg Am 2000;82-A:1387–1397.
23. Miller MA, Zachary JF. Mechanisms and morphology of cellular injury, adaptation and death. In: Zachary JF, ed. Pathologic basis of veterinary disease. 6th ed. St Louis: Elsevier, 2017;2–43.
24. Garner BC, Stoker AM, Kuroki K, et al. Using animal models in osteoarthritis biomarker research. J Knee Surg 2011;24:251–264.
25. Whitehair JG, Vasseur PB, Willits NH. Epidemiology of cranial cruciate ligament rupture in dogs. J Am Vet Med Assoc 1993; 203:1016–1019.
26. Witsberger TH, Villamil JA, Schultz LG, et al. Prevalence of and risk factors for hip dysplasia and cranial cruciate ligament deficiency in dogs. J Am Vet Med Assoc 2008;232:1818–1824.
27. Comerford EJ, Tarlton JF, Wales A, et al. Ultrastructural differences in cranial cruciate ligaments from dogs of two breeds with a differing predisposition to ligament degeneration and rupture. J Comp Pathol 2006;134:8–16.
