Inflammatory changes in ruptured canine cranial and human anterior cruciate ligaments

OBJECTIVE: To compare expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K and histologic changes in canine cranial cruciate ligaments (CCLs) and human anterior cruciate ligaments (ACLs). STUDY POPULATION: Sections of cruciate ligaments from 15 dogs with ruptured CCLs, 8 aged dogs with intact CCLs, 14 human beings with ruptured ACLs, and 11 aged human beings with intact ACLs. PROCEDURE: The CCLs and ACLs were evaluated histologically, and cells containing TRAP and cathepsin K were identified histochemically and immunohistochemically, respectively. RESULTS: The proportion of ruptured CCLs that contained TRAP+ cells was significantly higher than the proportion of intact ACLs that did but similar to proportions of intact CCLs and ruptured ACLs that did. The proportion of ruptured CCLs that contained cathepsin K+ cells was significantly increased, compared with all other groups. Numbers of TRAP+ and cathepsin K+ cells were significantly increased in ruptured CCLs, compared with intact ACLs. The presence of TRAP+ cells was correlated with inflammatory changes, which were most prominent in ruptured CCLs. CONCLUSION AND CLINICAL RELEVANCE: Results suggest that synovial macrophage-like cells that produce TRAP are an important feature of the inflammation associated with CCL rupture in dogs. Identification of TRAP and cathepsin K in intact CCLs and ACLs from aged dogs suggests that these enzymes have a functional role in cruciate ligament remodeling and repair. We hypothesize that recruitment and activation of TRAP+ macrophage-like cells into the stifle joint synovium and CCL epiligament are critical features of the inflammatory arthritis that promotes progressive degradation and eventual rupture of the CCL in dogs.     

Collagen fragmentation in ruptured canine cranial cruciate ligament explants

Collagen fragmentation in cranial cruciate ligament (CCL) explants and stifle synovial fluid was investigated in dogs with ruptured and intact CCL. Cathepsin K and tartrate-resistant acid phosphatase (TRAP) activities were determined in CCL explant supernatants. Formation of collagen fragments was determined in explant supernatants and stifle synovial fluid. Cathepsin K(+) and TRAP(+) cells were stained specifically in histological sections of CCL. Formation of telopeptide collagen fragments was increased in ruptured CCL explants and stifle synovial fluid from dogs with ruptured CCL. In ruptured CCL explants, release of collagen fragments was associated with extracellular release of TRAP and the presence of cathepsin K(+) cells within CCL tissue. Cathepsin K(+) and TRAP(+) cells were only seen in ruptured CCL. It was concluded that infiltration of the CCL with TRAP(+) cells in dogs with CCL rupture is associated with increased collagenolysis. It is hypothesized that recruitment and activation of TRAP(+) mononuclear cells within the synovium and CCL precipitates CCL rupture through upregulation of collagenolytic enzymes and collagen degradation.     

Evaluation of tartrate-resistant acid phosphatase and cathepsin K in ruptured cranial cruciate ligaments in dogs

OBJECTIVE: To determine localization of tartrate-resistant acid phosphatase (TRAP) and cathepsin K in ruptured and healthy cranial cruciate ligaments (CCL) in dogs. ANIMALS: 30 dogs with ruptured CCL, 8 aged dogs without ruptured CCL, and 9 young dogs without ruptured CCL. PROCEDURE: The CCL was examined histologically and cells containing TRAP and cathepsin K were identified histochemically and immunohistochemically, respectively. RESULTS: Cathepsin K and TRAP were detected within the same cells, principally within the epiligamentous region and to a lesser extent in the core region of ruptured CCL. Numbers of cells containing TRAP and cathepsin K were significantly greater in ruptured CCL, compared with CCL from young or aged dogs, and numbers of such cells were greater in CCL from aged dogs, compared with those of young dogs. In aged dogs, small numbers of cells containing TRAP and cathepsin K were seen in intact CCL associated with ligament fascicles in which there was chondroid transformation of ligament fibroblasts and disruption of the extracellular matrix. CONCLUSIONS AND CLINICAL RELEVANCE: Ruptured CCL contain greater numbers of cells with the proteinases TRAP and cathepsin K than CCL from healthy, young, or aged dogs. Results suggest that cell-signaling pathways that regulate expression of these proteinases may form part of the mechanism that leads to upregulation of collagenolytic ligament remodeling and progressive structural failure of the CCL over time.     

Expression of immune response genes in the stifle joint of dogs with oligoarthritis and degenerative cranial cruciate ligament rupture

Dysregulation of immune responses within joints plays an important role in development of inflammatory arthritis. We determined expression of a panel of immune response and matrix turnover genes in synovial fluid collected from a group of dogs with stifle oligoarthritis and associated degenerative cranial cruciate ligament (CCL) rupture (n=27). We also studied synovial fluid gene expression in dogs affected with other forms of degenerative arthritis (n=9) and in the stifle joint of healthy dogs with intact CCL (n=14). After collection, synovial cells were pelleted and RNA was isolated. Relative expression of cathepsin K, cathepsin S, tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), invariant chain (li), toll-like receptor-2 (TLR-2), and TLR-9 was determined using real-time quantitative RT-PCR. Data were normalized to peripheral blood mononuclear cells (PBMC) as an internal control. Relative expression of cathepsin K, MMP-9, TRAP, and li was increased in the stifle synovial fluid of dogs with oligoarthritis, when compared with the stifles of healthy dogs (P<0.05). In contrast, relative expression of all of the genes-of-interest in synovial fluid from joints affected with other forms of arthritis was not significantly different from the stifles of healthy dogs. TRAP expression was also significantly increased in the stifle joints of dogs with oligoarthritis, when compared to joint expression of TRAP in dogs with other forms of degenerative arthritis (P<0.05). In the dogs with stifle oligoarthritis, expression of both matrix turnover and immune response genes was increased in stifle synovial fluid, when compared with the internal PBMC control, whereas in healthy dogs and dogs with other forms of arthritis, only expression of matrix turnover genes was increased in synovial fluid, when compared with the internal PBMC control (P<0.05). Taken together, these findings suggest that antigen-specific immune responses within the stifle joint may be involved in the pathogenesis of persistent synovitis and associated joint degradation in dogs with oligoarthritis and degenerative CCL rupture.     

Histologic changes in ruptured canine cranial cruciate ligament

OBJECTIVES: To determine changes to the cells and collagenous and amorphous extracellular matrix (ECM) structure in ruptured canine cranial cruciate ligaments (CCL). STUDY DESIGN: Prospective clinical study. ANIMALS: CCL specimens obtained from 29 dogs with ruptured CCL and 6 young dogs with intact CCL. METHODS: Ligament fibroblast number density and phenotype were determined in the core and epiligamentous regions. ECM birefringence and crimp structure in the core region were also studied. RESULTS: Loss of fibroblasts from the core region of ruptured CCL was seen (P <.001), whereas, in the epiligamentous region, cell number densities were similar in ruptured and intact CCL (P =.7). In ruptured CCL, numbers of typical ligament fibroblasts (fusiform and ovoid cells) were decreased, and numbers of cells exhibiting chondroid transformation (spheroid cells) were increased in the core region (P <.001). Expansion of the volume of the epiligamentous region was also seen, although bridging scar tissue was not seen between the ends of ruptured CCL. The structure of the ECM collagen in the core region was extensively disrupted in ruptured CCL. This was, in part, because of decreased birefringence and elongation of the crimp in the remaining collagen fibers when compared with intact CCL (P <.01). CONCLUSIONS: Extensive alterations to the cell populations and collagenous ECM structure were seen in ruptured CCL. Although a proliferative epiligamentous repair response was seen in ruptured CCL, there was a lack of any bridging scar between the ruptured ends of the CCL. CLINICAL RELEVANCE: The cellular and ECM changes in ruptured CCL that we have described appear to result from the cumulative effects of remodeling and adaptation to mechanical loading and microinjury. Treatment of early cruciate disease in dogs will need to inhibit or reverse these progressive changes to CCL tissue, which are directly associated with partial or complete structural failure of the CCL under conditions of normal activity.     

Immunohistochemical characterization of inflammatory cell populations and adhesion molecule expression in synovial membranes from dogs with spontaneous cranial cruciate ligament rupture

This study describes the distribution of CD4+ and CD8alpha+ T lymphocytes, B lymphocytes, macrophages, MHC class II antigens, immunoglobulin (IgG, IgM, IgA)-containing cells and of adhesion molecules belonging to the CD11/CD18 family in synovial membrane biopsies from 28 dogs with spontaneous rupture of the cranial cruciate ligament (CCL). Synovial membranes from 11 dogs without evidence of joint lesions were used as control tissues. The main cell types in synovial membranes from dogs with CCL rupture were B lymphocytes and plasma cells belonging to the IgG isotype. The severity of inflammatory cell infiltration in CCL cases was positively correlated with the expression of adhesion molecules. Double immunofluorescence labelling of frozen sections revealed that in the inflamed synovium of dogs with CCL rupture numerous dendritic cells expressing MHC class II antigen and canine CD1c were present. The findings further support the view that in the synovium of dogs with CCL rupture an immunologic response is going on in which dendritic cells are possibly involved by presenting hitherto unknown antigens to T lymphocytes.     

Cranial cruciate ligament pathophysiology in dogs with cruciate disease: a review

Cruciate disease is a common cause of chronic lameness in dogs. Midsubstance rupture of the cranial cruciate ligament (CCL) arises from progressive pathological failure, often under conditions of normal loading in adult dogs with CCL instability. A high risk of rupture is associated with inflammation of the synovium and adaptive or degenerative changes in the cells and matrix of the CCL. In contrast, CCL rupture in puppies is usually associated with traumatic injury and avulsion of the CCL from its sites of attachment.     

Vascular distribution in ruptured canine cranial cruciate ligament

Objective: To (1) determine the microanatomic vascular distribution in ruptured canine cranial cruciate ligaments (CCL) using specific vascular immunohistochemical techniques, and (2) compare vessel density between ruptured and intact canine CCL and between different areas of interest in ruptured CCL using histomorphometric analysis. Study Design: In vitro study. Animals: Dogs (n=41) admitted for surgical treatment of ruptured CCL and 19 dogs euthanatized for nonorthopedic conditions. Methods: Diseased (variable CCL rupture) and intact (normal control) CCL were processed for immunohistochemical staining specific to vessels (factor VIII, laminin). Mean vascular density was assessed and compared in areas of interest (torn end versus remaining core regions of CCL, proximal femoral versus distal tibial core CCL regions). Results: Ruptured CCL was more vascular than intact CCL; however there was no difference in vascular density between the torn end and the remaining core area of the ruptured CCL. Ruptured CCL was vascularized to a greater degree at the proximal portion than the distal portion of the CCL. Partially ruptured CCLs had a higher vessel density than completely ruptured CCLs. Conclusions: Vascular density is increased in diseased CCL compared with intact CCL. It remains to be determined whether this finding is associated with the cause of CCL rupture or is a result of CCL degeneration and rupture.     

Breed, sex, and body weight as risk factors for rupture of the cranial cruciate ligament in young dogs.

OBJECTIVE: To describe clinical features of dogs < 2 years old with rupture of the cranial cruciate ligament (CCL) and to evaluate breed, sex, and body weight as risk factors. DESIGN: Case-control study. ANIMALS: 201 dogs < 2 years old with rupture of the CCL and 804 age-matched control dogs. PROCEDURE: Medical records were reviewed for breed, sex, and body weight, and results were compared with results of age-matched control dogs. RESULTS: Breed predisposition was detected for Neapolitan Mastiff, Akita, Saint Bernard, Rottweiler, Mastiff, Newfoundland, Chesapeake Bay Retriever, Labrador Retriever, and American Staffordshire Terrier. Increased risk was detected for neutered males and neutered females, compared with sexually intact males and sexually intact females, respectively. Differences in prevalence of rupture of the CCL were not detected between all males and females, sexually intact males and sexually intact females, or neutered males and neutered females. Body weights of dogs with ruptured CCL were significantly greater than those of control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Several large breeds of dogs are predisposed to rupture of the CCL at a young age.     

Detection and distribution of apoptotic cell death in normal and diseased canine cranial cruciate ligaments

One of the possible initiating factors in canine cranial cruciate ligament (CCL) rupture could be an abnormal pattern of ligament cell death. This study compared apoptotic cell death in sections of ruptured CCLs and normal controls, and examined nitric oxide (NO) production in joint tissues and correlated this to apoptosis. CCLs and cartilage from the lateral femoral condyle were harvested from 10 healthy dogs and 15 dogs with CCL rupture and ligaments were further processed to detect cleaved caspase-3 and to determine supernatant NO production in explant cultures. Apoptotic activity was greater in ruptured ligaments compared to controls. NO in ligaments showed a moderate but significant positive correlation with caspase-positive cells. The results suggest that increased apoptosis has a role in CCL rupture and that apoptosis may be influenced by local NO production.     

Nitric oxide metabolite production in the cranial cruciate ligament, synovial membrane, and articular cartilage of dogs with cranial cruciate ligament rupture

OBJECTIVE: To measure concentrations of nitric oxide metabolites (nitrite-nitrate [NOt]) in cartilage, synovial membrane, and cranial cruciate ligament (CCL) in dogs and evaluate associations with osteoarthritis in dogs with CCL rupture. ANIMALS: 46 dogs with CCL rupture and 54 control dogs without joint disease. PROCEDURE: Tissue specimens for histologic examination and explant culture were harvested during surgery in the CCL group or immediately after euthanasia in the control group; NOt concentrations were measured in supernatant of explant cultures and compared among dogs with various degrees of osteoarthritis and between dogs with and without CCL rupture. RESULTS: Osteoarthritic cartilage had significantly higher NOt concentration (1,171.6 nmol/g) than did healthy cartilage (491.0 nmol/g); NOt concentration was associated with severity of macroscopic and microscopic lesions. Synovial membrane NOt concentration did not differ between dogs with and without CCL rupture. Ruptured CCL produced less NOt than did intact ligaments. In control dogs, NOt concentrations were similar for intact ligaments (568.1 nmol/g) and articular cartilage (491.0 nmol/g). Synthesis of NOt was inhibited substantially by coincubation with inhibitors. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that NOt in canine joint tissues originates from the inducible nitric oxide synthase pathway. Nitric oxide metabolite production in cartilage was greater in dogs with osteoarthritis than in healthy dogs and was associated with lesion severity, suggesting that nitric oxide inhibitors may be considered as a treatment for osteoarthritis. The CCL produces substantial concentrations of NOt; the importance of this finding is unknown.     

Evaluation of ligament fibroblast viability in ruptured cranial cruciate ligament of dogs

OBJECTIVE: To determine fibroblast viability, assess development of apoptosis, and evaluate tissue hypoxia via histochemical, in-situ hybridization, or immunohistochemical staining in ruptured and intact cranial cruciate ligaments (CCLs) of dogs. ANIMALS: 32 dogs with ruptured CCLs, and 8 aged and 19 young dogs with intact CCLs. PROCEDURE: Markers of cell viability (lactate dehydrogenase [LDH]), apoptosis (terminal deoxynucleatidyl transferase-mediated deoxyuridine triphosphate-nick end labeling [TUNEL] method), and hypoxia (hypoxia-inducible factor-1alpha [HIF-1alpha] monoclonal antibody) were applied to CCL specimens; positive cells were assessed objectively (LDH) and subjectively (TUNEL and HIF-1alpha) in the main axial tissue component (core) and synovial intima and subintima (epiligamentous tissue). RESULTS: Viable fibroblasts were seen in all intact and ruptured CCLs. More nonviable cells were found in the core regions of ruptured CCLs and intact CCLs of young dogs than in the epiligamentous regions. Number of nonviable cells in the core region of ruptured CCLs was greater than that in intact CCLs of young and aged dogs, whereas the number in the epiligamentous region was similar in all specimens. The TUNEL and HIF-1alpha staining was only found in the epiligamentous region of ruptured CCLs. CONCLUSIONS AND CLINICAL RELEVANCE: Ruptured CCLs contained a high number of nonviable cells but not a great number of apoptotic cells. Repair processes in the epiligamentous region of the CCL include a metabolic response to hypoxia, suggesting that necrosis of ligament fibroblasts and transformation of surviving cells to a spheroid phenotype may be a response to hypoxia cause by microinjury or inadequate blood flow.     

COL-3 inhibition of collagen fragmentation in ruptured cranial cruciate ligament explants from dogs with stifle arthritis

Inhibition of collagen fragment generation in canine cranial cruciate ligament (CCL) explant cultures by the matrix metalloprotease inhibitor (6-demethyl)-6-deoxy-4-dedimethylamino tetracycline (COL-3) was studied. Cranial cruciate ligament specimens were collected from dogs with inflammatory stifle arthritis/CCL rupture and dogs with normal stifles. Explant cultures from each CCL specimen included one COL-3 treated explant and a baseline control; explants from 12 ruptured CCLs were prepared in triplicate and a protease inhibitor cocktail positive control was used. Explant supernatants were analyzed for generation of collagen fragments after two days. Treatment of ruptured CCL explants with 10(-4)M COL-3 decreased generation of collagen fragments. The extent of this inhibition was increased in explants treated with a protease inhibitor cocktail. Generation of collagen fragments was increased in ruptured CCLs, when compared with intact CCLs. It is concluded that generation of collagen fragments was increased in pathological ruptured CCL explants. This degradation could be significantly inhibited in vitro by 10(-4)M COL-3.     

Cartilage-derived biomarkers of osteoarthritis in synovial fluid of dogs with naturally acquired rupture of the cranial cruciate ligament

OBJECTIVE: To compare synovial fluid biomarkers of cartilage metabolism in joints with naturally acquired or experimentally induced cranial cruciate ligament (CCL) rupture and determine correlations with stage and severity of disease in dogs. ANIMALS: 95 dogs with ruptured CCL, 8 dogs with experimentally ruptured CCL, and 24 healthy dogs. PROCEDURES: Synovial fluid was assayed for chondroitin sulfate neo-epitopes 3B3(-) and 7D4 and glycosaminoglycan (GAG) concentration. Results were correlated with demographic data, duration of lameness, radiographic osteoarthritis score, and intra-articular lesions. RESULTS: The 7D4 concentrations and 7D4:GAG in synovial fluid from joints with naturally acquired CCL rupture and experimental CCL transection were similar and significantly greater than values for healthy control joints. The 3B3(-) concentrations in the CCL-deficient groups were not significantly different, although only values in the naturally acquired CCL rupture group were significantly greater than those in the healthy control group. Within the naturally acquired CCL rupture group there was a significant correlation between 3B3(-) and 7D4 concentrations. However, there were no significant correlations between biomarker concentrations and continuous demographic or disease-related variables or differences in biomarker concentrations with different categories of disease. CONCLUSIONS AND CLINICAL RELEVANCE: Synovial fluid biomarker concentrations were significantly increased in joints with secondary osteoarthritis associated with naturally acquired or experimental CCL rupture; however, lack of apparently simple relationships with demographic variables or stage or severity of disease limits their clinical usefulness.     

Morphologic and functional features of the canine cruciate ligaments

OBJECTIVE: To review the gross, microscopic, and functional anatomy of the cranial cruciate ligament (CCL) in dogs. STUDY DESIGN: Literature review. METHODS: Reports of the anatomy and function of the cruciate ligaments in dogs were retrieved by search of the 1975-2005 PubMed database. RESULTS: The CCL has an important biomechanical function resisting cranial drawer, hyperextension, and internal rotation and acts to fine tune and guide the stifle through its rolling and sliding motion. It has a complex architecture, and distinct geographic regions within the ligament have different functional roles depending on the angle and loading conditions. Collagen type I is the main component of the extracellular matrix; the fibrils have a crimped structure. The cruciate ligaments are almost completely covered by synovium, protecting them from synovial fluid. Cruciate blood supply is mainly of soft tissue origin. The intraligamentous network is relatively limited whereas the core of the middle third of the CCL is even less well vascularized. Neurohistologic studies are very limited in the dog. Various mechanoreceptors and proprioceptive receptors have been identified within the substance of the cruciate ligaments. CONCLUSIONS: CCL structural characteristics play an important part in its complex behaviour with the crimped pattern of the collagen fibrils being an important determinant of its biomechanical properties. In contrast to reports of managing CCL rupture, there are few reports describing the microanatomy and neurovascular morphology of the cruciate ligaments. CLINICAL RELEVANCE: Cruciate disease is likely multi-factorial. Improved understanding of CCL degradation leading to CCL rupture is critical to development of new diagnostic tests for cruciate disease in dogs. Appropriate intervention during the early stages of disease process might preserve CCL structural properties by preventing further collagen degradation. Accurate knowledge of functional and fiber bundle anatomy is imperative for reconstruction and restoration of normal stifle joint physiology. Reconstructive goals should alleviate existing instability and mimic normal kinematics. Knowledge of the exact function of the CCL in the neuromuscular control around the stifle joint could possibly explain osteoarthritis progression after CCL damage.     

Apoptosis of ligamentous cells of the cranial cruciate ligament from stable stifle joints of dogs with partial cranial cruciate ligament rupture

OBJECTIVE: To describe the presence and amount of apoptotic ligamentous cells in different areas of partially ruptured canine cranial cruciate ligaments (prCCLs) and to compare these findings with apoptosis of ligamentous cells in totally ruptured cranial cruciate ligaments (trCCLs). ANIMALS: 20 dogs with prCCLs and 14 dogs with trCCLs. PROCEDURES: Dogs with prCCLs or trCCLs were admitted to the veterinary hospital for stifle joint treatment. Biopsy specimens of the intact area of prCCLs (group A) and the ruptured area of prCCLs (group B) as well as specimens from trCCLs (group C) were harvested during arthroscopy. Caspase-3 and poly (ADP-ribose) polymerase (PARP) detection were used to detect apoptotic ligamentous cells by immunohistochemistry. RESULTS: No difference was found in the degree of synovitis or osteophytosis between prCCLs and trCCLs. No difference was found in degenerative changes in ligaments between groups A and B. A substantial amount of apoptotic cells could be found in > 90% of all stained slides. A correlation (r(s) = 0.71) was found between the number of caspase-3-and PARP-positive cells. No significant difference was found in the amount of apoptotic cells among the 3 groups. No significant correlation could be detected between the degree of synovitis and apoptotic cells or osteophyte production and apoptotic cells. CONCLUSIONS AND CLINICAL RELEVANCE: The lack of difference between the 3 groups indicates that apoptosis could be a factor in the internal disease process leading to CCL rupture and is not primarily a consequence of the acute rupture of the ligament.     

Comparison of tibial plateau angles in dogs with unilateral versus bilateral cranial cruciate ligament rupture: 150 cases (2000-2006)

OBJECTIVE: To compare tibial plateau angle (TPA) in dogs with unilateral versus bilateral cranial cruciate ligament (CCL) rupture, to compare right versus left TPA in dogs with bilateral CCL rupture, and to determine whether TPA can be used to predict whether a dog with unilateral CCL rupture would subsequently rupture the contralateral CCL. DESIGN: Retrospective case series. ANIMALS: 150 dogs with unilateral (n=58) or bilateral (92) CCL rupture. PROCEDURES: Medical records were reviewed and TPA was recorded. Dogs with unilateral CCL rupture that were not known to have ruptured the contralateral ligament were followed up for a minimum of 2 years. RESULTS: Dogs with unilateral CCL rupture were significantly older (median, 7.0 years) than dogs with bilateral CCL rupture (median, 4.5 years). Median TPA for dogs with unilateral CCL rupture (26 degrees) was not significantly different from median TPA for dogs with bilateral rupture (27 degrees in both the right and left limbs), and right and left TPAs were not significantly different in dogs with bilateral CCL rupture. There was no correlation between TPA and the time interval between diagnosis of the initial and subsequent CCL ruptures in dogs with bilateral CCL rupture. CONCLUSION AND CLINICAL RELEVANCE: Results suggested that TPA in the range studied (mostly<35 degrees) was not a useful predictor of contralateral CCL rupture among dogs with unilateral CCL rupture, although age may be a risk factor for development of bilateral CCL rupture. The incidence of bilateral CCL rupture may be higher than previously reported.     

Investigation of the composition, turnover, and thermal properties of ruptured cranial cruciate ligaments of dogs

OBJECTIVE: To assess different components of the extracellular matrix with regard to their thermal properties, composition, and turnover in ruptured cranial cruciate ligaments (CCLs) of dogs, compared with components of intact CCLs from a breed predisposed to CCL failure. SAMPLE POPULATION: Ruptured CCLs obtained from 8 dogs of breeds predisposed to ruptured CCLs and intact CCLs from 12 cadaveric Labrador Retrievers. PROCEDURE: Ruptured and intact CCLs were analyzed for water content; collagen content and collagen cross-links were evaluated via hydroxyproline and amino-acid analyses, respectively. Glycosaminoglycan (GAG) content was analyzed via dimethylmethylene blue and uronic acid assays. Matrix metalloproteinases (MMPs)-2 and -9 and the tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 were detected via gelatin SDS-PAGE zymography and reverse gelatin zymography. Thermal analysis of ligaments was performed by use of differential scanning calorimetry. RESULTS: Ruptured CCLs had significantly higher lamounts of immature cross-links, total and sulfated GAGs, and water content, compared with that of the intact ligaments. Compared with intact CCLs, concentration of pro-MMP-2 was significantly higher in ruptured CCLs; the maximum temperature of collagen denaturation was significantly lower in the ruptured CCLs. CONCLUSIONS AND CLINICAL RELEVANCE: The extracellular matrix of ruptured CCLs had an increased matrix turnover indicated by increased collagen and GAG synthesis, compared with that of intact CCLs. Although the extracellular matrix changes may have occurred before ligament rupture, it is possible that these observed changes may be part of a reparative process after rupture.     

Caudal cruciate ligament damage in dogs with cranial cruciate ligament rupture

Objective: To investigate the incidence of caudal cruciate ligament (CaCL) damage in dogs with cranial cruciate ligament rupture (CCLR). Study Design: Prospective clinical study. Animals: Dogs (n=24) admitted for surgical stabilization of the stifle after CCLR and 8 healthy dogs with intact cranial cruciate ligament (CCL) and CaCL studied as controls. Methods: Preoperative radiographs and stifle joint images (arthrotomy, 6; arthroscopy, 18) were collected from dogs with CCLR. Severity of arthritis, synovitis, CCL damage, and CaCL damage were assessed using numerical rating scales. The CaCL was probed to determine whether minor fraying or a full thickness defect in the ligament was present. Data collected from the study population were compared with the control population of dogs. Results: The CaCL was damaged in 21/24 (88%) of dogs with CCLR; 6/24 (25%) had a full thickness defect in the CaCL. Severity of stifle synovitis and severity of damage to the CaCL were positively correlated (P<.05). Conclusions: The CaCL is damaged in a high percentage of dogs with CCLR. A significant and positive correlation exists between the degree of synovitis present and the extent of CaCL damage. Clinical Relevance: In dogs with CCLR, cruciate ligament pathology typically involves both the CCL and CaCL. As the severity of synovitis and the extent of CaCL damage are related, this observation supports the hypothesis that stifle synovitis may contribute to CCL and CaCL degeneration and subsequent damage.