Bone and lean tissue changes following cranial cruciate ligament transection and stifle stabilization

Following cranial cruciate ligament transection and extracapsular stabilization, dual-energy X-ray absorptiometry was used to analyze bone mineral content and lean tissue mass in the surgical and nonsurgical legs (n=14) at 0, 2, 4, and 8 weeks, and to evaluate bone mineral content and bone mineral density (BMD) of the proximal, mid-, and distal tibia of both the surgical and nonsurgical legs (n=15) at 0, 5, and 10 weeks. There was significant loss of bone mineral content and lean tissue in the surgical leg compared to the nonsurgical leg. Significant loss in bone mineral content and BMD was detected in the tibia of the surgical leg and was most pronounced in the metaphyseal region.     

Correlation of prostaglandin E2 concentrations in synovial fluid with ground reaction forces and clinical variables for pain or inflammation in dogs with osteoarthritis induced by transection of the cranial cruciate ligament

OBJECTIVE: To evaluate the temporal pattern of prostaglandin (PG) E2 concentrations in synovial fluid after transection of the cranial cruciate ligament (CCL) in dogs and to correlate PGE2 concentrations with ground reaction forces and subjective clinical variables for lameness or pain. ANIMALS: 19 purpose-bred adult male Walker Hounds. PROCEDURE: Force plate measurements, subjective clinical analysis of pain or lameness, and samples of synovial fluid were obtained before (baseline) and at various time points after arthroscopic transection of the right CCL. Concentrations of PGE2 were measured in synovial fluid samples, and the PGE2 concentrations were correlated with ground reaction forces and clinical variables. RESULTS: The PGE2 concentration increased significantly above the baseline value throughout the entire study, peaking 14 days after transection. Peak vertical force and vertical impulse significantly decreased by day 14 after transection, followed by an increase over time without returning to baseline values. All clinical variables (eg, lameness, degree of weight bearing, joint extension, cumulative pain score, effusion score, and total protein content of synovial fluid, except for WBC count in synovial fluid) increased significantly above baseline values. Significant negative correlations were detected between PGE2 concentrations and peak vertical force (r, -0.5720) and vertical impulse (r, -0.4618), and significant positive correlations were detected between PGE2 concentrations and the subjective lameness score (r, 0.5016) and effusion score (r, 0.6817). CONCLUSIONS AND CLINICAL RELEVANCE: Assessment of the acute inflammatory process by measurement of PGE2 concentrations in synovial fluid may be correlated with the amount of pain or lameness in dogs.     

Matrix metalloproteinase activity in stifle synovial fluid of cranial cruciate ligament deficient dogs and effect of postoperative doxycycline treatment

This prospective clinical study investigated the activity of matrix metalloproteinases (MMPs) in stifle synovial fluid (SF) of 13 dogs with acute cranial cruciate ligament (CCL) rupture, and the effect of a postoperative doxycycline treatment. MMP-2, 3, 9 and 13 activities were compared with respect to the time of sampling (preoperatively or 1 month after surgical stabilisation) and the type of postoperative adjuvant treatment (doxycycline or not). No significant activity was detected for both MMP-3 and MMP-13. MMP-2 and MMP-9 activities were found to be significantly highly increased in SF of CCL ruptured stifles compared to control stifles of unaffected dogs. No significant effect from surgical stabilisation and postoperative doxycycline treatment on MMP-2 and MMP-9 activities was found, indicating that doxycycline may not be an appropriate postoperative medical treatment after CCL rupture.     

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.     

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.     

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.     

Osteophyte formation in the canine stifle joint following treatment for rupture of the cranial cruciate ligament

A clinical and radiological study of osteophyte formation following surgical or conservative treatment for rupture of the cranial cruciate ligament (C.C.L.), was carried out in thirty-eight dogs. Osteophyte formation had occurred in all of the affected stifle joints, in the period between treatment and the re-examination, irrespective of the method of treatment. In general, the degree of formation was less than that which had occurred before treatment. In the larger and more obese dogs, osteophyte formation was more extensive. The degree of lameness did not appear to be related to the degree of osteophyte formation radiographically evident at re-examination. No direct correlation could be established between the degree of joint instability, assessed at the re-examination, the degree of osteophyte formation following treatment, or the function of the limb.     

Radiographic assessment of the progression of osteoarthrosis in the contralateral stifle joint of dogs with a ruptured cranial cruciate ligament

The formation and progression of osteoarthrosis in the unaffected contralateral stifle joints of 14 dogs with a unilateral cranial cruciate ligament rupture were monitored radiographically in terms of a global score and the scores for 10 parameters specific for the stifle joint. The dogs were examined initially and six and 12 months later by three observers, and the variability between the observers' scores was also assessed. The score for osteophytes at the tibial attachment site of the ligament was the most reliable parameter, and that for the increase in femoropatellar joint space was the least reliable. In the contralateral stifle joints there were significant increases after six and 12 months in osteophyte formation caudal to the tibial plateau, and in subchondral sclerosis of the tibial plateau and of the long digital extensor muscle groove. These three parameters progressed more regularly during the disease process than the other parameters. The global osteoarthrosis score of the contralateral stifle joint was an important risk factor for sustaining a rupture of the cranial cruciate ligament in that joint during the next six months.     

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.     

Evaluation of anticollagen type I antibody titers in synovial fluid of both stifle joints and the left shoulder joint of dogs with unilateral cranial cruciate disease

OBJECTIVE: To evaluate anticollagen type I antibodies in synovial fluid of the affected stifle joint, the contralateral stifle joint, and the left shoulder joint of dogs with unilateral cranial cruciate ligament (CrCL) rupture during an extended period of 12 to 18 months. ANIMALS: 13 client-owned dogs with CrCL rupture and 2 sham-operated dogs. PROCEDURES: All dogs were examined and arthrocentesis of all 3 joints was performed every 6 months after surgery. Synovial fluid samples were tested for anticollagen type I antibodies by use of an ELISA. RESULTS: Dogs with partial CrCL rupture had higher antibody titers than dogs with complete rupture. Six of 13 dogs ruptured the contralateral CrCL during the study, whereby higher antibody titers were found for the stifle joints than for the shoulder joint. Seronegative dogs or dogs with extremely low antibody titers and 2 dogs with high antibody titers did not sustain a CrCL rupture in the contralateral stifle joint. CONCLUSIONS AND CLINICAL RELEVANCE: In most dogs that had a CrCL rupture of the contralateral stifle joint, a distinct antibody titer gradient toward the stifle joints was detected, suggesting that there was a local inflammatory process in these joints. However, only a small number of sham-operated dogs were used to calculate the cutoff values used to determine the anticollagen type I antibody titers in these patients. Synovial fluid antibodies against collagen type I alone do not initiate CrCL rupture because not all dogs with high antibody titers sustained a CrCL rupture in the contralateral stifle joint.     

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.     

Synovial membrane changes after experimental transection of the cranial cruciate ligament in dogs

Degenerative joint disease and inflammation of the synovial membrane were produced in the left stifle of 16 dogs by severing the cranial cruciate ligament. Arthrotomy only was performed on the right stifle. Synovial membrane from these joints was histologically examined at 1, 2, 8, and 13 weeks after surgical operation. Similar tissue was obtained from 4 healthy dogs for comparison. Inflammatory changes in the synovium of the left stifle progressed with time and were prominent at 8 weeks postoperatively; subsynovial fibrosis was greatest at 13 weeks. Inflammation of the synovial membrane and subsynovial tissue was characterized by synovial cell hypertrophy and hyperplasia, plasma cell and lymphocyte infiltration, and increased vascularization of the subsynovial region.     

Detection of synovial macrophages in the joint capsule of dogs with naturally occurring rupture of the cranial cruciate ligament

OBJECTIVE: To test the hypotheses that the densities of macrophages in the synovial membranes and capsules of stifle joints in dogs with ruptured cranial cruciate ligaments are greater than those of normal joints and that those densities in affected joints are positively correlated with the chronicity and severity of the disease. ANIMALS: 17 dogs with naturally occurring rupture of the cranial cruciate ligament and 5 healthy control dogs. PROCEDURE: All dogs underwent orthopedic and radiographic evaluations. In affected dogs, duration of clinical signs was used as an indicator of disease chronicity and the severity of osteoarthritis in the stifle joint was determined radiographically. Joint capsule specimens were evaluated histologically; macrophages, interleukin-6, and tumor necrosis factor-alpha were identified by use of immunocytochemical techniques. RESULTS: Compared with unaffected joints, macrophage density was increased in all affected joints. Duration of disease was significantly associated with radiographic severity of osteoarthritis and synovial macrophage density. Synovial macrophage density was significantly associated with severity of osteoarthritis and with the presence of interleukin-6 and tumor necrosis factor-a. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that synovial macrophages may be involved in the development of pathologic changes (including osteophyte formation) in the stifle joints of dogs with osteoarthritis secondary to rupture of the cranial cruciate ligament. Determination of the importance of synovial macrophages in the development of changes in osteoarthritic joints may result in new treatment strategies that involve elimination of the deleterious effects of those cells.     

Synovitis in dogs with stable stifle joints and incipient cranial cruciate ligament rupture: a cross-sectional study

Objective: To evaluate stifle joints of dogs for synovitis, before development of joint instability and cranial cruciate ligament rupture (CrCLR). Study Design: Cross‐sectional study. Animals: Dogs (n=16) with CrCLR and stable contralateral stifles; 10 control dogs with intact CrCL. Methods: Arthritis and tibial translation were graded radiographically. Synovitis severity and cruciate pathology were assessed arthroscopically. Presence of inflammatory cells in synovial membrane biopsies was scored histologically. CrCLR stifle pairs and control stifles were compared. Results: Radiographic evidence of arthritis, cranial tibial translation, and arthroscopic synovitis were increased in unstable stifles, when compared with stable contralateral stifles in CrCLR dogs (P<.05). Arthroscopic synovitis in both joints of CrCLR dogs was increased compared with controls, was correlated with radiographic arthritis (S_R=0.71, P<.05), and was present in all stable contralateral stifles. Arthroscopically, 75% of stable stifle joints had CrCL fiber disruption, which correlated with severity of synovitis (S_R=0.56, P<.05). Histologic evidence of synovitis was identified in all CrCLR dogs, but was only significantly correlated with arthroscopic observations in stable stifles (r²=0.57, P<.005). Conclusion: Synovitis is an early feature of the CrCLR arthropathy in dogs before development of joint instability clinically. Severity of synovitis is correlated with radiographic arthritis in joints with minimal to no clinically detectable CrCL damage.     

Prevalence and relevance of antibodies to type-I and -II collagen in synovial fluid of dogs with cranial cruciate ligament damage

OBJECTIVE: To measure and compare synovial fluid antibody titers to type-I and -II collagen in stifle joints with instability caused by complete or partial cranial cruciate ligament (CCL) rupture and joints with osteoarthrosis secondary to other pathologic changes in dogs. ANIMALS: 82 dogs with diseased stifle joints. PROCEDURE: Synovial fluid samples were collected from 7 dogs with clinically normal stifles (control group) and 82 dogs with diseased joints (50 stifle joints with complete rupture of the CCL, 20 with partial damage of the CCL, and 12 joints with radiographic signs of osteoarthritis secondary to other arthropathies). Synovial fluid samples were tested for autoantibodies to type-I and -II collagen by an ELISA. RESULTS: In dogs with complete and partial CCL rupture, synovial fluid antibody titers to type-I and -II collagen were significantly increased, compared with control dogs. Forty-eight percent (24/50) of samples from dogs with complete CCL rupture and 35% (7/20) of samples from dogs with partial CCL rupture had antibody titers to type-I collagen that were greater than the mean plus 2 standard deviations of the control group titers. Synovial fluid antibody titers to type-II collagen were high in 40% of the dogs with partial or (8/20) complete (20/50) CCL rupture. Dogs with osteoarthrosis secondary to other pathologic changes had significantly increased synovial fluid antibodies to type-I and -II collagen, compared with control dogs. CONCLUSION: Increases in autoantibodies to collagen in synovial fluid are not specific for the type of joint disorder. It is unlikely that the anticollagen antibodies play an active role in the initiation of weakening of the CCL.     

Changes in the canine stifle joint following rupture of the anterior cruciate ligament

Sixty–four dogs with rupture of the anterior cruciate ligament in one or both stifle joints were referred to the Royal Veterinary College, London for treatment. Details of the pathological changes in the joints were recorded at the time of open surgery in fifty–seven and these changes are discussed. A post–mortem examination was made on one dog and the joint lesions were studied more closely.