Detection and evaluation of matrix metalloproteinases involved in cruciate ligament disease in dogs using multiplex bead technology.

OBJECTIVE: To measure matrix metalloproteinases (MMPs) suspected to be involved in the initiation or progression of osteoarthritis (OA) in cranial cruciate ligament (CCL) explant culture media using multiplex bead technology. STUDY DESIGN: In vitro experimental study. ANIMALS: Adult dogs with (n=10) and without (n=10) CCL deficiency. METHODS: Based on clinical, radiographic, and gross evidence of CCL deficiency, excised CCL were classified as normal and intact (n=10) or partially torn (n=10). The ligament was excised and immediately placed in tissue culture. Culture media were sampled and replaced on days 3 and 6. MMP-1, 2, 3, 9, and 13 were quantified in explant media using a multiplexing machine that uses flow cytometry, microspheres, spectral dyes, lasers, digital signal processing, and traditional chemistry. MMP concentrations were determined using a standard curve constructed from the serial dilution of positive controls. Media MMP concentrations comparing the type of ligament and the time frame were analyzed using a Mann-Whitney rank sum test. RESULTS: Media exposed to intact ligaments had >3 times the amount of MMP-2 than for partially torn ligaments on day 6 (P=.006). Media exposed to intact ligaments also had significantly higher levels of MMP-3 than for partially torn ligaments on day 3 (P=.035) and on day 6 (P=.05). CONCLUSIONS: MMP multiplexing is a reliable, cost-effective, efficient, and sample-sparing method of MMP quantification. MMP-2, 3, 9, and 13 are released from CCL explants exposed to culture media and can be detected using multiplex bead technology. CLINICAL RELEVANCE: CCL remnants exposed to the intra-articular environment may release degradative enzymes in vivo similar to that demonstrated in this in vitro study. Because MMPs are known to be involved in the initiation and progression of OA, debridement of these remnants as a component of treatment for cruciate disease in dogs deserves consideration.

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.

Anatomic double-bundle anterior cruciate ligament reconstruction.

Anterior cruciate ligament (ACL) reconstruction is one of the most common procedures performed by orthopaedic surgeons. The procedure has improved significantly since its inception in 1900 and continues to be intensively studied with outcomes receiving considerable attention. Traditional ACL reconstruction techniques have focused on one portion of the ACL--the anteromedial bundle. Single-bundle ACL reconstruction is the criterion standard and has provided good to excellent results, with many athletes being able to return to sports; however, recently, some authors have noted persistent instability with functional testing and degenerative radiographic changes after single-bundle ACL reconstruction. As a result, several centers have attempted to improve upon the single-bundle technique by reconstructing both the anteromedial and posterolateral bundles of the ACL. This article will present the embryologic, anatomic, and biomechanical rationale for double-bundle ACL reconstruction. In addition, the latest outcomes of double-bundle ACL reconstruction will be presented.

Localization of cathepsin K and tartrate-resistant acid phosphatase in synovium and cranial cruciate ligament in dogs with cruciate disease.

OBJECTIVE: To localize cathepsin K and tartrate-resistant acid phosphatase (TRAP) in synovium and cranial cruciate ligament (CCL) of dogs with cruciate disease. ANIMALS: Dogs (n=15) with cruciate disease and ruptured CCL, and 12 dogs with intact CCL. METHODS: Synovium and CCL were examined histologically and cells containing cathepsin K or TRAP were identified immunohistochemically and histochemically, respectively. RESULTS: Increased cellular localization of cathepsin K and TRAP was detected in synovium and ruptured CCL in dogs with cruciate disease, when compared with tissues from dogs with intact CCL. Inflammation of synovium with TRAP+ macrophage-like cells was seen in 73% of dogs with CCL disease, but was not seen in dogs with intact CCL. The presence of cathepsin K and TRAP protein in synovium and CCL tissues was significantly correlated in dogs with CCL rupture. CONCLUSION: Inflammation of the epiligament of ruptured CCL with cathepsin K+ and TRAP+ macrophage-like cells forms part of a similar, more generalized chronic inflammatory change within the periarticular tissues of the stifle of a large proportion of dogs with CCL rupture. CLINICAL RELEVANCE: Production of matrix-degrading enzymes by the synovium may induce progressive pathologic rupture of the CCL. Therefore, these collagenolytic pathways may offer a novel target for medical therapy of joint inflammation in canine patients with cruciate disease.

Characterization of sulfate, proline, and glucose transport systems in anterior cruciate and medial collateral ligament cells.

The present study was undertaken to define the nature of key transport processes for sodium, glucose, proline, and sulfate in primary culture of canine anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells. Uptake studies using radiolabeled isotopes were performed and Na,K-ATPase activity was determined in cell lysates. At 25 degrees C both ACL and MCL cells showed a significant uptake of 86Rb. Ouabain inhibited Rb uptake by 55% in ACL cells and by 60% in MCL cells. The transport activity of Na,K-ATPase in intact cells was calculated to be 57 and 71 nmol.(mg protein)-1.(15 min)-1, respectively. The enzymatic activity of Na,K-ATPase in cell lysates was observed to be 104 for ACL cells and 121 nmol.(mg protein)-1.(15 min)-1 for MCL cells. Cytochalasin B, a known inhibitor of sodium-independent D-glucose transport, completely inhibited D-glucose uptake in ACL and MCL cells. Removal of Na+ or addition of 10-5 mol/L phlorizin, a potent inhibitor of the sodium-D-glucose cotransporter, did not alter D-glucose uptake, suggesting that glucose entered the cells using a sodium-independent pathway. Both ACL and MCL cells exhibited high sulfate uptake that was not altered by replacement of Na+ by N-methyl-D-glucamine, whereas DIDS, an inhibitor of sulfate/anion exchange abolished sulfate uptake in both cell types. Thus, neither cell type seems to possess a sodium-sulfate cotransport system. Rather, sulfate uptake appeared to be mediated by sulfate/anion exchange. Proline was rapidly taken up by ACL and MCL cells and its uptake was reduced by 85% when Na+ was replaced by N-methyl-D-glucamine, indicating that proline entered the cells via sodium-dependent cotransport systems. The data demonstrate that both ACL and MCL cells possess a highly active sodium pump, a secondary active sodium-proline cotransport system, and sodium-independent transport systems for D-glucose and sulfate.