Characterizing osteochondrosis in the dog: potential roles for matrix metalloproteinases and mechanical load in pathogenesis and disease progression.

OBJECTIVE: To address possible roles of matrix metalloproteinases (MMPs) and mechanical stress in the pathogenesis of osteochondrosis (OC). METHODS: Naturally-occurring canine OC lesions (n=50) were immunohistochemically analyzed for MMP-1, -3, and -13, and normal canine articular cartilage explants (n=6) cultured under 0-, 2-, or 4-MPa compressive loads (0.1 Hz, 20 min every 8 h up to 12 days) were compared to OC samples (n=4) biochemically and molecularly. RESULTS: MMP-1 and -3 immunoreactivities were readily detected in both OC samples and control tissues obtained from age-matched dogs (n=11) whereas MMP-13 was only detectable in OC samples. MMP-13 gene expression as determined by real-time reverse transcription polymerase chain reaction was elevated in OC samples and cartilage explants cultured without mechanical stimuli (0 MPa groups) compared to normal cartilage (day 0 controls). Glycosaminoglycan content (per weight) in cartilage explants cultured under no load was significantly (P<0.05) lower on day 12 than in the day 0 controls. Gene expression levels of aggrecan and type II collagen in OC samples were lower than those in the day 0 controls. High levels of aggrecan and collagen II expression were seen in the 2 MPa groups. CONCLUSIONS: These findings imply that impaired biochemical characteristics in OC-affected cartilage may be attributable to decreased extracellular matrix production that may stem from disruption of normal weight bearing forces.

The effects of TIMP-1 and -2 on canine chondrocytes cultured in three-dimensional agarose culture system.

OBJECTIVE: To investigate the effects of tissue inhibitor of metalloproteinase (TIMP)-1 and -2 on chondrocytes cultured with or without interleukin (IL)-1 beta. DESIGN: Canine articular chondrocytes were cultured in three-dimensional (3-D) agarose constructs. Cells were distributed into each of the two groups, those without IL-1 beta and those with IL-1 beta added to the liquid media. Each group was subdivided into three groups, based on the presence of TIMP-1 or -2. IL-1 beta and TIMPs were added to liquid media bathing the 3-D constructs beginning on day 3. The liquid media and the 3-D constructs were collected on days 9, 15, and 24, and analyzed histologically, biochemically, and immunohistochemically. RESULTS: Addition of TIMP-1 or -2 resulted in decreases in matrix metalloproteinase (MMP)-3 concentrations of 37 and 41%, and MMP-1 immunoreactivity of 32 and 36%, respectively, compared with the IL-1 beta group, on day 9. Chondrocytes in groups without IL-1 beta maintained viability and produced abundant extracellular matrix (ECM). Chondrocytes in IL-1 beta groups appeared less viable and produced less ECM compared with those without IL-1 beta. Glycosaminoglycan (GAG) concentrations in 3-D constructs (GAG/weight) were significantly (P<0.001) higher in groups without IL-1 beta than in those with IL-1 beta, on days 15 and 24. CONCLUSIONS: The addition of TIMP was not detrimental to chondrocytes, as used in this study. Despite evidence of decreased MMP levels, TIMPs did not prevent IL-1 beta-associated changes in cellular or ECM characteristics. Further study is necessary before clinically relevant conclusions can be drawn regarding the use of TIMPs in the treatment of osteoarthritis.

Kinetic study of the replacement of porcine small intestinal submucosa grafts and the regeneration of meniscal-like tissue in large avascular meniscal defects in dogs.

Porcine small intestinal submucosa (SIS) was used to replace large, avascular defects in the medial menisci of dogs. Twelve dogs received SIS grafts and 3 dogs were left untreated as controls. Dogs were evaluated at 4, 8, and 12 weeks by means of lameness scoring and ultrasonography. Dogs were sacrificed at 1, 6, or 12 weeks after implantation, and the tissue at the site of meniscal resection was evaluated for gross and histologic appearance, cross-sectional and surface area, and collagen types I and II. The femoral and tibial condyles were assessed for articular cartilage damage. Control dogs were significantly more lame than grafted dogs 8 and 12 weeks after instrumentation. Grafted dogs' replacement tissue appeared meniscal-like when evaluated grossly and ultrasonographically 12 weeks after instrumentation. The amount of replacement tissue was significantly greater in both cross-sectional and surface area for grafted dogs than for controls at all time points. Histologically, the SIS biomaterial could be identified in all grafted dogs at 1 week post-implantation, but in none at 6 weeks post-implantation. Subjectively, grafted dogs' replacement tissue was histologically superior to that of controls with respect to tissue type, organization, and architecture. Collagen types I and II immunoreactivity in grafted menisci were similar to that of normal menisci. Control dogs had significantly more articular cartilage damage than grafted dogs. SIS appears to induce regeneration of meniscal-like tissue in large, avascular meniscal defects in dogs, resulting in superior clinical function and articular cartilage protection compared to ungrafted controls.

Biochemical characterization of cartilage affected by osteochondritis dissecans in the humeral head of dogs.

OBJECTIVE: To determine glycosaminoglycan (GAG) concentration and immunohistochemical staining characteristics of type-I, -II, and -X collagen from cartilage affected by osteochondritis dissecans (OCD) in dogs. ANIMALS: 31 dogs with OCD and 11 clinically normal purpose-bred dogs. PROCEDURE: Cartilage samples were evaluated microscopically, and GAG content was determined. Immunohistochemical staining was performed for type-I, -II, and -X collagen. Sections were subjectively evaluated for location and intensity of staining. RESULTS: Cartilage affected by OCD had a variety of pathologic changes and significantly lower GAG concentrations than did normal cartilage. Normal cartilage had no detectable type-I collagen. For dogs < 9 months of age, cartilage affected by OCD had significantly more type-I collagen but significantly less type-X collagen than did control cartilage. For dogs > 12 months of age, cartilage affected by OCD contained significantly more type-I collagen than did control cartilage. There was a significant negative correlation between immunoreactivity of type-I collagen and that of type-II and -X collagen. A significant positive correlation was found between immunoreactivity of type-II and -X collagen. CONCLUSIONS AND CLINICAL RELEVANCE: Cartilage affected by OCD contains less GAG, more type-I collagen, and less type-X collagen, compared with normal cartilage. A direct correlation between these changes and the etiopathogenesis of OCD was not established.

Arthroscopic removal and curettage of osteochondrosis lesions on the lateral and medial trochlear ridges of the talus in two dogs.

Osteochondrosis lesions in the tibiotarsal joint were treated arthroscopically in two dogs. One dog had unilateral osteochondrosis of the dorsal aspect of the lateral trochlear ridge of the talus. The second dog had bilateral osteochondrosis of the plantar aspect of the medial trochlear ridge of the talus. Removal of all osteochondral fragments (i.e., joint mice) and debridement and curettage of the remaining talar defects were accomplished arthroscopically. Both dogs had excellent short-term outcomes.

Effect of ascorbate and two different media on canine chondrocytes in three-dimensional culture.

The objective of this study was to determine the effects of ascorbate and two different culture media on cell morphology and extracellular matrix formation of canine chondrocytes grown in a three-dimensional (3-D) culture system. Articular cartilage harvested from the humeral head of three adult canine cadavers was used to obtain chondrocytes for primary culture. Subcultured chondrocytes were seeded in a 3-D medium of RPMI-1640 (R), RPMI-1640 with 50 microg/mL ascorbate (RA), Ham's F-12 (F), or Ham's F-12 with 50 microg/mL ascorbate (FA) in agarose. Samples were harvested at 5, 10, 15, and 20 days of 3-D culture and analyzed for histologic appearance and proteoglycan staining, electron microscopic appearance, dimethylmethylene blue assay for glycosaminoglycan (GAG) content, and immunohistochemical staining for collagen type II production. Chondrocytes in all four groups maintained appropriate morphology and produced matrix over the entire study period. Chondrocytes from groups R and RA produced more GAG and collagen type II than did those from groups F and FA on days 10 (P = .00791) and 15 (P = .0173). Chondrocytes from group RA produced more GAG on days 5 (P = .0154) and 20 (P = .0044) than did those in groups R, F, and FA. With respect to matrix production, RPMI-1640 is superior to Ham's F-12 for 3-D culture of canine chondrocytes. The addition of ascorbate at 50 microg/mL to RPMI-1640 did have a positive effect on the production of GAG but had minimal effect on type II collagen production. Determining the most ideal in vitro microenvironment for canine chondrocytes grown in a 3-D culture system has important implications to the in vivo application of this technique.

Quantification of measurement of femoral head coverage and Norberg angle within and among four breeds of dogs.

OBJECTIVE: To report values for percentage coverage of the femoral head (PC) and Norberg angle (NA) in 4 common breeds of dogs and to determine values for each that distinguish between normal and dysplastic hip status on the basis of Orthopedic Foundation for Animals (OFA) hip evaluation. ANIMALS: 1,841 dogs 24 to 48 months of age that were Labrador Retrievers (455), Golden Retrievers (423), Rottweilers (545), or German Shepherd Dogs (418). PROCEDURE: Retrospective analysis of NA and PC measured from standard OFA ventrodorsal pelvic radiographs from 4 breeds of dog. RESULTS: Norberg angle ranged from 67.4 to 124.4 degrees for Labrador Retrievers, 59.7 to 128.6 degrees for Rottweilers, 70.2 to 119.4 degrees for Golden Retrievers, and 55.3 to 121.3 degrees for German Shepherd Dogs. The PC ranged from 6.5 to 79.9% for Labrador Retrievers, 5.7 to 79.5% for Rottweilers, 8.3 to 79.3% for Golden Retrievers, and 5.4 to 83.7% for German Shepherd Dogs. On the basis of logistic regression modeling for determining normal versus abnormal hip status for all 4 breeds, cutoff points for NA were <105 degrees and PC were <50%. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study indicate that cutoff points of NA of 105 degrees and PC of 50% do not differentiate normal versus dysplastic hip status. Each of the 4 breeds had different values for NA and PC that distinguished normal from dysplastic hip status.

Blood plasma chemistries from wild mourning doves held in captivity.

Despite the extensive amount of research conducted on mourning doves (Zenaida macroura), no biochemical reference values exist for this species. Our objective, therefore, was to establish base line clinical chemistry reference values for mourning doves to assist with establishing clinical diagnoses. Wild mourning doves were captured 19 March 1996 to 8 August 1996, and 6 February 1998 to 12 May 1998; blood samples were collected from 382 mourning doves. Plasma biochemical values were established for glucose, sodium, potassium, chloride, enzymatic CO2, albumin, total protein, globulin, calcium, phosphorus, cholesterol, magnesium, aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH), and uric acid. These reference values are invaluable for determining diagnosis of diseases of the gastrointestinal, hepatic, renal, cardiovascular, musculoskeletal, and endocrine systems.

Effects of human recombinant interleukin-1beta on canine articular chondrocytes in three-dimensional culture.

OBJECTIVE: To determine the effects of interleukin (IL)-1beta on matrix synthesis and degradation by chondrocytes cultured in a 3-dimensional (3-D) gel medium. SAMPLE POPULATION: Chondrocytes from 7 dogs. PROCEDURE: Articular chondrocytes were harvested and cultured in 3-D gel medium alone or with 10 or 20 ng IL-1beta/ml that was added beginning on day 0, 3, 6, or 9. On days 3, 6, 12, and 20 of 3-D culture, samples of the liquid medium were evaluated for glycosaminoglycan (GAG), prostaglandin E2 (PGE2), and matrix metalloprotease (MMP)-3 content. The 3-D plug in each well was evaluated for histologic characteristics of viability, cell morphology, and proteoglycan staining, immunohistochemically stained for collagen type II, and spectrophotometrically analyzed for GAG content. RESULTS: Significant differences for all variables were detected between controls and each IL-1beta group, among groups with different IL-1beta concentrations, and among groups with IL-1beta added at various time points. Chondrocytes exposed to IL-1beta had loss of GAG, increased PGE2 and MMP-3 concentrations, and lack of collagen type-II synthesis. These IL-1beta effects appeared to be time and concentration dependent. CONCLUSIONS: Addition of IL-1beta to chondrocytes in 3-D gel medium results in time- and concentration-dependent effects on matrix synthesis and degradation and provides an appropriate in vitro model for many of the pathophysiologic events associated with osteoarthritis.

Fluoroscopically guided closed reduction and internal fixation of fractures of the lateral portion of the humeral condyle: prospective clinical study of the technique and results in ten dogs.

OBJECTIVE: To report a technique for fluoroscopically guided closed reduction with internal fixation of fractures of the lateral portion of the humeral condyle (FLHC) and determine the long-term results in 10 clinical cases. STUDY DESIGN: Prospective clinical case study. ANIMALS: Ten dogs with 11 fractures. METHODS: Fractures of the lateral portion of the humeral condyle were stabilized with transcondylar screws and Kirschner wires. Closed reduction and implant placement were achieved using intraoperative fluoroscopic guidance. After fracture repair, postoperative radiographs were evaluated for articular alignment and implant placement. Dogs were evaluated after surgery by means of lameness scores, elbow range of motion (ROM), radiographic assessment, and owner evaluation of function. RESULTS: Postoperative reduction was considered anatomic in 6 fractures with all other fractures having <1.5 mm of malreduction. Follow-up was available for 9 patients from 9 to 21 months after surgery. All of the fractures had healed. One minor (wire migration) and one major (implant failure) complication occurred. Mean lameness scores were 0 (n = 6), 0.5 (n = 2), and 1 (n = 1) at the time of final follow-up. No significant differences were found in follow-up ROM values between affected and unaffected elbows. All of the dogs in this study regained 90-100% of full function, based on owner assessment. CONCLUSIONS AND CLINICAL RELEVANCE: Fluoroscopic guidance for closed reduction and internal fixation of FLHC in dogs is an effective technique.

Induction of meniscal regeneration in dogs using a novel biomaterial.

A unique biomaterial, porcine small intestinal submucosa, was used to construct grafts for implantation into surgically created medial meniscal defects in dogs. Five dogs received grafts and two were left untreated as controls. All dogs were evaluated at 4, 8, and 12 weeks by means of lameness scoring, force plate analysis, and ultrasonography. Twelve weeks after implantation the dogs were sacrificed and the replacement tissue was evaluated for gross and histologic appearance, amount, glycosaminoglycan content, and type II collagen immunoreactivity. Four weeks after instrumentation, both groups had lameness scores that were significantly higher than preoperative scores, but at the 8- and 12-week evaluations, scores for the grafted dogs were not different from preoperative values. The ultrasonographic appearance of replacement tissue in grafted defects resembled normal meniscus. In the untreated defects, only unorganized tissue was present. In control dogs, replacement tissue resembled fibrous tissue and cartilage erosions were visible on the medial femoral condyles. In four of the five grafted dogs, replacement tissue was grossly indistinguishable from normal meniscus. The amount of tissue in the defect was significantly greater for the grafted dogs. Histologically, replacement tissue in control dogs was composed of vascularized connective tissue with no evidence of chondroid differentiation. Replacement tissue in grafted dogs closely resembled normal meniscal tissue with respect to chondroid differentiation, collagen content, and zonal architecture. Porcine small intestinal submucosa appeared to have beneficial effects on meniscal regeneration.

Closed reduction and lag screw fixation of sacroiliac luxations and fractures.

OBJECTIVE: To describe a technique for closed reduction and percutaneous insertion of a lag screw for fixation of sacroiliac fracture-luxations, and to report the success of this technique in stabilizing sacroiliac fracture-luxations. STUDY DESIGN: A retrospective clinical study. STUDY POPULATION: 13 consecutive client-owned dogs with sacroiliac fracture-luxations. METHODS: Sacroiliac fracture-luxations were stabilized by using a closed reduction and percutaneous lag screw fixation technique. Preoperative, postoperative, and last re-examination radiographs were used to assess the location and number of pelvic injuries, other orthopedic injuries, percent reduction of the sacroiliac joint, percent sacral width screw depth, position of the screw, pelvic canal diameter ratio, hemipelvic canal width ratio, and complications. Information on signalment, weight, weight-bearing status, neurologic status, and complications was obtained from the medical record. RESULTS: Mean percent reduction of the sacroiliac joint was 92.33%. All screws were placed within the sacral body with a mean screw depth/sacral width of 79.03%. No screw loosening occurred. Mean pelvic canal diameter ratios were 0.99, 1.20, and 1.14 preoperatively, immediately postoperatively, and at the last re-examination, respectively. Nine of 13 dogs were willing to walk on the ipsilateral rear leg the day after surgery. CONCLUSIONS: Closed reduction and percutaneous insertion of a lag screw for stabilization of fracture-luxation of the sacroiliac joint is an acceptable method of repair. CLINICAL RELEVANCE: Sacroiliac fracture-luxations can be successfully reduced and stabilized using a minimally invasive technique.

In vitro effects of glucosamine and acetylsalicylate on canine chondrocytes in three-dimensional culture.

OBJECTIVE: To determine effects of glucosamine and acetylsalicylate on canine chondrocytes in 3-dimensional culture. SAMPLE POPULATION: Chondrocytes isolated from articular cartilage of 2 adult female dogs recently euthanatized for reasons unrelated to orthopedic abnormalities. PROCEDURE: Chondrocytes were cultured in a 3-dimensional agarose-based medium alone (control), with glucosamine (100 microg/ml; GL), or with acetylsalicylate (18 microg/ml; AS). Supernatant and agarose plugs from 4 wells/group/d were collected on days 3, 6, and 12 of culture. Agarose plugs were evaluated for percentage of viable cells, percentage of cells producing pericellular or territorial matrix, glycosaminoglycan (GAG) concentration, and type-II collagen production. Prostaglandin E2 concentration in supernatants was determined. RESULTS: Chondrocytes in all groups had characteristics indicative of viability and differentiation; however, on day 12, a lower percentage of viable cells was detected in the AS group, compared with the other 2 groups. On day 6, GAG concentration in the AS group was significantly greater than concentrations in the other 2 groups. On day 12, GAG concentrations in the GL and AS groups were significantly less than in the control group. Within the GL and AS groups, cell viability was significantly less on day 12, compared with day 3. Significant differences in PGE2 concentration among or within groups and evidence of type II collagen production were not detected. CONCLUSIONS: 3-dimensional culture of canine chondrocytes allows for production of hyaline cartilage matrix constituents and growth of cells with morphologic characteristics similar to those of articular cartilage. Acetylsalicylate and glucosamine, at the single concentration evaluated, had detrimental effects on chondrocyte viability, GAG production, or both.

Biomechanical evaluation of a crimp clamp system for loop fixation of monofilament nylon leader material used for stabilization of the canine stifle joint.

OBJECTIVE: To test a crimp clamp system designed to secure monofilament nylon leader (MNL) material commonly used as lateral fabellotibial sutures (LFS) in extra-articular stabilization of the canine stifle joint. STUDY DESIGN: In vitro biomechanical tests of MNL loops secured with either the crimp clamp system or knots were performed. Suture loops (n = 94) were created from 27.3 kg tensile strength MNL and fastened with knots or crimp clamps. Tests were conducted on steam-sterilized, ethylene-oxide-sterilized, and nonsterilized MNL sutures. Loops were evaluated in single load tests and cycled tests. Values for load to failure, initial loop tension, loop elongation, mode of failure, and point of failure were determined. RESULTS: Crimp-clamped loops were superior to knotted loops in all parameters tested in both cycled and noncycled tests. Loop failure generally occurred by breaking within 3 mm of the fixation in both clamped and knotted tests. Loop elongation after cycling was greater in the knotted loops compared with clamped loops (P < .001). Load to failure was greater in clamped tests than in knotted tests (P < .001), regardless of sterilization technique used. Significantly higher initial loop tension could be achieved with the clamp system compared with knot fixation (P < .001). CONCLUSIONS: The crimp clamp system provides superior in vitro loop fixation characteristics compared with knot fixation in 27.3 kg test MNL. CLINICAL RELEVANCE: Based on the results of biomechanical testing and the known biocompatibility of the system's implant components, clinical trials using the crimp clamp system are warranted.

Fibrosarcoma in the distal radius and carpus of a four-year-old Persian.

A four-year-old Persian was presented for evaluation of a nonweight-bearing, left forelimb lameness. Radioulnar and pancarpal osteolysis with minimal periosteal reaction were seen on radiographs of the antebrachium. Cytological examinations of fine-needle aspirates and impression smears were suggestive of sarcoma. After forequarter amputation, histopathological examination provided a diagnosis of fibrosarcoma with axillary lymph-node metastasis.

Scapular fractures in dogs: epidemiology, classification, and concurrent injuries in 105 cases (1988-1994).

A retrospective study of canine scapular fractures diagnosed and treated from 1988 through 1994 at four veterinary teaching hospitals was performed. Dogs (n = 105) with 109 scapular fractures were included. Most scapular fractures occurred in young (i.e., less than four years of age), male, medium- to large-breed (i.e., greater than 10 kg) dogs as the result of vehicular trauma. Concurrent injuries (primarily thoracic trauma) occurred in approximately 70% of cases. In-house follow-up evaluations were considered adequate in only 17% of the cases. A classification system that includes biomechanical principles for categorization is described to avoid discrepancies between various traditional classification systems.

Three-dimensional culture of canine articular chondrocytes on multiple transplantable substrates.

OBJECTIVE: To determine the effects of transplantable substrates on canine chondrocytes grown in three-dimensional culture. ANIMALS: 3 canine cadavers. PROCEDURE: Articular cartilage harvested from canine cadavers was used to obtain chondrocytes for primary culture. Subcultured chondrocytes were grown in agarose alone (AG), or in agarose on canine cancellous bone (CB), polypropylene mesh, or oxidized regenerated cellulose substrate. Cell proliferation, proteoglycan and glycosaminoglycan (GAG) production, and collagen production were assessed on days 3, 6, 10, 15 and 20. RESULTS: Chondrocytes from groups AG and CB proliferated and produced matrix over the entire 20-day study period. Group-CB chondrocytes had significantly more GAG than did chondrocytes of all other groups on days 6 (P = 0.0297) and 15 (P = 0.00272). Those of groups AG and CB contained significantly (P = 0.0235) more GAG on day 20. Chondrocytes of the polypropylene mesh group proliferated and produced matrix through day 10 in culture, but were no longer viable and had no matrix production on days 15 and 20. Regenerated cellulose appeared to be toxic to canine chondrocytes during all stages of in vitro three-dimensional culture. CONCLUSIONS: Three-dimensional culture of canine chondrocytes in agarose appears to produce favorable results with respect to chondrocyte proliferation and matrix production. Canine CB appears to have beneficial effects with regard to early GAG synthesis. Polypropylene mesh and oxidized regenerated cellulose had detrimental effects on cellular proliferation and matrix production.

Modified distraction-stabilization technique using an interbody polymethyl methacrylate plug in dogs with caudal cervical spondylomyelopathy.

A modified technique for distraction-stabilization that used an interbody polymethyl methacrylate plug was performed in 22 dogs with confirmed caudal cervical spondylomyelopathy. Myelographically, all compressive lesions were dynamic and predominantly located ventral to the spinal cord. Nineteen of 21 (90%) dogs for which success/failure could be determined had a successful outcome, and 11 of 22 (50%) dogs attained normal neurologic status. The 2 cases that were considered failures involved dogs that were nonambulatory tetraparetic prior to surgery and failed to improve to a functional status. Complications were self-limiting and included ventral displacement of the cement without loss of distraction in 1 dog and diskosponsylitis at an adjacent disk space in another dog. Evidence of fusion of the affected vertebrae, in the distracted position, was radiographically evident in all dogs. Use of the interbody polymethyl methacrylate plug appears to be a viable surgical treatment of caudal cervical spondylomyelopathy in dogs.

Articular fractures of the distal portion of the humerus in Vietnamese pot-bellied pigs: six cases (1988-1992)

Medical records of 6 Vietnamese pot-bellied pigs with articular fractures of the distal portion of the humerus were reviewed. Evaluation of the medical records did not reveal a sex predilection. All fractures were associated with minor traumatic episodes in young pigs. Of 6 fractures involving the humeral condyle, 4 involved the medial portion, 1 involved the lateral portion, and 1 was a Y-shaped fracture. Five of the pigs underwent surgical repair of the fracture, and all 5 did not have signs of lameness at follow-up evaluations (mean, 11 months). Of 4 pigs that had follow-up radiography, all had evidence of mild to moderate degenerative joint disease. Articular fractures of the distal portion of the humerus should be considered as a differential diagnosis in all Vietnamese pot-bellied pigs with forelimb lameness, even if the trauma sustained appeared mild. Surgical repair in Vietnamese pot-bellied pigs is straightforward, and excellent clinical results can be expected.