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in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association
in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine the quantity (concentration) and quality (molecular weight) of synovial fluid hyaluronan with respect to presence and severity of osteoarthritis in stifle joints of dogs.

Animals—21 purpose-bred dogs and 6 clinically affected large-breed dogs (cranial cruciate ligament [CrCL] disease with secondary osteoarthritis).

Procedures—Research dogs underwent arthroscopic surgery in 1 stifle joint to induce osteoarthritis via CrCL transection (CrCLt; n = 5 stifle joints), femoral condylar articular cartilage groove creation (GR; 6), or meniscal release (MR; 5); 5 had sham surgery (SH) performed. Contralateral stifle joints (n = 21) were used as unoperated control joints. Synovial fluid was obtained from research dogs at time 0 and 12 weeks after surgery and from clinically affected dogs prior to surgery. All dogs were assessed for lameness, radiographic signs of osteoarthritis, and pathologic findings on arthroscopy as well as for quantity and quality of hyaluronan.

Results—Clinically affected dogs had significantly greater degrees of pathologic findings, compared with dogs with surgically induced osteoarthritis (ie, those with CrCLt, GR, and MR stifle joints), and with respect to lameness scores, radiographic signs of osteoarthritis, pathologic findings on arthroscopy, and synovial fluid hyaluronan concentration. Synovial fluid from stifle joints of dogs with surgically induced osteoarthritis had hyaluronan bands at 35 kd on western blots that synovial fluid from SH and clinically affected stifle joints did not.

Conclusions and Clinical Relevance—Synovial fluid hyaluronan quantity and quality were altered in stifle joints of dogs with osteoarthritis, compared with control stifle joints. A specific hyaluronan protein fragment may be associated with early pathologic changes in affected joints.

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in American Journal of Veterinary Research
in Journal of the American Veterinary Medical Association

Abstract

Objective—To characterize chondrocytes from naturally occurring osteochondrosis (OC) lesions of the humeral head of dogs.

Sample Population—15 cartilage specimens from 13 client-owned dogs with humeral head OC and 10 specimens from the humeral head of healthy dogs (controls).

Procedure—Chondrocytes were isolated and cultured in a 3-dimensional system. On days 7, 10, 15, 20, and 25, glycosaminoglycan and hydroxyproline content and cytologic characteristics were evaluated. Expression of collagen types I, II, and X was assessed by use of immunohistochemistry.

Results—Chondrocytes from OC lesions were less viable, compared with control chondrocytes. Glycosaminoglycan content in the OC group was significantly less than in the control group on all days except day 20. Hydroxyproline content was also significantly less in the OC group on days 10, 20, and 25. Expression of collagen type II was significantly less in the OC group, compared with the control group on all days, whereas expression of collagen type I was significantly greater in the OC group on days 20 and 25. Expression of collagen type X was significantly less in the OC group on all days except day 25.

Conclusions and Clinical Relevance—Chondrocytes from naturally occurring OC lesions of the humeral head of dogs cultured in a 3-dimensional system were less viable and less capable of producing appropriate extracellular matrix molecules than chondrocytes from unaffected dogs. Alterations in the synthetic capabilities of chondrocytes from OC-affected cartilage may be a cause or an effect of the disease process. (Am J Vet Res 2002;63:186–193)

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in American Journal of Veterinary Research

Abstract

Objective—To determine the effects of interleukin (IL)-1β 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-1βml 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-1β group, among groups with different IL-1β concentrations, and among groups with IL-1β added at various time points. Chondrocytes exposed to IL-1β had loss of GAG, increased PGE2 and MMP-3 concentrations, and lack of collagen type-II synthesis. These IL-1β effects appeared to be time and concentration dependent.

Conclusions—Addition of IL-1β to chondrocytes in 3- D gel medium results in time- and concentrationdependent effects on matrix synthesis and degradation and provides an appropriate in vitro model for many of the pathophysiologic events associated with osteoarthritis. (Am J Vet Res 2000;61:766–770)

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in American Journal of Veterinary Research

Abstract

Objective—To assess the cellular, biochemical, and histologic effects of bipolar radiofrequency-generated heat on canine articular cartilage.

Sample Population—Articular cartilage explants (n = 72) from 6 canine cadavers and cultured articular chondrocytes from 5 canine cadavers.

Procedure—Cartilage explants were randomly assigned to receive no treatment or treatment with focal (3 seconds) or diffuse bipolar radiofrequency. Following treatment, methylene blue permeability assay was performed (n = 12) and remaining samples (60) were cultured. Immediately and 5, 10, and 20 days after treatment, cultured explants were assessed for glycosaminoglycan (GAG) and collagen contents, type II collagen and matrix metalloproteinase (MMP)-13 immunoreactivity, and modified Mankin histologic scores. Liquid culture media were collected every 4 days and GAG content measured. Additionally, cultured chondrocytes were exposed for 3 seconds to media preheated to 37°, 45°, or 55°C. Cell viability was determined via 2 different assays immediately and 24 hours after treatment.

Results—Radiofrequency-treated cartilage had reduced permeability and considerable histologic damage, compared with control samples; most treated samples had reduced collagen II staining and increased MMP-13 immunostaining. Compared with other treatments, less GAGs were released from cartilage after diffuse radiofrequency treatment throughout the study period. Cell viability was significantly different between controls and cells treated at 55°C immediately and 24 hours after heat treatment.

Conclusions and Clinical Relevance—In this study, bipolar radiofrequency treatment had detrimental effects on normal articular cartilage cells and extracellular matrix with probable long-term clinical consequences. The usefulness of radiofrequency for treatment of osteoarthritic articular cartilage requires further investigation. ( Am J Vet Res 2004;65:604–609)

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in American Journal of Veterinary Research

Abstract

Objective—To elucidate tissue inhibitor of metalloproteinase (TIMP)-mediated effects on chondrocytes.

Sample Population—Articular cartilage from humeral heads of 6 dogs.

Procedure—Chondrocytes from harvested specimens were cultured in 3-dimensional (3-D) agarose at 106 cells/mL. We prepared 3-D constructs exposed to only tumor necrosis factor (TNF)-α (50 ng/mL). Recombinant human TIMP-1 (255nM), -2 (285nM), or -3 (250nM) was added to liquid media bathing 3-D constructs cultured with TNF-α. Chondrocytes cultured without TIMP or TNF-α served as control samples. Samples of liquid media were collected on days 6, 9, 15, and 21 of culture for evaluation of glycosaminoglycan (GAG) and nitric oxide concentrations. The 3-D constructs were collected on days 9, 15, and 21 for evaluation of GAG, hydroxyproline (HP), and DNA contents.

Results—GAG content in control samples increased significantly during the study, whereas GAG content in 3-D constructs cultured with TNF-α or TNF-α plus TIMP did not increase. On day 9, GAG release from 3-D constructs cultured with TNF-α was significantly higher than that in other constructs. The HP content in control samples increased during the study and was significantly higher than that in all other constructs on day 21. Concentrations of nitric oxide were significantly lower in control samples on day 6, compared with concentrations for all other constructs.

Conclusions and Clinical Relevance—Addition of TIMPs did not counteract suppression of GAG and HP accumulation in 3-D constructs exposed to TNF-α. Apparently, adverse effects on chondrocytes exposed to TNF-α cannot be prevented by addition of TIMP alone. (Am J Vet Res 2004;65:1611–1615)

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in American Journal of Veterinary Research