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- Author or Editor: Keiichi Kuroki x
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Abstract
Objective—To determine the effects of interleukin (IL)-1 and tumor necrosis factor (TNF)-α on canine chondrocytes cultured in an agarose-based 3-dimensional (3-D) system.
Sample Population—Humeral head articular cartilage chondrocytes obtained from 6 adult dogs.
Procedure—Chondrocytes were cultured in a 3-D system for ≤ 12 days in serum-free medium with IL-1α, IL-1β, or TNF-α at concentrations of 20, 50, or 100 ng/mL. After 1, 3, 6, and 12 days, glycosaminoglycan (GAG) concentrations in 3-D constructs; nitric oxide and prostaglandin E2 (PGE2) concentrations in media samples; and relative expressions of selected genes, including metalloproteinase (MMP)-13 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, were evaluated. Control specimens were comprised of chondrocytes cultured without proinflammatory cytokines.
Results—In control 3-D constructs, GAG content was significantly higher than for all other constructs. Compared with control values, relative expressions of MMP-13, TIMP-1, and TIMP-2 genes in the IL-1β (50 ng/mL) group were significantly higher at day 1; at all evaluations, media concentrations of nitric oxide were significantly higher in all TNF-α–treated cultures; and concentrations of PGE2 in media samples were significantly higher in the IL-1β (50 ng/mL) and IL-1β (100 ng/mL) groups at days 1 and 3, in the IL-1β (100 ng/mL) group at day 6, and in all TNF-α groups at days 1, 3, and 6.
Conclusions and Clinical Relevance—Results suggested that TNF-α more readily induces production of nitric oxide and PGE2 by canine chondrocytes, compared with IL-1β. In vitro, IL-1α appeared to have a minimal effect on canine chondrocytes. (Am J Vet Res 2005;66:1187–1196)
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)
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)
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)
Abstract
Objective—To investigate the presence or absence of Toll-like receptor (TLR)-2 and TLR-4 in synovial tissues collected from stifle joints (SJs) of dogs with or without osteoarthritis.
Animals—21 purpose-bred research dogs, 3 client-owned dogs with SJ osteoarthritis, and 3 dogs without SJ osteoarthritis.
Procedures—Research dogs underwent arthroscopic surgery in 1 SJ to induce osteoarthritis via cranial cruciate ligament transection (CrCLt; n = 5), femoral condylar articular cartilage groove creation (6), or release of the caudal horn of the medial meniscus (5); 5 dogs underwent sham surgery. Synovial tissue specimens were obtained from both stifle joints of each dog 12 weeks after surgery, and TLR-2 and TLR-4 gene expression were determined via real-time reverse transcription PCR assays. Expression of TLR-4 protein was determined via an immunofluorescence technique in additional specimens obtained from osteoarthritic SJs of dogs with cranial cruciate ligament insufficiency and from dogs with nonosteoarthritic SJs.
Results—Synovial tissues from CrCLt-treated joints had significantly higher TLR-4 gene expression, compared with the contralateral control SJs or any other joint group. TLR-2 gene expression did not differ significantly among groups. Toll-like receptor-4 protein was detected in synovial tissues of osteoarthritic SJs but was rarely evident in nonosteoarthritic SJs.
Conclusions and Clinical Relevance—Increased TLR-4 gene expression in the synovial tissue of SJs with osteoarthritis secondary to CrCLt suggests that activation of innate immunity may play a role in the pathophysiology of SJ osteoarthritis in at least a subset of dogs.
