The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform
A C Hall - One of the best experts on this subject based on the ideXlab platform.
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alpha toxin of staphylococcus aureus is the major cause of in situ chondrocyte death in a bovine Cartilage Explant model of septic arthritis
Orthopaedic Proceedings, 2018Co-Authors: Smith I D M, K M Milto, C Doherty, Amyes S G B, Simpson A H R W, A C HallAbstract:Staphylococcus aureus is a highly virulent pathogen and implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections suggest that alpha-(Hl...
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rapid in situ chondrocyte death induced by staphylococcus aureus toxins in a bovine Cartilage Explant model of septic arthritis
Osteoarthritis and Cartilage, 2013Co-Authors: Idm Smith, K M Milto, S G B Amyes, Ahrw Simpson, J P Winstanley, C Doherty, E Czarniak, A C HallAbstract:Summary Objective To assess in situ chondrocyte viability following exposure to a laboratory strain and clinical isolates of Staphylococcus aureus . Methods Bovine Cartilage Explants were cultured in the presence of S. aureus 8325-4 (laboratory strain), clinical S. aureus isolates or non-infected culture medium of pH values 7.4, 6.4 and 5.4. All clinical isolates were isolated from the joint aspirates of patients presenting with S. aureus -induced septic arthritis (SA). At designated time points, in situ chondrocyte viability was assessed within defined regions-of-interest in the axial and coronal plane following live- and dead-cell image acquisition using the fluorescent probes 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), respectively, and confocal laser-scanning microscopy (CLSM). Cartilage water content, following S. aureus 8325-4 exposure, was obtained by measuring Cartilage wet and dry weights. Results S. aureus 8325-4 and clinical S. aureus isolates rapidly reduced in situ chondrocyte viability (>45% chondrocyte death at 40 h). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins found SZ chondrocytes to be more susceptible to the toxins than DZ chondrocytes. Cartilage water content was not significantly altered compared to non-infected controls. Conclusions Toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes in this experimental model of SA. These data advocate the prompt and thorough removal of bacteria and their toxins during the treatment of SA.
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alpha toxin of staphylococcus aureus is the major cause of in situ chondrocyte death in a bovine Cartilage Explant model of septic arthritis
Journal of Bone and Joint Surgery-british Volume, 2013Co-Authors: Idm Smith, K M Milto, S G B Amyes, Ahrw Simpson, C Doherty, A C HallAbstract:Staphylococcus aureus is a highly virulent pathogen and implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections suggest that alpha-(Hla), beta-(Hlb) and gamma-(Hlg) toxins are key virulence factors, with the ‘pore-forming’ alpha-toxin considered the most potent. Here, we have assessed the influence of alpha-toxin alone on in situ chondrocyte viability. Osteochondral Explants were harvested from the metacarpophalangeal joints of 3-year-old cows and cultured in Dulbecco9s Modified Eagle9s Medium. The flasks were then inoculated with isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-: alpha-toxin only strain) or DU1090 (Hla-Hlb+Hlg+: beta- and gamma-toxin only strain). Explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein-di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death determined. Alpha-toxin-producing S. aureus caused 24.8+/−3.7% chondrocyte death at 18hrs and 44.6+/−7.2% death at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%;p
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a bovine Cartilage Explant model for the study of staphylococcus aureus induced septic arthritis
Orthopaedic Proceedings, 2012Co-Authors: Idm Smith, K M Milto, S G B Amyes, Ahrw Simpson, A C HallAbstract:Staphylococcus aureus is the most common bacterial isolate in septic arthritis. From studies on isolated Cartilage cells, the ‘pore-forming’ alpha and gamma toxins are considered the most virulent factors. However, understanding the response of in situ chondrocytes is important in order to identify new treatments to reduce the extent of Cartilage damage during, and following, episodes of septic arthritis. Animal models can give useful information; however the interpretation of data can be complex because of the strong immune response. Thus, to clarify the role of S. aureus toxins on in situ chondrocytes we have developed a bovine Cartilage Explant model. Metacarpophalangeal joints, from 3-year-old cows, were opened under sterile conditions within 6hrs of slaughter and Cartilage Explants harvested. Explants were placed into flasks containing Dulbecco9s Modified Eagle Medium (DMEM). Aspirates from a patient with septic arthritis of the hip, containing S. aureus, were compared to negative aspirates (no bacterial growth) from a patient with an inflamed knee joint (controls). The Explants were incubated at 37 degrees Celsius and stained after 18, 24 and 40hrs with the fluorescent probes chloromethylfluorescein di-acetate and propidium iodide (10 micromolar each) to label living chondrocytes green and dead cells red respectively. Following imaging of Cartilage by confocal laser scanning microscopy, the percentage cell death at each time point was obtained using Volocity 4 software. There was no detectable change in chondrocyte viability ( These results show that a strain of S. aureus capable of manifesting clinical disease exerts a potent effect on in situ chondrocytes. In the absence of an immune response, chondrocyte death was purely the result of the bacteria and their products. This bovine Cartilage Explant model could therefore be useful for studying the effects of S. aureus on chondrocyte behaviour and, ultimately, Cartilage integrity.
A P Hollander - One of the best experts on this subject based on the ideXlab platform.
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retinoic acid induced type ii collagen degradation does not correlate with matrix metalloproteinase activity in Cartilage Explant cultures
Arthritis & Rheumatism, 1999Co-Authors: J S Price, S Wangweigand, R Bohne, L D Kozaci, A P HollanderAbstract:OBJECTIVE: To determine the role of matrix metalloproteinases (MMPs) in retinoic acid (RetA)-induced degradation of type II collagen in Cartilage. METHODS: Bovine nasal Cartilage Explants were cultured with 1 microM RetA or in 3 nM interleukin-1alpha (IL-1alpha). Release of proteoglycan and type II collagen into the medium was measured by colorimetric assay and immunoassay, respectively. MMP activity in the medium was determined using a quenched fluorescent substrate assay, while specific collagenases were identified by Western immunoblotting. In some cases the effects of low molecular mass synthetic MMP inhibitors and serum on collagen degradation were studied. RESULTS: RetA promoted maximal breakdown of type II collagen after 4 or 5 weeks in culture, compared with 3 weeks in culture with IL-1alpha. In IL-1alpha-stimulated cultures, collagen degradation was coincident with a large increase in MMP activity in the culture medium, whereas in RetA-stimulated cultures, there was only a small increase. In Western immunoblots of culture media containing RetA, prointerstitial collagenase and active collagenase 3 were sometimes detected, but not in all experiments. In IL-1alpha cultures, active interstitial collagenase was always detected, and active collagenase 3 was detectable in some experiments. Neutrophil collagenase was not detected in any cultures. IL-1alpha-stimulated collagen degradation was effectively inhibited by a potent, broad-spectrum inhibitor of MMPs, whereas it was poorly inhibited by a weak MMP inhibitor. The same 2 compounds were both only weak inhibitors of RetA-induced collagen degradation. When fetal calf serum was included in Cartilage cultures, MMP activity in the culture medium was reduced to low levels. This resulted in a marked inhibition of IL-1alpha-induced type II collagen degradation, whereas there was no inhibition of RetA-induced collagen degradation. CONCLUSION: Unlike IL-1alpha, RetA induces degradation of type II collagen in Cartilage Explants by a mechanism that is mainly independent of those MMPs that can be detected in the culture medium.
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degradation of type ii collagen but not proteoglycan correlates with matrix metalloproteinase activity in Cartilage Explant cultures
Arthritis & Rheumatism, 1997Co-Authors: Didem L Kozaci, David J Buttle, A P HollanderAbstract:OBJECTIVE: To determine the contribution of certain matrix metalloproteinases (MMPs) to the degradation of proteoglycan and type II collagen in Cartilage. METHODS: Bovine nasal and articular Cartilage Explants were cultured with recombinant human interleukin-1 alpha (IL-1 alpha) for up to 4 weeks. Release of proteoglycan and type II collagen into the medium was determined by colorimetric assay and immunoassay, respectively. The activity of MMPs in the medium was assayed using a quenched fluorescent substrate, as well as with a collagen fibril assay, by zymography, and in Western immunoblots. In some experiments, the effects of specific MMP inhibitors on type II collagen degradation were studied. RESULTS: In cultures of nasal Cartilage with IL-1 alpha, almost all the proteoglycan was released within the first week, whereas there was no detectable release of type II collagen for the first 2 weeks of culture. A rapid period of almost complete dissolution of the collagen occurred in the third or fourth week. MMP activity measured using a quenched fluorescent substrate was negligible during the first 2 weeks of culture but was substantially increased in the third week of culture, at the time of collagen degradation. Similarly, there was a large increase in collagenolytic activity (by collagen fibril assay) and gelatinolytic activity (by zymography) during the third week of culture. Articular Cartilage cultured with IL-1 alpha lost proteoglycan progressively during the 4-week period; however, there was no loss of type II collagen from the matrix in that time and no significant increase in MMP activity. The loss of type II collagen from nasal Cartilage stimulated with IL-1 alpha was inhibited by BB87, an inhibitor of both collagenases and gelatinases, and by BB3003, a selective inhibitor of gelatinase A. In Western immunoblots, procollagenase and active interstitial collagenase could be readily detected in nasal Cartilage cultures. Some procollagenase 3 and active collagenase 3 was also shown to be present. CONCLUSION: MMP activity correlates with degradation of type II collagen, but not proteoglycan, in Cartilage cultures. Interstitial collagenase, collagenase 3, and gelatinases are all likely to contribute to cleavage and removal of collagen from the Cartilage matrix. The proteinase(s) responsible for aggrecan breakdown remains unclear.
Christopher J Handley - One of the best experts on this subject based on the ideXlab platform.
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distribution of newly synthesized aggrecan in Explant cultures of bovine Cartilage treated with retinoic acid
Matrix Biology, 2002Co-Authors: Christopher J Handley, Mirna Z Ilic, Gavin M Winter, J M Ross, Anthony C Poole, Clem H RobinsonAbstract:This paper describes temporal changes in the metabolism and distribution of newly synthesized aggrecan and the organization of the extracellular matrix when Explant cultures of articular Cartilage maintained in the presence of fetal calf serum were exposed to retinoic acid for varying periods of time. Explant cultures of articular Cartilage were incubated with radiolabeled sulfate prior to exposure to retinoic acid. The radiolabeled and chemical aggrecan present in the tissue and appearing in the culture medium was studied kinetically. Changes in the localization of radiolabeled aggrecan within the extracellular matrix were monitored by autoradiography in relation to type VI collagen distribution in the extracellular matrix. In control cultures where tissue levels of aggrecan remain constant the newly synthesized aggrecan remained closely associated with the territorial matrix surrounding the chondrocytes. Exposure of cultures to retinoic acid for the duration of the experiment, resulted in the extensive loss of aggrecan from the tissue and the redistribution of the remaining radiolabeled aggrecan from the chondron and territorial matrix into the inter-territorial matrix. These changes preceded alterations in the organization of type VI collagen in the extracellular matrix that involved the remodeling of the chondron and the appearance of type VI collagen in the inter-territorial matrix; there was also evidence of chondrocyte proliferation and clustering. In Cartilage Explant cultures exposed to retinoic acid for 24 h there was no loss of aggrecan from the matrix but there was an extensive redistribution of the radiolabeled aggrecan into the inter-territorial matrix. This work shows that maintenance of the structure and organization of the extracellular matrix that comprises the chondron and pericellular microenvironment of chondrocytes in articular Cartilage is important for the regulation of the distribution of newly synthesized aggrecan monomers within the tissue.
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highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular Cartilage Explant cultures
Matrix Biology, 2002Co-Authors: Shannon E Munteanu, Mirna Z Ilic, Christopher J HandleyAbstract:: The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular Cartilage Explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular Cartilage Explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by Explant cultures of articular Cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular Cartilage Explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular Cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.
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calcium pentosan polysulfate inhibits the catabolism of aggrecan in articular Cartilage Explant cultures
Arthritis & Rheumatism, 2000Co-Authors: Shannon E Munteanu, Mirna Z Ilic, Christopher J HandleyAbstract:Objective. The catabolism of aggrecan and loss of aggrecan fragments from articular Cartilage is a key event in the pathogenesis of arthritic diseases such as osteoarthritis. The catabolism of aggrecan is mediated by the specific proteolytic activity termed aggrecanase. The aim of this study was to investigate the effect of the chondroprotective agent calcium pentosan polysulfate (CaPPS) on the aggrecanase-mediated catabolism of aggrecan. Methods. The catabolism of 35 S-labeled aggrecan and loss of tissue glycosaminoglycans (GAGs) were investigated using bovine articular Cartilage Explant cultures maintained in medium containing varying concentrations of CaPPS (1‐100 mg/ml) in the presence or absence of 10 26 M retinoic acid or 7 ng/ml recombinant human interleukin-1a (rHuIL-1a). In addition, the effect of CaPPS on the degradation of aggrecan monomers by aggrecanase activity present in conditioned medium from joint capsule Explant cultures was investigated. Results. CaPPS inhibited the catabolism of 35 Slabeled aggrecan in a dose-dependent manner, particularly when retinoic acid or rHuIL-1a was used to stimulate aggrecan catabolism. These effects were reflected in the tissue levels of GAG remaining in these cultures at the end of the experiment. CaPPS inhibited the degradation of aggrecan monomers by soluble aggrecanase activity. Conclusion. CaPPS inhibits the catabolism of aggrecan by articular Cartilage in a dose-dependent manner, particularly when the processes responsible for aggrecan loss are stimulated. This effect occurs, at least in part, through direct inhibition of aggrecanase activity. CaPPS did not adversely affect overall chondrocyte metabolism, as shown by the incorporation of 35 Ssulfate and 3 H-leucine into macromolecules and by lactate production in Cartilage Explant cultures.
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metabolic processing of newly synthesized link protein in bovine articular Cartilage Explant cultures
Matrix Biology, 1999Co-Authors: Angus M Tester, Mirna Z Ilic, Clem H Robinson, Christopher J HandleyAbstract:Abstract In Explant cultures of articular Cartilage from cattle of different ages radiolabeled leucine was shown to be incorporated into link proteins 1, 2 and 3. The newly synthesized link proteins were incorporated into and lost from the Cartilage extracellular matrix with time. The levels of radiolabeled link proteins 1 and 2 remaining in the matrix declined over the culture period, but there was an initial increase in the amount of radiolabeled link protein 3, before its level declined. The turnover time of the radiolabeled link proteins 1 and 2 were similar, indicating that neither link protein was preferentially processed to generate link protein 3, nor lost from the extracellular matrix. The majority of the radiolabeled link protein lost from the Cartilage matrix could not be recovered from the culture medium, suggesting that turnover of the radiolabeled aggrecan complexes involves the newly synthesized link protein being internalized by the chondrocytes. Inclusion of cytotoxic proteinase inhibitors to the culture medium resulted in a marked decrease in the rate of loss of link protein from the Cartilage, suggesting that the catabolism of link protein is cell-mediated and dependent on metabolically active cells.
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effect of insulin like growth factor i on the synthesis and distribution of link protein and hyaluronan in Explant cultures of articular Cartilage
Biochimica et Biophysica Acta, 1992Co-Authors: Andrea J Curtis, Christopher J Handley, Chee Keng Ng, Clem H RobinsonAbstract:Abstract Addition of 20% (v/v) fetal calf serum or insulin-like growth factor-I (IGF-I; 20 ng/ml) to the medium of Explant cultures of adult articular Cartilage resulted in an increased rate of synthesis of the three components of the proteoglycan aggregate — namely link protein, hyaluronan and aggrecan. Fetal calf serum also stimulated the synthesis of other matrix proteins by articular Cartilage compared with tissue mantained in medium alone or medium containing IGF-I. Although addition of fetal calf serum or IGF-I to the culture medium of Cartilage Explant cultures stimulated both hyaluronan and aggrecan synthesis, no change in the distribution of these two macromolecules between tissue and medium was observed. Approx. 50% of the newly synthesized hyaluronan was retained by the tissue compared to 93% of the labelled aggrecan. Culture conditions had some influence on the distribution of link protein, in cultures maintained in medium alone or in medium containing IGF-I, less than 12% of the newly synthesized link protein was lost to the medium of the cultures. However, in cultures maintained with fetal calf serum between 25% and 19% of the radiolabelled link protein was lost from the matrix of the Explants. This work suggests that the chondrocyte synthesizes the macromolecules that make up the proteoglycan aggregate in a co-ordinated manner, thereby retaining the relative amounts of each component of this functionally important complex.
Idm Smith - One of the best experts on this subject based on the ideXlab platform.
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rapid in situ chondrocyte death induced by staphylococcus aureus toxins in a bovine Cartilage Explant model of septic arthritis
Osteoarthritis and Cartilage, 2013Co-Authors: Idm Smith, K M Milto, S G B Amyes, Ahrw Simpson, J P Winstanley, C Doherty, E Czarniak, A C HallAbstract:Summary Objective To assess in situ chondrocyte viability following exposure to a laboratory strain and clinical isolates of Staphylococcus aureus . Methods Bovine Cartilage Explants were cultured in the presence of S. aureus 8325-4 (laboratory strain), clinical S. aureus isolates or non-infected culture medium of pH values 7.4, 6.4 and 5.4. All clinical isolates were isolated from the joint aspirates of patients presenting with S. aureus -induced septic arthritis (SA). At designated time points, in situ chondrocyte viability was assessed within defined regions-of-interest in the axial and coronal plane following live- and dead-cell image acquisition using the fluorescent probes 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), respectively, and confocal laser-scanning microscopy (CLSM). Cartilage water content, following S. aureus 8325-4 exposure, was obtained by measuring Cartilage wet and dry weights. Results S. aureus 8325-4 and clinical S. aureus isolates rapidly reduced in situ chondrocyte viability (>45% chondrocyte death at 40 h). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins found SZ chondrocytes to be more susceptible to the toxins than DZ chondrocytes. Cartilage water content was not significantly altered compared to non-infected controls. Conclusions Toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes in this experimental model of SA. These data advocate the prompt and thorough removal of bacteria and their toxins during the treatment of SA.
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alpha toxin of staphylococcus aureus is the major cause of in situ chondrocyte death in a bovine Cartilage Explant model of septic arthritis
Journal of Bone and Joint Surgery-british Volume, 2013Co-Authors: Idm Smith, K M Milto, S G B Amyes, Ahrw Simpson, C Doherty, A C HallAbstract:Staphylococcus aureus is a highly virulent pathogen and implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections suggest that alpha-(Hla), beta-(Hlb) and gamma-(Hlg) toxins are key virulence factors, with the ‘pore-forming’ alpha-toxin considered the most potent. Here, we have assessed the influence of alpha-toxin alone on in situ chondrocyte viability. Osteochondral Explants were harvested from the metacarpophalangeal joints of 3-year-old cows and cultured in Dulbecco9s Modified Eagle9s Medium. The flasks were then inoculated with isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-: alpha-toxin only strain) or DU1090 (Hla-Hlb+Hlg+: beta- and gamma-toxin only strain). Explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein-di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death determined. Alpha-toxin-producing S. aureus caused 24.8+/−3.7% chondrocyte death at 18hrs and 44.6+/−7.2% death at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%;p
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a bovine Cartilage Explant model for the study of staphylococcus aureus induced septic arthritis
Orthopaedic Proceedings, 2012Co-Authors: Idm Smith, K M Milto, S G B Amyes, Ahrw Simpson, A C HallAbstract:Staphylococcus aureus is the most common bacterial isolate in septic arthritis. From studies on isolated Cartilage cells, the ‘pore-forming’ alpha and gamma toxins are considered the most virulent factors. However, understanding the response of in situ chondrocytes is important in order to identify new treatments to reduce the extent of Cartilage damage during, and following, episodes of septic arthritis. Animal models can give useful information; however the interpretation of data can be complex because of the strong immune response. Thus, to clarify the role of S. aureus toxins on in situ chondrocytes we have developed a bovine Cartilage Explant model. Metacarpophalangeal joints, from 3-year-old cows, were opened under sterile conditions within 6hrs of slaughter and Cartilage Explants harvested. Explants were placed into flasks containing Dulbecco9s Modified Eagle Medium (DMEM). Aspirates from a patient with septic arthritis of the hip, containing S. aureus, were compared to negative aspirates (no bacterial growth) from a patient with an inflamed knee joint (controls). The Explants were incubated at 37 degrees Celsius and stained after 18, 24 and 40hrs with the fluorescent probes chloromethylfluorescein di-acetate and propidium iodide (10 micromolar each) to label living chondrocytes green and dead cells red respectively. Following imaging of Cartilage by confocal laser scanning microscopy, the percentage cell death at each time point was obtained using Volocity 4 software. There was no detectable change in chondrocyte viability ( These results show that a strain of S. aureus capable of manifesting clinical disease exerts a potent effect on in situ chondrocytes. In the absence of an immune response, chondrocyte death was purely the result of the bacteria and their products. This bovine Cartilage Explant model could therefore be useful for studying the effects of S. aureus on chondrocyte behaviour and, ultimately, Cartilage integrity.
Clem H Robinson - One of the best experts on this subject based on the ideXlab platform.
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distribution of newly synthesized aggrecan in Explant cultures of bovine Cartilage treated with retinoic acid
Matrix Biology, 2002Co-Authors: Christopher J Handley, Mirna Z Ilic, Gavin M Winter, J M Ross, Anthony C Poole, Clem H RobinsonAbstract:This paper describes temporal changes in the metabolism and distribution of newly synthesized aggrecan and the organization of the extracellular matrix when Explant cultures of articular Cartilage maintained in the presence of fetal calf serum were exposed to retinoic acid for varying periods of time. Explant cultures of articular Cartilage were incubated with radiolabeled sulfate prior to exposure to retinoic acid. The radiolabeled and chemical aggrecan present in the tissue and appearing in the culture medium was studied kinetically. Changes in the localization of radiolabeled aggrecan within the extracellular matrix were monitored by autoradiography in relation to type VI collagen distribution in the extracellular matrix. In control cultures where tissue levels of aggrecan remain constant the newly synthesized aggrecan remained closely associated with the territorial matrix surrounding the chondrocytes. Exposure of cultures to retinoic acid for the duration of the experiment, resulted in the extensive loss of aggrecan from the tissue and the redistribution of the remaining radiolabeled aggrecan from the chondron and territorial matrix into the inter-territorial matrix. These changes preceded alterations in the organization of type VI collagen in the extracellular matrix that involved the remodeling of the chondron and the appearance of type VI collagen in the inter-territorial matrix; there was also evidence of chondrocyte proliferation and clustering. In Cartilage Explant cultures exposed to retinoic acid for 24 h there was no loss of aggrecan from the matrix but there was an extensive redistribution of the radiolabeled aggrecan into the inter-territorial matrix. This work shows that maintenance of the structure and organization of the extracellular matrix that comprises the chondron and pericellular microenvironment of chondrocytes in articular Cartilage is important for the regulation of the distribution of newly synthesized aggrecan monomers within the tissue.
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metabolic processing of newly synthesized link protein in bovine articular Cartilage Explant cultures
Matrix Biology, 1999Co-Authors: Angus M Tester, Mirna Z Ilic, Clem H Robinson, Christopher J HandleyAbstract:Abstract In Explant cultures of articular Cartilage from cattle of different ages radiolabeled leucine was shown to be incorporated into link proteins 1, 2 and 3. The newly synthesized link proteins were incorporated into and lost from the Cartilage extracellular matrix with time. The levels of radiolabeled link proteins 1 and 2 remaining in the matrix declined over the culture period, but there was an initial increase in the amount of radiolabeled link protein 3, before its level declined. The turnover time of the radiolabeled link proteins 1 and 2 were similar, indicating that neither link protein was preferentially processed to generate link protein 3, nor lost from the extracellular matrix. The majority of the radiolabeled link protein lost from the Cartilage matrix could not be recovered from the culture medium, suggesting that turnover of the radiolabeled aggrecan complexes involves the newly synthesized link protein being internalized by the chondrocytes. Inclusion of cytotoxic proteinase inhibitors to the culture medium resulted in a marked decrease in the rate of loss of link protein from the Cartilage, suggesting that the catabolism of link protein is cell-mediated and dependent on metabolically active cells.
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effect of insulin like growth factor i on the synthesis and distribution of link protein and hyaluronan in Explant cultures of articular Cartilage
Biochimica et Biophysica Acta, 1992Co-Authors: Andrea J Curtis, Christopher J Handley, Chee Keng Ng, Clem H RobinsonAbstract:Abstract Addition of 20% (v/v) fetal calf serum or insulin-like growth factor-I (IGF-I; 20 ng/ml) to the medium of Explant cultures of adult articular Cartilage resulted in an increased rate of synthesis of the three components of the proteoglycan aggregate — namely link protein, hyaluronan and aggrecan. Fetal calf serum also stimulated the synthesis of other matrix proteins by articular Cartilage compared with tissue mantained in medium alone or medium containing IGF-I. Although addition of fetal calf serum or IGF-I to the culture medium of Cartilage Explant cultures stimulated both hyaluronan and aggrecan synthesis, no change in the distribution of these two macromolecules between tissue and medium was observed. Approx. 50% of the newly synthesized hyaluronan was retained by the tissue compared to 93% of the labelled aggrecan. Culture conditions had some influence on the distribution of link protein, in cultures maintained in medium alone or in medium containing IGF-I, less than 12% of the newly synthesized link protein was lost to the medium of the cultures. However, in cultures maintained with fetal calf serum between 25% and 19% of the radiolabelled link protein was lost from the matrix of the Explants. This work suggests that the chondrocyte synthesizes the macromolecules that make up the proteoglycan aggregate in a co-ordinated manner, thereby retaining the relative amounts of each component of this functionally important complex.