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P46. Changes in glycosaminoglycan epitopes in acute knee trauma synovial fluids and in a lapine model of osteoarthritis (OA)
Abstracts
from the Joint Meeting,
September
1992
127
not HAP. Furthermore, a trace of magnesium was identified in the crystals. A method to isolate the crystals without modification of the mineral phase was developed to facilitate mineral phase identification. Initial results from analytical studies of these preparations using electron and x-ray diffraction and raman spectroscopy, suggest the crystals are a magnesium whitlockite. The rhombohedral habit of this mineral would describe the mcrphology of these crystals. This mineral phase has been reported in a number of tissues including dental calculi, kidney stones and, more rarely, fibrocartilage. This would be however, the first report in articular cartilage. It is anticipated that with knowledge of the mineral phase it will now be possible to determine their mode of formation and deposition.
P43. The effects of impact loading chondmcytes in vitro LA Thomson, JE Jeffrey and RM Aspden Department of Orthopaedics, University
on articular
of Aberdeen,
cartilage
Aberdeen
Joint impact injuries are commonly encountered following falls from heights or car accidents in which the knee of an occupant strikes the dashboard. Secondary arthritis is a common consequence of such injuries. This study is a preliminary investigation into the effects of a single impact load on metabolism of articular cartilage chondrocytes. Full-depth samples of bovine articular cartilage are maintained in culture. Atter 24 hours they are subjected to a single impact load from a falling mass of between 100 g to 1 kg dropped from a height of up to 50 cm. To study the breakdown of matrix components following impact the samples are preincubated in medium containing 3H-leucine and %Odand media collected at intervals over the days succeeding impact. Incorporation of proteins and glycosaminoglycans is studied in different experiments using the same isotopes added as a 4 h pulse at intervals of up to 2 weeks following impact. Surprisingly we have so far found very little response in the impacted samples compared with unloaded controls. The release of prelabelled components appears to be reduced in the impacted samples. This reduction increases with height of drop for a given mass and a greater reduction is found for a larger mass. Incorporation rates following impact are little changed and no change has been found in the viability of the chondrocytes. Despite gross disruption of the tissue there appears to be no change in the cells. If confirmed, these results suggest that the chondrocyte is remarkably insensitive to its physical environment.
Novel articular cartilage structure P44. domestica M Morrison, MT Bayliss’, MWJ Ferguson** Department of Anatomy, UWCC, Cardiff, Rheumatology, Hnmmersmith, London and Structural
Biology,
University
in Monodelphis and C W Archer ‘Kennedy “Depf of
Inst of Cell and
of Manchester
It is traditionally held that adult articular cartilage presents a smooth lubricated surface facilitating good joint mobility. Here, we show that in the distal joints of the limbs of the South American Opposum Monodelphis domestica that this notion doe not appear to hold. Sections taken from wax, fresh frozen and resin embedded matr:ial show that the articular surface possesses a series of nodules, each enclosing a single rounded chondrocyte. There is an absence of flat discoid chondrocytes normally present in articular surfaces. These nodules have also been observed using the scanning electron microscope and the Electroscan. It is interesting to note, however, that the joints more proximal to the body axis, ie. knee, show a traditional articular cartilage structure. It is difficult to envisage the mechanica of articulation in these distal joints and, indeed, the selective advantage that such a structure may achieve.
P45. Localiaation and activity of catheapin B and ita modulation by ILlg in human articular chondrogtcs in vitro using fluorescence methods MHM Meijjrs, MEJ Billingham’, RGG Russell and RAD gunning” Department of Human Metabolism and Clinical Biochemistry, Sheffield University Medical School, Sheffield, *Osteoarthritis Research, Lilly Research Centre Ltd, Windleshnm, Surrey and “Division of Biomedical Sciences, Sheffield City Polytechnic The regulation of cysteine proteinases, such as cathepsin B, has received great attention in recent years because of their possible role in the pathogenesis of rheumatic diseases, This study shows the use of fluorescence methods to investigate cathepsin B activity in human articular chondrocytes in vitro. Cathepsin B activity was Iocalised in unfixed cultured chondrocytes by formation of a final fluorescent reaction product using 5nitrosalicylaldehyde as coupling agent and N-CBZ-Ala-Arg-Argrnethoxy-2-naphtylamide as substrate for cathepsin B after 30 min incubation. Addition of E-64 &nM), a specific cysteine proteinase inhibitor, verified the specificity of this reaction. Cathepsin B activity in cell lysates and supernatants was measured using the substrate N-CBZ-Arg-Arg-4-methyl-7coumarin. Addition of ILlb significantly stimulated cathepsin B activity in cell lysates over the range of 10 U/ml to 100 U/ml while enzyme activity in cell supernatants was not detectable, even on stimulation with ILlb. E-64 (5mM) completely inhibited both basal and ILlb-induced cathepsin B activity. This observation suggests a possible involvement of ILlb in the regulation of cathepsin B activity in human articular cartilage in vitro. Increase of such proteolytic enzymes might lead to destruction of bone and cartilage associated with arthritis.
P46. Changes in glycosaminoglycan epitopes in acute knee trauma synovial fluids and in a lapine model of osteoarthritis @AI P Hazell, J Lewthwaite, C Dent’, M Bayliss and T Hardingham Kennedy Institue of Rheumatology, London W6 7DW and ‘University of Wules College of Medicine, Curdiff CF2 ISZ Proteoglycans (PCs) are present in high concentrations in articular cartilage and are essential for its biomechanical properties. In previous work it was shown that in early experimental canine OA there was an increase in the expression of chondroitin sulphate (CS) epitopes in the cartilage of operated compared with contralateral control joints [ll. In this study epitopes of CS and keratan sulphate (KS) were detected and measured using ELISA techniques in paired synovial fluid samples from the traumatised (ACL rupture and/or meniscal tears) and ‘normal’ knee joints of patients. Elevated levels of CS epitopes were detected in proteoglycans in samples from the traumatised knee in the majority of patients together with a decrease in KS epitope levels. In addition, comparisons were made with levels of CS epitopes in an experimental model of OA achieved by partial medial meniscectomy in the rabbit. An increase in intensity was observed on immunoblots for PGs extracted from the operated joint with antibodies 383 and M022.5. In both areas of study, the changes in CS epitope expression may reflect the state of hypermetabolic activity seen in the early stages of articular cartilage responses in OA. These epitopes may therefore be useful as markers in following the progression of joint disease or monitoring the results of pharmacological or surgical intervention. 1. Caterson B et al. (1990) J Cell Sci 97:411417.
from the Joint Meeting,
September
1992
127
not HAP. Furthermore, a trace of magnesium was identified in the crystals. A method to isolate the crystals without modification of the mineral phase was developed to facilitate mineral phase identification. Initial results from analytical studies of these preparations using electron and x-ray diffraction and raman spectroscopy, suggest the crystals are a magnesium whitlockite. The rhombohedral habit of this mineral would describe the mcrphology of these crystals. This mineral phase has been reported in a number of tissues including dental calculi, kidney stones and, more rarely, fibrocartilage. This would be however, the first report in articular cartilage. It is anticipated that with knowledge of the mineral phase it will now be possible to determine their mode of formation and deposition.
P43. The effects of impact loading chondmcytes in vitro LA Thomson, JE Jeffrey and RM Aspden Department of Orthopaedics, University
on articular
of Aberdeen,
cartilage
Aberdeen
Joint impact injuries are commonly encountered following falls from heights or car accidents in which the knee of an occupant strikes the dashboard. Secondary arthritis is a common consequence of such injuries. This study is a preliminary investigation into the effects of a single impact load on metabolism of articular cartilage chondrocytes. Full-depth samples of bovine articular cartilage are maintained in culture. Atter 24 hours they are subjected to a single impact load from a falling mass of between 100 g to 1 kg dropped from a height of up to 50 cm. To study the breakdown of matrix components following impact the samples are preincubated in medium containing 3H-leucine and %Odand media collected at intervals over the days succeeding impact. Incorporation of proteins and glycosaminoglycans is studied in different experiments using the same isotopes added as a 4 h pulse at intervals of up to 2 weeks following impact. Surprisingly we have so far found very little response in the impacted samples compared with unloaded controls. The release of prelabelled components appears to be reduced in the impacted samples. This reduction increases with height of drop for a given mass and a greater reduction is found for a larger mass. Incorporation rates following impact are little changed and no change has been found in the viability of the chondrocytes. Despite gross disruption of the tissue there appears to be no change in the cells. If confirmed, these results suggest that the chondrocyte is remarkably insensitive to its physical environment.
Novel articular cartilage structure P44. domestica M Morrison, MT Bayliss’, MWJ Ferguson** Department of Anatomy, UWCC, Cardiff, Rheumatology, Hnmmersmith, London and Structural
Biology,
University
in Monodelphis and C W Archer ‘Kennedy “Depf of
Inst of Cell and
of Manchester
It is traditionally held that adult articular cartilage presents a smooth lubricated surface facilitating good joint mobility. Here, we show that in the distal joints of the limbs of the South American Opposum Monodelphis domestica that this notion doe not appear to hold. Sections taken from wax, fresh frozen and resin embedded matr:ial show that the articular surface possesses a series of nodules, each enclosing a single rounded chondrocyte. There is an absence of flat discoid chondrocytes normally present in articular surfaces. These nodules have also been observed using the scanning electron microscope and the Electroscan. It is interesting to note, however, that the joints more proximal to the body axis, ie. knee, show a traditional articular cartilage structure. It is difficult to envisage the mechanica of articulation in these distal joints and, indeed, the selective advantage that such a structure may achieve.
P45. Localiaation and activity of catheapin B and ita modulation by ILlg in human articular chondrogtcs in vitro using fluorescence methods MHM Meijjrs, MEJ Billingham’, RGG Russell and RAD gunning” Department of Human Metabolism and Clinical Biochemistry, Sheffield University Medical School, Sheffield, *Osteoarthritis Research, Lilly Research Centre Ltd, Windleshnm, Surrey and “Division of Biomedical Sciences, Sheffield City Polytechnic The regulation of cysteine proteinases, such as cathepsin B, has received great attention in recent years because of their possible role in the pathogenesis of rheumatic diseases, This study shows the use of fluorescence methods to investigate cathepsin B activity in human articular chondrocytes in vitro. Cathepsin B activity was Iocalised in unfixed cultured chondrocytes by formation of a final fluorescent reaction product using 5nitrosalicylaldehyde as coupling agent and N-CBZ-Ala-Arg-Argrnethoxy-2-naphtylamide as substrate for cathepsin B after 30 min incubation. Addition of E-64 &nM), a specific cysteine proteinase inhibitor, verified the specificity of this reaction. Cathepsin B activity in cell lysates and supernatants was measured using the substrate N-CBZ-Arg-Arg-4-methyl-7coumarin. Addition of ILlb significantly stimulated cathepsin B activity in cell lysates over the range of 10 U/ml to 100 U/ml while enzyme activity in cell supernatants was not detectable, even on stimulation with ILlb. E-64 (5mM) completely inhibited both basal and ILlb-induced cathepsin B activity. This observation suggests a possible involvement of ILlb in the regulation of cathepsin B activity in human articular cartilage in vitro. Increase of such proteolytic enzymes might lead to destruction of bone and cartilage associated with arthritis.
P46. Changes in glycosaminoglycan epitopes in acute knee trauma synovial fluids and in a lapine model of osteoarthritis @AI P Hazell, J Lewthwaite, C Dent’, M Bayliss and T Hardingham Kennedy Institue of Rheumatology, London W6 7DW and ‘University of Wules College of Medicine, Curdiff CF2 ISZ Proteoglycans (PCs) are present in high concentrations in articular cartilage and are essential for its biomechanical properties. In previous work it was shown that in early experimental canine OA there was an increase in the expression of chondroitin sulphate (CS) epitopes in the cartilage of operated compared with contralateral control joints [ll. In this study epitopes of CS and keratan sulphate (KS) were detected and measured using ELISA techniques in paired synovial fluid samples from the traumatised (ACL rupture and/or meniscal tears) and ‘normal’ knee joints of patients. Elevated levels of CS epitopes were detected in proteoglycans in samples from the traumatised knee in the majority of patients together with a decrease in KS epitope levels. In addition, comparisons were made with levels of CS epitopes in an experimental model of OA achieved by partial medial meniscectomy in the rabbit. An increase in intensity was observed on immunoblots for PGs extracted from the operated joint with antibodies 383 and M022.5. In both areas of study, the changes in CS epitope expression may reflect the state of hypermetabolic activity seen in the early stages of articular cartilage responses in OA. These epitopes may therefore be useful as markers in following the progression of joint disease or monitoring the results of pharmacological or surgical intervention. 1. Caterson B et al. (1990) J Cell Sci 97:411417.