Effect of collagen turnover and matrix metalloproteinase activity on healing of venous leg ulcers.

BACKGROUND: The presence of fibrous tissue in poorly healing venous leg ulcers suggests abnormal collagen metabolism. The aim was to determine whether there were differences in collagen turnover and matrix metalloproteinase (MMP) activity between ulcers that healed, those that did not heal and normal skin. METHODS: Biopsies were taken from the ulcers of 12 patients whose venous ulcers went on to heal and 15 patients whose ulcers failed to heal despite 12 months of compression bandaging. Biopsies were taken from 15 normal controls. Collagen turnover (collagen III N-terminal propeptide (PIIINP) and degraded collagen), and total MMP, MMP-1 and MMP-3 activities were measured. RESULTS: PIIINP and degraded collagen levels were higher in ulcers that healed compared with lesions that failed to heal (P = 0.005 and P < 0.001 respectively) and normal skin (P = 0.003 and P < 0.001). MMP-1 activity was also higher in healing ulcers than resistant ulcers (P < 0.001) and normal skin (P < 0.001). Significantly more total MMP activity was present in all ulcers than in normal skin (P < 0.001), but there was no difference in total MMP (and MMP-3 activity) between ulcers that healed and those that did not. CONCLUSION: Rapidly healing venous leg ulcers had increased collagen turnover and MMP-1 activity, which appeared to differentiate them from those that failed to heal within 12 months.

Pyridinium cross-links in bone of patients with osteogenesis imperfecta: evidence of a normal intrafibrillar collagen packing.

The brittleness of bone in patients with osteogenesis imperfecta (OI) has been attributed to an aberrant collagen network. However, the role of collagen in the loss of tissue integrity has not been well established. To gain an insight into the biochemistry and structure of the collagen network, the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) and the level of triple helical hydroxylysine (Hyl) were determined in bone of OI patients (types I, III, and IV) as well as controls. The amount of triple helical Hyl was increased in all patients. LP levels in OI were not significantly different; in contrast, the amount of HP (and as a consequence the HP/LP ratio and the total pyridinoline level) was significantly increased. There was no relationship between the sum of pyridinolines and the amount of triple helical Hyl, indicating that lysyl hydroxylation of the triple helix and the telopeptides are under separate control. Cross-linking is the result of a specific three-dimensional arrangement of collagens within the fibril; only molecules that are correctly aligned are able to form cross-links. Inasmuch as the total amount of pyridinoline cross-links in OI bone is similar to control bone, the packing geometry of intrafibrillar collagen molecules is not disturbed in OI. Consequently, the brittleness of bone is not caused by a disorganized intrafibrillar collagen packing and/or loss of cross-links. This is an unexpected finding, because mutant collagen molecules with a random distribution within the fibril are expected to result in disruptions of the alignment of neighboring collagen molecules. Pepsin digestion of OI bone revealed that collagen located at the surface of the fibril had lower cross-link levels compared with collagen located at the inside of the fibril, indicating that mutant molecules are not distributed randomly within the fibril but are located preferentially at the surface of the fibril.

Collagen structure regulates fibril mineralization in osteogenesis as revealed by cross-link patterns in calcifying callus.

Although >80% of the mineral in mammalian bone is present in the collagen fibrils, limited information is available about factors that determine a proper deposition of mineral. This study investigates whether a specific collagen matrix is required for fibril mineralization. Calcifying callus from dog tibias was obtained at various times (3-21 weeks) after fracturing. At 3 weeks, hydroxylysine (Hyl) levels were almost twice as high as in control bone, gradually reaching normal levels at 21 weeks. The decrease in Hyl levels can only be the result of the formation of a new collagen network at the expense of the old one. The sum of the cross-links hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) in callus matched that of bone at all stages of maturation. However, the ratio HP/LP was 2.5-4.5 times higher in callus at 3-7 weeks than in normal bone and was normalized at 21 weeks. Some 40% of the collagen was nonmineralized at the early stages of healing, reaching control bone values (approximately 10%) at 21 weeks. In contrast, only a small increase in callus mineral content from 20.0 to 22.6 (% of dry tissue weight) from week 3 to 21 was seen, indicating that initially a large proportion of the mineral was deposited between, and not within, the fibrils. A strong relationship (r = 0.80) was found between the ratio HP/LP and fibril mineralization; the lower the HP/LP ratio, the more mineralized the fibrils were. Because the HP/LP ratio is believed to be the result of a specific packing of intrafibrillar collagen molecules, this study implies that mineralization of fibrils is facilitated by a specific orientation of collagen molecules in the fibrils.

Age-related accumulation of the advanced glycation endproduct pentosidine in human articular cartilage aggrecan: the use of pentosidine levels as a quantitative measure of protein turnover.

During aging, non-enzymatic glycation results in the formation and accumulation of the advanced glycation endproduct pentosidine in long-lived proteins, such as articular cartilage collagen. In the present study, we investigated whether pentosidine accumulation also occurs in cartilage aggrecan. Furthermore, pentosidine levels in aggrecan subfractions of different residence time were used to explore pentosidine levels as a quantitative measure of aggrecan turnover. In order to compare protein turnover rates, protein residence time was measured as racemization of aspartic acid. As has previously been shown for collagen, pentosidine levels increase with age in cartilage aggrecan. Consistent with the faster turnover of aggrecan compared to collagen, the rate of pentosidine accumulation was threefold lower in aggrecan than in collagen. In the subfractions of aggrecan, pentosidine levels increased with protein residence time. These pentosidine levels were used to estimate the half-life of the globular hyaluronan-binding domain of aggrecan to be 19.5 years. This value is in good agreement with the half-life of 23.5 years that was estimated based on aspartic acid racemization. In aggrecan from osteoarthritic (OA) cartilage, decreased pentosidine levels were found compared with normal cartilage, which reflects increased aggrecan turnover during the OA disease process. In conclusion, we showed that pentosidine accumulates with age in aggrecan and that pentosidine levels can be used as a measure of turnover of long-lived proteins, both during normal aging and during disease.

Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay.

Stromelysin-1 (MMP-3) is an important member of the matrix metalloproteinase family. In joint-degrading diseases like arthritis, elevated levels of MMP-3 protein are detected in synovial fluid using immunological methods. However, these methods do not discriminate between active and inactive enzyme. In the present study, a specific stromelysin activity assay was developed using the selective fluorogenic substrate TNO003 (Dabcyl-Gaba-Arg-Pro-Lys-Pro-Val-Glu / Nva-Trp-Arg-Glu-(EDANS)-Ala-Lys-NH2, / =cleavage site). For its use in biological media, cleavage of TNO003 by enzymes other than stromelysin was effectively blocked by a proteinase inhibitor cocktail. Spiking of MMP-3 to synovial fluid resulted in an MMP-3 concentration-dependent linear increase in activity. The measured MMP-3 activity was not affected by the addition of MMP-13, even in a 5-fold excess over MMP-3. Synovial fluid from rheumatoid arthritis patients demonstrated 100-fold higher levels of active stromelysin than control synovial fluids.

Temporal changes in collagen composition and metabolism during rodent palatogenesis.

Cleft lip and palate is a common craniofacial malformation in man. The aetiology is multifactorial and not known. Since collagen is a major structural component of the developing palate, we studied its composition and metabolism during palate shelf formation and elevation in the rat. Palatal shelves were harvested at embryonic days (E) 15, 16 and 17 as well as post-partum. Palatal collagen increased threefold from E15 to E17 and tenfold from E17 to 5-day-old pups. Palatal calcification was seen in the main, post-partum. Collagen cross-linking, which may be important in shelf elevation and union, varied. The concentration of hydroxylysyl-pyridinolone cross-links was greatest prior to shelf elevation, declining thereafter. Similarly, the highest concentration of dihydroxylysinononorleucine was seen at E16 and this supports the concept of a compliant mesenchymal shelf responding to an intrinsic elevating force. We then determined if enzymes responsible for matrix degradation, matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMPs) altered over the same time periods. MMP-2, and TIMP-1 and TIMP-2 were identified by gelatin zymography and reverse zymography, respectively. MMP-3 activity was determined with a fluorogenic substrate assay. TIMP-1, TIMP-2 and MMP-3 levels remained constant from E15 to E17. The MMP-2 levels showed a significant elevation from E15 to E16 and E16 to E17. This suggests the regulation of extracellular matrix is likely to be of importance in palate morphogenesis.

Distinct associations of HbA1c and the urinary excretion of pentosidine, an advanced glycosylation end-product, with markers of endothelial function in insulin-dependent diabetes mellitus.

Dysfunction of the vascular endothelium is considered an early step in the development of diabetic angiopathy. Hyperglycaemia results in endothelial dysfunction, both through direct effects of glucose and through formation of advanced glycosylation end-products (AGEs). We hypothesized that the effects of glucose and AGEs on endothelial function in insulin-dependent diabetes mellitus (IDDM) are distinct and are reflected by distinct plasma markers of endothelial function. We therefore measured plasma levels of von Willebrand factor (vWF), soluble (s) E-selectin and vascular cell adhesion molecule-1 (sVCAM-1), and evaluated the relationship with HbA1c and urinary excretion of pentosidine, an AGE product, in 56 patients with IDDM. Urinary pentosidine excretion was higher in the diabetic than in a control group (n = 60) of similar age (P < 0.0001) and showed a steeper increase with age (P < 0.02 vs controls). In the diabetic group, sE-selectin was correlated to HbA1c (r = 0.52, P < 0.0001), whereas sVCAM-1 was not (r = 0.11, P = 0.47). In contrast, sVCAM-1 showed a trend towards a correlation with log (pentosidine excretion) (r = 0.27, P = 0.06), whereas sE-selectin did not (r = -0.16, P = 0.27). Log(vWF) was correlated to HbA1c (r = 0.50, P < 0.0001) and tended to correlate with log (pentosidine excretion) (r = 0.25, P = 0.07). Multivariate analyses with both pentosidine and HbA1c as independent variables showed significant associations of sE-selectin with HbA1c, of sVCAM-1 with pentosidine, and of log(vWF) with both HbA1c and pentosidine (all P-values < 0.02). Our results imply that the effects of glucose and AGEs on the endothelium can be reflected by distinct endothelial markers. Plasma sE-selectin may reflect short-term effects of glucose on the endothelium, sVCAM-1 the effects of AGEs, and vWF the combined effect of glucose and AGEs.

Fluorogenic MMP activity assay for plasma including MMPs complexed to alpha 2-macroglobulin.

Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). Reconstituted MMP-13/alpha 2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by alpha 2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW approximately 28,000) was unable to inhibit MMP/alpha 2M enzyme activity, whereas the low-MW inhibitor BB94 (MW approximately 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/alpha 2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by alpha 2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.

The role of collagen in determining bone mechanical properties.

The hypothesis of this study was that collagen denaturation would lead to a significant decrease in the toughness of bone, but has little effect on the stiffness of bone. Using a heating model, effects of collagen denaturation on the biomechanical properties of human cadaveric bone were examined. Prior to testing, bone specimens were heat treated at varied temperatures (37-200 degrees C) to induce different degrees of collagen denaturation. Collagen denaturation and mechanical properties of bone were determined using a selective digestion technique and three-point bending tests, respectively. The densities and weight fractions of the mineral and organic phases in bone also were determined. A repeated measures analysis of variance showed that heating had a significant effect on the biomechanical integrity of bone, corresponding to the degree of collagen denaturation. The results of this study indicate that the toughness and strength of bone decreases significantly with increasing collagen denaturation, whereas the elastic modulus of bone is almost constant irrespective of collagen denaturation. These results suggest that the collagen network plays an important role in the toughness of bone, but has little effect on the stiffness of bone, thereby supporting the hypothesis of this study.

Compositional and metabolic changes in damaged cartilage are peak-stress, stress-rate, and loading-duration dependent.

The first objective of this study was to determine if the cumulative effects of impact or smoothly arising compression would damage the matrix of articular cartilage. Canine cartilage explants were subjected to repeated impacts or to smoothly arising compressions of as much as 20 MPa at 0.3 Hz for as long as 120 minutes. An increase in the water content of the loaded core compared with the surrounding ring was considered indicative of matrix damage. The results showed that damage to cartilage required repeated impacts with a peak stress of at least 2.5 MPa and a stress rate of at least 30 MPa/sec for 2 minutes or longer. This suggested that impact damage is cumulative and stress-rate dependent. The second objective was to identify biosynthetic and compositional changes in impact-damaged cartilage over a period of time after loading. Accordingly, canine cartilage explants were subjected to repetitive impacts of 5 MPa at 0.3 Hz for 2, 20, and 120 minutes. The loaded explants were then cultured for as long as 10 days. The increase in water content (1.9-3.8%) in the core region relative to the surrounding ring persisted during the 10-day culture. A significant increase in fibronectin synthesis (22-47%) was found in the core region of impact-damaged cartilage. Proteoglycan synthesis was increased by 41-104%. An increase in denatured collagens (11-70%) in the loaded cores substantiated damage to the collagen network. Denatured collagens stained with COL2-3/4m monoclonal antibody were consistent with the compositional findings and were mainly located near the articular surface and in the deep zone. These changes were consistent with early osteoarthritis and suggested the induction of the initial stages of osteoarthritis in the impact-damaged cartilage.

Active MMPs captured by alpha 2 macroglobulin as a marker of disease activity in rheumatoid arthritis.

OBJECTIVE: The aim of the present study was to analyze alpha 2 Macroglobulin/MMP (alpha 2M/MMP) complex formation and to investigate whether MMP activity in alpha 2M/MMP complexes in serum can be used as a disease marker in rheumatoid arthritis (RA). METHODS: High and low molecular weight (H/LMW) substrates and inhibitors and size exclusion were used to analyze alpha 2M/MMP complex formation. LMW fluorogenic substrates were used to quantify the level of MMPs in alpha 2M/MMP complexes in the serum of RA patients and healthy controls. RESULTS: Active MMPs were fully inhibited by LMW inhibitor BB94 in the presence of alpha 2M, whereas no inhibition was achieved by HMW inhibitor TIMP-1. Size exclusion analysis showed alpha 2M/MMP complex formation in buffer and in normal plasma spiked with activated MMPs, which indicated alpha 2M/MMP complex formation in the systemic circulation. MMP activity in alpha 2M/MMP complexes in the serum of RA patients was significantly higher than in the serum of healthy controls (P < 0.001). MMP activity levels in the serum of RA patients were correlated with ESR (r = 0.72, P < 0.001). CONCLUSION: In the systemic circulation of RA patients, active MMPs form complexes with alpha 2M and can be detected using LMW fluorogenic substrates. MMP activity measurements in serum allow discrimination between RA patients and healthy controls and provide a new tool for the assessment of the disease process in RA.

No therapeutic effect of plasmin antagonist tranexamic acid in rheumatoid arthritis. A double-blind placebo-controlled pilot study.

OBJECTIVE: In the present study, the effects of plasmin antagonist tranexamic acid (TEA) on urinary pyridinoline excretion rates were investigated in rheumatoid arthritis (RA) patients. METHODS: The study was set up as a double-blind placebo-controlled pilot study. Ten patients received tranexamic acid and 9 received placebo for 12 weeks. Urinary excretion rates of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were used as molecular markers of articular cartilage and bone degradation. In addition, clinical parameters of disease activity were assessed and CRP levels were measured. RESULTS: Treatment with TEA did not reduce pyridinoline excretion, nor was any effect observed on clinical parameters of disease activity or on CRP levels. CONCLUSION: The results of the present pilot study show no beneficial effect of TEA as adjuvant therapy in RA patients with respect to joint destruction or disease activity.

Biochemical development of subchondral bone from birth until age eleven months and the influence of physical activity.

Subchondral bone provides structural support to the overlying articular cartilage, and plays an important role in osteochondral diseases. There is growing insight that the mechanical features of bone are related to the biochemistry of the collagen network and the mineral content. In the present study, part of the normal developmental process and the influence of physical activity on biochemical composition of subchondral bone was studied. Water content, calcium content and characteristics of the collagen network (collagen, hydroxylysine, lysylpyridinoline (LP) and hydroxylysylpyridinoline (HP) crosslinking) of subchondral bone were measured in newborn foals, 5-month-old foals (pasture-grown and box-confined) and 11-month-old foals at 2 differently loaded sites of the proximal articular surface of the first phalanx. During the first 5 months postpartum, water and hydroxylysine content decreased significantly while calcium and collagen content and the amount of HP and LP crosslinks increased significantly. The withholding of physical activity during this developmental phase affected the biochemical characteristics of subchondral bone only at the site that is loaded during physical exercise. At this site, calcium content and both HP and LP crosslink levels increased significantly less than in pasture-raised animals. During development from 5-11 months, measured parameters remained essentially constant, except for water content, which decreased further. It is concluded that substantial changes, presumed to be largely exercise-driven, take place during the normal process of development in the biochemical composition of equine subchondral bone. Normal development of subchondral bone is presumably important for the normal functional adaptation of this bone to the loading conditions it is subjected to and therefore essential to resist the future biomechanical challenges the horse will encounter during its athletic career. The findings from this study and the assumed important role of subchondral bone quality in the pathogenesis of osteochondral disease merit more attention to the role of the collagen network in subchondral bone.

Functional adaptation of equine articular cartilage: the formation of regional biochemical characteristics up to age one year.

Biochemical heterogeneity of cartilage within a joint is well known in mature individuals. It has recently been reported that heterogeneity for proteoglycan content and chondrocyte metabolism in sheep develops postnatally under the influence of loading. No data exist on the collagen network in general or on the specific situation in the horse. The objective of this study was to investigate the alterations in equine articular cartilage biochemistry that occur from birth up to age one year, testing the hypothesis that the molecular composition of equine cartilage matrix is uniform at birth and biochemical heterogeneity is formed postnatally. Water content, DNA content, glycosaminoglycan content (GAG) and biochemical characteristics of the collagen network (collagen content, hydroxylysine content and hydroxylysylpyridinoline [HP] crosslinks) were measured in immature articular cartilage of neonatal (n = 16), 5-month-old foals (n = 16) and yearlings (n = 16) at 2 predefined differently loaded sites within the metacarpophalangeal joint. Statistical differences between sites were analysed by ANOVA (P<0.01), and age correlation was tested by Pearson's product moment correlation analysis (P<0.01). In neonatal cartilage no significant site differences were found for any of the measured biochemical parameters. This revealed that the horse has a biochemically uniform joint (i.e. the cartilage) at birth. In the 5-month-old foals and yearlings, significant site differences, comparable to those in the mature horse, were found for DNA, GAG, collagen content and hydroxylysine content. This indicates that functional adaptation of articular cartilage to weight bearing for these biochemical parameters takes place during the first months postpartum. Water content and HP crosslinks showed no difference between the 2 sites from neonatal horses, 5-month-old animals and yearlings. At both sites water, DNA and GAG decreased during maturation while collagen content, hydroxylysine content and HP crosslinks increased. We propose that a foal is born with a uniform biochemical composition of cartilage in which the functional adaptation to weight bearing takes place early in life. This adaptation results in biochemical and therefore biomechanical heterogeneity and is thought to be essential to resist the different loading conditions to which articular cartilage is subjected during later life. As collagen turnover is extremely low at mature age, an undisturbed functional adaptation of the collagen network of articular cartilage at a young age may be of significant importance for future strength and resistance to injury.

Development of biochemical heterogeneity of articular cartilage: influences of age and exercise.

The objective of this study was to document the development of biochemical heterogeneity from birth to maturity in equine articular cartilage, and to test the hypothesis that the amount of exercise during early life may influence this process. Neonatal foals showed no biochemical heterogeneity whatsoever, in contrast to a clear biochemical heterogeneity in mature horses. The process of formation of site differences was almost completed in exercised foals age 5 months, but was delayed in those deprived of exercise. For some collagen-related parameters, this delay was not compensated for after an additional 6 month period of moderate exercise. It is concluded that the functional adaptation of articular cartilage, as reflected in the formation of biochemical heterogeneity in the horse, occurs for the most part during the first 5 months postpartum. A certain level of exercise seems essential for this process and withholding exercise in early life, may result in a delay in the adaptation of the cartilage.

Training affects the collagen framework of subchondral bone in foals.

Subchondral bone provides structural support to the overlying articular cartilage and plays an important role in osteochondral diseases. There is growing insight that the mechanical features of bone are related to the biochemistry of the collagen network. In this study the effect of exercise on water, calcium and the collagen network (total collagen, lysyl-hydroxylation, hydroxylysylpyridinoline, and lysylpyridinoline crosslinks) of subchondral bone at two differently loaded sites (site 1: intermittently loaded; site 2: constantly loaded) is investigated in foals. Exercise influenced calcium content and levels of both types of crosslinks at site 1, but had no influence on site 2. There was no concomitant increase in lysyl-hydroxylation level with the rise in crosslinks. Levels of lysyl-hydroxylation and lysylpyridinoline crosslinking were lower at site 1 than at site 2. It is concluded that exercise affects the post-translational modifications of the collagen component of subchondral bone. Loading also appears to play a role in site-related topographical differences. The lack of any relation between the sum of pyridinoline crosslinks and the amount of triple helical hydroxylysine gives support to a recent hypothesis that lysyl-hydroxylation of the triple helix and the telopeptides are under separate control.

The influence of strenuous exercise on collagen characteristics of articular cartilage in Thoroughbreds age 2 years.

In order to assess the influence of strenuous exercise on collagen characteristics of articular cartilage, the response of the collagen network was studied in seven 2-year-old Thoroughbreds subjected to strenuous exercise compared to 7 nontrained individuals. After 13 weeks, the animals were subjected to euthanasia, fetlock joints of the forelimbs were scored macroscopically after Indian Ink staining, and articular cartilage from different locations of the articular surface of the proximal first phalanx was sampled and analysed for water content, collagen content, hydroxylysine content and amount of hydroxylysylpyridinoline (HP) crosslinks. Gross lesions were significantly more severe in the exercised than in the nonexercised group. In the control animals, the characteristic site-specific differences in collagen parameters were found as described earlier, but in the strenuously exercised animals this physiological biochemical heterogeneity had disappeared. In the exercised animals, an increase in water content and a sharp decrease in HP crosslinking was found that was correlated with the presence of wear lines. It is concluded that the strenuous exercise provoked significant alterations in the characteristics of the collagen network of the articular cartilage of the fetlock joint which were suggestive of microdamage and loosening of the collagen network. The collagen component of cartilage, in contrast to the proteoglycan component, is known to have a very limited capacity for repair and remodelling due to an extremely low turnover rate. Therefore, alterations within the articular collagen network might be expected to play an important role in the pathophysiology of degenerative joint disorders.

Topographical mapping of biochemical properties of articular cartilage in the equine fetlock joint.

The aim of this study was to evaluate topographical differences in the biochemical composition of the extracellular matrix of articular cartilage of the normal equine fetlock joint. Water content, DNA content, glycosaminoglycan (GAG) content and a number of characteristics of the collagen network (total collagen content, levels of hydroxylysine- (Hyl) and the crosslink hydroxylysylpyridinoline, (HP) of articular cartilage in the proximal 1st phalanx (P1), distal 3rd metacarpal bone (MC), and proximal sesamoid bones (PSB) were determined in the left and right fetlock joint of 6 mature horses (age 5-9 years). Twenty-eight sites were sampled per joint, which included the clinically important areas often associated with pathology. Biochemical differences were evaluated between sampling sites and related with the predisposition for osteochondral injury and type of loading. Significant regional differences in the composition of the extracellular matrix existed within the joint. Furthermore, left and right joints exhibited biochemical differences. Typical topographic distribution patterns were observed for each parameter. In P1 the dorsal and palmar articular margin showed a significantly lower GAG content than the more centrally located sites. Collagen content and HP crosslinks were higher at the joint margins than in the central area. Also, in the MC, GAG content was significantly lower at the (dorsal) articular margin compared with the central area. Consistent with findings in P1, collagen and HP crosslinks were significantly lower in the central area compared to the (dorsal) articular margin. Biochemical and biomechanical heterogeneity of articular cartilage is supposed to reflect the different functional demands made at different sites. In the present study, GAG content was highest in the constantly loaded central areas of the joint surfaces. In contrast, collagen content and HP crosslinks were higher in areas intermittently subjected to peak loading which suggests that the response to a certain type of loading of the various components of the extracellular matrix of articular cartilage are different. The differences in biochemical characteristics between the various sites may help to explain the site specificity of osteochondral lesions commonly found in the equine fetlock joint. Finally, these findings emphasise that the choice of sampling sites may profoundly influence the outcome of biochemical studies of articular cartilage.

Influence of site and age on biochemical characteristics of the collagen network of equine articular cartilage.

OBJECTIVE: To determine variations in biochemical characteristics of equine articular cartilage in relation to age and the degree of predisposition for osteochondral disease at a specific site. SAMPLE POPULATION: Articular cartilage specimens from 53 horses 4 to 30 years old. PROCEDURE: Healthy specimens were obtained from 2 locations on the proximal articular surface of the first phalanx that had different disease prevalences (site 1 at the mediodorsal margin and site 2 at the center of the medial cavity). Water, total collagen, and hydroxylysine contents and enzymatic (hydroxylysylpyridinoline [HP]) and nonenzymatic (pentosidine) crosslinking were determined at both sites. Differences between sites were analyzed by ANOVA (factors, site, and age), and age correlation was tested by Pearson's product-moment correlation analysis. Significance was set at P< 0.01. RESULTS: Correlation with age was not found for water, collagen, hydroxylysine contents, and enzymatic cross-linking. Nonenzymatic crosslinking was higher in older horses and was linearly related to age (r = 0.94). Water and collagen contents and HP and pentosidine crosslinks were significantly higher at site 1. Hydroxylysine content was significantly lower at site 1. CONCLUSIONS: Except for nonenzymatic glycation, the composition of articular cartilage collagen does not change significantly in adult horses. A significant topographic variation exists in biochemical characteristics of the articular cartilage collagen network in equine metacarpophalangeal joints. These differences may influence local biomechanical properties and, hence, susceptibility to osteochondral disease, as will greater pentosidine crosslinks in older horses that are likely to cause stiffer and more brittle cartilage.

MMP protein and activity levels in synovial fluid from patients with joint injury, inflammatory arthritis, and osteoarthritis.

OBJECTIVE: To determine protein and activity levels of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) in synovial fluid of patients with knee joint injury, primary osteoarthritis, and acute pyrophosphate arthritis (pseudogout). METHODS: Measurements were done on knee synovial fluid obtained in a cross sectional study of cases of injury (n = 283), osteoarthritis (n = 105), and pseudogout (n = 65), and in healthy controls (n = 35). Activity of MMP-1 and MMP-3 in alpha(2) macroglobulin complexes was measured using specific low molecular weight fluorogenic substrates. ProMMP-1, proMMP-3, and TIMP-1 (tissue inhibitor of metalloproteinase 1) were quantified by immunoassay. RESULTS: Mean levels of proMMP-1, proMMP-3, and TIMP-1 were increased in injury, osteoarthritis, and pseudogout compared with controls. MMP-1 activity was increased in pseudogout and injury groups over control levels, whereas MMP-3 activity was increased only in the pseudogout group. The increase in MMP-1 activity coincided with a decrease in TIMP-1 levels in the injury group. CONCLUSIONS: Patients with joint injury have a persistent increase in proMMP-1 and proMMP-3 in synovial fluid and an increase in activated MMPs, which are not inhibited by TIMP. The differences in activation and inhibition patterns between the study groups are consistent with disease specific patterns of MMP activation and/or inhibition in joint pathology.