Profiling the secretion of soluble mediators by end stage osteoarthritis synovial tissue explants reveals a reduced responsiveness to an inflammatory trigger.

OBJECTIVE: Evidence is accumulating that synovial tissue plays an active role in osteoarthritis (OA), however, exact understanding of its contribution is lacking. In order to further elucidate its role in the OA process, we aimed to identify the secretion pattern of soluble mediators by synovial tissue and to assess its ability to initiate cartilage degeneration. METHODS: Synovial tissue explants (STEs) obtained from donors without history of OA (n = 8) or from end stage OA patients (n = 16) were cultured alone or together with bovine cartilage explants in the absence or presence of IL-1α. The secretion of 48 soluble mediators was measured and the effect on glycosaminoglycan (GAG) release and matrix metalloproteinase (MMP) activity was determined. RESULTS: Normal and OA STEs secreted comparable levels of almost all measured soluble mediators. However, in the presence of IL-1α these mediators were less secreted by OA than by normal STEs of which 15 differed significantly (p<0.01). No effect of normal or OA STEs on GAG release from the cartilage explants was observed, and no differences in MMP activity between OA and normal STEs were detected. CONCLUSIONS: Unexpectedly, a comparable secretion profile of soluble mediators was found for OA and normal STEs while the reduced responsiveness of OA STEs to an inflammatory trigger indicates a different state of this tissue in OA patients. The effects could be the result of prolonged exposure to an inflammatory environment in OA development. Further understanding of the pro-inflammatory and inflammation resolving mechanisms during disease progression in synovial tissue may provide valuable targets for therapy in the future.

Longevity of elastin in human intervertebral disc as probed by the racemization of aspartic acid.

BACKGROUND: Aging and degeneration of human intervertebral disc (IVD) are associated with biochemical changes, including racemization and glycation. These changes can only be counteracted by protein turnover. Little is known about the longevity of IVD elastin in health or disease. Yet, such knowledge is important for a quantitative understanding of tissue synthesis and degradation. METHODS: We have measured the accumulation of d-Asp and pentosidine in IVD elastin. Samples representing a broad range of ages (28-82years) and degeneration grades (1-5) were analyzed. RESULTS: d/l-Asp for elastin increased linearly with age from 3.2% (early 30s) to 14.8% (early 80s) for normal tissue (grades 1-2) and from 1.7% (late 20s) to 6.0% (until the mid 50s) for degenerate tissue (grades 3-5) with accumulation rates of 16.2±3.1×10(-4) and 11.7±3.8×10(-4)year(-1), respectively; no significant difference was found between these values (p<0.05). Above the mid 50s, a decrease in d-Asp accumulation was recorded in the degenerate tissue. d-Asp accumulation correlated well with pentosidine content for elastin from healthy and degenerate tissues combined. We conclude that IVD elastin is metabolically-stable and long-lived in both healthy and degenerate human IVDs, with signs of new synthesis in the latter. The correlation of d-Asp with pentosidine content suggests that both these agents may be used as markers in the overall aging process of IVD. GENERAL SIGNIFICANCE: Accumulation of modified IVD elastin argues for its longevity and may have a negative impact on its role in disc function. Weak signs of newly synthesized molecules may act to counteract this effect in degenerate tissue.

Integration of efficacy, pharmacokinetic and safety assessment of interleukin-1 receptor antagonist in a preclinical model of arthritis.

Pharmacokinetic properties and safety profile of a drug are likely influenced by the disease state of a patient. In this study, we investigated the influence of arthritic processes on pharmacokinetics and immunotoxicity of interleukin-1 receptor antagonist (Anakinra) in the rat adjuvant arthritis model. Anakinra dose-dependently suppressed joint inflammation and degradation as demonstrated by reduced clinical arthritis score, paw thickness, synovial infiltration and bone degradation. In addition, plasma levels of chemokines MCP-1 and GRO/KC were reduced. Pharmacokinetic behaviour of Anakinra was influenced by disease state of the rats as judged from a decrease in C(max) and an increase of the MRT as the disease progressed at a dose of 24 and 72 mg Anakinra/kg body weight. The pharmacokinetic parameters increased dose-dependently, but non-proportionally with increasing dose. Low level anti-Anakinra antibody formation was observed at prolonged exposure to the biologic. Safety parameters, including haematology, splenic lymphocyte subset analysis, ex vivo stimulation of spleen cells and histopathology of immune system organs were affected by the disease itself to such extent that no additional effects of Anakinra could be observed. In conclusion, we demonstrated that pharmacokinetic behaviour of Anakinra was influenced by the arthritis background of the rats resulting in decreased internal exposure.

Does loading influence the severity of cartilage degeneration in the canine Groove-model of OA?

Many animal models are used to study osteoarthritis (OA). In these models the role of joint loading in the development of OA is not fully understood. We studied the effect of loading on the development of OA in the canine Groove-model. In ten female beagle dogs OA was induced in one knee according to the Groove-model. The animals were divided in groups with and without forced-loading. Forced-loading was achieved by fixing the contra-lateral limb to the trunk 3 times a week for 4 hours. After 20 weeks joint tissues of all dogs were evaluated. Subjective evaluation revealed less movement with more loading in the forced-loading-group compared to the group without forced-loading. In both groups induction of OA resulted in macroscopical and microscopical OA changes as well as alterations in cartilage metabolism characteristics for OA. Although differences were small, for some parameters they were statistically significant for the forced-loading-group. There were no differences between the contra-lateral healthy joints of both groups. The present study demonstrates that in the Groove-model intensified loading is not a prerequisite for the development of OA, although it adds to some extent to the severity of the OA.

Profiling of endogenous peptides in human synovial fluid by NanoLC-MS: method validation and peptide identification.

Synovial fluid potentially contains markers for early diagnosis and disease progression in degenerative joint diseases such as osteoarthritis. Here, a method is described for profiling endogenous peptides in human synovial fluid, using ultrafiltration, solid-phase extraction, nanoscale liquid chromatography, and high-resolution mass spectrometry. Synovial fluid is characterized by its high viscosity, caused by the presence of the lubricant hyaluronic acid. The method proved to be capable of eliminating the high concentrations of hyaluronic acid, which appeared to be necessary to obtain satisfactory analytical performance, that is, within-day relative standard deviations of 5-15%, between-day relative standard deviations of 6-16%, a linear response of R2=0.994, a limit of detection in the femtomole range, and reproducible recoveries of 14-67%. With the developed method, in a synovial fluid sample from an osteoarthritis patient and a healthy control, in total, 501 peptides originating from 40 proteins were identified. Peptide cleavage products from six proteins that have been associated with osteoarthritis in earlier studies (collagen II, proteoglcycan 4, serum amyloid A, tubulin, vimentin, and Matrix Gla) could also be identified with our profiling method. The robustness of the method indicates that it can be applied in systems biology approaches for further studies on degenerative joint disease, eventually leading to a better understanding of the disease and its therapy, as well as the development of novel biomarkers to monitor these processes.

Achilles tendinosis: changes in biochemical composition and collagen turnover rate.

BACKGROUND: Understanding biochemical and structural changes of the extracellular matrix in Achilles tendinosis might be important for developing mechanism-based therapies. HYPOTHESIS: In Achilles tendinosis, changes occur in biochemical composition and collagen turnover rate. STUDY DESIGN: Descriptive laboratory study. METHODS: From 10 patients undergoing surgery for Achilles tendinopathy, 1 tendinosis biopsy specimen and 1 biopsy specimen of macroscopically healthy tendon tissue adjacent to the lesion were collected. Furthermore, biopsy samples were collected from 3 donors with asymptomatic Achilles tendons. Water content, collagen content, percentage of denatured collagen, amount of lysine hydroxylation, number of enzymatic and nonenzymatic crosslinks, matrix metalloproteinase activity, and matrix metalloproteinase and collagen gene-expression levels were analyzed. RESULTS: In tendinotic lesions, the water content was highest, and collagen content was subnormal with higher amounts of denatured/damaged collagen. Low pentosidine levels in tendinotic tissue indicated the presence of relatively young collagenous matrix. More hydroxylated lysine residues were present in tendinotic samples, but enzymatic crosslinks revealed no differences between tendinotic, adjacent, and healthy samples. In tendinotic specimens, matrix metalloproteinase activity was higher, matrix metalloproteinase gene-expression profile was altered, and collagen type I and III gene expression were upregulated. CONCLUSION: In Achilles tendinosis, the collagen turnover rate is increased, and the natural biochemical composition of the collagenous matrix is compromised. CLINICAL RELEVANCE: Although tendon tissue directly adjacent to an Achilles tendinosis lesion looks macroscopically healthy, histological and biochemical degenerative changes in adjacent tissue are evident, which may have implications for surgical interventions.

Corticosteroids reduce the tensile strength of isolated collagen fascicles.

BACKGROUND: Overuse tendon injuries are frequent. Corticosteroid injections are commonly used as treatment, although their direct effects on the material properties of the tendon are poorly understood. PURPOSE: To examine the influence of corticosteroids on the tensile strength of isolated collagen fascicles. STUDY DESIGN: Controlled laboratory study. METHODS: Single strands (300-500 mum) of rat-tail collagen fascicles were incubated in either high (1 mL of 40 mgmL(-1) mixed with 0.5 mL saline 9%) or low (1 mL of 40 mgmL(-1) mixed with 2 mL saline 9%) concentration of methylprednisolone acetate (Depomedrol) for 3 or 7 days, while the control segment from the same fascicle was kept in saline (N = 64). After the incubation period, the fascicles underwent displacement to failure in a mechanical test rig at 0.13 mm/s, and thereafter hydroxylysyl pyridinoline and lysyl pyridinoline cross-link content was evaluated in a high-performance liquid chromatography system. Data for each group were analyzed with a 2-way analysis of variance (time x incubation) for ultimate stress (mean +/- standard deviation). RESULTS: In the high-concentration groups, strength was reduced after 3 (16.6 +/- 4.6 MPa) and 7 (8.6 +/- 1.7 MPa) days compared to the controls (30.2 +/- 5.0 MPa and 25.6 +/- 4.6 MPa, respectively; P < .05). In the low-concentration groups, strength was reduced after 3 (12.0 +/- 3.1 MPa) and 7 days (10.9 +/- 2.5 MPa) compared to the controls (31.5 +/- 5.0 MPa and 32.4 +/- 5.6 MPa, respectively; P < .05). The amount of cross-linking was unaffected by the intervention. CONCLUSION: Data show that the tensile strength of isolated fascicles is markedly reduced after 3- and 7-day incubation in both high and low concentration of corticosteroids, although the observed effect on whole tendon remains unknown. CLINICAL RELEVANCE: Corticosteroids may weaken specific regions of the injected tendon and leave it more prone to rupture. This weakening effect is manifested in the individual collagen fascicles that constitute the tendon.

Accumulation of advanced glycation end products as a molecular mechanism for aging as a risk factor in osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is one of the most prevalent and disabling chronic conditions affecting the elderly. Its etiology is largely unknown, but age is the most prominent risk factor. The current study was designed to test whether accumulation of advanced glycation end products (AGEs), which are known to adversely affect cartilage turnover and mechanical properties, provides a molecular mechanism by which aging contributes to the development of OA. METHODS: The hypothesis that elevated AGE levels predispose to the development of OA was tested in the canine anterior cruciate ligament transection (ACLT) model of experimental OA. Cartilage AGE levels were enhanced in young dogs by intraarticular injections of ribose. This mimics the accumulation of AGEs without the interference of other age-related changes. The severity of OA was then assessed 7 weeks after ACLT surgery in dogs with normal versus enhanced AGE levels. RESULTS: Intraarticular injections of ribose enhanced cartilage AGE levels approximately 5-fold, which is similar to the normal increase that is observed in old dogs. ACLT surgery resulted in more-pronounced OA in dogs with enhanced AGE levels. This was observed as increased collagen damage and enhanced release of proteoglycans. The attempt to repair the matrix damage was impaired; proteoglycan synthesis and retention were decreased at enhanced AGE levels. Mankin grading of histology sections also revealed more-severe OA in animals with enhanced AGE levels. CONCLUSION: These findings demonstrate increased severity of OA at higher cartilage AGE levels and provide the first in vivo experimental evidence for a molecular mechanism by which aging may predispose to the development of OA.

Matrix metalloproteinase activities and their relationship with collagen remodelling in tendon pathology.

Our aim was to correlate the activity of matrix metalloproteinases (MMPs) with denaturation and the turnover of collagen in normal and pathological human tendons. MMPs were extracted from ruptured supraspinatus tendons (n=10), macroscopically normal ("control") supraspinatus tendons (n=29) and normal short head of biceps brachii tendons (n=24). Enzyme activity was measured using fluorogenic substrates selective for MMP-1, MMP-3 and enzymes with gelatinolytic activity (MMP-2, MMP-9 and MMP-13). Collagen denaturation was determined by alpha-chymotrypsin digestion. Protein turnover was determined by measuring the percentage of D-aspartic acid (% D-Asp). Zymography was conducted to identity specific gelatinases. MMP-1 activity was higher in ruptured supraspinatus compared to control supraspinatus and normal biceps brachii tendons (70.9, 26.4 and 11.5 fmol/mg tendon, respectively; P<0.001). Gelatinolytic and MMP-3 activities were lower in normal biceps brachii and ruptured supraspinatus compared to control supraspinatus (gelatinase: 0.18, 0.23 and 0.82 RFU/s/mg tendon respectively; P<0.001; MMP-3: 9.0, 8.6 and 55 fmol/mg tendon, respectively; P<0.001). Most gelatinase activity was shown to be MMP-2 by zymography. Denatured collagen was increased in ruptured supraspinatus compared to control supraspinatus (20.4% and 9.9%, respectively; P<0.001). The % D-Asp content increased linearly with age in normal biceps brachii but not in control supraspinatus and was significantly lower in ruptured supraspinatus compared to age-matched control tendons (0.33 and 1.09% D-Asp, respectively; P<0.01). We conclude that the short head of biceps brachii tendons show little protein turnover, whereas control supraspinatus tendons show relatively high turnover mediated by the activity of MMP-2, MMP-3 and MMP-1. This activity is thought to represent a repair or maintenance function that may be associated with an underlying degenerative process caused by a history of repeated injury and/or mechanical strain. After tendon rupture, there was increased activity of MMP-1, reduced activity of MMP-2 and MMP-3, increased turnover and further deterioration in the quality of the collagen network. Tendon degeneration is shown to be an active, cell-mediated process that may result from a failure to regulate specific MMP activities in response to repeated injury or mechanical strain.