Matrix metalloproteinase activity and prostaglandin E2 are elevated in the synovial fluid of meniscus tear patients.

PURPOSE: Meniscus tears are a common knee injury and are associated with the development of post-traumatic osteoarthritis (OA). The purpose of this study is to evaluate potential OA mediators in the synovial fluid and serum of meniscus tear subjects compared to those in the synovial fluid of radiographic non-OA control knees. MATERIALS AND METHODS: Sixteen subjects with an isolated unilateral meniscus injury and six subjects who served as reference controls (knee Kellgren-Lawrence grade 0-1) were recruited. Twenty-one biomarkers were measured in serum from meniscus tear subjects and in synovial fluid from both groups. Meniscus tear subjects were further stratified by tear type to assess differences in biomarker levels. RESULTS: Synovial fluid total matrix metalloproteinase (MMP) activity and prostaglandin E2 (PGE2) were increased 25-fold and 290-fold, respectively, in meniscus tear subjects as compared to reference controls (p < 0.05). Synovial fluid MMP activity and PGE2 concentrations were positively correlated in meniscus tear subjects (R = 0.83, p < 0.0001). In meniscus tear subjects, synovial fluid levels of MMP activity, MMP-2, MMP-3, sGAG, COMP, IL-6, and PGE2 were higher than serum levels (p < 0.05). Subjects with complex meniscus tears had higher synovial fluid MMP-10 (p < 0.05) and reduced serum TNFα and IL-8 (p < 0.05) compared to other tear types. CONCLUSIONS: Given the degradative and pro-inflammatory roles of MMP activity and PGE2, these molecules may alter the biochemical environment of the joint. Our findings suggest that modulation of PGE2 signaling, MMP activity, or both following a meniscus injury may be targets to promote meniscus repair and prevent OA development.

ADAMTS-4 activity in synovial fluid as a biomarker of inflammation and effusion.

OBJECTIVE: To evaluate the potential of ADAMTS-4 (aggrecanase -1) activity in synovial fluid (SF) as a biomarker of knee injury and joint disease. DESIGN: We have measured ADAMTS-4 activity in the synovial fluid of 170 orthopaedic patients with different degrees of joint pathology, using a commercial ADAMTS-4 fluorescence resonance energy transfer (FRET) substrate assay. Patients were classified at arthroscopy as (i) macroscopically normal, (ii) with an injury of the meniscus, anterior cruciate ligament or chondral/osteochondral defects or (iii) with osteoarthritis, and the influence of independent factors (age, patient group, effusion and synovial inflammation) on ADAMTS-4 activity levels was assessed. RESULTS: In most patients (106/170) ADAMTS-4 activity was undetectable; ADAMTS-4 ranged from 0 to 2.8 ng/mL in synovial fluid from patients with an injury, 0-4.1 ng/mL in osteoarthritic patients and 4.0-12.3 ng/mL in patients with large effusions. Four independent variables each significantly influenced ADAMTS-4 activity in synovial fluid (all P < 0.001): age (concordance = 0.69), presence of osteoarthritis (OA) (concordance = 0.66), level of effusion (concordance = 0.78) and inflammation (concordance = 0.68). Not only did effusion influence the amount of ADAMTS-4 activity most strongly, but it also did this in an ordered manner (P < 0.001). CONCLUSIONS: The main finding of this study is that ADAMTS-4 levels in synovial fluid are most strongly correlated with inflammation and severity of effusion in the knee. Further study is required to determine if it could provide a useful tool to aid clinical diagnoses, indicate treatment, to monitor progression of joint degeneration or OA or alternatively the success of treatment.

In vivo cartilage strain increases following medial meniscal tear and correlates with synovial fluid matrix metalloproteinase activity.

Meniscal tears are common injuries, and while partial meniscectomy is a frequent treatment option, general meniscus loss is a risk factor for the development of osteoarthritis. The goal of this study was to measure the in vivo tibiofemoral cartilage contact patterns in patients with meniscus tears in relation to biomarkers of cartilage catabolism in the synovial fluid of these joints. A combination of magnetic resonance imaging and biplanar fluoroscopy was used to determine the in vivo motion and cartilage contact mechanics of the knee. Subjects with isolated medial meniscus tears were analyzed while performing a quasi-static lunge, and the contralateral uninjured knee was used as a control. Synovial fluid was collected from the injured knee and matrix metalloproteinase (MMP) activity, sulfated glycosaminoglycan, cartilage oligomeric matrix protein, prostaglandin E2, and the collagen type II cleavage biomarker C2C were measured. Contact strain in the medial compartment increased significantly in the injured knees compared to contralateral control knees. In the lateral compartment, the contact strain in the injured knee was significantly increased only at the maximum flexion angle (105°). The average cartilage strain at maximum flexion positively correlated with total MMP activity in the synovial fluid. These findings show that meniscal injury leads to loss of normal joint function and increased strain of the articular cartilage, which correlated to elevated total MMP activity in the synovial fluid. The increased strain and total MMP activity may reflect, or potentially contribute to, the early development of osteoarthritis that is observed following meniscal injury.

In-vitro and in-vivo imaging of MMP activity in cartilage and joint injury.

Non-destructive detection of cartilage-degrading activities represents an advance in osteoarthritis (OA) research, with implications in studies of OA pathogenesis, progression, and intervention strategies. Matrix metalloproteinases (MMPs) are principal cartilage degrading enzymes that contribute to OA pathogenesis. MMPSense750 is an in-vivo fluorimetric imaging probe with the potential to continuously and non-invasively trace real-time MMP activities, but its use in OA-related research has not been reported. Our objective is to detect and characterize the early degradation activities shortly after cartilage or joint injury with MMPSense750. We determined the appropriate concentration, assay time, and linear range using various concentrations of recombinant MMPs as standards. We then quantified MMP activity from cartilage explants subjected to either mechanical injury or inflammatory cytokine treatment in-vitro. Finally, we performed in-vivo MMP imaging of a mouse model of post-traumatic OA. Our in-vitro results showed that the optimal assay time was highly dependent on the MMP enzyme. In cartilage explant culture media, mechanical impact or cytokine treatment increased MMP activity. Injured knees of mice showed significantly higher fluorescent signal than uninjured knees. We conclude that MMPSense750 detects human MMP activities and can be used for in-vitro study with cartilage, as well as in-vivo studies of knee injury, and can offering real-time insight into the degradative processes that occurring within the joint before structural changes become evident radiographically.

Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis.

OBJECTIVE: To characterize mitogen activated protein (MAP) kinase activity and chondrocyte apoptosis in an in vitro model of cartilage mechanical injury as a function of tissue depth and time post-injury. DESIGN: Mechanically injured osteochondral explants were assessed for cell viability, MAP kinase and caspase-3 activity over 15 days using immunofluorescence microscopy and Western blot. Zonal distributions of cell viability and apoptosis were quantified in the presence of specific mitogen activated protein kinase inhibitors. RESULTS: Viability rapidly decreased post-injury, most significantly in the superficial zone, with some involvement of the middle and deep zones, which correlated with increased caspase-3 activity. Transient and significant increases in extracellular-regulated protein kinase (ERK) activity were observed in middle and deep zones at 1 and 6 days post-injury, while c-Jun-amino terminal protein kinase activity increased in the deep zone at 1 and 6 days compared to uninjured controls. Changes in p38 activity were particularly pronounced, with significant increases in all three zones 30 min post-injury, but only in the middle and deep zones after 1 and 6 days. Inhibition of ERK and p38 increased chondrocyte viability which correlated with decreased apoptosis. CONCLUSIONS: Spatiotemporal patterns of MAP kinase signalling in cartilage after mechanical injury strongly correlate with changes in cell viability and chondrocyte apoptosis. Importantly, these signals may be pro-survival or pro-apoptotic depending on zonal location and time post-injury. These data yield mechanistic insights which may improve the diagnosis and treatment of cartilage injuries.

MMP proteolysis of the human extracellular matrix protein aggrecan is mainly a process of normal turnover.

Although it has been shown that aggrecanases are involved in aggrecan degradation, the role of MMP (matrix metalloproteinase) aggrecanolysis is less well studied. To investigate MMP proteolysis of human aggrecan, in the present study we used neoepitope antibodies against MMP cleavage sites and Western blot analysis to identify MMP-generated fragments in normal and OA (osteoarthritis/osteoarthritic) cartilage, and in normal, knee injury and OA and SF (synovial fluid) samples. MMP-3 in vitro digestion showed that aggrecan contains six MMP cleavage sites, in the IGD (interglobular domain), the KS (keratan sulfate) region, the border between the KS region and CS (chondroitin sulfate) region 1, the CS1 region, and the border between the CS2 and the G3 domain, and kinetic studies showed a specific order of digestion where the cleavage between CS2 and the G3 domain was the most preferred. In vivo studies showed that OA cartilage contained (per dry weight) 3.4-fold more MMP-generated FFGV fragments compared with normal cartilage, and although aggrecanase-generated SF-ARGS concentrations were increased 14-fold in OA and knee-injured patients compared with levels in knee-healthy reference subjects, the SF-FFGV concentrations did not notably change. The results of the present study suggest that MMPs are mainly involved in normal aggrecan turnover and might have a less-active role in aggrecan degradation during knee injury and OA.

Elevated aggrecanase activity in a rat model of joint injury is attenuated by an aggrecanase specific inhibitor.

OBJECTIVE: To evaluate aggrecanase activity after traumatic knee injury in a rat model by measuring the level of aggrecanase-generated Ala-Arg-Gly-aggrecan (ARG-aggrecan) fragments in synovial fluid, and compare with ARG-aggrecan release into joint fluid following human knee injury. To evaluate the effect of small molecule inhibitors on induced aggrecanase activity in the rat model. METHOD: An enzyme-linked immunosorbent assay (ELISA) was developed to measure ARG-aggrecan levels in animal and human joint fluids. A rat model of meniscal tear (MT)-induced joint instability was used to assess ARG-aggrecan release into joint fluid and the effects of aggrecanase inhibition. Synovial fluids were also obtained from patients with acute joint injury or osteoarthritis and assayed for ARG-aggrecan. RESULTS: Joint fluids from human patients after knee injury showed significantly enhanced levels of ARG-aggrecan compared to uninjured reference subjects. Similarly, synovial fluid ARG-aggrecan levels increased following surgically-induced joint instability in the rat MT model, which was significantly attenuated by orally dosing the animals with AGG-523, an aggrecanase specific inhibitor. CONCLUSIONS: Aggrecanase-generated aggrecan fragments were rapidly released into human and rat joint fluids after injury to the knee and remained elevated over a prolonged period. Our findings in human and preclinical models strengthen the connection between aggrecanase activity in joints and knee injury and disease. The ability of a small molecule aggrecanase inhibitor to reduce the release of aggrecanase-generated aggrecan fragments into rat joints suggests that pharmacologic inhibition of aggrecanase activity in humans may be an effective treatment for slowing cartilage degradation following joint injury.

Coordinated expression of MMPs and TIMPs in rat knee intra-articular tissues after ACL injury.

Anterior cruciate ligament (ACL) has poor healing ability and an injured ACL would induce the degeneration of other intra-articular connective tissues. However, the coordinated expression and activities of matrix metalloproteinase (MMPs) in intra-articular tissues induced by ACL rupture were poorly understood. With a rat ACL rotating injury model, we found that after ACL injury, the mRNA levels of MMP-13, TIMP-1, and CD147 were significantly elevated in ACL, posterior cruciate ligament (PCL), synovium, meniscus, and cartilage. Also, MMP-2 activity was also elevated significantly in a time-dependent manner in all intra-articular tissues. Synovium showed the most capability to release MMPs, whereas ACL showed the highest MMP-13/TIMP-1 ratio. Generic MMP activity assay and zymography showed time dependent elevation of MMP activities in synovial fluids (SF). We concluded that the ACL injury would induce a coordinated response of intra-articular tissues to express MMPs, TIMPs, and CD147. The MMP activities in the microenvironment in SF would accumulate, released by all the intra-articular tissues, which would contribute to the knee damage and degeneration induced by ACL injury.

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.

[Nitric oxide synthase and apoptosis activity in chondrocytes of articular cartilage in posttraumatic knee instability]. / Aktivnost' nitroksidsintazy i apoptoza v khondrotsitakh sustavnogo khriashcha pri posttravmaticheskoi nestabil'nosti kolennogo sustava.

The dynamics of nitric oxide synthase (NOS) and apoptosis activity was studied in the in chondrocytes of articular cartilage of the patients with posttraumatic knee instability. Statistically significant increase of chondrocyte NOS activity was detected in the earliest period after the trauma (1.5 months). This was accompanied by the appearance of a great number of apoptotic cells in the zones of the cartilage with high NOS activity. Remarkably, these changes developed at the same time with statistically significant decrease of total matrix glycosaminoglycan content. These results suggest that there is a NOS-dependent signaling way of apoptosis that could participate in the development of early dystrophic changes in the cartilage.

Matrix metalloproteinase-13 expression in rabbit knee joint connective tissues: influence of maturation and response to injury.

The hypothesis of the present work was that expression of matrix metalloproteinase-13 (MMP-13, collagenase-3) would be induced during conditions involving important matrix remodeling such as ligament maturation, scar healing and joint instability. Therefore, MMP-13 expression in the medial collateral ligament (MCL) during the variable situations of tissue maturation and healing was assessed. MMP-13 expression in three intra-articular connective tissues of the knee (i.e. articular cartilage, menisci and synovium) following the transection of the anterior cruciate ligament of the knee was evaluated at 3 and 8 weeks post-injury. MMP-13 mRNA (semi-quantitative RT-PCR) and protein (immunohistochemistry and Western blotting) were detected in all of the tissues studied. Significantly higher MCL mRNA levels for MMP-13 were detected during the early phases of tissue maturation (i.e. 29 days in utero and 2-month-old rabbits) compared to later phases (5- and 12-month-old rabbits). This pattern of expression was recapitulated following MCL injury, with very high levels of expression in scar tissue at 3 weeks post-injury and then a decline to levels not significantly different from control values by 14 weeks. Elevated mRNA levels correlated with increased protein levels for MMP-13 in both menisci and synovium following the transection of the anterior cruciate ligament and during medial collateral ligament healing. These results indicate that MMP-13 expression is regulated by a number of variables and that high levels of expression occur in situations when connective tissue remodeling is very active.

Development of osteoarthritis in the knee joints of Wistar rats after strenuous running exercise in a running wheel by intracranial self-stimulation.

The influence of excessive running load on the development of knee osteoarthritis (OA) was investigated in male Wistar rats. Running exercises were performed in a running wheel using intracranial self-stimulation to motivate Wistar rats to run daily distances of 500 m at 5 days/week. Hereby, ten rats ran a distance of 15 km within three weeks while a further ten rats run a total of 30 km within six weeks. Thirteen Wistar rats without running exercises served as controls. Complete knee joint sections of all rats were evaluated histologically using MANKINs grading system with categorization of the findings into non, mild moderate, and severe osteoarthritis. In addition, immunoreactivity of the chondrocytes to MMP-3 as an important cartilage degrading enzyme in OA was assessed by immunostaining with monoclonal MMP-3 IgG antibodies. Histological assessment of the knee joint sections revealed a significant increase in osteoarthritic changes with higher running load. While in rats with 15 km running all but two knee joints showed mild OA, moderate OA was the predominant finding in rats with 30 km running. In contrast, no OA was found in the controls. Immunostaining for MMP-3 revealed a significant increase in immunoreactivity of the chondrocytes to MMP-3 with higher running load, indicating a running load-depending production of this cartilage-degrading enzyme in the course of increasing OA. Compared to 47.4% immunoreactive chondrocytes to MMP-3 in the controls, this ratio rose to 70.4% in rats with 15 km running and even up to 89.9% in rats with 30 km running. In conclusion, in Wistar rats, excessive running load leads to marked, running distance-depending osteoarthritic changes which are caused, at least in part, by an increase in MMP-3 production rising with greater running distance. Within this exercise model of OA, intracranial self-stimulation is an effective method to motivate Wistar rats to extremely excessive running in a running wheel. This model offers a wide range of further approaches to studying different processes of the development of OA.

Differential patterns of PMN-elastase and type III procollagen peptide in knee joint effusions due to acute and chronic sports injuries.

In 38 traumatic knee joint effusions the proteolytic enzyme PMN-elastase (PMN-E) and the repair marker procollagen III aminoterminal peptide (PIIINP) were determined. According to the period between trauma and first aspiration of the effusion, the patients were divided into 3 groups. Group I (17 patients; period between trauma and first aspiration not longer than 72 hours) showed high concentrations of PMN-E (up to 5400 ng/ml) and low concentrations of PIIINP (less than 13 U/ml). Group II (11 patients; aspiration within 4 to 14 days) had mean PMN-E and PIIINP concentrations of 125.6 ng/ml and 52.1 U/ml, respectively. In group III (10 patients, aspiration after 14 days) mean PMN-E concentration was 123.8 ng/ml and mean PIIINP concentration was 63.4 U/ml. Graphic depiction of PMN-E and PIIINP levels in each individual sample as a function of time between trauma and fluid collection revealed highly increasing PMN-E levels during the first 24 posttraumatic hours, followed by rapidly decreasing levels within 72 hours post trauma, and no change after the 4th posttraumatic day. In contrast, PIIINP increased continuously up to the first posttraumatic week and stayed at high levels up to 90 days (end of the observation period). The differential patterns of PMN-E and PIIINP concentration in knee joint effusions may be useful in estimating the period between trauma and first treatment (aspiration of effusion) and should, therefore, be helpful in detecting degenerative lesions, which seem to be characterized by low PMN-E concomitantly with high PIIINP levels.

Injury of the anterior cruciate ligament: the role of collagenase in ligament degeneration.

Rapid degeneration of the anterior cruciate ligament (ACL) has been observed following acute ACL rupture. An understanding of this process might explain some of the poor clinical results of primary ACL repair. We created a surgical rabbit model of acute ACL injury and developed an in vitro assay for collagenase activity in the ACL and menisci. Microscopic evaluation revealed a rapidly degenerative process in injured ACLs, with loss of cellularity and matrix organization. This was associated with a significant increase in collagenase activity and a decrease in total collagen of the injured ACLs as compared with sham-operated controls. These findings confirm the observation that cut ACL ligament ends rapidly degenerate. This degenerative process might be partly due to a response of cells intrinsic to the ACL to injury. Left unchecked, this process may be detrimental to surgical attempts for primary ACL repair.

Histological and enzyme histochemical study on the injured knee meniscus in human.

The activity and distribution of 10 enzymes was determined in the ruptured knee meniscus of 23 patients, when meniscus was operatively removed. The activities of NADH and SDH indicating oxydative energy metabolism were low in the ruptured meniscus as well as in the synovium close to it. On the contrary, NADPH and LDH, indicating anaerobic energy metabolism and G-6-PDH as an indicator of pentose-phosphate shunt, showed moderate or high activity. The activities of GLDH, ATPase, AcPase, AlPase, and LAPase were low in the meniscus tissue, but moderate and sometimes high in synovial tissue and fibroblasts close to the meniscus. In the vascular walls these enzyme activities all were moderate or high indicating reparative capacity in the peripheral, vascularized part of meniscus. The age of the patients as well as the time interval between the trauma and the operation was not in relationship with enzyme activities studied.

[Role of cathepsin D of synovial fluid of the knee joint in the pathogenesis of traumatic arthritis]. / Rol' katepsina D sinovial'noi zhidkosti kolennogo sustava v patogeneze travmaticheskogo artrita.

Activity of cathepsin D in synovial fluid of the knee joint and blood of 113 patients with injuries of this joint was studied. A conclusion is made about pathogenetic role of cathepsin D in the development of reactive arthritis.

[Dynamics of lysozyme activity in the synovial fluid of patients with knee joint injuries]. / Dinamika aktivnosti lizotsima v sinovial'noi zhidkosti u bol' nykh s povrezhdeniiami kolennogo sustava.

The activity of lysozyme in the sinovial fluid and blood serum was studied by the authors in 69 patients with injuries of the knee joint and after operations on the joint. The lysozyme activity was higher in the blood serum of patients as compared with normality, that in the sinovial fluid was higher than in the blood serum and found to decrease in time. The lysozyme activity in the sinovial fluid was found to correspond to the degree of the inflammatory process in the joint. The authors made a conclusion that lysozyme on a level with hialuronidase takes part in the development of the inflammatory process in the joint injuries.

Acute effect of open joint wounds on articular cartilage and synovium in rabbits.

Open joint wounds were made in immature and mature rabbit knees by surgical arthrotomy. The wounds of the right knees were packed open for 5 days and the left knee wounds were closed primarily. The biochemical studies were selected to determine the effect of our treatment regimen on changes in the metabolism of articular cartilage and synovium. Neither closed nor open treatment produced significant changes in enzyme activities measured in the articular cartilage as compared to the controls in either immature or mature rabbits. Most of the synovial enzymes were elevated in the injured joints. There were, however, no significant differences in enzyme activity between the joints treated by either open or closed methods. Our findings suggest that short-term open treatment wounds does not cause matrix degradation in the cartilage nor affect the synovium more than simple arthrotomy.