Markers of joint tissue turnover in joint fluids from hips with osteonecrosis of the femoral head.

Osteonecrosis of the femoral head often results in secondary osteoarthritis of the hip joint; however, the pathologic processes underlying the destruction of articular cartilage are not fully understood. Molecular markers in the hip joint fluids were measured to examine the changes in turnover of cartilage and other joint tissues. Marker data were related to clinical, radiological, and histopathological changes in the articular cartilage of the hip. Forty-five patients (median age: 43 years) were studied. The median time between the onset of symptoms and sampling of hip synovial fluid was 6 months. Aggrecan fragments, C-propeptide of type-II collagen, matrix metalloproteinase-3, and tissue inhibitor of metalloproteinases-1 levels in joint fluid were determined by immunoassay. Osteonecrosis of the femoral head was graded by radiology as minimal collapse of the femoral head (stage 2: 26 patients), severe collapse (stage 3: 15 patients), or severe collapse with osteoarthritis (stage 4: four patients). Histological changes of the articular cartilage, consistent with early-stage osteoarthritis, were evident at stage 3 and were more advanced at stage 4. The average concentrations of proteoglycan fragments and C-propeptide of type-II collagen were 207 (SD 182) microg/ml and 19.6 (SD 19.3) ng/ml, respectively. The average concentrations of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 were 177 (SD 291) nM and 23.0 (SD 9.9) nM, respectively. Measurable levels for all markers assayed were noted in the earliest stage of the disease, only a few months after the onset of symptoms and well before the appearance of radiological changes. Levels of matrix metalloproteinase-3 and molar ratios of matrix metalloproteinase-3/tissue inhibitor of metalloproteinases-1 were higher in early stage disease than in later stage disease.

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

Use of an antibody against the matrix metalloproteinase-generated aggrecan neoepitope FVDIPEN-COOH to assess the effects of stromelysin in a rabbit model of cartilage degradation.

OBJECTIVE: To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo. METHODS: The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescence. Proteoglycan fragments in joint fluids were quantified by a dimethylmethylene blue dye-binding assay. RESULTS: Stromelysin cleavage of rabbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN: An anti-FVDIPEN antibody recognized the FMDIPEN neoepitope in situ in cartilage from stromelysin-injected joints. The appearance of the FMDIPEN neoepitope corresponded to the release of cartilage proteoglycan fragments into the joint fluid, and could be inhibited by pretreatment of the rabbits with a synthetic stromelysin inhibitor. CONCLUSION: These results indicate that the anti-FVDIPEN antibody can be used to assess the role of MMPs in cartilage degradation in vivo.

The potential role of fibroblasts in periprosthetic osteolysis: fibroblast response to titanium particles.

Periprosthetic osteolysis with or without aseptic loosening is a major clinical problem in total hip arthroplasty. While the macrophage response to prosthetic wear debris and its role in periprosthetic osteolysis has been extensively studied, information regarding other cell types (fibroblasts, osteoblasts) is limited. This study explored the response of fibroblasts to particulate wear debris. Fibroblasts isolated from interfacial membranes of patients with failed total hip replacements and normal synovial tissue, when challenged with small-sized ( < 3 microns) titanium (Ti) particles, responded with significantly enhanced expressions of collagenase, stromelysin and, to a much lesser extent, their tissue inhibitor of metalloproteinases (TIMP). These "regulated" expressions at both mRNA and protein levels were correlated with the size and composition of particles. De novo protein synthesis was required for the regulation of these mRNAs. A similar effect could be induced by the treatment of the cells with particle-free conditioned medium from Ti particle-stimulated fibroblasts. Furthermore, this conditioned medium significantly suppressed the mRNA levels of procollagen alpha 1 (I) and alpha 1 (III) in osteoblast-like MG-63 cells. It is concluded that fibroblasts stimulated with certain particle debris may play an important role in periprosthetic osteolysis by releasing bone-resorbing metalloproteinases and mediator(s) which resulted in suppressed collagen synthesis in osteoblasts.

Markers of cartilage matrix metabolism in human joint fluid and serum: the effect of exercise.

The concentrations of cartilage proteoglycan (aggrecan), stromelysin-1, tissue inhibitor of metalloproteinases-1 (TIMP-1) and procollagen II C-propeptide in knee joint fluid and the levels of aggrecan, hyaluronan and keratan sulfate in serum were measured before and after exercise in 33 healthy athletes. The samples before exercise were obtained after 24 h rest from running or soccer and the samples after exercise were obtained 30-60 min after the exercise. Nine athletes ran on a treadmill for 60 min, 16 ran on road for 80 min and 8 played one soccer game (90 min). A reference group of 28 patients with knee pain but not evidence of joint pathology or injury was used for comparison. In joint fluid no single marker from the degradative processes in cartilage matrix changed significantly with exercise but all showed a rising trend. All markers except stromelysin showed lower concentrations in athletes at rest compared to the reference group. In serum from runners before exercise the concentration of keratan sulfate was significantly higher than in both the soccer and reference groups and further increased after exercise. The increase in markers after exercise may reflect an effect of mechanical loading in combination with a possible high turnover rate of body cartilage matrix in these individuals.

Cartilage metabolism in the injured and uninjured knee of the same patient.

OBJECTIVE: To examine if unilateral knee injury affects the synovial fluid concentrations of aggrecan fragments, cartilage oligomeric matrix protein (COMP) fragments, stromelysin-1, and tissue inhibitor of metalloproteinases-1 (TIMP-1) in the contralateral uninjured knee. METHODS: Synovial fluids from the injured and uninjured knees were obtained at different times in a group of patients after unilateral knee trauma. Serum samples were obtained on the same occasion. Concentrations of aggrecan fragments were determined by precipitation with Alcian Blue; those of COMP fragments, stromelysin-1, and TIMP-1 were measured by immunoassay. Concentrations were compared with those in a reference group of 10 healthy volunteers. RESULTS: Immediately after knee injury, concentrations of aggrecan fragments, COMP fragments, stromelysin-1 and TIMP-1 were increased in the synovial fluid of the injured knee. However, concentrations of aggrecan and COMP fragments, and stromelysin-1 increased also in the contralateral uninjured knee immediately after injury, but less than in the injured knee. Subsequently, the concentrations of all markers decreased in the synovial fluid of the injured knee, but remained unchanged in the uninjured knee. The concentration of aggrecan fragments in the injured knee decreased to less than that in the uninjured knee in the chronic phase. Serum concentrations of COMP were much smaller than those in synovial fluid. CONCLUSIONS: The increased concentrations of aggrecan and COMP fragments and stromelysin-1 in the joint fluid of the contralateral, uninjured knee following unilateral knee injury, compared with concentrations in healthy reference knees, suggest changes in joint cartilage metabolism in both knees following unilateral knee injury. The mechanisms for these changes are unclear. The low serum concentration of COMP makes it less likely that there is any significant 'exchange' of molecular makers between the knees. A further consequence of these findings is that the contralateral knee cannot be recommended as the only control joint in studies of matrix metabolism in patients with unilateral knee injury.

Induction of increased levels of proteoglycan fragments in synovial fluid (SF) and increased levels of stromelysin in cartilage, synovium and SF by intraarticular injection of canine monocyte conditioned medium into dogs.

OBJECTIVE: To study the effects of the intraarticular injection of canine monocyte conditioned medium (cMCM) into dogs on proteoglycan fragment and stromelysin levels in the joint. METHODS: cMCM was injected intraarticularly into dogs, and the levels of proteoglycan fragments in synovial fluid (SF) as well as stromelysin levels in cartilage, synovium, and SF were assessed after 12 h. RESULTS: There was a 4-fold increase of proteoglycan fragment levels and a 6-fold increase in stromelysin levels in SF, and a 4.4-fold increase in stromelysin levels in cartilage extracts. Elevated mRNA levels were detected in both synovium and cartilage. By immunofluorescence staining, stromelysin was localized in chondrocytes throughout the cartilage and in synovial cells. CONCLUSION: Intraarticular injection of cMCM stimulated the expression of stromelysin mRNA and protein in cartilage and synovium and caused marked increases in stromelysin protein and proteoglycan fragment levels in SF.

Temporal patterns of stromelysin-1, tissue inhibitor, and proteoglycan fragments in human knee joint fluid after injury to the cruciate ligament or meniscus.

Stromelysin-1, tissue inhibitor of metalloproteinases-1 (TIMP-1), and proteoglycan fragments were quantified in knee synovial fluid samples in a cross-sectional study of patients who had injury to the anterior cruciate ligament or the meniscus. The average concentrations of stromelysin-1 and TIMP-1 increased 25-fold and 10-fold within the first day after the trauma, respectively, and the concentration of proteoglycan fragments increased 4-fold. From approximately 1-6 months after injury, the levels of these markers were higher after injury to the cruciate ligament than after injury to the meniscus. From 6 months to 18 years after trauma, however, the levels of stromelysin-1 and TIMP-1 in patients who had an injury to the ligament were the same as the levels in patients who had a meniscal lesion, but the levels were increased compared with those for a reference group of healthy volunteers. The molar balance of stromelysin-1 to TIMP-1 in synovial fluid in both groups of injured joints changed from a balance representing an excess of free inhibitor in the normal joint to one representing an excess of free enzyme in the injured joint. The increased release of these markers to joint fluid both early and late after trauma may be caused by a change in the loading patterns in the knee with an injured ligament or meniscus or by synovitis induced by bleeding. The increased release may be associated with the frequent development of posttraumatic osteoarthritis in patients with these injuries.

Differential in vivo expression of collagenase messenger RNA in synovium and cartilage. Quantitative comparison with stromelysin messenger RNA levels in human rheumatoid arthritis and osteoarthritis patients and in two animal models of acute inflammatory arthritis.

OBJECTIVE: To compare quantitatively the in vivo expression of collagenase messenger RNA (mRNA) and stromelysin mRNA in the joint tissues of human osteoarthritis (OA) and rheumatoid arthritis (RA) patients and in two animal models of acute inflammatory arthritis. METHODS: In vivo levels of metalloproteinase mRNA and protein were determined by quantitative Northern hybridization and by enzyme-linked immunosorbent assay, respectively. RESULTS: In synovium, mean levels of collagenase mRNA were similar to those of stromelysin mRNA; however, in cartilage, mean levels of collagenase mRNA were significantly lower. The ratios of collagenase mRNA to stromelysin mRNA levels in RA and OA cartilage reflected similar ratios of collagenase protein to stromelysin protein levels in synovial fluid. CONCLUSION: The regulation of collagenase mRNA expression in cartilage is distinct from that of stromelysin, suggesting distinct roles for these two metallo-proteinases in normal and abnormal physiologic functioning of cartilage.

Stromelysin, tissue inhibitor of metalloproteinases and proteoglycan fragments in human knee joint fluid after injury.

OBJECTIVE: To determine in a cross sectional study the concentrations of stromelysin, tissue inhibitor of metalloproteinases (TIMP), and proteoglycan fragments in knee synovial fluid (SF) at different times after injury to cruciate ligament or meniscus. METHODS: Joint fluid samples were obtained from patients with knee injury diagnosed by arthroscopy. Concentrations of stromelysin-1 and TIMP-1 were determined by immunoassay with monoclonal and polyclonal antibodies. Cartilage proteoglycan fragments were quantified by immunoassay with polyclonal antibodies or by dye precipitation. RESULTS: Average concentrations of stromelysin increased 40-fold in association with injury, and after about 6 months decreased to a plateau level about 10-fold increased compared to a reference group with healthy knees. TIMP and proteoglycan levels also increased in similar temporal patterns, but less markedly. Increased average SF levels of these markers were maintained for at least 17 years after injury. SF from knees with injury contained a 1.5 to 2.5 molar excess of stromelysin over TIMP, while reference joint fluids contained a 2-fold molar excess of TIMP over stromelysin. CONCLUSION: The persistent changes in SF markers after joint injury may be associated with the cartilage destruction and frequent development of posttraumatic osteoarthritis in this group of patients.

Metalloproteinases, tissue inhibitor, and proteoglycan fragments in knee synovial fluid in human osteoarthritis.

OBJECTIVE: To determine the concentrations of human stromelysin-1, collagenase, tissue inhibitor of metalloproteinases (TIMP), and proteoglycan fragments in knee synovial fluid in patients with injury to the meniscus or anterior cruciate ligament, posttraumatic osteoarthritis, primary osteoarthritis, or pyrophosphate arthritis. METHODS: Synovial fluid samples were collected from patients with knee disease diagnosed arthroscopically and radiologically. Concentrations of stromelysin-1, collagenase, and TIMP-1 were determined by sandwich immunoassay, using monoclonal and polyclonal antibodies. Fragments of cartilage proteoglycan containing the chondroitin sulfate-binding region were determined by immunoassay with a polyclonal antibody. RESULTS: Average concentrations of metalloproteinases, TIMP, and proteoglycan fragments in joint fluid were significantly elevated in patients from all disease groups as compared with volunteers with healthy knees (reference group). Stromelysin concentrations in disease groups averaged 15-45 times that of the reference group. The molar ratios between stromelysin and collagenase varied between 10 and 150. The molar ratio between total stromelysin and free TIMP was 0.5 in the reference group and between 1.6 and 5.3 in the disease groups. CONCLUSION: Stromelysin concentration in joint fluid is a parameter that distinguishes diseased joints from healthy joints, with a sensitivity of 84% and a specificity of 90%. The high concentrations of metalloproteinase relative to TIMP in joint fluid from patients with the conditions studied may be associated with cartilage matrix degradation in these arthritides.

In vivo expression of stromelysin in synovium and cartilage of rabbits injected intraarticularly with interleukin-1 beta.

OBJECTIVE: To examine the in vivo expression of the matrix metalloproteinase stromelysin in the synovium and articular cartilage of rabbits injected intraarticularly with recombinant human interleukin-1 beta (IL-1). METHODS: The direct isolation of messenger RNA (mRNA) from articular cartilage without the prior isolation of chondrocytes is described. The in vivo expression of stromelysin was examined at the mRNA level by Northern blot analysis, and at the protein level by in situ immunolocalization and by enzyme-linked immunosorbent assay. RESULTS: In the synovium of IL-1-injected joints, stromelysin mRNA levels were highest at 4 hours and declined to background levels within 24 hours. In the cartilage of IL-1-injected joints, stromelysin mRNA was elevated at 4 hours and continued to increase until 8 hours, before declining. Stromelysin mRNA expression preceded a similar increase in stromelysin protein levels in both synovium and cartilage. CONCLUSION: Intraarticular injection of IL-1 induced the endogenous expression of stromelysin mRNA and protein in both synovium and cartilage. The kinetics of stromelysin expression correlated well with the accumulation of stromelysin and proteoglycan in synovial fluids. Therefore, the de novo synthesis of stromelysin in cartilage may have contributed to the loss of proteoglycan from that tissue.

Recombinant human interleukin-1 beta-induced increase in levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid.

OBJECTIVE: To evaluate the effects of intraarticular injection of recombinant human interleukin-1 beta (IL-1 beta) on levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid (SF), and to determine the effects of leukocyte depletion on SF proteoglycan and stromelysin levels. METHODS: Levels of leukocytes and of proteoglycans, stromelysin, and collagenase were evaluated 12 hours after the intraarticular injection of various doses of IL-1, and over a 24-hour period after injection at a single dose level. We used a monoclonal antibody (MAb) against leukocyte integrins, which markedly depressed leukocyte accumulation in SF, to evaluate the role of synovial leukocytes on IL-1-induced increases in SF proteoglycan and stromelysin levels. RESULTS: Levels of both proteoglycans and stromelysin increased in the IL-1-injected joints between 4 hours and 24 hours after the injection of a single 200-ng dose of IL-1. The highest levels of stromelysin and proteoglycans were achieved with IL-1 doses greater than or equal to 100 ng. Infiltration of polymorphonuclear cells (PMN) into the joint fluid of the IL-1-injected rabbits also increased, in a dose-dependent manner. Treatment of rabbits with MAb 1B4 markedly reduced infiltration of PMN into the joint, without affecting either stromelysin or proteoglycan levels. CONCLUSION: Taken together, the data suggest that there is a coordinate increase in SF stromelysin and proteoglycan levels in rabbits injected with IL-1, and that leukocytes play a minimal role in the accumulation of proteoglycans and stromelysin in the SF.