David S. Howell

Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage.

Cartilage specimens from tibial plateaus, obtained from 13 osteoarthritic (OA) patients and seven controls, were selected from three regions: zone A, center of fibrillated area; zone B, area adjacent to fibrillation, and zone C, remote region of plateau. Acid and neutral metalloproteinases and tissue inhibitor of metalloproteinase (TIMP) were extracted with 2 M guanidine. Methods were developed to selectively destroy either proteinases or TIMP to prevent cross-reaction during assay. Acid and neutral proteinases were elevated approximately 150% in OA; TIMP was elevated approximately 50%. A positive correlation (r = 0.50) was found between acid and neutral proteinase activities in OA, but not in controls. Both proteinases were elevated two-to threefold in zones A, B, and C. However, the self-active form of the acid metalloproteinase was elevated only in zones A and B (200%); it correlated well with the Mankin scores, whereas the total activities did not. TIMP was elevated (50%) only in zones A and B. Both the proteinase levels and the Mankin score were elevated to a greater extent in the medial, than in the lateral, compartment. Titration of TIMP against the two metalloproteinases indicates that there is a small excess of inhibitor over enzymes in normal cartilage. In OA, TIMP does not increase to the same extent as the proteinases; the resultant excess of proteinases over TIMP may contribute to cartilage breakdown.

Pathogenesis of osteoarthritis

This article reviews the etiology and pathogenesis of osteoarthritis, particularly one of several current concepts concerning the possible central mechanisms regulating degradation of cartilage. According to this theory, degradation involves diffuse or focal exposure of the extracellular matrix to active neutral metalloproteinases, which then results in injury as well as initiation of repair processes. Diffuse matrix exposure is probably not a physiologic aberrancy but rather a pathologic result of either physical injury to local chondrocytes or inflammatory mediators.

Partition of calcium, phosphate, and protein in the fluid phase aspirated at calcifying sites in epiphyseal cartilage

A reproducible method, adapted from renal micropuncture techniques, was developed for sampling 10-40 mmul of a clear fluid from epiphyseal cartilage of normal or rachitic rats in vivo, either from the hypertrophic cell zone (C(f1)) or surface resting cell cartilage (L(f1)). Characterization of this fluid depended upon quantitation of protein, total inorganic phosphate (P(it)), total calcium (Ca(t)), nucleotide, and hemoglobin in volumes of 20 mmul. Established methods for macroscale measurements of each of these parameters have been modified to permit direct spectrophotometric readings on samples of 10(-10)-10(-11) g. The fluid from hypertrophic and peripheral resting cell cartilage was of an extracellular nature as evidenced by a high chloride and sodium, as well as low potassium, protein, and nucleotide content. The pH of fluid isolated from endochrondral plates in vivo was measured under oil as a function of P(CO2) and the computed bicarbonate was elevated above concurrent serum levels. After ultracentrifugation of C(f1) of normal, rachitic, and healing rachitic animals, nonprotein-bound calcium (Ca(f)) and phosphate (P(if)) were determined on supernatant fluids. The hypertrophic cell cartilage fluid of rachitic rats was distinguished by a high ratio C(f1)/serum of P(if). This ratio returned to normal during treatment of rickets. The upper limit for ionic activity A(1) Ca(++) x A HPO(4) (=) was too low to initiate precipitation of brushite or dicalcium phosphate but was in a range of supersaturation in respect to crystalline apatites. Thus these data are consistent with initiation of calcification by heterogeneous nucleation of mineral in the septal matrix but can be reconciled alternately with a precipitation mechanism only if the site of initial mineral phase separation is outside the septal matrix.

Metalloproteases of human articular cartilage that digest cartilage proteoglycan at neutral and acid pH.

Extracts of human articular cartilage contain proteases capable of degrading the proteoglycan component of cartilage matrix at neutral and acid pH. These enzymes have been partially purified by ion exchange chromotography and characterized by disc electrophoresis, inhibition patterns, and action of proteoglycan. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit optimally at pH 7.25 has been purified up to 900-fold. It is strongly inhibited by o-phenanthroline, alpha-2-macroglobulin, and egg white, and to a lesser extent by D-penicillamine and EDTA. Inhibition by chelating agents is reversed by cobalt, zinc, and ferrous ions. Two acid metalloproteases, distinct from cathespins B1, D, and F, digest proteoglycan subunit at pH 4.5 and 5.5. Both are inhibited by o-phenanthroline and activity is restored by cobalt, zinc, or ferrous ions. With electron microscopy, it was found that cartilage slices were depleted of ruthenium red-staining matrix proteoglycan after incubation in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immuno-diffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteoglycan subunit, producing fragments with 5 to 12 chondroitin sulfate chains. The link proteins were not digested.

Treatment of canine osteoarthritis with insulin-like growth factor-1 (IGF-1) and sodium pentosan polysulfate.

The potential therapeutic effects of insulin-like growth factor-1 (IGF-1) and sodium pentosan polysulfate (PPS) were evaluated in an anterior cruciate ligament-deficient canine model of osteoarthritis (OA). A control group of animals received no treatment or surgery (N). The remaining four groups of animals received anterior cruciate transection and either no treatment (OA), intra-articular IGF-1 (IGF-1), intra-muscular PPS (PPS), or a combination of intra-articular IGF-1 and intra-muscular PPS (IGF-1/PPS). All therapy was begun 3 weeks after surgery and continued for 3 weeks. At 6 weeks, articular cartilage from the femoral condyle was evaluated for anatomy, histology (Mankin grade) and biochemistry. Anatomically, only cartilage from dogs in the IGF-1/PPS group approximated that found in N. Mankin scores indicated less severe disease in both PPS and IGF-1/PPS groups compared with the OA group. Consistent with histology, the level of active neutral metalloproteinase was lower in cartilage from the PPS group compared with the OA group. Active and total neutral metalloproteinase, tissue inhibitor of metalloproteinases (TIMP), total collagenase, uronate and hydroxyproline contents were all near normal in the IGF-1/PPS group. In a model of mild OA, therapeutic intervention with IGF-1 and PPS appeared to successfully maintain cartilage structure and biochemistry. From these data, it is hypothesized that proteinase activity was successfully blocked by PPS, and that this allowed the observed growth factor induced effects. As we unravel the various factors that regulate cartilage metabolism, it is becoming apparent that combinations of agents will be needed to effectively control cartilage repair in OA. The addition of PPS to IGF-1 shows promise as a therapeutic intervention and introduces a new rational approach to therapy of OA.

Biomechanical and biochemical properties of dog cartilage in experimentally induced osteoarthritis.

The finding of other investigators that increased water content is often associated with signs of a torn collagen network in human osteoarthritic (OA) cartilage led to this study. In the Pond-Nuki model of post-traumatic OA experimental but not control femoral condylar cartilage showed evidence of breakdown and stiffening of collagen network as assessed by measurement of swelling properties and indentation behaviour respectively. These changes in the unstable knees occurred despite lack of erosion of that surface cartilage ascertained from carbon black mapping and history. The stiffening rather than softening change was therefore attributed to cartilage oedema of the middle and deep certilagenous zones, wherein breakdown of collagen network has been postulated to occur. Because of insignificant reduction of total hexuronate in these cartilages, a proteoglycan (PG) profile of sedimentation coefficients for aggregate (PGA) and subunit species (PGS) was analysed to see if collagen network changes in the dog preceded PG alteration. Despite minimal histological changes our results confirmed previous findings in the tibial plateau cartilage in this model, that PGA was reduced in size and PGS increased in amount. Slight enzymatic breakdown of PGs, or altered synthesis due to cellular responses to either the injury directly or to synovial inflammation, seems necessary to explain such changes in the absence of cartilage erosion.

Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of β-glycerophosphate and ascorbic acid

Matrix vesicles, media vesicles, and plasma membranes from three well-characterized, osteoblast-like cells (ROS 17/2.8, MG-63, and MC-3T3-E1) were evaluated for their content of enzymes capable of processing the extracellular matrix. Matrix vesicles were enriched in alkaline phosphatase specific activity over the plasma membrane and contained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collagenase, lysozyme, hyaluronidase, and tissue inhibitor of metalloproteinases (TIMP) were not found in any of the membrane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 μg/ml), β-glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcification. Ascorbate or β-glycerophosphate alone had no effect, but in combination produced significant increases in both active and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix vesicles being the most dramatic. This correlated with an increase in the formation of von Kossa-positive nodules. The results of the present study indicate that osteoblast-like cells produce matrix vesicles enriched in proteoglycan-degrading metalloproteinases. In addition, the observation that matrix vesicles contain significantly increased metalloproteinases under conditions favorable for mineralization in vitro lends support to the hypothesis that matrix vesicles play an important role in extracellular matrix processing and calcification in bone.

Pyridoxine deficiency affects biomechanical properties of chick tibial bone

The mechanical integrity of bone is dependent on the bone matrix, which is believed to account for the plastic deformation of the tissue, and the mineral, which is believed to account for the elastic deformation. The validity of this model is shown in this study based on analysis of the bones of vitamin B6-deficient and vitamin B6-replete chick bones. In this model, when B6-deficient and control animals are compared, vitamin B6 deficiency has no effect on the mineral content or composition of cortical bone as measured by ash weight (63 +/- 6 vs. 58 +/- 3); mineral to matrix ratio of the FTIR spectra (4.2 +/- 0.6 vs. 4.5 +/- 0.2), line-broadening analyses of the X-ray diffraction 002 peak (beta 002 = 0.50 +/- 0.1 vs. 0.49 +/- 0.01), or other features of the infrared spectra. In contrast, collagen was significantly more extractable from vitamin B6-deficient chick bones (20 +/- 2% of total hydroxyproline extracted vs. 10 +/- 3% p < or = 0.001). The B6-deficient bones also contained an increased amount of the reducible cross-links DHLNL, dehydro-dihydroxylysinonorleucine, (1.03 +/- 0.07 vs. 0.84 +/- 0.13 p < or = 0.001); and a nonsignificant increase in HLNL, dehydro-hydroxylysinonorleucine, (0.51 +/- 0.03 vs. 0.43 +/- 0.03, p < or = 0.10). There were no significant changes in bone length, bone diameter, or area moment of inertia. In four-point bending, no significant changes in elastic modulus, stiffness, offset yield deflection, or fracture deflection were detected. However, fracture load in the B6-deficient animals was decreased from 203 +/- 35 MPa to 151 +/- 23 MPa, p < or = 0.01, and offset yield load was decreased from 165 +/- 9 MPa to 125 +/- 14 MPa, p < or = 0.05. Since earlier histomorphometric studies had demonstrated that the B6-deficient bones were osteopenic, these data suggest that although proper cortical bone mineralization occurred, the alterations of the collagen resulted in changes to bone mechanical performance.

Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans

SummaryThis study examined the presence of extracellular matrix processing enzymes in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. Optimum procedures for the extraction of each enzyme activity were determined. Enzyme activity associated with chondrocyte plasma membrane microsomes was used for comparison. There was a differential distribution of the enzyme activities related to the cartilage zone from which the cells were isolated. Acid and neutral metalloproteinase (TIMP), plasminogen activator, and betaglucuronidase were highest in the growth zone chondrocyte (GC) membrane fractions when compared with matrix vesicles and plasma membranes isolated from resting zone chondrocyte (RC) cultures. There was a threefold enrichment of total and active acid metalloproteinase in GC matrix vesicles, whereas no enrichment in enzyme activity was observed in RC matrix vesicles. Total and active neutral metalloproteinase were similarly enriched twofold in GC matrix vesicles. TIMP, plasminogen activator, and betaglucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found in any of the membrane fractions. The data indicate that matrix vesicles are selectively enriched in enzymes which degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

Mechanical behavior and biochemical composition of canine knee cartilage following periods of joint disuse and disuse with remobilization

The mechanical behavior and biochemical composition of articular cartilage were studied in an experimental model of joint disuse, in which the canine knee was immobilized in a sling at 90 degrees of flexion. Articular cartilage from the surface zone of the femur was tested in an isometric tensile test and full-thickness cartilage on the tibial plateau was tested in a compressive indentation test. Water, proteoglycan and collagen contents were measured in site-matched samples. Site-specific increases in the tensile moduli (approximately 88% above control values in distal femoral groove) were observed in cartilage after 8 weeks of joint disuse, and after 3 weeks of remobilization following either 4 (approximately 140%, distal and proximal femoral groove) or 8 weeks (approximately 140%, distal femoral groove) of joint disuse. In contrast, the compressive properties of cartilage determined in the indentation test exhibited no change from control values with joint disuse or disuse followed by remobilization. Water contents increased at some sites on the tibia after 8 weeks of joint disuse (approximately 6% of tissue wet weight, posterior site), but not in the surface zone tissue of the femur. Proteoglycan/collagen and cartilage thickness were not found to change with disuse or disuse followed by remobilization. Reduced values for the ratio of proteoglycan:water were observed in the surface zone tissue of the femur (approximately 23%, distal femoral groove) and in the full-thickness tissue of the tibia (approximately 21%, anterior and posterior sites) after periods of joint disuse. In this study, the measured material properties suggest that the articular surface remains intact following periods of disuse or disuse with remobilization. This finding suggests one important difference between this model of joint disuse and other experimental models in which cartilage changes are both progressive and degenerative, such as surgically-induced joint instability.