David D. Dean

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.

Elevated tissue levels of collagenase during dilation of uterine cervix in human parturition

Seven biopsy specimens from the cervix and 17 from the lower uterine segment were obtained in 24 women at term (37 to 42 weeks). Collagenase was extracted and assayed on telopeptide-free [3H]collagen; typical collagen cleavage products were found on sodium dodecyl sulfate-gel electrophoresis. There was no significant difference between collagenase levels in the cervix and in the lower uterine segment in women not in labor and with the cervix closed. Levels of active and latent collagenase in 11 such specimens were 0.14 +/- 0.03 and 0.64 +/- 0.90 U/gm wet weight, respectively (mean +/- SEM; 1 U = 1 micrograms collagen digested per minute at 30 degrees C). Thirteen women at term in active labor with cervical dilation of 4 to 8 cm exhibited a thirteenfold increase in mean collagenase activity in the lower uterine segment. Active and latent collagenase increased to 2.06 +/- 0.92 and 8.64 +/- 2.87 U/gm, respectively. This is the first direct evidence that interstitial collagenase increases markedly during cervical dilation in human parturition.

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.

Preliminary observations of chondral abrasion in a canine model.

Articular cartilage repair was followed for one year in skeletally mature dogs after destabilisation by anterior cruciate ligament transection of the stifle joint (CT), abrasion of the inferior medial condyle (ABR) to bleeding bone, or anterior cruciate transection followed by chondral abrasion (CT/ABR). ABR animals formed repair cartilage at the abrasion site (ABR and CT/ABR) at six months as determined by arthroscopy and at necropsy. CT and CT/ABR animals had an additional cartilage ulcer on the superior aspect of the medial condyle. The abraded site extended in CT/ABR condyles. Repair cartilage (ABR and CT/ABR) contained reduced amounts of proteoglycan as seen by histological loss of safranin O staining and reduced uronic acid content. Fibrocartilage was suggested by histological appearance, hypocellularity, and a higher hydroxyproline content. In contrast with ABR animals, the repair cartilage in the CT/ABR animals contained near normal amounts of hydroxyproline. Collagen profiles of abrasion site repair cartilage in ABR animals had more types I and V collagens, similar amounts of type VI collagen, and decreased amounts of types II, IX, and XI collagens than CT/ABR animals. The results of this study are consistent with abrasion chondroplasty leading to a repair cartilage. Despite extended ulcers, repair cartilage from the destabilised joint (CT/ABR) animals was more hyaline-like in its hydroxyproline content and collagen composition than repair cartilage from the stable joint (ABR animals). In these models additional measures appear to be needed as the defects induced by abrasion chondroplasty did not form a functional hyaline cartilage.

Proteinase-mediated cartilage degradation in osteoarthritis

Osteoarthritis (OA) is a disease of complex etiology that results in articular cartilage breakdown. Current experimental evidence strongly suggests that proteinases may be involved in this loss of cartilage matrix. The present review summarizes the evidence for implicating proteinases in the etiopathogenesis of OA and suggests that blockade of proteinase activity may provide a rational basis for new therapeutic agents.

Localization of collagenase in the growth plate of rachitic rats.

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.

A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen

Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T.E. Cawston and A.J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryhänen et al. (L. Ryhänen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.

The Titanium-Bone Cell Interface In Vitro: The Role of the Surface in Promoting Osteointegration

One of the major advances in the use of metals for long-term implants was the serendipitous use of titanium (Ti). The nonreactive properties of this metal [1] made it an ideal material for the aerospace industry. Many of these same properties made it equally ideal for use in the body. Unlike stainless steel, Ti forms a surface oxide that prevents leaching of ions [2]. The oxide is biocompatible in that there is little, if any, immune response [3]. Early literature noted the lack of an immune response and defined Ti as biologically inert [4]. While we now know that Ti is not inert, there is no question that it is exceptionally well-tolerated by the body. Consequently, Ti has become the material of choice for dental implants [5] (See Chaps. 24 and 25) and cardiovascular stents [6] (See Chap. 26), and its alloys are commonly used in orthopaedics where greater strength is needed [7] (See Chap. 21).

Association of collagenase and tissue inhibitor of metalloproteinases (TIMP) with hypertrophic cell enlargement in the growth plate

In the transition from proliferating to hypertrophic cell zones in the growth plate, there is an increased in chondrocyte cell volume and a corresponding decrease in collagen content to allow for cell enlargement. To substantiate our hypothesis that collagenase is responsible for these changes, growth plates from rats treated with bisphosphonate (HEBP) were compared histologically and biochemically with growth plates from normal and vitamin D and phosphate deficient (-VDP) rats. HEBP-treated rats developed an expanded hypertrophic cell zone (HCZ) characterized by the presence of two distinct populations of hypertrophic cells. The proximal hypertrophic cells were only 2-fold enlarged compared to the proliferating cells, whereas 1/6 of the distal hypertrophic cells were enlarged almost 5-fold and appeared morphologically identical with hypertrophic cells from normal and -VDP rats. The HEBP growth plates were divided into cross-sectional thirds and analyzed for active and latent collagenase. The juxta-metaphyseal (lower 1/3) cartilage contained 100% of the fully enlarged hypertrophic cells and appeared identical to those found in normal and -VDP growth plates, along with 81% of the active and 77% of the total collagenase. Collagenase and tissue inhibitor of metalloproteinases (TIMP) were measured in extracts of similarly divided tissues. The presence of true collagenas was confirmed by using [3H]-telopeptide-free collagen. TIMP levels were inversely related to the presence of active collagenase and cellular hypertrophy. Substantial levels of latent collagenase were found in the extracellular fluid at sites of active collagenolysis, but not in the fluid phase surrounding the 2-fold enlarged hypertrophic cells. It is postulated that increased amounts of active collagenase and insufficient levels of TIMP may account for the reduced collagen content seen in the lower HCZ of both -VDP and HEBP rickets. Unlike active collagenase, which remains localized by binding to collagen, latent enzyme is probably restricted in its mobility throughout the extracellular space by diffusion, itself, or the interstices of the extracellular matrix.

Potential Mechanisms for the Plasmin-Mediated Release and Activation of Latent Transforming Growth Factor-β1 from the Extracellular Matrix of Growth Plate Chondrocytes

Chondrocytes produce latent transforming growth factor-β1 (TGF-β1) in a small, circulating form of 100 kDa and also store latent TGF-β1 in their matrix in a large form of 290 kDa containing the latent TGF-β1 binding protein 1. As growth plate cartilage cells are exceptionally sensitive to TGF-β1 and are known to produce plasminogen activator, the role of plasmin in the activation of soluble and matrix-bound latent TGF-β1 was examined. As is true for other cell types, low-dose plasmin (0.01 U/ml) was found to release both active and latent TGF-β1 from chondrocyte matrix in a time-dependent manner over 3 h. However, high-dose plasmin (1.0 U/ml) was found to release active TGF-β1 more rapidly than low-dose plasmin, and this release ceased within 30 min; latent complex continued to be released over time (3 h). When high-dose plasmin was titrated against the serine protease inhibitors, aprotinin and α-(2-aminoethyl)benzenesulfonyl fluoride, results similar to low-dose plasmin were obtained, indicating that the...