Effects of proteoglycan extraction on the tensile behavior of articular cartilage.

We undertook an interdisciplinary biomechanical and biochemical study to explore the extent and manner in which the total pool of proteoglycans influences the kinetic and static behavior of bovine articular cartilage in tension. Two biomechanical tests were used: (a) the viscoelastic creep test and (b) a slow constant-rate uniaxial tension test; and two enzymatic proteoglycan extraction procedures were used: (a) chondroitinase ABC treatment and (b) a sequential enzymatic treatment with chondroitinase ABC, trypsin, and Streptomyces hyaluronidase. We found that the viscoelastic creep response of all cartilage specimens may be divided into two distinct phases: an initial phase (less than 15 s), characterized by a rapid increase in strain following load application, and a late phase (15 s less than or equal to t less than 25,000 s), characterized by a more gradual increase in strain. A major finding of this study is that the kinetics of the creep response is greatly influenced by the glycosaminoglycan content of the tissue. For untreated and control specimens, the initial response comprises about 50% of the total strain, while for chondroitinase ABC and sequentially extracted specimens, the initial response comprises up to 83% of the total strain. Furthermore, most untreated and control specimens did not reach equilibrium within the 25,000 s test period, while enzymatically digested specimens often reached equilibrium in less than 100 s. Thus, we conclude that through their physical restraints on collagen, the bulk of proteoglycan present in the tissue acts to retard fibrillar reorganization and alignment under tensile loading, thereby effectively preventing sudden extension of the collagen network. In contrast, the results of our slow constant-rate uniaxial tension experiment show that essentially complete extraction of proteoglycan glycosaminoglycans does not affect the intrinsic tensile stiffness and strength of cartilage specimens or the collagen network in a significant manner. Hence, an important function of the bulk proteoglycans (i.e., the large aggregating type) in cartilage is to retard the rate of stretch and alignment when a tensile load is suddenly applied. This mechanism may be useful in protecting the cartilage collagen network during physiological situations, where sudden impact forces are imposed on a joint.

Induction of macrophage C-C chemokine expression by titanium alloy and bone cement particles.

Particulate wear debris is associated with periprosthetic inflammation and loosening in total joint arthroplasty. We tested the effects of titanium alloy (Ti-alloy) and PMMA particles on monocyte/macrophage expression of the C-C chemokines, monocyte chemoattractant protein-1 (MCP-1), monocyte inflammatory protein-1 alpha (MIP-1alpha), and regulated upon activation normal T expressed and secreted protein (RANTES). Periprosthetic granulomatous tissue was analysed for expression of macrophage chemokines by immunohistochemistry. Chemokine expression in human monocytes/macrophages exposed to Ti-alloy and PMMA particles in vitro was determined by RT-PCR, ELISA and monocyte migration. We observed MCP-1 and MIP-1alpha expression in all tissue samples from failed arthroplasties. Ti-alloy and PMMA particles increased expression of MCP-1 and MIP-1alpha in macrophages in vitro in a dose- and time-dependent manner whereas RANTES was not detected. mRNA signal levels for MCP-1 and MIP-1alpha were also observed in cells after exposure to particles. Monocyte migration was stimulated by culture medium collected from macrophages exposed to Ti-alloy and PMMA particles. Antibodies to MCP-1 and MIP-1alpha inhibited chemotactic activity of the culture medium samples. Release of C-C chemokines by macrophages in response to wear particles may contribute to chronic inflammation at the bone-implant interface in total joint arthroplasty.

Quantitation of hyaluronic acid in tissues by ion-pair reverse-phase high-performance liquid chromatography of oligosaccharide cleavage products.

A method for quantifying hyaluronic acid in biological tissues and fluids is described. The assay uses ion-pair HPLC to resolve and quantify the oligosaccharide end products of Streptomyces hyaluronidase digestion. Tissue samples were solubilized by papain, and the nondiffusate after dialysis was exhaustively digested with Streptomyces hyaluronidase. The resulting tetrasaccharide and hexasaccharide cleavage products were resolved by reverse-phase high-performance liquid chromatography in the presence of the ion-pairing agent, tetrabutylammonium phosphate. The saccharides were detected and quantified by their absorbance at 232 nm due to the alpha, beta-unsaturated carboxyl group generated by the eliminase reaction. In control experiments 93 +/- 3% of a hyaluronic acid standard so treated was reproducibly recovered as its tetra- and hexasaccharide cleavage products. As little as 0.5 microgram of the oligosaccharides could be quantified with no interference from a vast excess of chondroitin sulfate or other tissue components. The assay was applied to various types of human, bovine, and rabbit cartilage and to samples of other tissues including nucleus pulposus, annulus fibrosus, skin, aorta, cervix, cockscomb, synovial fluid, and vitreous humor. Results on human articular cartilage showed a linear increase in the content of hyaluronate from 0.1 to 0.5% of tissue dry weight between birth and 80 years of age.

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.

Sites of stromelysin cleavage in collagen types II, IX, X, and XI of cartilage.

Human recombinant stromelysin-1 was shown to cleave four types of collagen (types II, IX, X, and XI) prepared from bovine and rat cartilages at specific sites. Stromelysin-1 cleaved salt-soluble native molecules of type IX collagen into two main triple-helical fragments, COL1 and COL2,3. Protein microsequencing identified the exact cleavage sites in the NC2 domain of all three chains, alpha 1(IX), alpha 2(IX), and alpha 3(IX). Stromelysin-1 also acted as a "telopeptidase," in that it efficiently clipped intact molecules of types II and XI collagens at sites just inside their terminal cross-linking hydroxylysine residues. Native molecules of type X collagen were cleaved by stromelysin-1 within their triple helical domains at a COOH-terminal site that reduced the alpha 1(X) chain size by 10 kDa. These findings suggest an important role for stromelysin in the turnover and remodeling of the collagenous matrix of cartilage both normally and in degenerative joint disease.

Ultrasonic propagation properties of articular cartilage at 100 MHz.

A pilot study on articular cartilage assessed the contributions of individual matrix components to ultrasound propagation. The influence of collagen fibril orientation and collagen cross linking was also assessed. Sections of adult bovine articular cartilage cut both parallel and perpendicular to the articular surface were examined using the scanning laser acoustic microscope (SLAM) operating at an ultrasonic frequency of 100 MHz. A set of samples was evaluated that had been sequentially treated by enzymes to (1) remove 85% of the chondroitin sulfate; (2) remove remaining glycosaminoglycans, glycoproteins, and other noncollagen proteins, leaving only the collagen fibril network; and (3) disrupt the collagen intermolecular cross links. Two striking observations were made: a profound effect of the "preferred" collagen fibril orientation on ultrasonic speed and a marked increase in attenuation coefficient when intermolecular cross links were broken in the collagen.