Cahir A. McDevitt

Interaction of intact type VI collagen with hyaluronan.

The capacity of non‐pepsinyzed type VI collagen to oind to hyaluronan was investigated. Type VI collagen was extracted from bovine meniscal cartilage with 6 M GuHCl and purified by extraction of PEG precipitates and dissociative Sephacryl S‐500 HR chromatography. Type VI collagen, detected with a monoclonal antibody, bound in 0.5 M NaCl to hyaluronan‐coated micro‐wells, the degree of binding being higher at 37°C than 23°C and 4°C. Incubation of type VI collagen in competitive inhibition assays with testicular hyaluronidase digests of hyaluronan in liquid phase, reduced binding of the protein to hyaluronan‐coated microwells to background levels. Thus, non‐pepsinyzed type VI collagen binds to hyaluronan in vitro.

The Concentration, Gene Expression, and Spatial Distribution of Aggrecan in Canine Articular Cartilage, Meniscus, and Anterior and Posterior Cruciate Ligaments: A New Molecular Distinction Between Hyaline Cartilage and Fibrocartilage in the Knee Joint

The concentration, spatial distribution, and gene expression of aggrecan in meniscus, articular cartilage, and the anterior and posterior cruciate ligaments (ACL and PCL) was determined in the knee joints of five mature dogs. An anti-serum against peptide sequences specific to the G1 domain of aggrecan was employed in competitive-inhibition ELISA of guanidine HCl extracts and immunofluorescence microscopy. Gene expression was determined by Taqman real-time PCR. The concentration of aggrecan in articular cartilage (240.1 ± 32 nMol/g dry weight) was higher than that in meniscus (medial meniscus: 33.4 ± 4.3 nMol/g) and ligaments (ACL: 6.8 ± 0.9 nMol/g). Aggrecan was more concentrated in the inner than the outer zone of the meniscus. Aggrecan in meniscus showed an organized, spatial network, in contrast to its diffuse distribution in articular cartilage. Thus, differences in the concentration, gene expression, and spatial distribution of aggrecan constitute another molecular distinction between hyaline cartilage and fibrocartilage of the knee.

Cell, matrix changes and alpha-smooth muscle actin expression in repair of the canine meniscus

Processes in the repair of a crevice in the knee joint meniscus were investigated in 10 dogs. Two 2‐mm cylindrical plugs from each medial meniscus were removed, rendered acellular by freezing and thawing, and then reinserted into the meniscus. Dogs were euthanized at intervals of 3–52 weeks after surgery. The crevice between the plug and meniscus at 3 weeks after surgery was filled with a tissue containing α‐smooth muscle actin‐positive cells. One year after surgery, the plug had remodeled and was populated with spindle‐shaped and fibrochondrocyte‐like cells. The plug had an appearance intermediate between that of hyaline and fibrocartilage at this time, with a seamless integration in sites between the remodeled plug and the surrounding meniscus. α‐smooth muscle actin‐positive cells were concentrated at the interface of the remodeled plug and adjacent meniscus and at the surface of the plug. Therefore, remodeling of both the plug and meniscal tissue and the participation of α‐smooth muscle actin‐positive cells appear essential for integration of the plug into the adjacent meniscal tissue. Cells in the superficial zone of the meniscus seem to be active in the repair process. A change in both the phenotype of the cells and the quality of the matrix toward a more hyaline state appears to be an integral part of the remodeling process in the meniscus.

Attachment of articular cartilage chondrocytes to the tissue form of type VI collagen

Type VI collagen is composed of a short triple helix rich in RGD sequences with globular domains at each extremity of the helix. Disulfide-bonded tetramers of the monomeric molecule associate non-covalently to form networks of microfibrils in connective tissues, including cartilage. The disulfide-bonded tetramer can be extracted with 6 M guanidine HCl and purified without pepsin digestion and is referred to here as the tissue form of type VI collagen. Type VI collagen in mature articular cartilage appears to be concentrated pericellularly. We undertook a systematic investigation using solid phase assays to establish the nature of the attachment of bovine articular cartilage chondrocytes to the intact, tissue form of bovine type VI collagen. The tissue form of type VI collagen was extracted from bovine meniscus cartilage with 6 M guanidine HCl and purified by polyethylene glycol precipitation. When equal molar quantities were coated on microwells, the tissue form of type VI collagen attached more cells than the pepsin-digested form of the molecule that lacked the globular domains. The attachment to the intact, tissue form was dose-dependent and saturable and was not inhibited by heparin or type II collagen. A linear GRGDSP peptide failed to inhibit attachment of the chondrocytes to the intact, tissue or pepsin-digested forms of type VI collagen, but totally inhibited the interaction when the intact molecule was reduced and alkylated. In contrast, a cyclic C*GRGDSPC* peptide inhibited attachment to the tissue form of type VI collagen, but not to fibronectin. The attachment had a metal ion dependence that could be satisfied by MnCl2, slightly less by MgCl2, but not at all by CaCl2. A direct interaction between the tissue form of type VI collagen and a chondrocyte cell surface receptor or receptors is a structural feature of the pericellular matrix in cartilage.

Experimental osteoarthritic articular cartilage is enriched in guanidine-soluble type VI collagen

Experimental osteoarthritis was surgically induced in the right knee joint of dogs; the left knee served as a control. Articular cartilage was extracted with 4 M guanidinium chloride, 0.05 M sodium acetate, pH 6.0, containing proteinase inhibitors and the proteins purified by associative CsCl density gradient centrifugation. Equal quantities of protein were electrophoresed in agarose-acrylamide gradient gels and the high molecular weight type VI collagen bands detected in immunoblots with a polyclonal antiserum. Type VI collagen bands between 185 and 220 kDa were evident in the pathological specimens of dogs sacrificed 3, 5, and 7 months after surgery and were either absent or only very weakly visible in the controls. These results demonstrate that experimental osteoarthritic cartilage is enriched in 4 M guanidine-soluble type VI collagen.

Thrombospondin is present in articular cartilage and is synthesized by articular chondrocytes.

Thrombospondin, a multifunctional adhesive glycoprotein originally identified in platelets, was isolated and identified from an extract of ovine articular cartilage. Immunoreactive material from a cartilage extract comigrated on gel electrophoresis with purified human platelet thrombospondin. When articular chondrocytes were cultured in the presence of 35S-methionine, metabolically labeled thrombospondin was immunoprecipitated from the culture medium and cell layer extract. These results demonstrate that thrombospondin is present in articular cartilage and is synthesized by articular chondrocytes.

Emerging concepts of the cell biology of the meniscus

Three distinct populations of cells are now recognized in the knee joint meniscus: (a) round or oval-shaped fibrochondrocytes; (b) fibroblast-like cells; (c) superficial zone cells. Injury to the meniscus causes apoptosis in specific sites and an upregulation of gene expression for type I, II, III,

Thrombospondin in ligament, meniscus and intervertebral disc

The presence of thrombospondin in extracts of ligament, meniscus and human nucleus pulposus was demonstrated by Western blot analysis using an anti-human thrombospondin antibody. Metabolic labeling of ligament fibroblast and meniscal fibrochondrocyte cultures followed by immunoprecipitation with anti-human thrombospondin revealed a single band corresponding to the molecular weight of thrombospondin when analyzed by SDS-PAGE and fluorography under reduced and non-reduced conditions. Thrombospondin, therefore, is a matrix constituent of ligament, knee joint meniscus and the nucleus pulposus and is synthesized by ligament fibroblasts and meniscal fibrochondrocytes in vitro.

Cigarette smoke degrades hyaluronic acid

Cigarette smoke has been implicated in the pathogenesis of a variety of diseases that affect connective tissues. The effect of the gas phase of cigarette smoke on hyaluronic acid was investigated in vitro. The smoke markedly reduced the chain length of purified hyaluronic acid as assessed by both viscometry and gel chromatography. The hyaluronate exposed to smoke, nevertheless, could aggregate proteoglycans, although the viscosity of the aggregates was lower than in those formed with the unexposed material. The degradation of the hyaluronate was a rapid phenomenon and was inhibited by dimethylsulfoxide, a known scavenger of hydroxyl radicals. We conclude that free radicals in the gas phase of cigarette smoke degrade hyaluronic acid in vitro.

Absolute concentrations of mRNA for type I and type VI collagen in the canine meniscus in normal and ACL‐deficient knee joints obtained by RNase protection assay

Relatively little is known about the cellular and molecular responses of the knee joint meniscus to joint injury, despite the functional importance of the tissue. We investigated how meniscus cells respond to joint injury in the early stages of post‐traumatic osteoarthritis by characterizing the changes in matrix gene expression in menisci at 3 and 12 weeks post‐surgery in dogs in which the anterior cruciate ligament (ACL) in one joint was transected and the other unoperated joint served as a control. Changes in the total RNA and DNA concentrations of the menisci were determined. Absolute concentrations of the mRNA of the COL1A1 gene of type 1 collagen, the major fibrillar collagen of the meniscus, and the COL6A3 gene of type VI collagen, a major repair molecule, were determined by quantitative ribonuclease (RNase) protection assay. The concentration of total RNA in medial and lateral menisci increased from 40 to 60 μg RNA/g wet wt in unoperated, control joints to 200—350 μg RNA/g wet wt in ACL‐deficient joints. No significant changes were detected in the concentration of DNA (900—1200 μg DNA/g wet wt). Low concentrations of COL1A1 (2—3 pmol mRNA/g DNA) and COL6A3 (0.3–0.6 pmol mRNA/g DNA) mRNA transcripts were measured in normal menisci. ACL‐deficiency induced a 20–38 fold increase in COL1A1 and COL6A3 mRNA concentration at 3 weeks, and an 11–19 fold increase at 12 weeks post‐surgery. In general, the increase in COL1A1 and COL6A3 mRNA concentrations was greater in medial menisci than in lateral menisci. These results demonstrate that the menisci initiate a vigorous biosynthetic response to transection of the ACL.