No abstract

studies on collagen in bovine articular cartilage. APMIS 101: 133-140, 1993. In an ultrastructural stereologic study on bovine articular cartilage we found that collagen volume density increased with increasing distance from the joint surface and from the chondrocyte. These results not only corroborate previous biomechanical data of a vertical stiffness gradient, but they also suggest that the mechanical forces are unevenly distributed horizontally. On the other hand, although mean collagen fibril diameter showed large differences between the interterritorial compartments of the three zones, there was a population of slender fibrils in all zones and compartments. Since the coarser fibrils provide the high tensile strength (Nimni 1988), the role of the slender fibrils may be to enhance the deformability of the tissue. Moreover, in spite of substantial differences in mean fibril diameter, collagen surface densities were in the same order of magnitude in the territorial and interterritorial compartments, and only slightly lower in the pericellular compartments. The surface parameter may be important for specific molecular interactions. The collagen fibrils have different polarity, i.e. the direction of the fibrils appears to be parallel and antiparallel, about 50% running in each direction. This, together with the very high length/diameter ratio (Clark 1985), may indicate that each fibril is assembled by the concerted action of many cells. The characteristic properties of articular cartilage depend on interactions between its macromolecular components, and the present quantitative data form a basis for discussions on the specificity and regulation of such interactions.

Aggrecan, the predominant large proteoglycan of cartilage, is a multidomain macromolecule with each domain contributing specific functional properties. One of the domains contains the majority of the keratan sulfate (KS) chain substituents and a protein segment with a proline-rich hexapeptide repeat sequence. The function of this domain is unknown but the primary structure suggests a potential for binding to collagen fibrils. We have examined binding of aggrecan fragments encompassing the KS-rich region in a solid-phase assay. A moderate affinity (apparent K d ‫؍‬ 1.1 M) for isolated collagen II, as well as collagen I, was demonstrated. Enzymatic digestion of the KS chains did not alter the capacity of the peptide to bind to collagen, whereas cleavage of the protein core abolished the interaction. The distribution of the aggrecan KS-rich region in bovine tarsometatarsal joint cartilage was investigated using immunoelectron microscopy. Immunoreactivity was relatively low in the superficial zone and higher in the intermediate and deep zones of the uncalcified cartilage. Within the pericellular and territorial matrix compartments the epitopes representing the aggrecan KSrich region were detected preferentially near or at collagen fibrils. Along the fibrils, epitope reactivity was non-randomly distributed, showing preference for the gap region within the D-period. Our data suggest that collagen fibrils interact with the KS-rich regions of several aggrecan monomers aligned within a proteoglycan aggregate. The fibril could therefore serve as a backbone in at least some of the aggrecan complexes.Articular cartilage matrix can be regarded as a fiber-reinforced composite material (1), where aggrecan complexes are entangled within a network of collagen fibrils. The aggrecan complexes, constituting about 90% of the proteoglycan content (2), endow the matrix with high osmotic pressure, compressive stiffness, and resilience, whereas collagen is essential for the tensile strength of the tissue (3). Different mechanical properties of the composite depend on these major constituents and how they are assembled and stabilized by intermolecular interactions. The capacity of cartilage to withstand mechanical stress depends upon its structural integrity and, hence, numerous interactions between the matrix components. Indeed, the molecules are so tightly associated that most of the tissue constituents require denaturing solvents or proteases for extraction. This has hampered studies of molecular function. To gain insight into the physiology of articular cartilage, it is necessary to identify and characterize interactions between the matrix constituents, particularly those involving the collagen.In the present study we focused on a domain of the aggrecan molecule containing the majority of the keratan sulfate (KS) 1 chain substituents and a protein segment with a hexapeptide repeat sequence (4). The second globular domain (G2) is localized adjacent to this KS-rich region, on the N-terminal side. The first globular domain (G1), which r...

Fibronectin is a well known glycoprotein of extracellular connective tissue matrices due to a specific amino acid-sequence (RGD) suggested to act as an attachment factor in cell-cell or cell-matrix interactions. Although also present in bone, little is known about the role of fibronectin in this tissue. To obtain data for discussions on function we used ultrastructural immunolocalization techniques to quantitatively examine the distribution of fibronectin in various bone matrix compartments. The study was focused on three different stages of endochondral ossification in growing long bones of young rats. The results show large amounts of fibronectin in mature bone tissue. At a higher magnification, an obvious fibronectin association to individual fibrils of collagen type I was demonstrated. Intracellular labeling was observed in Golgi-related vesicles in some active osteoblasts of metaphyseal bone, indicating local synthesis of fibronectin. In contrast to previous suggestions based on light microscopic observations, the labeling of bone or cartilage matrices facing the surface of all cell types were low. The pattern is clearly different from that of osteopontin and bone sialoprotein, two other bone matrix proteins with the same cell-binding sequence. Our results indicate that fibronectin at these stages of development participates in matrix organization rather than being an important link between cartilage or bone matrix and adjacent cells.

Focusing on resorption processes, we have extended our previous studies on chondroclasts and osteoclasts in normally developing tissues, using a model of nutritionally induced vitamin D-deficiency rickets. To analyze the resorption process, we investigated the matrix-resorbing cells in this modified and poorly mineralized tissue regarding morphological features and expression of tartrate-resistant acid phosphatase (TRAP) at the subcellular level. Our goal was to test the hypotheses that initiation of resorption is impaired with unmineralized matrix, and that such alterations involve changes in the subcellullar distribution of TRAP, implicating a role for this enzyme in the resorption process. Our results reveal distinctly different morphological appearances of clast-like cells in rickets compared with normal osteoclasts and chondroclasts. Ordinary resorption structures of osteoclasts and chondroclasts at the cell-matrix border, i.e., ruffled borders and clear zones, are profoundly altered in favor of a less well-defined intermediate zone. TRAP distribution at the subcellullar level is also clearly different from that in osteoclasts and chondroclasts from normal rodents, with impaired secretion; consequently, the enzyme is unable to function in the matrix outside the ruffled border. Our ultrastructural observations demonstrate that in rickets, the clasts are incapable of degrading the poorly mineralized cartilage and bone efficiently. Rachitic clasts seem to be recruited to the matrix surface and interaction between cell and matrix is also initiated, but definitive resorption structures at the cell-matrix border are not normally developed. Whether resorption is inhibited by the mere lack of mineral or mineral-associated proteins, or by other mechanisms remains to be settled.