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In this study, the influence of nonenzymatic glycation (NEG) on the mechanical properties of bone and bone collagen were investigated. Bovine cortical bone specimens were incubated in ribose to cause collagen cross-links in vitro, and nondestructive mechanical testing was used to determine tensile and compressive elastic modulus as a function of incubation time. Mechanical properties associated with yield, postyield, and final fracture of bone were determined at the end of the incubation period. The stiffness of the collagen network was measured using stress relaxation tests of demineralized bone cylinders extracted periodically throughout the incubation period. It was found that accumulation of nonenzymatic glycation end-products in cortical bone caused stiffening of the type I collagen network in bone (r2 = 0.92; p < 0.001) but did not significantly affect the overall stiffness of the mineralized bone (p = 0.98). The ribosylated group had significantly more NEG products and higher yield stress and strain than the control group (p < 0.05). Postyield properties including postyield strain and strain energy were lower in the ribosylated group but were not significantly different from the control group (p = 0.24). Compared with the control group, the ribosylated group was characterized by significantly higher secant modulus and lower damage fraction (p < 0.05). Taken together, the results of this study suggest that collagen in bone is susceptible to the same NEG-mediated changes as collagen in other connective tissues and that an increased stiffness of the collagen network in bone due to NEG may explain some of the age-related increase in skeletal fragility and fracture risk.

Articular cartilage damage and eventual loss is the primary pathological change seen in osteoarthrosis (OA). In this study we have investigated the link between turnover of the collagen matrix and changes in chondrocytes. The background fluorescence of articular cartilage, as indicated by its emission spectrum and resistance to extraction was generated by the slow non-enzymic modification of the collagen matrix by advanced glycation end products (AGEs). Assessment of changes in background fluorescence in sections of articular cartilage provided a narrative of collagen degradation. Patients without OA pathology typically had a uniform strong background fluorescence throughout the depth of the cartilage. Cartilage from OA patients showed a range of changes in background fluorescence dependent on depth from the articular surface and proximity to overt lesions. Loss of background fluorescence was centered on chondrocytes, more extensive near the surface and associated with detection of the proteoglycan epitope 7D4. Expression of type X collagen was seen in articular cartilage in the region of the interface of with subchondral bone in most OA patients but was not associated with prominent, pericellular, loss of background fluorescence. These observations are consistent with progressive cartilage damage in OA, whereby collagen turnover and loss of surface integrity is associated with chondrocyte activity similar to that seen in immature articular cartilage.

In this report, the susceptibility of type VIII collagen to human neutrophil elastase is compared to other extracellular matrix components. Type X collagen is degraded to specific fragments at a substrate to enzyme ratio of 5:1 after 20 h at room temperature, but type VIII collagen is almost completely degraded after only 4 h incubation at a substrate to enzyme ratio of 50:1 and partly degraded after only 15 min. Laminin, merosin and types I, III, IV and V collagen exhibit no susceptibility to neutrophil elastase under the latter conditions, while fibronectin is degraded.

Direct comparison of type X collagen synthesized by human, sheep and chick growth-plate cartilage has shown that the human type X collagen is similar to the chick in both its molecular mass, containing component alpha-chains of 59 kDa with helical regions of 45 kDa, and apparent absence of disulphide-stabilized aggregates, whereas the sheep type X collagen has slightly larger alpha-chains (63 kDa) accounted for by a longer helical region (49 kDa) that contains cystine residues essential for the formation of the high-molecular-mass aggregates found with this species. Type X collagen from all three species showed heterogeneity in primary collagen structure as revealed by Staphylococcus aureus V8 proteinase-generated peptide maps. Collagen synthesis by growth-plate cartilage in culture, particularly synthesis of type IX and X collagen, was shown to be very sensitive to prior storage and suggests caution in the interpretation of changes detected when examining collagen synthesis by growth plates in culture.

The mature chick sternum is divisible almost equally into cephalic calcified and caudal cartilagenous regions. Isolation and culture of cells derived from embryonic precursors of these regions has revealed two discrete populations of cells with distinct morphological features and synthetic capabilities. Both cell populations grew well in culture within or upon collagen gels or upon plastic and maintained morphologies similar to those observed in the parent tissue. Polyacrylamide gel electrophoresis of radiolabeled proteins synthesized by the cells in culture demonstrated large differences in the types of collagens synthesized. Both chondrocyte populations synthesized type II and minor cartilage collagens but only chondrocytes isolated from the presumptive calcification region synthesized the previously identified, low molecular weight collagen, termed G collagen. Synthesis of G collagen was stimulated by culture within or upon collagen gels such that it represented an average of 65% of the total collagen synthesized by presumptive calcification region chondrocytes after 7 d of culture within collagen gels. Light and scanning electron microscopy demonstrated that the two chondrocyte types exhibited distinct morphological features and accumulated different extracellular matrices in culture.

We have localized type X collagen in chick cartilage and lung by use of affinity-purified antibodies raised against the purified protein. Examination of the centers of primary endochondral development in the embryonic sternum, developing cartilage in the embryonic tubular bones and growth plate cartilages demonstrated the specific association of type X collagen with regions of hypertrophic chondrocytes in these tissues. Furthermore these studies revealed a tendency for type X collagen to accumulate adjacent to regions of active vascular invasion and an apparent lag between the initial intracellular accumulation and the matrix deposition of type X collagen. A lag in the matrix accumulation of type X collagen was also shown biochemically and immunohistochemically in chondrocytes in culture. In chick lung type X collagen was localized to the smooth muscle of the blood vessels and to smooth muscle surrounding alveolar ducts.

Chick-embryo sternal chondrocytes have been cultured within three-dimensional collagen gels as part of a study concerned with the effects of extracellular matrix macromolecules on chondrocyte gene expression. Data are presented indicating that chondrocytes cultured within such a collagenous environment synthesize significantly more of an hitherto unidentified, low molecular weight collagen species than do cells grown on plastic tissue-culture dishes in the conventional manner. This low molecular weight collagen species contains noncollagenous domains (as indicated by its decreased molecular size after mild pepsin digestion), is distinct from the known collagen types (as judged by CNBr peptide analysis), and forms part of the insoluble collagenous matrix produced by the chondrocytes. Cells growing within the gel tend to form colonies consisting of a linear array of cells reminiscent of the cellular organization in growth cartilage.

Culture of chick-embryo sternal-cartilage chondrocytes within three-dimensional collagen gels promotes the synthesis of three low-molecular-weight collagenous polypeptides. The proportions of these novel collagens synthesized and released into the medium are markedly influenced by the presence or the absence of fibronectin in the serum supplement. Chondrocytes cultured on plastic dishes appear to synthesize only small amounts of these low-molecular-weight species. The three species (designated G, H and J) were characterized with respect to the proportion of [14C]proline incorporated into each polypeptide occurring as hydroxy[14C]proline and with respect to their susceptibilities to bacterial collagenase. On the basis of their electrophoretic mobilities under reducing conditions, the G, H and J polypeptides were calculated to have Mr 59 000, 69 000 and 84 000 respectively. Chymotrypsin digestion converted the G collagen into a species containing polypeptides of Mr 45 000, whereas the H and J polypeptides yielded a single band of Mr 53 000. The H and J polypeptides were found to occur as disulphide-linked aggregates, as was the chymotrypsin-digestion product. Peptide 'mapping' has shown that G, H and J polypeptides show no common identity and are distinct from the known interstitial collagens. Native G collagen was digested by human collagenase to discrete products, whereas H and J chains were not cleaved under identical conditions.

Our morphological studies have demonstrated that the appearance of localized, paired zones of primary calcification on either side of the midline of the 19-d embryonic chick sternum is heralded by the development of paired, translucent zones 2 d previously. Histological studies demonstrated that the majority of chondrocytes within these translucent zones are hypertrophic, and that the zones are surrounded by a margin of flattened nonhypertrophic cells. The discrete localization of these paired areas of hypertrophic chondrocytes and subsequent endochondral bone development allows for the direct correlation of the histological and biochemical characteristics of the zones sequentially during development and makes it possible to precisely match the synthetic activity to the cellular morphology, thereby eliminating possible minor but critical variations in developmental staging that could otherwise arise. Our studies have demonstrated that there is a direct spatial and temporal correlation between the degree of cellular maturation and the synthesis of type X collagen, and that the sudden and profound initiation of type X collagen synthesis on days 16-17 of development occurs concurrently with the attainment of hypertrophic characteristics by the majority of cells within the translucent zone. Before acquisition of these hypertrophic characteristics, the cells of this precalcification zone synthesize only type II and the minor cartilage collagens. Chondrocytes isolated from these regions in more immature sternae (i.e., 11+ d embryos) were found to synthesize high levels of type X collagen within 4 d of culture within collagen gels even though hypertrophic development and type X collagen synthesis by cells within this region would not normally have been apparent in ovo for several more days. These data indicate that there is a direct correlation between the development of hypertrophic characteristics and the synthesis of type X collagen, and that the maturation of chondrocytes in precalcification zones may be regulated by matrix components and/or stimulated by culture within collagen gels.