Decreased collagen thermal stability as a response to the loss of structural integrity of thyroid cartilage

The amino acid composition, thermal behavior and birefringence properties of thyroid cartilage tissues have been studied. A collagen component in perichondrium consists of type-I and type-II collagens whose fibers form a highly ordered anisotropic structure with a birefringence of 4.75 × 10^?3 and a melting (denaturation) temperature of 65°C. The hyaline constituent, which is visualized as a quasi-anisotropic medium, contains of only type-II collagen, which does not denature in intact tissues at temperatures up to 100°C. However, in tissues whose proteoglycane subsystem is damaged by trypsin, the denaturation of collagen takes place at 60°C. In the integral perichondrium-hyaline system, the temperature of collagen denaturation in the perichondrium reaches 75°C, which indicates the immobilization of collagen in this tissue by the extracellular matrix of the hyaline constituent.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence: II. Localization of type I and type II collagen during long bone development

The transition of type I and type II collagens during cartilage and bone development in the chick embryo was studied by immunofluorescence using antibodies against type I or type II collagens. Type II collagen was found in all cartilaginous structures which showed metachromatic staining. Type I collagen appeared in the perichondrium of the tibia at stage 28 and was also found in osteoid, periosteal and enchondral bone after decalcification, periosteum, and tendons, ligaments, and capsules.Using the immunohistological method it was possible to identify specific collagen types in areas undergoing rapid proliferation and collagen transition, such as diaphyseal and epiphyseal perichondrium, or in enchondral osteogenesis. During enchondral ossification type I collagen is deposited onto the eroded surface of cartilage. It partially diffuses into the cartilage matrix forming a “hybrid” collagen matrix with type II collagen, which is a site for subsequent ossification. During appositional growth of diaphyseal cartilage and differentiation of epiphyseal perichondrium into articular cartilage, perichondral cells switch from type I to type II collagen synthesis when differentiating into chondroblasts. In the transition zones, chondroblasts are imbedded in a “hybrid” matrix consisting of a mixture of type I and type II collagens.     

Distribution of the collagen-binding integrin α10β1 during mouse development

We have previously identified and characterised the collagen type II-binding integrin subunit alpha10, which is a member of the beta1 family and is expressed by chondrocytes. In the present study, we examined the expression of the alpha10 integrin in various mouse tissues. Immunohistochemical analysis of alpha10 on cryosections from 3-day-old mice demonstrated that alpha10beta1 was present in the hyaline cartilage of joints, vertebral column, trachea and bronchi. In addition, alpha10 was found in the ossification groove of Ranvier, in the aortic and atrioventricular valves of the heart and in the fibrous tissue lining skeletal muscle and ligaments. Overall, the distribution was distinct from that of the collagen-binding integrins alpha1beta1 and alpha2beta1. We also found that alpha10beta1was the dominating collagen-binding integrin during cartilage development. Expression of alpha10 appeared at embryonic day 11.5 (E11.5) at the same time as chondrogenesis started as judged by collagen type II expression. At E13.5, alpha10 was present throughout the anlage as well as in the perichondrium and in mesenchyme just outside the perichondrium, where it localised with collagen type I. Four weeks after birth, alpha10 was prominent both at the articular surface and in the growth plate. In conclusion, we found that integrin alpha10beta1 was a major collagen-binding integrin during cartilage development and in mature hyaline cartilage. In addition, we found that alpha10beta1 was present in some fibrous tissues.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.     

The distribution of different molecular species of collagen in fibrous, elastic and hyaline cartilages of the pig.

The distribution of type II collagen, considered to be characteristic of cartilaginous tissues, was determined in various specialized cartilages of the mature pig. The tissues examined were: (1) fibrocartilage of the semilunar meniscus of the knee; (2) elastic cartilage of the external ear; (3) hyaline cartilage of (a) the synovial joint (b) the thyroid plate of the larynx, and (c) the nasal septum. The predominant species of collagen in each tissue, whether type I or type II, was appraised semi-quantitatively by analysis of purified collagen solubilized by pepsin and of peptide fragments produced by cyanogen bromide. Cyanogen bromide-derived peptides were characterized by column chromatography on CM-cellulose and by electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The proportion of each type of collagen was determined precisely by isolating the homologous small peptides alpha1(II)CB6 [nomenclature of Miller (1973) Clin. Orthop. 92, 260-280], by column chromatography on phosphocellulose and determining their relative proportions by amino acid analysis. Thus collagen of the fibrocartilage of the meniscus proved to be all type I; type II was not detected. In contrast, collagen of elastic cartilage of the outer ear, after rigorous exclusion of perichondrium, was type II. Similarly, type II was the only collagen detected in all the mature hyalline cartilages examined.     

Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.     

Localization of collagens and alkaline phosphatase activity during mineralization and ossification of human first rib cartilage

The localization of type X collagen and alkaline phosphatase activity was examined in order to gain a better understanding of tissue remodelling during development of human first rib cartilage. First rib cartilages from children and adolescents showed no staining for type X collagen and alkaline phosphatase activity. After onset of mineralization in the late second decade, a peripheral ossification process preceded by mineralized fibrocartilage could be distinguished from a more central one preceded by mineralized hyaline cartilage. No immunostaining for type X collagen was found in either type of cartilage. However, strong staining for alkaline phosphatase activity was detected around chondrocyte-like cells within fibrocartilage adjacent to the peripheral mineralization front, while a weaker staining pattern was observed around chondrocytes of hyaline cartilage near the central mineralization front. In addition, the territorial matrix of some chondrocytes within the hyaline cartilage revealed staining for type I collagen, suggesting that these cells undergo a dedifferentiation process, which leads to a switch from type II to type I collagen synthesis. The study provides evidence that mineralization of the hyaline cartilage areas in human first rib cartilage occurs in the absence of type X collagen synthesis but in the presence of alkaline phosphatase. Thus, mineralization of first rib cartilage seems to follow a different pattern from endochondral ossification in epiphyseal discs.     

Abnormal development of the os penis in male mice treated neonatally with tamoxifen

The os penis of male C57BL/Tw mice given 5 daily injections of 100 micrograms tamoxifen (Tx) starting on the day of birth (day 0) was examined at ages of 5-60 days; the bones of males given Tx injections for 5 days starting at 0-10 days and of those given neonatal injections of 100 micrograms clomiphene or nafoxidine were examined at 60 days. In the control males given the vehicle alone, the proximal segment of the os penis, composed of a compact cell mass found at day 0, developed at 5 days into the membrane bone with bone marrow and hyaline cartilage; the distal segment, composed of mesenchymatous cells until 10 days, developed at 30 days into fibrocartilage characterized by a distribution of type I collagen. By contrast, in Tx mice, fibrocartilage in the distal segment, and hyaline cartilage characterized by a distribution of type II collagen, and bone marrow in the proximal segment disappeared by 30 days. The maximum area of the proximal and distal segments gradually increased with age in control mice, whereas the proximal segment area remained unchanged in Tx mice. In clomiphene and nafoxidine mice at 60 days, the proximal segment was composed of hyaline cartilage; however, the distal segment lacked fibrocartilage. Hyaline cartilage in the proximal segment and fibrocartilage in the distal segment disappeared in all 60-day-old mice given Tx starting within 5 days. Neonatal castration did not suppress the formation of bone marrow and fibrocartilage in the os penis, though the bone size was smaller than in the intact controls. Formation of spines on the glans penis skin was suppressed by Tx given within 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bortezomib prevents the expression of MMP-13 and the degradation of collagen type 2 in human chondrocytes

The structural backbone of extracellular matrix in cartilage is the collagen fibril, which is mainly composed of type II collagen. A measurable increase in type II collagen denaturation and degradation has been found in early Osteoarthritis (OA). Pro-inflammatory cytokine such as TNF-α produced in OA cartilage induced the expression of matrix metalloproteinase-13 (MMP-13), which targets and degrades type II collagen. Bortezomib is a proteasome inhibitor approved by the FDA for treatment of multiple myeloma and mantel cell lymphoma. The effects of bortezomib in OA have not been reported before. In this study, we found that bortezomib is able to suppress the degradation of type II collagen induced by TNF-α in human chondrocytes. Mechanistically, bortezomib treatment inhibits the expression of IRF-1 through blunting JAK2/STAT1 pathway, thereby prevents the induction of MMP-13 as well as the degradation of type II collagen. Our findings suggest the therapeutic potentials of bortezomib in patients with OA.     

Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear "lucent" after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded "lucent" fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the "lucency" of the fibrils indicated that they were composed of collagen I, whereas the "lucency" of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.     

Ultrastructure of calcified cartilage in the endoskeletal tesserae of sharks

The tesserate pattern of endoskeletal calcification has been investigated in jaws, gill arches, vertebral arches and fins of the sharks Carcharhinus menisorrah, Triaenodon obesus and Negaprion brevirostris by techniques of light and electron microscopy. Individual tesserae develop peripherally at the boundary between cartilage and perichondrium. An inner zone, the body, is composed of calcified cartilage containing viable chondrocytes separated by basophilic contour lines which have been called Liesegang waves or rings. The outer zone of tesserae, the cap, is composed of calcified tissue which appears to be produced by perichondrial fibroblasts more directly, i.e., without first differentiating as chondroblasts. Furthermore, the cap zone is penetrated by acidophilic Sharpey fibers of collagen. It is suggested that scleroblasts of the cap zone could be classified as osteoblasts. If so, the cap could be considered a thin veneer of bone atop the calcified cartilage of the body of a tessera. By scanning electron microscopy it was observed that outer and inner surfaces of tesserae differ in appearance. Calcospherites and hydroxyapatite crystals similar to those commonly seen on the surface of bone are present on the outer surface of the tessera adjacent to the perichondrium. On the inner surface adjoining hyaline cartilage, however, calcospherites of variable size are the predominant surface feature. Transmission electron microscopy shows calcification in close association with coarse collagen fibrils on the outer side of a tessera, but such fibrils are absent from the cartilaginous matrix along the under side of tesserae. Calcified cartilage as a tissue type in the endoskeleton of sharks is a primitive vertebrate characteristic. Calcification in the tesserate pattern occurring in modern Chondrichthyes may be derived from an ancestral pattern of a continuous bed of calcified cartilage underlying a layer of perichondral bone, as theorized by Ørvig (1951); or the tesserate pattern in these fish may itself be primitive.     

An immunofluorescence study of chondrogenesis in murine mandibular ectomesenchyme

The temporal and spatial distribution of type I collagen, type II collagen, cartilage-specific proteoglycan (CSPG) and fibronectin in mouse mandible is described. CD-1 mouse embryos of 12-, 15-, and 18-day gestation were used, and matrix molecules were localized using indirect immunofluorescence. On day 12, accumulation of type II collagen, CSPG, and fibronectin within regions of condensed mesenchyme was noted. On day 15, intense staining for type II collagen and CSPG occurred. Fibronectin was less brilliant with its greatest concentration near the perichondrium. On day 18, the cartilage matrix was undergoing osseous replacement concurrent with loss of type II collagen and CSPG. Type I collagen was seen in the perichondrium, membranous bone and sub-basement membrane region in specimens of all ages. Synthesis and expression of extracellular matrix molecules reflect patterns of differentiation in mandibular mesenchyme.     

Discrete reduction of type I collagen thermal stability upon oxidation

The oxidation of acid-soluble calf skin collagen type I caused by metal-dependent free radical generating systems, Fe(II)/H2O2 and Cu(II)/H2O2, was found to bring down in a specific, discrete way the collagen thermal stability, as determined by microcalorimetry and scanning densitometry. Initial oxidation results in splitting of the collagen denaturational transition into two components. Along with the endotherm at 41 degrees C typical for non-oxidized collagen, a second, similarly cooperative endotherm appears at 35 degrees C and increases in enthalpy with the oxidant concentration and exposure time, while the first peak correspondingly decreases. The two transitions at 35 and 41 degrees C were registered by densitometry as stepwise increases of the collagen-specific volume. Further oxidation results in massive collagen destruction manifested as abolishment of both denaturational transitions. The two oxidative systems used produce identical effects on the collagen stability but at higher concentrations of Cu(II) in comparison to Fe(II). The discrete reduction of the protein thermal stability is accompanied by a decrease of the free amino groups, suggestive of an oxidation attack of the side chains of lysine residues. Since the denaturation temperature of collagen shifts from above to below body temperature (41 degrees C-35 degrees C) upon oxidation, it appears important to account for this effect in a context of the possible physiological implications of collagen oxidation.     

Type III Collagen,a Fibril Network Modifier in Articular Cartilage

The collagen framework of hyaline cartilages, including articular cartilage, consists largely of type II collagen that matures from a cross-linked heteropolymeric fibril template of types II, IX, and XI collagens. In the articular cartilages of adult joints, type III collagen makes an appearance in varying amounts superimposed on the original collagen fibril network. In a study to understand better the structural role of type III collagen in cartilage, we find that type III collagen molecules with unprocessed N-propeptides are present in the extracellular matrix of adult human and bovine articular cartilages as covalently cross-linked polymers extensively cross-linked to type II collagen. Cross-link analyses revealed that telopeptides from both N and C termini of type III collagen were linked in the tissue to helical cross-linking sites in type II collagen. Reciprocally, telopeptides from type II collagen were recovered cross-linked to helical sites in type III collagen. Cross-linked peptides were also identified in which a trifunctional pyridinoline linked both an α1(II) and an α1(III) telopeptide to the α1(III) helix. This can only have arisen from a cross-link between three different collagen molecules, types II and III in register staggered by 4D from another type III molecule. Type III collagen is known to be prominent at sites of healing and repair in skin and other tissues. The present findings emphasize the role of type III collagen, which is synthesized in mature articular cartilage, as a covalent modifier that may add cohesion to a weakened, existing collagen type II fibril network as part of a chondrocyte healing response to matrix damage.     

Retention of the native chondrocyte pericellular matrix results in significantly improved matrix production.

The interaction of the cell with its surrounding extracellular matrix (ECM) has a major effect on cell metabolism. We have previously shown that chondrons, chondrocytes with their in vivo-formed pericellular matrix, can be enzymatically isolated from articular cartilage. To study the effect of the native chondrocyte pericellular matrix on ECM production and assembly, chondrons were compared with chondrocytes isolated without any pericellular matrix. Immediately after isolation from human cartilage, chondrons and chondrocytes were centrifuged into pellets and cultured. Chondron pellets had a greater increase in weight over 8 weeks, were more hyaline appearing, and had more type II collagen deposition and assembly than chondrocyte pellets. Minimal type I procollagen immunofluorescence was detected for both chondron and chondrocyte pellets. Chondron pellets had a 10-fold increase in proteoglycan content compared with a six-fold increase for chondrocyte pellets over 8 weeks (P<0.0001). There was no significant cell division for either chondron or chondrocyte pellets. The majority of cells within both chondron and chondrocyte pellets maintained their polygonal or rounded shape except for a thin, superficial edging of flattened cells. This edging was similar to a perichondrium with abundant type I collagen and fibronectin, and decreased type II collagen and proteoglycan content compared with the remainder of the pellet. This study demonstrates that the native pericellular matrix promotes matrix production and assembly in vitro. Further, the continued matrix production and assembly throughout the 8-week culture period make chondron pellet cultures valuable as a hyaline-like cartilage model in vitro.     

Isolation of human nasoseptal chondrogenic cells: a promise for cartilage engineering

In cartilaginous tissues, perichondrium cambium layer may be the source of new cartilage. Human nasal septal perichondrium is considered to be a homogeneous structure in which some authors do not recognize the perichondrium internal zone or the cambium layer as a layer distinct from adjacent cartilage surface. In the present study, we isolated a chondrogenic cell population from human nasal septal cartilage surface zone. Nasoseptal chondrogenic cells were positive for surface markers described for mesenchymal stem cells, with exception of CD146, a perivascular cell marker, which is consistent with their avascular niche in cartilage. Although only Sox-9 was constitutively expressed, they also revealed osteogenic and chondrogenic, but not adipogenic, potentials in vitro, suggesting a more restricted lineage potential compared to mesenchymal stem cells. Interestingly, even in absence of chondrogenic growth factors in the pellet culture system, nasoseptal chondrogenic cells had a capacity to synthesize sulfated glycosaminoglycans, large amounts of collagen type II and to a lesser extent collagen type I. The spontaneous chondrogenic potential of this population of cells indicates that they may be a possible source for cartilage tissue engineering. Besides, the pellet culture system using nasoseptal chondrogenic cells may also be a model for studies of chondrogenesis.     

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate

Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.