Type X collagen alterations in rachitic chick epiphyseal growth cartilage

We examined collagens of both normal and vitamin D-deficient chick epiphyseal growth cartilage. Special emphasis was placed on the study of Type X collagen, a recently described product of hypertrophic chondrocytes. Scanning electron microscopy of the epiphyseal growth cartilage of vitamin D-deficient chickens showed an enlarged growth cartilage with a disorganized extracellular matrix. The cartilage collagens were solubilized by proteolytic digestion and disulfide bond reduction of both normal and rachitic growth tissues. Sequential extraction with neutral salt and acetic acid buffers followed by pepsin digestion at 4 degrees C solubilized about 12% of normal tissues and about 7% of collagen from rachitic growth cartilage. Treatment of the pepsin-resistant collagens with neutral salt-dithiothreitol buffer under nondenaturing conditions and a subsequent pepsin digestion increased the yield of solubilized collagen to greater than 95% of the total tissue collagen. Results of the biochemical studies showed a marked increase in the relative proportion of Type X collagen (from 5.6 to 27.9%), a corresponding decrease in the proportions of Types II and IX collagens, and a moderate increase in Type XI collagen in rachitic cartilage. Amino acid analysis indicated that there were no differences in the Types II and X collagens of normal and rachitic cartilage. However, an abnormality in the relative proportions of the CNBr peptides of Type X collagen was detected in the rachitic cartilage. We suggest that the increase in collagen in the rachitic state may reflect increased levels of Type X collagen synthesis by cells in the hypertrophic region. It is likely that in rickets the overproduction of Type X collagen may be a compensatory mechanism by which the hypertrophic chondrocyte attempts to provide a maximum area of calcifiable matrix for the calcium-depleted serum.     

Immunolocalization of type X collagen before and after mineralization of human thyroid cartilage

In this study the distribution of type X collagen in thyroid cartilages of various ages is described. Fetal and juvenile thyroid cartilage was negative for type X collagen, but showed a strong staining reaction for type II collagen. Type X collagen and calcium deposition were first detected in thyroid cartilage of 18-to 21-year-old adults. Type X collagen was restricted to large chondrocytes near or in mineralized cartilage, confirming the notion that type X collagen precedes mineralization. From these observations it was concluded that chondrocytes in thyroid cartilage undergo differentiation steps that are similar, but much slower, compared to cells in growth plate and sternal cartilage. Some type X collagen-positive areas also showed staining for type I collagen, suggesting that there is a further differentiation of chondrocytes to cells which are characterized by the simultaneous synthesis of type X and I collagen. However, a dedifferentiation process during aging of thyroid cartilage where cells switch from synthesis of type II to type I collagen cannot be excluded.     

Expression of type X collagen mRNA levels in embryonic chick sternum during development

Embryonic chick sternum cartilage exhibits profound spatial and temporal changes in Type X collagen biosynthesis during development. Production of this collagen is confined to the presumptive calcification region and its expression is not acquired until stage 43. To examine the mechanisms responsible for regulation of developmental changes in biosynthetic expression of Type X collagen, we determined the levels of translatable Type X procollagen mRNA employing a cell-free translation system. We found that mRNA capable of directing Type X collagen synthesis was present exclusively in cartilage destined to undergo calcification and that its levels were nearly equivalent at all stages of development. These findings suggest that expression of Type X collagen in embryonic chick sternum is determined at the translational level.     

Synthesis of type I and type II collagen by embryonic chick cartilage

Embryonic chick articular and keel cartilage was found to synthesize two types of collagen. The amount of Type I collagen synthesis decreased from 60% to nearly 10% during the embryonic period studied, thus suggesting not only coexistence of both collagen types in the same tissue, but also a developmental transformation from predominantly Type I synthesis to Type II synthesis with cartilage development and maturation. Radioautographs suggested that all chondrocytes were equally active in collagen synthesis and failed to show any significant non-cartilagenous tissue contamination. Therefore variation in collagen type synthesis must be a product of some unknown genetic regulatory mechanism within the cartilage tissue.     

Extracellular matrix synthesis by articular chondrocytes and synovial fibroblasts in long-term monolayer culture

Synthesis of collagen and proteoglycan by rabbit articular chondrocytes and synovial fibroblasts has been studied over a 12-week period in primary monolayer culture. Chondrocytes, but not fibroblasts, accumulate large quantities of proteoglycan over the culture period studied. Radiolabeling studies with [35S]sulfate have shown that the major proteoglycan synthesized by cultured chondrocytes is similar to the proteoglycan of cartilage matrix. Chondrocytes also synthesize a smaller dermatan sulfate proteoglycan, which is apparently the only proteoglycan species produced by synovial fibroblasts. Collagen synthesis was studied by radiolabeling with [3H]proline. Cultured chondrocytes produce mainly Type II collagen, with lesser amounts of Type I, whereas synovial fibroblasts produce Type I collagen and some low molecular weight collagenous species. Therefore, long-term monolayer culture permits the production of extensive chondroid matrix by chondrocytes, but not fibroblasts.     

Quantitative analysis of type X-collagen biosynthesis by embryonic-chick sternal cartilage.

We have performed a quantitative analysis of the various collagens biosynthesized by organ cultures of whole embryonic-chick sternum and its separate anatomical regions corresponding to the zones of permanent hyaline and presumptive-calcification cartilages. Our studies demonstrated that embryonic-chick sternum devotes a large portion of its biosynthetic commitment towards production of Type X collagen, which represented approx. 18% of the total newly synthesized collagen. Comparison of the collagens biosynthesized by the permanent hyaline cartilage and by the cartilage from the presumptive-calcification zone demonstrated that Type X-collagen production was strictly confined to the presumptive-calcification region. Sequential extraction of the newly synthesized Type X collagen demonstrated the existence of two separate populations. One population (approx. 20%) was composed of easily extractable molecules that were solubilized with 1.0 m-NaCl/50 mM-Tris/HCI buffer, pH 7.4. The second population was composed of molecules that were not extractable even after repeated pepsin digestion, but became completely solubilized after treatment with 20 mM-dithiothreitol/0.15 M-NaCl buffer at neutral pH. These results suggest that most of the Type X collagen normally exists in the tissue as part of a pepsin-resistant molecular aggregate that may be stabilized by disulphide bonds. Quantitative analysis of the proportion of Type X collagen relative to the other collagens synthesized in the cultures indicated that this collagen was a major biosynthetic product of the presumptive-calcification cartilage, since it represented about 35% of the total collagen synthesized by this tissue. In contrast, the permanent hyaline cartilage did not display any detectable synthesis of Type X collagen. When compared on a per-cell basis, the chondrocytes from the presumptive-calcification zone synthesized approx. 33% more Type X collagen than the amount of Type II collagen synthesized by the chondrocytes from the permanent-hyaline-cartilage zone. Subsequently, it was demonstrated that Type X collagen is a structural component of chick sternum matrix, since quantitative amounts could be extracted from the region of presumptive calcification of 17-day-old chick-embryo sterna and from the calcified portion of adult-chick sterna. The strict topographic distribution in the expression of Type X collagen biosynthesis to the zone of presumptive calcification suggests that this collagen may play an important role in initiation or progression of tissue calcification.     

Immunochemical and mechanical characterization of cartilage subtypes in rabbit.

Cartilage is categorized into three general subgroups, hyaline, elastic, and fibrocartilage, based primarily on morphologic criteria and secondarily on collagen (Types I and II) and elastin content. To more precisely define the different cartilage subtypes, rabbit cartilage isolated from joint, nose, auricle, epiglottis, and meniscus was characterized by immunohistochemical (IHC) localization of elastin and of collagen Types I, II, V, VI, and X, by biochemical analysis of total glycosaminoglycan (GAG) content, and by biomechanical indentation assay. Toluidine blue staining and safranin-O staining were used for morphological assessment of the cartilage subtypes. IHC staining of the cartilage samples showed a characteristic pattern of staining for the collagen antibodies that varied in both location and intensity. Auricular cartilage is discriminated from other subtypes by interterritorial elastin staining and no staining for Type VI collagen. Epiglottal cartilage is characterized by positive elastin staining and intense staining for Type VI collagen. The unique pattern for nasal cartilage is intense staining for Type V collagen and collagen X, whereas articular cartilage is negative for elastin (interterritorially) and only weakly positive for collagen Types V and VI. Meniscal cartilage shows the greatest intensity of staining for Type I collagen, weak staining for collagens V and VI, and no staining with antibody to collagen Type X. Matching cartilage samples were categorized by total GAG content, which showed increasing total GAG content from elastic cartilage (auricle, epiglottis) to fibrocartilage (meniscus) to hyaline cartilage (nose, knee joint). Analysis of aggregate modulus showed nasal and auricular cartilage to have the greatest stiffness, epiglottal and meniscal tissue the lowest, and articular cartilage intermediate. This study illustrates the differences and identifies unique characteristics of the different cartilage subtypes in rabbits. The results provide a baseline of data for generating and evaluating engineered repair cartilage tissue synthesized in vitro or for post-implantation analysis.     

Coordinate regulation of collagen and alkaline phosphatase levels in chick embryo chondrocytes

Chick embryo tibial chondrocytes release into their extracellular matrix several species of proteochondroitin sulfate and collagen as well as matrix vesicles that are rich in Ca2+ and alkaline phosphatase and that appear to play a role in the calcification of cartilage. To determine whether there was any parallel regulation of the production of these products, the rates of collagen synthesis by cultured chick embryo tibial chondrocytes were altered, and the resulting changes in proteochondroitin sulfate synthesis and alkaline phosphatase levels in the cells were measured. As the rate of collagen synthesis was increased by adding increasing amounts of ascorbic acid to the culture medium, there was a parallel increase in the level of alkaline phosphatase. Similarly, when the rate of collagen synthesis was inhibited by adding 3,4-dehydroproline to the culture medium, the levels of alkaline phosphatase fell. The alkaline phosphatase in the culture medium was associated with vesicles which appeared to be matrix vesicles. It was recovered quantitatively by filtration through membranes with a pore size of 0.1 mu and measured by solubilizing the alkaline phosphatase from the membrane with detergent and assaying with 4-methylumbelliferyl phosphate as the substrate. When the matrix vesicles from the culture medium were analyzed for collagen types, it was found that only Type X collagen was recovered in this fraction. The implications of the association of Type X collagen and the matrix vesicles, both of which are found primarily in growth plate cartilage in the zone of hypertrophied chondrocytes which is in the process of mineralization, are discussed.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.     

Identification of a disulfide-bonded 70 Kd type X procollagen in embryonic chick sternum cartilage

Chondrocytes from the presumptive calcification region of 20 day old embryonic chick sternum were found to synthesize a 70 Kd Type X procollagen precursor in addition to the previously described 59 Kd Type X collagen molecules. The 70 Kd molecules exhibited an additional cyanogen bromide peptide, contained a disulfide-bonded domain, and were converted into the 59 Kd moieties during pulse-chase experiments. The conversion of the 70 Kd to the 59 Kd Type X collagen was prevented upon microtubular transport inhibition with colchicine and resulted in tissue accumulation of the 70 Kd Type X procollagen.     

Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes

We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckel's cartilage which does not hypertrophy in situ, environmental influences, probably inhibitory in nature, must regulate chondrogenesis in mesectodermal derivatives. (ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoperoxidase localization of type X collagen in chick tibiae

Type X collagen was prepared from medium of long-term cultures of embryonic chick tibiotarsal chondrocytes. Antibodies to type X collagen were raised and used in immunoperoxidase localization studies with embryonic and growing chick tibiotarsus. Strong anti-type X collagen reactivity was detected mainly in the region of hypertrophic chondrocytes, and to a lesser extent in the zone of calcified cartilage. No reactivity was detected in the proliferative zone nor the superficial layer of the cartilage growth plate. These results suggest that type X collagen may play a key role in matrix calcification during growth and development of the skeletal system.     

Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture.

Rabbit articular chondrocytes in suspension culture synthesize Type II collagen [3alpha1(II)] in the absence of extracellular Ca2+ and Type I collagen [2alpha1(I) - alpha2] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca2+ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl2. If added directly to the suspension culture medium containing no CaCl2, calcitonin stimulates an active efflux of Ca2+ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.     

Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium

Epiphyseal chondrocytes cultured in a medium containing 10% serum may be maintained as three dimensional aggregates and differentiate terminally into hypertrophic cells. There is an attendant expression of genes encoding type X collagen and high levels of alkaline phosphatase activity. Manipulation of the serum concentration to optimal levels of 0.1 or 0.01% in this chondrocyte pellet culture system results in formation of features of developing cartilage architecture which have been observed exclusively in growth cartilage in vivo. Cells are arranged in columns radiating out from the center of the tissue, and can be divided into distinct zones corresponding to the recognized stages of chondrocyte differentiation. Elimination of the optimal serum concentration in a chemically defined medium containing insulin eliminates the events of terminal differentiation of defined cartilage architecture. Chondrocytes continue to enlarge into hypertrophic cells and synthesize type X collagen mRNA and protein, but in the absence of the optimal serum concentration, alkaline phosphatase activity does not increase and the cells retain a random orientation. Addition of thyroxine to the chemically defined medium containing insulin and growth hormone results in dose-dependent increases in both type X collagen synthesis and alkaline phosphatase activity, and reproduces the optimal serum-induced morphogenesis of chondrocytes into a columnar pattern. These experiments demonstrate the critical role of thyroxine in cartilage morphogenesis.     

The developmentally regulated type X collagen gene contains a long open reading frame without introns

Type X collagen is a recently discovered product of hypertrophic chondrocytes that is localized to presumptive mineralization zones of hyaline cartilage. Thus, in the epiphyseal growth plate of long bones it is present only in the zone of hypertrophic chondrocytes and absent in the resting and rapidly growing cartilage and in bone. Type X collagen represents, therefore, a transient and developmentally regulated collagen which is synthesized by a subpopulation of chondrocytes. We report here the isolation and characterization of cDNA and genomic clones specific for the chicken protein. The results demonstrate that the polypeptide chains of this collagen contain three distinct domains: a short non-collagenous, amino-terminal region, a collagenous domain of 460 amino acid residues, and a non-collagenous, carboxyl-terminal domain of 170 amino acid residues. The nucleotide sequence of the gene shows that these domains are encoded by a long open reading frame that is not interrupted by introns. Examination of the amino acid sequence derived from this nucleotide sequence reveals the presence of a hydrophobic segment localized 10 amino acid residues upstream from the translational stop codon. The length and sequence characteristics of this segment raise the intriguing possibility that Type X collagen polypeptides may contain a transmembrane segment.     

Isolation of human type X collagen and immunolocalization in fetal human cartilage

Type X collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion-exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 66,000 under reducing conditions, and as a high-Mr oligomer under non-reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4 degrees C decreases the Mr of the monomer to 53,000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage.     

Type X collagen, a product of hypertrophic chondrocytes.

The synthesis of collagen types IX and X by explants of chick-embryo cartilages was investigated. When sternal cartilage labelled for 24h with [3H]proline was extracted with 4M-guanidinium chloride, up to 20% of the 3H-labelled collagen laid down in the tissue could be accounted for by the low-Mr collagenous polypeptides (H and J chains) of type IX collagen; but no type X collagen could be detected. Explants of tibiotarsal and femoral cartilages were found to synthesize type IX collagen mainly in zones 1 and 2 of chondrocyte proliferation and elongation, whereas type X collagen was shown to be a product of the hypertrophic chondrocytes in zone 3. Pulse-chase experiments with tibiotarsal (zone-3) explants demonstrated a time-dependent conversion of type X procollagen into a smaller species whose polypeptides were of Mr 49 000. The processed chains [alpha 1(X) chains] were shown by peptide mapping techniques to share a common identity with the pro alpha 1(X) chains of Mr 59 000. No evidence for processing of type IX collagen was obtained in analogous pulse-chase experiments with sternal tissue. When chondrocytes from tibiotarsal cartilage (zone 3) were cultured on plastic under standard conditions for 4-10 weeks they released large amounts of type X procollagen into the medium. However, 2M-MgCl2 extracts of the cell layer were found to contain mainly the processed collagen comprising alpha 1(X) chains. The native type X procollagen purified from culture medium was shown by rotary shadowing to occur as a short rod-like molecule 148 nm in length with a terminal globular extension, whereas the processed species comprising alpha 1(X) chains of Mr 49 000 was detected by electron microscopy as the linear 148 nm segment.     

Type X collagen synthesis by chick sternal cartilage and its relationship to endochondral development

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.