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Type II collagen deposition in cruciate ligament precedes osteoarthritis in the guinea pig knee.

OBJECTIVE: To examine the collagens in cruciate ligaments of young Dunkin-Hartley guinea pigs, to determine whether a change in specific collagen types is an early feature of the spontaneous osteoarthritis (OA), which consistently develops in the medial compartment of the knee in this strain. DESIGN: Collagen types I, II, III, IX, and XI were detected by immunofluorescence microscopy in the anterior and posterior cruciate ligaments of animals at 3, 4-5 and 12 weeks of age. Type II collagen in PCL was further analysed by confocal microscopy or biochemical assay after cyanogen bromide digestion, SDS-PAGE and immunoblotting. Interfibrillar proteoglycans were visualized by transmission electron microscopy. RESULTS: Collagen types I and III formed the bulk of fibrous mid-ligament tissue in all animals. Typical cartilage collagens, types II, IX and XI, were identified by immunolabeling where ligaments attached to tibial bone. Type II collagen, normally restricted to the fibrocartilage attachment sites, was also found at separate foci in anterior fiber bundles of the posterior cruciate ligament in 12-week-old animals. Biochemical data confirmed these observations which, together with electron microscopy showing large atypical proteoglycan structures, suggested the deposition of fibrocartilage within the fibrous mid-ligament. CONCLUSIONS: Cruciate ligaments, especially posterior cruciate ligament in Dunkin-Hartley guinea pigs synthesize cartilage-like matrix in mid-ligament prior to the appearance of classical signs of OA.

Aging↗

Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens.

The collagens of growth plate and articular cartilage from 5-6 month old commercial pigs were characterised. Growth plate cartilage was found to contain less total collagen than articular cartilage as a proportion of the dry weight. Collagen types I, II, VI, IX and XI are present in both growth plate and articular cartilage whereas type X is found exclusively in growth plate cartilage. Types III and V collagen could not be detected in either cartilage. Type I collagen makes up at least 10% of the collagenous component of both cartilages. There are significant differences in the ratios of the quantifiable collagen types between growth plate and articular cartilage. Collagen types I, II, and XI were less readily extracted from growth plate than from articular cartilage following pepsin treatment, although growth plate cartilage contains less of the mature collagen cross-links, hydroxylysyl-pyridinoline and lysyl-pyridinoline. Both cartilages contain significant amounts of the divalent reducible collagen cross-links, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine. Immunofluorescent localisation indicated that type I collagen is located predominantly at the surface of articular cartilage but is distributed throughout the matrix in growth plate. Types II and XI are located in the matrix of both cartilages whereas type IX is predominantly pericellular in the calcifying region of articular cartilage and the hypertrophic region of the growth plate. Collagen type VI is located primarily as a diffuse area at the articular surface.

Animals↗

Gene expression profile of bovine bone marrow mesenchymal stem cell during spontaneous chondrogenic differentiation in pellet culture system.

Bovine bone marrow mesenchymal stem cells (MSCs) cultured in condensate culture, spontaneous and independent for any external biostimulants, undergo chondrogenic differentiation. In the present study, the bovine MSC chondrogenesis pathway was studied by analyzing stage-specific gene expression using quantitative "Real Time" reverse transcriptase polymerase chain reaction (qRT-PCR). Results showed that bovine MSCs underwent complete chondrogenesis; the initial stage was characterized by expression. of sox9 messenger ribonucleic acid (mRNA), followed by high transcription of chondrocyte specific genes, collagen type II and IX, biglycan and cartilage oligomeric matrix protein, and the final prehypertrophic and/or hypertrophic stage was distinguished by increased expression of collagen type X. From day 7 to day 14 of differentiation increased mRNA expression of the transforming growth factors beta1 and beta2, basic fibroblast growth factor (FGF 2), bone morphogenic protein 6 (BMP 6), insulin-like growth factors 1, parathyroid hormone related peptide and indian hedgehog (Ihh) were detected. These results suggest that these well know chondrogenic growth factors may play a role in bovine chondrogenesis in autocrine and/or paracrine manner. On day 21 of the culture, FGF 2, BMP 6 and Ihh were highly expressed, compared to cells cultured in monolayer manner, which suggests a possible function in maintaining the terminal stage of differentiation. This data extends our knowledge about the unusual species-specific bovine MSC chondrogenesis, allowing us to define the phenotype of the differentiated cells. Furthermore, this study contributes to our in understanding of known chondrogenic-growth factors in autocrine and/or paracrine manner playing a role in the spontaneous differentiation.

Animals↗

Etodolac preserves cartilage-specific phenotype in human chondrocytes: effects on type II collagen synthesis and associated mRNA levels.

We have shown that interleukin-1 (IL-1) suppresses expression of cartilage-specific types II and IX collagens by cultured human chondrocytes. This inhibition is potentiated by agents which block IL-1-stimulated PGE2 production (J. Clin. Invest. 82:2026, 1988). In contrast, expression of types I and III collagens and fibronectin, matrix components produced by chondrocytes that have lost cartilage-specific phenotype, is increased by IL-1, particularly when IL-1-stimulated synthesis of PGE2 is blocked by a prostaglandin synthetase inhibitor. Etodolac is a new NSAID which is an effective inhibitor of PGE2 synthesis. The enhanced potency of etodolac in chondrocytes (compared with macrophages) suggests that this drug may have selective effects on different target cell types. The present studies were undertaken to compare the effects of etodolac and other nonsteroidal anti-inflammatory drugs (NSAIDs) on IL-1-induced modulation of chondrocyte phenotype. Juvenile human costal chondrocytes or adult articular chondrocytes in primary culture were incubated with etodolac, indomethacin or ketoprofen in the absence or presence of IL-1 beta. After treatment the [3H] proline-labelled collagens were analyzed by SDS-PAGE and type I and type II collagen mRNAs were analyzed by Northern or dot hybridization. Indomethacin (0.3-300 nM) or ketoprofen (2-2000 nM) produced a dose-dependent suppression of type II collagen synthesis associated with decreased levels of type II collagen mRNA in the absence of IL-1, while they potentiated the inhibitory effects of IL-1. In contrast, etodolac (2-2000 nM) maintained expression of type II collagen protein and mRNA. Etodolac unmasked a stimulatory effect of IL-1 on synthesis of type I collagen and fibronectin and levels of type I collagen mRNA, but to a lesser extent than indomethacin. These results suggest that, despite equipotent inhibitory effects of etodolac (IC50 congruent to 10 nM) on PGE2 biosynthesis compared with indomethacin (IC50 congruent to 1.0 nM) or ketoprofen (IC50 congruent to 10 nM), etodolac may be capable of maintaining type II collagen expression by chondrocytes. In vivo this could help to prevent the detrimental effects of mediators such as IL-1 on cartilage matrix synthesis in inflammatory joint diseases.

Anti-Inflammatory Agents, Non-Steroidal↗

Divide, accumulate, differentiate: cell condensation in skeletal development revisited.

Cell condensation is a pivotal stage in skeletal development. Although prechondrogenic condensations normally exist for some 12 h, duration can vary. Variation is seen both between condensations for different cartilages (Meckel's vs. elastic ear cartilage) and within a single condensation from which more than one skeletal element will form, as in the three components of the single first arch chondrogenic condensation. Understanding how duration of the condensation phase is established--how the condensation phase is entered and exited during cell differentiation--remains a major area for future study. During chondrogenesis, cell-specific products such as collagen types II and IX and cartilage proteoglycan appear concomitant with condensation. Therefore, during chondrogenesis, condensation precedes commitment of cells as prechondroblasts. During osteogenesis, however, differentiation of preosteoblasts precedes condensation. Therefore, during osteogenesis, condensation amplifies the number of committed osteogenic cells. Further comparative analysis of skeletogenesis should provide us with a more rigorous understanding of cell commitment, when differentiation is initiated, how commitment and differentiation are measured and the relationship of condensation to onset of differentiation. Current knowledge of molecules characteristic of condensations focused attention on extracellular matrix and cell surface components on the one hand, and on growth factors homeobox genes and transcription factors on the other. We have drawn together the molecular data for pre-chondrogenic condensations in diagrammatic form in Figure 2. Three major phases of chondrogenesis are identified: (a) epithelial-mesenchymal interactions that precede condensation, (b) condensation itself, and (c) cell differentiation. Although we label the third phase differentiation, it is important to recognize that phases a and b also constitute aspects of chondroblast cell differentiation (see Dunlop and Hall, 1995 for a discussion of this point. The pre-condensation phase is characterized by expression of Hox genes, growth factors (TGF-beta and BMP-2) and the cell surface proteoglycan receptor, syndecan-1. Expression of Msx-1 and Msx-2, growth factors and syndecan continues into the condensation phase. Other molecules, such as versican, syndecan-3 and tenascin, present in low concentrations before condensation, are up-regulated during condensation. Yet other molecules--Hox genes, transcription factors, growth factors (activin, BMP-4 and -5, GDF-5), cell adhesion molecules and proteoglycans--are only expressed during the condensation phase, while the transcription factor Pax-1, fibronectin, hyaluronan and hyaladherin are expressed both during and after condensation. During condensation mRNAs for collagen types II and IX and for the core protein of cartilage proteoglycan are up-regulated. Late in condensation and increasingly thereafter, the protein products of these genes accumulate as chondroblasts differentiate (see Fig. 2 for details). Not all the molecules present before, during of after condensation can be placed into causal sequences. Some however can. In Figure 3 we summarize the causal sequences discussed in this paper as they relate to initiation of condensation and to transit from condensation to overt differentiation during chondrogenesis. Condensations form following activation of at least three pathways: (1) Initiation of epithelial-mesenchymal interactions by tenascin, BMP-2, TGF beta-1 and Msx-1 and -2. (2) Up-regulation of N-CAM by activin. (3) Up-regulation of fibronectin by TGF-beta, further enhancing N-CAM accumulation (Fig. 3). It is by these three pathways that condensations are initiated and grow. Transition from condensation to overt cell differentiation is under both positive and negative control (Fig. 3). Syndecan blocks fibronectin and so blocks N-CAM accumulation, preventing accumulation of additional cell

Animals↗

The alpha 1 (IX) collagen gene gives rise to two different transcripts in both mouse embryonic and human fetal RNA.

We have isolated and characterized portions of the alpha 1 (IX) collagen gene from mouse and human DNA. Nucleotide sequence analysis and comparison with the chicken gene suggest that the mammalian genes contain an alternative exon that is located within the intron between exons 6 and 7. Using oligonucleotide primers specific for exons 4, 8, and the alternative exon (exon 1*), we demonstrated by the polymerase chain reaction that embryonic mouse and fetal human RNAs contain two types of alpha 1(IX) collagen transcripts. One type of transcript does not contain the sequence encoded by exon 1*; the second type of transcript contains this exon. Both mouse and human alpha 1(IX) collagen genes give rise, therefore, to (at least) two mRNA transcripts.

Amino Acid Sequence↗

Retarded chondrogenesis in transgenic mice with a type II collagen defect results in fracture healing abnormalities.

We have examined the biological and biomechanical consequences of defective type II collagen production for fracture repair employing a genetically engineered mouse line Del1 which was generated by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7 (Metsäranta et al. [1992] J. Cell Biol. 118:203-212). Standardized tibial fractures were produced in transgenic Del1 mice and their nontransgenic littermates were used as controls. The fracture callus tissues were analyzed at days 7, 9, 14, 28, and 42 using radiography, histomorphometry, biomechanical testing, and Northern analysis of mRNAs for several tissue-specific matrix components. Deficient production of cartilage in Del1 mice resulted in reduced radiographic callus size, smaller cross-sectional area, and impaired biomechanical properties when compared with fractures of nontransgenic control mice. The differences were most evident in 14-day fracture calluses. Consequently mRNAs for cartilage-specific type IX and X collagens and aggrecan were also reduced in Del1 calluses. Levels of type II collagen mRNAs were unaffected since the mutated transgene produced additional type II collagen mRNA molecules. Further abnormalities in the fracture repair process of Del1 mice were observed in callus remodeling. In the control animals a typical feature of external callus remodeling was reduction of callus size during endochondral ossification between days 14 and 28. Such reduction was not observed in the transgenic mice. Histological examination of fracture calluses suggested also a reduction in trabecular surface area, which was found to be even more pronounced in metaphyseal bone of Del1 mice. Despite these differences the biomechanical properties of the calluses in the two groups became similar by day 28 of fracture healing. The results thus suggest that reduced chondrogenesis due to the presence of mutated transgenes in Del1 mice not only causes a temporary impairment in biomechanical properties of healing fractures but also affects later stages of callus remodeling.

Animals↗

Selective intracellular retention of extracellular matrix proteins and chaperones associated with pseudoachondroplasia.

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.

Aggrecans↗

Human and sheep growth-plate cartilage type X collagen synthesis and the influence of tissue storage.

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.

Aging↗

Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria.

Osteogenetic protein-1 (OP-1), a member of the TGF-beta superfamily, induces endochondrial bone formation at subcutaneous sites in vivo and stimulates osteoblastic phenotypic expression in vitro. Primary cultures of newborn rat calvarial cells contain a spectrum of osteogenic phenotypes ranging from undifferentiated mesenchymal osteoprogenitor cells to parathyroid hormone (PTH)-responsive osteoblasts. We examined whether treatment of this cell population with recombinant human osteogenic protein-1 could induce chondrogenesis in vitro. Markers of chondroblastic versus osteoblastic differentiation included alcian blue staining at pH 1, alkaline phosphatase-specific activity, osteocalcin radioimmunoassay, and expression of collagen mRNAs. 6 d of treatment (culture days 1-7) with 4-100 ng OP-1/ml caused dose-dependent increases in alcian blue staining intensity and alkaline phosphatase activity (4.7- and 3.4-fold, respectively, at 40 ng/ml), while osteocalcin production decreased twofold. Clusters of round, refractile, alcian blue-stained cells appeared by day 3, increased in number until day 7, and then became hypertrophic and gradually became less distinct. Histochemically, the day 7 clusters were associated with high alkaline phosphatase activity and became mineralized. mRNA transcripts for collagen types II and IX were increased by OP-1, peaking at day 4, while type X collagen mRNA was detectable only on day 7 in OP-1-treated cultures. Delay of OP-1 exposure until confluence (day 7) amplifies expression of the normal osteoblastic phenotype and accelerates its developmental maturation. In contrast, early OP-1 treatment commencing on day 1 strongly amplifies chondroblastic differentiation. In the same protocol, TGF-beta 1 alone at 0.01-40 ng/ml fails to induce any hypertrophic chondrocytes, and in combination with OP-1, TGF-beta 1 blocks OP-1-dependent chondroinduction. OP-1 is believed to act on a subpopulation of primitive osteoprogenitor cells to induce endochondrial ossification, but does not appear to reverse committed osteoblasts to the chondrocyte phenotype.

Alkaline Phosphatase↗

Preliminary observations of chondral abrasion in a canine model.

Articular cartilage repair was followed for one year in skeletally mature dogs after destabilisation by anterior cruciate ligament transection of the stifle joint (CT), abrasion of the inferior medial condyle (ABR) to bleeding bone, or anterior cruciate transection followed by chondral abrasion (CT/ABR). ABR animals formed repair cartilage at the abrasion site (ABR and CT/ABR) at six months as determined by arthroscopy and at necropsy. CT and CT/ABR animals had an additional cartilage ulcer on the superior aspect of the medial condyle. The abraded site extended in CT/ABR condyles. Repair cartilage (ABR and CT/ABR) contained reduced amounts of proteoglycan as seen by histological loss of safranin O staining and reduced uronic acid content. Fibrocartilage was suggested by histological appearance, hypocellularity, and a higher hydroxyproline content. In contrast with ABR animals, the repair cartilage in the CT/ABR animals contained near normal amounts of hydroxyproline. Collagen profiles of abrasion site repair cartilage in ABR animals had more types I and V collagens, similar amounts of type VI collagen, and decreased amounts of types II, IX, and XI collagens than CT/ABR animals. The results of this study are consistent with abrasion chondroplasty leading to a repair cartilage. Despite extended ulcers, repair cartilage from the destabilised joint (CT/ABR) animals was more hyaline-like in its hydroxyproline content and collagen composition than repair cartilage from the stable joint (ABR animals). In these models additional measures appear to be needed as the defects induced by abrasion chondroplasty did not form a functional hyaline cartilage.

Animals↗

[Cartilage-specific autoimmunity in otosclerosis].

The otic capsule of patients with otosclerosis contains premature bone with numerous cartilaginous remnants. Some investigators have proposed that the pathogenesis of otosclerosis is related to these cartilaginous rests in the otic capsule. In this study we investigated the presence of a humoral immune reaction against cartilage-specific antigens using ELISA-methods in patients with otosclerosis. Concomitantly, 8 age- and sex-matched healthy blood donors, free of any symptoms of autoimmune disease, served as controls. The following antigen substrates were used: collagen (I, II, III, VI, IX and XI), chondrocytes and chondrocyte membranes. Findings then showed that the levels of antibodies to collagens type II and IX as well as to chondrocytes were higher in the otosclerosis patients than in the control subjects. The high titer of antibodies against chondrocytes was not accompanied by an increase in antibodies against the chondrocyte membranes. To our knowledge these observations represent the first evidence for the existence of autoantibodies against minor collagens and chondrocyte-specific antigens and support a possible role for a cartilage-specific autoimmunity in the etiopathogenesis of otosclerosis.

Adult↗

The mouse col11a2 gene. Some transcripts from the adjacent rxr-beta gene extend into the col11a2 gene.

Type XI collagen is present in small amounts in cartilage, together with small amounts of type IX and type V collagens and large amounts of type II collagen. Here, primers based on the nucleotide sequences of partial human cDNAs and mouse genomic DNAs that were analyzed by other investigators were used to isolate a cDNA for the mouse col11a2 gene. Cosmid clones for the mouse col11a2 gene were isolated, and 12.4 kb of the nucleotide sequences were defined. Analysis of the genomic sequences identified three exons in the mouse gene that were recently shown to undergo alternative splicing (Tsumaki and Kimura, J. Biol. Chem. 270, 2372-2378, 1995; Zhidkova et al., J. Biol. Chem. 270, 94886-9493, 1995). In addition, analysis of the cosmid clones revealed that the 5' end of the mouse col11a2 gene was located head-to-tail with the mouse retinoic X receptor beta gene. RT-PCR assays demonstrated that some transcripts from the retinoic X receptor beta gene extend into the col11a2 gene. Therefore, there may be coordinate expression of the two genes.

Animals↗

Musculoskeletal differentiation of cells derived from human embryonic germ cells.

Stem cells have the potential to significantly improve cell and tissue regeneration therapies, but little is understood about how to control their behavior. We investigated the potential differentiation capability of cells derived from human embryonic germ (EG) cells into musculoskeletal lineages by providing a three-dimensional environment with increased cell-cell contact and growth factors. Cells were clustered into pellets to mimic the mesenchyme condensation process during limb development. LVEC cells, an embryoid body-derived (EBD) cell culture generated from EG cells, were cultured in micromass pellets for 21 days in the presence of bone morphogenetic protein 2 (BMP2) and/or transforming growth factor beta-3 (TGFbeta3). Gene expression for cartilage-, bone-, and muscle-specific matrix proteins--including collagen types I, II, III, IX, X; aggrecan; cartilage proteoglycan link protein; cartilage oligomeric protein; chondroitin sulfate-4-S; and myf5--was upregulated in the pellets treated with TGFbeta3, while mRNAs for neurofilament heavy (NFH), a neuron marker, and flk-1, a hematopoietic marker, decreased. Total collagen and proteoglycan production exhibited a time-dependent increase in the pellets treated with TGFbeta3, further confirming the expression of characteristic musculoskeletal markers. Furthermore, our results indicate the ability to select or differentiate stem cells toward a musculoskeletal lineage from a heterogenous EBD cell line.

Cartilage↗

Immunoassays for collagens in chondrocyte and cartilage explant cultures.

Quantitative immunoassays have been developed to measure the content, degradation, and synthesis of types II and IX collagens in hyaline cartilages. Some of these assays and their applications are described in this chapter. These and other assays are commercially available. The applications of these assays are discussed with examples from recent publications.

Amino Acid Sequence↗

Role of basement membrane collagen and elastin in the autofluorescence spectra of the colon.

BACKGROUND: Autofluoresence can be used to detect neoplasia in the colon. Two known fluorophores, collagen and elastin, are probably partly responsible for colonic emission spectra. Their contribution to colonic autofluorescence was investigated. METHODS: Autofluorescence spectra of normal, dysplastic, and malignant colonic tissue were studied by using excitation wavelengths from 280 nm to 350 nm. The wavelengths of peak emission and their widths at half maximum intensity were measured. Similar measurements were performed on collagen types I, III, IV, V, IX, and elastin. Colonic spectra were compared to those of collagen and elastin. Spectral differences between collagen types IV (basement membrane) I, III, V, and IX were studied. RESULTS: Four major emission peaks were noted whose wavelength of peak emission and full widths at half maximum intensity were independent of tissue histology. The emission spectra of type IV collagen differed markedly from that of nonbasement membrane collagens and elastin. CONCLUSIONS: Type IV (basement membrane) collagen is most likely responsible for the emission peak at 365 nm. The spectra of basement membrane collagen and not other types of collagen should be used in studies of epithelial tissue spectra. Elastin did not appear to be responsible for any of the four autofluorescence peaks observed in colonic tissue.

Adenocarcinoma↗

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.

Amino Acids↗

Underhydroxylated minor cartilage collagen precursors cannot form stable triple helices.

Matrix-free cells from chick-embryo sterna were incubated with various concentrations of 2,2'-bipyridyl, an iron chelator that inhibits prolyl hydroxylase and lysyl hydroxylase. At concentrations in the region of 0.1 mM, significant effects on cartilage collagen hydroxylation and secretion were observed. When the underhydroxylated collagens were subsequently digested with chymotrypsin or chymotrypsin plus trypsin at 4 degrees C for 15 min, the minor cartilage collagen precursors (namely types IX and XI) were extensively degraded; type II procollagen was only partially susceptible and was converted into underhydroxylated collagen. The results demonstrate that there were significant differences in triple-helix stability among cartilage collagens such that the underhydroxylated minor collagen precursors were unable to attain a native structure under conditions where type II procollagen was successful.

2,2'-Dipyridyl↗