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

Summary 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.     

A comparison of the immunohistochemical localization of type I and type II collagens in craniofacial cartilages of the rat.

The immunohistochemical localization of types I and II collagen was examined in the following 4 cartilaginous tissues of the rat craniofacial region: the nasal septal cartilage and the spheno-occipital synchondrosis (primary cartilages), and the mandibular condylar cartilage and the cartilage at the intermaxillary suture (secondary cartilages). In both primary cartilages, type II collagen was present in the extracellular matrix (ECM) of the whole cartilaginous area, but type I collagen was completely absent from the ECM. In the secondary cartilages, type I collagen was present throughout the cartilaginous cell layers, and type II collagen was restricted to the ECM of the mature and hypertrophic cell layers. These observations indicate differences in the ECM components between primary and secondary craniofacial cartilages, and that these differences may contribute to their modes of chondrogenesis.     

Localization of CD44 (hyaluronan receptor) and hyaluronan in rat mandibular condyle.

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.     

Immunohistochemical localization of type II and type I collagens in articular cartilage of the femoral head of dexamethasone-treated rats

The immunohistochemical localization of type II and type I collagens was examined in the articular cartilage of the femoral head of growing rats injected systemically with 5 mg kg−1 dexamethasone for 2 weeks every other day. The intensities of immunostaining for type II collagen, measured by microphotometry, was highest in the flattened cell layer and high in the hypertrophic cell layer, moderate in the proliferative cell and transitional cell layers and low in the superficial layer. After dexamethasone administration, the intensities decreased markedly in the flattened cell layer and slightly in the hypertrophic cell layer, although the decreases in other layers were negligible. The staining intensities for type I collagen were highest in the flattened cell layer, low in the superficial and transitional cell layers and very low in the proliferative and hypertrophic cell layers. After dexamethasone administration, the intensities increased markedly in the flattened cell layer and slightly in the superficial and proliferative cell layers, but did not change in the transitional and hypertrophic cell layers. Thus, dexamethasone administration caused a decrease in type II collagen and an increase in type I collagen in the matrix of the surface portion of articular cartilage. The composition of isoforms of collagen in the matrix changed after the steroid administration. The results strongly suggest that the shift in collagen composition from type II to type I predominance is a cause of the degeneration of the articular cartilage after glucocorticoid administration. This revised version was published online in November 2006 with corrections to the Cover Date.     

Collagen I and II mRNA distribution in the rat temporomandibular joint region during growth

The distribution of type I and II collagen synthesis in the temporomandibular joint (TMJ) area of 1- to 28-day-old rats was studied after hybridization with probes to pro alpha1(I) and pro alpha1(II) collagen mRNA, and stain intensity through the various cartilaginous zones of the mandibular condyle and other areas of TMJ was assessed. The pro alpha(I) collagen mRNA was detected in the perichondrium/periosteum, in the fibrous and undifferentiated cell layers of the mandibular condyle, in the articular disc, and in all bone structures and muscles. The pro alpha1(II) collagen mRNA was found in the condylar cartilage and the articular fossa. Intensity in the condyle was highest in the chondroblastic layer and decreased towards the lower hypertrophic layer. In the condylar cartilage of the 21- to 28-day-old rats the chondroblastic cell zone was relatively narrow compared with the younger animals, whereas the reverse seems to be the case in the cartilage of the articular fossa. Changes in the pro alpha1(II) collagen mRNA were observed in the osseochondral junction area of the primary spongiosa, in that at the age of 5 days intense staining was found, whereas no staining was observed by 14 days. In the mineralizing zone, however, the majority of osteoblastic cells gave a positive signal with the pro alpha1(I) collagen probe. In conclusion, type II collagen synthesis of the mandibular condyle is restricted to its upper area. This differs from the long bone epiphyseal plate, where this type of collagen is produced virtually throughout the cartilage. Type II collagen synthesis of the fossal cartilage seems to increase as a function of age.     

An Immunohistochemical Study of Matrix Proteins in the Craniofacial Cartilage in Midterm Human Fetuses

Immunohistochemical localization of collagen types I, II, and X, aggrecan, versican, dentin matrix protein (DMP)-1, martix extracellular phosphoprotein (MEPE) were performed for Meckel’s cartilage, cranial base cartilage, and mandibular condylar cartilage in human midterm fetuses; staining patterns within the condylar cartilage were compared to those within other cartilaginous structures. Mandibular condylar cartilage contained aggrecan; it also had more type I collagen and a thicker hypertrophic cell layer than the other two types of cartilage; these three characteristics are similar to those of the secondary cartilage of rodents. MEPE immunoreactivity was first evident in the cartilage matrix of all types of cartilage in the human fetuses and in Meckel’s cartilage of mice and rats. MEPE immunoreactivity was enhanced in the deep layer of the hypertrophic cell layer and in the cartilaginous core of the bone trabeculae in the primary spongiosa. These results indicated that MEPE is a component of cartilage matrix and may be involved in cartilage mineralization. DMP-1 immunoreactivity first became evident in human bone lacunae walls and canaliculi; this pattern of expression was comparable to the pattern seen in rodents. In addition, chondroid bone was evident in the mandibular (glenoid) fossa of the temporal bone, and it had aggrecan, collagen types I and X, MEPE, and DMP-1 immunoreactivity; these findings indicated that chondroid bone in this region has phenotypic expression indicative of both hypertrophic chondrocytes and osteocytes.Key words: condylar cartilage, human fetus, extracellular matrix, MEPE, DMP-1     

Comparison of age-dependent expression of aggrecan and ADAMTSs in mandibular condylar cartilage, tibial growth plate, and articular cartilage in rats

A disintegrin and metalloproteinase with thrombospondin motif (adamalysin–thrombospondins, ADAMTS) degrades aggrecan, one of the major extracellular matrix (ECM) components in cartilage. Mandibular condylar cartilage differs from primary cartilage, such as articular and growth plate cartilage, in its metabolism of ECM, proliferation, and differentiation. Mandibular condylar cartilage acts as both articular and growth plate cartilage in the growing period, while it remains as articular cartilage after growth. We hypothesized that functional and ECM differences between condylar and primary cartilages give rise to differences in gene expression patterns and levels of aggrecan and ADAMTS-1, -4, and -5 during growth and aging. We employed in situ hybridization and semiquantitative RT-PCR to identify mRNA expression for these molecules in condylar cartilage and primary cartilages during growth and aging. All of the ADAMTSs presented characteristic, age-dependent expression patterns and levels among the cartilages tested in this study. ADAMTS-5 mainly contributed to ECM metabolism in growth plate and condylar cartilage during growth. ADAMTS-1 and ADAMTS-4 may be involved in ECM turn over in articular cartilage. The results of the present study reveal that ECM metabolism and expression of related proteolytic enzymes in primary and secondary cartilages may be differentially regulated during growth and aging.     

Expression of collagen type I,II, X and Ki-67 in osteochondroma compared to human growth plate cartilage

In order to characterize the consequences for the process of endochondral ossification we performed an immunohistochemical study and compared the expression of collagen type I, II and X as markers of cartilage differentiation and Ki-67 as a marker of cell proliferation in solitary (7-26 years, n=9) and multiple (11-42 years, n=6) osteochondromas with their expression in human fetal and postnatal growth plates. In fetal and young postnatal controls, we found a thin superficial layer of articular cartilage that stained positive for collagen type I while collagen II was expressed in the rest of the cartilage and collagen type X was restricted to the hypertrophic zone. Osteochondromas from children showed lobular collagen type II-positive areas surrounded by collagen type I. In adults, the separation of collagen type I- and type II-positive areas was more blurred, or the cartilaginous cap was missing. Collagen type X was detected in a pericellular distribution pattern within hypertrophic zones but also deeper between bone trabecula. The proliferative activity of osteochondromas from children younger than 14 years of age was comparable to postnatal growth plates, whereas in cartilage from individuals older than 14 years of age, we could not detect significant proliferative activity.     

Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.     

Histochemical and immunohistochemical distribution of glycosaminoglycans, type II collagen, and fibronectin in developing fetal cartilage of congenital osteochondrodysplasia rat (ocd/ocd).

The osteochondrodysplasia rat (ocd/ocd) is a lethal dwarfism. The ocd/ocd shows histological abnormalities of the epiphysis, characterized by a decrease in amount of glycosaminoglycans (GAGs) in the extracellular matrix (ECM). The present study describes histochemical and immunohistochemical distributions of GAGs, type II collagen, and fibronectin (FN) in abnormal humeral cartilage of the ocd/ocd fetuses on days 16-21 of gestation. A wide-spread region with severe necrosis was observed in the cartilage on days 20 and 21. The affected cartilage has small amounts of ECM, irregular columnizations, thinner hypertrophic zones, and expanded and pyknotic chondrocytes on days 16-21 of gestation. The severely expanded chondrocytes did not have cytoplasmic glycogens on days 19-21. Reactions for chondroitin sulfate (CS) and hyaluronic acid (HA) in the ECM were consistently lower in ocd/ocd than in +/+ during the entire period of observation, although there were granules immunoreactive to CS within the chondrocytes of ocd/ocd. The distribution of type II collagen seemed normal in relatively normal regions in the affected cartilage. Strong reactions for CS, HA, type II collagen, and FN were present in the necrotic region on days 20 and 21 of gestation. These findings suggest that the affected chondrocyte may have some defects in releasing ECM substances, which may be released by the process of cell rupture. We hypothesize that some defects in releasing processes inherent to the ocd/ocd cartilage may relate to cellular differentiation and cell death.     

Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured with degraded collagen

The functional integrity of articular cartilage is dependent on the maintenance of the extracellular matrix (ECM), a process which is controlled by chondrocytes. The regulation of ECM biosynthesis is complex and a variety of substances have been found to influence chondrocyte metabolism. In the present study we have investigated the effect of degraded collagen on the formation of type II collagen by mature bovine chondrocytes in a cell culture model. The culture medium was supplemented with collagen hydrolysate (CH) and biosynthesis of type II collagen by chondrocytes was compared to control cells treated with native type I and type II collagen and a collagen-free protein hydrolysate. The quantification of type II collagen by means of an ELISA technique was confirmed by immunocytochemical detection as well as by the incorporation of (14)C-proline in the ECM after a 48 h incubation. Chondrocytes in the control group were maintained in the basal medium for 11 days. The presence of extracellular CH led to a dose-dependent increase in type II collagen secretion. However, native collagens as well as a collagen-free hydrolysate of wheat proteins failed to stimulate the production of type II collagen in chondrocytes. These results clearly indicate a stimulatory effect of degraded collagen on the type II collagen biosynthesis of chondrocytes and suggest a possible feedback mechanism for the regulation of collagen turnover in cartilage tissue.     

Altered endochondral bone development in matrix metalloproteinase 13-deficient mice

The assembly and degradation of extracellular matrix (ECM) molecules are crucial processes during bone development. In this study, we show that ECM remodeling is a critical rate-limiting step in endochondral bone formation. Matrix metalloproteinase (MMP) 13 (collagenase 3) is poised to play a crucial role in bone formation and remodeling because of its expression both in terminal hypertrophic chondrocytes in the growth plate and in osteoblasts. Moreover, a mutation in the human MMP13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia. Inactivation of Mmp13 in mice through homologous recombination led to abnormal skeletal growth plate development. Chondrocytes differentiated normally but their exit from the growth plate was delayed. The severity of the Mmp13- null growth plate phenotype increased until about 5 weeks and completely resolved by 12 weeks of age. Mmp13-null mice had increased trabecular bone, which persisted for months. Conditional inactivation of Mmp13 in chondrocytes and osteoblasts showed that increases in trabecular bone occur independently of the improper cartilage ECM degradation caused by Mmp13 deficiency in late hypertrophic chondrocytes. Our studies identified the two major components of the cartilage ECM, collagen type II and aggrecan, as in vivo substrates for MMP13. We found that degradation of cartilage collagen and aggrecan is a coordinated process in which MMP13 works synergistically with MMP9. Mice lacking both MMP13 and MMP9 had severely impaired endochondral bone, characterized by diminished ECM remodeling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation (resulting in drastically shortened bones). These data support the hypothesis that proper ECM remodeling is the dominant rate-limiting process for programmed cell death, angiogenesis and osteoblast recruitment during normal skeletal morphogenesis.     

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 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.     

Immunochemical and immunocytochemical studies of the C-propeptide of type II procollagen in chondrocytes of the growth plate.

The C-propeptide of type II procollagen has previously been implicated in cartilage calcification. To further characterize this propeptide, we have investigated its molecular status and intracellular distribution in bovine fetal growth plate chondrocytes, particularly within the calcifying zone, using cell isolation, Western blotting, and localization with immunofluorescence and immunogold techniques. We found that in all cells freshly isolated by collagenase digestion the C-propeptide was a component of type II pro-alpha chains. No free C-propeptide was detected intracellularly. In situ localization of the C-propeptide by immunostaining employing immunofluorescence revealed the presence of procollagen in most growth plate cells, staining being most intense in hypertrophic cells. In the latter, large dilations of the rough endoplasmic reticulum were observed. These were not found in proliferating cells and had an approximate diameter of 5 microns. With immunogold localization these, together with Golgi-derived secretory granules, stained for the C-propeptide. These combined results suggest that in all cells of the growth plate the C-propeptide is a constituent part of type II collagen pro-alpha chains, and that it is usually segregated in the rough endoplasmic reticulum at a time when, according to other studies, collagen synthesis ceases in the lower hypertrophic zone and calcification of the extracellular matrix ensues. This suggests that the intracellular translocation of type II collagen pro-alpha chains may change in hypertrophic cells at this time.     

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.     

Immunohistochemical study of collagens of the extracellular matrix in cartilage of Sepia officinalis

We used various anti-collagen antibodies to perform indirect immunofluorescent staining of cartilage sections from cuttlefish (S. officinalis). On ultrathin sections and collagen fibril preparations from the same tissue, we performed immunostaining with colloidal gold. The extracellular matrix (ECM) of S. officinalis cartilage reacted intensely and homogeneously with an antibody directed against type I-like collagen isolated from the cartilage of cuttlefish and with anti-rat type V collagen antibody. A weak reaction was observed with anti-fish and anti-chicken type I collagen antibodies, while no reaction was observed with anti-rat type I and anti calf type II collagen antibodies. Anti-chicken type II, anti calf type IX and type XI collagen antibodies reacted weakly with ECM, while stained cell bodies and cell processes reacted more intensely. A similar pattern of reaction was observed on cartilage section and isolated collagen fibrils prepared for electron microscopy. These findings suggest that ECM of cuttlefish cartilage may be composed of molecules similar to the type I, type V, type IX and type XI collagen molecules of vertebrates. Cephalopods have evolved a cartilage of structure and macromolecular organisation similar to that of vertebrate cartilage. However, the main molecular components of S. officinalis cartilage--type I-like and type V collagens--differ from those of vertebrate cartilage. We suggest that this type I-like collagen can be considered an initial step toward the evolution of type II collagen typical of vertebrates.     

Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.