Immunohistochemical localization of fibronectin and chondronectin in canine articular cartilage

We compared the distribution of fibronectin and chondronectin within the matrix of canine articular cartilage. Fibronectin was found throughout the matrix as well as pericellularly. In contrast, chondronectin was observed predominantly associated with the cell or pericellular matrix. Interactions of these molecules with matrix components in the pericellular matrix probably differs, however, since concentrations of hyaluronidase which prevented detection of pericellular fibronectin allowed detection of chondronectin. Chondronectin and fibronectin were detected in osteoarthritic cartilage as well as in disease-free cartilage. Penetration of biotinylated fibronectin into cartilage from the external medium occurred only in osteoarthritic cartilage and proceeded only from the articular surface. Disease-free cartilage appeared to maintain a barrier to fibronectin penetration from the articular surface which was sustained even after the proteoglycan content was markedly depleted by incubation of cartilage with catabolin or lipopolysaccharide. In cartilage that was proteoglycan-depleted, the only detectable penetration of external fibronectin was from the cut surface.     

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.     

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.     

Neutral protease inhibitors from human intervertebral disc and femoral head articular cartilage

Assays of several proteases, incorporating guanidinium chloride extracts of human femoral head cartilage and intervertebral disc, demonstrated that both tissues contain inhibitors of certain serine proteases. Trypsin, chymotrypsin and a granule extract of human polymorphonuclear leukocyte containing elastase and cathepsin G activities, were inhibited by low molecular weight fractions prepared by Sephadex G-75 chromatography. Using a radioassay, it was further shown that these fractions inhibit proteolysis of cartilage proteoglycan. The inhibitor in intervertebral disc is concentrated in the nucleus pulposus, with a decreasing gradient to the periphery of the annulus fibrosus.It is proposed that these inhibitors confer at least partial protection against pathological proteolysis of the proteoglycans in human articular cartilage and nucleus pulposus.     

Neutral protease inhibitors from human intervertebral disc and femoral head articular cartilage.

Assays of several proteases, incorporating guanidinium chloride extracts of human femoral head cartilage and intervertebral disc, demonstrated that both tissues contain inhibitors of certain serine proteases. Trypsin, chymotrypsin and a granule extract of human polymorphonuclear leukocytes containing elastase and cathepsin G activities, were inhibited by low molecular weight fractions prepared by Sephadex G-75 chromatography. Using a radioassay, it was further shown that these fractions inhibit proteolysis of cartilage proteoglycan. The inhibitor in intervertebral disc is concentrated in the nucleus pulposus, with a decreasing gradient to the periphery of the annulus fibrosus. It is proposed that these inhibitors confer at least partial protection against pathological proteolysis of the proteoglycans in human articular cartilage and nucleus pulposus.     

Immunohistochemical localization of collagens and fibronectin in human breast neoplasms

Forty four specimens from neoplastic, hyperplastic and normal human breast tissues were studied for localization of collagens and fibronectin. Affinity purified antihuman type I, III and IV collagens and antifibronectins were utilized by the indirect immunoperoxidase technique on fixed and paraffin-embedded sections. 86% of the cell cytoplasm of infiltrating ductal and 83% of the lobular cancers were positively stained for collagen type I and III. Collagen type IV, however, was detected in 100% of infiltrating ductal and 83% of lobular carcinomas. Focal cytoplasmic staining is a predominant feature for all antigens in the intraduct carcinoma while a diffuse pattern is encountered in the infiltrating types. Intact basement membranes in various lesions always stained for type IV collagen and showed variable staining for type III collagen and fibronectin. Epithelia of normal, benign, hyperplastic breast and most medullary carcinoma were negative for the three collagen types. Our results are in favour of the view that infiltrating breast carcinoma cells produce inappropriately the majority of collagens and inconsistently other proteins such as fibronectin.     

Chromatographically different type II collagens from human normal and osteoarthritic cartilage.

The genetic type of collagen was examined in both fresh and 2 to 4 month $\text{in vitro}$ cultured human normal and osteoarthritic articular cartilage. Disc electrophoresis indicated that all cartilage samples examined contained only type II collagen, however CM cellulose chromatography revealed chromatographic differences between the normal and pathological tissues.     

Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone,cartilage and giant cell tumor

Summary Carbonic anhydrase isoenzymes I and II have been localized in human bone and cartilage. Osteoclasts are strongly positive for carbonic anhydrase II but very little if any reaction is observed for carbonic anhydrase I. In tendon giant cell tumor osteoclastlike-giant cells contained high amounts of carbonic anhydrase II suggesting the close relation of these cells to normal osteoclasts. In growth plate cartilage strong staining was obtained in late proliferative and hypertrophic chondroxytes as well as in extracellular matrix of hypertrophic zone also only with anti-human carbonic anhydrase II.     

Immunohistochemical analysis of ageing and osteoarthritic articular cartilage

Articular cartilage degeneration seen in osteoarthritis is primarily the consequence of events within the articular cartilage that leads to the production of proteases by chondrocytes. 22 osteoarthritic cartilage specimens were obtained from patients with primary osteoarthritis (46–81 years) undergoing total knee replacement. 12 age-matched (41–86 years) and 16 young (16–40 years) non-osteoarthritic control cartilage specimens were obtained from the cadavers in the department of Anatomy and from patients undergoing lower limb amputation in Trauma center of PGIMER, Chandigarh. 5 μ thick paraffin sections were stained for osteocalcin, osteopontin, osteonectin and alkaline phosphatase to analyze their expression in hypertrophied chondrocytes and osteoarthritic cartilage matrix and to compare the staining intensity with that of normal ageing articular cartilage. Immunohistochemical staining of tissue sections revealed moderate to strong cytoplasmic staining for all four stains in all the specimens of the osteoarthritic group compared to age-matched control. The immunohistochemical scores were significantly higher in the osteoarthritic group for all four stains. The features of the osteoarthritic articular cartilage were markedly different from the non-osteoarthritic age-matched articular cartilage suggesting that osteoarthritis is not an inevitable feature of aging.     

Immunohistochemical localization of articular cartilage proteoglycan and link protein in situ using monoclonal antibodies and lectin-binding methods

Lectins have specificity for certain carbohydrate structures in macromolecules. Lectins are, therefore, useful histochemical tools for demonstrating the composition and localization of components of connective tissue matrices, such as articular cartilage. In order to assess the significance of observed lectin-binding patterns, experiments were performed in which monoclonal antibodies against chondroitin sulphate- and keratan sulphate-containing proteolgycans and link proteins were applied to sections of bovine articular cartilage after enzymatic digestion with chondroitinase ABC and keratanase. The following conclusions were made: (1) Binding of peanut agglutinin (PNA) in the interterritorial matrix predominantly indicates the presence of keratan sulphate, but may also detectO-linked oligosaccharides of proteoglycans. (2) In normal cartilage wheat germ agglutinin (WGA) binds nearly exclusively to keratan sulphate. In cartilage degraded with chondroitinase ABC and keratanase this lectin may also detect carbohydrates in link protein due to enhanced accessibility. Binding of WGA toO-linked oligosaccharides may eventually occur. (3) In enzymatically digested cartilage matrix, staining with soybean agglutinin (SBA) may be due to link protein, but not to chondroitin sulphate, because specific breakdown of the glycosaminoglycan chain is required for binding of SBA. (4)Ulex europaeus agglutinin I (UEA I) binding sites are only detectable in digested cartilage matrix.     

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.     

Immunohistochemical localization of osteogenetic protein (OP-1) and its receptors in rabbit articular cartilage.

We assessed the distribution and relative immunohistochemical staining intensity of the bone morphogenetic protein-7, osteogenic protein-1 (OP-1), in its pro- and mature forms, and four of its receptors, type I (ALK-2, ALK-3, and ALK-6) and type II in normal adolescent New Zealand White rabbit articular cartilage. Expression of the protein and its receptors was also examined in cartilage from joints that had been previously subjected to cartilage matrix degradation. Pro-OP-1 was moderately expressed in chondrocytes of the superficial, middle, and deep cartilage zones and in the osteocytes. The expression of mature OP-1 was similar, with the exception of less staining in the superficial zone of cartilage. Expression of these two forms of OP-1 was enhanced in the middle and deep cartilage zones after catabolic challenge. The type I receptor, ALK-6, displayed the strongest staining of the receptors in both cartilage and bone, whereas ALK-2 displayed the weakest staining. No differences were observed in the receptor staining levels after catabolic challenge. This study shows that OP-1 and its receptors have been identified in rabbit articular cartilage and bone, suggesting a possible role for this pathway in cartilage and bone homeostasis.     

Peptides of type II collagen can induce the cleavage of type II collagen and aggrecan in articular cartilage.

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195-218), but not synthetic peptide CB12-IV (residues 231-254), potently and consistently induced in explant cultures at 10 microM and 25 microM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues

Monoclonal antibodies were produced against the recently described short chain cartilage collagen (type X collagen), and one (AC9) was extensively characterized and used for immunohistochemical localization studies on chick tissues. By competition enzyme-linked immunosorbent assay, antibody AC9 was observed to bind to an epitope within the helical domain of type X collagen and did not react with the other collagen types tested, including the minor cartilage collagens 1 alpha, 2 alpha, 3 alpha, and HMW-LMW. Indirect immunofluorescence analyses with this antibody were performed on unfixed cryostat sections from various skeletal and nonskeletal tissues. Only those of skeletal origin showed detectable reactivity. Within the cartilage portion of the 13-d-old embryonic tibiotarsus (a developing long bone) fluorescence was observed only in that region of the diaphysis containing hypertrophic chondrocytes. None was detectable in adjacent regions or in the epiphysis. Slight fluorescence was also present within the surrounding sleeve of periosteal bone. Consistent with these results, the antibody did not react with the cartilages of the trachea and sclera, which do not undergo hypertrophy during the stages examined. It did, however, lightly react with the parietal bones of the head, which form by intramembranous ossification. These results are consistent with our earlier biochemical analyses, which showed type X collagen to be a product of that subpopulation of chondrocytes that have undergone hypertrophy. In addition, either it or an immunologically cross-reactive molecule is also present in bone, and exhibits a diminished fluorescent intensity as compared with hypertrophic cartilage.