Itaru Mizoguchi

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

SummaryImmunohistochemical 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 regional differences in the distribution of type I and type II collagens in rat mandibular condylar cartilage

The mammalian temporomandibular joint is a highly specialized diarthrodial joint under multidirectional compressive and tensile forces. In such a complicated biomechanical environment, the phenotypic expression of extracellular matrix may vary in different regions of the mandibular condylar cartilage. To test this hypothesis, immunohistochemical techniques were used to examine the localization of type I and type II collagens in various anterioposterior regions of the condylar cartilage of 4-week-old rats. In the posterosuperior region, which is mainly subjected to compressive forces, a strong reaction for type II collagen was observed in the cartilaginous layer (maturative and hypertrophic cell layers), and a rather weak reaction was observed for type I collagen in the precartilaginous and cartilaginous layers, compared with the reactions in other peripheral regions. Proceeding anteriorly, staining for type I collagen increased, while that for type II collagen decreased. In posteroinferior cartilage, which is subjected mainly to tensile forces because of its direct attachment to the retrodiscal pad, staining for type I collagen was strong, and that for type II collagen was faint in the cartilaginous layer. These results demonstrate that marked regional differences exist in the phenotypic expression of two major collagen components in mandibular condylar cartilage, which may reflect the local functional environment and cellular response.

Effects of expansive force on the differentiation of midpalatal suture cartilage in rats

In an attempt to clarify the effects of biomechanical tensional force on chondrogenic and osteogenic differentiation of secondary cartilage, the midpalatal sutures of 4-week-old Wistar male rats were expanded by orthodontic wires which applied 20 g force for 4, 7, 10, and 14 days. The differentiation pathways in the midpalatal suture cartilage were examined by immunohistochemistry for osteocalcin, type I and type II collagen, and von Kossa histochemistry. Although the midpalatal sutures of the control animals consisted mainly of two separate secondary cartilages with mesenchyme-like cells at their midlines, type I collagen-rich fibrous tissue began to appear at day 4 and increased at the midline of the cartilage with days of experiment. At the end of the experiment, type I collagen-rich and calcified bone matrix appeared at the boundary between the precartilaginous and the cartilaginous cell layers. Most of the cartilaginous tissues were separated from each other and the midpalatal suture was replaced by osteocalcin-positive intramembranous bone and fibrous sutural tissue. These results strongly suggest that tensional force changed the phenotypic expression of collagenous components in secondary cartilage, which may reflect the differentiation pathway of osteochondro progenitor cells.

Chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of rat tibial articular cartilage.

The present study was designed to investigate whether or not chondrocytes in articular cartilage express type I collagen in vivo under physiological conditions. Expressions of the gene and the phenotype of type I collagen were examined in rat tibial articular cartilage in the knee joint during development. Knee joints of Wistar rats at 1, 5, and 11 weeks postnatal were fixed in 4% paraformaldehyde with or without 0.5% glutaraldehyde and decalcified in 10% EDTA. After the specimens were embedded in paraffin and serial sections made, adjacent sections were processed for immunohistochemistry and in situ hybridization for type I collagen. The epiphysis of the tibia was composed of cartilage in week-1 rats. Formation of articular cartilage was in progress in week 5 as endochondral ossification proceeded and was completed in week 11. Anti-type I collagen antibody stained only the superficial area of the epiphysis in week 1, but the immunoreactivity was expanded into the deeper region of the articular cartilage with development in weeks 5 and 11. Hybridization signals for pro-alpha 1 (I) collagen were seen in some of chondrocytes in the epiphysis of the week-1 tibia. The most intense signals were identified in chondrocytes in week 5 and the signals appeared weaker in week 11. The present study demonstrated that chondrocytes synthesize type I collagen and accumulate the protein in the matrix during development of the articular cartilage.

Localization of types I, II and X collagen and osteocalcin in intramembranous, endochondral and chondroid bone of rats

Abstractu2002Chondroid bone is a unique calcified tissue intermediate between bone and cartilage. To clarify its characteristics, we examined the distributions of the ECMs associated with chondrogenic differentiation and matrix calcification in the chondroid bone of the rat glenoid fossa, and compared them to those in two typical bone tissues, alveolar bone of the maxilla (intramembranous bone) and the growth plate of long bone (endochrondral bone), using immunofluorescence techniques. Morphologically, the glenoid fossa consisted of the fibrous, progenitor and cartilaginous cell layers and the cartilaginous cell layer was further divided into the superficial non-hypertrophic layers (secondary cartilage) and the deep hypertrophic cell layers (chondroid bone). The co-distribution of type I and type II collagens was observed in secondary cartilage and chondroid bone, whereas type X collagen was restricted to the pericellular matrix of hypertrophied cells (chondroid bone). Osteocalcin, which was absent from the calcified cartilage of endochondral bone formation, was also present in the ECM of the chondroid bone, but not in cells. These results demonstrate that chondroid bone of rats, which is adjacent to secondary-type cartilage in the glenoid fossa, has phenotypic expressions associated with both hypertrophied chondrocytes and osteocytes.

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.

Distribution of type I collagen, type II collagen and PNA binding glycoconjugates during chondrogenesis of three distinct embryonic cartilages

SummaryPrevious studies of chondrogenesis have been focused on limb bud cartilage, whereas little is known about chondrogenic processes of other cartilages with different developmental fates. We hypothesize that cartilages with various developmental fates might show identical characteristics of chondrogenesis. The chondrogenic processes in the nasal septum, the mandible, and the limb bud of the mouse were examined by means of PNA-binding glycoconjugate, and types I and II collagen expression. Swiss-Webster mouse embryos of 11 days (E11) to 14 days (E14) gestation were fixed and processed for imniuno- and lectin histochemistry. The blastema of mesenchymal cell aggregates stained positively with anti-type I collagen, but very weakly with anti-type II collagen in all three models at E12, whereas PNA bound to the blastema in the limb bud but not in nasal septum or mandible. Types I and II collagens coexisted in cartilages at E13. Type II collagen was predominant in E14; type I collagen was confined to the peripheral region. The synchronized transitional expression of the collagen phenotypes in all three embryonic cartilages may be systemically regulated. The presence or absence of the PNA-binding glycoconjugates may be involved in characterizing the nature of the cartilages.

BMPs induce endochondral ossification in rats when implanted ectopically within a carrier made of fibrous glass membrane

Bone morphogenetic proteins (BMPs) replicate the process of embryonic bone formation when implanted in ectopic sites. Our previous studies have indicated that BMPs can induce intramembranous ossification, i.e., direct bone formation without preexisting cartilage when implanted in rats subcutaneously by using the fibrous collagen membrane (FCM) as a carrier for implanting BMPs (Sasano et al. 1993. Anat. Rec., 236:373–380). The present study was designed to investigate how the physicochemical property of the carrier material influences the process of bone formation induced by BMPs, using a carrier made of fibrous glass membrane (FGM).

The process of calcification during development of the rat tracheal cartilage characterized by distribution of alkaline phosphatase activity and immunolocalization of types I and II collagens and glycosaminoglycans of proteoglycans

The rat tracheal cartilage was shown to calcify during development. The process of calcification was characterized in terms of distribution of alkaline phosphatase (ALP) activity and alterations to immunolocalization of types I and II collagens and glycosaminoglycans of proteoglycans during the development of the tracheal cartilage, in comparison with calcification of the epiphyseal growth plate cartilage. ALP activity was not identified in the tracheal cartilage in the course of calcification, which therefore differed from that in the growth plate. The tracheal cartilage matrix was not resorbed or invaded by type I collagen during calcification. This suggests that no osteogenesis is involved in calcification of the cartilage. Immunoreactivity for type II collagen became weaker in the central region of the tracheal cartilage during development. No net loss of proteoglycans was identified with Alcian blue staining after calcification of the tracheal cartilage. Immunoreactivity for chondroitin 4-sulphate increased in the calcified tracheal cartilage, while reactivity for chondroitin 6-sulphate was weaker in the calcified area than in the surrounding uncalcified region of the tracheal cartilage. The alteration of the extracellular matrices during development may be involved in the calcification of the rat tracheal cartilage.

Epithelial rests colocalize with cementoblasts forming acellular cementum but not with cementoblasts forming cellular cementum.

Epithelial rests of Malassez and cementoblasts were examined in the rat molars during the early stages of root formation using an antilaminin antibody and/or peanut agglutinin (PNA), and an antiosteocalcin (OC) antibody, respectively. The roots of the first molars were used for study. The antilaminin antibody stained the basement membrane surrounding the epithelial root sheath and epithelial rests. The basement membrane of the epithelial root sheath was continuous, but that of the epithelial rests was discontinuous. The cells of epithelial rests and epithelial root sheath were positive for PNA. The structural characteristics of the epithelial rests were seen in the sections stained doubly with PNA and the antilaminin antibody. The cells of epithelial rests were fibroblast-like and formed a fine mesh in 2-week-old rats. In 3-week-old rats, the epithelial rests were also present at the coronal half of root surface, showing typical cell cords, but were not present at the apical part of the root surface where the cellular cementum covered the root dentin. At the root apex of 3-week-old rats, the cells of epithelial rests forming fine meshes were seen near the epithelial root sheath. The anti-OC antibody stained cementoblasts lining acellular and cellular cementum. The sections doubly stained with the anti-OC and the antilaminin antibodies or PNA further revealed the close relation between epithelial rests and cementoblasts. The OC-positive cells lining acellular cementum or dentin were localized very close to the epithelial rests. In contrast, the OC-positive cells lining cellular cementum did not show close association with the epithelial cells, except the cells located most apically where the basement membrane of the epithelial root sheath is disrupted and the initial cellular cementum begins to be formed. The present results suggest that the epithelial rests and/or the discontinuous basement membrane of them may have a role for the acellular cementum formation at least in the early stage of root formation.