Manabu Kagayama

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.

Implantation of Octacalcium Phosphate (OCP) in Rat Skull Defects Enhances Bone Repair

Synthetic octacalcium phosphate (OCP) enhances bone formation if implanted into the subperiosteal region of murine bone. Such implanted OCP may be resorbed and replaced by bone with time. We hypothesized that OCP could be used as an effective bone substitute. To test this hypothesis, we designed the present study to investigate if bone repair in a rat skull defect is enhanced by the implantation of OCP. Rats were divided into two groups: OCPtreated animals and untreated controls. Six rats from each group were fixed at 4, 12, and 24 weeks after implantation. A full-thickness standardized trephine defect was made in the parietal bone, and synthetic OCP was implanted into the defect. After being examined radiographically, the specimens were decalcified and processed for histology. OCP implantation significantly promoted bone repair compared with the controls. A statistical analysis showed an increase in the area of radiopacity within the skull defect between week 4 and week 12. Histologically, bone was formed on the implanted OCP and along the defect margin at week 4. At week 12, the implanted OCP was surrounded by newly formed bone. At week 24, the defect was almost completely filled with bone. In the control, bone formation was observed only along the defect margin. The present results demonstrate that OCP could be used as an effective bone substitute.

Gene Expression of MMP8 and MMP13 During Embryonic Development of Bone and Cartilage in the Rat Mandible and Hind Limb

Matrix metalloproteinases (MMPs) 8 and 13 comprise the collagenase subfamily in rats and mice, and only MMP13 has been implicated in degradation of the collagenous matrices during development of bone and cartilage. On the hypothesis that MMP8 is also involved in bone and cartilage development, the present study was designed to investigate gene expression of MMP8 in rat embryonic mandibles and hind limbs. Expression of MMP8 was examined with in situ hybridization and RT-PCR and was compared with that of MMP13. Osteoblastic and chondrocytic cells expressing collagenous matrix molecules were identified using in situ hybridization for collagen Types I and II. The results demonstrated that MMP8 is expressed by osteoblastic progenitors, differentiated osteoblasts, osteocytes, and chondrocytes in the growth plate for the first time. Furthermore, the expression of MMP8 is much broader than that of MMP13, for which expression is confined to differentiated phenotypes of osteoblastic and chondrocytic lineage.

Maclura pomifera agglutinin-binding glycoconjugates on converted apatite from synthetic octacalcium phosphate implanted into subperiosteal region of mouse calvaria

We have previously shown that the mineral in granules of synthetic octacalcium phosphate (OCP) implanted subperiosteally in mouse calvariae was converted to apatitic crystals and that the OCP implantation stimulated bone formation. The matrix components accumulated on the converted apatite were very similar to those of bone nodules (starting locus of calcification) in intramembranous osteogenesis. In the present study, the nature of the matrices accumulated on OCP implants in calvariae was compared with that of the matrices accumulated in abdominal subcutaneous implants. The comparison was facilitated by the use of Maclura pomifera agglutinin (MPA) lectin which is known to have a high affinity for the primary intramembranous bone matrix. Micro-beam x-ray diffraction indicated conversion of the implanted OCP to apatitic crystals in situ, both in subperiosteal and subcutaneous sites, after 10 days. Additional bone formation was detected on the converted apatite after 13 days in subperiosteal implantation, whereas bone was not formed in the subcutaneous implantation. MPA reaction was strongly manifested after 10 days in matrices accumulated on the converted apatite in both subperiosteal and subcutaneous implantations. Biochemical data showed that intensely and weakly MPA-blotted molecules (53.0 and 152.6 kDa, respectively) were in all the mouse sera, in the guanidine HCl-EDTA extracts of mouse calvarial bone and in the extracts of the implanted OCP in both subperiosteal and subcutaneous sites. These findings indicated that the glycoconjugates accumulated on the converted apatite from OCP were similar to the glycoconjugates in the serum in terms of reactivity with MPA and molecular weights. Furthermore, the results suggest that MPA-binding glycoconjugates which had accumulated on the converted apatite may be a requisite for the differentiation of mesenchymal cells into osteoblasts in periosteum but not in subcutaneous sites.

Effect of stretching on gene expression of β1 integrin and focal adhesion kinase and on chondrogenesis through cell-extracellular matrix interactions

Differentiation of skeletal tissues, such as bone, ligament and cartilage, is regulated by complex interaction between genetic and epigenetic factors. In the present study, we attempted to elucidate the possible role of cell-extracellular matrix (ECM) adhesion on the inhibitory regulation in chondrogenesis responding to the tension force. The midpalatal suture cartilages in rats were expanded by orthopedic force. In situ hybridization for type I and II collagens, immunohistochemical analysis for fibronectin, alpha5 and beta1 integrins, paxillin, and vinculin, and cytochemical staining for actin were used to demonstrate the phenotypic change of chondrocytes. Immunohistochemical analysis for phosphorylation and nuclear translocation of extracellular signal-regulated kinase (ERK)-1/2 was performed. The role of the cell-ECM adhesion in the response of the chondroprogenitor cells to mechanical stress and the regulation of gene expression of focal adhesion kinase (FAK) and integrins were analyzed by using an in vitro system. A fibrous suture tissue replaced the midpalatal suture cartilage by the expansive force application for 14 days. The active osteoblasts that line the surface of bone matrix in the newly formed suture tissue strongly expressed the type I collagen gene, whereas they did not express the type II collagen gene. Although the numbers of precartilaginous cells expressing alpha5 and beta1 integrin increased, the immunoreactivity of alpha5 integrin in each cell was maintained at the same level throughout the experimental period. During the early response of midpalatal suture cartilage cells to expansive stimulation, formation of stress fibers, reorganization of focal adhesion contacts immunoreactive to a vinculin-specific antibody, and phosphorylation and nuclear translocation of ERK-1/2 were observed. In vitro experiments were in agreement with the results from the in vivo study, i.e. the inhibited expression of type II collagen and upregulation in integrin expression. The arginine-glycine-aspartic acid-containing peptide completely rescued chondrogenesis from tension-mediated inhibition. Thus, we conclude that stretching activates gene expression of beta1 integrin and FAK and inhibits chondrogenesis through cell-ECM interactions of chondroprogenitor cells.

Temporal and spatial gene expression of major bone extracellular matrix molecules during embryonic mandibular osteogenesis in rats

It is not known how gene expression of bone extracellular matrix molecules is controlled temporally and spatially, or how it is related with morphological differentiation of osteoblasts during embryonic osteogenesis in vivo. The present study was designed to examine gene expressions of type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin during mandibular osteogenesis using in situ hybridization. Wistar rat embryos 13–20 days post coitum were used. The condensation of mesenchymal cells was formed in 14-day rat embryonic mandibles and expressed genes of pro-α(I) collagen, osteonectin, bone sialoprotein and osteopontin. Cuboidal osteoblasts surrounding the uncalcified bone matrix were seen as early as in 15-day embryonic mandibles, while flat osteoblasts lining the surface of the calcified bone were seen from 16-day embryonic mandibles. Cuboidal osteoblasts expressed pro-α1(I) collagen, osteonectin and bone sialoprotein intensely but osteopontin very weakly. In contrast, flat osteoblasts expressed osteopontin very strongly. Osteocytes expressed the extracellular matrix molecules actively, in particular, osteopontin. The present study demonstrated the distinct gene expression pattern of type I collagen, osteonectin, bone sialoprotein, osteopontin and osteocalcin during embryonic mandibular osteogenesis in vivo.

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.

Expression of MMP-8 and MMP-13 mRNAs in Rat Periodontium during Tooth Eruption

The present study was designed to investigate mRNA expression of matrix metalloproteinase-8 (MMP-8) and MMP-13 in forming periodontium during tooth eruption in the rat. RT-PCR for the decalcified paraffin sections indicated expression of MMP-8 and MMP-13 in the periodontal tissues. In situ hydridization demonstrated expression of MMP-8 in osteoblasts, osteocytes, periodontal ligament cells, cementoblasts, and cementocytes along with collagen types I and III. In contrast, transcripts of MMP-13 were confined to a small population of osteoblasts and osteocytes in alveolar bone. The results suggested that MMP-8 may be involved in remodeling the periodontium during tooth eruption, and its expression may be coordinated with that of collagen types I and III, whereas the participation of MMP-13 may be rather limited.

Expression of major bone extracellular matrix proteins during embryonic osteogenesis in rat mandibles.

It is not known how bone proteins appear in the matrix before and after calcification during embryonic osteogenesis. The present study was designed to investigate expressions of the five major bone extracellular matrix proteins – i.e. type I collagen, osteonectin, osteopontin, bone sialoprotein and osteocalcin – during osteogenesis in rat embryonic mandibles immunohistochemically, and their involvement in calcification demonstrated by von Kossa staining. Wistar rat embryos 14 to 18 days post coitum were used. Osteogenesis was not seen in 14-day rat embryonic mandibles. Type I collagen was localized in the uncalcifed bone matrix in 15-day mandibles, where no other bone proteins showed immunoreactivity. Osteonectin, osteopontin, bone sialoprotein and osteocalcin appeared almost simultaneously in the calcified bone matrix of 16-day mandibles and accumulated continuously in 18-day mandibles. The present study suggested that type I collagen constitutes the basic framework of the bone matrix upon which the noncollagenous proteins are oriented to lead to calcification, whereas the noncollagenous proteins are deposited simultaneously by osteoblasts and are involved in calcification cooperatively.

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

Abstract Chondroid 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.