Yasuyuki Sasano

Expression of MMP-8 and MMP-13 Genes in the Periodontal Ligament during Tooth Movement in Rats

Periodontal ligament tissue is remodeled on both the tension and compression sides of moving teeth during orthodontic tooth movement. The present study was designed to clarify the hypothesis that the expression of MMP-8 and MMP-13 mRNA is promoted during the remodeling of periodontal ligament tissue in orthodontic tooth movement. We used the in situ hybridization method and semi-quantitative reverse-transcription/polymerase chain-reaction analysis to elucidate the gene expression of MMP-8 and MMP-13 mRNA. Expression of MMP-8 and MMP-13 mRNA transiently increased on both the compression and tension sides during active tooth movement in vivo. The gene expression of MMP-8 and MMP-13 was induced by tension, while compression indirectly promoted the gene expression of MMP-8 and MMP-13 through soluble factors in vitro. Thus, we concluded that the expression of MMP-8 and MMP-13 is differentially regulated by tension and compression, and plays an important role in the remodeling of the periodontal ligament.

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.

Expression of versican and ADAMTS1, 4, and 5 during bone development in the rat mandible and hind limb.

Extracellular matrix (ECM) remodeling is achieved by both production and degradation of ECM molecules during bone development. ADAMTS (a disintegrin and metalloprotease with thrombospondin type 1 motifs) constitutes a family of extracellular proteases which are implicated in cleaving the protein versican. The present study was designed to investigate the expression of versican and ADAMTS1, 4, and 5 mRNA during bone development in rat mandibles and hind limbs by RT-PCR and in situ hybridization. Versican was localized by immunohistochemistry. The process of bone development from day 14 postcoitum through week 6 postnatum was divided into the beginning of osteogenesis, woven bone, and lamellar bone stages. Versican protein was abundant in the woven bone matrix, but decreased in the lamellar bone matrix. Versican mRNA was prominent in some osteoblasts with corresponding localization of the cognate protein. The temporal and spatial mRNA expression pattern of ADAMTS1, 4, and 5 was comparable to that of versican. These results suggest that woven bone rich in versican alters into lamellar bone containing little versican during bone development in both mandibles and hind limbs, where some osteoblasts may be involved in production as well as degradation of versican by secreting ADAMTS1, 4, and 5.

Expression of genes for gelatinases and tissue inhibitors of metalloproteinases in periodontal tissues during orthodontic tooth movement.

Orthodontic tooth movement progresses by a combination of periodontal ligament (PDL) tissue and alveolar bone remodeling processes. Besides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a co-ordinated fashion to regulate the remodeling of periodontal tissues. We hypothesized that the expression levels of the genes for MMP-2, MMP-9, and TIMPs 1–3 are increased transiently in the periodontal tissue during orthodontic tooth movement. To test this hypothesis, we employed an animal model of tooth movement using rats, as well as in situ hybridization to analyze the expression levels of Mmp-2, Mmp-9, and Timps1-3. The expression levels of these genes increased transiently in cells of periodontal tissues, which include cementoblasts, fibroblasts, osteoblasts, and osteoclasts, at the compression side of the moving teeth. The transient increases in gene expression at the tension side were mainly limited to osteoblasts and cementoblasts. In conclusion, the expression levels of Mmp-2, Mmp-9, and Timps1-3 increase transiently during orthodontic tooth movement at both the tension and compression sides. The expression of these genes is regulated differentially in the periodontal tissue of the tension side and compression side. This altered pattern of gene expression may determine the rate and extent of remodeling of the collagenous ECM in periodontal tissues during orthodontic tooth movement.

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

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.

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.

Initiation of alveolar ridge augmentation in the rat mandible by subperiosteal implantation of octacalcium phosphate

The study was designed to investigate the process of bone formation caused by implantation of octacalcium phosphate as well as stability of the bone formed at the alveolar ridge. Synthetic octacalcium phosphate was implanted into a subperiosteal pocket in the rat mandible. Bone formation at the alveolar ridge was examined radiographically and histologically between 1 and 48 week(s) after implantation. Radiopacity of the octacalcium phosphate implant became obvious in week 2. Osteogenesis was initiated from the bone surface near the implantation site and multinucleated giant cells appeared on the implanted octacalcium phosphate in week 1. More apposition of new bone was observed on the implanted octacalcium phosphate in week 2 or later. Some implants were directly enclosed by newly formed bone and no cellular component was seen between the implant and the bone matrix. Many octacalcium phosphate implants were enclosed by bone, whereas the augmented ridge was not seen radiographically in week 24 or later. If its persistence can be improved, octacalcium phosphate could be used to augment atrophic alveolar ridges.

An immunohistochemical study of the localization of biglycan, decorin and large chondroitin-sulphate proteoglycan in adult rat temporomandibular joint disc.

To analyse regional variations in extracellular matrix components of adult rat temporomandibular joint discs, immunohistochemical techniques were used to examine the localization of two small dermatan-sulphate proteoglycans, biglycan and decorin, and a large chondroitin-sulphate proteoglycan. Staining for biglycan was intense in the posterior band, although it had a rather weak and even distribution throughout the disc. In contrast, staining for decorin was faint in the intermediate zone and the central part of the posterior band, moderate in the anterior and posterior attachments and most intense in the junction between the anterior band and attachment. The upper surface of the disc stained more intensely than the lower. Similarly, there was intense staining for large chondroitin-sulphate proteoglycan in the peripheral band, but both the anterior and the temporal parts of the posterior attachments were faintly stained. These results demonstrate marked regional differences in the expression of biglycan, decorin and large chondroitin-sulphate proteoglycan in the temporomandibular joint discs of adult rats. These variations probably reflect the different biomechanical environments caused by the complicated articulatory functions of the temporomandibular joint.

Implantation of octacalcium phosphate combined with transforming growth factor-β1 enhances bone repair as well as resorption of the implant in rat skull defects

In our previous study, we reported that synthetic octacalcium phosphate (OCP) enhances bone repair if implanted in rat skull defects. We hypothesized that OCP can be used as an effective carrier for transforming growth factor-beta1 (TGF-beta1) to promote bone repair. We designed the present study to investigate histomorphometrically whether combination with recombinant human TGF-beta1 could promote bone repair caused by OCP per se (Control/OCP). A full-thickness standardized trephine defect was made in the rat parietal bone and OCP combined with recombinant human TGF-beta1 (TGF-beta1/OCP) or Control/OCP was implanted into the defect. Four rats from each group were fixed at 2, 4, and 8 weeks after implantation. Histomorphometrical analysis of the percentage of newly formed bone (n-Bone %) and remaining implants (r-Imp %) in the defect was performed. The statistical analysis showed the n-Bone % of TGF-beta1/OCP was significantly higher than that of the Control/OCP in week 4, whereas the r-Imp % of TGF-beta1/OCP was significantly lower than that of the Control/OCP. The present study demonstrated that OCP can be used as an effective carrier for TGF-beta1 and their combination enhances bone repair as well as resorption of the carrier OCP in the early stage of bone formation.