Biomedical subjects

M Kagayama

Publications and source records attributed to M Kagayama.

At least 55 records · Page 3Linked to original sources

PubMed1996

An improved mounting method for observation of thick specimens using confocal microscopy.

When applying aqueous media to mount thick specimens, air bubbles occur frequently between coverslips and slide glasses. This results in an increase of background signal by confocal microscopy. Using Spurr's resin as a mounting medium, we could observe thick specimens with oil immersion objective lens without the use of coverslips, then avoid air bubbles near the specimen. Fading of the fluorescence appeared to be less in specimens mounted by the resin than in specimens mounted by aqueous media. Further, the new method increased depth of field so that more planes of specimens could be analyzed.

Animals↗

PubMed1995

Effects of lateral pterygoid muscle hyperactivity on differentiation of mandibular condyles in rats.

BACKGROUND: The effects of biomechanical stress on the growth and development of the mandibular condyle have been studied by many investigators. However, the role of the lateral pterygoid muscle in this development is not clear. METHODS: Hyperfunction of the lateral pterygoid muscles of male 3-week-old Sprague-Dawley rats was induced by electrical stimulation, and the responses of the mandibular condyles were compared to control tissues by a double-fluorescent staining technique using polyclonal antibodies against type I and type II collagen. Electrical stimulation consisted of repeated application (5 seconds on/5 seconds off) of a 5 Hz current for up to 7 days. RESULTS: In the first 2 days, cartilaginous tissues rich in type II collagen disappeared in the anterior and posterior areas, which were loaded by tensional force due to direct and indirect attachment of the lateral pterygoid muscles. Tissues in these areas were replaced by intramembranous bone that was reactive for type I collagen at 7 days. By the end of the experiment, the trabecula of the condyle was remodeled more perpendicularly, thus resisting the compressive force due to hyperfunction of the lateral pterygoid muscles. CONCLUSIONS: These results suggest that the activity of the lateral pterygoid muscle might play a significant role in the differentiation of progenitor cells and in the maturation and calcification of chondrocytes in mandibular condyles.

Animals↗

PubMed1995

Subperiosteal implantation of octacalcium phosphate (OCP) stimulates both chondrogenesis and osteogenesis in the tibia, but only osteogenesis in the parietal bone of a rat.

BACKGROUND: It is not known whether long bones and calvaria have distinct biological characteristics. Octacalcium phosphate (OCP), which is a precursor phase of the hydroxyapatite, has been reported to stimulate bone formation if implanted in the subperiosteal region of mouse calvaria. The present study was designed to investigate how the long bone and the calvarium respond to OCP implantation and to compare their biological characteristics. METHODS: The synthetic OCP was implanted into the subperiosteal region of rat tibiae and parietal bones being mixed with bovine type I collagen treated by pepsin (Atelocollagen). The biological response was examined histologically and immunohistochemically for collagen matrix phenotypes of types I and II to identify bone and cartilage formation. RESULTS: Both chondrogenesis and osteogenesis were initiated in the tibia 1 week after implantation of OCP and most of the cartilage was replaced by bone at week 2. However, the parietal bone did not show osteogenesis responding to OCP implantation until week 3, and no cartilage formation was associated with the osteogenesis. CONCLUSIONS: The present study demonstrated the distinct characteristics of biological response to OCP implantation between the long bone and the calvarium in terms of whether or not cartilage formation is involved in the stimulated osteogenesis by OCP, and in terms of timing of the stimulated chondrogenesis and/or osteogenesis, i.e., the parietal bone takes more time to respond to OCP implantation than the tibia.

Animals↗

PubMed1995

Immunohistochemical localization of connexin 43 in the developing tooth germ of rat.

Distribution of gap junction protein in maxillary tooth germs of 1-day-old rats was examined by immunohistochemistry, using an affinity-purified antibody specific to residues 360-376 of rat connexin (CX) 43. In 1-day-old rats, the maxillary second molar formed the shape of the cusp, but neither dentine nor enamel was formed between the cells of the dental papilla and the inner enamel epithelium. In the tooth germ, CX 43 was expressed in the cells of the stratum intermedium and the inner enamel epithelium. Labelling in the stratum intermedium was extensive and showed an increasing gradient from peripheral to cuspal regions. CX 43 detected in the inner enamel epithelium was at cell surfaces facing the interface between the dental papilla and the inner enamel epithelium. The cells of the dental papilla and the inner enamel epithelium began differentiation as odontoblasts and secretory ameloblasts respectively, in the cusps of the first molars, where predentine and dentine were formed but enamel matrix was not secreted. CX 43 was present in the stratum intermedium, inner enamel epithelium, preodontoblasts, odontoblasts and subodontoblasts. The incisors showed the most advanced stage of development, where the enamel matrix and calcified dentine were formed in the labial part of the teeth. The CX 43 epitope was seen in the stratum intermedium, inner enamel epithelium, preameloblasts, preodontoblasts, odontoblasts, and subodontoblasts. Immunolabelling was more extensive in the stratum intermedium and subodontoblasts than in preameloblasts, preodontoblasts, and odontoblasts. The immunolabelling in preameloblasts and predontoblasts was accumulated at cell surfaces facing the predentine.(ABSTRACT TRUNCATED AT 250 WORDS)

Ameloblasts↗

PubMed1995

Time and position-specific expression of glycosaminoglycans in rat molar cementum related to physiological tooth movement.

The role of glycosaminoglycans (GAGs) and proteoglycans during cementogenesis is not known. In this study, we have analysed the temporal and spacial expression of GAGs in the cellular cementum of 10-30 weeks old rats, immunohistochemically using monoclonal antibodies 2B6 and 3B3, specific for chondroitin 4-sulfate/dermatan sulfate and chondroitin 6-sulfate, respectively. Both 2B6- and 3B3-epitopes were expressed at similar position and time in the rat cellular cementum. Two types of cellular cementum were identified; GAG-positive and GAG-negative cementum. The former corresponded to the lightly stained and the latter to the darkly stained cementum in sections stained with haematoxylin and eosin. The GAG-positive cementum was seen at the distal side of dentine surface and appeared most thick at, middle of the apical half roots, whereas the other parts of the cementum were the GAG-negative. Distribution of GAG-positive cementum showed changes with age of animals. In 10-15 week old rats, the GAG-positive cementum occupied most of the cementum layer, covering a thin layer of the GAG-negative cementum. The cellular cementum of 20-30 week old rats consisted of three layers; GAG-negative, GAG-positive and GAG-negative cementum from dentine to cementum surface, reducing the GAG-positive area. Because our previous study has demonstrated that the lightly stained cementum is uncalcified, the present result suggests a correlation between calcification and contents of GAGs in the cellular cementum. Further, time- and position-specific expression of GAGs indicates their relation to the physiological tooth movement, which has been known in the rat molars.

Age Factors↗

PubMed1995

On the developing periodontal ligament of rats, using a new specimen preparation method for SEM in comparison with histochemistry.

To visualize the cells and fibers of the developing periodontal ligament (PDL) by scanning electron microscope (SEM), we examined a new tissue preparation method including decalcification, sectioning by cryomicrotome, and chemical treatment for removal of cells or collagen fibers. The advantages of this method were as follows: (1) it was possible to expose the restricted area, (2) it caused no damage by heat or various embedding agents such as paraffin or resin, and (3) it was possible to make comparisons the SEM observation with histochemical or immunohistochemical observation using the neighboring sections. We could classify the development of PDL into three stages by alkaline phosphatase (ALPase) activity and observe each stage by this method. Stage I was the zone of dental follicle proper that showed negative ALPase activity. Stage II was the tissue surrounding the disrupted Hertwig's epithelial root sheath (HERS) which evinced intense ALPase activity, and stage III was the further advanced zone of differentiation that displayed moderate ALPase activity. Using this new method for SEM, cells with many processes and thin fibers were seen irregularly at stage II. On the other hand, at stage III, fibers were seen as interconnecting meshworks of thick bundles and cells that showed regularly arranged rows running obliquely to the surface of the root and alveolar bone. At the transition between stages II and III, the thickness and orientation of fibers changed abruptly.

Animals↗

PubMed1994

Localization of uncalcified cementum in adult rat molar roots and its relation to physiological tooth movement.

The study was designed to elucidate the effect of physiological tooth movement on cellular cementum, using the upper molar roots of 10-week-old rats. Paraffin sections stained with haematoxylin and eosin displayed two types of cellular cementum, lightly and darkly staining. The lightly stained was present on the distal half of all molar roots except the mesial root of the first molar. The alveolar bone facing the lightly stained cementum showed resorption lacunae and multinucleated osteoclasts, while the opposite bone surface was lined with osteoblasts. In contact microradiographs of undemineralized ground sections, the X-ray density of the lightly stained cementum was similar to that of the periodontal ligament and pulp, while the X-ray density of the darkly stained cementum was similar to that of alveolar bone. Tetracycline labelling lines were seen at the interface between the two types of cellular cementum as well as on surfaces of bone and cementum located mesially to the root dentine. The results suggest that the mechanical stress of tooth movement differently affects the alveolar bone and cellular cementum; the bone is resorbed whereas the cementum resists resorption and its calcification is inhibited under the compressive force of tooth movement.

Absorptiometry, Photon↗

PubMed1993

BMPs induce direct bone formation in ectopic sites independent of the endochondral ossification in vivo.

Bone formation in vivo occurs via two major processes, one of which depends on pre-existing cartilage, and the other does not. Bone morphogenetic proteins (BMPs) have been suggested to induce cartilage formation from non-skeletogenic mesenchymal cell population, which results in osteogenesis through the endochondral sequence. In the present study we examined if BMPs could cause direct bone formation independent of pre-existing cartilage using bovine fibrous collagen membrane (FCM) as a carrier for BMPs. Bovine metatarsal bone was extracted in 4 M guanidine HC1 and BMPs were partially purified through the hydroxyapatite chromatography and the Heparin-Sepharose CL6B chromatography. The carrier was loaded with BMPs and then implanted in Wistar rats subcutaneously. The implants were fixed together with surrounding tissue every week after implantation and processed for von Kossa stain, immunohistochemistry, and electron microscopy. The phenotypes of bone and cartilage were identified histologically and immunohistochemically using antibodies against type I and type II collagen. Cartilage and bone were independently induced by 2 weeks. The bone formed directly on the collagen substrate of FCM without pre-existing cartilage. Calcification occurred in the carrier as well as the cartilage and bone matrix. The present study suggests that the BMPs induce osteogenesis in vivo independent of the endochondral sequence.

Animals↗

PubMed1993

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.

Alkaline Phosphatase↗

PubMed1993

Presence of chondroid bone on rat mandibular condylar cartilage. An immunohistochemical study.

Immunohistochemical techniques were used to examine the locations of type I and type II collagens in the the most anterior and the posterosuperior regions of the mandibular condylar cartilages of young and adult rats. Large ovoid and polygonal cells, which were morphologically different from any of the neighboring cells, e.g., mature or hypertrophied chondrocytes, osteoblasts, or fibroblasts, were observed at the most anterior margin of the young and adult condylar cartilages. In the extracellular matrix (ECM) of this area, an eosinophilic staining pattern similar to that in bone matrix was observed, while the peripheral ECM showed basophilic staining and very weak reactivity to Alcian blue. Immunohistochemical examination showed that the ECM was stained heavily and diffusely for type I collagen, while a staining for type II collagen was faint and limited to the peripheral ECM. Two different staining patterns for type II collagen could be recognized in the ECM: one pattern revealed a very faint and diffuse reaction while the other showed a wak rim-like reaction. These staining patterns were markedly different from those in the cartilaginous cell layer in the posterosuperior area of the condylar secondary cartilage, which showed faint staining for type I collagen and a much more intense staining for type II collagen. These observations reveal the presence of chondroid bone, a tissue intermediate between bone and cartilage tissues, in the mandibular condylar cartilage, and suggest the possibility of osteogenic transdifferentiation of mature chondrocytes.

Animals↗

PubMed1993

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.

Animals↗

PubMed1993

Lectin binding in bone matrix of adult rats with special reference to cement lines.

The present study was designed to localize glycoconjugates recognized by four lectins (LFA, MPA, WGA, and ConA) in adult rat bone, histochemically and biochemically. LFA, a lectin recognizing sialic acid, stained intensely the cement lines of calvariae and the interface between cartilage and bone of mandibular condyles, which were also stained with WGA, and intermittently stained with ConA. The irregular bone surface of calvariae displayed intense positive reaction with LFA and WGA, while the osteoid of mandibular condyles and the smooth bone surface of calvariae were intensely stained with WGA and ConA, but weakly stained with LFA and MPA. MPA recognized osteoclasts. At the superficial layer of mandibular condyles, the bone matrix appeared positive as granules with LFA and as patches with MPA, WGA and ConA. Lectin blotting analysis of bone extracts revealed two LFA positive bands at molecular weights of 18 kDa and 45 kDa. The former was positive with WGA, ConA, and MPA, while the latter was positive with MPA. The result suggests that the LFA-binding glycoconjugates, possibly 18 kDa and/or 45 kDa sialoglycoconjugates, may contribute to cementing mechanism of cement lines and/or to the bone formation in remodeling process as well as in intramembranous and intracartilaginous osteogenesis.

Animals↗

PubMed1992

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

Previous 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 immuno- 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.

Animals↗

PubMed1992

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.

Animals↗

PubMed1992

Distribution of glycoconjugates localized by peanut and Maclura pomifera agglutinins during mouse molar root development.

Glycolipids, glycoproteins, glycosaminoglycans and sialoglycoproteins have all been implicated in a number of developmentally significant processes related to complex interactions between cell surfaces and the extracellular matrix. The present study was designed to localize glycoconjugates recognized by peanut agglutinin (PNA) and Maclura pomifera (MPA) lectins during mouse molar root development. Postnatal ICR mice at 10, 15, 21, 28 and 42 days were used. Lower jaws were dissected, fixed in 4% paraformaldehyde, decalcified in 5% EDTA and embedded in paraffin. Serial sections were made and stained with FITC-conjugated PNA or MPA. beta-Lactose was used as an inhibitory sugar for PNA, and alpha-D-melibiose for MPA. PNA specifically stained Hertwig's epithelial root sheath (HERS), whereas MPA stained a number of tissues. The outermost layer of root dentin, forming cellular cementum, alveolar bone and HERS showed positive reactions with MPA. Glycoconjugates localized by the lectins may be functionally related to molecules which contribute to root formation and cemento-genesis.

Animals↗

PubMed1991

Immunohistochemical and immunochemical detection of a similar epitope in enamel proteins and monocyte-macrophage protein recognized by mouse monoclonal antibody MOMA-2.

These studies were made as a first step in elucidating unknown functions of enamel proteins in odontogenesis. The cytoplasm of ameloblasts and the proteins in dentine matrix before mineralization were stained with this monoclonal antibody. SDS-PAGE and Coomassie blue staining of proteins extracted from enamel showed several protein bands. Immunoblotting revealed that proteins recognized by this antibody were situated between 20-30 kDa. These results indicate that enamel proteins, presumably amelogenins, have an epitope resembling monocyte-macrophage protein. The findings suggest that epithelial-mesenchymal interaction in odontogenesis and preosteoblast-preosteoclast interaction in osteogenesis may be similar.

Ameloblasts↗

PubMed1991

Bone formation on synthetic precursors of hydroxyapatite.

The aim of this study was to investigate the reaction of skeletal tissue to various synthetic calcium phosphate (Ca-P) compounds in vivo. Five synthetic Ca-P compounds were implanted into the subperiosteal area of the calvaria of 7-week-old BALB/c mice for one to 15 weeks. Synthetic compounds were dicalcium phosphate (DCP), octacalcium phosphate (OCP), amorphous calcium phosphate (ACP), Ca-deficient hydroxyapatite and hydroxyapatile (HA). Implanted DCP, OCP and ACP were found to be converted to apatitic phase by x-ray microdiffraction analysis using undecalcified specimens. Structure of bone was found out on all of Ca-P compounds eventually at late stage under the light microscope, but the rate of bone formation calculated from a number of experiments varied on respective synthetic Ca-P compound. It was high as 80% for DCP, OCP and ACP, but was low as 5.6% for Ca-deficient HA, and no reaction was found for HA at the stage of 3 weeks. Fine filaments and granular materials in the newly formed bone matrix were detected at 7 days around the remnants of OCP particles which already converted to apatitic phase by ultrastructural study of decalcified specimens. These structures were very similar to the components of bone nodules seen in intramembranous osteogenesis. It is postulated that the precursors of HA have an important role in intramembranous osteogenesis.

Animals↗

PubMed1990

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.

Animals↗