Biomedical subjects

Y Sasano

Publications and source records attributed to Y Sasano.

At least 55 records · Page 3Linked to original sources

PubMed1996

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.

Age Factors↗

PubMed1996

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.

Animals↗

PubMed1996

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.

Alveolar Process↗

PubMed1996

Localization of glycosaminoglycans in periodontal ligament during physiological and experimental tooth movement.

Localization of chondroitin sulphates in periodontal ligaments (PDL) of rat molar roots during physiological and experimental tooth movement were analysed immunohistochemically with the use of monoclonal antibodies, 3B3 and 2B6, specific to chondroitin 6-sulphate (CH-6S) and chondroitin 4-sulphate/dermatan sulfate (CH-4S/DS), respectively. The maxillary first molars of experimental animals were forced to move laterally with a 10 g weight by U-shaped wires for 3 and 7 d. In control animals, 3B3 epitope was seen in the PDL near to the bone surface facing the distal half of roots, which corresponded to the compressive side during physiological tooth movement. Immunoreactivity for 2B6 was weak or negative in the PDL. Both epitopes were present at osteoid, precementum, lacunae and canaliculli of osteocytes and cementocytes. In 3-d-treated animals, the early stage of hyalinization characterized with visible cells and fibres was observed in the PDL at the buccal side of the mesial root, which showed intense immunoreactivity for 3B3. Further 3B3 positive area seen in control animals changed its position from the distal to the buccal side of the PDL. Immunoreactivity for 2B6 did not change in the PDL of 3-d-treated animals. In 7-d-treated animals, the typical hyalinization characterized with no visible cells and fibres was seen in the PDL at the buccal sides of both mesial and disto-buccal roots, where both epitopes were present at the peripheral part of the tissue. Observation of serial sections suggested that the 3B3-positive area was present at the peripheral part of the 2B6-positive area. The present results suggest that the expression of CH-6S is related to the compressive force in non-hyalinized and hyalinized PDL, whereas that of CH-4S/DS is not influenced by the mechanical stress.

Alveolar Process↗

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

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↗

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

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

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↗

PubMed1992

Endogenous epidermal growth factor regulates the timing and pattern of embryonic mouse molar tooth morphogenesis.

The tooth organ provides a model for discrete patterns of morphogenesis over short periods of developmental time. Studies were designed to test the hypothesis that endogenous epidermal growth factor (EGF) functions to regulate multiple cusp molar tooth morphogenesis during embryonic mouse development. The relative levels of endogenous EGF and EGF receptor (EGFR) transcripts were determined in both enamel organ epithelia and dental ectomesenchyme by reverse transcription-polymerase chain reaction (RT-PCR) assays. EGF and EGFR were localized by immunohistochemistry; both antigenic determinants were demonstrated on the same odontogenic cells in cultured tooth explants. To examine EGF-mediated signal transduction, cap stage mouse molar tooth organs (E16) were cultured in serumless, chemically-defined medium as either (i) controls, or supplemented with (ii) tryphostin (an EGF receptor kinase inhibitor), (iii) tyrphostin plus exogenous EGF, and (iv) exogenous EGF. Antisense oligodeoxynucleotide (ODN) strategy was used to investigate the functions of endogenous EGF employing (i) non-treated control, (ii) sense ODN control, (iii) antisense ODN, (iv) exogenous EGF, (v) sense ODN with exogenous EGF, and (vi) antisense ODN with exogenous EGF. Tyrphostin inhibited DNA synthesis and produced a significant decrease in the volume of the explants. These effects were recovered by addition of exogenous EGF. Antisense ODN inhibition resulted in abnormal cusp formations, decreased DNA synthesis, total DNA, RNA and protein content, and decreased stellate reticulum and tooth explant volumes. The decreased tooth size was not uniform, the most pronounced effect was in the stellate reticulum. This pattern of changes was not seen when antisense ODN treatment was supplemented with exogenous EGF. These results suggest that during cap stage of odontogenesis endogenous EGF acts to stimulate DNA synthesis, which increases the cell number of specific phenotypes within the enamel organ epithelia, and thereby regulates molar tooth morphogenesis.

Animals↗

PubMed1992

Gene expression, signal transduction and tissue-specific biomineralization during mammalian tooth development.

Tooth development provides a paradigm for intrinsic molecular controls for cell- and extracellular matrix (ECM)-mediated biomineralization. The intent of this review is to evaluate the sequential timing and positional information prerequisite for tissue-specific biomineralization. Recent investigations suggest that 1,25-dihydroxyvitamin D3 functions to up-regulate VDR (vitamin D receptor) that in turn could induce structural gene products, including calcium-binding proteins and several ECM proteins (e.g., enamelins, amelogenins, dentine sialoglycoproteins (DSP) and dentine phosphoproteins (DPP)), resulting in dentine and enamel formation. Inhibition of regulatory gene products and/or their receptors likely results in hypoplastic and/or hypomineralized ECM as a direct consequence of down-regulated (1) transcription and/or translation of structural and regulatory genes, (2) posttranslational modifications, (3) and/or decreased calcium transport to the forming dentine and enamel matrices. Advances in serumless in vitro culture methodology; computer-assisted access to nucleic acid sequences for probes to define when, where, and how many specific regulatory and structural gene products are expressed; antisense oligodeoxynucleotides to inhibit specific translation; and microtechniques to analyze biomineralization all provide additional avenues to investigate tissue-specific biomineralization.

Animals↗

PubMed1990

Cartilage, bone and tooth induction during early embryonic mouse mandibular morphogenesis using serumless, chemically-defined medium.

Studies were designed to test the hypothesis that plasma- and serum-deprived embryonic cells and tissues in vitro are capable of producing growth regulating factors which augment cartilage, bone and tooth induction during mouse mandibular process development. Embryonic mouse first branchial arch-derived mandibular processes (E11-E12, Theiler stages 18-19) or cap stage molar tooth (M1) organs (E15-E16, Theiler stage 23) expressed morphogenesis, histogenesis and cytodifferentiation (e.g., Meckel's cartilage and mandibular bone) when cultured as explants in permissive serumless and chemically-defined BGJB medium for periods up to 31 days in vitro. Organ cultures of early mandibular process explants in serumless conditions showed DNA synthesis comparable to the time- and position-restricted patterns characteristic for control in vivo development. As a paradigm for embryonic cell expression of putative growth factors, sense and antisense oligodeoxynucleotide probes corresponding to amino acids 1070-1081 for preproEGF, and antibodies directed against amino acids 348-691 of preproEGF, were used to identify and localize mRNA transcripts and translation products. Our preliminary evidence suggests that odontogenic epithelial and ectomesenchyme cells produce EGF-like products during instructive phases of tooth development. We suggest that plasma- and serum-deprived cells and tissues in vitro produce autocrine and/or paracrine growth factors which mediate embryonic mandibular morphogenesis, histogenesis and cytodifferentiation.

Animals↗