Expression and localization of CD63 in the intracellular vesicles of odontoblasts.

We hypothesized that odontoblasts release exosomes as well as dental pulp cells and focused on the exosome membrane marker CD63. Odontoblasts are well-differentiated mesenchymal cells that produce dentin. Dental pulp, a tissue complex formed with odontoblasts, releases exosomes to epithelial cells and stimulates their differentiation to ameloblasts. However, the localization of CD63 in differentiated odontoblasts is poorly understood. Therefore, herein, we aimed to reveal the expression of CD63 in odontoblasts during tooth development. We first investigated the localization of CD63 in mouse incisors and molars using immunofluorescence. In adult mouse incisors, the anti-CD63 antibody was positive in mature odontoblasts and dental pulp cells but not in pre-odontoblasts along the ameloblasts in the apical bud. Additionally, the anti-CD63 antibody was observed as a vesicular shape in the apical area of odontoblast cytosol and inside Tomes' fibers. The anti-CD63 antibody-positive vesicles were also observed using immunoelectron microscopy. Moreover, during mouse mandibular molar tooth morphogenesis (E16 to postnatal 6 weeks), labeling of anti-CD63 antibody was positive in the odontoblasts at E18. In contrast, the anti-CD63 antibody was positive in the dental pulp after postnatal day 10. Furthermore, anti-CD63 antibody was merged with the multivesicular body marker Rab7 in dental pulp tissues but not with the lysosome marker Lamp1. Finally, we determined the effect of a ceramide-generation inhibitor GW4869 on the mouse organ culture of tooth germ in vitro. After 28 days of GW4869 treatment, both CD63 and Rab7 were negative in Tomes' fibers, but were positive in control odontoblasts. These results suggest that CD63-positive vesicular organelles are important for mouse tooth morphogenesis.

An in situ hybridization study of syndecan family during the late stages of developing mouse molar tooth germ.

Expression of syndecan-1, 2, 3, and 4 mRNAs during the late stages of tooth germ formation was investigated by in situ hybridization, using [35S]-UTP-labeled cRNA probes. Syndecan-1 mRNA was mainly expressed in the stellate reticulum and stratum intermedium as well as at the cervical region of dental papilla/dental follicle during E18.5-P3.0. Expression in the dental epithelium was enhanced during the postnatal periods, which was supported by real-time RT-PCR analysis. These spatiotemporal expression patterns may suggest specific roles of syndecan-1 in tooth formation such as tooth eruption or root formation. Syndecan-3 mRNA expression became evident in odontoblasts at E18.5, but compared to collagen type I mRNA, which was strongly expressed at this stage, syndecan-3 expression in odontoblast was restricted in mature odontoblasts beneath the cusps during the postnatal periods. This result was also supported by real-time RT-PCR analysis, and indicated that syndecan-3 may be involved in the progress of dentinogenesis rather than in the initiation of it. Syndecan-4 mRNA roughly showed comparable expression patterns to those of syndecan-3. Syndecan-2 mRNA did not show significant expression during the experimental period, but real-time RT-PCR analysis suggested that syndecan-2 expression might be enhanced with hard tissue formation.

An in situ hybridization study of decorin and biglycan mRNA in mouse osteoblasts in vivo.

In situ hybridization of decorin and biglycan mRNA, principal members of small leucine-rich proteoglycan, was performed using [35S]-labeled RNA probes, in the context of the hypothesis that they show different expression patterns associated with osteoblast differentiation in mice. We adopted two ossifying sites that can clearly follow the developmental process of bone formation: ossifying tympanic ring and developing bone collar of mandibular condylar cartilage. Decorin mRNA was expressed in osteoblasts of developing tympanic ring at E14.0, as well as of developing bone collar at E15.0, but biglycan mRNA was not, indicating decorin mRNA was expressed earlier in newly differentiating osteoblasts than biglycan. With maturation of osteoblasts, biglycan mRNA became expressed and maintained its expression both in the outer region (periosteum) and in the interior region (endosteum) of bone. By contrast, decorin mRNA expression was maintained in the outer region but diminished in the interior region. These results indicate that decorin and biglycan show differential expression patterns in differentiating osteoblasts and play specific roles in bone formation.

An in situ hybridization study of the Syndecan family in the developing condylar cartilage of fetal mouse mandible.

Mandibular condylar cartilage is a representative secondary cartilage, differing from primary cartilage in various ways. Syndecan is a cell-surface heparan sulfate proteoglycan and speculated to be involved in chondrogenesis and osteogenesis. This study aimed to investigate the expression patterns of the syndecan family in the developing mouse mandibular condylar cartilage. At embryonic day (E)13.0 and E14.0, syndecan-1 and -2 mRNAs were expressed in the mesenchymal cell condensation of the condylar anlage. When condylar cartilage was formed at E15.0, syndecan-1 mRNA was expressed in the embryonic zone, wherein the mesenchymal cell condensation is located. Syndecan-2 mRNA was mainly expressed in the perichondrium. At E16.0, syndecan-1 was expressed from fibrous to flattened cell zones and syndecans-2 was expressed in the lower hypertrophic cell zone. Syndecan-3 mRNA was expressed in the condylar anlage at E13.0 and E13.5 but was not expressed in the condylar cartilage at E15.0. It was later expressed in the lower hypertrophic cell zone at E16.0. Syndecan-4 mRNA was expressed in the condylar anlage at E14.0 and the condylar cartilage at E15.0 and E16.0. These findings indicated that syndecans-1 and -2 could be involved in the formation from mesenchymal cell condensation to condylar cartilage. The different expression patterns of the syndecan family in the condylar and limb bud cartilage suggest the functional heterogeneity of chondrocytes in the primary and secondary cartilage.

Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption.

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1ß, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

Expression, localization and synthesis of small leucine-rich proteoglycans in developing mouse molar tooth germ.

The gene expression and protein synthesis of small leucine-rich proteoglycans (SLRPs), including decorin, biglycan, fibromodulin, and lumican, was analyzed in the context of the hypothesis that they are closely related to tooth formation. In situ hybridization, immunohistochemistry, and organ culture with metabolic labeling of [35S] were carried out in mouse first molar tooth germs of different developmental stages using ICR mice at embryonic day (E) 13.5 to postnatal day (P) 7.0. At the bud and cap stage, decorin mRNA was expressed only in the surrounding mesenchyme, but not within the tooth germ. Biglycan mRNA was then expressed in the condensing mesenchyme and the dental papilla of the tooth germ. At the apposition stage (late bell stage), both decorin and biglycan mRNA were expressed in odontoblasts, resulting in a switch of the pattern of expression within the different stages of odontoblast differentiation. Decorin mRNA was expressed earlier in newly differentiating odontoblasts than biglycan. With odontoblast maturation and dentin formation, decorin mRNA expression was diminished and localized to the newly differentiating odontoblasts at the cervical region. Simultaneously, biglycan mRNA took over and extended its expression throughout the new and mature odontoblasts. Both mRNAs were expressed in the dental pulp underlying the respective odontoblasts. At P7.0, both mRNAs were weakly expressed but maintained their spatial expression patterns. Immunostaining showed that biglycan was localized in the dental papillae and pulp. In addition, all four SLRPs showed clear immunostaining in predentin, although the expressions of fibromodulin and lumican mRNAs were not identified in the tooth germs examined. The organ culture data obtained supported the histological findings that biglycan is more predominant than decorin at the apposition stage. These results were used to identify biglycan as the principal molecule among the SLRPs investigated. Our findings indicate that decorin and biglycan show spatial and temporal differential expressions and play their own tissue-specific roles in tooth development.

Histochemical and Ultrastructural Study of Developing Gonial Bone With Reference to Initial Ossification of the Malleus and Reduction of Meckel's Cartilage in Mice.

Development of mouse gonial bone and initial ossification process of malleus were investigated. Before the formation of the gonial bone, the osteogenic area expressing alkaline phosphatase and Runx2 mRNA was widely recognized inferior to Meckel's cartilage. The gonial bone was first formed within the perichondrium at E16.0 via intramembranous ossification, surrounded the lower part of Meckel's cartilage, and then continued to extend anteriorly and medially until postnatal day (P) 3.0. At P0, multinucleated chondroclasts started to resorb the mineralized cartilage matrix with ruffled borders at the initial ossification site of the malleus (most posterior part of Meckel's cartilage). Almost all CD31-positive capillaries did not run through the gonial bone but entered the cartilage through the site where the gonial bone was not attached, indicating the forms of the initial ossification site of the malleus are similar to those at the secondary ossification center rather than the primary ossification center in the long bone. Then, the reducing process of the posterior part of Meckel's cartilage with extending gonial bone was investigated. Numerous tartrate-resistant acid phosphatase-positive mononuclear cells invaded the reducing Meckel's cartilage, and the continuity between the malleus and Meckel's cartilage was completely lost by P3.5. Both the cartilage matrix and the perichondrium were degraded, and they seemed to be incorporated into the periosteum of the gonial bone. The tensor tympani and tensor veli palatini muscles were attached to the ligament extending from the gonial bone. These findings indicated that the gonial bone has multiple functions and plays important roles in cranial formation. Anat Rec, 302:1916-1933, 2019. © 2019 American Association for Anatomy.

An in situ hybridization study of MMP-2, -9, -13, -14, TIMP-1, and -2 mRNA in fetal mouse mandibular condylar cartilage as compared with limb bud cartilage.

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.

An in situ hybridization study of Hyaluronan synthase (Has) mRNA in developing mouse molar and incisor tooth germs.

Hyaluronan (HA) is a major constituent molecule in most extracellular matrices and is synthesized by Hyaluronan synthase (Has). In the present study, we examined expression patterns of Has1, -2, -3 mRNA in developing mouse molar and incisor tooth germs from embryonic day (E) 11.5 to postnatal day (P) 7, focusing on Hertwig's epithelial root sheath (HERS) and the apical bud in particular. Has1 mRNA expression was not detected in all tooth germs examined. Has2 mRNA was expressed in the surrounding mesenchyme from E12.0 to 18.0 in both molar and incisor tooth germs, but disappeared after birth. Meanwhile, Has3 mRNA was exclusively expressed within the enamel organ, especially in the inner enamel epithelium (IEE), stellate reticulum (SR), and stratum intermedium (SI) until the early bell stage at E16.0. Has3 mRNA disappeared as IEE differentiated into differentiating ameloblasts (dABs), but remained in SI until the root developmental stage of the molar tooth germ at P7. Has3 mRNA was also expressed in HERS until P7. In incisors, Has3 mRNA was expressed in the apical bud, especially in the transit-amplifying (TA) cell region from E16.0 to P7, and in the papillary layer (PL) adjacent to the mature enamel. These gene expression patterns suggested that Has3 is the main control factor for prenatal and postnatal HA synthesis of the tooth germ, and may in part regulate crown and root formation of the tooth germ, maintenance of stem cell niches in the apical bud as well as mineral transport in PL.