Dental and craniofacial defects in the Crtap-/- mouse model of osteogenesis imperfecta type VII.

BACKGROUND: Inactivating mutations in the gene for cartilage-associated protein (CRTAP) cause osteogenesis imperfecta type VII in humans, with a phenotype that can include craniofacial defects. Dental and craniofacial manifestations have not been a focus of case reports to date. We analyzed the craniofacial and dental phenotype of Crtap-/- mice by skull measurements, micro-computed tomography (micro-CT), histology, and immunohistochemistry. RESULTS: Crtap-/- mice exhibited a brachycephalic skull shape with fusion of the nasofrontal suture and facial bones, resulting in mid-face retrusion and a class III dental malocclusion. Loss of CRTAP also resulted in decreased dentin volume and decreased cellular cementum volume, though acellular cementum thickness was increased. Periodontal dysfunction was revealed by decreased alveolar bone volume and mineral density, increased periodontal ligament (PDL) space, ectopic calcification within the PDL, bone-tooth ankylosis, altered immunostaining of extracellular matrix proteins in bone and PDL, increased pSMAD5, and more numerous osteoclasts on alveolar bone surfaces. CONCLUSIONS: Crtap-/- mice serve as a useful model of the dental and craniofacial abnormalities seen in individuals with osteogenesis imperfecta type VII.

Antagonistic interaction between Ezh2 and Arid1a coordinates root patterning and development via Cdkn2a in mouse molars.

Patterning is a critical step during organogenesis and is closely associated with the physiological function of organs. Tooth root shapes are finely tuned to provide precise occlusal support to facilitate the function of each tooth type. However, the mechanism regulating tooth root patterning and development is largely unknown. In this study, we provide the first in vivo evidence demonstrating that Ezh2 in the dental mesenchyme determines patterning and furcation formation during dental root development in mouse molars. Mechanistically, an antagonistic interaction between epigenetic regulators Ezh2 and Arid1a controls Cdkn2a expression in the dental mesenchyme to regulate dental root patterning and development. These findings indicate the importance of balanced epigenetic regulation in determining the tooth root pattern and the integration of roots with the jaw bones to achieve physiological function. Collectively, our study provides important clues about the regulation of organogenesis and has general implications for tooth regeneration in the future.

Fluoxetine effects on periodontogenesis: histomorphometrical and immunohistochemical analyses in rats.

OBJECTIVE: This study aimed to assess the effects of fluoxetine, a selective serotonin reuptake inhibitor, on the formation of the periodontal ligament during pregnancy and lactation in rat pups. MATERIAL AND METHODS: Twelve pregnant rats of Wistar lineage were divided into four study groups. In the control group, 0.9% sodium chloride solution was administered orally, throughout the entire period of the 21 days of pregnancy (CG group) and in the CGL group, it was administrated during pregnancy and lactation (from day 1 of pregnancy to the 21st day after birth). Fluoxetine was administered orally at the dose of 20 mg/kg in a group treated during pregnancy only (FG group), and during pregnancy and lactation (FGL group). Histometrical, histochemical and immunohistochemical analysis of the maxillary first molar periodontium region of the 24 rat pups was made under light microscopy, and periodontal ligament collagen was qualitatively evaluated under a polarizing light microscope. RESULTS: The quantity of fibroblasts (p=0.006), osteoblasts (p=0.027) and cementoblasts (p=0.001) was reduced in pups from the rats that received fluoxetine during pregnancy and lactation. No alterations were seen in the collagen fibers. CONCLUSION: These findings suggest that periodontal tissue may be sensitive to fluoxetine, and its interference in reducing periodontal cells depends on exposure time during lactation.

Fluoxetine effects on periodontogenesis: histomorphometrical and immunohistochemical analyses in rats

Abstract Reports have indicated that serotonin plays an important role in cell migration and differentiation during the organogenesis of several tissues, including the oral types. Administration of selective serotonin reuptake inhibitor (SSRI) drugs during pregnancy could affect the delivery of serotonin to embryonic tissues altering its development. Objective This study aimed to assess the effects of fluoxetine, a selective serotonin reuptake inhibitor, on the formation of the periodontal ligament during pregnancy and lactation in rat pups. Material and Methods Twelve pregnant rats of Wistar lineage were divided into four study groups. In the control group, 0.9% sodium chloride solution was administered orally, throughout the entire period of the 21 days of pregnancy (CG group) and in the CGL group, it was administrated during pregnancy and lactation (from day 1 of pregnancy to the 21st day after birth). Fluoxetine was administered orally at the dose of 20 mg/kg in a group treated during pregnancy only (FG group), and during pregnancy and lactation (FGL group). Histometrical, histochemical and immunohistochemical analysis of the maxillary first molar periodontium region of the 24 rat pups was made under light microscopy, and periodontal ligament collagen was qualitatively evaluated under a polarizing light microscope. Results The quantity of fibroblasts (p=0.006), osteoblasts (p=0.027) and cementoblasts (p=0.001) was reduced in pups from the rats that received fluoxetine during pregnancy and lactation. No alterations were seen in the collagen fibers. Conclusion These findings suggest that periodontal tissue may be sensitive to fluoxetine, and its interference in reducing periodontal cells depends on exposure time during lactation.

Expression of thymosin beta-4 in human periodontal ligament cells and mouse periodontal tissue and its role in osteoblastic/cementoblastic differentiation.

A recent report showed that thymosin beta-4 (Tß4) is expressed during the development of tooth germ, but its effect on osteoblastic/cementoblastic differentiation is a controversial topic. Furthermore, the precise expression and function of Tß4 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the immunolocalization of Tß4 in the developing periodontium of mouse, the function of Tß4 in osteoblastic/cementoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. Tß4 expression was observed in differentiating hPDLCs, osteoblasts of the periodontium during development, as well as in mature tissue. Higher Tß4 expression was observed in hPDLCs than in cementoblasts and osteoblasts in the developing periodontium. The expression of Tß4 mRNA and protein gradually increased during PDL cell differentiation. The downregulation of Tß4 expression by Tß4 siRNA transfection inhibited osteoblastic differentiation by decreasing calcium nodule formation, alkaline phosphatase (ALP) activity, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. In contrast, Tß4 activation using a Tß4 peptide, promoted these processes by activation of Akt, p38, ERK MAPKs, and the NF-κB pathway. The expression of nuclear NFATc1 was upregulated by Tß4 peptide in hPDLCs. Inhibition of the calcineurin/NFATc1 pathway by cyclosporin A and FK506, attenuated Tß4-induced osteoblastic differentiation and activation of Wnt-related genes, as well as nuclear ß-catenin in hPDLCs. In conclusion, this study demonstrates, for the first time, that Tß4 is expressed in developing periodontal tissue and that its expression is associated with osteoblastic/cementoblastic differentiation. These results suggests that Tß4 is a potential therapeutic target for periodontal regeneration or bone disease.

Semaphorin 3A induces mesenchymal-stem-like properties in human periodontal ligament cells.

Periodontal ligament stem cells (PDLSCs) have recently been proposed as a novel option in periodontal regenerative therapy. However, one of the issues is the difficulty of stably generating PDLSCs because of the variation of stem cell potential between donors. Here, we show that Semaphorin 3A (Sema3A) can induce mesenchymal-stem-like properties in human periodontal ligament (PDL) cells. Sema3A expression was specifically observed in the dental follicle during tooth development and in parts of mature PDL tissue in rodent tooth and periodontal tissue. Sema3A expression levels were found to be higher in multipotential human PDL cell clones compared with low-differentiation potential clones. Sema3A-overexpressing PDL cells exhibited an enhanced capacity to differentiate into both functional osteoblasts and adipocytes. Moreover, PDL cells treated with Sema3A only at the initiation of culture stimulated osteogenesis, while Sema3A treatment throughout the culture had no effect on osteogenic differentiation. Finally, Sema3A-overexpressing PDL cells upregulated the expression of embryonic stem cell markers (NANOG, OCT4, and E-cadherin) and mesenchymal stem cell markers (CD73, CD90, CD105, CD146, and CD166), and Sema3A promoted cell division activity of PDL cells. These results suggest that Sema3A may possess the function to convert PDL cells into mesenchymal-stem-like cells.

Developmentally regulated expression of Sema3A chemorepellant in the developing mouse incisor.

OBJECTIVE: Semaphorin 3A (Sema3A) is an essential chemorepellant controlling peripheral axon pathfinding and patterning, but also serves non-neuronal cellular functions. Incisors of rodent are distinctive from molars as they erupt continuously, have only one root and enamel is present only on the labial side. The aim of this study is to address putative regulatory roles of Sema3A chemorepellant in the development of incisor innervation and formation. MATERIALS AND METHODS: This study analyzed expression of Sema3A mRNAs during embryonic and early post-natal stages of mouse mandibular incisor using sectional radioactive in situ hybridization. RESULTS: Although Sema3A mRNAs were observed in condensed dental mesenchyme during the early bud stage, they were absent in dental papilla or pulp at later stages. Sema3A mRNAs were observed in the dental epithelium including the cervical loops and a prominent expression was also seen in alveolar bone. Interestingly, transcripts were absent from the mesenchymal dental follicle target area (future periodontal ligament) throughout the studied stages. CONCLUSION: The expression patterns of Sema3A indicate that it may control the timing and patterning of the incisor innervation. In particular, Sema3A appears to regulate innervation of the periodontal ligament, while nerve penetration into the incisor dental pulp appears not to be dependent on Sema3A. Moreover, Sema3A may regulate the functions of cervical loops and the development of alveolar bone. Future study with Sema3A deficient mice will help to elucidate the putative neuronal and non-neuronal functions of Sema3A in incisor tooth development.

Formation of the tooth-bone interface.

Not only are teeth essential for mastication, but also missing teeth are considered a social handicap due to speech and aesthetic problems, with a resulting high impact on emotional well-being. Several treatment procedures are currently available for tooth replacement with mostly inert prosthetic materials and implants. Natural tooth substitution based on copying the developmental process of tooth formation is particularly challenging and creates a rapidly developing area of molecular dentistry. In any approach, functional interactions among the tooth, the surrounding bone, and the periodontium must be established. Therefore, recent research in craniofacial genetics searches for mechanisms responsible for correct cell and tissue interactions, not only within a specific structure, but also in the context of supporting structures. A tooth crown that is not functionally anchored to roots and bone is useless. This review aims to summarize the developmental and tissue homeostatic aspects of the tooth-bone interface, from the initial patterning toward tooth eruption and lifelong interactions between the tooth and its surrounding alveolar bone.

Role of Wnt signaling in the biology of the periodontium.

Continuously erupting teeth have associated with them a continuously regenerating periodontal ligament, but the factors that control this amazing regenerative potential are unknown. We used genetic strategies to show that the periodontal ligament arises from the cranial neural crest. Despite their histological similarity, the periodontal ligament of continuously erupting incisor teeth differs dramatically from the periodontal ligament of molar teeth. The most notable difference was in the distribution of Wnt responsive cells in the incisor periodontal ligament, which coincided with regions of periodontal ligament cell proliferation. We discuss these findings in the context of dental tissue regeneration.

Postnatal expression of calretinin-immunoreactivity in periodontal Ruffini endings in the rat incisor: a comparison with protein gene product 9.5 (PGP 9.5)-immunoreactivity.

The postnatal expression of immunoreactivity for calretinin, one of the calcium binding proteins, and for protein gene product 9.5 (PGP 9.5), a general neuronal marker, was investigated in mechanoreceptive Ruffini endings in the periodontal ligament of the rat incisor. Age-related changes in the expression of these two proteins in periodontal nerves were further quantified with a computerized image analysis. At 1 day after birth, a few PGP 9.5-immunoreactive nerve fibers and a still smaller number of calretinin-positive fibers were found in the periodontal ligament: they were thin and beaded in appearance and no specialized nerve terminals were recognized. Tree-like terminals, reminiscent of immature Ruffini endings, were recognizable in 4-day-old rats by PGP 9.5-immunohistochemistry, while calretinin-immunostaining failed to reveal these specialized endings. At postnatal 7-11 days when PGP 9.5-immunostaining could demonstrate typical Ruffini endings, calretinin-immunopositive nerve fibers merely tapered off without forming the Ruffini type endings. A small number of Ruffini endings showing calretinin-immunoreactivity began to occur in the periodontal ligament at 24-26 days after birth when the occlusion of the first molars had been established. At the functional occlusion stage (60-80 days after birth), the Ruffini endings showing calretinin-immunoreactivity drastically increased in number and density, but less so than those positive for PGP 9.5-immunoreaction. The delayed expression of calretinin suggests that the function of the periodontal Ruffini endings is established after the completion of terminal formation because Ca2+, which binds to calcium binding proteins including calretinin with high affinity, plays an important role in mechano-electric transduction.

Immunolocalisation of collagens in the developing rat molar tooth.

This study identifies different types of collagens during tooth development, maturation and ageing. Tissues from the rat first molar (from animals ranging in age from E14 to 104 wk postnatally) were immunostained using a panel of mono- and polyclonal antibodies against types I, II, III, IV, VI, IX and X collagen, fibronectin and laminin. During tooth development, types I and III collagens were expressed in the dental papilla at all stages but were also unexpectedly observed in the stellate reticulum of the enamel organ. Transient expression of type II collagen was also observed in the stellate reticulum during the late bell stage. Types IV and VI collagens, with laminin and fibronectin, were located within the basement membranes of the tooth germ. Collagen types I and III were observed within the developing follicle/periodontal ligament, type III predominating where collagen fibres were inserting into the alveolar bone and cementum. The pattern of types I and III collagen labelling within the periodontal ligament and the dental pulp did not change with age. Thus, some unusual collagen localisations were observed in the tooth germ, particularly within the stellate reticulum.

Distribution of the neural cell adhesion molecule (NCAM) during pre- and postnatal development of mouse incisors.

Developmental changes in the distribution of the neural cell adhesion molecule (NCAM) were investigated in mouse incisors by means of the indirect immunofluorescence method. During the prenatal stages of development, NCAM was predominantly found in the dental follicle, but not in the dental papilla; the results were analogous to the distribution of NCAM during molar development. After birth, the expression of NCAM continued in the tissue between the enamel organ and the alveolar bone on the labial aspect. In contrast, the follicular tissue covering the lingual aspect of the incisor gradually lost NCAM immunoreactivity from its outer zone as it differentiated into the highly organized periodontal ligament. The intermediate zone of the ligament continued to express NCAM-immunoreactivity even in mice of 6 weeks of age. This pattern of NCAM expression was different from that found in molar teeth, where the organized peridontal ligament was NCAM-negative. The dental pulp, in which we previously reported that an NCAM-positive area appeared at later stages of molar tooth development, did not express NCAM immunoreactivity even at the latest stage of development covered in this study. These differences in the distribution of NCAM between the incisors and the molars might be related to the fact that rodent incisors continue to grow throughout the life of the animal.

Development and cell fate in interspecific (Mus musculus/Mus caroli) intraocular transplants of mouse molar tooth-germ tissues detected by in situ hybridization.

Mandibular first molar tooth germs were dissected from Mus musculus (CDI) and Mus caroli (age range: 14-day embryo to 1-day postnatal). Most of the tooth germs were separated enzymically into epithelial and mesenchymal components. Interspecific tissue recombinations and intact M. caroli tooth germs were grown in the anterior chamber of the eye of adult CDI mice for 24 weeks. Recombinations of M. caroli enamel-organ epithelium with M. musculus, dental papilla and follicle mesenchyme developed into normal teeth with advanced root, periodontal ligament and bone formation, thereby confirming extensive epithelial-mesenchymal interactions across the species barrier. Labelling sections by in situ hybridization with a M. musculus-specific DNA probe (pMSat5) showed that almost all cells in the pulp, periodontal ligament and bone were M. musculus, including cementoblasts. Reduced enamel epithelium and epithelial cell rests derived from donor M. caroli enamel organ were unlabelled. This indicates that any cementogenic role of Hertwig's epithelial root sheath must be short-lived. The immunological privilege of the intraocular transplantation site in M. musculus CDI mice did not extend to grafts including xenogeneic M. caroli dental mesenchyme. Thus, intact M. caroli tooth germs and recombinations of M. musculus enamel organ with M. caroli dental papilla and follicle showed limited development, with no root formation, and were populated almost exclusively with labelled host M. musculus lymphocytes.

The soft connective tissues of the gingiva and periodontal ligament: are they unique?

The connective tissues of the gingiva and periodontal ligament share a common embryonic development from cells of the cranial neural crest. This review paper describes the relationship of these tissues in tooth germ initiation, development and eruption.

Segregated localization of immunocompetent cells and osteoclasts in the periodontal ligament of the rat molar.

The spatial distribution of dendritic cells, macrophages, and their respective precursor cells in the periodontal ligament of rat molars was examined by means of ACPase enzyme histochemistry and immunohistochemistry. Intense reactions for ACPase were localized in both the multinucleated-and mononucleated cells of the periodontal ligament located exclusively in the portions of physiological bone resorption due to the physiological migration of the molar teeth. Immunohistochemical staining with OX6-monoclonal antibody that recognizes antigen-presenting cells such as dendritic cells and macrophages revealed the localization of immunopositive cells predominantly in the portions of the periodontal ligament that showed only trace reactions for ACPase. On the other hand, a large number of ED1-immunopositive cells, comprising a broad spectrum of cells of monocyte origin including dendritic cells and osteoclasts, displayed an almost even distribution throughout the periodontal ligament. Our current study is the first to show clear-cut in vivo morphological evidence that the cells of the bone-resorbing, osteoclastic cell lineage and those of the non-bone resorbing, macrophagic and/or dendritic cell lineages are exclusively localized in roughly the distal and proximal regions of the periodontal ligament of rat molars, respectively. An advantage is proposed for the use of the rat molar periodontal ligament as an in vivo model system for pursuing differentiation pathways of cells of the monocyte lineage, particularly of the osteoclastic cells.

[Odontogenic and nonodontogenic epithelial rests. Embryological origin]. / Resti epiteliali odontogeni e non odontogeni. Origine embriologica.

The close examination of the central osseous and/or parosteal lesions of the jaws must be reconducted to the study of the main fundamental embryonal processes that take place in the primordial stomodeus. Some pathological lesions can arise from the rests that survive after the disgregation of various odontogenic epithelial structures destined to disappear with growth: the epithelial root sheath, the dental lamina and the external epithelium of the enamel organ. Some osseous lesions can also originate from the nonodontogenic epithelial rests: this tissue comes from the uncompleted obliteration of the ectodermic layer during the processes of union and fusion of the various embryonal components of the maxillary structures.

Development of periodontal ligament and alveolar bone in homografted recombinations of enamel organs and papillary, pulpal and follicular mesenchyme in the mouse.

Mandibular first-molar tooth germs were dissected from 16-day-embryo and new-born CD1 mice. By incubation in collagenase they were separated into enamel organ, papilla and follicle. Dental pulp was obtained from mandibular first molars of 3-, 7- and 10-day-old mice. Various combinations of epithelial and mesenchymal tissues were grown for periods up to four weeks in the anterior chamber of the eye of homologous adult male mice. Recombinations of enamel organ and papilla formed teeth with regeneration of the investing layer of follicle and a root-related periodontal ligament, but no formation of alveolar bone. Bone only formed in those grafts which also included follicle. Recombinations of enamel organ and pulp produced dysplastic dentine with no enamel formation or proper tooth development. It was impossible, therefore, to assess whether the potential to regenerate an investing layer extends to the pulp later in development. At an earlier stage, however, the papillary mesenchyme has the ability to regenerate investing-layer cells which lack the capacity to form bone.

A mechanism for the regulation of ligament width based on the resonance frequency of ion concentration waves.

A model is developed for a mechanism for the regulation of the width of ligament spaces and of other tissue spaces bounded by calcified surfaces. The proposed mechanism involves the transmission, detection, and retransmission of ion concentration waves by cells located on the calcified surfaces. It is assumed that these cells can use the information regarding ligament width contained in the resonance frequency of the cell-concentration wave system. The assumptions of the proposed mechanism are supported by recent experimental evidence concerning the effect of electrical signals on bone cells, the use of frequency-encoded information by cells, and the production of low frequency K+ pulses by osteoblast-like cells. The relation between resonance frequency and ligament width is derived, and the resonance frequencies corresponding to measured ligament widths are shown to occur in the same frequency range as occur in the K+ pulses emitted by bone cells. The model suggests definite experimental tests that involve investigating the effect in vitro of ion concentration wave frequency on bone cell activity and hormone receptors.

Formation of the periodontal attachment in developing rabbit permanent incisors.

Permanent incisors from fetal New Zealand white rabbits aged 25-30 days in utero and from 1-20 days postnatally were processed for light microscopy. Examination of longitudinal and cross-sections revealed that cementum deposition and fiber attachment first occurred at 27 days in utero. A tooth-to-bone course of principal fibers confined to lateral aspects of the teeth was observed in 29-day fetal incisors, whereas lingually the periodontal ligament remained undeveloped as late as 5 days postnatally. In 20-day postnatal incisors, the development of the functional periodontal ligament was complete as evidenced by the fiber arrangement on all aspects of the root-analogue tooth surface. This study shows that 25-26-day fetal rabbit permanent incisors represent the end of the follicular phase, whereas 27-day fetal to 20-day postnatal teeth demonstrate distinct histologic characteristics of initial, incomplete and complete periodontal fiber attachment.

A new look at the rests of Malassez. A review of their embryological origin, anatomy, and possible role in periodontal health and disease.

The embryological formation and anatomy of the rests of Malassez are described and their possible roles of health and disease are discussed. It is emphasized that the rests are worthy of new interest and further study. They comprise a structure whose precise anatomy still is in need of accurate description and in which the presence or absence of a physiological role is unproven.