Signaling Pathways Critical for Tooth Root Formation.

Tooth is made of an enamel-covered crown and a cementum-covered root. Studies on crown dentin formation have been a major focus in tooth development for several decades. Interestingly, the population prevalence for genetic short root anomaly (SRA) with no apparent defects in crown is close to 1.3%. Furthermore, people with SRA itself are predisposed to root resorption during orthodontic treatment. The discovery of the unique role of Nfic (nuclear factor I C; a transcriptional factor) in controlling root but not crown dentin formation points to a new concept: tooth crown and root have different control mechanisms. Further genetic mechanism studies have identified more key molecules (including Osterix, ß-catenin, and sonic hedgehog) that play a critical role in root formation. Extensive studies have also revealed the critical role of Hertwig's epithelial root sheath in tooth root formation. In addition, Wnt10a has recently been found to be linked to multirooted tooth furcation formation. These exciting findings not only fill the critical gaps in our understanding about tooth root formation but will aid future research regarding the identifying factors controlling tooth root size and the generation of a whole "bio-tooth" for therapeutic purposes. This review starts with human SRA and mainly focuses on recent progress on the roles of NFIC-dependent and NFIC-independent signaling pathways in tooth root formation. Finally, this review includes a list of the various Cre transgenic mouse lines used to achieve tooth root formation-related gene deletion or overexpression, as well as strengths and limitations of each line.

Localization of RELM-ß/FIZZ2 Is Associated with Cementum Formation.

Resistin-like molecule-ß/found in inflammatory zone 2 (RELM-ß/FIZZ2) is a cysteine-rich secretory protein that is localized in the epithelium of the gastrointestinal tract and lung alveoli. Previous reports have suggested that this protein regulates glucose metabolism and inflammation. In the present study, to analyze the involvement of RELM-ß/FIZZ2 in tooth development, we immunohistochemically examined the localization of RELM-ß/FIZZ2 in tooth germs of embryonic days (E) 15-20 and postnatal days (P) 7-42 rats. RELM-ß/FIZZ2 was hardly detected in the tooth germ at the bud (E15) stage. However, at the cap (E17) and bell (E20) stages, this protein was detectable in the inner enamel epithelium; whereas cells in the other parts of the enamel organ including the outer enamel epithelium and stellate reticulum did not show the immunoreactivity. During the root formation stage (P14-28), cells in Hertwig's epithelial root sheath (HERS) localized RELM-ß/FIZZ2. Intense immunoreactivity was also seen in the matrix of the root dentin facing the HERS and the dental follicle. This reactivity was not present on the more upwardly located dentin surface. In contrast, cementum matrix positive for osteopontin and bone sialoprotein was observed on the dentin instead of immunoreactivity for RELM-ß/FIZZ2. Osterix-positive cells, indicating cementoblast progenitors, were also detected in the dental follicle near the matrix positive for RELM-ß/FIZZ2. These results suggest that RELM-ß/FIZZ2 secreted by the inner enamel epithelium was mainly localized in the matrix at the surface of the apical root dentin and might be involved in cementogenesis. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1865-1874, 2017. © 2017 Wiley Periodicals, Inc.

Immunohistochemical evidence for sclerostin during cementogenesis in mice.

The purpose of this study was to investigate systematically the expression of the glycoprotein sclerostin, the product of the SOST gene, in periodontal tissues, especially in the cementum of mice. Immunolocalization of sclerostin was performed in decalcified histological sections of the maxillary and mandibular jaws of 20 CB56BL/6 mice. For analysis, newborn mice as well as mice at the age of, 1, 2, 4 and 8 weeks were used to detect sclerostin in the cementum, periodontal ligament (PDL) and alveolar bone. For further characterization of the cells within the periodontium, antibodies for Runx2 and S100A4 were also applied. S100A4 as a marker for fibroblasts was used to characterize the fibroblasts, especially in the periodontal ligament. Runx2 as a marker for osteoblast-maturation was used to detect the osteoblasts in the alveolar bone. In addition to the detection in osteocytes, expression of sclerostin was observed in cementocytes of the cellular cementum. With regard to cementogenesis, positive identification of sclerostin could be verified in mice at the age of 4 and 8 weeks but not during the initial stages of cementogenesis. Positive immune reactions for Runx2 were observed in PDL cells, cementoblasts, cementocytes, osteoblasts and osteocytes. PDL cells generally showed positive immunoreactions for the S100A4 antibody. The main findings of this study were: (1) due to the fact that sclerostin was not identified in the initial stages of cementum development, its biological significance seems to be restricted to cementum homeostasis and possibly to regenerative processes; (2) verification of sclerostin only in cementocytes of cellular cementum points to biological similarity of cellular cementum and bone.

Constitutive stabilization of ß-catenin in the dental mesenchyme leads to excessive dentin and cementum formation.

Wnt/ß-catenin signaling plays an important role in morphogenesis and cellular differentiation during development. Essential roles of Wnt/ß-catenin signaling in tooth morphogenesis have been well known, but the involvement of Wnt/ß-catenin signaling in the dental hard tissue formation remains undefined. To understand roles of Wnt/ß-catenin signaling in dentin and cementum formation, we generated and analyzed the conditional ß-catenin stabilized mice in the dental mesenchyme. The OC-Cre;Catnb(lox(ex3)/+) mice exhibited malformed teeth characterized by aberrantly formed dentin and excessively deposited cementum. Large amount of dentin was rapidly formed with widened predentin and numerous globular calcifications in the crown. Whereas roots of molars were short and covered with the excessively formed cellular cementum. With age, the coronal pulp chamber and periodontal space were narrowed by the excessively formed dentin and cementum, respectively. To compare the changes of gene expression in the mutant mice, Col1a1 expression was increased but that of Dspp was decreased in the odontoblasts. However, both of Col1a1 and Bsp expression was increased in the cementoblasts. The gene expression changes were consistent with the localization of matrix proteins. Biglycan and PC-1 was increased but Phex was decreased in the odontoblasts and dentin matrix, respectively. TNAP was increased but Dmp1 and FGF23 was decreased in the cementoblasts and cementum matrix, respectively. Our results indicate that persistent stabilization of ß-catenin in the dental mesenchyme leads to premature differentiation of odontoblasts and differentiation of cementoblasts, and induces excessive dentin and cementum formation in vivo. These results suggest that temporospatial regulation of Wnt/ß-catenin signaling plays critical roles in the differentiation of odontoblasts and cementoblasts, and that inhibition of Wnt/ß-catenin signaling may be important for the formation of dentin and cementum during tooth development. Local modulation of Wnt/ß-catenin signaling has therapeutic potential to improve the regeneration of dentin and periodontium.

Hertwig's epithelial root sheath cells do not transform into cementoblasts in rat molar cementogenesis.

It is generally accepted that cementoblasts originate in the process of differentiation of the mesenchymal cells of the dental follicle. Recently, a different hypothesis for the origin of cementoblasts has been proposed. Hertwig's epithelial root sheath cells undergo the epithelial-mesenchymal transformation to differentiate into cementoblasts. To elucidate whether the epithelial-mesenchymal transformation occurs in the epithelial sheath, developing rat molars were examined by keratin-vimentin and Runx2 (runt-related transcription factor 2)-keratin double immunostaining. In both acellular and cellular cementogenesis, epithelial sheath and epithelial cells derived from the epithelial sheath expressed keratin, but did not express vimentin or Runx2. Dental follicle cells and cementoblasts, however, expressed vimentin and Runx2, but did not express keratin. No cells showed coexisting keratin-vimentin or Runx2-keratin staining. These findings suggest that there is no intermediate phenotype transforming epithelial to mesenchymal cells, and that epithelial sheath cells do not generate mineralized tissue. This study concludes that the epithelial-mesenchymal transformation does not occur in Hertwig's epithelial root sheath in rat acellular or cellular cementogenesis and that the dental follicle is the origin of cementoblasts, as has been proposed in the original hypothesis.

Advances in defining regulators of cementum development and periodontal regeneration.

Substantial advancements have been made in defining the cells and molecular signals that guide tooth crown morphogenesis and development. As a result, very encouraging progress has been made in regenerating crown tissues by using dental stem cells and recombining epithelial and mesenchymal tissues of specific developmental ages. To date, attempts to regenerate a complete tooth, including the critical periodontal tissues of the tooth root, have not been successful. This may be in part due to a lesser degree of understanding of the events leading to the initiation and development of root and periodontal tissues. Controversies still exist regarding the formation of periodontal tissues, including the origins and contributions of cells, the cues that direct root development, and the potential of these factors to direct regeneration of periodontal tissues when they are lost to disease. In recent years, great strides have been made in beginning to identify and characterize factors contributing to formation of the root and surrounding tissues, that is, cementum, periodontal ligament, and alveolar bone. This review focuses on the most exciting and important developments over the last 5 years toward defining the regulators of tooth root and periodontal tissue development, with special focus on cementogenesis and the potential for applying this knowledge toward developing regenerative therapies. Cells, genes, and proteins regulating root development are reviewed in a question-answer format in order to highlight areas of progress as well as areas of remaining uncertainty that warrant further study.

Amelogenin: a potential regulator of cementum-associated genes.

BACKGROUND: Studies suggest that enamel matrix proteins induce differentiation and mineralization of a variety of mesenchymal cells, including odontoblasts, osteoblasts, and cementoblasts. It has been postulated that this activity could be due to amelogenin-like proteins, known to be present in some mixtures of enamel matrix derivatives. Amelogenins have been reported to induce expression of a mineralized tissue-specific marker, bone sialoprotein (BSP), indicating that epithelial products can regulate the activity of mesenchyme-derived cells. METHODS: To explore the molecular mechanisms involved in BSP regulation, a clonal population of immortalized murine cementoblasts (OCCM-30) was exposed to full-length murine amelogenin protein (rp(H)M180), 0.1 microg/ml to 10.0 microg/ml, for 8 days in vitro. To further investigate the potential epithelial-mesenchymal interaction, an amelogenin knockout mouse model was used to examine expression of BSP and other markers, including Type I collagen, in tissue samples. RESULTS: The lowest dose of amelogenin slightly enhanced BSP expression, whereas at the highest dose, a dramatic decrease (three-fold) in BSP expression was observed. Parallel experiments showed a corresponding decrease in mineral nodule formation in vitro for cells treated with the higher dose of rp(H)M180. In situ hybridization and immunohistochemical analysis of sections from amelogenin null mice revealed a dramatic reduction in expression of BSP mRNA and protein in cementoblasts and surrounding osteoblasts in comparison to age-matched controls. In contrast, the expression of Type I collagen was not significantly different from controls. CONCLUSION: These data suggest that amelogenin may be a critical signaling molecule required for appropriate development of the periodontium.

APIÑAMIENTO DENTARIO ANTERIOR INFERIOR

Tratar el apiñamiento en la dentinción adulta joven es diferente al de la dentinción mixta. El apiñamiento es un resultado de una anormalidad que se debe tratar despues de presentada la consecuencia. El manejo del espacio es preventivo debiendo controlar el desarrollo de los maxilares. El apiñamiento unas veces es local. otras general. Se presenta con mayor frecuencia y es más grave en los sectores del arco donde los dientes son últimos del grupo en erupcionar, o sea en la región de los segundos premolares de ambos maxilares: superior e inferior. El segmento anterior del maxilar inferior es otra zona qu e merece atención especial donde a menudo se observan los incisivos apiñados. No obstante, se produce correcciones espontáneas, salvo que el aiñamiento sea muy pronunciado. Esto sucedepor el aumentodel ancho del proceso alveolar cuando erupcionan los incisivos, o mas tarde, como resultado de la diferencia entre los diametros de los molares temporarios y los premolares

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.

Hertwig's epithelial root sheath differentiation and initial cementum and bone formation during long-term organ culture of mouse mandibular first molars using serumless, chemically-defined medium.

Studies were designed to test the hypothesis that Hertwig's epithelial root sheath (HERS) synthesizes and secretes enamel-related proteins that participate in the process of acellular cementum formation. Our experimental strategy was to examine sequential root development of the mouse mandibular first molar in vivo and in long-term organ culture in vitro using serumless, chemically-defined medium. Using anti-amelogenin, anti-enamelin and anti-peptide antibodies, enamel-related antigens were localized within intermediate cementum during HERS differentiation and root formation in vivo. Cap stage molars maintained for periods of up to 31 days in organ culture expressed morphogenesis and cytodifferentiation as identified by tooth crown and initial root, cementum and bone formation. Metabolically-labeled HERS products were analyzed by immunodetection using enamel-related antibodies and one- and two-dimensional SDS gel electrophoresis. A 72 kDa and 26 kDa polypeptide were identified in forming mouse cementum. Both of these root putative cementum proteins yield similar (identical) amino acid compositions; however, both proteins differed from the compositions of either mouse crown enamelin or amelogenin proteins. This approach provides a new and novel in vitro model towards understanding HERS differentiation and functions related to root and bone formation. The data support the hypothesis that HERS cells synthesize polypeptides related to but also different from canonical crown enamel proteins.

Electron microscopic demonstration of non-mineralized and hypomineralized areas in dentin and cementum by silver methenamine staining of collagen.

An electron microscopic study on silver methenamine staining of hard dental tissues was made on a material that comprised human permanent teeth and primary tooth germs from human and porcine fetuses. It was demonstrated that silver-stained material consisted of collagen fibrils. In predentin and precementum all collagen fibrils were stained, while collagen fibrils of dentin and cementum were unstained except for some fibrils of minor special areas such as Owen's contour lines, interglobular dentin, Tomes's granular layer, and in cementum small "interglobular-like" areas. It is concluded that silver methenamine visualizes collagen fibrils of hypo- and unmineralized areas in dental hard tissues and therefore may be used to demonstrate abnormal patterns of mineralization. Finally variations of silver methenamine stainability in relation to differences in material and methods were studied and discussed.

Location and ultrastructure of the first cementum formed in rabbit incisors.

The morphology of the continuously erupting rabbit incisor differs characteristically from that of the rodent incisor and is incompletely known. In this study, permanent incisors from fetal New Zealand white rabbits aged 26-30 days in utero were processed for transmission electron microscopy. Examination of longitudinal and cross-sections revealed that disintegration of the epithelial sheath on root analog surfaces was followed by cementogenesis. The first layer of cementum matrix was seen on the mesial aspect and consisted of a finely granular ground substance and a few embedded collagen fibrils which extended to the cementodentinal junction. Within the period 27-30 days in utero, acellular cementum was formed on all root-analog surfaces and, in addition, formed a fine coating over the enamel. Although the initially formed cementum layer contained fewer collagen fibrils than subsequent layers, a layer of so-called intermediate cementum was not observed.

Considerations on the classification of odontogenic tumours.

The histogenetic, organogenetic and embryological aspects of tooth development are used in 4 hypotheses which form a basis for understanding the formal genesis of benign odontogenic tumours. The ectomesenchyme which gives rise to odontoblasts and cementoblasts is distinguished from mesenchymal tissue. Using 4 histogenetic cell groups (epithelial, ectomesenchymal, neuroectodermal and mesenchymal), the various types of odontogenic tumour are discussed. A short analysis is presented of different odontogenic tumour classifications, and some principle problems of terminology and taxonomy are examined.

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.

Presence of enamel on the lingual surface of rabbit permanent incisors.

Permanent incisors from fetal New Zealand white rabbits aged 25-30 d in utero were processed for light microscopy. Examination of transverse and longitudinal sections of demineralized specimens revealed the development of a complete enamel covering of the incisal tip and an enamel extension on the linguoincisal surface of unerupted teeth, as evidenced by the presence and distribution of ameloblasts, enamel matrix and enamel space. Formation of lingual enamel appeared to be completed within a few days before eruption of the incisor. The transition from the enamel-covered to enamel-free areas coincided with a gradual disintegration of the orderly ameloblast layer. The findings indicate that, histologically, the linguoincisal part of the rabbit permanent incisor is characterized by amelogenesis.

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.