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    Requirement of alveolar bone formation for eruption of rat molars. Wise Gary E,He Hongzhi,Gutierrez Dina L,Ring Sherry,Yao Shaomian European journal of oral sciences Tooth eruption is a localized event that requires a dental follicle (DF) to regulate the resorption of alveolar bone to form an eruption pathway. During the intra-osseous phase of eruption, the tooth moves through this pathway. The mechanism or motive force that propels the tooth through this pathway is controversial but many studies have shown that alveolar bone growth at the base of the crypt occurs during eruption. To determine if this bone growth (osteogenesis) was causal, experiments were designed in which the expression of an osteogenic gene in the DF, bone morphogenetic protein-6 (Bmp6), was inhibited by injection of the first mandibular molar of the rat with a small interfering RNA (siRNA) targeted against Bmp6. The injection was followed by electroporation to promote uptake of the siRNA. In 45 first molars injected, eruption was either delayed or completely inhibited (seven molars). In the impacted molars, an eruption pathway formed but bone growth at the base of the crypt was greatly reduced compared with the erupted first-molar controls. These studies show that alveolar bone growth at the base of the crypt is required for tooth eruption and that Bmp6 may be essential for promoting this growth. 10.1111/j.1600-0722.2011.00854.x
    Osteoclasts in Health and Disease. Lerner Ulf H Pediatric endocrinology reviews : PER Osteoclasts are multinucleated, giant cells originating from myeloid hematopoetic stem cells. These are the only cells in nature which can resorb bone. Differentiation of mononucleated osteoclast progenitor cells requires stimulation with M-CSF (macrophage colony-stimulating factor) for the cells to proliferate and survive and with RANKL (receptor activator of nuclear factor kappa B ligand) for differentiation along the osteoclastic lineage to cells which eventually fuse to the mature, multinucleated osteoclasts. Therefore, most hormones and cytokines stimulating osteoclastogenesis do so indirectly by regulating the expression in osteoblasts of RANKL and its inhibitory decoy receptor OPG. Antibodies neutralizing RANKL is a common therapy to inhibit excessive osteoclast formation in diseases such as osteoporosis and malignant tumors with skeletal metastasis. Mature osteoclasts resorb bone by stimulating acid release into the resorption lacunae, followed by proteolytic degradation of bone matrix proteins. Loss-of-function mutations of proteins involved in acidification and proteolysis cause osteopetrosis, a disease with sclerotic bone due to non-functional osteoclasts. Osteoclasts are important for a healthy skeleton by removing damaged bone during remodeling of the skeleton, but are also important for modeling of bone, calcium homeostasis and tooth eruption, and possibly also for glucose and fat metabolism. Loss of bone in inflammatory disease, metastasizing tumors and osteoporosis is due to increased RANKL expression and enhanced osteoclast formation. The present overview aims to summarize how osteoclasts are formed and resorb bone in health and disease. 10.17458/per.vol17.2019.l.osteoclastshealthdisease
    Osteoprotegerin expressed by osteoclasts: an autoregulator of osteoclastogenesis. Kang J H,Ko H M,Moon J S,Yoo H I,Jung J Y,Kim M S,Koh J T,Kim W J,Kim S H Journal of dental research Osteoprotegerin (OPG) is secreted by stromal and osteoblastic lineage cells and inhibits osteoclastogenesis by preventing the interaction of receptor activator of nuclear factor-κB ligand (RANKL) with receptor activator of nuclear factor-κB (RANK). In this study, the expression of OPG in osteoclasts themselves and its biological functions during osteoclastogenesis were investigated for the first time. OPG expression in vivo in the developing rat maxilla was examined by immunofluorescence analysis. OPG expression in osteoclasts during in vitro osteoclastogenesis was determined by reverse-transcription polymerase chain-reaction (RT-PCR), Western blot, and immunofluorescence staining. We determined the function of OPG produced by osteoclasts during osteoclastogenesis by silencing the OPG gene. The effects of OPG on bone-resorbing activity and apoptosis of mature osteoclasts were examined by the assay of resorptive pit formation on calcium-phosphate-coated plate and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. In the immunofluorescence findings, strong immunoreactivities were unexpectedly seen in multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts around the growing and erupting tooth germs in the rat alveolar bone. In vitro, OPG expression was significantly increased during the differentiation of osteoclasts from mouse bone-marrow-derived cells treated with a combination of macrophage colony-stimulating factor (M-CSF) and RANKL. Interestingly, it was found that OPG small interfering (si)RNA treatment during osteoclastogenesis enhanced the sizes of osteoclasts, but attenuated their bone-resorbing activity. Also, the increased chromosomal DNA fragmentation and caspase-3 activity in the late phase of osteoclastogenesis were found to be decreased by treatment with OPG siRNA. Furthermore, effects of OPG siRNA treatment on osteoclastogenesis and bone-resorbing activity were recovered by the treatment of exogenous OPG. These results suggest that OPG, expressed by the osteoclasts themselves, may play an auto-regulatory role in the late phase of osteoclastogenesis through the induction of apoptosis. 10.1177/0022034514552677
    Study on site-specific expression of bone formation and resorption factors in human dental follicles. Uribe Pamela,Plakwicz Pawel,Larsson Lena,Czochrowska Ewa,Westerlund Anna,Ransjö Maria European journal of oral sciences We sought to investigate site-specific expression of bone-regulatory factors expressed by human dental follicles and to compare the stimulated expression of tumour necrosis factor (ligand) superfamily, member 11/tumour necrosis factor receptor superfamily, member 11b (RANKL/OPG) in human dental follicle cells (HDFCs) from different patients. Analysis of bone-regulatory markers in follicles from 12 different study participants was performed using RT-qPCR and immunofluorescence; apical and coronal segments from each dental follicle were processed independently. Four additional dental follicles were used for cell cultures; HDFCs were precultured in osteogenic medium to initiate differentiation and thereafter cultured with 10 M forskolin (FSK) to activate the protein kinase cAMP (PKA/cAMP) signalling pathway and induce RANKL/OPG expression. We demonstrate that RANKL expression is significantly higher in the coronal part of follicles than in the apical part. High levels of collagen type 1 (COL1), alkaline phosphatase (ALP) and Gap-junction protein, alpha 1, 43 kDa (CX43) were expressed, whereas expression of Sp7 transcription factor (OSX), bone morphogenetic protein 2 (BMP2), colony-stimulating factor 1 (CSF-1), chemokine (C-C motif) ligand 2 (MCP1), and OPG was low in all samples. The immunofluorescence localization of CSF-1, MCP1, osteocalcin (OCN), RANKL, and BMP2 was not specific for either part of the follicles. In conclusion, a consistently high expression of CX43 suggests that gap-junction communication in HDFCs is essential for the eruption process. Furthermore, the induced expression of RANKL in HDFCs varies significantly between individuals and may relate to clinical variations in tooth eruption. 10.1111/eos.12568
    RUNX2 Mutation Impairs 1α,25-Dihydroxyvitamin D3 mediated Osteoclastogenesis in Dental Follicle Cells. Wang X Z,Sun X Y,Zhang C Y,Yang X,Yan W J,Ge L H,Zheng S G Scientific reports Cleidocranial dysplasia (CCD), a skeletal disorder characterized by delayed permanent tooth eruption and other dental abnormalities, is caused by heterozygous RUNX2 mutations. As an osteoblast-specific transcription factor, RUNX2 plays a role in bone remodeling, tooth formation and tooth eruption. To investigate the crosstalk between RUNX2 and 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) in human dental follicle cells (hDFCs) during osteoclast formation, we established a co-culture system of hDFCs from CCD patient and healthy donors with peripheral blood mononuclear cells (PBMCs). Expression of the osteoclast-associated genes and the number of TRAP(+) cells were reduced in CCD hDFCs, indicating its suppressed osteoclast-inductive ability, which was reflected by the downregulated RANKL/OPG ratio. In addition, 1α,25-(OH)2D3-stimulation elevated the expression of osteoclast-related genes, as well as RANKL mRNA levels and RANKL/OPG ratios in control hDFCs. Conversely, RUNX2 mutation abolished this 1α,25-(OH)2D3-induced RANKL gene activation and osteoclast formation in CCD hDFCs. Therefore, RUNX2 haploinsufficiency impairs dental follicle-induced osteoclast formation capacity through RANKL/OPG signaling, which may be partially responsible for delayed permanent tooth eruption in CCD patients. Furthermore, this abnormality was not rescued by 1α,25-(OH)2D3 application because 1α,25-(OH)2D3-induced RANKL activation in hDFCs is mediated principally via the RUNX2-dependent pathway. 10.1038/srep24225
    Effect of Runx2 silencing on autophagy and RANKL expression in osteoblasts. Qin Han,Cai Jun Archives of oral biology OBJECTIVE:This study aimed to investigate the effect of Runx2 silencing on autophagy and RANKL expression in mouse osteoblasts, and provide an experimental basis to assess obstacles in dental eruption. METHODS:In accordance with previously reported methods, LVpFU-GW-016PSC60109-1 virus was used to transfect mouse osteoblasts (MOI = 40). Target gene expression was assessed via cytometer, and the effect of silencing Runx2 was assessed via a two-step quantitative real-time polymerase chain reaction (qRT-PCR)-based method. Western blotting was performed to assess LC3, Beclin-1 and RANKL expression. RESULTS:As confirmed via qRT-PCR analysis, Runx2 was efficiently silenced in the experimental group (>90% efficiency). Western blotting revealed that LC3 and RANKL proteins were significantly down -regulated in the experimental group (group KD), their expression levels being particularly lower than those in the control group (group NC). However, Beclin-1 protein expression was not significantly different from that of the control. CONCLUSION:Upon Runx2 silencing, autophagy-related proteins and RANKL were repressed in osteoblasts, thereby potentially causing the tooth eruption disorder. 10.1016/j.archoralbio.2018.07.016
    Skeletal consequences of RANKL-blocking antibody (IK22-5) injections during growth: mouse strain disparities and synergic effect with zoledronic acid. Lézot Frédéric,Chesneau Julie,Navet Benjamin,Gobin Bérengère,Amiaud Jérome,Choi YongWon,Yagita Hideo,Castaneda Beatriz,Berdal Ariane,Mueller Christopher G,Rédini Françoise,Heymann Dominique Bone High doses of bone resorption inhibitors are currently under evaluation in pediatric oncology. Previous works have evidenced transient arrest in long bone and skull bone growth and tooth eruption blockage when mice were treated with zoledronic acid (ZOL). The question of potential similar effects with a RANKL-blocking antibody (IK22.5) was raised. Sensitivity disparities in these inhibitors between mouse strains and synergic effects of zoledronic acid and a RANKL-blocking antibody were subsidiary questions. In order to answer these questions, newborn C57BL/6J and CD1 mice were injected every two or three days (4 injections in total so 7 or 10 days of treatment length) with high doses of a RANKL-blocking antibody. The consequences on the tibia, craniofacial bones and teeth were analyzed by μCT and histology at the end of the treatment and one, two and three months later. The results obtained showed that RANKL-blocking antibody injections induced a transient arrest of tibia and skull bone growth and an irreversible blockage of tooth eruption in C57BL/6J mice. In CD1 mice, tooth eruption defects were also present but only at much higher doses. Similar mouse strain differences were obtained with zoledronic acid. Finally, a synergic effect of the two inhibitors was evidenced. In conclusion as previously observed for bisphosphonates (ZOL), a RANKL-blocking antibody induced a transient arrest in long bone and skull bone growth and a blockage of tooth eruption with however disparities between mouse strains with regard to this last effect. A synergic effect of both bone resorption inhibitors was also demonstrated. 10.1016/j.bone.2014.12.011
    Periodontal ligament-associated protein-1 gets involved in the development of osseous eruption canal. Yu Xijiao,Liu Hongmei,Li Chong,Du Yanmei,Du Yi,Zhang Shanyong Journal of molecular histology Osseous eruption is an important stage of tooth eruption process. The role of periodontal ligament-associated protein-1 (PLAP-1/asporin) in the development of osseous eruption canal remain undefined and were the focus of this study. C57BL/6 mice at postnatal days P11-13 and P 15-16 were chosen. The development of osseous eruption canal of lower first molar was observed and osteoclasts were detected by staining for tartrate-resistant acid phosphatase (TRAP). PLAP-1 expression in the process of osseous eruption (OE, P11-13) and post- osseous eruption (P-OE, P15-16) was assessed by immunohistochemistry, immunofluorescence and western blotting. Receptor activator of NF-κB ligand (RANKL) distribution in the process was also assessed by immunohistochemistry. A double immunofluorescence stain was used to reveal PLAP-1 in association with CD68 (osteoclast maker). Fresh occlusal tissues of erupting lower first molars at OE and P-OE were separated to detected RANKL/OPG ratio by western blotting to elucidate related mechanisms. At osseous eruption (OE), osseous and mucosal tissues could be observed on the occlusal side of lower first molar. Osseous eruption canal was developing. Many osteoclasts were found around occlusal alveolar bone in the development of osseous eruption canal. At post- osseous eruption (P-OE), osseous eruption canal had been built, only mucosal tissues were observed, and few osteoclasts were detected. More PLAP-1 expression was detected at OE, compared with that at P-OE. Similar distributions of PLAP-1 and RANKL in occlusal bone tissues of erupting lower first molars were detected at OE. Colocalization of PLAP-1 and CD68 revealed the positive relationship between PLAP-1 and osteoclasts in the development of osseous eruption canal. PLAP-1 positively correlated with RANKL and CD68+ osteoclasts, and areas of bone resorption. Higher RANKL/OPG ratio was detected at OE, compared with that at P-OE. PLAP-1 gets involved in the development of osseous eruption canal. 10.1007/s10735-018-9805-0
    A three-dimensional analysis of primary failure of eruption in humans and mice. Tokavanich Nicha,Gupta Aditi,Nagata Mizuki,Takahashi Akira,Matsushita Yuki,Yatabe Marilia,Ruellas Antonio,Cevidanes Lucia,Maki Koutaro,Yamaguchi Tetsutaro,Ono Noriaki,Ono Wanida Oral diseases OBJECTIVES:Primary failure of eruption (PFE) is a genetic disorder exhibiting the cessation of tooth eruption. Loss-of-function mutations in parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor (PTH/PTHrP receptor, PPR) were reported as the underlying cause of this disorder in humans. We showed in a PFE mouse model that PTHrP-PPR signaling is responsible for normal dental follicle cell differentiation and tooth eruption. However, the mechanism underlying the eruption defect in PFE remains undefined. In this descriptive study, we aim to chronologically observe tooth eruption and root formation of mouse PFE molars through 3D microCT analyses. SETTING AND SAMPLE POPULATION:Two individuals with PFE were recruited at Showa University. A mouse PFE model was generated by deleting PPR specifically in PTHrP-expressing dental follicle and divided into three groups, PPR ;R26R (Control), PTHrP-creER;PPR ;R26R (cHet), and PTHrP-creER;PRR ;R26R (cKO). MATERIALS AND METHODS:Images from human PFE subjects were acquired by CBCT. All groups of mouse samples were studied at postnatal days 14, 25, 91, and 182 after a tamoxifen pulse at P3, and superimposition of 3D microCT images among three groups was rendered. RESULTS:Mouse and human PFE molars exhibited a similar presentation in the 3D CT analyses. The quantitative analysis in mice demonstrated a statistically significant decrease in the eruption height of cKO first and second molars compared to other groups after postnatal day 25. Additionally, cKO molars demonstrated significantly shortened roots with dilacerations associated with the reduced interradicular bone height. CONCLUSIONS:Mouse PFE molars erupt at a much slower rate compared to normal molars, associated with shortened and dilacerated roots and defective interradicular bones. 10.1111/odi.13249
    PTH intermittent administration may be a useful therapeutic agent to avoid premature eruption of the tooth. Vasconcelos Daniel Fernando Pereira,Vasconcelos Any Carolina Cardoso Guimarães Medical hypotheses Parathyroid hormone (PTH) acts as a controller of bone remodeling and has influence on periodontal tissues. In addition to the well-established catabolic effects (activation of bone resorption) of PTH, it is recognized that the PTH intermittent administration has anabolic effects (promotion of bone formation). However, there is no information regarding the effects of the PTH intermittent administration on the eruption tooth rate. Studies have shown that tooth eruption depends on the presence of osteoclasts to create an eruption pathway through the alveolar bone. It may also be controlled by osteoblast, precursor of osteoclast, and cells of periodontal ligament. Our hypothesis is based on previous studies showing that the PTH intermittent administration can promote bone formation, particularly in the areas around which the tooth erupts. Furthermore, the PTH intermittent administration influenced periodontal ligament fiber, what may be seen as greater organization, and isomerization, as well as higher birefringence of the periodontal ligament fiber, which then offers increased resistance to the process, delaying tooth eruption. Thus, this article opens new perspectives for the treatment and maintenance of teeth that can erupt early. 10.1016/j.mehy.2015.12.025
    Failure of tooth eruption and brachydactyly in pseudohypoparathyroidism are not related to plasma parathyroid hormone-related protein levels. Reis Mariana Tenorio Antunes,Matias Diogo Toledo,Faria Maria Estela Justamante de,Martin Regina Matsunaga Bone BACKGROUND:Pseudohypoparathyroidism (PHP) is a genetic disorder characterized by resistance to the peripheral action of PTH due to maternally inherited heterozygous inactivating mutations in the coding sequence of Gsα or intronic regions of GNAS leading to aberrant splice variants (PHP1A), or methylation defects at GNAS (PHP1B). Brachydactyly is a clinical feature associated with both PHP1A and PHP1B, although it is more frequent in PHP1A patients. Loss-of-function mutations in PTHLH, the gene coding for parathyroid hormone related protein (PTHrP) were previously described in some patients with brachydactyly. Primary failure of tooth eruption (PFE) is related to some syndromes involving skeletal development, but it is also known as a nonsyndromic autosomal dominant condition. Previous studies showed that familial nonsyndromic PFE is caused by heterozygous mutations in the gene encoding the G protein-coupled receptor (PTH1R) for PTH and PTHrP. Thus, we hypothesized that PTHrP resistance could result in failure of tooth eruption (FTE) and/or brachydactyly in PHP. SUBJECTS AND METHODS:Nineteen patients with a molecular diagnosis of PHP underwent dental panoramic radiography (DPR), hand radiography and had their PTHrP levels measured. Patients with alterations at DPR were submitted to clinical dental evaluation. RESULTS:Nine patients had FTE and 7 patients had brachydactyly; 4 patients presented both features and none of them presented high PTHrP levels. Fourteen patients had PTHrP levels within the normal range and only one patient had slightly elevated PTHrP levels. Additionally, three novel GNAS mutations were described. CONCLUSION:We described the dental abnormalities in a large series of PHP patients that were followed in a single tertiary center. No relationship between plasma PTHrP levels and failure of tooth eruption, dental manifestations of PHP or brachydactyly was found. It is important that doctors pay attention to dental manifestations of the disease in order to refer patients to a proper care with dentists. 10.1016/j.bone.2016.02.002
    Autocrine regulation of mesenchymal progenitor cell fates orchestrates tooth eruption. Takahashi Akira,Nagata Mizuki,Gupta Aditi,Matsushita Yuki,Yamaguchi Tetsutaro,Mizuhashi Koji,Maki Koutaro,Ruellas Antonio C,Cevidanes Lucia S,Kronenberg Henry M,Ono Noriaki,Ono Wanida Proceedings of the National Academy of Sciences of the United States of America Formation of functional skeletal tissues requires highly organized steps of mesenchymal progenitor cell differentiation. The dental follicle (DF) surrounding the developing tooth harbors mesenchymal progenitor cells for various differentiated cells constituting the tooth root-bone interface and coordinates tooth eruption in a manner dependent on signaling by parathyroid hormone-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR). However, the identity of mesenchymal progenitor cells in the DF and how they are regulated by PTHrP-PPR signaling remain unknown. Here, we show that the PTHrP-PPR autocrine signal maintains physiological cell fates of DF mesenchymal progenitor cells to establish the functional periodontal attachment apparatus and orchestrates tooth eruption. A single-cell RNA-seq analysis revealed cellular heterogeneity of PTHrP cells, wherein PTHrP DF subpopulations abundantly express PPR. Cell lineage analysis using tamoxifen-inducible mice revealed that PTHrP DF cells differentiate into cementoblasts on the acellular cementum, periodontal ligament cells, and alveolar cryptal bone osteoblasts during tooth root formation. PPR deficiency induced a cell fate shift of PTHrP DF mesenchymal progenitor cells to nonphysiological cementoblast-like cells precociously forming the cellular cementum on the root surface associated with up-regulation of and matrix proteins, resulting in loss of the proper periodontal attachment apparatus and primary failure of tooth eruption, closely resembling human genetic conditions caused by PPR mutations. These findings reveal a unique mechanism whereby proper cell fates of mesenchymal progenitor cells are tightly maintained by an autocrine system mediated by PTHrP-PPR signaling to achieve functional formation of skeletal tissues. 10.1073/pnas.1810200115
    Null mutation of chloride channel 7 (Clcn7) impairs dental root formation but does not affect enamel mineralization. Guo Jing,Bervoets Theodore J M,Henriksen Kim,Everts Vincent,Bronckers Antonius L J J Cell and tissue research ClC-7, located in late endosomes and lysosomes, is critical for the function of osteoclasts. Secretion of Cl(-) by the ruffled border of osteoclasts enables H(+) secretion by v-H(+)-ATPases to dissolve bone mineral. Mice lacking ClC-7 show altered lysosomal function that leads to severe lysosomal storage. Maturation ameloblasts are epithelial cells with a ruffled border that secrete Cl(-) as well as endocytose and digest large quantities of enamel matrix proteins during formation of dental enamel. We tested the hypothesis that ClC-7 in maturation ameloblasts is required for intracellular digestion of matrix fragments to complete enamel mineralization. Craniofacial bones and developing teeth in Clcn7(-/-) mice were examined by micro-CT, immunohistochemistry, quantified histomorphometry and electron microscopy. Osteoclasts and ameloblasts in wild-type mice stained intensely with anti-ClC-7 antibody but not in Clcn7(-/-) mice. Craniofacial bones in Clcn7(-/-) mice were severely osteopetrotic and contained 1.4- to 1.6-fold more bone volume, which was less mineralized than the wild-type littermates. In Clcn7(-/-) mice maturation ameloblasts and osteoclasts highly expressed Ae2 as in wild-type mice. However, teeth failed to erupt, incisors were much shorter and roots were disfigured. Molars formed a normal dental crown. In compacted teeth, dentin was slightly less mineralized, enamel did not retain a matrix and mineralized fairly normal. We concluded that ClC-7 is essential for osteoclasts to resorb craniofacial bones to enable tooth eruption and root development. Disruption of Clcn7 reduces bone and dentin mineral density but does not affect enamel mineralization. 10.1007/s00441-015-2263-z
    Root and Eruption Defects in c-Fos Mice Are Driven by Loss of Osteoclasts. Alfaqeeh S,Oralova V,Foxworthy M,Matalova E,Grigoriadis A E,Tucker A S Journal of dental research c-Fos homozygous mice lack osteoclasts with a failure of the teeth to erupt and with an arrest of root development. Here, we characterize the defects associated with the failure in root development and the loss of the tooth-bone interface, and we investigate the underlying causes. We show that, while homozygous c-Fos mice have no multinucleated osteoclasts, heterozygous mice have a reduction in the number of osteoclasts with a reduction in the tooth-bone interface during development and subtle skeletal defects postnatally. In the homozygous mutants bone is found to penetrate the tooth, particularly at the apical end, physically disrupting the root forming HERS (Hertwig's epithelial root sheath) cells. The cells of the HERS continue to proliferate but cannot extend downward due to the presence of bone, leading to a loss of root formation. Tooth germ culture showed that the developing tooth invaded the static bone in mutant tissue, rather than the bone encroaching on the tooth. Although c-Fos has been shown to be expressed in developing teeth, the defect in maintenance of the tooth-bone interface appears to be driven solely by the lack of osteoclasts, as this defect can be rescued in the presence of donor osteoclasts. The rescue suggests that signals from the tooth recruit osteoclasts to clear the bone from around the tooth, allowing the tooth to grow, form roots, and later erupt. 10.1177/0022034515608828
    Loss of epithelial FAM20A in mice causes amelogenesis imperfecta, tooth eruption delay and gingival overgrowth. Li Li-Li,Liu Pei-Hong,Xie Xiao-Hua,Ma Su,Liu Chao,Chen Li,Qin Chun-Lin International journal of oral science FAM20A has been studied to a very limited extent. Mutations in human FAM20A cause amelogenesis imperfecta, gingival fibromatosis and kidney problems. It would be desirable to systemically analyse the expression of FAM20A in dental tissues and to assess the pathological changes when this molecule is specifically nullified in individual tissues. Recently, we generated mice with a Fam20A-floxed allele containing the beta-galactosidase reporter gene. We analysed FAM20A expression in dental tissues using X-Gal staining, immunohistochemistry and in situ hybridization, which showed that the ameloblasts in the mouse mandibular first molar began to express FAM20A at 1 day after birth, and the reduced enamel epithelium in erupting molars expressed a significant level of FAM20A. By breeding K14-Cre mice with Fam20A(flox/flox) mice, we created K14-Cre;Fam20A(flox/flox) (conditional knock out, cKO) mice, in which Fam20A was inactivated in the epithelium. We analysed the dental tissues of cKO mice using X-ray radiography, histology and immunohistochemistry. The molar enamel matrix in cKO mice was much thinner than normal and was often separated from the dentinoenamel junction. The Fam20A-deficient ameloblasts were non-polarized and disorganized and were detached from the enamel matrix. The enamel abnormality in cKO mice was consistent with the diagnosis of amelogenesis imperfecta. The levels of enamelin and matrix metalloproteinase 20 were lower in the ameloblasts and enamel of cKO mice than the normal mice. The cKO mice had remarkable delays in the eruption of molars and hyperplasia of the gingival epithelium. The findings emphasize the essential roles of FAM20A in the development of dental and oral tissues. 10.1038/ijos.2016.14
    Gene-Expression Analysis Identifies IGFBP2 Dysregulation in Dental Pulp Cells From Human Cleidocranial Dysplasia. Greene Stephen L,Mamaeva Olga,Crossman David K,Lu Changming,MacDougall Mary Frontiers in genetics Cleidocranial dysplasia (CCD) is an autosomal dominant disorder affecting osteoblast differentiation, chondrocyte maturation, skeletal morphogenesis, and tooth formation. Dental phenotype in CCD include over-retained primary teeth, failed eruption of permanent teeth, and supernumerary teeth. The underlying mechanism is unclear. We previously reported one CCD patient with allelic deletion (CCD-011). In the current study, we determined the transcriptomic profiles of dental pulp cells from this patient compared to one sex-and-age matched non-affected individual. Next Generation RNA sequencing revealed that 60 genes were significantly dysregulated (63% upregulated and 27% downregulated). Among them, (insulin-like growth factor binding protein-2) was found to be upregulated more than twofold in comparison to control cells. Stable overexpression of RUNX2 in CCD-011 pulp cells resulted in the reduction of . Moreover, expression was up-regulated in CCD-011 pulp cells after introduction of normal RUNX2. Promoter analysis revealed that there are four proximal putative RUNX2 binding sites in -1.5 kb promoter region. Relative luciferase assay confirmed that is a direct target of RUNX2. Immunohistochemistry demonstrated that IGFBP2 was expressed in odontoblasts but not ameloblasts. This report demonstrated the importance of RUNX2 in the regulation of gene profile related to dental pulp cells and provided novel insight of RUNX2 into the negative regulation of IGFBP2. 10.3389/fgene.2018.00178
    Bone resorption deficiency affects tooth root development in RANKL mutant mice due to attenuated IGF-1 signaling in radicular odontoblasts. Huang Hong,Wang Jue,Zhang Yan,Zhu Guochun,Li Yi-Ping,Ping Ji,Chen Wei Bone The tooth root is essential for normal tooth physiological function. Studies on mice with mutations or targeted gene deletions revealed that osteoclasts (OCs) play an important role in tooth root development. However, knowledge on the cellular and molecular mechanism underlying how OCs mediate root formation is limited. During bone formation, growth factors (e.g. Insulin-like growth factor-1, IGF-1) liberated from bone matrix by osteoclastic bone resorption stimulate osteoblast differentiation. Thus, we hypothesize that OC-osteoblast coupling may also apply to OC-odontoblast coupling; therefore OCs may have a direct impact on odontoblast differentiation through the release of growth factor(s) from bone matrix, and consequently regulate tooth root formation. To test this hypothesis, we used a receptor activator of NF-κB ligand (RANKL) knockout mouse model in which OC differentiation and function was entirely blocked. We found that molar root formation and tooth eruption were defective in RANKL mice. Disrupted elongation and disorganization of Hertwig's epithelial root sheath (HERS) was observed in RANKL mice. Reduced expression of nuclear factor I C (NFIC), osterix, and dentin sialoprotein, markers essential for radicular (root) odontogenic cell differentiation indicated that odontoblast differentiation was disrupted in RANKL deficient mice likely contributing to the defect in root formation. Moreover, down-regulation of IGF/AKT/mTOR activity in odontoblast indicated that IGF signaling transduction in odontoblasts of the mutant mice was impaired. Treating odontoblast cells in vitro with conditioned medium from RANKL OCs cultured on bone slices resulted in inhibition of odontoblast differentiation. Moreover, depletion of IGF-1 in bone resorption-conditioned medium (BRCM) from wild-type (WT) OC significantly compromised the ability of WT osteoclastic BRCM to induce odontoblast differentiation while addition of IGF-1 into RANKL osteoclastic BRCM rescued impaired odontoblast differentiation, confirming that root and eruption defect in RANKL deficiency mice may result from failure of releasing of IGF-1 from bone matrix through OC bone resorption. These results suggest that OCs are important for odontoblast differentiation and tooth root formation, possibly through IGF/AKT/mTOR signaling mediated by cell-bone matrix interaction. These findings provide significant insights into regulatory mechanism of tooth root development, and also lay the foundation for root regeneration studies. 10.1016/j.bone.2017.12.026
    Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age-Dependent Change in Osteoclast Number, and a Reduced Number of Osteoblasts In Vivo. Zhu Meiling,Sun Ben-Hua,Saar Katarzyna,Simpson Christine,Troiano Nancy,Dallas Sarah L,Tiede-Lewis LeAnn M,Nevius Erin,Pereira João P,Weinstein Robert S,Tommasini Steven M,Insogna Karl L Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism. 10.1002/jbmr.2733
    Differential diagnosis of primary failure of eruption (PFE) with and without evidence of pathogenic mutations in the PTHR1 gene. Pilz P,Meyer-Marcotty P,Eigenthaler M,Roth H,Weber B H F,Stellzig-Eisenhauer A Journal of orofacial orthopedics = Fortschritte der Kieferorthopadie : Organ/official journal Deutsche Gesellschaft fur Kieferorthopadie BACKGROUND:Primary failure of eruption (PFE) may be associated with pathogenic mutations in the PTHR1 gene. It has numerous manifestations and is characterized by severe posterior open bite. However, there are also phenotypically similar types of eruption anomalies not associated with a known pathogenic PTHR1 mutation. The purpose of this study was to evaluate whether a distinction can be made between PTHR1-mutation carriers and noncarriers based on clinical and radiological findings. PATIENTS AND METHODS:A total of 36 patients with suspected PFE diagnoses were included and analyzed in accordance with specific clinical and radiographic criteria. In addition, all patients underwent Sanger DNA sequencing analysis of all coding sequences (and the immediate flanking intronic sequences) of the PTHR1 gene. RESULTS:Of these patients, 23 exhibited a heterozygous pathogenic mutation in the PTHR1 gene (PTHR1-mutation carriers), while molecular genetic analysis revealed nosequence alteration in the other 13 patients (non-PTHR1-mutation carriers). Relevant family histories were obtained from 5 patients in the carrier group; hence, this group included a total of 13 familial and 10 simplex cases. The group of noncarriers revealed no relevant family histories. All patients in the carrier group met six of the clinical and radiographic criteria explored in this study: (1) posterior teeth more often affected; (2) eruption disturbance of an anterior tooth in association with additional posterior-teeth involvement; (3) affected teeth resorbing the alveolar bone located coronal to them; (4) involvement of both deciduous and permanent teeth; (5) impaired vertical alveolar-process growth; and (6) severe subsequent finding of posterior open bite. None of the analyzed criteria were, by contrast, met by all patients in the noncarrier group. All patients in the carrier group could be assigned to one of three classifications indicating the extent of eruption disturbance, whereas 4 of the 13 noncarriers presented none of these three patterns. The clinical and radiographic criteria employed in this study would have correctly identified 10 of the 13 PFE patients in the noncarrier group as possessing no detectable PTHR1 mutation. CONCLUSION:The evaluation of clinical and radiographic characteristics can heighten the specificity of ruling out suspected PTHR1 involvement in PFE patients. A hereditary element of PTHR1-associated PFE is clearly identifiable. More studies with more patients are needed to optimize the sensitivity of this preliminary approach on the differential identification of PTHR1-mutation carriers versus noncarriers by multivariate analysis. 10.1007/s00056-014-0215-y
    Primary failure of eruption of teeth in two siblings with a novel mutation in the PTH1R gene. Aziz S,Hermann N V,Dunø M,Risom L,Daugaard-Jensen J,Kreiborg S European archives of paediatric dentistry : official journal of the European Academy of Paediatric Dentistry BACKGROUND:Primary failure of tooth eruption (PFE) is a rare non-syndromic disorder with prematurely ceased eruption of the posterior teeth, despite clearance by bone resorption of the eruption path. It is generally agreed that most of the impacted teeth are second molars that are deeply seated, and without symptoms. Traditionally, patients with failure of tooth eruption undergo surgical and/or orthodontic treatment. However, patients with PTH1R mutations have no beneficial effect of such a regime and PFE is therefore important to diagnose. CASE REPORT AND FOLLOW-UP:A family with three PFE affected members in two generations, involving both the primary and permanent dentitions, and a novel mutation in the PTH1R gene are reported. Furthermore, the treatment of the eruption failure was documented in one of the cases. CONCLUSION:In the present study, the proband initially only had a minor clinical problem, lack of eruption of the primary second left mandibular molar. However, over time several problems appeared in the permanent dentition. Clinical signs of PFE should lead one to look for similar dental problems in related family members and to molecular DNA testing. Confirmation of the diagnosis PFE in young children has the advantage that unnecessary treatment can be avoided, since early orthodontic intervention for these patients is futile. Once growth is complete, several multidisciplinary treatment strategies can partially solve the posterior open bite malocclusion that is characteristic of this disorder. Treatment should be planned in cooperation with specialists who are used to treating PFE patients. 10.1007/s40368-018-00410-8
    [Innovations in diagnosis and treatment about a case of primary failure eruption linked to a PTHR1 gene mutation]. Raberin Monique,Diesmusch Caroline,Cordier Marie-Pierre,Farges Jean-Christophe L' Orthodontie francaise Primary failure of eruption is a rare condition marked by non-eruption of the posterior teeth due to mutation of a gene responsible for tooth eruption. Today, this anomaly can be detected early using innovative 3D-imaging techniques. Genetic and histologic testing will confirm the diagnosis and unfavorable prognosis. Alveolar growth must be followed in other areas too in order to avoid structural and functional asymmetry. An analysis of the diagnostic and therapeutic options using bone-borne anchorage is presented via the long-term monitoring of a female patient presenting primary failure of eruption linked to mutation of the PTHR1 gene. 10.1051/orthodfr/2015025
    Receptor activator of nuclear factor-kappa ligand, OPG, and IGF-I expression during orthodontically induced inflammatory root resorption in the recombinant human growth hormone-treated rats. Hu Yajun,Liu Wentao,Liu Zhijian,Kuang Wenying,He Hong The Angle orthodontist OBJECTIVE:To investigate the effects of growth hormone (GH) on local receptor activator of nuclear factor-kappa ligand (RANKL), OPG, and IGF-I expression during orthodontically induced inflammatory root resorption in rats. MATERIALS AND METHODS:Forty Wistar rats (gender: male; age: 7 weeks) were randomly divided into control and experimental groups. A force of 50 g was applied to move the right upper first molars mesially. The experimental and control groups received daily subcutaneous injections of recombinant human growth hormone (GH; 2 mg/kg) and equivalent volumes of saline, respectively. The rats were sacrificed on days 1, 3, 7, and 14. Micro-computed tomography-reconstructed images of the upper right first molars were used to survey root resorption and tooth movement. Horizontal sections of the maxillae were prepared for hematoxylin and eosin, tartrate-resistant acid phosphatase, and immunohistochemical staining. RESULTS:Resorption lacunae appeared on the compressed side of the distal buccal root of the right first molar on days 7 and 14. Compared with the control groups, GH-treated groups showed more RANKL-positive cells and osteoclasts on day 3 and more OPG- and IGF-I-positive cells and fewer odontoclasts on days 7 and 14. Indexes of root resorption were lower and tooth movement was faster in the GH-treated groups than in the control groups on days 7 and 14. CONCLUSIONS:The inhibitory effect of GH on root resorption by heavy force might be mediated by RANKL/OPG and IGF-I. Short-term GH administration may be a method with which to reduce root resorption and shorten treatment time, especially in patients who are susceptible to root resorption. 10.2319/052014-361.1
    Growth hormone receptor gene is related to root length and tooth length in human teeth. Hikita Yu,Yamaguchi Tetsutaro,Tomita Daisuke,Adel Mohamed,Nakawaki Takatoshi,Katayama Koshu,Maki Koutaro,Kimura Ryosuke The Angle orthodontist OBJECTIVES:To examine the relationship between tooth length and growth hormone receptor (GHR) gene variants in a healthy Japanese population. MATERIALS AND METHODS:The subjects consisted of 193 Japanese adults (69 men, 124 women), aged 13 to 56 years. Genomic DNA was extracted from saliva and genotyped GHR rs6184 and rs6180 variants using the Taqman genotyping. Computed tomography (CT) images were acquired using a dental cone-beam CT scanner and reconstructed using open-source OsiriX medical image processing software. The maxillary (upper; U) and mandibular (lower, L) central incisors (1), lateral incisors (2), canines (3), first premolars (4), second premolars (5), first molars (6), and second premolars (7) were evaluated. Teeth were assessed for crown height (CH), root length (RL), overall tooth length (C+R), and crown to root ratio (C/R). The relationships between GHR variants and CH, RL, C+R, and C/R were statistically examined. RESULTS:The GHR variant rs6184 was associated with the root lengths and tooth length for the upper and lower lateral incisors and upper canines (U2 RL; U3 RL, C+R; L2 RL [ P < .05]). CONCLUSIONS:The results indicate that the GHR rs6184 variant is associated with tooth length and ratio dimensions in a Japanese cohort. Further studies utilizing a larger sample size are needed to confirm this finding. 10.2319/092917-659.1
    Primary failure of eruption: Clinical and genetic findings in the mixed dentition. Grippaudo Cristina,Cafiero Concetta,D'Apolito Isabella,Ricci Beatrice,Frazier-Bowers Sylvia A The Angle orthodontist OBJECTIVE:To test the hypothesis that mutations in the parathyroid hormone 1 receptor ( PTH1R) include effects in both primary and permanent teeth. MATERIALS AND METHODS:DNA was extracted from saliva samples of 29 patients (8 familial and 21 sporadic) who presented with clinical evidence of infraoccluded teeth, and their unaffected relatives (N = 22). Sequencing followed by mutational analysis of the coding regions of PTH1R gene was completed for all individuals (N = 29). RESULTS:Eight of 29 cases revealed a heterozygous pathogenic variant in the PTH1R gene; five of eight variants represented distinct mutations based on comparison with the dbSNP, HGMD, and ESP databases. One mutation (c.1765 T>C p.Trp89Arg) was found to segregate within a family (n = 3). In silico analyses for all variants revealed a putative pathogenic effect. A genotype-phenotype correlation was reported as defined by a functional mutation in PTH1R and corresponding effects on one or more posterior teeth only; unilateral or bilateral involvement, infraoccluded primary teeth. CONCLUSIONS:Novel mutations were reported in the PTH1R gene that included PFE-affected primary molars, thus providing the basis for using a genetic diagnostic tool for early diagnosis leading to proper management. 10.2319/062717-430.1
    Twenty-year follow-up of a familial case of PTH1R-associated primary failure of tooth eruption. Kanno Cláudia Misue,de Oliveira José Américo,Garcia José Fernando,Roth Helmut,Weber Bernhard H F American journal of orthodontics and dentofacial orthopedics : official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics INTRODUCTION:Nonsyndromic primary failure of eruption (PFE) is a rare autosomal dominant disorder of dental eruption with no obvious dental or soft tissue interference. The purposes of this study were to genetically and clinically characterize a family with many members affected by PFE and to describe the natural evolution of the disorder. METHODS:Three generations of a family with 18 members, 10 of them clinically affected by PFE, were evaluated periodically during 20 years of clinical follow-up. PFE was observed in varying degrees of severity in both sexes. Clinical presentation became more severe in adulthood. One patient had spontaneous reeruption of 2 posterior teeth. Cervical root resorptions were observed in 3 members. Genetic analysis showed a deleterious heterozygous mutation in intron 9 of the PTH1R gene (c.639-2A>G) and diagnosed an additional affected member. CONCLUSIONS:The long-term follow-up of PFE cases in this family permitted the following observations: (1) the onset occurred from the preemergence to the postemergence phases, (2) PFE appeared to be closely related to ankylosis, (3) affected teeth maintained the eruptive potential even in adulthood, (4) the earlier the onset the more severe the open bite, and (5) cervical root resorptions occurred in 3 affected members. 10.1016/j.ajodo.2016.09.012
    Detection of the gubernacular canal and its attachment to the dental follicle may indicate an abnormal eruption status. Gaêta-Araujo Hugo,da Silva Matheus Bronetti,Tirapelli Camila,Freitas Deborah Queiroz,de Oliveira-Santos Christiano The Angle orthodontist OBJECTIVES:To evaluate and compare the detection of gubernacular canals (GC) and their characteristics in normal and abnormal tooth eruption. MATERIALS AND METHODS:Patients with unerupted teeth were classified according to sex and age. Each tooth was classified according to dental group, eruption status, formation status, angulation, and GC detection. The opening of the GC in the alveolar crest and the attachment sites in relation to the dental follicle were assessed. Data were analyzed by the chi-square and Kruskal-Wallis tests, with a significance level of 5%. RESULTS:Cone-beam computed tomography scans of 159 patients were evaluated. The final sample (N = 598) consisted of 423 teeth with normal eruption, 140 impacted teeth, and 35 teeth with delayed eruption. The overall detection rate of GC was 90.6%. These rates were 94.1%, 87.1%, and 62.9% for normal eruption, impacted teeth, and delayed eruption, respectively. GC detection rates were higher in the early stages of tooth formation in normal tooth eruption and in impacted teeth. The rate of GC detection was even lower in delayed teeth when they were angulated. Unusual attachment sites of the GC to the dental follicle were associated with abnormal eruption status. CONCLUSIONS:The results of the present study suggest that GC characteristics may indicate an abnormal eruption status. 10.2319/090518-651.1
    RUNX2 mutation reduces osteogenic differentiation of dental follicle cells in cleidocranial dysplasia. Liu Yang,Wang Yixiang,Sun Xiangyu,Zhang Xianli,Wang Xiaozhe,Zhang Chenying,Zheng Shuguo Mutagenesis Disturbed permanent tooth eruption is common in cleidocranial dysplasia (CCD), a skeletal disorder caused by heterozygous mutation of RUNX2, but the mechanism underlying is still unclear. As it is well known that dental follicle cells (DFCs) play a critical role in tooth eruption, the changed biological characteristics of DFCs might give rise to disturbance of permanent tooth eruption in CCD patients. Thus, primary DFCs from one CCD patient and normal controls were collected to investigate the effect of RUNX2 mutation on the bone remodeling activity of DFCs and explore the mechanism of impaired permanent tooth eruption in this disease. Conservation and secondary structure analysis revealed that the RUNX2 mutation (c.514delT, p.172fs) found in the present CCD patient was located in the highly conserved RUNT domain and converted the structure of RUNX2. After osteogenic induction, we found that the mineralised capacity of DFCs and the expression of osteoblast-related genes, including RUNX2, ALP, OSX, OCN and Col Iα1, in DFCs was severely interfered by the RUNX2 mutation found in CCD patients. To investigate whether the osteogenic deficiency of DFCs from the CCD patient can be rescued by RUNX2 restoration, we performed 'rescue' experiments. Surprisingly, the osteogenic deficiency and the abnormal expression of osteoblast-associated genes in DFCs from the CCD patient were almost rescued by overexpression of wild-type RUNX2 using lentivirus. All these findings indicate that RUNX2 mutation can reduce the osteogenic capacity of DFCs through inhibiting osteoblast-associated genes, thereby disturbing alveolar bone formation, which serves as a motive force for tooth eruption. This effect may provide valuable explanations and implications for the mechanism of delayed permanent tooth eruption in CCD patients. 10.1093/mutage/gey010
    Dental and Cranial Pathologies in Mice Lacking the Cl(-) /H(+) -Exchanger ClC-7. Wen Xin,Lacruz Rodrigo S,Paine Michael L Anatomical record (Hoboken, N.J. : 2007) ClC-7 is a 2Cl(-) /1H(+) -exchanger expressed at late endosomes and lysosomes, as well as the ruffled border of osteoclasts. ClC-7 deficiencies in mice and humans lead to impaired osteoclast function and therefore osteopetrosis. Failure of tooth eruption is also apparent in ClC-7 mutant animals, and this has been attributed to the osteoclast dysfunction and the subsequent defect in alveolar bone resorptive activity surrounding tooth roots. Ameloblasts also express ClC-7, and this study aims to determine the significance of ClC-7 in enamel formation by examining the dentitions of ClC-7 mutant mice. Micro-CT analysis revealed that the molar teeth of 3-week old ClC-7 mutant mice had no roots, and the incisors were smaller than their age-matched controls. Despite these notable developmental differences, the enamel and dentin densities of the mutant mice were comparable to those of the wild-type littermates. Scanning electron microscopy showed normal enamel crystallite and prismatic organization in the ClC-7 mutant mice, although the enamel was thinner (hypoplastic) than in controls. These results suggested that ClC-7 was not critical to enamel and dentin formation, and the observed tooth defects may be related more to a resulting alveolar bone phenotype. Micro-CT analysis also revealed abnormal features in the calvarial bones of the mutant mice. The cranial sutures in ClC-7 mutant mice remained open compared to the closed sutures seen in the control mice at 3 weeks. These data demonstrate that ClC-7 deficiency impacts the development of the dentition and calvaria, but does not significantly disrupt amelogenesis. 10.1002/ar.23118
    Hmga2 regulation of tooth formation and association with Sox2 and Nanog expression. Kodama Yuki,Harinath Devipriya,Mihara-Tomiyama Nozomi,Tominaga Noriko,Ide Yoshiaki,Nakahara Taka,Maeda Munehiro,Igarashi Masaru,D'Armiento Jeanine,Chada Kiran,Imai Kazushi Biochemical and biophysical research communications Tooth formation is accomplished under strict genetic programs. Although patients with chromosome 12q14 aberration shows tooth phenotype including the size and eruption timing with bone growth anomaly, its etiology is uncertain. Here, we examined expression of Hmga2, which is encoded at chromosome 12q14, in mouse tooth germs and analyzed the involvement in lower first molar (M1) and mandibular bone development. Hmga2 expression was immunohistochemically detected at enamel organ and the surrounding mesenchyme of the M1 germs. The expression was dynamically changed with gestation and rapidly decreased in postnatal mice. In Hmga2 mice, the M1 germs and crowns were diminished in size, and formation and eruption of molars were delayed with mandibular bone growth retardation. Hmga2 cDNA or siRNA transfection showed that Hmga2 transcriptionally up-regulates expression of stem cell factors, Sox2 and Nanog. They were co-localized with Hmga2 in the germs, but differentially distributed at enamel organ and mesenchyme in Hmga2 mice. These results demonstrate that Hmga2 expressed in tooth germs regulates the growth, sizing and eruption and stem cell factor expression in different compartment of the germ and associates with mandibular bone growth. Although future studies are needed, the present study demonstrates HMGA2 regulation of tooth genesis with skeletal development. 10.1016/j.bbrc.2019.01.017
    Biological Effects of Anti-RANKL Antibody and Zoledronic Acid on Growth and Tooth Eruption in Growing Mice. Isawa Motoki,Karakawa Akiko,Sakai Nobuhiro,Nishina Saki,Kuritani Miku,Chatani Masahiro,Negishi-Koga Takako,Sato Masashi,Inoue Mitsuko,Shimada Yukie,Takami Masamichi Scientific reports The anti-bone resorptive drugs denosumab, an anti-human-RANKL antibody, and zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, have recently been applied for treatment of pediatric patients with bone diseases, though details regarding their effects in growing children have yet to be fully elucidated. In the present study, we administered these anti-resorptive drugs to mice from the age of 1 week and continued once-weekly injections for a total of 7 times. Mice that received the anti-RANKL antibody displayed normal growth and tooth eruption, though osteopetrotic bone volume gain in long and alveolar bones was noted, while there were nearly no osteoclasts and a normal of number osteoblasts observed. In contrast, ZOL significantly delayed body growth, tooth root formation, and tooth eruption, with increased osteoclast and decreased osteoblast numbers. These findings suggest regulation of tooth eruption via osteoblast differentiation by some types of anti-resorptive drugs. 10.1038/s41598-019-56151-1
    Interaction between fibronectin and β1 integrin is essential for tooth development. Saito Kan,Fukumoto Emiko,Yamada Aya,Yuasa Kenji,Yoshizaki Keigo,Iwamoto Tsutomu,Saito Masahiro,Nakamura Takashi,Fukumoto Satoshi PloS one The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation. 10.1371/journal.pone.0121667
    Dental Follicle Cells Participate in Tooth Eruption via the RUNX2-MiR-31-SATB2 Loop. Ge J,Guo S,Fu Y,Zhou P,Zhang P,Du Y,Li M,Cheng J,Jiang H Journal of dental research Cleidocranial dysplasia (CCD) is characterized by the runt-related transcription factor 2 (RUNX2) mutation, which results in delayed tooth eruption due to disturbed functions of dental follicle. Accumulating evidence has revealed a key regulatory circuit, including RUNX2, miR-31, and special AT-rich binding protein 2 (SATB2) acting in concert in mesenchymal stem cell homeostasis and functions. However, whether such a regulatory loop works in dental follicle cells (DFCs) remains unknown. Herein, we investigated the roles of RUNX2-miR-31-SATB2 in DFCs from patients with CCD (DFCs-CCD) to advance our understanding regarding physical tooth eruption. We identified a novel mutation on exon 5 (c.634T>G, p.T212P) in RUNX2 via exome sequencing in the CCD patient with typical clinical presentations. Compared with DFCs from healthy donors, DFCs-CCD displayed significantly lower osteogenic, osteoclast-inductive, and matrix-degrading capacities and had lower RUNX2 (a transcriptional inhibitor of miR-31), higher miR-31, and downregulated SATB2. Lower ratios of RANKL/OPG and RANKL/RANK, as well as decreased expression of matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 2 (MMP2), would lead to inactivation of osteoclasts and suppression of bone matrix remodeling in DFCs-CCD. Furthermore, the roles of the RUNX2-miR-31-SATB2 loop in DFCs-CCD were revealed by endogenous miR-31 knockdown, which resulted in increased SATB2 and RUNX2, as well as osteoclast-inductive and matrix degradation capacities. Conversely, SATB2, RUNX2, MMP9, MMP2, and osteoclast-inductive factors expression declined upon ectopic miR-31 overexpression in normal DFCs. Importantly, neonatal mice with in vivo siRUNX2 delivery exhibited less activated osteoclasts around dental follicles and delayed tooth eruption. Together, these results suggest that RUNX2 mutation/haploinsufficiency disturbs osteoclast-inductive signaling in DFCs, which may be responsible for delayed tooth eruption in CCD patients. Manipulation of the RUNX2-miR-31-SATB2 loop may be a potential way to facilitate tooth eruption in CCD patients. 10.1177/0022034515578908
    Essential Roles of Bone Morphogenetic Protein-1 and Mammalian Tolloid-like 1 in Postnatal Root Dentin Formation. Wang Jun,Muir Alison M,Ren Yinshi,Massoudi Dawiyat,Greenspan Daniel S,Feng Jian Q Journal of endodontics INTRODUCTION:Mutations in the proteinase bone morphogenetic protein-1 (BMP1) were recently identified in patients with osteogenesis imperfecta, which can be associated with type 1 dentinogenesis imperfecta. BMP1 is co-expressed in various tissues and has overlapping activities with the closely related proteinase mammalian tolloid-like 1 (TLL1). In this study we investigated whether removing the overlapping activities of BMP1 and TLL1 affects the mineralization of tooth root dentin. METHODS:Floxed alleles of the BMP1 and TLL1 genes were excised via ubiquitously expressed Cre induced by tamoxifen treatment beginning at 3 days of age (harvested at 3 weeks of age) or beginning at 4 weeks of age (harvested at 8 weeks of age). Multiple techniques, including x-ray analysis, double-labeling with calcein and alizarin red stains for measurement of dentin formation rate, and histologic and immunostaining assays, were used to analyze the dentin phenotype. RESULTS:BMP1/TLL1 double knockout mice displayed short and thin root dentin, defects in dentin mineralization, and delayed tooth eruption. Molecular mechanism studies revealed accumulation of collagens in dentin and a sharp reduction in non-collagenous proteins such as dentin matrix protein 1 and dentin sialophosphoprotein. Furthermore, we found a strong reduction in tartrate-resistant acid phosphatase, which is likely caused by defects in bone cells. CONCLUSIONS:BMP1/TLL1 appear to play crucial roles in maintaining extracellular matrix homeostasis essential to root formation and dentin mineralization. 10.1016/j.joen.2016.09.007
    Impairment of Bony Crypt Development Associated With Hexavalent Chromium Exposure During Tooth Eruption. Sánchez Luciana M,Lewicki Marianela,De Lucca Romina C,Ubios Ángela M Acta odontologica latinoamericana : AOL Improperly treated hexavalent chromium-containing industrial wastes contaminate drinking water, potentially affecting children taking breast milk or baby bottles prepared with infant formula. Thus, the aim of the present work was to determine the effect of this toxic on bone activity in the developing alveolus during tooth eruption of suckling Wistar rats intoxicated with potassium dichromate. Experimental animals received a daily dose of 12.5mg/kg body weight of potassium dichromate by gavage for 10 days; controls received an equivalent volume of saline solution. Histologic and histomorphometric studies of the mandible were performed. The data were statistically analyzed using Student's t test; statistical significance was set at a value of p <0.05. Experimental animals exhibited delayed tooth eruption, decreased periodontal width and bone volume, a lower percentage of bone formation surfaces, and higher percentage of quiescent surfaces (p<0.05) compared to controls. The delay in tooth eruption observed after exposure to hexavalent chromium is the result of a lower rate of bone remodeling in the developing alveolus. The obtained results show the importance of controlling toxic substances in drinking water, since their effects may alter the growth and development of subjects who were exposed during early infancy.
    Modulation of incisor eruption in rats by sympathetic efferents. Chidiac José Johann,Kassab Ammar,Rifai Khaldoun,Saadé Nayef E,Al Chaer Elie D Archives of oral biology INTRODUCTION:Intact neural supply is necessary for tooth eruption. Sympathetic denervation accelerates or decelerates the eruption rate depending on the tooth condition (intact or injured). The aim of this study is to reexamine the role of the sympathetic innervation, through the observation of the effects of pre or post ganglionic chemical sympathectomy on the eruption of intact rat incisors. MATERIALS AND METHODS:Different groups of rats were subjected to either ganglionic or peripheral chemical sympathectomy and the observed effects on incisor eruption were compared to those made on intact/sham groups or on rats subjected to inferior alveolar nerve (IAN) lesion. RESULTS:The total amount of eruption in control/naïve rats, measured over a total period of 144 h, was 3 ± 0.15 mm and decreased to 2.57 ± 0.06 mm (n = 8; p < 0.01) or 2.8 ± 0.10 mm (n = 8; p < 0.05) following treatment with guanethidine and hexamethonium, respectively. This amount decreased to 1.8 ± 0.14 mm (p < 0.001 vs. control, n = 7; or p < 0.01 vs. sham, n = 5) in rats subjected to IAN lesion. CONCLUSION:Sympathectomy delayed tooth eruption. Blocking the sympathetic effectors with guanethidine exerted more potent effects than ganglionic block with hexamethonium. Intact sympathetic supply is required for tooth growth under normal conditions. 10.1016/j.archoralbio.2018.02.003
    RUNX2 mutation impairs osteogenic differentiation of dental follicle cells. Liu Yang,Sun Xiangyu,Zhang Xianli,Wang Xiaozhe,Zhang Chenying,Zheng Shuguo Archives of oral biology OBJECTIVES:Cleidocranial dysplasia (CCD), mainly caused by RUNX2 mutation, is a dominantly inherited skeletal disorder with many dental abnormalities, characterized by delayed permanent tooth eruption. In this study, we explored a novel RUNX2 mutation and the effect of RUNX2 mutation on osteogenic differentiation of dental follicle cells (DFCs). DESIGN:A CCD patient with typical clinical features was involved in this study. Conservation and secondary structural analysis of the RUNX2 mutation was first performed. Then DFCs that stably expressing wild-type or mutant RUNX2 were established using lentiviruses. Cell Counting Kit 8 (CCK8) assays were performed to test the proliferation of DFCs. Measurement of alkaline phosphatase (ALP) activity, ALP staining, alizarin red staining and determination of osteoblast-specific genes expression were performed to assess osteogenic capacity of DFCs. RESULTS:A missense mutation (c.674 G > T, p. R225 L) of RUNX2 gene was identified in the CCD patient. Conservation and secondary structural analysis revealed that the mutation was located in highly conserved Runt domain and altered secondary structure of RUNX2. CCK8 assays showed that mutant RUNX2 increased the proliferation rate of DFCs compared to wild-type RUNX2. ALP activity, ALP staining and alizarin red staining results indicated that mutant RUNX2 decreased the mineralization ability of DFCs. In addition, mutant RUNX2 significantly down-regulated the expression of osteoblast-associated genes. CONCLUSIONS:RUNX2 mutation can reduce the osteogenic capacity of DFCs by inhibiting osteoblast-associated genes and then affecting bone formation, which participates in bone remodeling during tooth eruption. These effects may be partly responsible for the defects in permanent tooth eruption of CCD patients. 10.1016/j.archoralbio.2018.10.029
    4H syndrome with late-onset growth hormone deficiency caused by POLR3A mutations. Potic Ana,Brais Bernard,Choquet Karine,Schiffmann Raphael,Bernard Geneviève Archives of neurology OBJECTIVE:To report a novel clinical and genetic presentation of a patient with 4H syndrome, which is a recently described leukodystrophy syndrome characterized by ataxia, hypomyelination, hypodontia, and hypogonadotropic hypogonadism. DESIGN:Case report. SETTING:University teaching hospital. PATIENT:A 20-year-old male patient with 4H syndrome. RESULTS:The patient was found to have delayed tooth eruption and a late-onset growth hormone deficiency without overt growth failure. He was a compound heterozygote for the novel missense mutations R1005H and A1331T of POLR3A, which codes for the largest subunit of RNA polymerase III. CONCLUSION:This is the first report of this type of leukodystrophy from southeastern Europe, which suggests that POLR3A mutations should be suspected in patients with hypomyelination and various central nervous system–based endocrine abnormalities. 10.1001/archneurol.2011.1963
    [Tooth and bone research using genetically modified mice]. Suda Naoto Clinical calcium Teeth and bone are both hard tissues and composed of hydroxyapatite. Tooth development initiates with the invasination of oral epithelium, followed by aggregation of supporting ectomesenchymal cells. From mouse study, numbers of molecules have been discovered to relate tooth development. These discoveries have helped to clarify the responsible genes of human genetic disorders with abnormal tooth number and structure. During tooth development, teeth erupt into the outer environment, oral cavity. From this point, teeth are completely different from bone which is always covered by soft tissues. Tooth eruption is composed of two different processes, that is, eruption pathway formation and vertical tooth movement. In this review, mutant mice with abnormal tooth development and eruption are introduced, and molecular mechanism required for this process is discussed. CliCa12012731
    [Primary failure of eruption: a French prospective survey among the orthodontists from the Grand Est and Bourgogne-Franche-Comté regions]. Strub Marion,Kramer Elie,Manière Marie-Cécile,Wagner Delphine L' Orthodontie francaise INTRODUCTION: Primary failure of eruption (PFE) can be defined as the partial or complete failure of eruption of at least one posterior tooth, without any mechanical obstacle; isolated and syndromic forms exist. PFE results from an abnormal dental eruption process that can affect temporary teeth and / or permanent teeth. Molars are the main affected teeth, inducing posterior infraclusions. Orthodontists are the specialists most often concerned by this rare pathology. Unsuccessful orthodontic-surgical traction therapies are commonly reported. MATERIALS AND METHODS: The aim of our study using a prospective survey was to evaluate the level of knowledge reported by the orthodontists and the therapeutic difficulties they reported. An anonymous questionnaire was submitted to practitioners practicing in north-eastern France (Grand Est and Bourgogne-Franche-Comté regions). RESULTS: The participation rate was 33.5%. In France, until 2015, specialization in orthodontics was obtained thanks to a local qualification, the Certificat d'Etudes Cliniques Spéciales - Mention Orthodontie (CECSMO), which has now been replaced by specialization following a national ranking competition. Most respondents obtained their qualification between 1980 and 2009 (80%), via the CECSMO (87%). Eighty-six per cent were aware of PFE but only 20% of them knew that PTHR1 (Parathyroid Hormone Receptor 1) gene could be involved in this pathology. The wide range of proposed therapies and the variable satisfaction rates highlight the difficulties encountered by practitioners. DISCUSSION: Phenotypic variability complicates the diagnosis and makes any therapeutic systematization uncertain. CONCLUSION: New clinical research projects, particularly in the field of molecular diagnosis, may improve understanding of genotype-phenotype correlations, and may potentially pilot therapeutic management. 10.1051/orthodfr/2019013
    The etiology of eruption disorders - further evidence of a 'genetic paradigm' Frazier-Bowers Sylvia A,Puranik Chaitanya P,Mahaney Michael C Seminars in orthodontics The clinical spectrum of tooth eruption disorders includes both syndromic and non-syndromic problems ranging from delayed eruption to a complete failure of eruption. A defect in the differential apposition/resorption mechanism in alveolar bone can cause conditions such as tooth ankylosis, primary failure of eruption, failure of eruption due to inadequate arch length and canine impaction. As our knowledge of the molecular events underlying normal tooth eruption has increased, so too has our understanding of clinical eruption disorders. The recent finding that one gene, parathyroid hormone receptor 1 (PTH1R), is causative for familial cases of primary failure of eruption (PFE) suggests that other disturbances in tooth eruption may have a genetic etiology. In this report, we evaluated the current terminology (ankylosis, PFE, secondary retention, etc.) used to describe non-syndromic eruption disorders, in light of this genetic discovery. We observed that some individuals previously diagnosed with ankylosis were subsequently found to have alterations in the PTH1R gene, indicating the initial misdiagnosis of ankylosis and the necessary re-classification of PFE. We further investigated the relationship of the PTH1R gene, using a network pathway analysis, to determine its connectivity to previously identified genes that are critical to normal tooth eruption. We found that PTH1R acts in a pathway with genes such as PTHrP that have been shown to be important in bone remodeling, hence eruption, in a rat model. Thus, recent advances in our understanding of normal and abnormal tooth eruption should allow us in the future to develop a clinical nomenclature system based more on the molecular genetic cause of the eruption failures versus the clinical appearance of the various eruption disorders. 10.1053/j.sodo.2010.05.003
    Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormone-related peptide. Schipani E,Lanske B,Hunzelman J,Luz A,Kovacs C S,Lee K,Pirro A,Kronenberg H M,Jüppner H Proceedings of the National Academy of Sciences of the United States of America Mice in which the genes encoding the parathyroid hormone (PTH)-related peptide (PTHrP) or the PTH/PTHrP receptor have been ablated by homologous recombination show skeletal dysplasia due to accelerated endochondral bone formation, and die at birth or in utero, respectively. Skeletal abnormalities due to decelerated chondrocyte maturation are observed in transgenic mice where PTHrP expression is targeted to the growth plate, and in patients with Jansen metaphyseal chondrodysplasia, a rare genetic disorder caused by constitutively active PTH/PTHrP receptors. These and other findings thus indicate that PTHrP and its receptor are essential for chondrocyte differentiation. To further explore the role of the PTH/PTHrP receptor in this process, we generated transgenic mice in which expression of a constitutively active receptor, HKrk-H223R, was targeted to the growth plate by the rat alpha1 (II) collagen promoter. Two major goals were pursued: (i) to investigate how constitutively active PTH/PTHrP receptors affect the program of chondrocyte maturation; and (ii) to determine whether expression of the mutant receptor would correct the severe growth plate abnormalities of PTHrP-ablated mice (PTHrP-/-). The targeted expression of constitutively active PTH/PTHrP receptors led to delayed mineralization, decelerated conversion of proliferative chondrocytes into hypertrophic cells in skeletal segments that are formed by the endochondral process, and prolonged presence of hypertrophic chondrocytes with delay of vascular invasion. Furthermore, it corrected at birth the growth plate abnormalities of PTHrP-/- mice and allowed their prolonged survival. "Rescued" animals lacked tooth eruption and showed premature epiphyseal closure, indicating that both processes involve PTHrP. These findings suggest that rescued PTHrP-/- mice may gain considerable importance for studying the diverse, possibly tissue-specific role(s) of PTHrP in postnatal development. 10.1073/pnas.94.25.13689
    Role of PTH1R Signaling in Prx1 Mesenchymal Progenitors during Eruption. Cui C,Bi R,Liu W,Guan S,Li P,Song D,Xu R,Zheng L,Yuan Q,Zhou X,Fan Y Journal of dental research Tooth eruption is a complex process requiring precise interaction between teeth and adjacent tissues. Molecular analysis demonstrates that bone remodeling plays an essential role during eruption, suggesting that a parathyroid hormone 1 receptor (PTH1R) gene mutation is associated with disturbances in bone remodeling and results in primary failure of eruption (PFE). Recent research reveals the function of PTH1R signaling in mesenchymal progenitors, whereas the function of PTH1R in mesenchymal stem cells during tooth eruption remains incompletely understood. We investigated the specific role of PTH1R in Prx1 progenitor expression during eruption. We found that Prx1-progenitors occur in mesenchymal stem cells residing in alveolar bone marrow surrounding incisors, at the base of molars and in the dental follicle and pulp of incisors. Mice with conditional deletion of PTH1R using the promoter exhibited arrested mandibular incisor eruption and delayed molar eruption. Micro-computed tomography, histomorphometry, and molecular analyses revealed that mutant mice had significantly reduced alveolar bone formation concomitant with downregulated gene expression of key regulators of osteogenesis in PTH1R-deficient cells. Moreover, culturing orofacial bone-marrow-derived mesenchymal stem cells (OMSCs) from mice or from transfecting Cre recombinase adenovirus in OMSCs from mice suggested that lack of expression inhibited osteogenic differentiation in vitro. However, bone resorption was not affected by PTH1R ablation, indicating the observed reduced alveolar bone volume was mainly due to impaired bone formation. Furthermore, we found irregular periodontal ligaments and reduced Periostin expression in mutant incisors, implying loss of PTH1R results in aberrant differentiation of periodontal ligament cells. Collectively, these data suggest that PTH1R signaling in Prx1 progenitors plays a critical role in alveolar bone formation and periodontal ligament development during eruption. These findings have implications for our understanding of the physiologic and pathologic function of PTH1R signaling in tooth eruption and the progression of PFE. 10.1177/0022034520934732
    [Effects of idiopathic hypoparathyroidism on tooth eruption of rats]. Zhao D F,Guan S Y,Fan Y,Zheng L W Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology To explore the effects of reduced parathyroid function in early growth and development on tooth eruption and enamel development by establishing an animal model of idiopathic hypoparathyroidism (IHP) and to explore the mechanism of IHP affecting tooth eruption with a view to provide experimental basis for early diagnosis and clinical treatment of IHP. Forty-eight SD rats at postnatal day 7 were randomly and equally divided into sham operation group and IHP group. The bilateral parathyroidectomy (PTX) was performed by using carbon nanoparticles technique to establish an IHP rat model, while no parathyroids were removed in the sham operation group using the same technique. Serum was extracted after surgery, serum calcium, serum phosphorus, and serum parathyroid hormone (PTH) concentrations were detected in order to verify the success of the modeling. At postnatal day 14, day 25 and day 38 (P14, P25 and P38) the rats were sacrificed to collect the mandible samples (six from each group) and to analyze the volume of enamel, the height of the tooth eruption and the bone microarchitecture parameters of the root-oriented alveolar bone of mandibular third molar quantitatively by micro-CT scanning. Histological sections were prepared. The distribution and expression levels of osteoblast differentiation markers runt-related transcription factor 2 (RUNX2) and osterix (OSX) in the alveolar bone around the third molar were detected by immunohistochemical staining and the osteoclast activity was detected by tartrate-resistant acid phosphatase (TRAP) staining. After each of the third molars was isolated, the microhardness of the enamel was measure by using a microhardness tester and the enamel microstructure was photographed by using scanning electron microscope. Primary dental follicle stem cells were isolated from other six mandibulars from each group at P14 and cultured . The cell proliferation activity was tested by cell colony forming units detection. After induction of dental follicle stem cells into osteogenic differentiation, the degree of mineralization was detected by using alkaline phosphatase staining and alizarin red staining. The mRNA of mandibular tissues and dental follicle cells were extracted, the expression of genes related to osteoblasts and osteoclast differentiations and parathyroid hormone receptor 1 (PTH1R) were detected by real-time quantitative PCR. Bilateral parathyroidectomy was successfully performed on rats with the help of carbon nanoparticles under stereomicroscope. After surgery, the serum calcium concentration reduced, the serum phosphorus concentration increased and the serum PTH concentration distinctly reduced (<0.01). The volume of enamel [(4.58±0.24) mm] and the microhardness [(167.76±21.86) MPa] in IHP group were significantly lower than that in sham operation group [(5.22±0.46) mm, <0.05; (223.92±10.94) MPa, <0.01, respectively]. The eruption height of the mandibular third molar in the IHP group was respectively lower than that in the sham operation group (<0.05). The bone volume over total volume and trabecular number of the root-oriented alveolar bone of the mandibular third molars in the IHP group were respectively lower than that in sham operation group (<0.05). The expression levels of RUNX2 and OSX proteins in the root-oriented alveolar bone of the mandibular third molars in the IHP group respectively reduced, compared to that in sham operation group (<0.05). Furthermore, the number of osteoclasts (3.86±1.07) in crown-oriented alveolar bone in the IHP group was respectively lower than that in sham operation group (6.43±1.27) (<0.01). The proliferative activity of dental follicle stem cells in the IHP group respectively decreased (<0.01). After the induction of osteogenic differentiation, the mineralization ability of dental follicle stem cells in the IHP group was weakened. In the mandibular tissues of IHP group, the expression levels of osteogenesis related genes such as RUNX2 and OSX and the the expression of PTH1R significantly reduced (<0.05). The receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio reduced significantly (<0.01) compared to those of sham operation group. Also in the dental follicle cells of IHP group, the expression levels of osteogenesis related genes such as RUNX2 and OSX, the RANKL/OPG ratio and the expression of PTH1R significantly decreased simultaneously compared to that in sham operation group (<0.01). Under the condition of idiopathic hypoparathyroidism, the weakening of PTH/PTH1R signaling may reduce the proliferative activity of dental follicle stem cells, inhibit their regulation for osteoblast and osteoclast differentiations and functions, thereby interfere the bone remodeling of alveolar bone around the tooth germ during tooth eruption, which eventually leads to delayed tooth eruption. 10.3760/cma.j.cn112144-20210510-00221
    A novel homozygous PTH1R variant identified through whole-exome sequencing further expands the clinical spectrum of primary failure of tooth eruption in a consanguineous Saudi family. Jelani Musharraf,Kang Changsoo,Mohamoud Hussein Sheikh Ali,Al-Rehaili Rayan,Almramhi Mona Mohammad,Serafi Rehab,Yang Huanming,Al-Aama Jumana Yousuf,Naeem Muhammad,Alkhiary Yaser Mohammad Archives of oral biology OBJECTIVES:The present study aimed to identify the genetic cause of non-syndromic primary failure of tooth eruption in a five-generation consanguineous Saudi family using whole-exome sequencing (WES) analysis. DESIGN:The family pedigree and phenotype were obtained from patient medical records. WES of all four affected family members was performed using the 51 Mb SureSelect V4 library kit and then sequenced using the Illumina HiSeq2000 sequencing system. Sequence alignment, variant calling, and the annotation of single nucleotide polymorphisms and indels were performed using standard bioinformatics pipelines. The genotype of candidate variants was confirmed in all available family members by Sanger sequencing. RESULTS:Pedigree analysis suggested that the inheritance was autosomal recessive. WES of all affected individuals identified a novel homozygous variant in exon 8 of the parathyroid hormone 1 receptor gene (PTH1R) (NM_000316: c.611T>A: p.Val204Glu). CONCLUSION:To the best of our knowledge, this is the first report of primary failure of eruption caused by a homozygous mutation in PTH1R. Our findings prove the application of WES as an efficient molecular diagnostics tool for this rare phenotype and further broaden the clinical spectrum of PTH1R pathogenicity. 10.1016/j.archoralbio.2016.03.012
    PTH1R Mutants Found in Patients with Primary Failure of Tooth Eruption Disrupt G-Protein Signaling. Subramanian Hariharan,Döring Frank,Kollert Sina,Rukoyatkina Natalia,Sturm Julia,Gambaryan Stepan,Stellzig-Eisenhauer Angelika,Meyer-Marcotty Philipp,Eigenthaler Martin,Wischmeyer Erhard PloS one AIM:Primary failure of tooth eruption (PFE) is causally linked to heterozygous mutations of the parathyroid hormone receptor (PTH1R) gene. The mutants described so far lead to exchange of amino acids or truncation of the protein that may result in structural changes of the expressed PTH1R. However, functional effects of these mutations have not been investigated yet. MATERIALS AND METHODS:In HEK293 cells, PTH1R wild type was co-transfected with selected PTH1R mutants identified in patients with PFE. The effects on activation of PTH-regulated intracellular signaling pathways were analyzed by ELISA and Western immunoblotting. Differential effects of wild type and mutated PTH1R on TRESK ion channel regulation were analyzed by electrophysiological recordings in Xenopus laevis oocytes. RESULTS:In HEK293 cells, activation of PTH1R wild type increases cAMP and in response activates cAMP-stimulated protein kinase as detected by phosphorylation of the vasodilator stimulated phosphoprotein (VASP). In contrast, the PTH1R mutants are functionally inactive and mutant PTH1R/Gly452Glu has a dominant negative effect on the signaling of PTH1R wild type. Confocal imaging revealed that wild type PTH1R is expressed on the cell surface, whereas PTH1R/Gly452Glu mutant is mostly retained inside the cell. Furthermore, in contrast to wild type PTH1R which substantially augmented K+ currents of TRESK channels, coupling of mutated PTH1R to TRESK channels was completely abolished. CONCLUSIONS:PTH1R mutations affect intracellular PTH-regulated signaling in vitro. In patients with primary failure of tooth eruption defective signaling of PTH1R mutations is suggested to occur in dento-alveolar cells and thus may lead to impaired tooth movement. 10.1371/journal.pone.0167033
    [Effects of post-natal inhibition of RANKL on molar eruption and root formation in C57BL/6 mice]. Gama Andrea,Perea Linamary,Yepes Catalina,Betancur Jhon J,Vargas Jorge,Amiaud Jerôme,Babajko Sylvie,Lezot Frédéric,Castaneda Beatriz L' Orthodontie francaise INTRODUCTION:Recent observations performed in the orthodontic department of La Pitié-Salpêtrière hospital in Paris reported an increase of non-familial eruption defects of permanent molars. Our recent data have evidenced the involvement of osteoclasts (OC) in both the eruption and the dental retention processes through the RANKL/RANK/OPG signaling pathway. These facts are at the origin of the hypothesis of the existence of an environmental etiology for those eruption defects that would correspond to the perturbation of cellular autocrine/paracrine signaling pathways as the RANKL/ RANK/OPG. MATERIALS AND METHODS:C57BL/6 mice were submitted to repeated injections with anti-RANKL neutralizing antibody during the nine days following birth. A phenotypic comparison with transgenic mice overexpressing RANK was performed for the functional characterization of the RANKL/RANK/OPG pathway. The dento-alveolar complex was analyzed using micro-CT for bone density and Masson's trichrome staining for histological examination. RESULTS:The RANKL transient invalidation of RANKL stopped the molar root development and tooth eruption contrary to transgenic mice overexpressing RANK. The recruitment and the OC activity were strongly impacted. DISCUSSION:This research is of direct clinical interest in understanding the pathology of eruption as indirect in establishing orthodontic treatment protocols for particular cases. 10.1051/orthodfr/2019008
    Parathyroid hormone-related peptide (1-34) promotes tooth eruption and inhibits osteogenesis of dental follicle cells during tooth development. Zhang Jiawei,Liao Lijun,Li Yuyu,Xu Yang,Guo Weihua,Tian Weidong,Zou Shujuan Journal of cellular physiology Dental follicle cells (DFCs) activate and recruit osteoclasts for tooth development and tooth eruption, whereas DFCs themselves differentiate into osteoblasts to form alveolar bone surrounding tooth roots through the interaction with Hertwig's epithelial root sheath (HERS). Also during tooth development, parathyroid hormone-related peptide (PTHrP) is expressed surrounding the tooth germ. Thus, we aimed to investigate the effect of PTHrP (1-34) on bone resorption and osteogenesis of DFCs in vitro and in vivo. In vitro studies demonstrated that DFCs cocultured with HERS cells expressed higher levels of BSP and OPN than the DFCs control group, whereas cocultured DFCs treated with PTHrP (1-34) had lower expressions of ALP, RUNX2, BSP, and OPN than the cocultured DFCs control group. Moreover, we found PTHrP (1-34) inhibited osteogenesis of cocultured DFCs by inactivating the Wnt/β-catenin pathway. PTHrP (1-34) also increased the expression of RANKL/OPG ratio in DFCs. Consistently, in vivo study found that PTHrP (1-34) accelerated tooth eruption and inhibited alveolar bone formation. Therefore, these results suggest that PTHrP (1-34) accelerates tooth eruption and inhibits osteogenesis of DFCs by inactivating Wnt/β-catenin pathway. 10.1002/jcp.27857
    Semaphorin 3A gets involved in the establishment of mouse tooth eruptive pathway. Yu Xijiao,Zheng Fuju,Du Yi,Tang Kailiang,Wang Wei,Zhang Shanyong Journal of molecular histology The accurately establishment of the eruptive pathway is of vital importance. The mechanisms governing tooth eruption pathway remain little known. This study is to elucidate the roles of Semaphorin 3A (Sema 3A) in mouse tooth eruptive pathway. C57BL/6 mice (11-13 and 15-17 days after birth) were chosen to observe eruptive pathway of mouse lower first molar. Expressions of Sema 3A and its receptor neuropilin 1 and plexin A1 were detected. Osteoclasts were identified by TRAP staining. Co-localization of Sema 3A and osteoclast maker CD68 was detected by double immunofluorescence staining. Picrosirius red staining was applied to observe collagen fibers during mucosal penetration phase. In vitro, Bone marrow-derived macrophages (BMMs) were prepared from 4 week C57BL/6 mice to observe the effect of Sema 3A on the differentiation of BMMs into osteoclasts by TRAP staining. Expressions of Sema 3A was observed by immunofluorescence and western blotting. At osseous eruption phase, many TRAP-positive multi-nucleated cells were distributed around occlusal alveolar bone. The positive expressions of Sema 3A were observed in the multi-nucleated cells. Fluorescence double staining showed that Sema 3A and CD68 were co-expressed in osteoclasts. Its receptor neuropilin 1 and plexin A1 were also found in osteoclasts. In vitro, Sema3A negatively regulated osteoclast differentiation. At mucosal penetration, occlusal alveolar bone had been completely resorbed and collagen fires were gradually degraded for eruptive pathway. Similar positive expressions of Sema 3A and its receptor neuropilin 1 and plexin A1 were also found in the mucosal penetration pathway. Sema 3A gets involved in the establishment of mouse tooth eruptive pathway by modulating osteoclast activity. Sema3A should be considered as a novel nervous agent or a potential biomarker for mouse tooth eruptive pathway. 10.1007/s10735-019-09838-8
    Notch Coordinates Periodontal Ligament Maturation through Regulating Lamin A. Denes B J,Bolton C,Illsley C S,Kok W L,Walker J V,Poetsch A,Tredwin C,Kiliaridis S,Hu B Journal of dental research Tooth eruption is a continuous biological process with dynamic changes at cellular and tissue levels, particularly within the periodontal ligament (PDL). Occlusion completion is a significant physiological landmark of dentition establishment. However, the importance of the involvement of molecular networks engaging in occlusion establishment on the final PDL maturation is still largely unknown. In this study, using rat and mouse molar teeth and a human PDL cell line for RNAseq and proteomic analysis, we systematically screened the key molecular links in regulating PDL maturation before and after occlusion establishment. We discovered Notch, a key molecular pathway in regulating stem cell fate and differentiation, is a major player in the event. Intercepting the Notch pathway by deleting its key canonical transcriptional factor, , using a conditional knockout strategy in the mice delayed PDL maturation. We also identified that Lamin A, a cell nuclear lamina member, is a unique marker of PDL maturation, and its expression is under the control of Notch signaling. Our study therefore provides a deep insight of how PDL maturation is regulated at the molecular level, and we expect the outcomes to be applied for a better understanding of the molecular regulation networks in physiological conditions such as tooth eruption and movement and also for periodontal diseases. 10.1177/0022034519871448
    Biomechanical stress regulates mammalian tooth replacement via the integrin β1-RUNX2-Wnt pathway. Wu Xiaoshan,Hu Jinrong,Li Guoqing,Li Yan,Li Yang,Zhang Jing,Wang Fu,Li Ang,Hu Lei,Fan Zhipeng,Lü Shouqin,Ding Gang,Zhang Chunmei,Wang Jinsong,Long Mian,Wang Songlin The EMBO journal Renewal of integumentary organs occurs cyclically throughout an organism's lifetime, but the mechanism that initiates each cycle remains largely unknown. In a miniature pig model of tooth development that resembles tooth development in humans, the permanent tooth did not begin transitioning from the resting to the initiation stage until the deciduous tooth began to erupt. This eruption released the accumulated mechanical stress inside the mandible. Mechanical stress prevented permanent tooth development by regulating expression and activity of the integrin β1-ERK1-RUNX2 axis in the surrounding mesenchyme. We observed similar molecular expression patterns in human tooth germs. Importantly, the release of biomechanical stress induced downregulation of RUNX2-wingless/integrated (Wnt) signaling in the mesenchyme between the deciduous and permanent tooth and upregulation of Wnt signaling in the epithelium of the permanent tooth, triggering initiation of its development. Consequently, our findings identified biomechanical stress-associated Wnt modulation as a critical initiator of organ renewal, possibly shedding light on the mechanisms of integumentary organ regeneration. 10.15252/embj.2019102374
    Mesenchymal Progenitor Regulation of Tooth Eruption: A View from PTHrP. Nagata M,Ono N,Ono W Journal of dental research Tooth eruption is a unique biological process by which highly mineralized tissues emerge into the outer world, and it occurs concomitantly with tooth root formation. These 2 processes have been considered independent phenomena; however, recent studies support the theory that they are indeed intertwined. Dental mesenchymal progenitor cells in the dental follicle lie at the heart of the coupling of these 2 processes, providing a source for diverse mesenchymal cells that support formation of the highly functional tooth root and the periodontal attachment apparatus, while facilitating formation of osteoclasts. These cells are regulated by autocrine signaling by parathyroid hormone-related protein (PTHrP) and its parathyroid hormone/PTHrP receptor PPR. This PTHrP-PPR signaling appears to crosstalk with other signaling pathways and regulates proper cell fates of mesenchymal progenitor cell populations. Disruption of this autocrine PTHrP-PPR signaling in these cells leads to defective formation of the periodontal attachment apparatus, tooth root malformation, and failure of tooth eruption in molars, which essentially recapitulate primary failure of eruption in humans, a rare genetic disorder exclusively affecting tooth eruption. Diversity and distinct functionality of these mesenchymal progenitor cell populations that regulate tooth eruption and tooth root formation are beginning to be unraveled. 10.1177/0022034519882692
    Apoptosis and reduced microvascular density of the lamina propria during tooth eruption in rats. de Pizzol Júnior José Paulo,Sasso-Cerri Estela,Cerri Paulo Sérgio Journal of anatomy During tooth eruption, structural and functional changes must occur in the lamina propria to establish the eruptive pathway. In this study, we evaluate the structural changes that occur during lamina propria degradation and focus these efforts on apoptosis and microvascular density. Fragments of maxilla containing the first molars from 9-, 11-, 13- and 16-day-old rats were fixed, decalcified and embedded in paraffin. The immunohistochemical detection of vascular endothelial growth factor (VEGF), caspase-3 and MAC387 (macrophage marker), and the TUNEL method were applied to the histological molar sections. The numerical density of TUNEL-positive cells and VEGF-positive blood vessel profiles were also obtained. Data were statistically evaluated using a one-way anova with the post-hoc Kruskal-Wallis or Tukey test and a significance level of P ≤ 0.05. Fragments of maxilla were embedded in Araldite for analysis under transmission electron microscopy (TEM). TUNEL-positive structures, fibroblasts with strongly basophilic nuclei and macrophages were observed in the lamina propria at all ages. Using TEM, we identified processes of fibroblasts or macrophages surrounding partially apoptotic cells. We found a high number of apoptotic cells in 11-, 13- and 16-day-old rats. We observed VEGF-positive blood vessel profiles at all ages, but a significant decrease in the numerical density was found in 13- and 16-day-old rats compared with 9-day-old rats. Therefore, the establishment of the eruptive pathway during the mucosal penetration stage depends on cell death by apoptosis, the phagocytic activity of fibroblasts and macrophages, and a decrease in the microvasculature due to vascular cell death. These data point to the importance of vascular rearrangement and vascular neoformation during tooth eruption and the development of oral mucosa. 10.1111/joa.12359
    ClC-7 Deficiency Impairs Tooth Development and Eruption. Wang He,Pan Meng,Ni Jinwen,Zhang Yanli,Zhang Yutao,Gao Shan,Liu Jin,Wang Zhe,Zhang Rong,He Huiming,Wu Buling,Duan Xiaohong Scientific reports CLCN7 gene encodes the voltage gated chloride channel 7 (ClC-7) in humans. The mutations in CLCN7 have been associated with osteopetrosis in connection to the abnormal osteoclasts functions. Previously, we found that some osteopetrosis patients with CLCN7 mutations suffered from impacted teeth and root dysplasia. Here we set up two in vivo models under a normal or an osteoclast-poor environment to investigate how ClC-7 affects tooth development and tooth eruption. Firstly, chitosan-Clcn7-siRNA nanoparticles were injected around the first maxillary molar germ of newborn mice and caused the delay of tooth eruption and deformed tooth with root dysplasia. Secondly, E13.5 molar germs infected with Clcn7 shRNA lentivirus were transplanted under the kidney capsule and presented the abnormal changes in dentin structure, periodontal tissue and cementum. All these teeth changes have been reported in the patients with CLCN7 mutation. In vitro studies of ameloblasts, odontoblasts and dental follicle cells (DFCs) were conducted to explore the involved mechanism. We found that Clcn7 deficiency affect the differentiation of these cells, as well as the interaction between DFCs and osteoclasts through RANKL/OPG pathway. We conclude that ClC-7 may affect tooth development by directly targeting tooth cells, and regulate tooth eruption through DFC mediated osteoclast pathway. 10.1038/srep19971
    Multiple essential MT1-MMP functions in tooth root formation, dentinogenesis, and tooth eruption. Xu H,Snider T N,Wimer H F,Yamada S S,Yang T,Holmbeck K,Foster B L Matrix biology : journal of the International Society for Matrix Biology Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a transmembrane zinc-endopeptidase that breaks down extracellular matrix components, including several collagens, during tissue development and physiological remodeling. MT1-MMP-deficient mice (MT1-MMP(-/-)) feature severe defects in connective tissues, such as impaired growth, osteopenia, fibrosis, and conspicuous loss of molar tooth eruption and root formation. In order to define the functions of MT1-MMP during root formation and tooth eruption, we analyzed the development of teeth and surrounding tissues in the absence of MT1-MMP. In situ hybridization showed that MT1-MMP was widely expressed in cells associated with teeth and surrounding connective tissues during development. Multiple defects in dentoalveolar tissues were associated with loss of MT1-MMP. Root formation was inhibited by defective structure and function of Hertwig's epithelial root sheath (HERS). However, no defect was found in creation of the eruption pathway, suggesting that tooth eruption was hampered by lack of alveolar bone modeling/remodeling coincident with reduced periodontal ligament (PDL) formation and integration with the alveolar bone. Additionally, we identified a significant defect in dentin formation and mineralization associated with the loss of MT1-MMP. To segregate these multiple defects and trace their cellular origin, conditional ablation of MT1-MMP was performed in epithelia and mesenchyme. Mice featuring selective loss of MT1-MMP activity in the epithelium were indistinguishable from wild type mice, and importantly, featured a normal HERS structure and molar eruption. In contrast, selective knock-out of MT1-MMP in Osterix-expressing mesenchymal cells, including osteoblasts and odontoblasts, recapitulated major defects from the global knock-out including altered HERS structure, short roots, defective dentin formation and mineralization, and reduced alveolar bone formation, although molars were able to erupt. These data indicate that MT1-MMP activity in the dental mesenchyme, and not in epithelial-derived HERS, is essential for proper tooth root formation and eruption. In summary, our studies point to an indispensable role for MT1-MMP-mediated matrix remodeling in tooth eruption through effects on bone formation, soft tissue remodeling and organization of the follicle/PDL region. 10.1016/j.matbio.2016.01.002
    The parathyroid hormone-related protein is secreted during the osteogenic differentiation of human dental follicle cells and inhibits the alkaline phosphatase activity and the expression of DLX3. Klingelhöffer C,Reck A,Ettl T,Morsczeck C Tissue & cell The dental follicle is involved in tooth eruption and it expresses a great amount of the parathyroid hormone-related protein (PTHrP). PTHrP as an extracellular protein is required for a multitude of different regulations of enchondral bone development and differentiation of bone precursor cells and of the development of craniofacial tissues. The dental follicle contains also precursor cells (DFCs) of the periodontium. Isolated DFCs differentiate into periodontal ligament cells, alveolar osteoblast and cementoblasts. However, the role of PTHrP during the human periodontal development remains elusive. Our study evaluated the influence of PTHrP on the osteogenic differentiation of DFCs under in vitro conditions for the first time. The PTHrP protein was highly secreted after 4days of the induction of the osteogenic differentiation of DFCs with dexamethasone (2160.5pg/ml±345.7SD. in osteogenic differentiation medium vs. 315.7pg/ml±156.2SD. in standard cell culture medium; Student's t Test: p<0.05 (n=3)). We showed that the supplementation of the osteogenic differentiation medium with PTHrP inhibited the alkaline phosphatase activity and the expression of the transcription factor DLX3, but the depletion of PTHrP did not support the differentiation of DFCs. Previous studies have shown that Indian Hedgehog (IHH) induces PTHrP and that PTHrP, in turn, inhibits IHH via a negative feedback loop. We showed that SUFU (Suppressor Of Fused Homolog) was not regulated during the osteogenic differentiation in DFCs. So, neither the hedgehog signaling pathway induced PTHrP nor PTHrP suppressed the hedgehog signaling pathway during the osteogenic differentiation in DFCs. In conclusion, our results suggest that PTHrP regulates independently of the hedgehog signaling pathway the osteogenic differentiated in DFCs. 10.1016/j.tice.2016.05.007
    Primary failure of eruption (PFE): a systematic review. Hanisch Marcel,Hanisch Lale,Kleinheinz Johannes,Jung Susanne Head & face medicine BACKGROUND:Primary failure of eruption (PFE) is a rare disease defined as incomplete tooth eruption despite the presence of a clear eruption pathway. Orthodontic extrusion is not feasible in this case because it results in ankylosis of teeth. To the best of our knowledge, besides the study of Ahmad et al. (Eur J Orthod 28:535-540, 2006), no study has systematically analysed the clinical features of and factors associated with PFE. Therefore, the aim of this study was to systematically evaluate the current literature (from 2006 to 2017) for new insights and developments on the aetiology, diagnosis, genetics, and treatment options of PFE. METHODS:Following the PRISMA guidelines, a systematic search was performed using the PubMed/Medline database for studies reporting on PFE. The following terms were used: "primary failure of tooth eruption", "primary failure of eruption", "tooth eruption failure", and "PFE". RESULTS:Overall, 17 articles reporting clinical data of 314 patients were identified. In all patients, the molars were affected. In 81 reported cases, both the molars and the premolars were affected by PFE. Further, 38 patients' primary teeth were also affected. In 27 patients, no family members were affected. Additional dental anomalies were observed in 39 patients. A total of 51 different variants of the PTH1R gene associated with PFE were recorded. CONCLUSIONS:Infraocclusion of the posterior teeth, especially if both sides are affected, is the hallmark of PFE. If a patient is affected by PFE, all teeth distal to the most mesial tooth are also affected by PFE. Primary teeth can also be impacted; however, this may not necessarily occur. If a patient is suspected of having PFE, a genetic test for mutation in the PTH1R gene should be recommended prior to any orthodontic treatment to avoid ankylosis. Treatment options depend on the patient's age and the clinical situation, and they must be evaluated individually. 10.1186/s13005-018-0163-7
    Gene expression profiles in dental follicles from patients with impacted canines. Uribe Pamela,Larsson Lena,Westerlund Anna,Ransjö Maria Odontology Animal studies suggest that the dental follicle (DF) plays a major role in tooth eruption. However, the role of the DF during tooth impaction and related root resorptions in adjacent teeth is not clear. The hypothesis for the present study is that expression of regulatory factors involved in the bone remodelling process necessary for tooth eruption may differ between dental follicles from teeth with different clinical situations. We have analysed the gene expression profiles in the DF obtained from impacted canines, with (N = 3) or without (N = 5) signs of root resorption, and from control teeth (normal erupting teeth, mesiodens) (N = 3). DF from 11 patients (mean age: 13 years) obtains at the time of surgical exposure of the tooth. Due to the surgical time point, all teeth were in a late developmental stage. Gene expression related to osteoblast activation/bone formation, osteoclast recruitment and activation was analysed by RTqPCR. Genes related to bone formation (RUNX2, OSX, ALP, OCN, CX43) were highly expressed in all the samples, but osteoclast recruitment/activation markers (OPG, RANKL, MCP-1, CSF-1) were negligible. No apparent patterns or significant differences in gene expression were found between impacted canines, with or without signs of root resorption, or when compared to control teeth. Our results suggest the DF regulation of osteoclastic activity is limited in the late pre-emergent stage of tooth development, irrespective if the tooth is normally erupting or impacted. We suggest that the follicle may have an important regulatory function for alveolar bone formation in the final eruption process and CX43-gap junction communication could be an important signalling pathway. 10.1007/s10266-018-0342-9
    Matrix Metalloproteinase-1 and Acid Phosphatase in the Degradation of the Lamina Propria of Eruptive Pathway of Rat Molars. de Pizzol Júnior José Paulo,Sasso-Cerri Estela,Cerri Paulo Sérgio Cells The comprehension of dental pathogenesis and disorders derived from eruption failure requires a deep understanding of the molecular mechanisms underlying normal tooth eruption. As intense remodelling is needed during tooth eruption, we hypothesize that matrix metalloproteinase-1 (MMP-1) and acid phosphatase (ACP) play a role in the eruptive pathway degradation. We evaluated MMP-1-immunoexpression and the collagen content in the lamina propria at different eruptive phases. Immunohistochemistry and ultrastructural cytochemistry for detection of ACP were also performed. In the maxillary sections containing first molars of 9-, 11-, 13-, and 16-day-old rats, the birefringent collagen of eruptive pathway was quantified. MMP-1 and ACP-2 immunohistochemical reactions were performed and the number of MMP-1-immunolabelled cells was computed. Data were analyzed by one-way ANOVA and Tukey post-test ( ≤ 0.05). ACP cytochemistry was evaluated in specimens incubated in sodium β-glycerophosphate. In the eruptive pathway of 13- and 16-day-old rats, the number of MMP-1-immunolabelled cells increased concomitantly to reduction of collagen in the lamina propria. Enhanced ACP-2-immunolabelling was observed in the lamina propria of 13- and 16-day-old rats. Fibroblasts and macrophages showed lysosomes and vacuoles containing fragmented material reactive to ACP. MMP-1 degrades extracellular matrix, including collagen fibers, being responsible for the reduction in the collagen content during tooth eruption. The enhanced ACP activity at the mucosal penetration stage indicates that this enzyme plays a role in the degradation of remnant material, which is engulfed by macrophages and fibroblasts of the eruptive pathway. Therefore, enzymatic failure in the eruptive pathway may disturbs tooth eruption. 10.3390/cells7110206
    Abnormal bone remodelling activity of dental follicle cells from a cleidocranial dysplasia patient. Liu Yang,Zhang Xianli,Sun Xiangyu,Wang Xiaozhe,Zhang Chenying,Zheng Shuguo Oral diseases OBJECTIVES:To explore the role of dental follicle cells (DFCs) with a novel cleidocranial dysplasia (CCD) causative gene RUNX2 mutation (DFCs ) in delayed permanent tooth eruption. MATERIALS AND METHODS:A CCD patient with typical clinical features was involved in this study. DFCs were cultured and DNA was extracted for RUNX2 mutation screening. Measurements of cell proliferation, alkaline phosphatase (ALP) activity, alizarin red staining and osteoblast-specific genes expression were performed to assess osteogenesis of DFCs . Co-culture of DFCs and peripheral blood mononuclear cells (PBMCs), followed tartrate-resistant acid phosphatase (TRAP) staining, real-time PCR and western blot were performed to evaluate osteoclast-inductive capacity of DFCs . RESULTS:A missense RUNX2 mutation (c. 557G>C) was found in DFCs from the CCD patient. Compared with normal controls, this mutation did not affect the proliferation of DFCs , but down-regulated the expression of osteogenesis-related genes, leading to a decrease in ALP activity and mineralisation. Co-culture results showed that DFCs reduced the formation of TRAP multinucleated cells and the expression of osteoclastogenesis-associated genes. Furthermore, the mutation reduced the ratio of RANKL/OPG in DFCs . CONCLUSIONS:DFCs disturbs bone remodelling activity during tooth eruption through RANK/RANKL/OPG signalling pathway and may thus be responsible for impaired permanent tooth eruption in CCD patients. 10.1111/odi.12900
    Regulatory roles of microRNAs in human dental tissues. Sehic Amer,Tulek Amela,Khuu Cuong,Nirvani Minou,Sand Lars Peter,Utheim Tor Paaske Gene MicroRNAs (miRNAs) are a class of small, non-coding RNAs that provide an efficient pathway for regulation of gene expression at a post-transcriptional level. Tooth development is regulated by a complex network of cell-cell signaling during all steps of organogenesis. Most of the congenital dental defects in humans are caused by mutations in genes involved in developmental regulatory networks. Whereas the developmental morphological stages of the tooth development already are thoroughly documented, the implicated genetic network is still under investigation. The involvement of miRNAs in the regulation of tooth genetic network was suggested for the first time in 2008. MiRNAs regulate tooth morphogenesis by fine-tuning the signaling networks. Unique groups of miRNAs are expressed in dental epithelium compared with mesenchyme, as well as in molars compared with incisors. The present review focuses on the current state of knowledge on the expression and function of miRNAs in human dental tissues, including teeth and the surrounding structures. Herein, we show that miRNAs exhibit specific roles in human dental tissues and are involved in gingival and periodontal disease, tooth movement and eruption, dental pulp physiology including repair and regeneration, differentiation of dental cells, and enamel mineralization. In light of similarities between the tooth development and other organs originating from the epithelium, further understanding of miRNAs` function in dental tissues may have wide biological relevance. 10.1016/j.gene.2016.10.009
    MicroRNA control of tooth formation and eruption. Jin Ying,Wang Chenglin,Cheng Si,Zhao Zhihe,Li Juan Archives of oral biology Tooth development involves epithelium invagination, mesenchyme aggregation, and epithelium-mesenchyme communication. A sophisticated signaling pathway network regulates the differentiation and crosstalk of multiple cell types in tooth germs and coordinates the broad spectrum of complex processes. MicroRNAs (miRNAs), a class of small non-coding RNA species that have been relatively well studied over the last few years, are now proposed as important regulators of tooth developmental signaling pathways as they repress cellular protein levels to provide a posttranscriptional gene regulation. In this review, we summarize the current knowledge of miRNA characteristics in regulating morphogenesis, amelogenesis, dentin formation, and tooth eruption and how they interplay with the signaling molecules during these processes. 10.1016/j.archoralbio.2016.08.026
    Delayed tooth eruption and suppressed osteoclast number in the eruption pathway of heterozygous Runx2/Cbfa1 knockout mice. Yoda Shuichi,Suda Naoto,Kitahara Yutaka,Komori Toshihisa,Ohyama Kimie Archives of oral biology Genetic studies have recently identified a mutation of one allele of runt-related gene 2 (RUNX2/CBFA1) as the cause for an autosomal-dominant skeletal disorder, cleidocranial dysplasia (CCD), which is characterised by hypoplasia of the clavicles and calvariae and widened sutures and fontanelles. In addition, CCD is frequently affected with multiple supernumerary teeth and the impaction and delayed eruption of teeth, the causes of all these dental abnormalities are still unknown. To clarify the cellular mechanism of the delayed tooth eruption in CCD, the process of tooth eruption was examined in heterozygous Runx2/Cbfa1 (mouse homolog of RUNX2/CBFA1) knockout mice, known to mimic most of the bone abnormalities of CCD. The timing of the appearance of maxillary and mandibular teeth into the oral cavity was significantly delayed in heterozygous mutant mice compared with wild-type mice. From postnatal days 8 to 10, an active alveolar bone resorption and a marked increase of the osteoclast surfaces was observed in the eruption pathway of both genotypes, but this increase was significantly suppressed in the mutant mice. In contrast, the osteoclast surfaces did not show a significant difference between the two genotypes in the future cortical area of femora. These results suggest that haploinsufficiency of Runx2/Cbfa1 does not effect the femoral bone remodelling but is insufficient for the active alveolar bone resorption essential for the prompt timing of tooth eruption. These results also suggest the possibility that impaired recruitment of osteoclasts is one of the cellular mechanisms of delayed tooth eruption in CCD patients. 10.1016/j.archoralbio.2004.01.010
    The regulatory role of the RANKL/RANK/OPG signaling pathway in the mechanisms of tooth eruption in patients with impacted teeth. Brodetska Ludmila,Natrus Larysa,Lisakovska Olha,Kaniura Olexandr,Iakovenko Liudmyla,Skrypnyk Irina,Flis Petro BMC oral health BACKGROUND:Tooth impaction is a common problem in orthodontic practice and in some cases accompanied by pain and pathological changes of surrounding teeth. Understanding the cellular and molecular mechanisms underlying tooth impaction allows finding the most effective orthodontic treatment for patients with impacted teeth (IT). RANK (receptor activator of NF-κB) / RANKL (RANK ligand) / OPG (osteoprotegerin) signaling pathway controls bone resorption and may be involved in the regulation of tooth eruption. The study aimed to evaluate bone remodeling based on the assessment of the RANKL/RANK/OPG status in patients with IT. METHODS:Bone samples from 18 patients (mean age 25.27 ± 3.34) were divided into 3 groups: 1 - bone tissue of healthy persons (control group); 2 - bone tissue, that was taken near the healthy tooth in patients with tooth impaction; 3 - bone tissue, that was collected near the IT. Levels of RANKL, RANK, OPG, osteocalcin (OC), NF-κB p65 subunit, NFATc1, and caspase-3 were determined by western blotting. The difference between groups was assessed using ANOVA followed by Tukey's post-hoc test. P-value ≤0.05 was considered statistically significant. RESULTS:We established a 1.73-fold elevation of RANK level in the IT area vs. control, indicating the recruitment of preosteoclasts. An increase in RANKL, OPG, and OC content was demonstrated (1.46-, 1.48-, and 1.42-fold respectively), reflecting the high activity of osteoblasts near the IT. Despite the activation of the RANKL/RANK/OPG system in the impaction area, NF-κB and NFATc1 levels did not change compared vs. control, indicating a blocked/delayed process of osteoclastogenesis. We found a decrease in the content of procaspase-3 (1.28-fold), while the level of its active form p17 increased by 2.26 folds near the healthy tooth in patients with IT compared with control. In the area of ​​IT, we observed an increase in procaspase-3 and p17 levels (1.32 and 1.78 folds). This reflects impairments of caspase-3 activation and accumulation of its inactive form in the IT area that may contribute to the tooth eruption failure. CONCLUSIONS:Tooth impaction may be associated with the disturbances in the caspase-3 cascade activation and the imbalance in the RANKL/RANK/OPG system, and as a result, blocked bone resorption. 10.1186/s12903-020-01251-y
    Pathognomonic oral profile of Enamel Renal Syndrome (ERS) caused by recessive FAM20A mutations. de la Dure-Molla Muriel,Quentric Mickael,Yamaguti Paulo Marcio,Acevedo Ana-Carolina,Mighell Alan J,Vikkula Miikka,Huckert Mathilde,Berdal Ariane,Bloch-Zupan Agnes Orphanet journal of rare diseases Amelogenesis imperfecta (AI) is a genetically and clinically heterogeneous group of inherited dental enamel defects. Commonly described as an isolated trait, it may be observed concomitantly with other orodental and/or systemic features such as nephrocalcinosis in Enamel Renal Syndrome (ERS, MIM#204690), or gingival hyperplasia in Amelogenesis Imperfecta and Gingival Fibromatosis Syndrome (AIGFS, MIM#614253). Patients affected by ERS/AIGFS present a distinctive orodental phenotype consisting of generalized hypoplastic AI affecting both the primary and permanent dentition, delayed tooth eruption, pulp stones, hyperplastic dental follicles, and gingival hyperplasia with variable severity and calcified nodules. Renal exam reveals a nephrocalcinosis which is asymptomatic in children affected by ERS. FAM20A recessive mutations are responsible for both syndromes. We suggest that AIGFS and ERS are in fact descriptions of the same syndrome, but that the kidney phenotype has not always been investigated fully in AIGFS. The aim of this review is to highlight the distinctive and specific orodental features of patients with recessive mutations in FAM20A. We propose ERS to be the preferred term for all the phenotypes arising from recessive FAM20A mutations. A differential diagnosis has to be made with other forms of AI, isolated or syndromic, where only a subset of the clinical signs may be shared. When ERS is suspected, the patient should be assessed by a dentist, nephrologist and clinical geneticist. Confirmed cases require long-term follow-up. Management of the orodental aspects can be extremely challenging and requires the input of multi-disciplinary specialized dental team, especially when there are multiple unerupted teeth. 10.1186/1750-1172-9-84
    Correlation between genotype and supernumerary tooth formation in cleidocranial dysplasia. Suda N,Hattori M,Kosaki K,Banshodani A,Kozai K,Tanimoto K,Moriyama K Orthodontics & craniofacial research INTRODUCTION:Cleidocranial dysplasia (CCD, MIM#119600), for which the responsible gene is RUNX2, is a genetic disorder characterized by hypoplasia or aplasia of the clavicles, patent fontanelles, and a short stature. Supernumerary teeth and delayed eruption and impaction of permanent teeth are frequently associated with CCD. Our previous study reported wide intrafamilial variation in supernumerary tooth formation associated with a mutation in the RUNT-domain of RUNX2, suggesting a low correlation between the genotype and supernumerary tooth formation. To further clarify this point, a more precise evaluation was performed. DESIGN:Gene mutational analysis of nine Japanese individuals with CCD was performed. Dental and skeletal characteristics were examined based on patient examinations and radiographs. RESULTS:Four different gene mutations, including one novel mutation in RUNX2 gene (NM_001024630), were identified. Among them, four individuals had the R225Q mutation, three siblings had the P224S mutation, and the other two individuals had different frame-shift mutations. Wide variations in supernumerary tooth formation were observed in individuals with identical gene mutations, and discordance was seen between monozygotic twins. Asymmetric supernumerary tooth formation was noted in five out of the nine individuals. CONCLUSION:Individuals with identical gene mutations showed a wide variation in the supernumerary tooth formation. Not only the genotype but also environmental factors and a complex system including epigenetics and copy number variation might regulate supernumerary tooth formation in CCD. 10.1111/j.1601-6343.2010.01495.x
    Downregulation of matrix metalloproteinases in hyperplastic dental follicles results in abnormal tooth eruption. Kim Seong-Gon,Kim Myung-Hee,Chae Chang-Hoon,Jung Youn-Kwan,Choi Je-Yong BMB reports In this study, we compared the gene expression profiles of non-syndromic hyperplastic dental follicle (HDF) fibroblasts and normal dental follicle (NDF) fibroblasts using cDNA microarrays, quantitative PCR, and immunohistochemical staining. Microarray analysis showed that several collagens genes were upregulated in the HDFos, including collagen types I, IV, VIII, and XI and TIMP-1, -3, and -4 (fold ratio > 2.0). In contrast, the expression of MMP-1, -3, -10, and -16 together with IL-8 was more than two fold downregulated. The differential expression of the genes encoding alkaline phosphatase, MMP-1, -3, -8, and IL-8 was confirmed by quantitative RT-PCR, while that of 24 HDFs and 18 NDFs was confirmed by immunohistochemical analysis. However, HDFs showed stronger expression of MMP-3 than NDFs (P < 0.001). Collectively, these results indicate that defective regulation of MMPs mediating connective tissue remodeling may be responsible for abnormal tooth eruption. 10.5483/bmbrep.2008.41.4.322
    Nicotinamide Improves Delayed Tooth Eruption in Mice. Yoon H,Kim H J,Shin H R,Kim B S,Kim W J,Cho Y D,Ryoo H M Journal of dental research Patients with cleidocranial dysplasia (CCD) caused by mutations in RUNX2 have severe dental anomalies, including delayed or absent eruption of permanent teeth. This requires painful and expensive surgical/orthodontic intervention because of the absence of medicine for this condition. Here, we demonstrate that nicotinamide, a vitamin B3 and class III histone deacetylase inhibitor, significantly improves delayed tooth eruption in mice, a well-known CCD animal model, through the restoration of decreased osteoclastogenesis. We also found that mRNA and protein levels were significantly reduced in osteoblasts as compared with wild type whereas RANKL and OPG levels had no significant difference between wild type and osteoblasts. The nicotinamide-induced restoration of osteoclastogenesis of bone marrow-derived macrophages in mice was due to the increased expression of RUNX2 and CSF1 and increased RANKL/OPG ratio. RUNX2 directly regulated mRNA expression via binding to the promoter region of the gene. In addition, nicotinamide enhanced the RUNX2 protein level and transacting activity posttranslationally with Sirt2 inhibition. Taken together, our study shows the potential and underlying molecular mechanism of nicotinamide for the treatment of delayed tooth eruption by using the murine model, suggesting nicotinamide as a candidate therapeutic drug for dental abnormalities in patients with CCD. 10.1177/0022034520970471