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Objectives: This systematic review aimed at evaluating the reliability of dental maturation (DM) according to Demirjian method compared to hand and wrist maturation (HWM) to assess skeletal maturity (SM) in growing subjects, to identify the teeth and the corresponding mineralisation stages related to the pubertal growth spurt (PGS)., Materials and Methods: PubMed, Scopus, and Web of Science were systematically searched until January 5th, 2024, to identify observational cross-sectional studies that assessed the reliability of Demirjian method compared to the HWM methods (i.e., Grave and Brown and Fishman) in growing subjects. The quality assessment was evaluated using the Joanna Briggs Institute (JBI) Critical Appraisal Checklist., Results: Out of 136 papers suitable for title/abstract screening, 19 included studies. Of them, 17 papers showed the reliability of Demirjian DM method compared to HWM Fishman and Grave and Brown methods to assess SM in growing subjects. According to JBI Critical Appraisal Checklist, 12 papers were high-quality studies and 7 papers were medium-quality studies.  Conclusions: The mandibular second molar might be considered as the best indicator compared to other teeth and that the peak of growth occurs no earlier than stage F in females and stage G in males according to Demirjian method. Also, the mandibular canine might be analysed as indicator of SM in males, and results suggest that the peak of growth occurs no earlier than maturation stage F according to Demirjian method, only in male subjects. Further studies are needed to confirm these findings.

Context: Human growth shows considerable variation. In addition to constitutional differences in the timing of maturation, there are diseases, systemic disorders and environmental factors that may affect a child's physiologic maturity. Interrelationships among skeletal, somatic and sexual maturity have been shown to be consistently strong. The study was conducted to evaluate the relationship between dental calcification stages and skeletal maturity indicators., Materials and Methods: The hand wrist radiographs, the lateral cephalogram and the orthopantomogram (OPG) of 120 patients (60 males and 60 females, age group 8-14 years) were taken. The hand wrist radiographs were analyzed using Fishman's skeletal maturity index (SMI), and the cephalograms analyzed by cervical vertebrae maturation (CVM) by Baccetti. OPG was analyzed according to the Demirjian's system., Result: On assessment, cervical vertebrae maturation indicator (CVMI) staging showed significant results with a correlation coefficient of 0.716 being the highest in canines in males while assessment of dental calcification stages and its comparison with skeletal maturity indicator showed insignificant results. The correlation coefficient values were 0.11, 0.09, 0.09 and 0.13 for canine, first premolar, second premolar and second molar respectively., Conclusion: There is a strong correlation between the maturation stage of cervical vertebrae and calcification stages of canine, first premolar, second premolar and second molar as determined by Demirjian's method., Competing Interests: None

In addition to bone, the dentin-pulp complex is also influenced by menopause, showing a decreased regenerative capacity. High levels of follicle-stimulating hormone (FSH) during menopause could directly regulate bone metabolism. Here, the role of FSH in the odontogenic differentiation of the dentin-pulp complex was investigated. Dental pulp stem cells (DPSCs) were isolated. CCK-8 assays, cell apoptosis assays, Western blotting (WB), real-time RT-PCR, alkaline phosphatase activity assays, and Alizarin Red S staining were used to clarify the effects of FSH on the proliferation, apoptosis and odontogenic differentiation of the DPSCs. MAPK pathway-related factors were explored by WB assays. FSH and its inhibitor were used in OVX rats combined with a direct pulp-capping model. HE and immunohistochemistry were used to detect reparative dentin formation and related features. The results indicated that FSH significantly decreased the odontogenic differentiation of the DPSCs without affecting cell proliferation and apoptosis. Moreover, FSH significantly activated the JNK signalling pathway, and JNK inhibitor partly rescued the inhibitory effect of FSH on DPSC differentiation. In vivo, FSH treatment attenuated the dentin bridge formation and mineralization-related protein expression in the OVX rats. Our findings indicated that FSH reduced the odontogenic capacity of the DPSCs and was involved in reparative dentinogenesis during menopause., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The cartilaginous fishes (Chondrichthyes) have a rich fossil record which consists mostly of isolated teeth and, therefore, phylogenetic relationships of extinct taxa are mainly resolved based on dental characters. One character, the tooth histology, has been examined since the 19 th century, but its implications on the phylogeny of Chondrichthyes is still in debate. We used high resolution micro-CT images and tooth sections of 11 recent and seven extinct lamniform sharks to examine the tooth mineralization processes in this group. Our data showed similarities between lamniform sharks and other taxa (a dentinal core of osteodentine instead of a hollow pulp cavity), but also one feature that has not been known from any other elasmobranch fish: the absence of orthodentine. Our results suggest that this character resembles a synapomorphic condition for lamniform sharks, with the basking shark, Cetorhinus maximus, representing the only exception and reverted to the plesiomorphic tooth histotype. Additionally, †Palaeocarcharias stromeri, whose affiliation still is debated, shares the same tooth histology only known from lamniform sharks. This suggests that †Palaeocarcharias stromeri is member of the order Lamniformes, contradicting recent interpretations and thus, dating the origin of this group back at least into the Middle Jurassic.

Objective: The aim of this study was to investigate the possibility of evaluating the relationship between dental maturation and skeletal maturation without taking a hand-wrist radiogram outside of panoramic radiogram., Materials and Methods: Panoramic and hand-wrist radiographs of 717 patients (383 girls and 334 boys), with ages ranging from 10 to 15 years, were evaluated. Dental maturity stages of the mandibular canine, first premolar, second premolar and second molar teeth were determined by Demirjian's and Nolla's methods. The skeletal maturation was determined by Fishman method and bone age was determined through the Greulich and Pyle method., Results: All teeth showed positive and statistically significant correlations, the highest correlation was between the mandibular second premolar calcification stages and hand-wrist maturation stages. According to both dental age determination methods, mandibular second premolar was prominent in determining to the pubertal growth stages., Conclusions: There was a positive relationship between dental calcification stages and skeletal maturation stages, in the study population. Dental calcification stages of the second mandibular premolar showed the highest positive correlation with the skeletal maturation stages.

Objective: The aim of this study was to investigate the spatiotemporal expression and potential role of p75NTR in tooth morphogenesis and tissue mineralization., Materials and Methods: The dynamic morphology of the four stages (from the beginning of E12.5 d, then E13.5 d and E15.5 d, to the end of E18.5 d) was observed, and the expressions of p75NTR and Runx2 were traced. The ectomesenchymal stem cells (EMSCs) were harvested in vitro, and the biological characteristics were observed. Moreover, the mineralization capability of EMSCs was evaluated. The relations between p75NTR and ALP, Col-1 and Runx2 were investigated., Results: The morphologic results showed that the dental lamina appeared at E12.5 d, the bud stage at E13.5 d, the cap stage at E15.5 d and the bell stage at E18.5 d. p75NTR and Runx2 showed the similar expression pattern. EMSCs from the four stages showed no significant difference in proliferation. But the positive rate of p75NTR in the E12.5 d cells was significantly lower than that in the other three stages (P < 0.05). Moreover, the higher positive rate of p75NTR the cells were, the stronger mineralization capability they showed. p75NTR was well positively correlated with the mineralization-related markers ALP, Col-1 and Runx2, which increased gradually with the mature of dental germs., Conclusion: p75NTR might play an important role in the regulation of tooth morphogenesis, especially dental hard tissue formation., (© 2018 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Objectives: Soluble IL-33 (interleukin (IL)-1-like cytokine) acts as endogenous alarm signal (alarmin). Since alarmins, besides activating immune system, act to restore tissue homeostasis, we investigated whether IL-33 exerts beneficial effects on oral stem cell pull., Materials and Methods: Clonogenicity, proliferation, differentiation and senescence of stem cells derived from human periodontal ligament (PDLSCs) and dental pulp (DPSCs) were determined after in vitro exposure to IL-33. Cellular changes were detected by flow cytometry, Western blot, immunocytochemistry and semiquantitative RT-PCR., Results: IL-33 stimulated proliferation, clonogenicity and expression of pluripotency markers, OCT-4, SOX-2 and NANOG, but it inhibited ALP activity and mineralization in both PDLSCs and DPSCs. Higher Ki67 expression and reduced β-galactosidase activity in IL-33-treated cells were demonstrated, whereas these trends were more conspicuous in osteogenic medium. However, after 7-day IL-33 pretreatment, differentiation capacity of IL-33-pretreated cells was retained, and increased ALP activity was observed in both cell types. Results showed that IL-33 regulates NF-κB and β-catenin signalling, indicating the association of these molecules with changes observed in IL-33-treated PDLSCs and DPSCs, particularly their proliferation, pluripotency-associated marker expression and osteogenesis., Conclusions: IL-33 treatment impairs osteogenesis of PDLSCs and DPSCs, while increases their clonogenicity, proliferation and pluripotency marker expression. After exposure to IL-33, osteogenic capacity of cells stayed intact. NF-κB and β-catenin are implicated in the effects achieved by IL-33 in PDLSCs and DPSCs., (© 2018 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Third molar development of 705 White and 563 Black South African individuals aged between 15 and 25 years was assessed from panoramic radiographs obtained from the School of Dentistry, University of Pretoria, South Africa. The maxillary and mandibular left third molars were scored according to a ten-stage scoring system. Ancestry and sex differences in dental maturity were assessed, and the likelihood of an individual being 18 years of age was determined for each developmental stage. Statistically significant differences were noted among ancestry groups for most developmental stages, with South African Black individuals consistently maturing earlier than the White individuals. Statistically significant differences were noted among sex groups for some of the stages, mostly those near the final stages of root development. The results indicate that male third molars completed their development faster than that of females. The likelihood of an individual being 18 years of age based on the third molar development stage for the maxilla and mandible on its own was also determined. Combined likelihood results, for the maxillary and mandibular left third molars for stage H, increased the likelihood of being 18 years to 95% for all the studied ancestry and sex groups.

Shark jaws exhibit teeth that are arranged into distinct series and files and display great diversities in shapes and structures, which not only is related to their function (grasping, cutting, crushing) during feeding, but also bear a strong phylogenetic signal. So far, most research on the relationship between shark teeth and feeding ecology and systematics focused on the external tooth morphology only. Although the tooth histology of sharks has been examined since the early 19th century, its functional and systematic implications are still ambiguous. Shark teeth normally consist of either a porous, cellular dentine, osteodentine (in lamniform sharks and some batoids) or a dense layer of orthodentine (known from different sharks). Sharks of the order Carcharhiniformes, comprising ca. 60% of all extant shark species, are known to have orthodont teeth, with a single exception-the snaggletooth shark, Hemipristis elongata. High resolution micro-CT images of jaws and teeth from selected carcharhiniform sharks (including extant and fossil snaggletooth sharks) and tooth sections of teeth of Hemipristis, other carcharhiniform and lamniform sharks, have revealed that (1) Hemipristis is indeed the only carcharhiniform shark filling its pulp cavity with osteodentine in addition to orthodentine, (2) the tooth histology of Hemipristis elongata differs from the osteodont histotype, which evolved in lamniform sharks and conversely represents a modified orthodonty, and (3) this modified orthodonty was already present in extinct Hemipristis species but the mineralization sequence has changed over time. Our results clearly show the presence of a third tooth histotype-the pseudoosteodont histotype, which is present in Hemipristis. The unique tooth histology of lamniform sharks might provide a phylogenetic signal for this group, but more research is necessary to understand the phylogenetic importance of tooth histology in sharks in general., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Knowledge of the growth status of patients is essential to formulate and initiate a precise treatment plan. This study aimed at determining the role of calcification of permanent mandibular teeth for the assessment of skeletal maturity., Methods: A cross-sectional study was conducted using lateral cephalograms and dental panoramic radiographs of 360 patients (ages 7-18 years) equally divided into six groups according to cervical vertebral maturation stages. Skeletal age was determined using Baccetti et al. method and dental age was calculated using Nolla and Demirjian methods., Results: Mean chronological stage at CS5 revealed a significant difference between male and female subjects (p= 0.003), which showed that the latter achieved skeletal maturity one year earlier than the former. A significant difference (p= 0.007) was found for dental age using Nolla's stages at CS3, which showed females demonstrated a dental age of 1.4 years less than males. Mandibular canine showed the highest correlation with Demirjian index (DI) in males (rho = 0.818) and females (rho = 0.833). Mandibular second premolar showed the highest correlation with Nolla's stages in males (rho = 0.654) and females (rho = 0.664)., Conclusion: Comparisons between sexes revealed that females are skeletally and dentally advanced. The DI indicated stage F and Nolla's stages identified stages 9, 10 to be indicative of CS2-3 for the mandibular canine and stages F and G and 9-10 for CS2-3 for the first premolars, second premolars and second molars, respectively.

Formation of highly organized dental hard tissues is a complex process involving sequential and ordered deposition of an extracellular scaffold, followed by its mineralization. Odontoblast and ameloblast differentiation involves reciprocal and sequential epithelial-mesenchymal interactions. Similar to early tooth development, various Bmps are expressed during this process, although their functions have not been explored in detail. Here, we investigated the role of odontoblast-derived Bmp2 for tooth mineralization using Bmp2 conditional knockout mice. In developing molars, Bmp2LacZ reporter mice revealed restricted expression of Bmp2 in early polarized and functional odontoblasts while it was not expressed in mature odontoblasts. Loss of Bmp2 in neural crest cells, which includes all dental mesenchyme, caused a delay in dentin and enamel deposition. Immunohistochemistry for nestin and dentin sialoprotein (Dsp) revealed polarization defects in odontoblasts, indicative of a role for Bmp2 in odontoblast organization. Surprisingly, pSmad1/5/8, an indicator of Bmp signaling, was predominantly reduced in ameloblasts, with reduced expression of amelogenin ( Amlx), ameloblastin ( Ambn), and matrix metalloproteinase ( Mmp20). Quantitative real-time polymerase chain reaction (RT-qPCR) analysis and immunohistochemistry showed that loss of Bmp2 resulted in increased expression of the Wnt antagonists dickkopf 1 ( Dkk1) in the epithelium and sclerostin ( Sost) in mesenchyme and epithelium. Odontoblasts showed reduced Wnt signaling, which is important for odontoblast differentiation, and a strong reduction in dentin sialophosphoprotein ( Dspp) but not collagen 1 a1 ( Col1a1) expression. Mature Bmp2-deficient teeth, which were obtained by transplanting tooth germs from Bmp2-deficient embryos under a kidney capsule, showed a dentinogenesis imperfecta type II-like appearance. Micro-computed tomography and scanning electron microscopy revealed reduced dentin and enamel thickness, indistinguishable primary and secondary dentin, and deposition of ectopic osteodentin. This establishes that Bmp2 provides an early temporal, nonredundant signal for directed and organized tooth mineralization.

FAM83H was identified as the major causative gene for autosomal dominant hypocalcified amelogenesis imperfect (ADHCAI). The pathogenic mechanism of FAM83H in ADHCAI remains elusive. The present study aims to investigate the effect of Fam83h mutation on the mineralization of mouse ameloblast cell line LS8 and to explore the possible pathogenesis of ADHCAI. Lentivirus package was performed for the plasmids with mouse Fam83h mutant cDNA (c.1186C > T, M3) and empty vector (Control) and transfected into LS8, which were divided into M3-FLAG and Control groups. Immunoprecipitation, western-blot and immunofluorescence were performed to detect the expression and subcellular localization of Fam83 h, CK1α and β-catenin. ALP activity, ALP staining, expression of the mineralization factors were detected in two groups during mineralization induction. Expression of the mineralization factors was also detected in M3-FLAG and LS8 exposing to pyrvinium pamoate. Compared with the Control, the Fam83h mutation altered the expression and localization of Fam83 h, CK1α and β-catenin in LS8, inhibited the mineralization and down-regulated the expression of mineralization factors in M3-FLAG. Pyrvinium pamoate, an inhibitor of the Wnt/β-catenin signaling pathway, up-regulated expression of mineralization factors in LS8 and rescued the inhibited mineralization in M3-FLAG. The results indicated that the Fam83h mutation could inhibit the mineralization in ameloblasts by activating Wnt/β-catenin signaling pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: This study aimed to search for scientific evidence concerning the accuracy of dental development for estimating the pubertal growth spurt., Methods: It was conducted according to the statements of PRISMA. An electronic search was performed in six databases, including the grey literature. The PICOS strategy was used to define the eligibility criteria and only observational studies were selected., Results: Out of 1,416 identified citations, 10 articles fulfilled the criteria and were included in this systematic review. The association between dental development and skeletal maturity was considered strong in seven studies, and moderate in two, although the association with the pubertal growth spurt had been verified in only four articles. According to half of the studies, the tooth that provided the greater association with the ossification centres was the lower canine. The meta-analysis performed also indicated a positive association, being stronger in females [0.725 (0.649-0.808)]. However, when the method used for dental evaluation was considered, it was possible to verify greater correlation coefficients for Nolla [0.736 (0.666-0.814)] than for Demirjian [0.631 (0.450-0.884)], at the boys sample. The heterogeneity test reached high values (Q = 51.00), suggesting a potential bias within the studies., Conclusions: Most of individual studies suggested a strong correlation between dental development and skeletal maturation, although the association with the peakof pubertal growth spurtwas clearly cited only in some of them. However, due to the high heterogeneity found among the studies included in this meta-analysis, a pragmatic recommendation about the use of dental stages is not possible.

Objective: This study aimed to compare the developmental timing and mineralization quality of mesiodentes, i.e., supernumerary teeth located mainly in the midline of the maxilla between the central incisors, with the developmental timing and mineralization quality of permanent and primary central incisors., Design: Sixteen mesiodentes, nine permanent and seven primary central incisors were collected. The location of the neonatal line was determined using a light microscope at 10× or 20× enlargements. Chemical composition of the enamel at two locations was analyzed using energy dispersive X-ray spectrometer., Results: Neonatal lines were observed in eight out of 16 mesiodentes, in all primary central incisors and in none of the permanent central incisors. Chemical analyses showed that mesiodentes mineralization was impaired, resulting in higher amount of organic ions and reduced inorganic ions. Discriminant analysis showed minimal overlap of mesiodentes with either primary or permanent centrals., Conclusions: Mesiodentes development begins before birth in 50% of the cases but later than the primary centrals. Mineralization of mesiodens is impaired with less mineral content and higher organic content. The results showed that mesiodentes are a special group of teeth with defective morpho-differentiation and mineralization, with little similarity to primary or permanent central incisors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

FAM20C mutations compromise the mineralization of skeleton and tooth in both human and mouse. Putatively, the mineralization disorder is attributed to the elevated fibroblast growth factor 23 (FGF23), which reduced the serum phosphorus by suppressing the reabsorption of phosphorus in kidney. Besides the regulation on systemic phosphorus homeostasis, FAM20C was also implicated to regulate cell behaviors and gene expression through a cell-autonomous manner. To identify the primary effects of Fam20c on dental mesenchymal cells, mouse Fam20c-deficient dental mesenchymal cells were generated by removing the floxed alleles from the immortalized mouse Fam20c f/f dental mesenchymal cells with Cre-expressing lentivirus. The removal of Fam20c exerted no impact on cell morphology, but suppressed the proliferation and mobility of the dental mesenchymal cells. Fam20c deficiency also significantly reduced the expression of Osterix, Runx2, type I Collagen a 1 (Col1a1), Alkaline phosphatase (Alpl) and the members of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family, but increased Fgf23 expression. Consistently, the in vitro mineralization of Fam20c-deficient dental mesenchymal cells was severely disabled. However, supplements of the non-collagenous proteins from wild type rat dentin failed to rescue the compromised mineralization, suggesting that the roles of FAM20C in tooth mineralization are more than phosphorylating local matrices and regulating systemic phosphorus metabolism. Moreover, the down-regulated BMP signaling pathways in the Fam20c deficient dental mesenchymal cells revealed that the kinase activity of FAM20C might be required to maintain BMP signaling. In summary, our study discloses that Fam20c indeed regulates cell behaviors and cell signaling pathway in a cell-autonomous manner., (Published by Elsevier Inc.)

Third molars are one of the few biological markers available for age estimation in undocumented juveniles close the legal age of majority, assuming an age of 18years as the most frequent legal demarcation between child and adult status. To obtain more accurate visualization and evaluation of third molar mineralization patterns from computed tomography images, a new software application, DentaVol©, was developed. Third molar mineralization according to qualitative (Demirjian's maturational stage) and quantitative parameters (third molar volume) of dental development was assessed in multi-slice helical computed tomography images of both maxillary arches displayed by DentaVol© from 135 individuals (62 females and 73 males) aged between 14 and 23years. Intra- and inter-observer agreement values were remarkably high for both evaluation procedures and for all third molars. A linear correlation between third molar mineralization and chronological age was found, with third molar maturity occurring earlier in males than in females. Assessment of dental development with both procedures, by using DentaVol© software, can be considered a good indicator of age of majority (18years or older) in all third molars. Our results indicated that virtual computed tomography imaging can be considered a valid alternative to orthopantomography for evaluations of third molar mineralization, and therefore a complementary tool for determining the age of majority., (Copyright © 2017 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.)

Forensic age estimation frequently relies upon the chronology of mineralization of the third molar teeth. However, even when present, third molar teeth cannot always be used for estimating age in people who are classified as minors. Seconds molars develop earlier and in a more predictable way, and therefore are often more reliable for age estimation in this age group. This study aims to contribute to forensic age estimation using an age threshold of 14-years, studying the stages of development of permanent mandibular second molar teeth mineralization. 367 orthopantograms of a Portuguese population group, aged between 3 and 19 years, were studied. The stages of mineralization of mandibular permanent second molar teeth were studied following the classification stages proposed by Demirjian et al. Stage descriptive analysis was performed, and associations between age and stage were studied. A logistic regression to determine age over 14 years, using maturation stages and sex as a predictive variables, was made. A second sample was used for testing the model. The significance level was set at 5%. The model correctly classified 92.0% of cases overall. The equation was tested in the second sample, and the results showed that there were no statistical significant differences between the binary real age (i.e. age < 14 and age ≥ 14 years) and the estimated age (p = 0.109). The developed model is useful for age estimation using 14-years as a threshold. However, stage maturation analyses showed that stage F, in males, and stages G and H, in both sexes, lead to an estimated age with significant statistical differences from chronological age.

Objective:: the objective of the study was to determine the relationship between dental calcification stages and skeletal maturation in a Peruvian sample., Methods:: panoramic, cephalometric and carpal radiographs of 78 patients (34 girls and 44 boys) between 7 and 17 years old (9.90 ± 2.5 years) were evaluated. Stages of tooth calcification of the mandibular canine, first premolar, second premolar, and second molar and the skeletal maturation with a hand-wrist and a cervical vertebrae method were assessed. The relationships between the stages were assessed using Spearman's correlation coefficient. Additionally, the associations of mandibular and pubertal growth peak stages with tooth calcification were evaluated by Fisher's exact test., Results:: all teeth showed positive and statistically significant correlations, the highest correlation was between the mandibular second molar calcification stages with hand-wrist maturation stages (r = 0.758, p < 0.001) and with vertebrae cervical maturation stages (r = 0.605, p < 0.001). The pubertal growth spurt was found in the G stage of calcification of the second mandibular molar, and the mandibular growth peak was found in the F stage of calcification of the second molar., Conclusion:: there was a positive relationship between dental calcification stages and skeletal maturation stages by hand-wrist and cervical vertebrae methods in the sample studied. Dental calcification stages of the second mandibular molar showed the highest positive correlation with the hand-wrist and cervical vertebrae stages.

Exposure of forming enamel to fluoride results into formation of hypomineralized enamel. We tested whether enamel hypomineralization was caused by lower expression of the NCKX4/SLC24A4 Ca 2+ -transporter by ameloblasts. Three commercial antibodies against NCKX4 were tested on enamel organs of wild-type and Nckx4-null mice, one of which (a mouse monoclonal) was specific. This antibody gave a prominent staining of the apical plasma membranes of maturation ameloblasts, starting at early maturation. The layer of immuno-positive ameloblasts contained narrow gaps without immunostaining or with reduced staining. In fluorotic mouse incisors, the quantity of NCKX4 protein in ameloblasts as assessed by western blotting was not different from that in non-fluorotic ameloblasts. However, immunostaining of the apical plasma membranes of fluorotic ameloblasts was strongly reduced or absent suggesting that trafficking of NCKX4 to the apical membrane was strongly reduced. Exposure to fluoride may reduce NCKX4-mediated transport of Ca 2+ by maturation stage ameloblasts which delays ameloblast modulation and reduces enamel mineralization., Competing Interests: A. L. J. J. Bronckers, R. Jalali, J. Lytton declare that they have no conflict of interest. Human and Animal Rights and Informed Consent The authors confirm that the animal studies were approved and carried out according to national guidelines.

Dental age determination methods that require the assessment of all teeth are tedious and time-consuming. Adapted methods that assess fewer teeth may be more easily applicable. The present study compared the applicability of two adapted Häävikko methods which evaluate seven mandibular teeth (HAM1) and four reference teeth (HAM2) in a population of 5 to 15 year-old Indian children. The HAM1 method underestimated age by -0.17 ± 0.80 years, -0.29 ± 0.83 years and -0.22 ± 0.82 years in boys, girls and the total sample respectively, while the HAM2 method underestimated age by -0.34 ± 0.88 years, -0.51 ± 0.82 years and -0.41 ± 0.86 years in boys, girls and the total sample, respectively. Significant gender-based differences were observed in mean DA-CA with both methods (p < 0.05). While both methods could be used for age estimation of the present population, the HAM1 method was the more accurate of the two.

Objective: The aims of this study were: to investigate the relationship between mandibular canine calcification stages and skeletal maturity; and to evaluate whether the mandibular canine calcification stages may be used as a reliable diagnostic tool for skeletal maturity assessment., Materials and Methods: This study included 151 subjects: 81 females and 70 males, with ages ranging from 9 to 16 years (mean age: 12.29±1.86 years). The inclusion criteria for subjects were as follows: age between 9 and 16 years; good general health without any hormonal, nutritional, growth or dental development problems. Subjects who were undergoing or had previously received orthodontic treatment were not included in this study. The calcification stages of the left permanent mandibular canine were assessed according to the method of Demirjian, on panoramic radiographs. Assessment of skeletal maturity was carried out using the cervical vertebral maturation index (CVMI), as proposed by the Hassel-Farman method, on lateral cephalograms. The correlation between the calcification stages of mandibular canine and skeletal maturity was estimated separately for male and female subjects., Results: Correlation coefficients between calcification stages of mandibular canine and skeletal maturity were 0.895 for male and 0.701 for female subjects., Conclusions: A significant correlation was found between the calcification stages of the mandibular canine and skeletal maturity. The calcification stages of the mandibular canine show a satisfactory diagnostic performance only for assessment of pre-pubertal growth phase., (Copyright © 2016 by Academy of Sciences and Arts of Bosnia and Herzegovina.)

Objective: To test in the maxilla and mandible for an association between stage of third-molar (M3) mineralization and space in the jaws for M3 eruption. Mineralization is hypothesized to be delayed not only for impacted M3s but also for M3s with eruption space less than their mesiodistal crown diameter., Material and Methods: Retrospective cone beam computed tomography (CBCT) scans of 37 females and 32 males aged 17-24 years, for a total sample of 197 upper and lower M3s, were used to assess the status of M3 eruption and measure the M3 crown diameter (CD) relative to the length of the retromolar space (RS). Stage of M3 mineralization was then compared between impacted and erupting M3s as well as between two conditions of relative eruption space (RS/CD ≥ 1 versus RS/CD < 1) using Mann-Whitney U tests., Results: Impacted M3s were at significantly earlier (delayed) stages of mineralization compared to erupting M3s. Mineralization was also delayed for M3s with eruption space less than their mesiodistal crown diameter (e.g. RS/CD < 1). A moderate positive correlation between stage of M3 mineralization and space was seen in both jaws, and was stronger in the mandible., Conclusion: Our study shows for the first time that stage of M3 mineralization is associated not only with impaction but also with amount of retromolar space, and that these associations are consistent in upper and lower jaws. Present findings underscore that M3 mineralization stage may be a clinically useful predictor of M3 impaction that thus merits further investigation.

Objective: The objective of this study was to investigate the diagnostic accuracy of dental maturation stages for identifying individual-specific skeletal maturation phases., Subjects and Methods: Prior to initiating this study, 255 orthodontic patients comprising 145 girls and 110 boys from the Department of Orthodontics, Aristotle University of Thessaloniki, Greece were identified. Lateral cephalometric and panoramic radiographs were evaluated. Dental calcification stages were assessed according to the Demirjian method and skeletal maturation according to the cervical vertebral maturation stage (CVMS) method. Statistical assessments included Spearman Brown formula, descriptive statistics, Spearman's rho correlation coefficient, and positive likelihood ratios (LHRs)., Results: The highest (r = 0.725) correlations were found for second molars and the lowest correlation for canines (r = 0.463, p < 0.001). Positive LHR values exceeding ten were found to identify the pre-peak growth phase in conjunction with the second molar (stage D), second premolar (stage E), and first premolar (stages D and E). Only the second molar (stage F) yielded positive LHR values for identifying the peak growth phase. The positive LHR values for the second molar also facilitated identification of the post-peak growth phase (stage H). Taking the clinical diagnostic efficacy of the second molar into account in identifying these growth phases, we calculated the positive LHRs of the second molar to determine dental maturation stages for diagnosing CVMS II and III. Positive LHR values greater than 10 identified CVMS II (stage D)., Conclusion: Evaluating dental maturation is a useful initial diagnostic step when assessing skeletal growth. The calcification stages of the second molar provide reliable diagnostic information with which to determine the pubertal growth spurt.

Chameleon teeth develop as individual structures at a distance from the developing jaw bone during the pre-hatching period and also partially during the post-hatching period. However, in the adult, all teeth are fused together and tightly attached to the jaw bone by mineralized attachment tissue to form one functional unit. Tooth to bone as well as tooth to tooth attachments are so firm that if injury to the oral cavity occurs, several neighbouring teeth and pieces of jaw can be broken off. We analysed age-related changes in chameleon acrodont dentition, where ankylosis represents a physiological condition, whereas in mammals, ankylosis only occurs in a pathological context. The changes in hard-tissue morphology and mineral composition leading to this fusion were analysed. For this purpose, the lower jaws of chameleons were investigated using X-ray micro-computed tomography, laser-induced breakdown spectroscopy and microprobe analysis. For a long time, the dental pulp cavity remained connected with neighbouring teeth and also to the underlying bone marrow cavity. Then, a progressive filling of the dental pulp cavity by a mineralized matrix occurred, and a complex network of non-mineralized channels remained. The size of these unmineralized channels progressively decreased until they completely disappeared, and the dental pulp cavity was filled by a mineralized matrix over time. Moreover, the distribution of calcium, phosphorus and magnesium showed distinct patterns in the different regions of the tooth-bone interface, with a significant progression of mineralization in dentin as well as in the supporting bone. In conclusion, tooth-bone fusion in chameleons results from an enhanced production of mineralized tissue during post-hatching development. Uncovering the developmental processes underlying these outcomes and performing comparative studies is necessary to better understand physiological ankylosis; for that purpose, the chameleon can serve as a useful model species., (© 2016 Anatomical Society.)

Objective: The objective of this study was to evaluate whether the calcification stages of maxillary canine, mandibular second molar, and mandibular third molar can be used for assessment of growth phase., Materials and Methods: The study group consisted of 274 subjects. Pre-treatment digital panoramic and lateral cephalometric radiographs of the patients were analysed. The patients' age was ranging from 7 to 19 years. Right maxillary canine, mandibular second molar and third molar were used as a sample. The teeth mineralization was assessed using modification of Gleiser and Hunt method. The skeletal maturation was assessed by the cervical vertebrae maturation (CVM) method., Results: A significant association was found between CVM stage 2 and maxillary canine (UC) stage 4, mandibular second molar (LM2) stage 4, and mandibular third molar (LM3) stage 1. CVM stage 3 corresponded with UC stage 5, LM2 stage 5, LM3 stage 2. CVM stage 4 matched with UC stage 5, LM2 stage 6 and LM3 stage 3. The highest correlations between CVM and calcification stages were in the group of the maxillary canine (r = 0.812, P < 0.01) and mandibular second molar (r = 0.824, P < 0.01)., Limitations: Limitation of our study was that the study sample was not very big and the distribution value in the groups was very high, so it was impossible to check more statistical parameters., Conclusions: The calcification stages of UC, LM2, and LM3 as indicators of skeletal maturity could be clinically used with caution, until this method is verified with a larger sample group., (© The Author 2015. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Phosphate is a key component of dental mineral composition. The physiological role of membrane proteins of dental cells is suspected to be crucial for mineralization mechanisms. Contrary to published data related to calcium, data on regulation of phosphate flux through membrane of mineralizing cells are scarce. To address this lack of data, we studied the expression of six membranous phosphate transporters in two dental cell lines: a rat odontoblastic cell line (M2H4) and a mouse ameloblastic cell line (ALC) for which we optimized the mineralizing culture conditions.

Introduction: Our aim in this study was to investigate the association between dental mineralization stages and the periods of the pubertal growth spurt (PGS)., Methods: The sample included panoramic and hand-wrist radiographs from 491 subjects (222 boys, 269 girls) aged 7 to 17 years. Dental development was rated, and skeletal maturation was evaluated. The relevant associations were investigated by analysis of ordinal multinomial logistic regression., Results: The second molar (odds ratio [OR] = 4.34) and the first premolar (OR = 2.45) were the best growth predictors for girls. For boys, the second molar (OR = 6.80), second premolar (OR = 2.41), and canine (OR = 3.21) proved to be the best predictors. Stages D and E of the second molar for girls, and stages E and F for boys, corresponded to the onset of the accelerated growth spurt. Stage F of the second molar for girls and stage G for boys corresponded to the peak of the PGS. At the end of the PGS, most teeth had already attained apical closure. In girls, however, most second molars were found at stage G., Conclusions: An association exists between the dental mineralization stages and the periods of the PGS, especially for second molars. Panoramic radiographs can be used as the first diagnostic tool to estimate the pubertal growth period., (Copyright © 2016 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Apatitic mineral of dentin forms within the collagenous matrix (intertubular dentin, ITD) secreted from the odontoblastic processes (OP). Highly calcified mineral (peritubular dentin, PTD) is deposited at the interface between the ITD and each process membrane, creating a tubular system penetrating the dentin that extends from the dentino-enamel junction to the predentin-dentin junction. We focus on determining the composition of the PTD both with regard to its organic matrix and the inorganic phase. A laser capture technique has been adapted for the isolation of the mineralized PTD free from the ITD, and for the analysis of the PTD by SEM, TEM, and energy dispersive spectrometry (EDS), these data were subsequently compared with similar analyses of intact dentin slices containing ITD bounded-PTD annuli. Elemental line scans reveal clearly marked boundaries between ITD, PTD, and OP components, and illustrate the differences in composition, and topographical surface roughness. The organic matrix of the PTD was shown to be sulfur rich, and further antibody labeling showed the sulfated organic component to be chondroitin sulfate [corrected]. In this PTD organic matrix the S/Ca and Ca/P ratios were distinctly higher than in the ITD, indicating that polysaccharide bound S supplies the anionic counterion facilitating the formation of the apatitic PTD mineral., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Most cases of hypophosphatasia (HPP) exhibit early loss of primary teeth. Results of micro-computed tomography (micro-CT) analysis of teeth with HPP have not yet been reported. The purpose of the present study was to describe the size and mineral density distribution and mapping of exfoliated teeth with HPP using micro CT., Study Design: Seven exfoliated teeth were obtained from a patient with HPP. Exfoliated teeth sizes were measured on micro CT images and mineral densities of the mandibular primary central incisors were determined., Results: Partial dentures were fabricated for the patient to replace the eight primary teeth which had exfoliated. Most primary teeth sizes were within the normal range. The mean values of enamel and dentin mineral densities in teeth with HPP were 1.35 and 0.88 g/cm 3 , respectively, in the mandibular primary central incisors., Conclusion: Mineral density distribution and mapping revealed that the values in teeth with HPP were lower than the homonymous teeth controls in all regions from the crown to apex. Furthermore, it was demonstrated that the differences between HPP and controls were larger on the crown side and the differences tended to converge on the apex side. These results suggested that the present patient showed mild hypomineralization in the primary dentition.

Objective: The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development., Materials and Methods: TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR., Results: Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration., Conclusion: These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.

This study investigated the relationships between the stages of calcification of teeth and cervical vertebral maturation. The sample consisted of 295 subjects (129 male and 166 female), mean age 13.36 ± 2.65 (range 7-18) years. Dental age was evaluated from panoramic radiographs according to the method of Demirjian. Cervical vertebral maturation was determined on lateral cephalometric radiographs using cervical vertebrae maturation stages (CVS). For assessing the relationship between cervical vertebral and dental maturation, percentage distributions of the stages of calcification for each studied tooth were calculated. Only in the first CVS stage, boys and girls were of the same age. In all other stages (CVS 2-CVS 6) girls were by 0.98 (range 0.23-1.86) younger than boys. Gender differences in the mineralization pattern were also observed. It was found that dental maturation was finished earlier in female subjects. The highest correlation coefficient between dental and skeletal maturity was found for second premolars. Mineralization pattern of second premolars could be considered as a guideline for prediction of the pubertal growth spurt. Dental maturation stages might be clinically useful as a reliable indicator of facial growth.

We report a novel method for the isolation of adult human epithelial stem cells (hEpiSCs) from the epithelial component of the periodontal ligament-the human epithelial cell rests of Malassez (hERM). hEpiSC-rich integrin-α6(+ve) hERM cells derived by fluorometry can be clonally expanded, can grow organoids, and express the markers of pluripotency (OCT4, NANOG, SOX2), polycomb protein RING1B, and the hEpiSC supermarker LGR5. They maintain the growth profile of their originating hERM in vitro. Subcutaneous cotransplantation with mesenchymal stem cells from the dental pulp on poly-l-lactic acid scaffolds in nude mice gave rise to perfect heterotopic ossicles in vivo with ultrastructure of dentin, enamel, cementum, and bone. These remarkable fully mineralized ossicles underscore the importance of epithelial-mesenchymal crosstalk in tissue regeneration using human progenitor stem cells, which may have already committed to lineage despite maintaining hallmarks of pluripotency. In addition, we report the clonal expansion and isolation of human LGR5(+ve) cells from the hERM in xeno-free culture conditions. The genetic profile of LGR5(+ve) cells includes both markers of pluripotency and genes important for secretory epithelial and dental epithelial cell differentiation, giving us a first insight into periodontal ligament-derived hEpiSCs., (© International & American Associations for Dental Research 2015.)

Introduction: The final stage of dental development of third molars is usually helpful to indicate whether or not a subject is aged over 18 years. A complexity is that the final stage of development is unlimited in its upper border. Investigators usually select an inappropriate upper age limit or censor point for this tooth development stage., Materials and Methods: The literature was searched for appropriate data sets for dental age estimation and those that provided the count (n), the mean (x¯), and the standard deviation (sd) for each of the tooth development stages. The Demirjian G and Demirjian H were used for this study. Upper and lower limits of the Stage G and Stage H data were calculated limiting the data to plus or minus three standard deviations from the mean. The upper border of Stage H was limited by appropriate censoring at the maximum value for Stage G., Results: The maximum age at attainment from published data, for Stage H, ranged from 22.60 years to 34.50 years. These data were explored to demonstrate how censoring provides an estimate for the correct maximum age for the final stage of Stage H as 21.64 years for UK Caucasians., Conclusion: This study shows that confining the data array of individual tooth developments stages to ± 3sd provides a reliable and logical way of censoring the data for tooth development stages with a Normal distribution of data. For Stage H this is inappropriate as it is unbounded in its upper limit. The use of a censored data array for Stage H using Percentile values is appropriate. This increases the reliability of using third molar Stage H alone to determine whether or not an individual is over 18 years old. For Stage H, individual ancestral groups should be censored using the same technique., (Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Objectives: Understanding variation in dental development among primates is important to accurately characterize species-specific sequences and times of tooth formation. Conventional approaches that summarize dental development data (i.e., dental maturity score, DMS) inherently omit information about the full range of variation in raw scores; thus, classic bivariate analyses are limited for exploring patterns of variation in detail. Here we report a new multivariate approach to simultaneously assess all raw dental scores, for all teeth, among all individuals for all groups, thus retaining much greater detail about population-specific patterns of variation., Methods: We scored (0-12) permanent tooth mineralization using radiographs of mandibles of captive-raised known-age chimpanzees (Pan troglodytes; n = 114) and free-lived age-unknown conspecifics (n = 54). As a test of our method we also scored free-lived baboons (Papio anubis, n = 50) because of well-described contrasting patterns of permanent molar initiation between Papio and Pan. Using principal component analysis (PCA), we investigated how crypt, crown and root formation scores covaried with each other in all three genera, and with chronological age in captive chimpanzees., Results: PCA successfully captured additional detail about variation among raw scores. Also, compared to DMS, PC1 scores correlated equivalently well with known ages but had lower prediction error., Conclusions: We found different patterns of variation in scores between younger juvenile free and captive chimpanzees but saw no wholesale differences between groups. Pan and Papio showed different patterns of variation, further validating this multivariate approach to visualize, quantify and compare raw dental score datasets among primate species., (© 2015 Wiley Periodicals, Inc.)

Objective: Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). PP which contains tandem serine/asparatic acid rich repeats (SDrr) is known to enhance dentin mineralization. The nucleotide sequences coding SDrr are identified in the DSPP genes of toothed animals and the length variations of SDrr between intra- and inter-species have been reported. However, it remains unknown about the relationship between the length variations in SDrr and the functions of PP in matrix mineralization., Design: By utilizing a mammalian expression system, we generated several recombinant PP proteins (rPP) containing SDrr of different lengths and analyzed their effects on the precipitation of calcium phosphate with an in vitro gel diffusion system., Results: rPP-Δ37.6 SDrr and rPP-Δ63.5 SDrr, which possessed shortened SDrr that accounted for 62.4 and 36.5% the length of SDrr in full-length rPP (rPP-full), respectively, induced the precipitation of calcium phosphate similar to that of rPP-full at the same molar concentration, whereas rPP-ΔSDrr, in which SDrr were flipped, did not. Furthermore, rPP-Δ63.5 SDrr significantly increased the accumulation of calcium compared with rPP-full at adjusted concentrations so that the same amounts of SDrr were embedded. The results of an ELISA analysis indicated that the amounts of rPP-Δ37.6 SDrr and rPP-Δ63.5 SDrr secreted from transfected cells were 5.2- and 7.1-fold greater than that of rPP-full, respectively., Conclusions: The generated rPP-Δ63.5 SDrr which can be substituted for rPP-full may be a candidate for a therapeutic molecule to facilitate hard tissue generation such as reparative dentin formation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Enamel is unique. It is the only epithelial-derived mineralized tissue in mammals and has a distinct micro- and nanostructure with nanofibrous apatite crystals as building blocks. It is synthesized by a highly specialized cell, the ameloblast, which secretes matrix proteins with little homology to any other known amino acid sequence, but which is composed of a primary structure that makes it competent to self-assemble and control apatite crystal growth at the nanometer scale. The end-product of ameloblast activity is a marvel of structural engineering: a material optimized to provide the tooth with maximum biting force, withstanding millions of cycles of loads without catastrophic failure, while also protecting the dental pulp from bacterial attack. This review attempts to bring into context the mechanical behavior of enamel with the developmental process of amelogenesis and structural development, since they are linked to tissue function, and the importance of controlling calcium phosphate mineralization at the nanometer scale. The origins of apatite nanofibers, the development of a stiffness gradient, and the biological processes responsible for the synthesis of a hard and fracture-resistant dental tissue are discussed with reference to the evolution of enamel from a fibrous composite to a complex, tough, and damage-tolerant coating on dentin., (© International & American Associations for Dental Research 2015.)

The molar tooth of the crayfish Cherax quadricarinatus is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of Cq-M13 in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, in vivo silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.

Enamel is a unique tissue in vertebrates, acellular, formed on a labile scaffolding matrix and hypermineralized. The ameloblasts are epithelial cells in charge of amelogenesis. They secrete a number of matrix proteins degraded by enzymes during enamel mineralization. This ordered cellular and extracellular events imply that any genetic or environmental perturbation will produce indelible and recognizable defects. The specificity of defects will indicate the affected cellular process. Thus, depending on the specificity of alterations, the teratogenic event can be retrospectively established. Advances in the field allow to use enamel defects as diagnostic tools for molecular disorders. The multifunctionality of enamel peptides is presently identified from their chemical roles in mineralization to cell signaling, constituting a source of concrete innovations in regenerative medicine., (© 2015 médecine/sciences – Inserm.)

Objectives: Understanding morphological changes and mineral content of tooth hard tissues may influence dental treatment. In this study, the effect of Type 1 Diabetes Mellitus (T1DM) on tooth structure was examined., Methods: Experimental T1DM was induced in 3-week old male Wistar rats (n=10) by a single dose of 60mg/kg body weight of Streprozotocin. All rats were injected with calcein twice during the experiment and sacrificed at the age of 7 weeks old. Micro-computed tomography (micro-CT) was used to determine the mineral density and thickness of enamel and dentine. Also, a histomorphometery study was conducted to detect the rates of dentine mineral apposition and formation. The examined area was in the crown analogue of the rat mandibular incisor parallel to the long axis of the mesial surface of the first molar. All results were compared using Students' t-test (p<0.05)., Results: Results showed that the enamel and dentine thickness were significantly reduced (hypoplasia) and there was a significant reduction of the rate of dentine mineral apposition and formation, while there was no significant effect of the T1DM condition on the mineral density of enamel and dentine., Conclusions: T1DM has a detrimental influence on the formation of enamel and dentine in the early growth stage., Clinical Significance: T1DM condition may alter treatment planning of orthodontic treatment as it is associated with decreased enamel and dentin thickness that may affect teeth size and their resistance to caries., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The dental pulp tissue is encased in hard tissue and surrounded by hard tissue-forming cells, but remains in a non-mineralized state itself, suggesting the presence of regulatory mechanisms precluding pulp mineralization. This study aimed to reveal the regulatory function of periostin (Postn), which is essential for osteoblast differentiation, for odontoblast differentiation/mineralization. We evaluated the effects of Postn overexpression and RNAi-mediated suppression in mouse dental papilla cells (MDPs) on the expression of odontoblastic markers and Notch signaling molecules, and on the formation of mineralized nodules. Localization of Postn in the dental pulp tissue of normal and cavity-prepared molars was observed immunohistologically. Enforced overexpression of Postn in MDPs induced down-regulation of odontoblastic markers and in vitro mineralization. Conversely, silencing of Postn mRNA in MDPs induced up-regulation of odontoblastic markers and ALP activity. Up- and down-regulation of Postn caused increased and decreased expression, respectively, of Notch signaling molecules. Postn expression was minimal in normal dental pulp, but was rapidly and globally increased in the whole pulp tissue of molar teeth at 1 day after cavity preparation, decreasing thereafter. These results indicate that Postn may be a negative regulator of odontoblast differentiation/mineralization, and that may exert its actions via Notch signals.

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr-null mice by quantitative electron probe microanalysis. Maturation-stage enamel from Cftr-null mice contained less chloride and calcium than did wild-type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr-null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr-deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion., (© 2014 Eur J Oral Sci.)

Objectives: The aim of the present study was to analyse the mineralization pattern of enamel and dentin in patients affected by X-linked hypophosphatemic rickets (XLHR) using micro-CT (µCT), and to associate enamel and dentin mineralization in primary and permanent teeth with tooth position, gender and the presence/absence of this disease., Methods: 19 teeth were collected from 5 individuals from the same family, 1 non-affected by XLHR and 4 affected by XLHR. Gender, age, tooth position (anterior/posterior) and tooth type (deciduous/permanent) were recorded for each patient. Following collection, teeth were placed in 0.1% thymol solution until µCT scan. Projection images were reconstructed and analysed. A plot profile describing the greyscale distance relationship in µCT images was achieved through a line bisecting each tooth in a region with the presence of enamel and dentin. The enamel and dentin mineralization densities were measured and compared. Univariate ANOVA and post hoc Tukey tests were used for all comparisons., Results: Teeth of all affected patients presented dentin with a different mineralization pattern compared with the teeth of healthy patients with dentin defects observed next to the pulp chambers. Highly significant differences were found for gray values between anterior and posterior teeth (p < 0.05), affected and non-affected (p < 0.05), as well as when position and disease status were considered (p < 0.05)., Conclusions: In conclusion, the mineralization patterns of dentin differed when comparing teeth from patients with and without FHR, mainly next to pulp chambers where areas with porosity and consequently lower mineral density and dentin defects were found.

Background: Age determination has great importance in many clinical decisions, being commonly used in odontopediatrics, orthodontics, pediatrics, and forensic medicine. The Nolla and Demirjian et al. methods have been used for these purposes. However, estimating chronological age by means of the dental mineralization stage is not a straightforward analysis, and it is fundamental to test the validity of these methods and their applicability to populations. In this article we intend to compare the accuracy of estimating chronological age from dental age measured with the Nolla and Demirjian methods in a Portuguese and Spanish sample, considering the variables of sex and age-group., Methods: The sample was composed of 821 orthopantomographs of healthy Portuguese (n = 270) and Spanish (n = 551) subjects from 4 to 34 years old. For the Nolla and Demirjian methods, seven mandibular left teeth were examined, staged according to the dental maturity scale of each method. We obtained a good index of inter-rater agreement, a good internal consistency for the teeth assessment, and a good temporal consistency., Results: Dental age was calculated for each method. The Demirjian et al. method tends to overestimate the real age of participants and the Nolla method tends to underestimate it. The accuracy of both methods varied between the sexes and age groups. Both methods were found to be more precise with males. As the age-group increases, the predictive capacities of both methods diminish. The Nolla method was more accurate than the Demirjian method in early and late childhood for both sexes. Neither method could predict chronological age in adults., Conclusions: We can estimate chronological age for early and late childhood, through the Nolla and Demirjian methods, with the former showing greater predictive capacities than the latter. The Demirjian method tends to overestimate age and the Nolla method tends to underestimate it, leading to the importance of forming regression equations adapted to the population studied. Nolla and Demirjian formulas adapted to our sample were created as a function of sex and age-group.

Objective: This study aimed to investigate how the structural stress distribution in human teeth could be affected by the presence of a graded material distribution often found in nature using the finite element (FE) method., Methods: Hydroxyapatite (HA) tablets with different densities were scanned using a Micro-CT scanner to obtain a relationship between the attenuation coefficient and the elastic modulus via the mineral density. Two maxillary premolars were scanned to provide the geometries and material distributions for constructing the FE models. Stress analyses were then performed to compare the stress distributions between the models with uniform material properties and those with a graded material layout., Results: The attenuation coefficients and densities of the HA tablets measured ranged from 109.77 to 175.01cm(-1) and 0.99 to 1.54gcm(-3), respectively. A linear relationship was found between them and applied to the premolars to derive the elastic modulus via the mineral density. Stress analysis showed that, with a graded material layout, the peak maximum principal stress in the enamel was reduced by about 50% and the overall stress distribution was more uniform. Along the DEJ, two stress peaks were found near the dentin horns, but again they were much lower in magnitude in the models with a graded material distribution., Significance: The results from this study support the hypothesis that the material layout in human enamel is optimized for distributing the external load evenly. They also point to the importance of taking into account the graded material distribution in nature when performing stress analysis for tooth structures., (Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Aim: The aim of this study was to investigate the relationship between the stages of calcification of the mandibular second molar and the stages of skeletal maturity among Italian children and young adults., Materials and Methods: Cross-sectional descriptive study. The samples were derived from panoramic radiographs and lateral cephalograms of 500 Italian subjects aged from 10 to 20 years. Dental maturity index (DM) was assessed by calcification stages of the mandibular second molars according to the Demirjian method; skeletal maturity was evaluated with cervical vertebrae maturation index (CVM) according to Hassel and Farman. The Pearson r coefficient and Spearman rank correlation coefficient were estimated to determine the relationship between DM and CVM., Results: Skeletal maturation stages occurred earlier in females (about 6 months). A highly significant correlation between CVM and DM (0.78-0.86 cor according to Pearson test and 0.81-0.85 rho according to Spearman test) was found. DM stage E corresponds to phase CVM 1 and 2 (the phase prior to the growth spurt), DM stage F corresponds to the phase of pubertal growth spurt, stages CVM 2 and CVM 3; DM stage G is indicator of the growth spurt underway (CVM 3-4), but it can still be found during CVM 5 in females., Conclusion: The second mandibular molars can be considered reliable indicators for the evaluation of the growth phases. In case of females the end of the growth spurt it is not clearly associated with a dental maturity stage and needs to be stated with further parameters such as CVM.

Objective: To assess the association between formation stages of the mandibular third molars and the space distal to the permanent molars (retromolar space)., Materials and Methods: The material included pretreatment lateral cephalographs of 96 orthodontic patients (49 males, 47 females; 8-18 years old). The molar formation stage was assessed through the method of Nolla, which rates the degree of calcification on a scale of 10 stages. The retromolar space was measured from the most concave point of the anterior border of the ramus to the distal surface of the first molar (used because the second molars had not yet erupted in the younger patients). Statistical analyses included t-tests and analyses of variance for group differences and the Pearson product moment to gauge associations among variables., Results: The formation stage advanced with age, but wide standard deviations were noted. Similarly, the retromolar distance increased with age and was greatest between 10 and 12 years. The correlation between retromolar space and developmental stage was high (r  =  0.85). On average, an increase of 5 mm of retromolar space corresponds to a 1.8 stage in tooth maturation., Conclusions: The correlation between third molar mineralization and available retromolar space essentially represents the association between one biologic age (dental formation) and another growth-related event (mandibular skeletal growth). The findings do not necessarily reflect successful emergence or nonimpaction of the molars. Longitudinal data are needed to determine such outcomes.

Objectives: This study aims to assess the accuracy of age estimation according to two cut-off points of Demirjian's developmental stages (G and H) in the wisdom teeth, using panoramic radiographs from Colombian and Mexican teenagers., Study Design: The degree of maturation of the third molars was classified according to Demirjian in 8 stages (from A to H) by a blinded trained assessor. The sensitivity, specificity and efficacy of two cut-off points (G and H) were calculated for both samples., Results: The orthopantomographies of 316 subjects, 171 Colombians (54.1%) and 145 Mexicans (45.9%), were analyzed. The stage H was found to be the best threshold for detecting juveniles (because the high specificity) in all the third molars assessed. The specificity was higher for lower third molars than for upper third molars, but no asymmetrical discrepancy was noted., Conclusions: The stage H is the best cut-off point for detecting the adulthood when a high-specificity test is required., (Copyright © 2014 Elsevier España, S.L.U. All rights reserved.)

Objectives: To investigate the relationship between cervical vertebrae maturation and mandibular second molar calcification stages., Study Design: The study was designed as a retrospective, descriptive and crosssectional research project. Pre-treatment lateral cephalograms and panoramic radiographs of 99 males and 110 females in the age range of 7 to 18 years 7 months were evaluated with Demirjian Index (DI) and cervical vertebrae maturation indicators (CVMI) of Hassel and Farman. A null hypothesis was proposed that there is no relation between CVMI and DI., Results: A highly significant association (Pearson's contingency coefficient 0.713 for males and 0.863 for females) was found between DI and CVMI. In males, the DI stage E corresponded to stage 2 of CVMI (pre-peak of pubertal growth spurt) and DI stages F and G corresponded to stages 3 and 4 of CVMI (peak of pubertal growth spurt). DI stage H was associated with stages 5 and 6 of CVMI (end of pubertal growth spurt). In females, the DI stages C, D corresponded to CVMI stages 1, 2; DI stages E, F with CVMI stages 3, 4; DI stages G, H with CVMI stages 5, 6., Conclusion: Mandibular second molar calcification stages can be used as indicators for assessment of skeletal maturity.

Dentin Sialophosphoprotein (DSPP) is the major non-collagenous protein of dentin and plays a significant role in dentin mineralization. Recently, animal models lacking DSPP have been developed and the DSPP KO phenotype has been characterized at the histological level. Little is known, however, about the DSPP KO dentin at nano- and meso-scale. Dentin is a hierarchical material spanning from nano- to macroscale, hence information on the effects of DSPP deficiency at the submicron scale is essential for understanding of its role in dentin biomineralization. To bridge this gap, we have conducted ultrastructural studies of dentin from DSPP KO animals. Transmission electron microscopy (TEM) studies of DSPP KO dentin revealed that although the overall ultrastructural organization was similar to the WT, the mineral particles were less organized. Scanning electron microscopy in the back-scattered mode (BS-SEM) of the DSPP KO dentin revealed that circumpulpal dentin comprises large areas of non-mineralized matrix, with numerous spherulitic mineralized inclusions, while the mantle dentin appeared largely unaffected. Analysis of the mineral distribution in the circumpulpal dentin of the DSPP KO mice suggests a reduction in the number of mineral nucleation sites and an increase in the nucleation barrier in DSPP KO dentin. These preliminary results indicate that in addition to the reduction of mineralized and total dentin volume in DSPP KO animals significant changes in the ultrastructural organization exist. These changes are likely related to the role of DSPP in the regulation of mineral formation and organization in dentin.

The objective of this study of the maturation of deciduous dentition was to offer a novel age-estimation method for Mediterranean populations, using the osteological collection of fetuses, infants, and young children in the Anthropology Laboratory of Granada University (Spain) as study material. After excluding premature newborns and infants with disease, the final study sample comprised 1303 deciduous teeth suitable for analysis from 138 individuals (80 male, 58 female) aged between 24 weeks in utero and 6 years. Eleven mineralization stages were defined for the dental maturation analysis, and the alveolar emergence was also studied. The criteria published by Demirjian et al. (Hum. Biol. 45 (1973) 211), Moorrees et al. (J. Dent. Res. 42 (1963a) 490), and Liversidge et al. (Am. J. Phys. Anthropol. 123 (2004) 172) were combined and modified for this purpose. The reproducibility of the proposed method is supported by the low intra- and inter-observer error in the identification of these development stages. The results provide information on the mean age of attainment of each of 11 mineralization stages and on the average age for each stage in each deciduous tooth type, considering each sex separately and both sexes combined., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)