Relationship between skeletal maturity indicators and dental calcification stages in a sample pediatric population.

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.

Skeletal, dental, and sexual maturation as an indicator of pubertal growth spurt.

OBJECTIVES: The aims of this study were to determine the skeletal, dental, and sexual maturation stages of individuals at the peak of the pubertal growth spurt and to analyze the correlations between these parameters. METHODS: The study included 98 patients, 49 females (mean chronological age 12.05 ± 0.96 years) and 49 males (mean chronological age: 13.18 ± 0.86 years), in the MP3cap stage. Skeletal maturation stages were determined using the cervical vertebral maturation (CVM) method on lateral cephalometric radiographs. The Demirjian index was used to determine dental maturation stages and dental ages on panoramic radiographs. The sexual maturation of the patients was evaluated in the pediatric endocrinology clinic by a pediatrician according to the Tanner stages. The frequencies of the variables were determined, and the Spearman rank correlation coefficients were used to analyze the correlations between the variables. RESULTS: It was determined that the cervical vertebral maturation stage was CS3 in 81.6% (n = 40) of both female and male patients, and 81.6% of the female and 89.8% of the male patients were in stage G in terms of mandibular second molar tooth development. According to the Tanner pubic hair staging, 73.5% of the male and 51.0% of the female patients were in Stage 3. A significant correlation was found between the cervical vertebra stages and mandibular second molar tooth development stages in both sexes and between the cervical vertebra and Tanner pubic hair stages only among the male patients (r = 0.357; p < .05). There was also a significant and strong correlation between the Tanner pubic hair stages and breast development stages (r = 0.715; p < .05). CONCLUSION: Cervical vertebral development in the CS3 stage and mandibular molar tooth development in the G stage can be considered the peak of the pubertal growth spurt. Tanner Stage 3 marks the peak of the pubertal growth spurt in males.

Mediator1 involved in functional integration of Smad3 and Notch1 promoting enamel mineralization.

Enamel hypoplasia is a tooth development defection due to the disruption of enamel matrix mineralization, manifesting as chalky white phenotype. Multiple genes may be involved in this tooth agenesis. It has been proved that ablation of coactivator Mediator1 (Med1) switches the cell fate of dental epithelia, resulting in abnormal tooth development via Notch1 signaling. Smad3 (-/-) mice displays the similar chalky white incisors. However, the expression of Smad3 in Med1 ablation mice and the impact of Med1 on functional integration between Smad3 and Notch1 remains unclear. Cre-loxP-based C57/BL6 mice with epithelial-specific Med1 knockout (Med1 KO) backgrounds were generated. Mandibles and dental epithelial stem cells (DE-SCs) from incisors cervical loop (CL) were isolated from wild-type (CON) mice and Med1 KO mice. Transcriptome sequencing was used to analyze the differences of CL tissue between KO and CON mice. The results revealed the enrichment of TGF-ß signaling pathway. qRT-PCR and western blot were performed to show the gene and protein expression of Smad3, pSmad3, Notch1 and NICD, the key regulators of TGF-ß and Notch1 signaling pathway. Expression of Notch1 and Smad3 was confirmed to be down-regulated in Med1 KO cells. Using activators of Smad3 and Notch1 on Med1 KO cells, both pSmad3 and NICD were rescued. Moreover, adding inhibitors and activators of Smad3 and Notch1 to cells of CON groups respectively, the protein expressions of Smad3, pSmad3, Notch1 and NICD were synergistically affected. In summary, Med1 participates in the functional integration of Smad3 and Notch1, thus promoting enamel mineralization.

Cone beam computed tomography assessment of the prevalence and association of pulp calcification with periodontitis.

Periodontitis has a known association with pathological calcification in the cardiovascular system. Considering the close anatomic and circulatory association between dental pulp and the periodontium, this study aimed to evaluate the prevalence of pulp calcification (PC) under different periodontal conditions, as well as the associations of PC with the degree of periodontal damage, via cone beam computed tomography (CBCT) examination. In this study, 55 patients were categorized into three groups according to periodontal condition: group 1 (healthy controls), group 2 (periodontitis stage I-II), and group 3 (periodontitis stage III-IV). PC and radiographic bone loss (RBL) was assessed by CBCT in sagittal, axial, and coronal views, and statistical analyses were conducted. PC was identified in 378 of 1170 teeth (32.3%). The prevalence significantly differed among the three groups (P < 0.001). Group 2 had a 2.43-fold (P < 0.001, 95% confidence interval [CI] 1.64-3.61) higher risk of PC than group 1; and the risk of PC was 3.04-fold (P < 0.001, 95% CI 2.06-4.48) higher in group 3 than group 1. Teeth with more severe RBL exhibited a higher prevalence of PC (P < 0.001). Molar teeth had a higher risk of PC than incisors and premolars. In conclusion, the occurrence of PC is related to the periodontal state, and the prevalence of PC is higher in teeth with periodontitis; tooth type and periodontitis status are important risk factors for PC.

Accuracy of dental calcification stages for the identification of craniofacial pubertal growth spurt: proposal of referral parameters.

PURPOSE: This study primarily aimed to assess the accuracy of radiographic dental calcification parameters for the identification of specific craniofacial growth stages. METHODS: Permanent mandibular canines, premolars, and second molars of 288 patients, from 6 to 15 years old, were analysed on randomly selected panoramic radiographs, and categorised according to two calcification methods. Reproducibility analyses and data derived from panoramic records were correlated with the gold-standard method, as evaluated from lateral cephalometric teleradiographs. Accuracy tests were finally calculated, considering several cutoff points. RESULTS: Dental calcification methods showed "strong" to "almost perfect" intra- and inter-examiner reproducibility. Significant, although weak correlations were observed for all parameters. Canine and first premolar calcification stage 8 and second premolar and second molar stage 7 showed higher sensitivity rates for identifying the pubertal growth spurt period, as well as the stage F for these teeth. Canine and first premolar stages 10 and H obtained higher specificity rates for identifying the absence of post-pubertal period. CONCLUSIONS: The dental calcification parameters showed adequate reproducibility, in addition to significant correlations with cervical vertebrae stages. Radiographic dental calcification parameters used for the mandibular first premolar obtained high accuracy rates and were recommended for identifying specific craniofacial growth periods.

CRISPR/Cas9-mediated deletion of Fam83h induces defective tooth mineralization and hair development in rabbits.

Family with sequence similarity 83 members H (Fam83h) is essential for dental enamel formation. Fam83h mutations cause human amelogenesis imperfecta (AI), an inherited disorder characterized by severe hardness defects in dental enamel. Nevertheless, previous studies showed no enamel defects in Fam83h-knockout/lacZ-knockin mice. In this study, a large deletion of the Fam83h gene (900 bp) was generated via a dual sgRNA-directed CRISPR/Cas9 system in rabbits. Abnormal tooth mineralization and loose dentine were found in homozygous Fam83h knockout (Fam83h-/- ) rabbits compared with WT rabbits. In addition, reduced hair follicle counts in dorsal skin, hair cycling dysfunction and hair shaft differentiation deficiency were observed in Fam83h-/- rabbits. Moreover, X-rays and staining of bone sections showed abnormal bending of the ulna and radius and an ulnar articular surface with insufficient trabecular bone in Fam83h-/- rabbits. Taken together, these data are the first report of defective hair cycling, hair shaft differentiation and abnormal bending of the ulna and radius in Fam83h-/- rabbits. This novel Fam83h-/- rabbit model may facilitate understanding the function of Fam83h and the pathogenic mechanism of the Fam83h mutation.

The Cameriere, Haavikko, Demirjian, and Willems methods for the assessment of dental age in Croatian children.

AIM: This study aimed to evaluate the accuracy and precision of the Cameriere European formula, Demirjian, Haavikko, and Willems methods for estimating dental age in a sample of children with permanent dentition in Croatia. MATERIAL AND METHODS: The study consisted of a sample of 1576 panoramic radiographs; a sub-sample of 84 OPGs, in which all first seven mandibular teeth were maturated, was excluded from the study. A final sample of 1492 (704 males and 788 females) aged 6.0 to 13.9 years was evaluated. Seven mandibular teeth from the left side of the mandible were analyzed, and dental age (DA) was determined by the Cameriere European formula, Demirjian method from 1976, Haavikko, and Willems methods and compared to chronological age (CA). In addition, the mean age difference (DA-CA), the mean absolute error (MAE) between dental and chronological age, the percentage of the individuals of dental age within ± 0.25 to ± 2 years of chronological age, and intra-observer and inter-observer statistics were calculated. RESULTS: The Cameriere European formula estimated the best dental age compared to the chronological age; the mean underestimation was - 0.4 years for both sexes, Haavikko underestimated by - 0.17 years, while Demirjian and Willems overestimated by 1.02 years and 0.48 years, respectively. The most significant difference showed the Demirjian method in 11-year-old and 12-year-old females. The MAE were 0.50 years, 1.01 years, 0.61 years, and 0.78 years in males and 0.51 years, 1.18 years, 0.61 years, and 0.70 years in females for the Cameriere European formula, Haavikko, Willems, and Demirjian methods, respectively. Furthermore, the Cameriere European formula showed the highest proportions of individuals with DA within ± 0.5 year difference of the CA, 61.5% in males and 59.6% in females. In addition, the Cameriere method showed the best intra-observer and inter-observer agreements. CONCLUSIONS: Although the Demirjian method was used previously in Croatian children for legal, medical, and clinical purposes, the Cameriere European formula, Haavikko, and Willems were more accurate in the tested sample. According to our findings, the Cameriere European formula showed the best accuracy and precision in dental age assessment in Croatian children following Haavikko, and we recommend it as the method of the first choice in forensic and clinical analyses.

Application of the ratio of the radiopaque calcified area to the dental follicle (RCA/DF) for dental age assessment on orthopantomograms.

OBJECTIVES: This study aimed at exploring a new tooth development evaluation method for age assessment and investigating the dynamic alteration and potential trend of tooth development by orthopantomograms (OPGs), in order to provide references for tooth development prediction and forensic purpose. MATERIALS AND METHODS: A total of 132 OPGs of children aged 3-8 years were collected. The developmental stages of the permanent mandibular second molar (M2)were evaluated by experienced examiners according to the Nolla method and Mimics software, respectively. Quantitative analysis of the ratio of the radiopaque calcified area to the dental follicle (RCA/DF) in different stages, ages, sexes and quadrants were evaluated and compared via descriptive statistics and Spearman's correlation coefficient analysis. RESULTS: There was a strong, positive correlation between the examiners' evaluation and mimics analysis results. With the age increased, the Nolla stage of M2 observed by OPGs increased, and the RCA/DF showed increased trends both in males and females. There were significant differences of the RCA/DF of the M2 at various ages. The tooth calcification development of female was 9.08% earlier than that of male between 3 and 8 years old. However, teeth of male seemed to develop faster than that of female during this period. There was no significant difference between left and right quadrant either according to the Nolla stage or RCA/DF. CONCLUSIONS: The RCA/DF value obtained from OPGs of the developmental mandibular second permanent molars could be used as a reliable indicator for tooth maturity and age estimation in children aged 3-8 years. CLINICAL RELEVANCE: Age assessment based on radiographs is considered as a reliable and efficient indicator for judging different types of malocclusion, making suitable orthodontic treatment plan, deciding the extraction time of retained deciduous teeth in clinic practice.

Influence of violet LED associated or not with peroxide gel on inflammation, mineralization, and collagen fiber maturation in dentin and pulp tissue.

OBJECTIVES: To evaluate the influence of violet LED, associated or not with a 17.5% hydrogen peroxide (HP) bleaching gel, on inflammation, mineralization in pulp tissue, and collagen fiber maturation in dentin and pulp tissue. MATERIALS AND METHODS: The maxillary molars of eighty Wistar rats were distributed into four groups (n = 10): CONT - without treatment; HP - 30 min application of 17.5% HP; LED - 20 min application of violet LED; and HP+LED - application of PH and violet LED. Rats were euthanized and jaws were processed for histologic and immunohistochemical evaluation (IL-17, IL-23, and osteocalcin) and picrosirius red immediately after (T0), and at 7 (T1), 15 (T2), and 30 days (T3) post-treatment, with Wilcoxon, Mann-Whitney, paired T-test, and T-test (α = 0.05). RESULTS: HP and HP+LED presented necrosis and severe inflammatory infiltrate. When compared to CONT group, LED presented severe osteocalcin (OCN) immunostaining in T2 and less immature fibers in T2 and T3. CONCLUSION: The violet LED caused no severe damage to the pulp tissue, increased IL-17 and IL-23 expression in T0 when associated with HP, and had no influence on pulp tissue mineralization, besides accelerating the maturation of collagen fibers of dentin. CLINICAL RELEVANCE: Violet LED therapy induced no inflammation in the pulp tissue of rats and played no role in pulp tissue fibrosis, besides accelerating the maturation of dentin collagen fibers.

Correlations between skeletal maturity and dental calcification stages in Korean children.

AIM: To evaluate the relationship between dental calcification and skeletal maturity and to identify the tooth with the highest correlation with skeletal maturity index in Korean children. MATERIALS: For 447 children (205 boys and 242 girls) aged between 5 and 13 years, hand-wrist and lateral cephalometric radiographs were taken to assess skeletal maturity by Fishman's skeletal maturity indicators (SMI) and Baccetti's cervical vertebrae maturation (CVM) stages. Dental panoramic radiographs were taken to assess dental maturity of the permanent mandibular canine, first and second premolar, and second molar using the method devised by Dermirjian. CONCLUSION: Dental calcification stages determined by panoramic radiographs can be clinically used as useful indices to predict skeletal maturity in Korean children.

Dental age estimation with fewer than mandibular seven teeth: An accuracy study of Bedek models in Turkish children.

OBJECTIVES: One or more missing teeth, such as hypodontia, make it difficult to obtain accurate results in age estimation methods. This paper aims to test the accuracy of the models developed by Bedek et al. that can estimate age with fewer than seven teeth for Turkish children. MATERIALS AND METHODS: Panoramic radiographs of 1118 children (540 girls and 578 boys) aged between 6 and 16 years were evaluated using the Willems method and Bedek models for the entire sample and for different ages and sexes. Differences between dental age (DA) and chronological age (CA) calculated for all methods for each sex and all age groups were analyzed the paired sample t-test and Wilcoxon signed rank test. The accuracy of the DA estimation methods was determined by the proximity of DA to CA by calculating mean absolute error (MAE). RESULTS: While all Bedek models underestimated CA by 0.2 to 0.1 years, the Willems method overestimated CA by 0.3 years. DA-CA difference in the total sample and girls was statistically significant in all methods. In the total sample, the seven- and four-teeth models had the lowest MAE, while the one-tooth model had the highest MAE. CONCLUSIONS: Seven- and four-teeth models were the most suitable for age estimation, and all models except the one-tooth model were found to be more accurate than the Willems method in northwestern Turkish children. CLINICAL RELEVANCE: Especially in children with hypodontia, it may be possible to estimate DA with the use of Bedek models.

Is there a correlation between dental and cervical vertebrae maturation stages in growing subjects? A systematic review with meta-analysis.

OBJECTIVE: To assess the evidence on the relationship between dental and cervical vertebrae maturation. MATERIALS AND METHODS: Searches were performed (up to December 2021) in seven databases, as well as in gray literature. Studies that included growing subjects, which evaluated the correlation between the stages of tooth formation and the maturation of cervical vertebrae, were eligible. The methodological quality of the selected reports was assessed using the JBI checklist for cross-sectional studies. Random-effects meta-analyses were implemented to calculate the pooled correlation coefficients between the maturation stages of cervical vertebrae and each tooth type. The GRADE approach was followed to assess the certainty of evidence. RESULTS: Seventy-seven studies were selected. Most of them had limitations related to sampling and reliability of the methods to evaluate maturation. The syntheses evidenced positive correlations between the maturation of cervical vertebrae and the formation/calcification stages of the following lower teeth: canine (n = 7318; r = 0.692; 95% CI: 0.656-0.724), first premolar (n = 6194; r = 0.689; 95% CI: 0.649-0.725), second premolar (n = 6194; r = 0.695; 95% CI: 0.659-0.729), and second molar (n = 7905; r = 0.698; 95% CI: 0.652-0.739). All estimates were affected by the risk of bias and the presence of unexplained statistical heterogeneity. CONCLUSIONS: The evidence points to an apparent positive correlation between dental and cervical vertebrae maturation. These results need to be carefully evaluated as the body of evidence is of very low quality. CLINICAL RELEVANCE: Dental maturation might be a potential indicator of skeletal craniofacial growth status. A routine dental x-ray could be sufficient, reducing radiation exposure by requesting additional radiographs.

Demirjian and Cameriere methods for age estimation in a Spanish sample of 1386 living subjects.

Currently, human identification is a challenge. Migration due to war, economic crisis or other factors is frequent. The wisdom teeth are the last teeth to initiate and complete development therefore, are fundamental for determining the legal age of majority in different countries. The aim of the study is to determine the validity of two methods based on mineralisation of the third molar to predict the ages of majority of individuals in a Spanish population. Orthopantomographies of 636 men and 750 women (mean age, 16.5 years) were analysed. The Demirjian and Cameriere methods were used, and each tooth was assigned a value according to the degree of mineralisation and maturation. The level of significance used in the analyses was 5% (α = 0.05), with a power of 96.2%. The predictive ability of the Demirjian method to determine 18 years of age in the lower wisdom teeth 93%, respectively. The Cameriere method has a predictive capacity of 88%. There are no statistically significant differences between men and women. Stage H and a cut-off point of 0.08 were the guiding values for determining the age of majority of the study population. For other proposed age ranges (14 and 16 years), both methods were useful in determining the actual age of individuals, with the Demirjian method having a sensitivity of 97.5% with and Cameriere having a predictive capacity of 95%. Both methods can be used with high reliability to determine the age of individuals where reliable documentation is unavailable. Stage H with the Demirjian method and a cut-off point of 0.08 with the Cameriere method can determine the age of majority of the Spanish population. The combination of the two methods does not substantially increase predictive ability.

Multi-Level Approach for Comprehensive Enamel Phenotyping.

Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 µm for in vivo imaging to 1.5 µm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium-carbon ratio. However, the methods mentioned above only show the result-already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels-starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high-resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: In vivo MicroCT scanning of mouse Basic Protocol 2: Ex vivo HR-MicroCT of the teeth Basic Protocol 3: SEM for teeth microstructure Basic Protocol 4: Stimulated emission depletion (STED) microscopy.

Guided endodontic access of severe calcified tooth without incisal edge ­ case report / Acesso endodôntico guiado de dente calcificado severamente sem borda incisal ­ relato de caso

Pulp tissue may suffer calcification because of trauma, operative procedures or carious lesions. This paper aimed to report and discuss the guided endodontic access as an alternative treatment. A 52 years old female patient had severe root canal calcification of tooth #11 associated with a radiolucent periapical lesion. Firstly, the crown and metal post and core were removed. A digital impression and cone-beam computed tomography "CBCT" scans were performed and imported to implant planning software (SimPlant Version 11; Materialise Dental, Leuven, Belgium).The guided endodontic access template was designed to allow the drill to reach a distance of 2 mm short of the apical foramen, once printed, it was tested in the mouth to evaluate its insertion and stability in the dental arch. The calcified root canal was penetrated using the access drill rotating by a low-speed hand-piece (10,000 rpm) under saline solution irrigation through advancing movements. Then, the apical foramen was negotiated with C-Pilot files #10 and #15. The working length was measured using the iPex-II apex locator. The instrumentation was carried out with Reciproc R50 and 2.5% sodium hypochlorite. One week later, a full-ceramic crown preparation was performed, and polyvinyl siloxane impression was carried out. A total of three follow-up sessions were performed after one week, one and twelve months. Bone neoformation was observed in the site of the periapical lesion and the patient had no signs or symptoms of any discomfort. Therefore, guided endodontics is indicated for severe calcified root canals.(AU)

O tecido pulpar pode sofrer calcificação por trauma, procedimentos cirúrgicos ou como resposta a lesões cariosas. Este trabalho teve como objetivo relatar e discutir o acesso endodôntico guiado como opção de tratamento. Paciente do sexo feminino, 52 anos, com calcificação severa do canal radicular do dente 11 associada a lesão periapical radiolucida. Na primeira intervenção clínica, a coroa e o pino de metal foram removidos. Uma impressão digital e imagens de CBCT foram realizadas e importadas para o software de planejamento de implante (SimPlant Versão 11; Materialize Dental, Leuven, Bélgica) tentando projetar um modelo de acesso endodôntico guiado para permitir que a broca alcance uma distância de 2 mm antes do forame apical, uma vez impresso, foi testado na boca para avaliar sua inserção e estabilidade na arcada dentária. O canal radicular calcificado foi penetrado com broca de acesso girando por peça de mão de baixa velocidade (10.000 rpm) sob irrigação com solução salina por meio de movimentos de avanço. Em seguida, o forame apical foi negociado com as limas C-Pilot nº 10 e nº 15. O comprimento de trabalho foi determinado usando o localizador de ápice iPex-II. A instrumentação foi realizada com Reciproc R50 e hipoclorito de sódio 2,5%. Uma semana depois, foi realizado o preparo da coroa total em cerâmica e a moldagem com polivinilsiloxano. Um total de três sessões de acompanhamento foram realizadas após uma semana, um e doze meses. A neoformação óssea foi observada no local da lesão periapical e a paciente não apresentava sinais ou sintomas de qualquer desconforto. Portanto, o acesso endodôntico guiado é indicado para canais radiculares calcificados severamente.(AU)

Hydroxypropylmethylcellulose as a film and hydrogel carrier for ACP nanoprecursors to deliver biomimetic mineralization.

Demineralization of hard tooth tissues leads to dental caries, which cause health problems and economic burdens throughout the world. A biomimetic mineralization strategy is expected to reverse early dental caries. Commercially available anti-carious mineralizing products lead to inconclusive clinical results because they cannot continuously replenish the required calcium and phosphate resources. Herein, we prepared a mineralizing film consisting of hydroxypropylmethylcellulose (HPMC) and polyaspartic acid-stabilized amorphous calcium phosphate (PAsp-ACP) nanoparticles. HPMC which contains multiple hydroxyl groups is a film-forming material that can be desiccated to form a dry film. In a moist environment, this film gradually changes into a gel. HPMC was used as the carrier of PAsp-ACP nanoparticles to deliver biomimetic mineralization. Our results indicated that the hydroxyl and methoxyl groups of HPMC could assist the stability of PAsp-ACP nanoparticles and maintain their biomimetic mineralization activity. The results further demonstrated that the bioinspired mineralizing film induced the early mineralization of demineralized dentin after 24 h with increasing mineralization of the whole demineralized dentin (3-4 µm) after 72-96 h. Furthermore, these results were achieved without any cytotoxicity or mucosa irritation. Therefore, this mineralizing film shows promise for use in preventive dentistry due to its efficient mineralization capability.

Identification of dental calcification stages as a predictor of skeletal development phase.

OBJECTIVE: This study aimed to establish a correlation between the stages of tooth calcification of mandibular canines and second molars with the phases of skeletal development. METHODS: In a consecutive series of panoramic, cephalometric and hand-wrist radiographs of 113 individuals (60 females and 53 males) with an average age of 12.24 ± 1.81 years, the stages of mandibular canine and second molar calcification, cervical vertebrae maturation indicators (CVMI) and skeletal maturity indicators (SMI) were classified. The variables were correlated by means of the Spearman's Rank test: chronological age, SMI, CVMI and tooth calcification stages. In order to assess whether the CVMI and tooth calcification stages were significant predictors of the SMI, an ordinal regression analysis was carried out. RESULTS: The stages of CVMI (OR = 16.92; CI 95% = 6.45-44.39; p< 0.001) and calcification of the second molars (OR = 3.22; CI 95% = 1.50-6.92; p= 0.003) were significant predictors of SMI, however similar result was not observed for canines (OR = 0.52, CI 95% = 0.18-1.54; p= 0.239). Calcification stage E for boys, and E and F for girls corresponded to the pre-peak phase of pubertal growth. Stages G and H for boys, and F and G for girls coincided with peak of growth. In the final growth phase, the majority of second molars presented with root apex closure (stage H). CONCLUSION: The stages of calcification of the second molar may be considered predictors of the stage of skeletal development in the population studied.

Plasma Membrane Ca2+-ATPase in Rat and Human Odontoblasts Mediates Dentin Mineralization.

Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+-Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1-4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4-8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

Ezh2 knockout in mesenchymal cells causes enamel hyper-mineralization.

Enhancer of zeste homolog 2 (EZH2) is the catalytic core of polycomb repressive complex 2 (PRC2), which primarily methylates lysine 27 on histone H3 (H2K27me3), generating transcriptionally suppressed heterochromatin. Since EZH2 suppresses expression of genes involved in dentin formation, we examined the role of EZH2 in tooth development. Intriguingly, microCT analysis of teeth from mice with conditional Ezh2 knockout in uncommitted mesenchymal cells showed hyper-mineralization of enamel, which is produced by the epithelial-lineage cells, ameloblasts. Scanning electron microscopy analysis and nano-indentation of the incisor enamel from knockout mice revealed smaller inter-rod spaces and higher hardness compared to wild type enamel, respectively. Interestingly, expression of the calcium channel subunit gene, Orai2, was decreased compared to its competitor, Orai1, both in knockout mouse incisors and the ex vivo culture of ameloblasts with the surrounding tissues under EZH2 inhibition. Moreover, histological analysis of incisor from knockout mice showed decreased ameloblastin and expedited KLK4 expression in the ameloblasts. These observations suggest that EZH2 depletion in dental mesenchymal cells reduces enamel matrix formation and increases enamel protease activity from ameloblasts, resulting in enamel hyper-mineralization. This study demonstrates the significant role of the suppressive H3K27me3 mark for heterochromatin on enamel formation.

Molecular and morphological findings in a sample of oral surgery patients: What can we learn for multivariate concepts for age estimation?

It has already been proposed that a combined use of different molecular and morphological markers of aging in multivariate models may result in a greater accuracy of age estimation. However, such an approach can be complex and expensive, and not every combination may be useful. The significance and usefulness of combined analyses of D-aspartic acid in dentine, pentosidine in dentine, DNA methylation in buccal swabs at five genomic regions (PDE4C, RPA2, ELOVL2, DDO, and EDARADD), and third molar mineralization were tested by investigating a sample of 90 oral surgery patients. Machine learning models for age estimation were trained and evaluated, and the contribution of each parameter to multivariate models was tested by assessment of the predictor importance. For models based on D-aspartic acid, pentosidine, and the combination of both, mean absolute errors (MAEs) of 2.93, 3.41, and 2.68 years were calculated, respectively. The additional inclusion of the five DNAm markers did not improve the results. The sole DNAm-based model revealed a MAE of 4.14 years. In individuals under 28 years of age, the combination of the DNAm markers with the third molar mineralization stages reduced the MAE from 3.85 to 2.81 years. Our findings confirm that the combination of parameters in multivariate models may be very useful for age estimation. However, the inclusion of many parameters does not necessarily lead to better results. It is a task for future research to identify the best selection of parameters for the different requirements in forensic practice.