Multivariate GWAS of Structural Dental Anomalies and Dental Caries in a Multi-Ethnic Cohort

Odontogenesis is a complex process, where disruption can result in dental anomalies and/or increase the risk of developing dental caries. Based on previous studies, certain dental anomalies tend to co-occur in patients, suggesting that these traits may share common genetic and etiological components. The main goal of this study was to implement a multivariate genome wide association study approach to identify genetic variants shared between correlated structural dental anomalies and dental caries. Our cohort (N = 3,579) was derived from the Pittsburgh Orofacial Clefts Study, where multiple dental traits were assessed in both the unaffected relatives of orofacial cleft (OFC) cases (n = 2,187) and unaffected controls (n = 1,392). We identified four multivariate patterns of correlated traits in this data: tooth agenesis, impaction, and rotation (AIR); enamel hypoplasia, displacement, and rotation (HDR); displacement, rotation, and mamelon (DRM); and dental caries, tooth agenesis and enamel hypoplasia (CAH). We analyzed each of these four models using genome-wide multivariate tests of association. No genome-wide statistically significant results were found, but we identified multiple suggestive association signals (P ≤ 10−5) near genes with known biological roles during tooth development, including ADAMTS9 and PRICKLE2 associated with AIR; GLIS3, WDR72, and ROR2 associated with HDR and DRM; ROBO2 associated with DRM; BMP7 associated with HDR; and ROBO1, SMAD2, and MSX2 associated with CAH. This is the first study to investigative genetic associations for multivariate patterns of correlated dental anomalies and dental caries. Further studies are needed to replicate these results in independent cohorts.

Assessment of Age at the Stages of the Eruption of Third Molar Teeth among the People of North-Eastern India

Background and Objectives. In the biological age determination of a person’s teeth at adolescence, the third molar (M3) or wisdom tooth development is considered a dependable method used over the years. The present research intended to evaluate the age from the eruption status of M3 and analyze and equivalence with a different quadrant of the jaws. Methods. A cross-sectional descriptive and analytical study was undertaken with 1060 Assamese individuals (642 males and 418 females) aged 14–26 years and was subjected to a clinical, dental, and general physical examination from January 2014 to December 2018. The data were statistically analyzed using Microsoft Excel and the Statistical Package for the Social Sciences (SPSS) version 22. The significant differences among variables were tested using the chi-square test and Student’s t -test, considering a p value < 0.05 as significant. Results. The carried-out research showed no eruption (NE) status of M3 with an overall mean (±SD) age at 17.39 (±2.273) years, although a significantly lower age among males with a mean age of 16.92 (±2.138) years ( p value < 0.001) was observed. The mean age (overall) for the complete eruption (CE) was observed at 20.33 (±2.566) years, which was seen earlier in males. The mandibular M3 appears earlier compared to the maxillary M3. The third molar eruption (TME) on both left and right quadrants of the jaw was observed substantially earlier in the lower jaw, compared to the upper jaw ( p value < 0.025). The earliest CE of M3 was marked at 15 years. The differences in the frequencies of TME in different chronological age groups were found significant ( p value < 0.001). A significant association between gender and TME ( p value < 0.045) in the current study is worth noting. Conclusion. Thus, determined by TME as a valid method, age can be used for various purposes to establish a person’s identity. Dental age estimated using third molar eruption status has a weighty association with chronological age. Thus, it should be utilized to determine the likely age of an individual.

PENENTUAN KECEPATAN PEMOTONGAN EFEKTIF BERDASARKAN NILAI KEKASARAN PERMUKAAN MATERIAL AA-7075 PADA PROSES FACE MILLING

During face milling machining, several machining parameters such as feed rate and cuttingspeed determine the surface quality of the workpiece produced by the process. The selection of the rightparameters will lead to the surface quality as planned. Therefore, to improve machining effectiveness, amethod is needed to determine the appropriate machining parameters to produce the desired surfacequality. This research was conducted using a milling machine, five variations of cutting speed and fivevariations of feed rate were used to cut the workpiece aluminum alloy 7075. After machining, the surfaceroughness was measured using a surface test. The surface roughness value is then substituted into thefeed rate equation and effective cutting speed. By finding effective cutting parameters, the machiningprocess will be more efficient and effective without using unnecessary resources. From the results of thestudy note that the development equation to determine the feed rate based on the value of surfaceroughness is ???? = 0,6????√???? ????????0.443mm/tooth. Development equation to determine the effective cutting speedbased on Surface roughness value is ???????? = 3.0686????????0.124 mm/min

Integration of Single-Cell RNA- and CAGE-seq Reveals Tooth-Enriched Genes

Organ development is dictated by the regulation of genes preferentially expressed in tissues or cell types. Gene expression profiling and identification of specific genes in organs can provide insights into organogenesis. Therefore, genome-wide analysis is a powerful tool for clarifying the mechanisms of development during organogenesis as well as tooth development. Single-cell RNA sequencing (scRNA-seq) is a suitable tool for unraveling the gene expression profile of dental cells. Using scRNA-seq, we can obtain a large pool of information on gene expression; however, identification of functional genes, which are key molecules for tooth development, via this approach remains challenging. In the present study, we performed cap analysis of gene expression sequence (CAGE-seq) using mouse tooth germ to identify the genes preferentially expressed in teeth. The CAGE-seq counts short reads at the 5′-end of transcripts; therefore, this method can quantify the amount of transcripts without bias related to the transcript length. We hypothesized that this CAGE data set would be of great help for further understanding a gene expression profile through scRNA-seq. We aimed to identify the important genes involved in tooth development via bioinformatics analyses, using a combination of scRNA-seq and CAGE-seq. We obtained the scRNA-seq data set of 12,212 cells from postnatal day 1 mouse molars and the CAGE-seq data set from postnatal day 1 molars. scRNA-seq analysis revealed the spatiotemporal expression of cell type–specific genes, and CAGE-seq helped determine whether these genes are preferentially expressed in tooth or ubiquitously. Furthermore, we identified candidate genes as novel tooth-enriched and dental cell type–specific markers. Our results show that the integration of scRNA-seq and CAGE-seq highlights the genes important for tooth development among numerous gene expression profiles. These findings should contribute to resolving the mechanism of tooth development and establishing the basis for tooth regeneration in the future.

Digitalised exercise material in forensic odontology

Introduction This paper presents digital educational material in forensic odontology, including dental identification after multiple fatalities and dental age estimation from different age groups. Material and method Electronic patient records consisting of intraoral scans of the dentition, digital radiographs, photographs and written dental records were collected. Exercises in age estimations contained digital radiographs and photographs of ground tooth sections, with digital measuring tools and tables according to age groups. The teaching material was organised as a module in an electronic Learning Management System with external links to all relevant teaching material. Results For the identification exercises, intraoral scans and the latest digital radiographs simulated the postmortem examination of the deceased. For comparison, all other radiographs, photographs and dental records were available as antemortem material. The exercise was to match postmortem findings with the antemortem records using the Interpol standard and reconciliation. Age assessment of children used designated tables to grade tooth development on digital radiographs. For adults, non-destructive methods, digital radiographs, photographs and measuring tools were used. Discussion The teaching concept was hybrid, but it can easily be adapted as a fully digital exercise. The instructions and written material can be translated into different languages. The level of difficulty in the exercises can be adjusted according to the participant’s level of knowledge. Conclusion The educational material embraces the new possibilities for digitalisation and intraoral scanning. This might be a valuable tool for motivating and engaging the students in their participation and understanding of the subject.

Gas1 Regulates Patterning of the Murine and Human Dentitions through Sonic Hedgehog

The mammalian dentition is a serially homogeneous structure that exhibits wide numerical and morphological variation among multiple different species. Patterning of the dentition is achieved through complex reiterative molecular signaling interactions that occur throughout the process of odontogenesis. The secreted signaling molecule Sonic hedgehog (Shh) plays a key role in this process, and the Shh coreceptor growth arrest-specific 1 (Gas1) is expressed in odontogenic mesenchyme and epithelium during multiple stages of tooth development. We show that mice engineered with Gas1 loss-of-function mutation have variation in number, morphology, and size of teeth within their molar dentition. Specifically, supernumerary teeth with variable morphology are present mesial to the first molar with high penetrance, while molar teeth are characterized by the presence of both additional and absent cusps, combined with reduced dimensions and exacerbated by the presence of a supernumerary tooth. We demonstrate that the supernumerary tooth in Gas1 mutant mice arises through proliferation and survival of vestigial tooth germs and that Gas1 function in cranial neural crest cells is essential for the regulation of tooth number, acting to restrict Wnt and downstream FGF signaling in odontogenic epithelium through facilitation of Shh signal transduction. Moreover, regulation of tooth number is independent of the additional Hedgehog coreceptors Cdon and Boc, which are also expressed in multiple regions of the developing tooth germ. Interestingly, further reduction of Hedgehog pathway activity in Shhtm6Amc hypomorphic mice leads to fusion of the molar field and reduced prevalence of supernumerary teeth in a Gas1 mutant background. Finally, we demonstrate defective coronal morphology and reduced coronal dimensions in the molar dentition of human subjects identified with pathogenic mutations in GAS1 and SHH/GAS1, suggesting that regulation of Hedgehog signaling through GAS1 is also essential for normal patterning of the human dentition.

Synergistic Mutations of LRP6 and WNT10A in Familial Tooth Agenesis

Familial tooth agenesis (FTA), distinguished by developmental failure of selected teeth, is one of the most prevalent craniofacial anomalies in humans. Mutations in genes involved in WNT/β-catenin signaling, including AXIN2 WNT10A, WNT10B, LRP6, and KREMEN1, are known to cause FTA. However, mutational interactions among these genes have not been fully explored. In this study, we characterized four FTA kindreds with LRP6 pathogenic mutations: p.(Gln1252*), p.(Met168Arg), p.(Ala754Pro), and p.(Asn1075Ser). The three missense mutations were predicted to cause structural destabilization of the LRP6 protein. Two probands carrying both an LRP6 mutant allele and a WNT10A variant exhibited more severe phenotypes, suggesting mutational synergism or digenic inheritance. Biallelic LRP6 mutations in a patient with many missing teeth further supported the dose-dependence of LRP6-associated FTA. Analysis of 21 FTA cases with 15 different LRP6 loss-of-function mutations revealed high heterogeneity of disease severity and a distinctive pattern of missing teeth, with maxillary canines being frequently affected. We hypothesized that various combinations of sequence variants in WNT-related genes can modulate WNT signaling activities during tooth development and cause a wide spectrum of tooth agenesis severity, which highlights the importance of exome/genome analysis for the genetic diagnosis of FTA in this era of precision medicine.

THE EFFECT OF PREGNANCY MILK ON THE EXPRESSION OF KALLIKREIN RELATED PEPTIDASE-4 (KLK-4) AND COLLAGEN TYPE 1 (Coll-1) IN AMELOGENESIS

Background: Tooth development during embryonic period is a complex process and requires adequate nutrients for the formation of healthy dental tissues. Kallikrein-related peptidase-4 (KLK-4) and collagen type 1 (Coll-1) are serine proteinases secreted by ameloblast during the transition and maturation stages of the amelogenesis processes, functioning to degrade the protein matrixes, so that the enamel can reach its final hardness. Pregnancy milk contains various nutrients expected to increase the KLK-4 expression of ameloblast cells in tooth development processes Purpose: This study aimed at determining the influence of pregnancy milk on the KLK-4 and collagen type 1 (Coll-1) expression of ameloblast cells in the tooth development processes.study Method The research subjects comprised of 10 pregnant female mice (Mus Musculus L.) that were divided into: control group (given sterile aquadest) and treatment group (given pregnancy milk + sterile aquadest) for 18 days followed by the collection of the tooth germ. The specimens were then stained using Imunnohistochemistry to see the KLK-4 and Coll-1 expressions. The data were analyzed using a pathway analysis. Result: The average KLK-4 and Coll-1 expression in the treatment group were higher than those in the control group. Based the pathway analysis, there were direct correlation of Pregnancy milk with Coll-1 expression and that with KLK-4 and Coll-1 expression as well as indirect correlation of pregnancy milk with KLK-4 expression. Conclusion: Pregnancy milk influences the Kallikrein-related peptidase-4 (KLK-4) and Coll-1 expression of ameloblast cells in the tooth development of the mice’s fetusesKeywords: Coll-1 pregnancy milk, Kallikrein-related peptidase-4 (KLK-4), Tooth development

Role of Primary Cilia in Bone and Cartilage

The primary cilium is a nonmotile microtubule-based organelle in most vertebrate cell types. The primary cilium plays a critical role in tissue development and homeostasis by sensing and transducing various signaling pathways. Ciliary proteins such as intraflagellar transport (IFT) proteins as well as ciliary motor proteins, kinesin and dynein, comprise a bidirectional intraflagellar transport system needed for cilia formation and function. Mutations in ciliary proteins that lead to loss or dysfunction of primary cilia cause ciliopathies such as Jeune syndrome and Ellis–van Creveld syndrome and cause abnormalities in tooth development. These diseases exhibit severe skeletal and craniofacial dysplasia, highlighting the significance of primary cilia in skeletal development. Cilia are necessary for the propagation of hedgehog, transforming growth factor β, platelet-derived growth factor, and fibroblast growth factor signaling during osteogenesis and chondrogenesis. Ablation of ciliary proteins such as IFT80 or IFT20 blocks cilia formation, which inhibits osteoblast differentiation, osteoblast polarity, and alignment and reduces bone formation. Similarly, cilia facilitate chondrocyte differentiation and production of a cartilage matrix. Cilia also play a key role in mechanosensing and are needed for increased bone formation in response to mechanical forces.

The concurrent stimulation of Wnt and FGF8 signaling induce differentiation of dental mesenchymal cells into odontoblast-like cells

Fibroblast growth factor 8 (FGF8) is known to be a potent stimulator of canonical Wnt/β-catenin activity, an essential factor for tooth development. In this study, we analyzed the effects of co-administration of FGF8 and a CHIR99021 (GSK3β inhibitor) on differentiation of dental mesenchymal cells into odontoblasts. Utilizing Cre-mediated EGFP reporter mice, dentin matrix protein 1 (Dmp1) expression was examined in mouse neonatal molar tooth germs. At birth, expression of Dmp1-EGFP was not found in mesenchymal cells but rather epithelial cells, after which Dmp1-positive cells gradually emerged in the mesenchymal area along with disappearance in the epithelial area. Primary cultured mesenchymal cells from neonatal tooth germ specimens showed loss of Dmp1-EGFP positive signals, whereas addition of Wnt3a or the CHIR99021 significantly regained Dmp1 positivity within approximately 2 weeks. Other odontoblast markers such as dentin sialophosphoprotein (Dspp) could not be clearly detected. Concurrent stimulation of primary cultured mesenchymal cells with the CHIR99021 and FGF8 resulted in significant upregulation of odonto/osteoblast proteins. Furthermore, increased expression levels of runt-related transcription factor 2 (Runx2), osterix, and osteocalcin were also observed. The present findings indicate that coordinated action of canonical Wnt/β-catenin and FGF8 signals is essential for odontoblast differentiation of tooth germs in mice.