The association between head injury and facial fracture treatment: an observational study of hospitalized bicyclists from a level 1 trauma centre.

PURPOSE: To compare the types of facial fractures and their treatment in bicyclists admitted to a level 1 trauma centre with major and minor-moderate head injury. METHODS: Retrospective analysis of data from bicycle-related injuries in the period 2005-2016 extracted from the Oslo University Hospital trauma registry. RESULTS: A total of 967 bicyclists with head injuries classified according to the Abbreviated Injury Scale (AIS) were included. The group suffering minor-moderate head injury (AIS Head 1-2) included 518 bicyclists, while 449 bicyclists had major head injury (AIS Head 3-6). The mean patient age was 40.2 years (range 3-91 years) and 701 patients (72%) were men. A total of 521 facial fractures were registered in 262 patients (on average 2 facial fractures per bicyclist). Bicyclists with major head injury exhibited increased odds for facial fractures compared to bicyclists with minor-moderate head injury (sex and age adjusted odds ratio (OR) 2.75, 95% confidence interval (CI) 2.03-3.72, p < 0.001. More specifically, there was increased odds for all midface fractures, but no difference for mandible fractures. There was also increased odds for orbital reconstruction in cyclist with major head injury compared to bicyclist with minor-moderate head injury (adjusted OR 3.34, 95% CI 1.30-8.60, p = 0.012). CONCLUSION: Bicyclists with more severe head injuries had increased odds for midface fractures and surgical correction of orbital fractures. During trauma triage, the head and the face should be considered as one unit.

Do Bicycle Helmets Protect Against Facial Fractures? An Observational Study From a Level 1 Trauma Centre.

This study investigates the impact of helmet use on the incidence of facial fractures in bicycle accidents. Analyzing data from hospitalized bicyclists between 2005 and 2016, the research focused on the correlation between helmet usage and various facial fractures. The study included 1256 bicyclists with known helmet use, among whom 277 individuals (22%) were identified with a total of 521 facial fractures. The findings revealed a significant reduction in the likelihood of facial fractures among helmeted cyclists compared with those without helmets (odds ratio, 0.65; confidence interval, 0.50-0.85; P=0.002). Specifically, the odds of sustaining fractures in the zygoma, orbit, nose, and maxilla were decreased by 47%, 46%, 43%, and 33%, respectively, among helmeted cyclists. However, helmet use did not significantly alter the odds of mandible fractures. Overall, the use of helmets in bicycling significantly lowered the risk of midface fractures but showed no notable effect on mandible fractures in severe cycling incidents.

Onset and progression of dental erosion in a mouse model.

OBJECTIVE: Purpose of this research was to examine the onset, progression and wear rates of dental erosion in an established mouse model. MATERIAL AND METHODS: Dental erosion in mice was experimentally induced, and the acidic effects of cola drink on their teeth after 2, 4 and 6-weeks were closely analysed by scanning electron microscopy. The tooth height and enamel or dentin loss were established.  Results: The dental erosion on the molars showed clear progression from 2 to 6 weeks. By the 2-week mark, a significant portion of enamel was already eroded, revealing the dentin on the lingual cusps. When adjusted for attritional wear, molars exposed to cola for 2 weeks showed a 35% drop in lingual tooth height compared to controls (533 µm vs. 818 µm). At 4 and 6 weeks, the cola-exposed group continued to display decreased lingual tooth heights by 40% (476 µm vs. 799 µm) and 43% (440 µm vs. 767 µm), respectively. CONCLUSION: This study revealed significant acidic effects of cola drink on mouse molars as early as 2 weeks. These findings highlight the challenge of monitoring dental erosion clinically and underscore the importance of early preventive and intervention measures.

Association of Locus Xq26.1-27.3 with Erosive Tooth Wear Phenotypes in a Group of Adolescents.

Erosive tooth wear is a multifactorial condition of an increasing prevalence. There is a need for discovering individual genetic predisposition for the development of this condition. Considering that the chromosome X locus was previously shown to be associated with dental caries, the aim of the present study was to look for the association between this locus and erosive tooth wear when dietary habits are considered as a co-factor. Saliva samples, erosive wear experience data, and dietary information from 16- to 18-year-old dental patients (n = 705) were used. Genotyping analyses were performed, and thereafter, analyses considering diet and oral hygiene data, using logistic regression, with the assumption that erosive tooth wear is a complex gene-environment model. Genotypic analyses revealed an association between chromosome X marker rs1324156 and erosive tooth wear phenotype. Logistic regression analysis showed that, in the presence of less common allele of rs12687601 and rs1324156, erosive tooth wear more likely develops when associated with numerous dietary variables from the questionnaire. These results indicate that erosive tooth wear may be the result of gene-environment interactions.

Facial Fractures and Their Relation to Head and Cervical Spine Injuries in Hospitalized Bicyclists.

Bicyclists are vulnerable road users. The authors aimed to characterise facial fractures and their association with head and neck injuries in bicyclists admitted to a Scandinavian Level 1 trauma center with a catchment area of ~3 million inhabitants. Data from bicycle-related injuries in the period 2005 to 2016 were extracted from the Oslo University Hospital trauma registry. Variables included were age; sex; date of injury; abbreviated injury scale (AIS) codes for facial skeletal, head and neck injuries; and surgical procedure codes for treatment of facial fractures. Anatomical injury was classified according to AIS98. A total of 1543 patients with bicycle-related injuries were included. The median age was 40 years (quartiles 53, 25), and 1126 (73%) were men. Overall, 652 fractures were registered in 339 patients. Facial fractures were observed in all age groups; however, the proportion rose with increasing age. Bicyclists who suffered from facial fractures more often had a concomitant head injury (AIS head >1) than bicyclists without facial fractures (74% vs. 47%), and the odds ratio for facial fracture(s) in the orbit, maxilla and zygoma were significantly increased in patients with AIS head >1 compared to patients with AIS head=1. In addition, 17% of patients with facial fractures had a concomitant cervical spine injury versus 12% of patients without facial fractures. This results showed that facial fractures were common among injured bicyclists and associated with both head and cervical spine injury. Thus, a neurological evaluation of these patients are mandatory, and a multidisciplinary team including maxillofacial and neurosurgical competence is required to care for these patients.

Anatomical distribution of mandibular fractures from severe bicycling accidents: A 12-year experience from a Norwegian level 1 trauma center.

BACKGROUND/AIM: The mandible makes up a substantial part of the lower face, and is susceptible to injury. Even in helmeted cyclists, accidents may lead to fractures of the mandible because conventional helmets provide little protection to the lower part of the face. In addition, some studies indicate that helmets may lead to an increased risk of mandibular fractures. Thus, the aim of this study was to examine the anatomic distribution of mandibular fractures in injured cyclists and to assess if helmet use influenced the fracture locations. MATERIAL AND METHODS: Data from a Norwegian Level 1 trauma center were collected in the Oslo University Hospital Trauma Registry over a 12-year period. Of 1543 injured cyclists, the electronic patient charts of 62 cyclists with fractures of the mandible were retrospectively evaluated in detail. Demographic data, helmet use, and fracture type were assessed. RESULTS: Sixty-two patients (4%) had fractures of the mandible, and women had an increased risk (OR 2.49, 95% CI 1.49-4.16, p < .001). The most common fracture site was the mandibular body, followed by the condyle. Isolated mandibular fractures occurred in 45% of the patients and 55% had other concomitant facial fractures. There were 42% of the patients with fractures in multiple sites of the mandible, and 42% had a concomitant dentoalveolar injury. Half of the cyclists were wearing a helmet at the time of the accident and 39% were not. There was no significant difference in fracture distribution between the helmeted and non-helmeted groups. CONCLUSIONS: Fracture of the mandibular body was the most prevalent mandibular fracture type following bicycle accidents. Women had an increased risk of mandibular fractures compared with men, whereas helmet wearing did not affect the anatomical fracture site.

Dentoalveolar injuries, bicycling accidents and helmet use in patients referred to a Norwegian Trauma Centre: A 12-year prospective study.

BACKGROUND/AIM: Despite its many benefits, bicycling carries the risk of accidents. Although numerous studies have reported the effect of helmet use on traumatic brain injury, it remains unclear if, and to what extent, helmet use reduces the risk of facial injuries. This is particularly true in regard to injuries of the lower face. In addition, there is limited evidence of the effect of helmet use on dentoalveolar injuries. Thus, the aim of this study was to determine the frequency and distribution of dentoalveolar injuries in bicycling accidents and to explore the influence of helmet use. MATERIAL AND METHODS: A total of 1543 bicyclists were included from the trauma registry of a Norwegian tertiary trauma center over a 12-year period. Data were collected prospectively, including patient characteristics, type of injury, and helmet use. The prevalence of dentoalveolar injuries was assessed in conjunction with helmet use and facial fractures. RESULTS: Twenty-five percent of the patients had maxillofacial injuries, and 18% of those with facial fractures exhibited concomitant dentoalveolar injuries. The most common type of dentoalveolar injury was tooth fracture (39%). The most frequent location of facial fractures with combined dentoalveolar injuries was the maxilla, which had fractured in 32 patients. Women had a higher risk of sustaining dentoalveolar injuries compared to men (odds ratio 1.50, 95% confidence interval 1.02-2.22). There were 1257 patients (81%) who had reliable registration of helmet use; 54% of these wore a helmet, while 46% did not. Helmet users had an increased risk of dentoalveolar injuries compared to non-helmeted bicyclists (adjusted odds ratio 1.54, 95% confidence interval 1.02-2.31). CONCLUSIONS: Dentoalveolar injuries are fairly common in trauma patients admitted to a trauma center following bicycling accidents. Bicycling helmets are associated with an increased risk of dentoalveolar injuries.

Aquaporins' Influence on Different Dental Erosive Wear Phenotypes in Humans.

Dental erosive wear is a multifactorial condition of high prevalence. Nowadays, there is an emphasis on discovering individual genetic predisposition for the development of this condition. Aquaporins (AQPs) are water channel proteins expressed in salivary glands, as well as during tooth development. They are involved in salivary secretion and composition and linked to physiological protection of the oral cavity. The aim of this study was to explore the relationship between different dental erosive wear phenotypes, AQP genes, and selected environmental factors. Data from 705 dental patients were used to investigate the association between dental erosive wear phenotypes and AQPs' single-nucleotide variants. Phenotypes were further analyzed considering diet and oral hygiene data, using logistic regression analysis, as implemented in PLINK, with the assumption that dental erosive wear is a complex gene-environment model. Associations were found between severe erosive tooth wear and rs2878771 (AQP2) for the genotypic (p = 0.02) and dominant (p = 0.03) models, and rs3736309 (AQP5) for the allelic model (p = 0.02). Logistic regression analyses, after implementing the Bonferroni correction, showed that several significant associations were present when covariates were included, suggesting that a strong environmental component is present. Our results show that dental erosive wear establishes under a gene-environmental complex model.

Dental erosion in mice with impaired salivary gland function.

Objective: Salivary flow rate exerts an essential impact on the development and progression of dental erosion. In this work, the experimental dental erosion in non-obese diabetic (NOD) mice with reduced salivary flow rate was induced, and the erosive effect of acidic drinks on their dentition was studied.Material and methods: Three acidic drinks (sports drink, cola light drink and sugar containing cola drink) were given to adult NOD mice (groups: N = 11) as the only drink for 6 weeks. Two control groups were included; wild type and NOD control (groups: N = 9). Experimental and control (water) teeth were dissected out and observed by scanning electron microscopy (SEM). Mandibular first molars were subsequently embedded in Epon, ground transversely, observed again by SEM, and the enamel thickness and tooth height were measured.Results: Mandibular molars were considerably more eroded than maxillary molars. The erosive process started at the top of the cusps and subsequently extended in the cervical, mesio-distal, and pulpal direction. Erosive lesions were evident in increased succession from sports drink, cola light to cola drink exposed mandibular molars, with the lingual tooth height being approximately 23%, 26%, and 37% lower, respectively, compared to the control. The lingual enamel was approximately 48% thinner in sports drink molars and 62% thinner in cola light molars. In cola drink molars, the lingual enamel was totally eroded, and significant erosion of dentine was evident.Conclusion: Reduced salivary flow, together with a high consumption of acidic drinks, results in severe erosion of NOD mice molars.

Tooth identification puzzle: A method of teaching and learning tooth morphology.

INTRODUCTION: The present report outlines a method of teaching/learning tooth morphology by tooth identification puzzle. MATERIALS AND METHODS: Students are presented with sets of extracted human teeth comprising complete dentitions except deciduous incisors and canines. The task is to place the teeth in correct positions in a schematic dentition diagram. The course, including 2-3 introductory lectures and a final test of one hour, has a time frame of 14-16 hours. A total of 506 2nd year students from several years participated. RESULTS: The course is much appreciated by the students who experience a marked progress in skills. In the final test, 51.8% of the students had no faults, whilst 3% failed (more than 12 faults). The average number of faults per student was 2.3. Of the 20 240 positioned teeth 5.7% were misplaced. The most frequently misplaced teeth were mandibular central incisors, maxillary second premolars and mandibular first premolars. The most common type of fault was inside determination. DISCUSSION: The course is cost-effective and facilitates learning through its multifaceted activity with involvement of many senses. An important asset is the appreciation of variations in tooth morphology. The course provides an arena for close and positive interaction between students and teachers.

Electrical Stimulation as a Means for Improving Vision.

Evolving research has provided evidence that noninvasive electrical stimulation (ES) of the eye may be a promising therapy for either preserving or restoring vision in several retinal and optic nerve diseases. In this review, we focus on minimally invasive strategies for the delivery of ES and accordingly summarize the current literature on transcorneal, transorbital, and transpalpebral ES in both animal experiments and clinical studies. Various mechanisms are believed to underlie the effects of ES, including increased production of neurotrophic agents, improved chorioretinal blood circulation, and inhibition of proinflammatory cytokines. Different animal models have demonstrated favorable effects of ES on both the retina and the optic nerve. Promising effects of ES have also been demonstrated in clinical studies; however, all current studies have a lack of randomization and/or a control group (sham). There is thus a pressing need for a deeper understanding of the underlying mechanisms that govern clinical success and optimization of stimulation parameters in animal studies. In addition, such research should be followed by large, prospective, clinical studies to explore the full potential of ES. Through this review, we aim to provide insight to guide future research on ES as a potential therapy for improving vision.

Circadian rhythms and gene expression during mouse molar tooth development.

OBJECTIVES: Incremental markings in dental enamel suggest that the circadian clock may influence the molecular underpinnings orchestrating enamel formation. The aim of this study was to investigate whether the genes and microRNAs (miRNAs) oscillate in a circadian pattern during tooth and enamel development. MATERIAL AND METHODS: Comparative gene and miRNA expression profiling of the first mandibular molar tooth germ isolated at different time-points during the light and night period was performed using microarrays and validated using real-time RT-PCR. Bioinformatic analysis was carried out using Ingenuity Pathway Analysis (IPA), and TargetScan software was used in order to identify computationally predicted miRNA-mRNA target relationships. RESULTS: In total, 439 genes and 32 miRNAs exhibited significantly different (p < 0.05) levels of expression in the light phase compared with the night phase tooth germs. Genes involved in enamel formation, i.e. Amelx, Ambn, Amtn, and Odam, oscillated in a circadian pattern. Furthermore, the circadian clock genes, in particular Clock and Bmal1, oscillated in mouse molar tooth germ during 24-h intervals. The expression of Clock and Bmal1 was inversely correlated with the expression of miR-182 and miR-141, respectively. CONCLUSIONS: MiRNAs, including miR-182 and miR-141, are involved in the control of peripheral circadian rhythms in the developing tooth by regulating the expression of genes coding for circadian transcription factors such as CLOCK and BMAL1. Regulation of circadian rhythms may be important for enamel phenotype, and the morphology of dental enamel may vary between individuals due to differences in circadian profiles.

Five-minute Apgar score ≤ 5 and Molar Incisor Hypomineralisation (MIH) - a case control study.

BACKGROUND: The aetiology of molar incisor hypomineralisation (MIH) is unclear. The asymmetric distribution of MIH in the dentition may indicate that an insult of short duration that affects ameloblasts at a vulnerable stage could be a causative factor. Apgar ≤ 5 at 5 min may indicate asphyxia (hypoxic-ischemic insult) during birth. It was hypnotised that low Apgar score during birth may cause MIH. The present study aimed to examine a possible association between Apgar ≤ 5 at 5 min and the occurrence of MIH. METHOD: Two study groups were selected for examination. The cases comprised 67 children aged 8-10 years born with Apgar score equal to or below 5 after 5 min. The control group comprised 157 age-matched healthy children. First permanent molars, second primary molars and all permanent incisors were examined in all children. Clinical examination was undertaken by two calibrated examiners and intraoral close-up photographs of the teeth were later evaluated by three calibrated and blinded clinicians. Demarcated opacities, post-eruptive breakdown, atypical restorations and extractions due to MIH, according to the criteria of the European Association of Paediatric Dentistry, were assessed. RESULTS: The prevalence of MIH did not differ between the two groups. A chi-square test failed to confirm any statistically significant relationship between 5-min Apgar scores and MIH occurrence. In addition, there was no statistically significant relationship between the number of affected first permanent molars in cases and controls. CONCLUSION: There was no association between Apgar ≤ 5 at 5 min and the occurrence of MIH.

Traditional Hands-On Puzzle Method versus Fully Digital Approach in Teaching Tooth Morphology: A Comparative Study of Educational Outcomes.

OBJECTIVE: Tooth morphology education is a critical component of dental curricula, providing a foundational understanding of the intricate structural anatomy of teeth. This study evaluates the learning outcomes in relation to tooth morphology of two student cohorts from different academic terms, comparing the traditional 'tooth puzzle' method to an alternative fully digital approach. MATERIALS AND METHODS: Two groups of Master of Dentistry students were retrospectively analyzed. The control group (55 students) was taught via the 'tooth puzzle' method in 2021, while the experimental group (55 students) underwent a fully digital course in 2020 due to COVID-19 restrictions. Both groups completed a digital examination involving the identification of 40 teeth, presented as images and videos. RESULTS: In the control group of 55 students, nearly half (49.1%) achieved faultless results, while 65.5% had at most two faults, and 74.5% had no more than four faults. The group had a total of 163 faults, averaging 3.0 per student, with only one student (1.8%) failing the test. In stark contrast, the experimental group had no students without faults, 9.1% had four or fewer faults, and a significant 61.8% made 10 or more faults, with 29.1% failing their first test attempt by exceeding 12 faults. Overall, the experimental group registered 582 faults, averaging 10.6 per student. CONCLUSIONS: The 'tooth puzzle' method, with its interactive and tactile elements, proved more effective in teaching tooth morphology than the digital-only approach. The increased number of faults and failed tests in the experimental group suggest that while digital tools offer meaningful support in learning tooth morphology, their main advantage is seen when coupled with traditional hands-on techniques, not unassisted and independently.

Different Methods of Teaching and Learning Dental Morphology.

Dental anatomy education is traditionally structured into theoretical and practical modules to foster both cognitive and psychomotor development. The theoretical module typically involves didactic lectures where educators elucidate dental structures using visual aids. In contrast, practical modules utilize three-dimensional illustrations, extracted and plastic teeth, and tooth carving exercises on wax or soap blocks, chosen for their cost, ease of handling, and fidelity in replication. However, the efficacy of these traditional methods is increasingly questioned. The criticism in this concern is that oversized carving materials may distort students' understanding of anatomical proportions, potentially affecting the development of necessary skills for clinical practice. Lecture-driven instruction, on the other hand, is also criticized for its limitations in fostering interactive learning, resulting in a gap between pre-clinical instruction and practical patient care. In this study, we review the various educational strategies that have emerged to enhance traditional dental anatomy pedagogy by describing the effectiveness of conventional didactic lectures, wax carving exercises, the use of real and artificial teeth, the flipped classroom model, and e-learning tools. Our review aims to assess each method's contribution to improving clinical applicability and educational outcomes in dental anatomy, with a focus on developing pedagogical frameworks that align with contemporary educational needs and the evolving landscape of dental practice. We suggest that the optimal approach for teaching tooth morphology would be to integrate the digital benefits of the flipped classroom model with the practical, hands-on experience of using extracted human teeth. To address the challenges presented by this integration, the creation and standardization of three-dimensional tooth morphology educational tools, complemented with concise instructional videos for a flipped classroom setting, appears to be a highly effective strategy.

Accumulation and removal of Streptococcus mutans biofilm on enamel and root surfaces in vitro.

Objective: This study aimed to quantitatively investigate the accumulation of Streptococcus mutans biofilm on enamel and root surfaces and assess the amount of biofilm removal using (1) experimental toothpaste and (2) water, in a closed system of flow chamber. Methods: Eight sound premolars were embedded in epoxy resin and polished with silicon carbide grinding papers to display enamel and root surfaces. To mimic biofilm, cultures of Streptococcus mutans were prepared and grown on the tooth surfaces over night before they were exposed to either 2 liters of Milli Q water or 2 liters of 40% experimental toothpaste in the flow chamber. The amount of biofilm was measured and quantified in Fluorescence microscopy. Mean fluorescence values were recorded and analysed using Microsoft® Excel® (MS Excel 2016). Results: The ability to grow biofilm was equally present at both the enamel and root surfaces. The use of water and 40% experimental toothpaste showed a significant reduction of areas covered with biofilm on both enamel and root dentin in comparison to untreated surfaces (p < 0.01). Significantly more biofilm was removed from enamel compared to root surfaces when treated with either water and toothpaste (p < 0.01). Slightly less biofilm was removed by the use of water compared to toothpaste on both enamel and root dentin surfaces, although the differences were not statistically significant. Conclusion: The results indicate that less biofilm is removed from the root surfaces than enamel by the use of water and 40% experimental toothpaste in flow chamber. Assessing oral biofilm accumulation and monitoring biofilm formation on enamel and root dentin surfaces give oral health professionals important directions that could strenghten the significance of dental caries prevention. Improving older individuals' oral hygiene practices should therefore be considered an important measure to prevent root caries.

Integrating Structured Digital Tools with the Traditional Hands-on Puzzle Method for Teaching Tooth Morphology: A Comparative Study of Educational Outcomes.

The study of tooth morphology is a critical component of the dental curriculum, highlighting the importance for dental students to acquire comprehensive and detailed knowledge of the complex structure of teeth. This study compared the educational outcomes of two student cohorts in a tooth morphology course, using traditional methods for the control group and additional digital video-based resources for the experimental group. We hypothesized that early integration of digital resources would significantly reduce the learning time. We retrospectively analyzed two groups of Master of Dentistry students. The control group (42 students) was taught using the traditional 'tooth puzzle' method, while the experimental group (42 students) supplemented traditional teaching with digital video-based tools developed by our department. Both groups' curricula culminated in a practical post-course test requiring the identification of 40 teeth, along with a mid-course test to track the students' learning progression. The number and type of incorrectly identified teeth were recorded. The mid-course test showed significant performance differences. The control group had a median (Q1, Q3) value of faults of 12.0 (7.8, 20.5), whereas the respective value for the experimental group was 4.0 (0.0, 8.0) (p < 0.001). In the control group, none achieved faultless results, with only two students (4.8%) having at most two faults, and six students (14.3%) having no more than four faults. The control group averaged 13.5 faults per student, with 19 students (45.2%) failing the test. Conversely, the experimental group showed improved performance: 12 students (28.6%) had no faults, and 25 students (59.5%) had four or fewer faults. The experimental group averaged 5.2 faults per student, with only four students (9.5%) failing. By the end of the course, both groups achieved commendable results on the practical tooth identification test. The experimental group slightly outperformed the control group, though the difference was not significant. The median (Q1, Q3) values were 0.0 (0.0, 2.5) and 1.0 (0.0, 4.5) for the experimental and control groups, respectively (p = 0.372). The students using both traditional and structured digital video-based tools showed greater learning advancement than those using only the traditional 'tooth puzzle' method.

Expression of Clu and Tgfb1 during murine tooth development: effects of in-vivo transfection with anti-miR-214.

Expression of clusterin (Clu) in the murine first molar tooth germ was markedly increased at postnatal developmental stages. The time-course of expression of this gene paralleled those of other genes encoding proteins involved during the secretory phase of odontogenesis, as described previously. Immunohistochemical studies of clusterin in murine molar tooth germs suggested this protein to be located in outer enamel epithelium, regressing enamel organ, secretory ameloblasts, and the dental epithelium connecting the tooth to the oral epithelium at an early eruptive stage. Immunolabelling of transforming growth factor beta-1 (TGF-ß1) revealed it to be located close to clusterin. The levels of expression of Clu and Tgfb1 were markedly decreased following in-vivo transfection with anti-miR-214. In contrast, the expression of several genes associated with regulation of growth and development were increased by this treatment. We suggest that clusterin has functions during secretory odontogenesis and the early eruptive phase. Bioinformatic analysis after treatment with anti-miR-214 suggested that, whilst cellular activities associated with tooth mineralization and eruption were inhibited, activities associated with an alternative developmental activity (i.e. biosynthesis of contractile proteins) appeared to be stimulated. These changes probably occur through regulation mediated by a common cluster of transcription factors and support suggestions that microRNAs (miRNAs) are highly significant as regulators of differentiation during odontogenesis.

Expression of delta-like 1 homologue and insulin-like growth factor 2 through epigenetic regulation of the genes during development of mouse molar.

Delta-like 1 homolog (Dlk1) and insulin-like growth factor 2 (Igf2) are two of six well-studied mouse imprinted gene clusters that are paternally expressed. Their expression is also linked to their maternally expressed non-coding RNAs, encoded by Gene trap locus 2 (Gtl2) and Imprinted maternally expressed transcript (H19), co-located as imprinted gene clusters. Using deoxyoligonucleotide microarrays and real-time RT-PCR analysis we showed Dlk1 and Gtl2 to exhibit a time-course of expression during tooth development that was similar to that of Igf2 and H19. Western blot analysis of proteins encoded by Dlk1 and Igf2 suggested that the levels of these proteins reflected those of the corresponding mRNAs. Immunohistochemical studies of DLK1 in murine molars detected the protein in both epithelial and mesenchymal regions, in developing cusp mesenchyme, and in newly synthesized enamel and dentin tubules. IGF2 protein was detected primarily at prenatal stages, suggesting that it may be active before birth. Analysis of methylation of cytosine-phosphate-guanine (CpG) islands in both Dlk1 and Igf2 suggested the presence of an increasing fraction of hypermethylated bases with increasing time of development. The increased levels of hypermethylation coincided both with the diminished levels of expression of Dlk1 and Igf2 and with decreased levels of DLK1 and IGF2 proteins in the tooth germ, suggesting that their expression is regulated via methylation of CpG islands present in these genes.

Effects of in vivo transfection with anti-miR-214 on gene expression in murine molar tooth germ.

MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that are believed to be important in many biological processes through regulation of gene expression. Little is known of their function in tooth morphogenesis and differentiation. MicroRNA-214 (miR-214), encoded by the polycistronic Dnm30os gene, is highly expressed during development of molar tooth germ and was selected as a target for silencing with anti-miR-214. Mandibular injection of 1-100 pmol of anti-miR-214 close to the developing first molar in newborn mice resulted in significant decrease in expression of miR-214, miR-466h, and miR-574-5p in the tooth germ. Furthermore, levels of miR-199a-3p, miR-199a-5p, miR-690, miR-720, and miR-1224 were significantly increased. Additionally, the expression of 863 genes was significantly increased and the expression of 305 genes was significantly decreased. Among the genes with increased expression was Twist-1 and Ezh2, suggested to regulate expression of miR-214. Microarray results were validated using real-time RT-PCR and Western blotting. Among genes with decreased expression were Amelx, Calb1, Enam, and Prnp; these changes also being reflected in levels of corresponding encoded proteins in the tooth germ. In the anti-miR-214-treated molars the enamel exhibited evidence of hypomineralization with remnants of organic material and reduced surface roughness after acid etching, possibly due to the transiently decreased expression of Amelx and Enam. In contrast, several genes encoding contractile proteins exhibited significantly increased expression. mRNAs involved in amelogenesis (Ambn, Amelx, Enam) were not found among targets of miRNAs that were differentially expressed following treatment with anti-miR-214. It is therefore suggested that effects of miR-214 on amelogenesis are indirect, perhaps mediated by the observed miR-214-dependent changes in levels of expression of numerous transcription factors.