Evaluation of hard palate and cleft morphology in neonates with Pierre Robin Sequence and Cleft Palate Only.

OBJECTIVES: This study aimed to establish a fully digital measurement protocol for standardizing the description of hard palate and cleft morphology in neonates with an isolated cleft palate (CPO) and Pierre Robin sequence (PRS). MATERIALS AND METHODS: A total of 20 digitized plaster models of neonates with CPO and 20 digitized plaster models of neonates with PRS were retrospectively investigated. For the control group, the hard palate was segmented from 21 pre-existing 1.5 T MRI datasets of neonates and exported as an STL file. The digital models were marked with predefined reference points by three raters. Distance, angular, and area measurements were performed using Blender and MeshLab. RESULTS: Neonates with CPO (20.20 ± 2.33 mm) and PRS (21.41 ± 1.81 mm) had a significantly shorter hard palate than the control group (23.44 ± 2.24 mm) (CPO vs. control: P < .001; PRS vs. control: P = .014). Notably, neonates with PRS (33.05 ± 1.95 mm) demonstrated a significantly wider intertuberosity distance than those with CPO (30.52 ± 2.28 mm) (P = .012). Furthermore, there were also significant differences measured between the cleft and control groups (25.22 ± 2.50 mm) (P < .001). CONCLUSIONS: The data from this study demonstrate the feasibility of using MRI datasets to generate digital models of the hard palate. The presence of a cleft palate leads to pronounced adaptations of the total palatal surface area, dorsal width, and length of the hard palate. Mandibular retrognathia and altered tongue position in PRS, as opposed to CPO, might further impact palatal morphology and intertuberosity distance.

Feasibility of 3 Tesla MRI for the assessment of mid-palatal suture maturation: a retrospective pilot study.

The maxilla occupies a key position in dentofacial orthopaedics, since its transversal development can be directly influenced by orthodontic therapy. The maturation stages of the mid-palatal suture, which are obtained from cone-beam computed tomography images (CBCT), present an addition to clinical decision-making in transversal discrepancies of the upper jaw. In an endeavour to reduce ionizing radiation in adolescents and young adults, who are particularly susceptible to long term stochastic irradiation effects, we investigated the feasibility of 3 Tesla (3T) MRI in detecting the maturation stages of the mid-palatal suture. A collective of 30 patients aged 24-93 years with routine neck MRI at 3T, underwent an additional three-dimensional isotropic T1 weighted study sequence of the midface. Image evaluation was performed on axial, multi-planar formatted reconstructions of the dataset aligned to the midline axis of the palate, and curved reconstructions aligned to the concavity of the palate. Inverted images helped to achieve an image impression similar to the well-known CBCT appearance. All datasets were reviewed by three readers and mid-palatal maturation was scored twice according to Angelieri et al. Intra- and inter-rater agreement were evaluated to measure the robustness of the images for clinical evaluation. 3T MRI deemed reliable for the assessment of mid-palatal suture maturation and hence for the appraisal of the hard palate and its adjacent sutures. The data of this pilot study display the feasibility of non-ionizing cross-sectional MRI for the determination of sutural maturation stages. These findings underline the potential of MRI for orthodontic treatment planning, further contributing to the avoidance of unnecessary radiation doses.

Clinical implementation of axial angulation of incisors in the course of routine fixed appliance treatment - a retrospective cohort study.

PURPOSE: To identify clinically relevant factors for changes in axial angulation of incisors during routine fixed appliance orthodontic treatment. METHODS: A total of 106 patients (grades 1-2 of IOTN, 64 females, 42 males; mean age: 15.5 years) from a private practice and treated with metal or ceramic brackets were included in this retrospective cohort study. The axial angulation of the upper and lower incisors was measured on lateral cephalograms before insertion of the first rectangular 0.016 × 0.022-in NiTi archwire (T0) and at the end of treatment about 8 weeks after insertion of the working 0.019 × 0.025-in stainless steel archwire (T1). Treatment-related changes according to bracket type, initial situation, premolar extraction, angle class, and skeletal vertical configuration were analyzed. RESULTS: Although statistically significant treatment-related changes were seen for both the upper incisors (+ 1.3°) and the lower incisors (- 5.2°), only in ten patients (9.4%) was the prescribed torque value of 17° for the upper incisors and in no patient for the lower incisors achieved. A negative association between the induced change of axial angulation of incisors and the initial values was detected for the upper incisors as well as for the lower incisors. A comparison of the angle classes revealed significant differences in incisor changes. At the end of therapy, only a slight change for the upper central incisors in patients in angle class I cases and a significantly greater change in patients with angle class II/2 was observed. Cases with premolar extraction ended with lower axial angulation of the incisor than cases without extraction. The individual analysis of possible influencing factors also revealed an association with the vertical skeletal configuration. CONCLUSIONS: For the first time, the presented data show clinically relevant influencing factors for incisor axial angulation changes of the upper and lower incisors in relation to the torque value of the applied brackets in the course of routine clinical practice. For the orthodontist, it remains mandatory to decide whether a customized system must be individualized in order to achieve individual therapy goals.

Impact of different cephalometric skeletal configurations on anatomic midface parameters in adults.

OBJECTIVES: Skull morphology and growth patterns are essential for orthodontic treatment, impacting clinical decision making. We aimed to determine the association of different cephalometric skeletal configurations on midface parameters as measured in 3D CT datasets. MATERIALS AND METHODS: After sample size calculation, a total of 240 fully dentulous patients between 20 and 79 years of age (mean age: 42 ± 15), who had received a CT of the skull within the scope of trauma diagnosis or intracranial bleeding, were retrospectively selected. On the basis of cephalometric analysis, using MPR reconstructions, patients were subdivided into three different vertical skull configurations (brachyfacial, mesofacial, dolichofacial) and the respective skeletal Class I, II, and III relationships. Anatomic parameters were measured using a three-dimensional post-processing console: the thickness of the maxillary and palatine bones as well as the alveolar crest, maxillary body and sutural length, width and height of the hard palate, maxillary facial wall thickness, and masseter muscle thickness and length. RESULTS: Individuals with brachyfacial configurations had a significantly increased palatal and alveolar ridge thicknesses compared to those with dolichofacial- or mesofacial configurations. Brachyfacial configurations presented a significantly increased length and thickness of the masseter muscle (4.599 cm; 1.526 cm) than mesofacial (4.431 cm; 1.466 cm) and dolichofacial configurations (4.405 cm; 1.397 cm) (p < 0.001). Individuals with a skeletal Class III had a significantly shorter palatal length (5.313 cm) than those with Class I (5.406 cm) and Class II (5.404 cm) (p < 0.01). Sutural length was also significantly shorter in Class III (p < 0.05). CONCLUSIONS: Skeletal configurations have an impact on parameters of the bony skull. Also, measurable adaptations of the muscular phenotype could result. CLINICAL RELEVANCE: The association between viscerocranial morphology and midface anatomy might be beneficial for tailoring orthodontic appliances to individual anatomy and planning cortically anchored orthodontic appliances.

The impact of passive alveolar molding vs. nasoalveolar molding on cleft width and other parameters of maxillary growth in unilateral cleft lip palate.

OBJECTIVE: Passive alveolar molding (PAM) and nasoalveolar molding (NAM) are established presurgical infant orthodontic (PSIO) therapies for cleft lip palate (CLP) patients. PAM guides maxillary growth with a modified Hotz appliance, while NAM also uses extraoral taping and includes nasal stents. The effects of these techniques on alveolar arch growth have rarely been compared. MATERIAL AND METHODS: We retrospectively compared 3D-scanned maxillary models obtained before and after PSIO from infants with unilateral, non-syndromic CLP treated with PAM (n = 16) versus NAM (n = 13). Nine anatomical points were set digitally by four raters and transversal/sagittal distances and rotations of the maxilla were measured. RESULTS: Both appliances reduced the anterior cleft, but NAM percentage wise more. NAM decreased the anterior and medial transversal width compared to PAM, which led to no change. With both appliances, the posterior width increased. The alveolar arch length of the great and small segments and the sagittal length of the maxilla increased with PAM but only partially with NAM. However, NAM induced a significant greater medial rotation of the larger and smaller segment compared to PAM with respect to the lateral angle. CONCLUSIONS: NAM and PAM presented some significant differences regarding maxillary growth. While NAM reduced the anterior cleft and effectively rotated the segments medially, PAM allowed more transversal and sagittal growth. CLINICAL RELEVANCE: The results of this study should be taken into consideration when to decide whether to use PAM or NAM, since they show a different outcome within the first few months. Further studies are necessary regarding long-term differences.

Influence of probiotics on the periodontium, the oral microbiota and the immune response during orthodontic treatment in adolescent and adult patients (ProMB Trial): study protocol for a prospective, double-blind, controlled, randomized clinical trial.

BACKGROUND: Orthodontic treatment with fixed appliances is often necessary to correct malocclusions in adolescence or adulthood. However, oral hygiene is complicated by appliances, and prior studies indicate that they may trigger oral inflammation and dysbiosis of the oral microbiota, especially during the first 3 months after insertion, and, thus, may present a risk for inflammatory oral diseases. In recent periodontal therapeutic studies, probiotics have been applied to improve clinical parameters and reduce local inflammation. However, limited knowledge exists concerning the effects of probiotics in orthodontics. Therefore, the aim of our study is to evaluate the impact of probiotics during orthodontic treatment. METHODS: This study is a monocentric, randomized, double blind, controlled clinical study to investigate the effectiveness of daily adjuvant use of Limosilactobacillus reuteri (Prodentis®-lozenges, DSM 17938, ATCC PTA 5289) versus control lozenges during the first three months of orthodontic treatment with fixed appliances. Following power analysis, a total of 34 adolescent patients (age 12-17) and 34 adult patients (18 years and older) undergoing orthodontic treatment at the University Hospital Erlangen will be assigned into 2 parallel groups using a randomization plan for each age group. The primary outcome measure is the change of the gingival index after 4 weeks. Secondary outcomes include the probing pocket depth, the modified plaque index, the composition of the oral microbiota, the local cytokine expression and-only for adults-serum cytokine levels and the frequencies of cells of the innate and adaptive immune system in peripheral blood. DISCUSSION: Preventive strategies in everyday orthodontic practice include oral hygiene instructions and regular dental cleaning. Innovative methods, like adjuvant use of oral probiotics, are missing. The aim of this study is to analyse, whether probiotics can improve clinical parameters, reduce inflammation and prevent dysbiosis of the oral microbiota during orthodontic treatment. If successful, this study will provide the basis for a new strategy of prophylaxis of oral dysbiosis-related diseases during treatment with fixed appliances. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov in two parts under the number NCT04598633 (Adolescents, registration date 10/22/2020), and NCT04606186 (Adults, registration date 10/28/2020).

Extending the allelic spectrum at noncoding risk loci of orofacial clefting.

Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.

Halitosis in Oral and Maxillofacial Surgery Patients - a Pilot Study.

BACKGROUND: Halitosis is a relatively inhomogeneous pathology with an extremely high prevalence in the population. Potential risk factors for bad breath include bacterial decomposition of organic material as well as numerous general and systemic diseases. The aim of the present study was to analyze whether certain subgroups of oral and maxillofacial surgery patients have a higher risk of halitosis. Further the impact of halitosis on the patient's quality of life was ascertained. METHODS: A total of 127 oral and maxillofacial patients aged between 19 and 86 years were enrolled in this study. On account of their underlining disease, patients were divided into five different investigation groups. The dental examination comprised tongue coating, periodontal screening index (PSI), gingival index (GI), PI (plaque index), DMF-T values as well as non-stimulated saliva flow rates. Halitosis was monitored both organoleptically according to Rosenberg and instrumentally by means of a Halimeter®, which records the volatile sulfur compounds (VSC values in ppm). Patients were further asked to fill out questionnaires regarding their medical history and oral hygiene, oral health (OHIP-14), and quality of life (BDI-II). RESULTS: Halitosis values, which were recorded by a Halimeter® correlated with the objective Rosenberg golden standard method. Furthermore, halitosis values correlated with elevated PSI, GI, and DMF-T values as well as the degree of tongue coating. Patients with oral cancer showed significantly higher VSC values compared to all other groups. No difference in VSC values could be found between all other patient groups. CONCLUSIONS: The Halimeter® could be validated as a suitable method for determining halitosis in oral and maxillofacial patients. The significantly increased halitosis values in cancer patients as opposed to all other patient groups suggests the potential of halitosis VSC values as a potential screening method. The development of non-invasive breath tests for diagnosis could be subject of future research.

Genotoxic and cytotoxic potential of methacrylate-based orthodontic adhesives.

OBJECTIVES: The biocompatibility of methacrylate-based adhesives is a topic that is intensively discussed in dentistry. Since only limited evidence concerning the cyto- and genotoxicity of orthodontic adhesives is available, the aim of this study was to measure the genotoxic potential of seven orthodontic methacrylate-based adhesives. MATERIALS AND METHODS: The XTT assay was utilized to determine the cytotoxicity of Assure Plus, Assure Bonding Resin, ExciTE F, OptiBond Solo Plus, Scotchbond Universal Adhesive, Transbond MIP, and Transbond XT after an incubation period of 24 h on human gingival fibroblasts. We also performed the γH2AX assay to explore the genotoxic potential of the adhesives within cytotoxic dose ranges after an incubation period of 6 h. RESULTS: The XTT assay showed a concentration-dependent reduction in cell viability. The decrease in cellular viability was in the same dose range most significant for Assure Plus, rendering it the adhesive material with the highest cytotoxicity. Employing the γH2AX assay, a concentration-dependent increase in H2AX phosphorylation was detected, indicating induction of DNA damage. CONCLUSIONS: For most products, a linear correlation between the material concentration and γH2AX foci was observed. The most severe effect on γH2AX focus induction was found for Transbond MIP, which was the only adhesive in the test group containing the co-initiator diphenyliodonium hexafluorophosphate (DPIHP). CLINICAL RELEVANCE: The data indicate that orthodontic adhesives, notably Transbond MIP, bear a genotoxic potential. Since the study was performed with in vitro cultivated cells, a direct translation of the findings to in vivo exposure conditions should be considered with great diligence.

Inflammation in the Human Periodontium Induces Downregulation of the α1- and ß1-Subunits of the sGC in Cementoclasts.

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α1- and ß1-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α1- and ß1-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α1- and ß1-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.

The Special Developmental Biology of Craniofacial Tissues Enables the Understanding of Oral and Maxillofacial Physiology and Diseases.

Maxillofacial hard tissues have several differences compared to bones of other localizations of the human body. These could be due to the different embryological development of the jaw bones compared to the extracranial skeleton. In particular, the immigration of neuroectodermally differentiated cells of the cranial neural crest (CNC) plays an important role. These cells differ from the mesenchymal structures of the extracranial skeleton. In the ontogenesis of the jaw bones, the development via the intermediate stage of the pharyngeal arches is another special developmental feature. The aim of this review was to illustrate how the development of maxillofacial hard tissues occurs via the cranial neural crest and pharyngeal arches, and what significance this could have for relevant pathologies in maxillofacial surgery, dentistry and orthodontic therapy. The pathogenesis of various growth anomalies and certain syndromes will also be discussed.

Cephalometry without complex dedicated postprocessing in an oriented magnetic resonance imaging dataset: a pilot study.

OBJECTIVES: Magnetic resonance imaging (MRI) enables a 3D-volume-imaging without ionizing radiation. Therefore, it was the aim of this study to present a post-processing-free method for cephalometric analysis of a MRI-dataset and to examine whether there is a significant difference between cephalometric analysis of conventional 2D cephalograms and MRI scans. METHODS: One MRI scan each was performed on three cadaver heads using a 3T-MR-scanner. Cephalometric analysis was conducted directly on the 3D dataset. All reference points were projected onto a virtual sagittal plane that was perpendicular to the Frankfort horizontal plane. Double-sided points were averaged. Cephalometric angles were measured from the projected points. Results were compared with cephalometric measurements on conventional lateral cephalometric radiographs (LCRs). The cephalometric analysis was performed by five raters. RESULTS: 390-angle measurements were obtained. The inter-rater reliability was high [intraclass correlation coefficients (ICCs) ≥ 0.74 for all angles]. Differences between the measurements on the cephalograms and MRI scans ranged between -0.91° (-1.88°, 0.07°) and 0.97° (-0.63°, 2.57°) on average and were equivalent with respect to a margin of [-2°, 2°] in all angles except L1-Me-Tgo (Bonferroni-Holm-corrected P < 0.05 in all angles except L1-Me-Tgo). The best match was found for the SNA angle. CONCLUSION: The clinical comparability of the MRI- and LCR-based cephalometry could be stated. Using MRI in orthodontics would reduce radiation exposure and the risk of stochastic radiation damage, which is of importance especially in younger patients.

Role of Oxygen Supply in Macrophages in a Model of Simulated Orthodontic Tooth Movement.

Apart from periodontal ligament fibroblasts, immune cells like macrophages also play an important mediating role in orthodontic tooth movement (OTM). Upon orthodontic force application to malpositioned teeth, macrophages in the periodontal ligament get exposed to both mechanical strain and hypoxic conditions (via a compression of blood vessels). In this study, we assessed the relative impact of orthodontically induced mechanical strain and hypoxic conditions on macrophages for the mediation and regulation of OTM. Macrophages were stimulated with physiological orthodontic compressive forces of 2 g/cm2 for 4 h and 24 h on gas-impermeable or gas-permeable cell culture plates under normoxic or hypoxic cell culture conditions. We quantified expression of genes involved in inflammation (Tnf, Il-6, and Cox-2), extracellular remodelling (Mmp-9), and angiogenesis (Vegf) by RT-qPCR. Furthermore, we analysed HIF-1α, prostaglandin-E2, and VEGF protein expression via immunoblotting or ELISA. Mechanical strain and oxygen supply both differentially affected expression of genes and proteins involved in inflammation and angiogenesis. In this context, we found that HIF-1α protein levels were elevated by combined mechanical strain and hypoxic conditions, whereas gas-permeable plates providing sufficient oxygen supply prevented HIF-1α stabilization at the protein level after pressure application on macrophages. Our results thus indicate that macrophages involved in the mediation of OTM are affected by and respond differently to hypoxic conditions and mechanical compressive strain, which occur concomitantly during OTM, than periodontal ligament fibroblasts (PDLF), thus indicating different roles of these cells in the regulation of OTM at the cellular-molecular level. We further observed that contrary to PDLF HIF-1α stabilization in macrophages is rather induced via the decreased oxygen supply associated with OTM than via mechanotransduction by mechanical strain.

Influence of Natural Killer Cells and Natural Killer T Cells on Periodontal Disease: A Systematic Review of the Current Literature.

Natural killer (NK) cells, as members of the innate immune system, and natural killer T (NKT) cells, bridging innate and adaptive immunity, play a prominent role in chronic inflammatory diseases and cancerogenesis, yet have scarcely been examined in oral diseases. Therefore, systematic research on the latest literature focusing on NK/NKT cell-mediated mechanisms in periodontal disease, including the time period 1988-2020, was carried out in MEDLINE (PubMed) using a predetermined search strategy, with a final selection of 25 studies. The results showed that NK cells tend to have rather proinflammatory influences via cytokine production, cytotoxic effects, dendritic-cell-crosstalk, and autoimmune reactions, while contrarily, NKT cell-mediated mechanisms were proinflammatory and immunoregulatory, ranging from protective effects via B-cell-regulation, specific antibody production, and the suppression of autoimmunity to destructive effects via cytokine production, dendritic-cell-crosstalk, and T-/B-cell interactions. Since NK cells seem to have a proinflammatory role in periodontitis, further research should focus on the proinflammatory and immunoregulatory properties of NKT cells in order to create, in addition to antibacterial strategies in dental inflammatory disease, novel anti-inflammatory therapeutic approaches modulating host immunity towards dental health.

A Human Periodontal Ligament Fibroblast Cell Line as a New Model to Study Periodontal Stress.

The periodontal ligament (PDL) is exposed to different kinds of mechanical stresses such as bite force or orthodontic tooth movement. A simple and efficient model to study molecular responses to mechanical stress is the application of compressive force onto primary human periodontal ligament fibroblasts via glass disks. Yet, this model suffers from the need for primary cells from human donors which have a limited proliferative capacity. Here we show that an immortalized cell line, PDL-hTERT, derived from primary human periodontal ligament fibroblasts exhibits characteristic responses to glass disk-mediated compressive force resembling those of primary cells. These responses include induction and secretion of pro-inflammatory markers, changes in expression of extracellular matrix-reorganizing genes and induction of genes related to angiogenesis, osteoblastogenesis and osteoclastogenesis. The fact that PDL-hTERT cells can easily be transfected broadens their usefulness, as molecular gain- and loss-of-function studies become feasible.

Defining Metaniches in the Oral Cavity According to Their Microbial Composition and Cytokine Profile.

The human oral microbiota consists of over 700 widespread taxa colonizing the oral cavity in several anatomically diverse oral niches. Lately, sequencing of the 16S rRNA genes has become an acknowledged, culture-independent method to characterize the oral microbiota. However, only a small amount of data are available concerning microbial differences between oral niches in periodontal health and disease. In the context of periodontitis, the cytokine expression in the gingival crevicular fluid has been studied in detail, whereas little is known about the cytokine profile in hard and soft tissue biofilms. In order to characterize oral niches in periodontal health, the oral microbiota and cytokine pattern were analyzed at seven different sites (plaque (P), gingival crevicular fluid (GCF), saliva (S), tongue (T), hard palate (HP), cheek (C) and sublingual area (U)) of 20 young adults using next-generation sequencing and multiplex immunoassays. Site-specific microbial compositions were detected, which clustered into three distinct metaniches ("P-GCF", "S-T-HP" and "C-U") and were associated with niche-/metaniche-specific cytokine profiles. Our findings allow the definition of distinct metaniches according to their microbial composition, partly reflected by their cytokine profile, and provide new insights into microenvironmental similarities between anatomical diverse oral niches.

Imputation of orofacial clefting data identifies novel risk loci and sheds light on the genetic background of cleft lip ± cleft palate and cleft palate only.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation.

Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate.

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO.

Autophagy in periodontal ligament fibroblasts under biomechanical loading.

Autophagy (cellular self-consumption) is an adaptive stress response and an important aspect of adaption to mechanical loading. If mechanical forces are associated with autophagy regulation in periodontal ligament (PDL) fibroblasts is still unknown. The aim of this study was to analyze the influence of force magnitude on autophagy regulation and subsequently on cell death in human PDL fibroblasts. Autophagy-associated genes were analyzed with a specific PrimePCR assay after 24 h of stimulation with high (STSH) and low magnitudes (STSL) of static tensile strain applied to PDL fibroblasts. Based on the results, targets were selected for further real-time PCR analysis. The autophagic flux was assessed by immunoblotting for autophagy marker microtubule-associated protein 1, light chain 3, and by autophagosome staining. Cell death was determined by TUNEL assay and Cell Death Detection ELISAPLUS. Autophagy was induced pharmacologically by rapamycin and inhibited by chloroquine. For statistical analysis, the Kruskal Wallis test followed by the post-hoc Dunnett's test was used. Static tensile strain had regulatory effects on mRNA expression of multiple autophagy-associated targets. Stimulation with STSH induced mRNA expression changes in more autophagy-associated targets than STSL. The autophagic flux was induced by STSH while STSL had no significant effect on autophagosome formation. Furthermore, autophagy inhibition led to increased cell death. Low magnitudes of tensile strain seem to have cell-protective properties. Taken together, our findings provide novel insights about autophagy regulation by biomechanical loading in human PDL fibroblasts. Our results suggest a gradual response of autophagy to static tensile strain in human PDL fibroblasts.

Effectiveness of early orthopaedic treatment with headgear: a systematic review and meta-analysis.

Background: Although the headgear appliance has been used extensively to correct anteroposterior discrepancies, its treatment effects have not yet been adequately assessed in an evidence-based manner. Objective: Aim of this systematic review was to assess the therapeutic and adverse effects of early headgear treatment from controlled clinical trials on human patients in an evidence-based manner. Search methods: An unrestricted electronic search of six databases from inception to December 2015. Selection criteria: Randomized and prospective non-randomized controlled trials assessing the effects of headgear treatment on human patients. Data collection and analysis: After duplicate study selection, data extraction, and risk of bias assessment according to the Cochrane guidelines, random-effects meta-analyses of mean differences (MDs) and relative risks (RRs), including their 95% confidence intervals (CIs) were performed, followed by subgroup and sensitivity analyses. Results: A total of 18 unique studies with a total of 930 (56% male/44% female) patients were included. Headgear treatment was associated with a posterior translation of the anterior maxilla border in the short term, as seen by the mean annualized change in the SNA angle (MD = -1.63°/year; 95% CI = -2.20 to -1.06°/year; high quality evidence) compared to untreated patients. This effect was independent of the rotation of the palatal plane and the inclination of the upper incisors, while a proportional relationship with the initial discrepancy in SNA was seen. The clinical significance of this improvement diminished in the long term, although only limited evidence existed. Additionally, early headgear treatment might decrease the risk of dental trauma during the following years (RR = 0.34; 95% CI = 0.14 to 0.80; moderate quality evidence). Low quality evidence on the effect of headgear on the rotation of the palatal plane, the nasolabial angle, the occlusal outcome, and signs of temporomandibular disorders precluded robust assessments, due to risk of bias, inconsistency, imprecision, and small-study effects. Conclusions: Based on existing trials, headgear is a viable treatment option to modify sagittal growth of the maxilla in the short term in Class II patients with maxillary prognathism. Registration: PROSPERO (CRD42015029837). Funding: None.