Recent Advances in In Vivo Somatic Cell Gene Modification in Newborn Pups.

Germline manipulation at the zygote stage using the CRISPR/Cas9 system has been extensively employed for creating genetically modified animals and maintaining established lines. However, this approach requires a long and laborious task. Recently, many researchers have attempted to overcome these limitations by generating somatic mutations in the adult stage through tail vein injection or local administration of CRISPR reagents, as a new strategy called "in vivo somatic cell genome editing". This approach does not require manipulation of early embryos or strain maintenance, and it can test the results of genome editing in a short period. The newborn is an ideal stage to perform in vivo somatic cell genome editing because it is immune-privileged, easily accessible, and only a small amount of CRISPR reagents is required to achieve somatic cell genome editing throughout the entire body, owing to its small size. In this review, we summarize in vivo genome engineering strategies that have been successfully demonstrated in newborns. We also report successful in vivo genome editing through the neonatal introduction of genome editing reagents into various sites in newborns (as exemplified by intravenous injection via the facial vein), which will be helpful for creating models for genetic diseases or treating many genetic diseases.

Recent Advances in the Production of Genome-Edited Rats.

The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide. The most important aspect of this process is the selection of the method used to deliver GE components to rat embryos. In earlier stages, the microinjection (MI) of GE components into the cytoplasm and/or nuclei of a zygote was frequently employed. However, this method is associated with the use of an expensive manipulator system, the skills required to operate it, and the egg transfer (ET) of MI-treated embryos to recipient females for further development. In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. Furthermore, in vitro transduction of rat embryos with adeno-associated viruses is potentially effective for obtaining GE rats. However, these two approaches also require ET. The use of gene-engineered embryonic stem cells or spermatogonial stem cells appears to be of interest to obtain GE rats; however, the procedure itself is difficult and laborious. Genome-editing via oviductal nucleic acids delivery (GONAD) (or improved GONAD (i-GONAD)) is a novel method allowing for the in situ production of GE zygotes existing within the oviductal lumen. This can be performed by the simple intraoviductal injection of GE components and subsequent in vivo EP toward the injected oviducts and does not require ET. In this review, we describe the development of various approaches for producing GE rats together with an assessment of their technical advantages and limitations, and present new GE-related technologies and current achievements using those rats in relation to human diseases.

RNA analysis based on a small number of manually isolated fixed cells (RNA-snMIFxC) to profile stem cells from human deciduous tooth-derived dental pulp cells.

BACKGROUND: Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette. RESULTS: Two or more colored cells (~ 10), after staining with a chromogen such a 3,3'-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells. CONCLUSION: These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.

Prevalence of an incompetent lip seal during growth periods throughout Japan: a large-scale, survey-based, cross-sectional study.

BACKGROUND: Systemic and local factors may lead to disruption of craniofacial growth and development, causing an imbalance between the orofacial skeleton, muscle and soft tissue, dental occlusion, and the dental arch during growth periods. We aimed to reveal whether the prevalence of incompetent lip seal (ILS) varies with age and region, as well as to clarify the factors related to an ILS, in a national, large-scale epidemiological study. METHODS: We surveyed 3399 children, from 3 to 12 years of age, visiting 66 pediatric dental clinics throughout Japan. For this survey, we employed a questionnaire consisting of 44 questions regarding daily health conditions and lifestyle habits. We evaluated the differences in ILS prevalence by age and region (using a Cochran-Armitage test for trend and a Kruskal-Wallis test), and the relationship between ILS and factors investigated in the questionnaire (using Spearman's rank correlation coefficient). RESULTS: We observed that 30.7% of Japanese children exhibited an ILS and that the ILS rate increased with age (p < 0.001). There were no regional differences in the rate of ILS in Japanese children (p = 0.506). We revealed that 12 of 44 survey items exhibited a statistically significant correlation with ILS (p < 0.001), using Spearman's rank correlation coefficient. These items involved orofacial morphology, mouth breathing, and possibly, allergic rhinitis. CONCLUSION: The rate of ILS seems to increase with age in children, throughout Japan. Therefore, this disorder may not self-correct during the growth periods in these children. Guidelines are required for pediatric dentists to recognize ILS among children aged 3-12 years.

In Vivo Piggybac-Based Gene Delivery towards Murine Pancreatic Parenchyma Confers Sustained Expression of Gene of Interest.

The pancreas is a glandular organ that functions in the digestive system and endocrine system of vertebrates. The most common disorders involving the pancreas are diabetes, pancreatitis, and pancreatic cancer. In vivo gene delivery targeting the pancreas is important for preventing or curing such diseases and for exploring the biological function of genes involved in the pathogenesis of these diseases. Our previous experiments demonstrated that adult murine pancreatic cells can be efficiently transfected by exogenous plasmid DNA following intraparenchymal injection and subsequent in vivo electroporation using tweezer-type electrodes. Unfortunately, the induced gene expression was transient. Transposon-based gene delivery, such as that facilitated by piggyBac (PB), is known to confer stable integration of a gene of interest (GOI) into host chromosomes, resulting in sustained expression of the GOI. In this study, we investigated the use of the PB transposon system to achieve stable gene expression when transferred into murine pancreatic cells using the above-mentioned technique. Expression of the GOI (coding for fluorescent protein) continued for at least 1.5 months post-gene delivery. Splinkerette-PCR-based analysis revealed the presence of the consensus sequence TTAA at the junctional portion between host chromosomes and the transgenes; however, this was not observed in all samples. This plasmid-based PB transposon system enables constitutive expression of the GOI in pancreas for potential therapeutic and biological applications.

piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential.

We aimed to immortalize primarily isolated human deciduous tooth-derived dental pulp cells (HDDPCs) by transfection with piggyBac (PB)-based transposon vectors carrying E7 from human papilloma virus 16 or complementary DNA (cDNA) encoding human telomerase reverse transcriptase (hTERT). HDDPCs were co-transfected with pTrans (conferring PB transposase expression) + pT-pac (conferring puromycin acetyltransferase expression) + pT-tdTomato (conferring tdTomato cDNA expression) and pT-E7 (conferring E7 expression) or pTrans + pT-pac + pT-EGFP (conferring enhanced green fluorescent protein cDNA expression) + pT-hTERT (conferring hTERT expression). After six days, these cells were selected in medium containing 5 µg/mL puromycin for one day, and then cultured in normal medium allowing cell survival. All resultant colonies were harvested and propagated as a pool. Stemness and tumorigenic properties of the established cell lines ("MT_E7" for E7 and "MT_hTERT" for hTERT) with untransfected parental cells (MT) were examined. Both lines exhibited proliferation similar to that of MT, with alkaline phosphatase activity and stemness-specific factor expression. They displayed differentiation potential into multi-lineage cells with no tumorigenic property. Overall, we successfully obtained HDDPC-derived immortalized cell lines using a PB-based transfection system. The resultant and parental cells were indistinguishable. Thus, E7 and hTERT could immortalize HDDPCs without causing cancer-associated changes or altering phenotypic properties.

Increased Expression of Cell Surface SSEA-1 is Closely Associated with Naïve-Like Conversion from Human Deciduous Teeth Dental Pulp Cells-Derived iPS Cells.

Stage-specific embryonic antigen 1 (SSEA-1) is an antigenic epitope (also called CD15 antigen) defined as a Lewis X carbohydrate structure and known to be expressed in murine embryonal carcinoma cells, mouse embryonic stem cells (ESCs), and murine and human germ cells, but not human ESCs/induced pluripotent stem cells (iPSCs). It is produced by α1,3-fucosyltransferase IX gene (FUT9), and F9 ECCs having a disrupted FUT9 locus by gene targeting are reported to exhibit loss of SSEA-1 expression on their cell surface. Mouse ESCs are pluripotent cells and therefore known as "naïve stem cells (NSCs)." In contrast, human ESCs/iPSCs are thought to be epiblast stem cells (EpiSCs) that are slightly more differentiated than NSCs. Recently, it has been demonstrated that treatment of EpiSCs with several reprograming-related drugs can convert EpiSCs to cells similar to NSCs, which led us to speculate that SSEA-1 may have been expressed in these NSC-like EpiSCs. Immunocytochemical staining of these cells with anti-SSEA-1 revealed increased expression of this epitope. RT-PCR analysis also confirmed increased expression of FUT9 transcripts as well as other stemness-related transcripts such as REX-1 (ZFP42). These results suggest that SSEA-1 can be an excellent marker for human NSCs.

Pilot Study of Visual and Quantitative Image Analysis of Facial Surface Asymmetry in Unilateral Complete Cleft Lip and Palate.

OBJECTIVE: To visualize and quantitatively analyze facial surface asymmetry following primary cleft lip repair in patients with unilateral cleft lip and palate (UCLP) and to compare this with noncleft controls. DESIGN: Retrospective comparative study. PATIENTS: Twenty-two patients with complete UCLP who underwent primary lip repair from 2009 to 2013 were enrolled in this study. The preserved 3-dimensional (3D) data of 23 healthy Japanese participants with the same age were used as controls. INTERVENTIONS: All patients had received primary labioplasty in accordance with Cronin triangular flap method with orbicular oris muscle reconstruction. MAIN OUTCOME MEASURES: Shadow and zebra images established from moiré images, which were reconstructed from 3D facial data using stereophotogrammetry, were bisected and reversed by the symmetry axes (the middle line of the face). The discrepancies of the gravity and density between cleft and noncleft sides in 2 regions of interest, facial and lip areas, were then calculated and compared with those of healthy participants. RESULTS: In the UCLP group, the mean discrepancies of gravity on shadow and zebra images were 1.76 ± 0.70 and 2.63 ± 1.72 pixels, respectively, in the facial area and 1.31 ± 0.36 and 3.83 ± 2.08 pixels, respectively, in the lip area. There was a significant difference in the mean discrepancies of gravity and density on zebra images in the lip area between the UCLP and control groups. CONCLUSIONS: Our image analysis of digital facial surface asymmetry in patients with UCLP provides visual and quantitative information, and it may contribute to improvements in muscle reconstruction on cleft lip repair.

Intrapancreatic Parenchymal Injection of Cells as a Useful Tool for Allowing a Small Number of Proliferative Cells to Grow In Vivo.

In vivo inoculation of cells such as tumor cells and induced pluripotent stem (iPS)/embryonic stem (ES) cells into immunocompromised mice has been considered as a powerful technique to evaluate their potential to proliferate or differentiate into various cell types originating from three germ cell layers. Subcutaneous grafting and grafting under the kidney capsule have been widely used for this purpose, but there are some demerits such as the requirement of a large number of tumor cells for inoculation and frequent failure of tumorigenesis. Therefore, grafting into other sites has been explored, including intratesticular or intramuscular grafting as well as grafting into the cochleae, liver, or salivary glands. In this study, we found that intrapancreatic parenchymal injection of cells is useful for allowing a small number of cells (~15 × 10³ cells or ~30 cell clumps µL-1·site-1) to proliferate and sometimes differentiate into various types of cells. It requires only surgical exposure of the pancreas over the dorsal skin and subsequent injection of cells towards the pancreatic parenchyma under dissecting microscope-based observation using a mouthpiece-controlled glass micropipette. We now name this technology "intrapancreatic parenchymal cell transplantation (IPPCT)", which will be useful, especially when only a small number of cells or colonies are available.

Relationships among nasal resistance, adenoids, tonsils, and tongue posture and maxillofacial form in Class II and Class III children.

INTRODUCTION: The purpose of this study was to clarify the relationships between upper airway factors (nasal resistance, adenoids, tonsils, and tongue posture) and maxillofacial forms in Class II and III children. METHODS: Sixty-four subjects (mean age, 9.3 years) with malocclusion were divided into Class II and Class III groups by ANB angles. Nasal resistance was calculated using computational fluid dynamics from cone-beam computed tomography data. Adenoids, tonsils, and tongue posture were evaluated in the cone-beam computed tomography images. The groups were compared using Mann-Whitney U tests and Student t tests. The Spearman rank correlations test assessed the relationships between the upper airway factors and maxillofacial form. RESULTS: Nasal resistance of the Class II group was significantly larger than that of the Class III group (P = 0.005). Nasal resistance of the Class II group was significantly correlated with inferior tongue posture (P <0.001) and negatively correlated with intermolar width (P = 0.028). Tonsil size of the Class III group was significantly correlated with anterior tongue posture (P <0.001) and mandibular incisor anterior position (P = 0.007). Anterior tongue posture of the Class III group was significantly correlated with mandibular protrusion. CONCLUSIONS: The relationships of upper airway factors differ between Class II and Class III children.

The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.

The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes.

Quantitative evaluation of toothbrush and arm-joint motion during tooth brushing.

OBJECTIVES: It is very difficult for dental professionals to objectively assess tooth brushing skill of patients, because an obvious index to assess the brushing motion of patients has not been established. The purpose of this study was to quantitatively evaluate toothbrush and arm-joint motion during tooth brushing. MATERIALS AND METHODS: Tooth brushing motion, performed by dental hygienists for 15 s, was captured using a motion-capture system that continuously calculates the three-dimensional coordinates of object's motion relative to the floor. The dental hygienists performed the tooth brushing on the buccal and palatal sides of their right and left upper molars. The frequencies and power spectra of toothbrush motion and joint angles of the shoulder, elbow, and wrist were calculated and analyzed statistically. RESULTS: The frequency of toothbrush motion was higher on the left side (both buccal and palatal areas) than on the right side. There were no significant differences among joint angle frequencies within each brushing area. The inter- and intra-individual variations of the power spectrum of the elbow flexion angle when brushing were smaller than for any of the other angles. CONCLUSIONS: This study quantitatively confirmed that dental hygienists have individual distinctive rhythms during tooth brushing. All arm joints moved synchronously during brushing, and tooth brushing motion was controlled by coordinated movement of the joints. The elbow generated an individual's frequency through a stabilizing movement. CLINICAL RELEVANCE: The shoulder and wrist control the hand motion, and the elbow generates the cyclic rhythm during tooth brushing.

Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations.

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.

Three-dimensional cone-beam computed tomography analysis of enlargement of the pharyngeal airway by the Herbst appliance.

INTRODUCTION: Pharyngeal airway size is increasingly recognized as an important factor in obstructive sleep apnea. However, few studies have examined the changes of pharyngeal airway form after dental procedures for treating obstructive sleep apnea during growth. The purpose of this study was to evaluate the effect of the Herbst appliance on the 3-dimensional form of the pharyngeal airway using cone-beam computed tomography. METHODS: Twenty-four Class II subjects (ANB, ≥5°; 11 boys; mean age, 11.6 years) who required Herbst therapy with edgewise treatment had cone-beam computed tomography images taken before and after Herbst treatment. Twenty Class I control subjects (9 boys; mean age, 11.5 years) received edgewise treatment only. The volume, depth, and width of the pharyngeal airway were compared between the groups using measurements from 3-dimensional cone-beam computed tomography images of the entire pharyngeal airway. RESULTS: The increase of the oropharyngeal airway volume in the Herbst group (5000.2 mm(3)) was significantly greater than that of the control group (2451.6 mm(3)). Similarly, the increase of the laryngopharyngeal airway volume in the Herbst group (1941.8 mm(3)) was significantly greater than that of the control group (1060.1 mm(3)). CONCLUSIONS: The Herbst appliance enlarges the oropharyngeal and laryngopharyngeal airways. These results may provide a useful assessment of obstructive sleep apnea treatment during growth.

Analysis of head motions during food intake in Japanese adults using a new motion capture system.

OBJECTIVE: A new motion capture system was developed to verify the hypothesis that "during food intake, head motion changes according to the properties of the food." DESIGN: Twenty healthy males and 20 healthy females with right-handed and normal occlusion participated in this study. The motion capture system used consisted of a Microsoft Xbox One Kinect Sensor® and a newly-developed program. Meatballs (solid), yogurt (paste), and water (fluid) were used as food samples. Head motion distance, head turning angle, and head forward angle were measured during food intake. Unpaired t-tests were used to analyze each head motion and compare the sexes. A one-way repeated measures analysis of variance was used to analyze each head motion for different food samples. RESULTS: Head motion distance was significantly smaller in females for the meatball and yogurt, but not for water. There were no significant differences between the sexes for head turning angle or head forward angle. Head motion distance and head forward angle were significantly larger for water than for meatballs and yogurt. The head turning angle was significantly smaller for the meatball than for yogurt and water. CONCLUSIONS: The results indicated that females tend to consume food without moving their heads when eating solid and paste foods. As the fluidity of the food increased, the head moved in a turning motion to avoid spilling the food, and the heads tilted forward. The motion capture system used in this study was also effective in analyzing head motion during eating.

Tongue posture improvement and pharyngeal airway enlargement as secondary effects of rapid maxillary expansion: a cone-beam computed tomography study.

INTRODUCTION: Rapid maxillary expansion (RME) is known to improve nasal airway ventilation. Recent evidence suggests that RME is an effective treatment for obstructive sleep apnea in children with maxillary constriction. However, the effect of RME on tongue posture and pharyngeal airway volume in children with nasal airway obstruction is not clear. In this study, we evaluated these effects using cone-beam computed tomography. METHODS: Twenty-eight treatment subjects (mean age 9.96 ± 1.21 years) who required RME treatment had cone-beam computed tomography images taken before and after RME. Twenty control subjects (mean age 9.68 ± 1.02 years) received regular orthodontic treatment. Nasal airway ventilation was analyzed by using computational fluid dynamics, and intraoral airway (the low tongue space between tongue and palate) and pharyngeal airway volumes were measured. RESULTS: Intraoral airway volume decreased significantly in the RME group from 1212.9 ± 1370.9 mm(3) before RME to 279.7 ± 472.0 mm(3) after RME. Nasal airway ventilation was significantly correlated with intraoral airway volume. The increase of pharyngeal airway volume in the control group (1226.3 ± 1782.5 mm(3)) was only 41% that of the RME group (3015.4 ± 1297.6 mm(3)). CONCLUSIONS: In children with nasal obstruction, RME not only reduces nasal obstruction but also raises tongue posture and enlarges the pharyngeal airway.

Smoothness of molar movement during gum chewing in children with primary dentition.

The purpose of this study was to test the null hypothesis that molar movement during gum chewing in children with primary dentition is as smooth as in adults. Twenty-two healthy children with primary dentition and 23 healthy adult females participated in this study. Mandibular movement during gum chewing was recorded using an optoelectronic analysis system with six degrees-of-freedom at 100 Hz, and 10 cycles were selected for analysis. Normalized jerk cost (NJC) at the incisors and working and balancing molars were calculated in each phase (i.e., opening, closing and occlusal level phases) for each chewing cycle. The NJC of the working side molar in children was larger than in adults in both the opening and occlusal phases. Inter-individual variances of the NJC in each phase in children and adults were smaller than corresponding intra-individual variances, except for the NJC during the occlusal phase of adults for the working and balancing side molars. The inter- and intra-individual variances of the NJC during the closing phase were the smallest in each phase for both children and adults. This indicates that the jaw movements of children with primary dentition are more variable, less smooth, and faster than that of adults.

Recent Genome-Editing Approaches toward Post-Implanted Fetuses in Mice.

Genome editing, as exemplified by the CRISPR/Cas9 system, has recently been employed to effectively generate genetically modified animals and cells for the purpose of gene function analysis and disease model creation. There are at least four ways to induce genome editing in individuals: the first is to perform genome editing at the early preimplantation stage, such as fertilized eggs (zygotes), for the creation of whole genetically modified animals; the second is at post-implanted stages, as exemplified by the mid-gestational stages (E9 to E15), for targeting specific cell populations through in utero injection of viral vectors carrying genome-editing components or that of nonviral vectors carrying genome-editing components and subsequent in utero electroporation; the third is at the mid-gestational stages, as exemplified by tail-vein injection of genome-editing components into the pregnant females through which the genome-editing components can be transmitted to fetal cells via a placenta-blood barrier; and the last is at the newborn or adult stage, as exemplified by facial or tail-vein injection of genome-editing components. Here, we focus on the second and third approaches and will review the latest techniques for various methods concerning gene editing in developing fetuses.

Lip and facial training improves lip-closing strength and facial morphology.

OBJECTIVE: Childhood is an important period for lip-closing strength (LCS) development, and failure to acquire LCS during childhood leads to various adverse health effects, such as mouth breathing. The purpose of this study was to examine the effectiveness of device-free lip and facial training in preschool children. DESIGN: The participants were divided into training and control groups. Both groups comprised 123 children aged 3-4 years, and only the training group received lip and facial training (i.e., opening and closing the lips and protruding the tongue) for 1 year. A two-way repeated measures analysis of variance was applied to compare the interaction effects of LCS and facial linear distance and angle by year (initial year vs. 1 year later) and group (training vs. control group). In addition, paired t-tests were used to test the changes in LCS and facial linear distance and angle after 1 year in both groups. Furthermore, the same analysis was performed in children with weak LCS in both groups (incompetent lip seal [ILS]). RESULTS: The LCS of children in the training group significantly increased after training compared with that in the control group, whether the analysis included all children or children with ILS alone. Lip and facial training for children with ILS reduced both the upper and lower lip protrusion; children with ILS without training had increased lip protrusion after 1 year. CONCLUSIONS: Lip and facial training for children with ILS effectively improved LCS and lip morphology, thereby preventing increased lip protrusion.

Improvement of nasal airway ventilation after rapid maxillary expansion evaluated with computational fluid dynamics.

INTRODUCTION: Rapid maxillary expansion is known to improve nasal airway ventilation. However, it is difficult to precisely evaluate this improvement with conventional methods. The purpose of this longitudinal study was to use computational fluid dynamics to estimate the effect of rapid maxillary expansion. METHODS: Twenty-three subjects (9 boys, 14 girls; mean ages, 9.74 ± 1.29 years before rapid maxillary expansion and 10.87 ± 1.18 years after rapid maxillary expansion) who required rapid maxillary expansion as part of their orthodontic treatment had cone-beam computed tomography images taken before and after rapid maxillary expansion. The computed tomography data were used to reconstruct the 3-dimensional shape of the nasal cavity. Two measures of nasal airflow function (pressure and velocity) were simulated by using computational fluid dynamics. RESULTS: The pressure after rapid maxillary expansion (80.55 Pa) was significantly lower than before rapid maxillary expansion (147.70 Pa), and the velocity after rapid maxillary expansion (9.63 m/sec) was slower than before rapid maxillary expansion (13.46 m/sec). CONCLUSIONS: Improvement of nasal airway ventilation by rapid maxillary expansion was detected by computational fluid dynamics.