Inhibition of compound action potentials in the frog sciatic nerve by inchinkoto, a traditional Japanese medicine used for oral mucositis.

OBJECTIVE: This study aimed to determine the effects of traditional Japanese (Kampo) medicines used to treat oral mucositis on nerve conduction. METHODS: The effects of Kampo medicines, crude drugs, and chemical compounds on compound action potentials (CAPs) were analyzed using extracellular recordings in frog sciatic nerves. RESULTS: Among the Kampo medicines, inchinkoto demonstrated the most significant reduction in CAP amplitude, with a half-maximal inhibitory concentration (IC50) of 5.4 mg/mL. Hangeshashinto, shosaikoto, hochuekkito, and juzentaihoto also showed a significant reduction. Regarding inchinkoto, Artemisiae Capillari Spica (artemisia) was the most effective crude drug, with an IC50 of 4.2 mg/mL for CAP amplitude reduction, whereas Gardeniae Fructus (gardenia) exerted no significant effect. However, the combined use of artemisia and gardenia reduced the CAP amplitude more effectively than artemisia alone, indicating a synergistic interaction. The chemical ingredient eugenol from artemisia administered at 1 and 3 mmol/L reduced CAP amplitude, whereas other chemical ingredients administered at 0.1 and 1 mmol/L had no significant effects. CONCLUSIONS: Inchinkoto exhibited the most effective reduction in CAP amplitude in the sciatic nerve of frogs, primarily through the action of artemisia, with potential synergistic interaction between artemisia and gardenia.

Inhibition of retinoid X receptor improved the morphology, localization of desmosomal proteins and paracellular permeability in three-dimensional cultures of mouse keratinocytes.

Retinoic acid (RA) plays an important role in epithelial homeostasis and influences the morphology, proliferation, differentiation and permeability of epithelial cells. Mouse keratinocytes, K38, reconstituted non-keratinized stratified epithelium in three-dimensional (3D) cultures with serum, which contains retinol (a source of RA), but the morphology was different from in vivo epithelium. The formed epithelium was thick, with loosened cell-cell contacts. Here, we investigated whether the inhibition of RA receptor (RAR)/retinoid X receptor (RXR)-mediated signaling by an RXR antagonist, HX 531, improved K38 3D cultures in terms of morphology and intercellular junctions. The epithelium formed by 0.5 µM HX531 was thin, and the intercellular space was narrowed because of the restoration of the layer-specific distribution of desmoglein (DSG)-1, DSG3 and plakoglobin (PG). Moreover, the levels of desmosomal proteins and tight junction proteins, including DSG1, DSG2, DSG3, PG, claudin (CLDN)-1 and CLDN4 increased, but the adherens junction protein, E-cadherin, did not show any change. Furthermore, CLDN1 was recruited to occludin-positive cell-cell contacts in the superficial cells and transepithelial electrical resistance was increased. Therefore, K38 3D cultures treated with 0.5 µM HX531 provides a useful in vitro model to study intercellular junctions in the non-keratinized epithelium.

Embedded Human Periodontal Ligament Stem Cells Spheroids Enhance Cementogenic Differentiation via Plasminogen Activator Inhibitor 1.

Spheroids reproduce the tissue structure that is found in vivo more accurately than classic two-dimensional (2D) monolayer cultures. We cultured human periodontal ligament stem cells (HPLSCs) as spheroids that were embedded in collagen gel to examine whether their cementogenic differentiation could be enhanced by treatment with recombinant human plasminogen activator inhibitor-1 (rhPAI-1). The upregulated expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP), established cementoblast markers, was observed in the 2D monolayer HPLSCs that were treated with rhPAI-1 for 3 weeks compared with that in the control and osteogenic-induction medium groups. In the embedded HPLSC spheroids, rhPAI-1 treatment induced interplay between the spheroids and collagenous extracellular matrix (ECM), indicating that disaggregated HPLSCs migrated and spread into the surrounding ECM 72 h after three-dimensional (3D) culture. Western blot and immunocytochemistry analyses showed that the CEMP1 expression levels were significantly upregulated in the rhPAI-1-treated embedded HPLSC spheroids compared with all the 2D monolayer HPLSCs groups and the 3D spheroid groups. Therefore, 3D collagen-embedded spheroid culture in combination with rhPAI-1 treatment may be useful for facilitating cementogenic differentiation of HPLSCs.

Maxillofacial morphological characteristics in growing orthodontic patients with non-syndromic oligodontia.

OBJECTIVE: Patients with oligodontia frequently show different types of malocclusions. However, how oligodontia affects the maxillofacial growth remains uncertain. This study aimed to investigate the maxillofacial morphological characteristics in growing patients with oligodontia. SETTING AND SAMPLE POPULATION: The study subjects included 33 Japanese children with non-syndromic oligodontia (14 boys and 19 girls; mean age: 10.2 years) who visited the orthodontic clinic of Fukuoka Dental College Medical and Dental Hospital from 1999 to 2019. MATERIALS AND METHODS: Cephalometric analyses were performed, and the variables measured in each subject were converted into Z scores in relation to the mean and standard deviation of the Japanese norms matched for growth stage. The one-sample t-test or Wilcoxon signed-rank test was performed to compare the mean scores in the patients with oligodontia with those of the Japanese norms. RESULTS: Compared with the Japanese norms, patients with oligodontia showed a smaller convexity and larger A-B plane and SNB angles. The Frankfort-mandibular plane and gonial angles were smaller, whereas the height of the ramus was larger. The vertical height of the alveolar bone in the maxillary and mandibular incisors and molar areas was smaller in patients with oligodontia. CONCLUSIONS: Patients with oligodontia showed Class III skeletal tendency with mandibular prognathism and flattened mandibular plane with a smaller gonial angle. These maxillofacial morphological features can be induced by a deficiency in the vertical growth of the alveolar bone in the maxillary and mandibular molar areas due to the lack of tooth germs.

SNAI2 is induced by transforming growth factor-ß1, but is not essential for epithelial-mesenchymal transition in human keratinocyte HaCaT cells.

Epithelial-mesenchymal transition (EMT) is a cellular process in which epithelial cells lose their epithelial traits and shift to the mesenchymal phenotype, and is associated with various biological events, such as embryogenesis, wound healing and cancer progression. The transcriptional program that promotes phenotype switching is dynamically controlled by transcription factors during EMT, including Snail (SNAI1), twist family bHLH transcription factor (TWIST) and zinc finger E-box binding homeobox 1 (ZEB1). The present study aimed to investigate the molecular mechanisms underlying EMT in squamous epithelial cells. Western blot analysis and immunocytochemical staining identified Slug (SNAI2) as a transcription factor that is induced during transforming growth factor (TGF)-ß1-mediated EMT in the human keratinocyte cell line HaCaT. The effect of SNAI2 overexpression and knockdown on the phenotypic characteristics of HaCaT cells was evaluated. Filamentous actin staining and western blot analysis revealed that the overexpression of SNAI2 did not induce the observed EMT-related phenotypic changes. In addition, SNAI2 knockdown demonstrated almost no impact on the EMT phenotypes induced by TGF-ß1. Notably, DNA microarray analysis followed by comprehensive bioinformatics analysis revealed that the differentially expressed genes upregulated by TGF-ß1 were significantly enriched in cell adhesion and extracellular matrix binding, whereas the genes downregulated in response to TGF-ß1 were significantly enriched in the cell cycle. No enriched gene ontology term and biological pathways were identified in the differentially expressed gene sets of SNAI2-overexpressing cells. In addition, the candidates for master transcription factors regulating the TGF-ß1-induced EMT were identified using transcription factor enrichment analysis. In conclusion, the results of study demonstrated that SNAI2 does not play an essential role in the EMT of HaCaT cells and identified candidate transcription factors that may be involved in EMT-related gene expression induced by TGF-ß1. These findings may enhance the understanding of molecular events in EMT and contribute to the development of a novel therapeutic approach against EMT in cancers and wound healing.

An orthodontic perspective on Larsen syndrome.

BACKGROUND: Larsen syndrome (LS) is a rare disorder of osteochondrodysplasia. In addition to large-joint dislocations, craniofacial anomalies are typical characteristics. In this report, we performed orthodontic analyses, including skeletal and occlusal evaluations, to examine whether the craniofacial skeletal morphology leads to the craniofacial anomalies in LS. CASE PRESENTATION: A 5 year old Japanese girl who was clinically diagnosed with LS was referred to the orthodontic clinic in the Fukuoka Dental College Medical and Dental Hospital because of a malocclusion. Clinical findings at birth were knee-joint dislocations, equinovarus foot deformities, and cleft soft palate. The patient showed craniofacial anomalies with hypertelorism, prominent forehead, depressed nasal bridge, and flattened midface. To evaluate the craniofacial skeletal morphology, cephalometric analysis was performed. In the frontal cephalometric analysis, the larger widths between bilateral points of the orbitale were related to hypertelorism. The lateral cephalometric analysis revealed the midface hypoplasia and the retrognathic mandible. These findings were responsible for the flattened appearance of the patient's face, even if the anteroposterior position of the nasion was normal. Her forehead looked prominent in relation to the face probably because of the retrognathic maxilla and mandible. Both the study model and the frontal cephalometric analysis indicated constriction of the upper and lower dental arches. The posterior crossbite facilitated by the premature contacts had developed in association with the constriction of the upper dental arch. CONCLUSIONS: This patient had some craniofacial anomalies with characteristic appearances in LS. It was evident that the underlying skeletal morphology led to the craniofacial dysmorphism.

Immunohistochemical study for the expression of leukocyte adhesion molecules, and FGF23 and ACE2 in P. gingivalis LPS-induced diabetic nephropathy.

OBJECTIVE: The present study aims to examine the expression of leukocyte adhesion molecules and renal metabolic factors in diabetic mouse kidneys with periodontal pathogen Pg-LPS-induced nephropathy. BACKGROUND: We recently reported that the glomerular endothelium expresses toll-like receptor (TLR)2 and TLR4 in diabetic environments and TLR2/4 ligand Porphyromonas (P.) gingivalis lipopolysaccharides (Pg-LPS) induce nephropathy in diabetic mice. It is thought that Pg-LPS promotes the chronic inflammation with the overexpression of leukocyte adhesion molecules and renal-specific metabolic enzymes by the recognition of Pg-LPS via TLR in the diabetic kidneys. There have been no reports of the effects of periodontopathic bacteria on the expression of leukocyte adhesion molecules and the accumulation of physiologically active substances in the kidney. METHODS: The immunohistochemical investigation was performed on diabetic mouse kidney with Pg-LPS-induced nephropathy with glomerulosclerosis in glomeruli. RESULTS: There were no vessels which expressed vascular cell adhesion molecule-1 (VCAM-1), E-selectin, or fibroblast growth factor (FGF) 23 in streptozotocin (STZ)-induced diabetic ICR mice (STZ-ICR), or in healthy ICR mice administered Pg-LPS (LPS-ICR). However, in diabetic ICR mouse kidneys with Pg-LPS-induced nephropathy (LPS-STZ) the expression of VCAM-1 and the accumulation of FGF23 were observed in renal tubules and glomeruli, and the expression of E-selectin was observed in renal parenchyma and glomeruli. The angiotensin-converting enzyme 2 (ACE2) was detected in the proximal tubules but not in other regions of ICR, STZ-ICR, or LPS-ICR. In LPS-STZ ACE2 was detected both in renal tubules as well as in glomeruli. The Mac-1 and podoplanin-positive cells increased in the renal parenchyma with diabetic condition and there was the distribution of a large number of Mac-1-positive cells in LPS-STZ. CONCLUSIONS: The Pg-LPS may induce diabetic renal inflammation such as glomerulosclerosis and tubulitis with infiltration of Mac-1/podoplanin positive macrophages via glomerular overexpression of VCAM-1 and E-selectin, resulting in accumulation of both ACE2 and FGF23 which were unmetabolized with the inflammation-induced kidney damage under the diabetic condition. Periodontitis may be a critical factor in the progress of nephropathy in diabetic patients.

Presurgical naso-alveolar molding paired with cheiloplasty to treat median cleft lip deformity in holoprosencephaly.

We report median cleft lip in an infant girl with lobar-typed holoprosencephaly who underwent presurgical naso-alveolar molding and subsequent cheiloplasty. At seven months postoperatively, we observed an upper lip with natural cupid-bow-shape formed with a nasal dome and two nostrils separated with reconstructed columella, which were maintained for eight years.

Immunohistochemical study of amelogenin binding proteins in an amelogenin point mutation mouse / Estudio inmunohistoquímico de proteínas de unión de la amelogenina en un ratón con mutación puntual de amelogenina

Amelogenin is one of the enamel matrices secreted by ameloblasts. A mutation of the amelogenin gene can cause hereditary dental enamel defects known as amelogenesis imperfecta (AI). Since lysosome-associated membrane protein-1 (LAMP-1), -3 (LAMP-3), and 78kDa glucose-related protein (Grp78) were identified as binding proteins of amelogenin, several studies have suggested the involvement of these binding proteins with the cell kinetics of ameloblasts in normal or abnormal conditions. The purpose of this study is to investigate the distribution of these amelogenin binding proteins in the ameloblast cell differentiation of mice with a point mutation of the amelogenin gene (Amelx*). The incisors of Amelx* mice had a white opaque color and the tooth surface was observed to be rough under a scanning electron microscope. Among the sequential ameloblast cell differentiation in the Amelx* mice, the shape of ameloblasts at the transition stage was irregular in comparison to those in wild-type (WT) mice. Immunostaining of Grp78 revealed that the whole cytoplasm of the transition stage ameloblasts was immunopositive for Grp78 antibody, while only the distal part of cell was positive in the WT mice. Furthermore, in the Amelx* mice, the cytoplasm of the transition stage ameloblasts was immunopositive for LAMP-1 and LAMP-3. These results suggest that Amelx* may cause the abnormal distribution of amelogenin binding proteins in the cytoplasm of ameloblasts.

La amelogenina es una de las matrices de esmalte secretadas por los ameloblastos. Una mutación del gen de amelogenina puede causar defectos hereditarios del esmalte dental conocidos como amelogénesis imperfecta (AI). Dado que la proteína de membrana asociada a lisosoma-1 (LAMP-1), -3 (LAMP-3) y la proteína relacionada con la glucosa de 78 kDa (Grp78) se identificaron como proteína de unión a amelogenina, varios estudios han sugerido la participación de estas proteínas con la cinética celular de los ameloblastos en condiciones normales o anormales. El objetivo del estudio fue investigar la distribución de LAMP-1, LAM-3 y Grp78 durante la diferenciación celular de ameloblastos de ratones con una mutación puntual del gen de amelogenina (Amelx*). Los incisivos de los ratones Amelx* presentaron un color blanco opaco y se observó en microscopio electrónico de barrido que la superficie del diente era áspera. La diferenciación celular secuencial y la forma de los ameloblastos en la etapa de transición en los ratones Amelx* fue irregular en comparación con los ratones silvestres (RS). La inmunotinción de Grp78 reveló que todo el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para el anticuerpo Grp78, mientras que solo la parte distal de la célula fue positiva en los ratones RS. Además, en ratones Amelx*, el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para LAMP-1 y LAMP-3. Estos resultados sugieren que Amelx* puede causar distribución anormal de proteínas de unión a amelogenina en el citoplasma de los ameloblastos.

Maxillofacial morphological factors related to acceleration of maxillary growth attributed to facial mask treatment: a structural superimposition study.

BACKGROUND: Anatomical textbooks mention that the contact between the pterygoid process and the palatine's pyramidal process is not a "suture" but "conjugation.".The aim was to evaluate the maxillofacial morphological factor responding most to the orthopedic force of facial mask treatment, using the structural superimposition analysis. METHODS: Thirty-one girls with Angle Class III malocclusion treated using a facial mask (FM group) and 11 girls with pseudo-Class III malocclusion (pseudo-III group) were examined. Lateral cephalograms at pre- and posttreatment were analyzed to evaluate maxillofacial changes. Cephalometric structural superimposition analysis originating with Björk and Skieller was also performed. RESULTS: In the FM group, a multiple linear regression model showed that maxillary sutural growth was significantly associated with counter-clockwise rotation of the maxilla and treatment changes in the anteroposterior distance from the pterygomaxillary fissure to the maxillary anterior alveolus, not changes in the distance from the nasion to the maxillary anterior alveolus. CONCLUSIONS: Structural superimposition analysis showed that counter-clockwise rotation of the maxilla and changes in the distance from the pterygomaxillary fissure to the maxillary anterior alveolus responded most to the orthopedic force of facial mask treatment. The analysis implicated that the pterygoid fissure-palatine's pyramidal process conjugation responds most to facial mask treatment among maxillofacial sutures and conjugation, and that the difference in the response induces maxillary counter-clockwise rotation.

TRPV2 channel inhibitors attenuate fibroblast differentiation and contraction mediated by keratinocyte-derived TGF-ß1 in an in vitro wound healing model of rats.

BACKGROUND: Keratinocytes release several factors that are involved in wound contracture and scar formation. We previously reported that a three-dimensional reconstruction model derived from rat skin represents a good wound healing model. OBJECTIVE: We characterized the role of transient receptor potential (TRP) channels in the release of transforming growth factor (TGF)-ß1 from keratinocytes and the differentiation of fibroblasts to identify possible promising pharmacological approaches to prevent scar formation and contractures. METHODS: The three-dimensional culture model was made from rat keratinocytes seeded on a collagen gel in which dermal fibroblasts had been embedded. RESULTS: Among the TRP channel inhibitors tested, the TRPV2 inhibitors SKF96365 and tranilast attenuated most potently keratinocyte-dependent and - independent collagen gel contraction due to TGF-ß signaling as well as TGF-ß1 release from keratinocytes and α-smooth muscle actin production in myofibroblasts. Besides the low amounts detected in normal dermis, TRPV2 mRNA and protein levels were increased after fibroblasts were embedded in the gel. TRPV2 was also expressed in the epidermis and keratinocyte layers of the model. Both inhibitors and TRPV2 siRNA attenuated the intracellular increase of Ca2+ induced by the TRPV agonist 2-aminoethoxydiphenyl borate in TGF-ß1-pretreated fibroblasts. CONCLUSION: This is the first study to show that compounds targeting TRPV2 channels ameliorate wound contraction through the inhibition of TGF-ß1 release and the differentiation of dermal fibroblasts in a culture model.

The promotion of nephropathy by Porphyromonas gingivalis lipopolysaccharide via toll-like receptors.

BACKGROUND: Recently, we reported that toll-like receptor (TLR)2 and TLR4 localized on the glomerular endothelium in the glomeruli of streptozotocin (STZ)-induced type 1 diabetic mice and high fat diet feed-induced type 2 diabetic mice, and that periodontal pathogen Porphyromonas gingivalis LPS (Pg-LPS) administration lowered the survival rate of diabetic mice. The present study aims to examine the effect of TLR4 blocking on the suppression of Pg-LPS-induced diabetic nephropathy. METHODS: The survival rate and morphological/biochemical features for streptozotocin-induced diabetic mice with Pg-LPS and TLR4 blocker eritoran administration were investigated by reporter gene assay, urine and blood analysis, immunohistochemistry, and real time-PCR. RESULTS AND CONCLUSIONS: All of the diabetic mice administered Pg-LPS were euthanized within the survival period of almost all of the diabetic mice. The blood urea nitrogen and creatinine, expression of TLR2 and TGF-b, and type 1 collagen accumulation, in the diabetic mice increased significantly with the Pg-LPS administration. In spite of the limited TLR4 activation with Pg-LPS, the TLR4 blocker eritoran decreased blood urea nitrogen and creatinine, and raised the survival rate of the Pg-LPS-administered diabetic mice slightly. The high expression levels of TLR2, TGF-b, and type 1 collagen in Pg-LPS-administered diabetic mice decreased with eritoran. Nuclear STAT3 which enhances TLR2 expression was detected in the TLR2-expressing glomeruli of diabetic mice. The TLR2 and STAT3 gene expression increased by the Pg-LPS administration but decreased with eritoran. These may suggest that Pg-LPS-induced diabetic nephropathy is mainly dependent on TLR2 signaling on glomerular endothelial cells, and that TLR4 blocker eritoran may play a role to slow the progress of diabetic nephropathy.

Quantitative evaluation of myofibroblast apoptosis during wound healing in rat palate after post-operative administration of basic fibroblast growth factor (bFGF).

OBJECTIVE: Excessive wound contraction apparently inhibits maxillary growth; thus, myofibroblast apoptosis needs to be accelerated in mucoperiosteal denudation after palatoplasty. The aim of this study was to evaluate myofibroblast apoptosis during wound healing in mucoperiosteal denudation of rat palates immediately after post-operative administration of basic fibroblast growth factor (bFGF). MATERIALS AND METHODS: A total of 100 male Wistar rats aged 20 days were divided into control, scar, sham and bFGF groups (n = 25 each). In the scar, sham and bFGF groups, mucoperiosteum was removed from the palate and fibrin glue was applied to the exposed bone surface immediately after surgery. In the bFGF group, 10 µL of 2 µg/µL bFGF solution was injected into the operated area beneath the fibrin glue. At 2, 5, 7, 14 and 28 days post-operatively, myofibroblast apoptosis during the wound healing process was investigated by double immunofluorescence staining. The apoptotic area of myofibroblasts was measured using image software. RESULTS: In the bFGF group, at 2 days, apoptosis of myofibroblasts in the lamina propria and submucosa was marked, as compared with the other three groups and apoptosis of myofibroblasts was scarcely seen at 5 days. At 5 and 7 days, the apoptotic area of myofibroblasts in the bFGF group was statistically significantly smaller when compared to the scar and sham groups. CONCLUSION: The results confirmed that bFGF injection immediately after surgery accelerated apoptosis of myofibroblasts in mucoperiosteal denudation of rats. This may reduce maxillary growth retardation due to excessive wound contraction.

Oxygen saturation and electromyographic changes in masseter muscle during experimental chewing of gum with harder texture.

OBJECTIVE: The purpose of this study was to clarify the relationship between changes in masseter muscle oxygenation measured by near-infrared spectroscopy (NIRS) and changes in the electromyographic (EMG) power spectrum during experimental chewing of gum with harder texture, to improve the understanding of the use of NIRS in assessing masseter muscle fatigue. MATERIAL AND METHODS: Ten female volunteers with normal occlusion were examined. Mean age (standard deviation) was 28.4 (3.8) years. Mean fracture stress of gum was 12.5 × 10(4) N/m(2). Subjects were instructed to chew gum for 60 s (75 strokes) on the voluntary chewing side at a pace of 1.25 strokes/s. Simultaneous recordings of NIRS and EMG signals from masseter muscle were performed during gum chewing. RESULTS: Oxygen saturation levels decreased from the start of chewing, then stabilized with a break point between the two phases. The normalized EMG amplitude increased and the mean frequency of the EMG power spectrum decreased during gum chewing. The timing of break point appearance was related to the timing of a significant decrease in median frequency, but no clear relationships were found between break point appearance and increased EMG amplitude. CONCLUSIONS: These results suggest that the break point of the oxygen saturation curve, as obtained from NIRS measurements, could be used as an indicator of masseter muscle fatigue as assessed by a shift in the EMG power spectrum to lower frequencies.