Cell lineage- and expression-based inference of the roles of forkhead box transcription factor Foxc2 in craniofacial development.

BACKGROUND: Foxc2 is a member of the winged helix/forkhead (Fox) box family of transcription factors. Loss of function of Foxc2 causes craniofacial abnormalities such as cleft palate and deformed cranial base, but its role during craniofacial development remains to be elucidated. RESULTS: The contributions of Foxc2-positive and its descendant cells to the craniofacial structure at E18.5 were examined using a tamoxifen-inducible Cre driver mouse (Foxc2-CreERT2) crossed with the R26R-LacZ reporter mouse. Foxc2 expression at E8.5 is restricted to the cranial mesenchyme, contributing to specific components including the cranial base, sensory capsule, tongue, upper incisor, and middle ear. Expression at E10.5 was still positively regulated in most of those regions. In situ hybridization analysis of Foxc2 and its closely related gene, Foxc1, revealed that expression domains of these genes largely overlap in the cephalic mesenchyme. Meanwhile, the tongue expressed Foxc2 but not Foxc1, and its development was affected by the neural crest-specific deletion of Foxc2 in mice (Wnt1-Cre; Foxc2fl/fl ). CONCLUSIONS: Foxc2 is expressed in cranial mesenchyme that contributes to specific craniofacial tissue components from an early stage, and it seems to be involved in their development in cooperation with Foxc1. Foxc2 also has its own role in tongue development.

Transmitted-light plethysmography detects changes in human pulpal blood flow elicited by innocuous tooth cooling and foot heating.

OBJECTIVES: The present study aimed firstly, to investigate whether pulpal circulatory changes elicited by innocuous tooth cooling and foot heating can be monitored with transmitted-light plethysmography (TLP), which detects pulpal blood volume changes, and secondly, to assess the effect of autonomic nervous control on TLP values. DESIGN: Thirty sound permanent maxillary incisors in 30 healthy volunteers (age: 25-35 years) were examined. The photodiode and 525-nm light-emitting diode of a prototype TLP system (J. Morita) were fixed onto the palatal and labial side, respectively, of each tooth with a custom-made acrylic cap. The subjects were stimulated for 10 min by cold (0 °C) water application to the experimental tooth or 30 min by foot heating with a footbath (43 °C). TLP and finger plethysmography were simultaneously recorded before (baseline), during and after stimulation. TLP values and autonomic nerve activity were statistically analyzed using a repeated measures one-way ANOVA followed by Tukey post-hoc test and partial correlation analysis. RESULTS: TLP values decreased significantly after both innocuous stimuli (P < 0.05), and returned to their baseline levels shortly after the removal of the stimuli. There was no significant serial correlation between the autonomic nervous activity and TLP values (P > 0.05). CONCLUSION: TLP was able to monitor the pulpal circulatory changes evoked by innocuous stimuli. Systemic autonomic nervous control mechanisms were not associated with the pulpal circulatory changes, suggesting the involvement of other mechanisms, such as somatosensory-sympathetic nervous control.

Replica-based inspection of enamel wear microfeatures.

BACKGROUND: Surface replication is a nondestructive evaluation technique applied in examining surface wear by recording surface irregularities, especially in conditions when surfaces of interest cannot be further manipulated to fit directly under a microscope to be examined. Enamel is the outermost protective layer of the human teeth and is constantly stressed by mastication forces which results in enamel wear. OBJECTIVE: To date, a procedure combining the clinical and microscopic examination of enamel surfaces is absent, which hinders the early diagnosis and comprehension of the wear process. METHODS: This study investigated the role of replication sheets in registering microscopic wear on human enamel surfaces by both negative and positive replication techniques. RESULTS: The sheets replicated wear features successfully. Sheets were compatible to use with multiple microscopes, with proper preparation, including high resolution microscopes such as the scanning electron microscope and transmitting electron microscope.

An oriented hydroxyapatite film with arrayed plate-like particles enhance chondrogenic differentiation of ATDC5 cells.

Osteochondral defects of articular cartilage cannot regenerate spontaneously. For its surgical treatment, advancements in cartilage tissue engineering have particularly focused on subchondral bone lesions that tend to delay healing. Therefore, it is important to understand interactions between subchondral bone and chondrocytes. This study aimed to investigate the behavior of chondrogenic ATDC5 cells on oriented hydroxyapatite (HAp) films that mimic bone surfaces. HAp nanoparticles prepared herein were needle like and plate like. HAp films were formed through self-organization of the nanoparticles and had 2D structures regularly arrayed with the particles. Both films prominently comprised a-plane orientation surfaces but differed in the degree of hydrophilicity because of the patterns of particle self-assembly. ATDC5 cells cultured on the HAp film with plate-like particles could adhered in a shorter period but could not spread. The adhesive force of cells was weaker with the hydrophilic surface than with other surfaces, as determined using a trypsin-based cell detachment assay. In addition, ATDC5 cells displayed enhanced proliferation and chondrogenic differentiation. Our results suggest that the oriented HAp film formed using plate-like particles provided chondrogenic cells with a desired scaffold as that of subchondral bone to increase cell proliferation and differentiation.

Plate-like hydroxyapatite synthesized from dodecanedioic acid enhances chondrogenic cell proliferation.

BACKGROUND: The scaffold for head and neck reconstruction needs mechanical strength to maintain specific forms. Hydroxyapatite (HA) enhances the mechanical strength of hydrogel and is routinely used for cartilage regeneration. However, there is a demand for hydroxyapatite that controls chondrogenic cell behavior. OBJECTIVE: Our aim was to regulate HA morphology through a hydrothermal process using organic acid and enhance chondrocyte proliferation and differentiation using shaped-regulated HA. METHODS: HA was synthesized from dodecanedioic acid (DD:HA) and oleic acid (OA:HA) by a hydrothermal method and then coated onto glass plates. Surface properties of the samples were compared by various techniques. Surface roughness and contact angles were calculated. Proliferation and differentiation of chondrogenic cells were measured by MTT assays and Alcian Blue staining, respectively, after various incubation periods. RESULTS: The morphological structures of DD:HA and OA:HA were different; however, the crystallinity and chemical structures were similar. Surface roughness and hydrophilic behavior were higher on DD:HA. DD:HA enhanced chondrogenic cell proliferation over time. The differentiation of ATDC5 cells was also increased on the DD:HA surface compared with those in other groups. CONCLUSIONS: DD:HA enhanced cell viability to a greater extent than OA:HA did, indicating its excellent potential as an inorganic material compatible with chondrocyte regeneration.

Silencing of PD-L2/B7-DC by Topical Application of Small Interfering RNA Inhibits Elicitation of Contact Hypersensitivity.

PD-L2 is a ligand for the immune checkpoint receptor PD-1; however, its regulatory function is unclear. We previously reported that silencing of CD86 in cutaneous dendritic cells by topical application of small interfering RNA (siRNA) inhibits the elicitation of contact hypersensitivity (CHS). Here, we investigated the effects of topical application of PD-L2 siRNA on allergic skin disease. PD-L2 was induced in dendritic cells concurrently with the elevation of major histocompatibility complex class II and CD86 expression. Topical application of PD-L2 siRNA inhibited the elicitation of CHS by suppressing early proinflammatory cytokine expression and migration of hapten-carrying dendritic cells into lymph nodes. Local injection of neutralizing anti-PD-L2 mAb inhibited CHS to the same extent. PD-L2 siRNA treatment inhibited CHS in PD-1/PD-L1 double knockout mice and in the sensitized T-cell-transferred skin. These results suggest that the effects of PD-L2 silencing are independent of PD-1 but dependent on local memory T cells. Most of the inhibitory effects of PD-L2 and CD86 silencing on CHS were comparable, but PD-L2 siRNA treatment did not inhibit atopic disease-like manifestations and T helper type 2 responses in NC/Nga mice. Our results suggest that PD-L2 in cutaneous dendritic cells acts as a costimulator rather than a regulator. Local PD-L2 silencing by topical application of siRNA represents a therapeutic approach for contact allergy.

Eruption disturbance caused by an odontoma that was partially attached to the crown of the adjacent maxillary first molar, and step lesion excavation for the tooth preservation: A case report.

Despite the partial attachment of a complex odontoma to an impacted tooth, it is possible to guide the tooth to erupt normally and preserve it by step lesion excavation and the prevention of infection.

The application of cellulose acetate replication sheets in enamel wear investigations.

Surface replication is a nondestructive evaluation technique applied in examining surfaces' cracks/wear. This is quite valuable when the surface of interest cannot be further manipulated by cutting or polishing to fit directly under a microscope. Replication by acetate sheets is one of the techniques applied in industrial, metallographic and anthropological wear/examination. Enamel is the outermost protective layer of human teeth and is subjected to mechanical stresses due to the masticatory function; thus, wear is inevitable in human teeth. This relatively inert hard tissue has been reported to possess some properties of both metals and glasses due to its unique delicate microstructure. This study investigated the applicability of replication sheets in registering wear induced features on human enamel surfaces. The sheets replicated wear features successfully with compatibility to use with multiple microscopes. Acetate sheets have a potential in enamel wear replication.

Enamel Microcracks Induced by Simulated Occlusal Wear in Mature, Immature, and Deciduous Teeth.

Enamel wear, which is inevitable due to the process of mastication, is a process in which the microcracking of enamel occurs due to the surface contacting very small hard particles. When these particles slide on enamel, a combined process of microcutting and microcracking in the surface and subsurface of the enamel takes place. The aim of this study was to detect microscopic differences in the microcrack behavior by subjecting enamel specimens derived from different age groups (immature open-apex premolars, mature closed-apex premolars, and deciduous molars) to cycles of simulated impact and sliding wear testing under controlled conditions. Our findings indicated that the characteristics of the microcracks, including the length, depth, count, orientation, and relation to microstructures differed among the study groups. The differences between the surface and subsurface microcrack characteristics were most notable in the enamel of deciduous molars followed by immature premolars and mature premolars whereby deciduous enamel suffered numerous, extensive, and branched microcracks. Within the limitations of this study, it was concluded that enamel surface and subsurface microcracks characteristics are dependent on the posteruptive age with deciduous enamel being the least resistant to wear based on the microcrack behavior as compared to permanent enamel.

Management of developmental enamel defects in the primary dentition.

This study attempted to identify appropriate materials for restoration of enamel defects in the primary dentition, which were classified by severity and region with the modified developmental defects of enamel index. To identify the most appropriate materials, we used restorative materials to protect teeth and evaluated clinical outcomes of restoration. Three materials were used for restoration or repair after dislodgement of restorations. Our findings in this case suggest that, because of its durability and esthetic advantages, adhesive resin is beneficial for patients with enamel defects, particularly for restorations of less than two-thirds of the extent of the defect.

Human pulpal blood flow in different root formation stages measured with transmitted-light plethysmography.

OBJECTIVES: To determine the pulp vitality after traumatic injury, dentists often use pulp sensitivity tests, which can be ambiguous in young permanent teeth with incomplete root formation. Transmitted-light plethysmography (TLP) is a non-invasive objective method that uses a 525-nm LED to detect blood volume change in the pulp. The present study aimed (1) to investigate pulpal blood flow with TLP and optical characteristics in healthy permanent maxillary incisors in different root formation stages, and (2) to assess the influences of body growth of the children and tooth color on the TLP amplitude. DESIGN: Seventy-eight fully erupted maxillary central incisors were divided into four groups, according to the root formation stages. Group 1: root with wide-open apex, Group 2: root completed in length with open apex, Group 3: root with half-closed apex, Group 4: root with complete formation. The TLP amplitude, optical density, electric pulp testing, and cervical tooth color measurements of each group were compared using a one-way analysis of variance followed by the Bonferroni method. The correlation between the weights/heights of children and TLP amplitudes was analyzed using Pearson coefficient. RESULTS: The TLP amplitude was significantly higher in Group 3 than in the other groups. The amplitude was correlated with the weights/heights of children, but not with the tooth color. Optical density and electric sensitivity increased with tooth maturation. CONCLUSION: The amplitude of TLP and optical density may be affected by growth and development in children and indicate changes in the vascular dynamics of the pulp and hard tissue maturation during root formation stages.

Synthesis and enhanced bone regeneration of carbonate substituted octacalcium phosphate.

Using a wet method, we have synthesized octacalcium phosphate carbonate, in which HPO42- in octacalcium phosphate is replaced with CO32-. The physical, crystal, and chemical properties of this new material were compared to octacalcium phosphate, Ca-deficient hydroxyapatite, and Ca-deficient carbonate apatite using X-ray diffraction, Fourier-transform infrared spectroscopy, inductively coupled plasma spectroscopy, and scanning electron microscopy. Surface roughness and morphology were also characterized, along with the ability to support proliferation and differentiation of MG63 cells, as measured by MTT and alkaline phosphatase assay. We found that octacalcium phosphate carbonate enhanced osteoblast proliferation more strongly than all other materials tested. Similarly, Ca-deficient carbonate apatite, a hydrolysate of octacalcium phosphate carbonate, stimulated osteoblast differentiation to a better extent than Ca-deficient hydroxyapatite, a carbonate-free hydrolysate of octacalcium phosphate. These results indicate that octacalcium phosphate carbonate has good biocompatibility and osteoconduction, and incorporation of carbonate into octacalcium phosphate and apatite enhances bone regeneration.

Wear characteristics of trimethylolpropane trimethacrylate filler-containing resins for the full crown restoration of primary molars.

Although the demand for aesthetic restoration of primary molars has increased, the full-crown restorations using resin and the details of the wear characteristics of trimethylolpropane trimethacrylate (TMPT) filler containing resins for primary molars are not well understood. This study was conducted to determine whether new light-cured composite resin (Fantasista) and 4-META/MMATBB resin (Bondfill SB) are appropriate for full crown restoration of primary molars by evaluating their wear characteristics. Both resins products contain TMPT filler. The properties of the resins were evaluated through in vitro impacting-sliding wear tests; the wear properties of the opposing enamel specimens used in the tests were also studied. The properties of the resins were compared with those of Litefill, MetafilC, and Clearfil FII, which had been evaluated previously. Fantasista exhibited simple shape of wear that was suggestive of a higher wear resistance than that of Litefill. Fantasista caused the least damage to the antagonistic primary enamel.

Assessment of occlusal fissure depth and sealant penetration using optical coherence tomography.

Swept source optical coherence tomography (SS-OCT) is an interferometric imaging technique. This study aimed to assess SS-OCT as a diagnostic tool for the evaluation of occlusal fissure depth and sealant penetration into fissure. Seventy investigation sites of occlusal fissures without visible evidence of caries were categorized into four groups (smooth, shallow, intermediate, and deep fissures). Categorization utilized two methods: 1) visually, using a computer screen, and 2) using SS-OCT images. After sealant placement, penetration was observed in SS-OCT. The results obtained from SS-OCT and visual inspections were compared with those of confocal laser scanning microscope (CLSM). The diagnostic power of SS-OCT was higher than that of visual inspection for fissure depth. Additionally, clear cross-sectional images of sealant penetration into fissures were observed with SS-OCT. SS-OCT can be used to evaluate fissure depth and monitor sealant penetration.

Detection of occlusal caries in primary teeth using swept source optical coherence tomography.

This study aimed to investigate swept source optical coherence tomography (SS-OCT) as a detecting tool for occlusal caries in primary teeth. At the in vitro part of the study, 38 investigation sites of occlusal fissures (noncavitated and cavitated) were selected from 26 extracted primary teeth and inspected visually using conventional dental equipment by six examiners without any magnification. SS-OCT cross-sectional images at 1330-nm center wavelength were acquired on the same locations. The teeth were then sectioned at the investigation site and directly viewed under a confocal laser scanning microscope (CLSM) by two experienced examiners. The presence and extent of caries were scored in each observation. The results obtained from SS-OCT and conventional visual inspections were compared with those of CLSM. Consequently, SS-OCT could successfully detect both cavitated and noncavitated lesions. The magnitude of sensitivity for SS-OCT was higher than those for visual inspection (sensitivity of visual inspection and SS-OCT, 0.70 versus 0.93 for enamel demineralization, 0.49 versus 0.89 for enamel cavitated caries, and 0.36 versus 0.75 for dentin caries). Additionally, occlusal caries of a few clinical cases were observed using SS-OCT in vivo. The results indicate that SS-OCT has a great detecting potential for occlusal caries in primary teeth.

Wear of resin composites and primary enamel and their applicability to full crown restoration of primary molars.

PURPOSE: To determine whether resin composites are appropriate for full crown restoration of primary molars by evaluating their wear characteristics. Specifically, the wear properties of resin composite specimens and the opposing enamel surfaces were characterized by means of impacting-sliding wear testing. METHODS: Three types of light-cured resin composites (Estelite Sigma quick, Litefill IIP, and Metafil C), one type of chemical-cured resin composite (Clearfil FII), and a hybrid composite (Estenia C&B) were tested in this study. The enamel sample was used as the control. The hemispherically prepared specimens were subjected to impacting-sliding wear testing against the flattened enamel of primary molars. The worn surfaces were examined by laser scanning microscopy and scanning electron microscopy. The volumetric loss was estimated by using micro-CT images. The areas of worn enamel surfaces were measured by 3D color laser microscopy. On most of the worn enamel surfaces, cracks appeared. Scatter plot analyses between their width and depth were carried out. Data for each specimen were statistically analyzed by multiple comparisons among the means of treatment by Bonferroni's method (P< 0.01). RESULTS: Clearfil showed significantly higher surface area wear, volumetric loss, and worn enamel surface area than did the other resin composites and the control enamel (P< 0.01). There was no significant difference among the worn surface areas ofEstelite, Litefill, Metafil, and Estenia (P< 0.01). The control enamel showed significantly lower worn surface area than did the resin composites (P< 0.01). There was no significant difference in volumetric loss and worn enamel surface areas among Estelite, Litefill, Metafil, Estenia, and the control enamel (P< 0.01). Cracks larger than that on the control enamel were seen on the worn enamel surface opposing Estenia.