Extracellular adenosine 5'-diphosphate promotes MCP-1/CCL2 expression via the P2Y13 purinergic receptor/ERK signaling axis in temporomandibular joint-derived mouse fibroblast-like synoviocytes.

BACKGROUND: Temporomandibular joint osteoarthritis (TMJ-OA) causes cartilage degeneration, bone cavitation, and fibrosis of the TMJ. However, the mechanisms underlying the fibroblast-like synoviocyte (FLS)-mediated inflammatory activity in TMJ-OA remain unclear. METHODS AND RESULTS: Reverse transcription-quantitative polymerase chain reaction analysis revealed that the P2Y1, P2Y12, and P2Y13 purinergic receptor agonist adenosine 5'-diphosphate (ADP) significantly induces monocyte chemotactic protein 1 (MCP-1)/ C-C motif chemokine ligand 2 (CCL2) expression in the FLS1 synovial cell line. In contrast, the uracil nucleotide UTP, which is a P2Y2 and P2Y4 agonist, has no significant effect on MCP-1/CCL2 production in FLS1 cells. In addition, the P2Y13 antagonist MRS 2211 considerably decreases the expression of ADP-induced MCP-1/CCL2, whereas ADP stimulation enhances extracellular signal-regulated kinase (ERK) phosphorylation. Moreover, it was found that the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 reduces ADP-induced MCP-1/CCL2 expression. CONCLUSION: ADP enhances MCP-1/CCL2 expression in TMJ FLSs via P2Y13 receptors in an MEK/ERK-dependent manner, thus resulting in inflammatory cell infiltration in the TMJ. Collectively, the findings of this study contribute to a partial clarification of the signaling pathway underlying the development of inflammation in TMJ-OA and can help identify potential therapeutic targets for suppressing ADP-mediated purinergic signaling in this disease.

Goltz Syndrome Combined with Triple X Syndrome, a Case Report.

Goltz syndrome is a rare X-linked dominant multisystem disorder that presents with ectoderm and mesoderm-derived symptoms. Skin manifestations including congenital patchy skin aplasia, congenital nodular fat herniation, congenital hypo- or hyperpigmentation along Blaschko's lines, telangiectasia, and congenital ridged dysplastic nails are typical in this disorder. Almost all cases of Goltz syndrome correspond to female newborns and that hemizygosis makes the syndrome fetal in males. Triple X syndrome is a relatively common congenital disorder that presents with mild to no symptoms in the developmental and psychiatric realm. The patient reported here was born with multisystem anomaly affecting the eyes, craniofacial region, cardiovascular system, skin, and limbs. A G-banding chromosomal study revealed 47, XXX. She was diagnosed with Goltz syndrome owing to her distinctive skin manifestations. The congenital cervical skin defect healed with conservative treatment. The facial cleft, cleft lip-palate, and syndactyly were successfully treated with multiple surgical treatments. The combination of triple X syndrome and Goltz syndrome is very rare. We describe the expression of presenting with both syndromes simultaneously.

Connective tissue growth factor enhances TGF-ß1-induced osteogenic differentiation via activation of p38 MAPK in mesenchymal stem cells.

OBJECTIVES: Cellular differentiation is based on the effects of various growth factors. Transforming growth factor (TGF)-ß1 plays a pivotal role in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the influence of connective tissue growth factor (CTGF), known to function synergistically with TGF-ß1, on osteogenic differentiation in MSCs. METHODS: UE7T-13 cells were treated with TGF-ß1 and/or CTGF. Subsequently, protein levels of intracellular signaling pathway molecules were determined through western blot analysis. The mRNA expression levels of osteogenic differentiation markers were investigated using reverse transcription-quantitative polymerase chain reaction. Bone matrix mineralization was evaluated through alizarin red staining. RESULTS: Co-treatment with TGF-ß1 and CTGF resulted in the suppression of TGF-ß1-induced phosphorylation of extracellular signal-regulated kinase 1/2, an intracellular signaling pathway molecule in MSCs, while significantly enhancing the phosphorylation of p38 mitogen-activated protein kinase (MAPK). In MSCs, co-treatment with CTGF and TGF-ß1 led to increased expression levels of alkaline phosphatase and type I collagen, markers of osteogenic differentiation induced by TGF-ß1. Osteopontin expression was observed only after TGF-ß1 and CTGF co-treatment. Notably, bone sialoprotein and osteocalcin were significantly upregulated by treatment with CTGF alone. Furthermore, CTGF enhanced the TGF-ß1-induced mineralization in MSCs, with complete suppression observed after treatment with a p38 MAPK inhibitor. CONCLUSIONS: CTGF enhances TGF-ß1-induced osteogenic differentiation and subsequent mineralization in MSCs by predominantly activating the p38 MAPK-dependent pathway.

Tensile and shear bond strengths of a stainless steel used in orthodontic brackets bonded to bovine enamel using two types of resin cement.

The purpose of this study was to examine the relationship between the bond strength of stainless steel with two types of resin cements (MMA- and composite-based) on bovine enamel depending on the directionality of the applied force. The specimens were either placed in water or subjected to thermal cycles (TC), and the shear or tensile bond strengths (SBS or TBS) were determined. The SBS showed significantly greater than the TBS for both types of cement, and the SBS and TBS for composite-based cement had larger than MMA-based one. No significant difference in SBS was observed in the cements even after being subjected to TC. Cohesive failures of the cement and bovine enamel in the composite-based group, while adhesive failures were observed in MMA-based one. Consequently, the direction of the force at both cements affected the retention of stainless steel, and MMA-based cement was preferred when prioritizing less enamel damages.

Classification of Maxillofacial Morphology by Artificial Intelligence Using Cephalometric Analysis Measurements.

The characteristics of maxillofacial morphology play a major role in orthodontic diagnosis and treatment planning. While Sassouni's classification scheme outlines different categories of maxillofacial morphology, there is no standardized approach to assigning these classifications to patients. This study aimed to create an artificial intelligence (AI) model that uses cephalometric analysis measurements to accurately classify maxillofacial morphology, allowing for the standardization of maxillofacial morphology classification. This study used the initial cephalograms of 220 patients aged 18 years or older. Three orthodontists classified the maxillofacial morphologies of 220 patients using eight measurements as the accurate classification. Using these eight cephalometric measurement points and the subject's gender as input features, a random forest classifier from the Python sci-kit learning package was trained and tested with a k-fold split of five to determine orthodontic classification; distinct models were created for horizontal-only, vertical-only, and combined maxillofacial morphology classification. The accuracy of the combined facial classification was 0.823 ± 0.060; for anteroposterior-only classification, the accuracy was 0.986 ± 0.011; and for the vertical-only classification, the accuracy was 0.850 ± 0.037. ANB angle had the greatest feature importance at 0.3519. The AI model created in this study accurately classified maxillofacial morphology, but it can be further improved with more learning data input.

Hydrogen peroxide-induced oxidative stress promotes expression of CXCL15/Lungkine mRNA in a MEK/ERK-dependent manner in fibroblast-like synoviocytes derived from mouse temporomandibular joint.

OBJECTIVES: Temporomandibular joint osteoarthritis (TMJ-OA) is a multifactorial disease caused by inflammation and oxidative stress. It has been hypothesized that mechanical stress-induced injury of TMJ tissues induces the generation of reactive oxygen species (ROS), such as hydroxyl radical (OH∙), in the synovial fluid (SF). In general, the overproduction of ROS contributes to synovial inflammation and dysfunction of the subchondral bone in OA. However, the mechanism by which ROS-injured synoviocytes recruit inflammatory cells to TMJ-OA lesions remains unclear. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the mRNA expression of chemoattractant molecules. The phosphorylation levels of intracellular signaling molecules were evaluated using western blot analysis. RESULTS: Hydrogen peroxide (H2O2) treatment significantly promoted mRNA expression of neutrophil chemoattractant CXCL15/Lungkine in a dose-dependent manner (100-500 µM) in fibroblast-like synoviocytes (FLSs) derived from mouse TMJ. H2O2 (500 µM) significantly upregulated the phosphorylation of extracellular signal-regulated kinase (ERK)1 and ERK2 in FLSs. Intriguingly, the mitogen-activated protein (MAP)/ERK kinase (MEK) inhibitor U0126 (10 µM) nullified H2O2-induced increase in CXCL15/Lungkine mRNA expression. Additionally, H2O2 (500 µM) administration significantly upregulated OH∙ production in FLSs, as assessed by live-cell permeant fluorescent probe targeted against OH∙ under fluorescence microscopy. Furthermore, the ROS inhibitor N-acetyl-l-cysteine (5 mM) partially but significantly reversed H2O2-mediated phosphorylation of ERK1/2. CONCLUSIONS: H2O2-induced oxidative stress promoted the expression of CXCL15/Lungkine mRNA in a MEK/ERK-dependent manner in mouse TMJ-derived FLSs, suggesting that FLSs recruit neutrophils to TMJ-OA lesions through the production of CXCL15/Lungkine and exacerbate the local inflammatory response.

Accuracy of a Method to Monitor Root Position Using a 3D Digital Crown/Root Model during Orthodontic Treatments.

This study aimed to assess the accuracy of a method of predicting post-movement root position during orthodontic treatment using a 3D digital crown/root model (3DCRM) created with pre-movement records of both cone-beam computed tomography (CBCT) and dental arch digital scans. Pre- and post-movement CBCT scans and dental arch digital scans of five patients who had completed orthodontic treatments were used in this study. The 3DCRM was superimposed onto the post-movement scanned dental arch to identify the post-movement root position (test method). Post-movement CBCT (referenced as the current method) served as the control to identify the actual post-movement root position. 3D-coordinate analysis revealed no significant differences between the test and current methods along the X and Y axes. However, the discrepancy on the Z axis (especially in cases of intrusion) was greater than that in all other directions for all three tooth types examined (p < 0.05). A strong positive correlation between the degree of discrepancy and the distance of tooth movement was observed on the Z axis (r = 0.71). The 3DCRM method showed promising potential to accurately predict root position during orthodontic treatments without the need for a second CBCT. However, root resorption, which affected the Z axis prediction, needs to be closely monitored using periapical radiographs to complement this method.

Receptor tyrosine kinase ligands and inflammatory cytokines cooperatively suppress the fibrogenic activity in temporomandibular-joint-derived fibroblast-like synoviocytes via mitogen-activated protein kinase kinase/extracellular signal-regulated kinase.

Osteoarthritis (OA)-related fibrosis is a possible cause of temporomandibular joint (TMJ) stiffness. However, the molecular mechanisms underlying the fibrogenic activity in fibroblast-like synoviocytes (FLSs) remain to be clarified. The present study examined the effects of receptor tyrosine kinase (RTK) ligands, such as fibroblast growth factor (FGF)-1 and epidermal growth factor (EGF), on myofibroblastic differentiation of the FLS cell line FLS1, which is derived from the mouse TMJ. The present study revealed that both FGF-1 and EGF dose-dependently suppressed the expression of the myofibroblast (MF) markers, including α-smooth muscle actin (α-SMA) and type I collagen, in FLS1 cells. Additionally, both FGF-1 and EGF activated extracellular signal-regulated kinase (ERK) in FLS1 cells. In addition, the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126 abrogated the FGF-1- and EGF-mediated suppression of MF marker expression. On the other hand, inflammatory cytokines, such as interleukin-1ß and tumor necrosis factor-α, also suppressed the expression of MF markers in FLS1 cells. Importantly, U0126 abrogated the inflammatory cytokine-mediated suppression of MF marker expression. Interestingly, RTK ligands and inflammatory cytokines additively suppressed the expression of type I collagen. These results suggested that RTK ligands and inflammatory cytokines cooperatively inhibited the fibrogenic activity in FLSs derived from the TMJ in a MEK/ERK-dependent manner. The present findings partially clarify the molecular mechanisms underlying the development of OA-related fibrosis in the TMJ and may aid in identifying therapeutic targets for this condition. Additionally, FGF-1 and EGF could be therapeutically utilized to prevent OA-related fibrosis around the inflammatory TMJ.

3D digital analysis of magnetic force-driven orthodontic tooth movement.

With the introduction of rare earth magnets like neodymium-iron-boron (NdFeB), it has become possible to produce small magnets with high forces, necessary for its usage in the field of dentistry, such as for orthodontic tooth movement. The ultimate goal of this project is to establish magnetic force-driven orthodontic treatment as a future treatment modality for comprehensive orthodontic treatment. In order to utilize magnets for orthodontic treatment, we must first understand the characteristics of tooth movement created by magnetic forces. In this study, we aimed to digitally assess the efficacy of magnetic attraction and repulsion forces by means of a 3D digital analysis of movement (distance, direction, angulation and duration) and rotation (yaw, pitch and roll) of the crown and root of teeth in an ex vivo typodont model. We performed space closure and space gain treatment of maxillary central incisors (n = 30) and analyzed the movement and rotation of the teeth and root apex with 3D digital analysis. The results of the typodont model indicated significant differences on amount, speed and rotation of tooth and root movement created by magnetic attraction and repulsion forces. We also mimicked a moderate crowding typodont case and successfully treated it with a combination of attraction and repulsion magnetic forces. The moderate crowding case utilized magnets and a titanium archwire to guide the planned tooth movements and prevent undesired or unexpected movement. Further ex vivo experiments and considerations for biosafety will be necessary to investigate magnet force-driven orthodontics as a future modality of orthodontic treatment.

Cephalometric Measurements Of Non-Syndromic Oligodontia In Early Dental Age In A Japanese Population.

PURPOSE: Oligodontia significantly affects oral function and esthetics. Recognition of skeletal and dental patterns may aid in proper diagnosis and development of appropriate interventions. The aim of this study was to analyze skeletal and dental patterns for pre-adolescent patients with a diagnosis of oligodontia. PATIENTS AND METHODS: This study included 19 oligodontia patients (age: 9.5±1.3, Hellman's developmental stage IIIA~IIIB) along with a control group that comprised of 19 participants (age: 9.9±1.6) without any skeletal disharmony or congenitally missing teeth, with an Angle class I relationship and general crowding. Average cephalometric measurements among the oligodontia group were compared to the control group. The correlation between number of congenitally missing teeth (CMT) and each measurement was investigated. Skeletal measurements for both male and female patients in the oligodontia group and the control group were also compared. RESULTS: No significant difference between the experimental and the control group was observed with respect to skeletal angular and linear measurements, except the gonial angle. Differences in dental pattern measurements were observed. The oligodontia group had significantly smaller Mo-Ms and Is-Mo than the control group (p<0.01). No correlation was detected between severity of oligodontia (number of CMT) and skeletal measurement except for SNB (R=-0.4). For females with oligodontia, Mo-Ms (eruption of maxillary first molar) and Is-Mo (mesial location of maxillary first molar) significantly differed from females in the control group (p<0.01). In contrast, no differences in Mo-Ms or Is-Mo were detected for male patients when oligodontia and control group were compared. CONCLUSION: Among pre-adolescent Japanese patients with oligodontia in Hellman's developmental age IIIA~IIIB, no significant differences in skeletal characteristics were established when compared to the control group. However, tooth position of maxillary first molars indicated smaller vertical descent and mesial shift, which may suggest weak maxillary vertical development.

Characterization of PAX9 variant P20L identified in a Japanese family with tooth agenesis.

Transcription factors PAX9 and MSX1 play crucial roles in the development of permanent teeth at the bud stage, and their loss-of-function variants have been associated with congenital tooth agenesis. We sequenced the coding regions of the PAX9 and MSX1 genes from nine patients with non-syndromic tooth agenesis, and identified a missense mutation, P20L, of PAX9 in a single familial case involving three patients in two generations. Identical mutation was previously reported by other authors, but has not been characterized in detail. The mutation was located in a highly conserved N-terminal subdomain of the paired domain and co-segregated as a heterozygote with tooth agenesis. The patients showed defects primarily in the first and second molars, which is typical for cases attributable to PAX9 mutation. Luciferase reporter assay using the 2.3-kb promoter region of BMP4 and electrophoretic mobility shift assay using the CD19-2(A-ins) sequence revealed that P20L substitution eliminated most of the transactivation activity and specific DNA binding activity of PAX9 under the experimental conditions we employed, while some residual activity of the mutant was evident in the former assay. The hypomorphic nature of the variant may explain the relatively mild phenotype in this case, as compared with other PAX9 pathogenic variants such as R26W.

SCRG1 suppresses LPS-induced CCL22 production through ERK1/2 activation in mouse macrophage Raw264.7 cells.

Recently, we identified the scrapie responsive gene 1 (SCRG1) secreted from mesenchymal stem cells (MSCs) and its receptor bone marrow stromal cell antigen 1 (BST1) as positive regulators of stem cell qualities such as self­renewal, migration abilities, and osteogenic differentiation potential. Here, we examined the effect of the paracrine activity of SCRG1 in macrophages. The mouse macrophage­like cell line Raw264.7 expressed BST1/ß1 or BST1/ß2 integrin as possible SCRG1 receptors. Unexpectedly, recombinant SCRG1 did not enhance cell proliferation, migration, or adhesion in these macrophages. However, further examination of the effect of SCRG1 in Raw264.7 cells did reveal a potent anti­inflammatory effect whereby SCRG1 suppressed LPS­induced CCL22 production. SCRG1 also induced the phosphorylation of extracellular signal­regulated kinase 1/2 (ERK1/2) in these cells and, moreover, a mitogen­activated protein kinase (MAPK)/ERK kinase inhibitor U0126 significantly suppressed the effect of SCRG1 on LPS­induced chemokine CCL22 production. Taken together, these data indicate that SCRG1 signals through the MAPK pathway and suppresses the LPS signaling pathway. CCL22 is generally known to be chemotactic for monocytes, dendritic cells, natural killer cells and chronically activated T lymphocytes, suggesting that MSC­derived SCRG1 may block infiltration of these cells. A mechanism is proposed by which MSCs play their immunosuppressive role through suppressing chemokine expression in monocyte/macrophage lineage cells.

ROCK/actin/MRTF signaling promotes the fibrogenic phenotype of fibroblast-like synoviocytes derived from the temporomandibular joint.

Malocclusion caused by abnormal jaw development or muscle overuse during mastication results in abnormal mechanical stress to the tissues surrounding the temporomandibular joint (TMJ). Excessive mechanical stress against soft and hard tissues around the TMJ is involved in the pathogenesis of inflammatory diseases, including osteoarthritis (OA). OA-related fibrosis is a possible cause of joint stiffness in OA. However, cellular and molecular mechanisms underlying fibrosis around the TMJ remain to be clarified. Here, we established a cell line of fibroblast­like synoviocytes (FLSs) derived from the mouse TMJ. Then, we examined whether the Rho­associated coiled­coil forming kinase (ROCK)/actin/myocardin-related transcription factor (MRTF) gene regulatory axis positively regulates the myofibroblast (MF) differentiation status of FLSs. We found that i) FLSs extensively expressed the MF markers α­smooth muscle actin (α­SMA) and type I collagen; and ii) an inhibitor against the actin­polymerizing agent ROCK, Y­27632; iii) an actin-depolymerizing agent cytochalasin B; iv) an inhibitor of the MRTF/serum response factor­regulated transcription, CCG­100602, clearly suppressed the mRNA levels of α­SMA and type I collagen in FLSs; and v) an MF differentiation attenuator fibroblast growth factor­1 suppressed filamentous actin formation and clearly suppressed the mRNA levels of α-SMA and type I collagen in FLSs. These results strongly suggest that the ROCK/actin/MRTF axis promotes the fibrogenic activity of synoviocytes around the TMJ. Our findings partially clarify the molecular mechanisms underlying the emergence of TMJ­OA and may aid in identifying drug targets for treating this condition at the molecular level.

Establishment of immortalized mesenchymal stem cells derived from the submandibular glands of tdTomato transgenic mice.

Transgenic mice that overexpress the red fluorescent protein tdTomato (tdTomato mice) are well suited for use in regenerative medicine studies. Cultured cells from this murine model exhibit strong red fluorescence, enabling real-time in vivo imaging through the body surface of grafted animals. Mesenchymal stem cells (MSCs) have marked potential for use in cell therapy and regenerative medicine; however, the mechanisms that regulate their dynamics in vivo are poorly understood. In the present study, an MSC line was derived from the submandibular gland fibroblasts of tdTomato mice. The fluorescent signal from this cell line was observed in organs throughout the body, as well as in salivary glands. Primary culture cells derived from the submandibular gland were immortalized with SV40 large T antigen (GManSV cells); these cells exhibited increased migratory ability, as compared with those isolated from the sublingual gland. GManSV cells were tdTomato-positive and exhibited spindle-shaped fibroblastic morphology; they also robustly expressed mouse MSC markers: Stem cell antigen-1 (Sca-1), CD44, and CD90. This cell line retained multipotent stem cell characteristics, as evidenced by its ability to differentiate into both osteogenic and adipogenic lineages. These results indicate that Sca-1+/CD44+/CD90+-GManSV cells may be useful for kinetic studies of submandibular gland-derived MSCs in the context of in vitro co-culture with other types of salivary gland-derived cells. These cells may also be used for in vivo imaging studies, in order to identify novel cell therapy and regenerative medicine for the treatment of salivary gland diseases.