Blockade of vasoactive intestinal peptide receptor 2 (VIPR2) signaling suppresses cyclin D1-dependent cell-cycle progression in MCF-7 cells.

Vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) is a G protein-coupled receptor that binds to Gαs, Gαi, and Gαq proteins to regulate various downstream signaling molecules, such as protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), and phospholipase C. In this study, we examined the role of VIPR2 in cell cycle progression. KS-133, a newly developed VIPR2-selective antagonist peptide, attenuated VIP-induced cell proliferation in MCF-7 cells. The percentage of cells in the S-M phase was decreased in MCF-7 cells treated with KS-133. KS-133 in the presence of VIP decreased the phosphorylation of extracellular signal-regulated kinase (ERK), AKT, and glycogen synthase kinase-3ß (GSK3ß), resulting in a decrease in cyclin D1 levels. In MCF-7 cells stably-expressing VIPR2, KS-133 decreased PI3K activity and cAMP levels. Treatment with the ERK-specific kinase (MEK) inhibitor U0126 and the class I PI3K inhibitor ZSTK474 decreased the percentage of cells in the S phase. KS-133 reduced the percentage of cells in the S phase more than treatment with U0126 or ZSTK474 alone and did not affect the effect of the mixture of these inhibitors. Our findings suggest that VIPR2 signaling regulates cyclin D1 levels through the cAMP/PKA/ERK and PI3K/AKT/GSK3ß pathways, and mediates the G1/S transition to control cell proliferation.

High-frequency low-intensity semiconductor laser irradiation enhances osteogenic differentiation of human cementoblast lineage cells.

PURPOSE: Laser irradiation activates a range of cellular processes in the periodontal components and promotes tissue repair. However, its effect on osteogenic differentiation of human cementoblast lineage cells remains unclear. This study aimed to examine the effects of high-frequency semiconductor laser irradiation on the osteogenic differentiation of human cementoblast lineage (HCEM) cells. METHODS: HCEM cells were cultured to reach 80% confluence and irradiated with a gallium-aluminum-arsenide (Ga-Al-As) semiconductor laser with a pulse width of 200 ns and wavelength of 910 at a dose of 0-2.0 J/cm2. The outcomes were assessed by analyzing the mRNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and type I collagen (COLL1) using real-time polymerase chain reaction (PCR) analysis 24 h after laser irradiation. Cell mineralization was evaluated using ALP activity, calcium deposition, and Alizarin Red staining. RESULTS: The laser-irradiated HCEM cells showed significantly enhanced gene expression levels of ALP, RUNX2, and COLL1 as well as ALP activity and calcium concentration in the culture medium compared with the non-irradiated cells. In addition, enhanced calcification deposits were confirmed in the laser-irradiated group compared with the non-irradiated group at 21 and 28 days after the induction of osteogenic differentiation. CONCLUSION: High-frequency semiconductor laser irradiation enhances the osteogenic differentiation potential of cultured HCEM cells, underscoring its potential utility for periodontal tissue regeneration.

Longitudinal effects of estrogen on mandibular growth and changes in cartilage during the growth period in rats.

Estrogen is a steroid hormone that induces skeletal growth and affects endochondral ossification of the long tubular bone growth plate during the growth period. However, the effects of estrogen on endochondral ossification of the mandibular condylar cartilage are unclear. In this study, ovariectomized Wistar/ST rats were used to investigate the longitudinal effects of estrogen on mandibular growth. The rats were administered different doses of estrogen. Longitudinal micro-computed tomographic scanning, histological staining and ELISA on plasma growth hormone were performed to examine the effects of estrogen on mandibular growth. The results showed that mandibular growth was suppressed throughout the growth period by estrogen in a dose-dependent manner. In addition, long-term administration of a high dose of estrogen to the rats resulted in significant increase in growth hormone throughout the growth period, significant circularization of cell nuclei in the proliferative layer, intensely staining cartilage matrix in the subchondral bone, and significant suppression of estrogen receptor (ER) alpha and beta expression in the mandibular cartilage. However, regardless of estrogen concentration, in the posterior part of the mandibular cartilage, ER expression extended to both the hypertrophic and proliferative layers. These results indicate that estrogen suppresses mandibular growth throughout the growth period. Additionally, it influences endochondral ossification via its effect on ERs.

Effect of CD146+ SHED on bone regeneration in a mouse calvaria defect model.

OBJECTIVE: Stem cells from human exfoliated deciduous teeth (SHED) have bone regeneration ability and potential therapeutic applications. CD146, a cell adhesion protein expressed by vascular endothelial cells, is involved in osteoblastic differentiation of stem cells. The effect of CD146 on SHED-mediated bone regeneration in vivo remains unknown. We aimed to establish efficient conditions for SHED transplantation. MATERIALS AND METHODS: SHED were isolated from the pulp of an extracted deciduous tooth and cultured; CD146-positive (CD146+ ) and CD146-negative (CD146- ) populations were sorted. Heterogeneous populations of SHED and CD146+ and CD146- cells were transplanted into bone defects generated in the skulls of immunodeficient mice. Micro-computed tomography was performed immediately and 4 and 8 weeks later. Histological and immunohistochemical assessments were performed 8 weeks later. RESULTS: Bone regeneration was observed upon transplantation with CD146+ and heterogeneous populations of SHED, with significantly higher bone regeneration observed with CD146+ cells. Bone regeneration was higher in the CD146- group than in the control group, but significantly lower than that in the other transplant groups at 4 and 8 weeks. Histological and immunohistochemical assessments revealed that CD146+ cells promoted bone regeneration and angiogenesis. CONCLUSION: Transplantation of CD146+ SHED into bone defects may be useful for bone regeneration.

High-frequency near-infrared semiconductor laser irradiation suppressed experimental tooth movement-induced inflammatory pain markers in the periodontal ligament tissues of rats.

High-frequency near-infrared (NIR) semiconductor laser-irradiation has an unclear effect on nociception in the compressed lateral periodontal ligament region, a peripheral nerve region. This study aimed to investigate the effects of NIR semiconductor laser irradiation, with a power of 120 J, on inflammatory pain markers and neuropeptides induced in the compressed lateral periodontal ligament area during ETM. A NIR semiconductor laser [910 nm wavelength, 45 W maximum output power, 300 mW average output power, 30 kHz frequency, and 200 ns pulse width (Lumix 2; Fisioline, Verduno, Italy)] was used. A nickel-titanium closed coil that generated a 50-g force was applied to the maxillary left-side first molars and incisors in 7-week-old Sprague-Dawley (280-300 g) rats to induce experimental tooth movement (ETM) for 24 h. Ten rats were divided into two groups (ETM + laser, n = 5; ETM, n = 5). The right side of the ETM group (i.e., the side without induced ETM) was evaluated as the untreated group. We performed immunofluorescent histochemistry analysis to quantify the interleukin (IL)-1ß, cyclooxygenase-2 (COX2), prostaglandin E2 (PGE2), and neuropeptide [calcitonin gene-related peptide (CGRP)] expression in the compressed region of the periodontal tissue. Post-hoc Tukey-Kramer tests were used to compare the groups. Compared with the ETM group, the ETM + laser group showed significant suppression in IL-1ß (176.2 ± 12.3 vs. 310.8 ± 29.5; P < 0.01), PGE2 (104.4 ± 14.34 vs. 329.6 ± 36.52; P < 0.01), and CGRP (36.8 ± 4.88 vs. 78.0 ± 7.13; P < 0.01) expression. High-frequency NIR semiconductor laser irradiation exerts significant effects on ETM-induced inflammation. High-frequency NIR semiconductor laser irradiation can reduce periodontal inflammation during orthodontic tooth movement.

The Physiological Function of Deglutition and Swallowing in Patients With Maxillary Protrusion After Mandibular Advancement.

The objective of this study was to determine the tongue-palatal contact changes in patients with skeletal maxillary protrusion after sagittal split ramus osteotomy (SSRO) during swallowing. In this study, 15 patients with maxillary protrusion and 10 normal subjects participated. Before and 3 months after surgery, tongue-palatal contact patterns during swallowing of patients with maxillary protrusion as well as controls were evaluated by electropalatography. The electrode contact number in the alveolar, palatal, and velar parts was examined. The swallowing duration of each phase was also evaluated. In the lateral area of the velar part, incomplete electrode contact was shown at 0.3 seconds in patients with maxillary protrusion. The electrode contact number in the velar part at 0.3 seconds before tongue-palatal complete contact was significantly less in the preoperative patients compared with the controls ( P < 0.05). A small increase in the electrode contact number of the velar part was shown in the postoperative patients at 0.3 and 0.2 seconds before tongue-palatal complete contact ( P < 0.05). The pharyngeal phase duration was significantly larger in the patients with maxillary protrusion before SSRO compared with the controls ( P < 0.05). After SSRO, the pharyngeal phase duration was significantly shortened. It was shown that the tongue-palatal contact pattern during swallowing in patients with maxillary protrusion improved after orthognathic surgery, and the pharyngeal phase duration was also shortened. It is suggested that the changes in the mesiodistal mandibular position by orthognathic surgery can improve tongue posture and movement during swallowing.

Bone Differentiation Ability of CD146-Positive Stem Cells from Human Exfoliated Deciduous Teeth.

Regenerative therapy for tissues by mesenchymal stem cell (MSCs) transplantation has received much attention. The cluster of differentiation (CD)146 marker, a surface-antigen of stem cells, is crucial for angiogenic and osseous differentiation abilities. Bone regeneration is accelerated by the transplantation of CD146-positive deciduous dental pulp-derived mesenchymal stem cells contained in stem cells from human exfoliated deciduous teeth (SHED) into a living donor. However, the role of CD146 in SHED remains unclear. This study aimed to compare the effects of CD146 on cell proliferative and substrate metabolic abilities in a population of SHED. SHED was isolated from deciduous teeth, and flow cytometry was used to analyze the expression of MSCs markers. Cell sorting was performed to recover the CD146-positive cell population (CD146+) and CD146-negative cell population (CD146-). CD146 + SHED without cell sorting and CD146-SHED were examined and compared among three groups. To investigate the effect of CD146 on cell proliferation ability, an analysis of cell proliferation ability was performed using BrdU assay and MTS assay. The bone differentiation ability was evaluated using an alkaline phosphatase (ALP) stain after inducing bone differentiation, and the quality of ALP protein expressed was examined. We also performed Alizarin red staining and evaluated the calcified deposits. The gene expression of ALP, bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN) was analyzed using a real-time polymerase chain reaction. There was no significant difference in cell proliferation among the three groups. The expression of ALP stain, Alizarin red stain, ALP, BMP-2, and OCN was the highest in the CD146+ group. CD146 + SHED had higher osteogenic differentiation potential compared with SHED and CD146-SHED. CD146 contained in SHED may be a valuable population of cells for bone regeneration therapy.

Head and neck cancer patients show poor oral health as compared to those with other types of cancer.

PURPOSE: Several studies have found associations between periodontitis and various types of cancer. Since the site of head and neck cancer (HNC) has contiguity or proximity to the oral cavity, it may be particularly influenced by oral inflammation. This study aimed to determine whether HNC patients have poor oral health as compared to those with other types of cancer. METHODS: This study retrospectively examined oral environmental factors including periodontal inflamed surface area (PISA), a new periodontal inflammatory parameter. A total of 1030 cancer patients were divided into the HNC (n = 142) and other cancer (n = 888) groups. Furthermore, the HNC group was divided into high (n = 71) and low (n = 71) PISA subgroups, and independent risk factors affecting a high PISA value were investigated. RESULTS: Multivariate logistic regression analysis showed that number of missing teeth (odds ratio 1.72, 95% CI 1.15-2.56, P < 0.01), PISA (odds ratio 1.06, 95% CI 1.03-1.06, P < 0.05), and oral bacterial count (odds ratio 1.02, 95% CI 1.01-1.03, P < 0.01) were independent factors related to HNC. In addition, multivariate logistic regression analysis indicated that current smoker (odds ratio 7.51, 95% CI 1.63-34.71, P < 0.01) and presence of untreated dental caries (odds ratio 3.33, 95% CI 1.23-9.00, P < 0.05) were independent risk factors affecting high PISA values in HNC patients. CONCLUSION: HNC patients have higher levels of gingival inflammation and poor oral health as compared to patients with other types of cancer, indicating that prompt oral assessment and an effective oral hygiene management plan are needed at the time of HNC diagnosis.

Correction of Severe Skeletal Class II High Angle with Mandibular Retrusion and Gummy Smile by Double-Jaw Surgery.

This report describes the treatment of severe skeletal Class II malocclusion in a young woman with a gummy smile and pronounced lower anterior facial height. Overjet and overbite were +12.0 mm and -1.0 mm, respectively. Cephalometric analysis revealed inferior positioning of the maxilla and severe mandibular retrusion with clockwise rotation. Both the upper and lower anterior teeth showed labial inclination. Based on a diagnosis of a skeletal Class II high angle with mandibular retrusion and a gummy smile, double-jaw orthognathic surgeries for upper and lower premolar extraction were chosen to gain ideal occlusion and an improvement in the esthetic facial profile. Le Fort I osteotomy was performed to move the anterior and posterior teeth upward by 4.0 mm and achieve mandibular counterclockwise rotation. Short lingual sagittal split ramus osteotomy was performed to move the mandible forward by 3.0 mm. As a result, normal overjet and overbite were achieved together with a straight profile and a good smile. After surgery, electromyographic evaluation of anterior temporal muscle activity showed an improvement in the percentage overlapping coefficient value (a symmetric index of bilateral muscle activity) from 28.1% to 63.2% compared to at pre-treatment. The pattern of jaw movement also showed an improvement. These results suggest that orthognathic surgery in skeletal Class II cases can improve not only malocclusion and the skeletal relationship of the jaws, but also masticatory function and jaw movement.

Imipramine prevents Porphyromonas gingivalis lipopolysaccharide-induced microglial neurotoxicity.

Porphyromonas gingivalis (P. gingivalis) is a Gram-negative anaerobe involved in the pathogenesis of chronic periodontitis, including local inflammation of the oral cavity. However, periodontal disease has recently been identified as a significant factor in the pathogenesis of neural diseases, including Alzheimer's disease. A virulence factor, P. gingivalis-lipopolysaccharide (LPS-PG), is involved in pro-inflammatory responses, not only in peripheral tissues but also in the brain. In this study, we examined whether P. gingivalis-induced brain inflammation could be ameliorated by pharmacotherapy, using in vivo and in vitro studies. In an animal experiment, peripheral administration of LPS-PG induced inflammation in the hippocampus via microglial activation, which was inhibited by pre-treatment with the antidepressant imipramine. Similarly, LPS-PG-induced inflammation in MG-6 cells, a mouse microglial cell line, was inhibited by pre-treatment with imipramine, which caused imipramine-induced inhibition of NF-κB signaling. Culture media obtained from LPS-PG-treated MG-6 cells induced neuronal cell death in Neuro-2A cells, a mouse neuroblastoma cell line, which was prevented by pre-treatment of MG-6 cells with imipramine. These results indicate that imipramine inhibits LPS-PG-induced inflammatory responses in microglia and ameliorates periodontal disease-related neural damage.

Effects of high-frequency near infrared laser irradiation on experimental tooth movement-induced pain in rats.

Discomfort and dull pain are known side effects of orthodontic treatment. Pain is expected to be reduced by near-infrared (NIR) lasers; however, the mechanism underlying effects of short-pulse NIR lasers in the oral and maxillofacial area remains unclear. This study aimed to examine the effects of high-frequency NIR diode laser irradiation on pain during experimental tooth movement (ETM) on 120 J. NIR laser with 910 nm wavelength, 45 W maximum output power, 300 mW average output power, and 200 ns pulse width (Lumix 2; (Lumix 2; Fisioline, Verduno CN, Italy) was used for the experiment. A nickel-titanium-closed coil was used to apply a 50-gf force between the maxillary left-side first molar and incisor in 7-week-old Sprague-Dawley rats (280-300 g) to induce ETM. We measured facial-grooming frequency and vacuous chewing movement (VCM) period between laser-irradiation and ETM groups. We performed immunofluorescent histochemistry analysis to quantify levels of Iba-1, astrocytes, and c-fos protein-like immunoreactivity (Fos-IR) in the trigeminal spinal nucleus caudalis (Vc). Compared with the ETM group, the laser irradiation group had significantly decreased facial-grooming frequency (P = 0.0036), VCM period (P = 0.043), Fos-IR (P = 0.0028), Iba-1 levels (P = 0.0069), and glial fibrillary acidic protein (GFAP) levels (P = 0.0071). High-frequency NIR diode laser irradiation appears to have significant analgesic effects on ETM-induced pain, which involve inhibiting neuronal activity, microglia, and astrocytes, and it inhibits c-fos, Iba-1, and GFAP expression, reducing ETM-induced pain in rats. High-frequency NIR diode laser application could be applied to reduce pain during orthodontic tooth movement.

High-frequency near-infrared diode laser irradiation suppresses IL-1ß-induced inflammatory cytokine expression and NF-κB signaling pathways in human primary chondrocytes.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are common inflammation-associated cartilage degenerative diseases. Recent studies have shown that low-level diode laser treatment can reduce inflammatory cytokine expressions in cartilage. We recently reported that high-frequency low-level diode laser irradiation attenuates matrix metalloproteinases (MMPs) expression in human primary chondrocytes. However, the molecular mechanism underlying the effect of high-frequency low-level diode laser on chondrocytes remains unclear. Therefore, we aimed to elucidate the effect of high-frequency low-level diode laser irradiation on inflammatory cytokine expression in human primary chondrocytes. Normal human articular chondrocytes were treated with recombinant interleukin-1 beta (IL-1ß) for 30 min or 24 h and irradiated with a high-frequency NIR diode laser at 8 J/cm2. The expression of IL-1ß, interleukin-6, and tumor necrosis factor-alpha was assessed using western blot analysis. To evaluate the nuclear factor-kappa B (NF-κB) signaling pathway, the phosphorylation, translocation, and DNA-binding activity of NF-κB were detected using western blot analysis, immunofluorescence analysis, electrophoretic mobility shift assay, and enzyme-linked immunosorbent assay analysis. High-frequency low-level diode laser irradiation decreased inflammatory cytokine expression in IL-1ß-treated chondrocytes. Moreover, high-frequency low-level diode laser irradiation decreased the phosphorylation, nuclear translocation, and DNA-binding activity of NF-κB in the IL-1ß-treated state. However, irradiation alone did not affect NF-κB activity. Thus, high-frequency low-level diode laser irradiation at 8 J/cm2 can reduce inflammatory cytokine expressions in normal human articular chondrocytes through NF-κB regulation. These findings indicate that high-frequency low-level diode laser irradiation may reduce the expression of inflammatory cytokines in OA and RA.

Masticatory Function Assessment of Adult Patients With Cleft Lip and Palate After Orthodontic Treatment.

OBJECTIVE: To determine whether orthodontically treated patients with cleft lip and palate (CLP) possess a different masticatory function than those of untreated patients with normal occlusion. DESIGN: Occlusal contact area, occlusal force, as well as masseter and anterior temporal muscular activity were measured during maximum voluntary clenching (MVC) tests. Mandibular left and right lateral movements during mastication were also assessed. To further elucidate the nature of masticatory function, especially to determine the rate of abnormal jaw movement patterns, a parametric error index (EI) was set. Finally, masticatory efficiency was evaluated with a glucose sensitive measuring device. PARTICIPANTS: Fifteen patients with CLP who had previously completed the orthodontic treatments required to achieve an acceptable and more harmonious occlusion accepted to volunteer in this study along with 21 untreated patients who already possessed a normal occlusion. RESULTS: Patients with CLP showed a significantly lower occlusal force, reduced occlusal contact area, and decreased masticatory efficiency as well as significantly higher EI value when compared with controls. However, there was no significant difference when analyzing muscle activity, although masticatory efficiency was significantly different between the 2 groups. Despite this result, the scores obtained by the patients with CLP in the masticatory efficiency tests were still in the normal range. CONCLUSIONS: Orthodontic treatment for adult patients with CLP provides a satisfactory result for the patients' masticatory ability albeit significantly less ideal compared with untreated patients with normal occlusion.

An Investigation of Tongue-Palatal Contact Changes in Patients With Skeletal Mandibular Lateral Deviation After Sagittal Split Ramus Osteotomy.

ABSTRACT: This study aimed to investigate the changes in tongue-palatal contact patterns in patients with mandibular lateral deviation by electropalatography (EPG) before and after sagittal split ramus osteotomy (SSRO). Ten mandibular asymmetry patients who underwent SSRO participated in the study. Tongue-palatal contact patterns for the production of /t/ and /s/ sounds were observed using EPG before surgery and 3 months after surgery, and the changes in EPG pattern were examined. The number of electrode contacts in the 2 vertical columns of the EPG plate was calculated both in the mandibular deviation side and the nondeviation side. The EPG patterns for /t/ and /s/ showed asymmetry before surgery but became normal after surgery. Before surgery, the number of electrode contacts in the 2 vertical columns in the mandibular deviation side was significantly lower than that in the nondeviation side and the normal participants during /t/ and /s/ articulation. However, the number of electrode contacts in the deviation side significantly increased after surgery. This study demonstrated that the tongue-palatal contact patterns for /t/ and /s/ articulation shifted to the direction of mandibular deviation and improved after SSRO.

Overexpression of miR-125b in Osteoblasts Improves Age-Related Changes in Bone Mass and Quality through Suppression of Osteoclast Formation.

We recently reported an unexpected role of osteoblast-derived matrix vesicles in the delivery of microRNAs to bone matrix. Of such microRNAs, we found that miR-125b inhibited osteoclast formation by targeting Prdm1 encoding a transcriptional repressor of anti-osteoclastogenesis factors. Transgenic (Tg) mice overexpressing miR-125b in osteoblasts by using human osteocalcin promoter grow normally but exhibit high trabecular bone mass. We have now further investigated the effects of osteoblast-mediated miR-125b overexpression on skeletal morphogenesis and remodeling during development, aging and in a situation of skeletal repair, i.e., fracture healing. There were no significant differences in the growth plate, primary spongiosa or lateral (periosteal) bone formation and mineral apposition rate between Tg and wild-type (WT) mice during early bone development. However, osteoclast number and medial (endosteal) bone resorption were less in Tg compared to WT mice, concomitant with increased trabecular bone mass. Tg mice were less susceptible to age-dependent changes in bone mass, phosphate/amide I ratio and mechanical strength. In a femoral fracture model, callus formation progressed similarly in Tg and WT mice, but callus resorption was delayed, reflecting the decreased osteoclast numbers associated with the Tg callus. These results indicate that the decreased osteoclastogenesis mediated by miR-125b overexpression in osteoblasts leads to increased bone mass and strength, while preserving bone formation and quality. They also suggest that, in spite of the fact that single miRNAs may target multiple genes, the miR-125b axis may be an attractive therapeutic target for bone loss in various age groups.

Epidermal growth factor-immobilized surfaces for the selective expansion of neural progenitor cells derived from induced pluripotent stem cells.

Neural progenitor cells (NPCs) are considered to be a promising source for stem cell-based regenerative therapy for central nervous disorders. However, the widespread clinical application of NPCs requires another technology that permits the efficient production of pure NPCs in large quantities. In this study, culture substrates were designed by immobilizing epidermal growth factor (EGF) onto the substrate and evaluated for their feasibility of expanding NPCs obtained through the neurosphere culture of induced pluripotent stem (iPS) cells. After three passages we obtained neurospheres that contained cells abundantly expressing an EGF receptor. The neurospheres were dissociated into single cells and seeded onto the EGF-immobilized substrates. It was observed that neurosphere-forming cells seeded and cultured on the EGF-immobilized surface formed a two-dimensional cellular network characteristic of NPCs. These cells were found to be capable of being subcultured, while remaining their proliferation potential. Furthermore, a majority of cells (~99% of total cells) on the substrate was shown to express an NPC marker, nestin, whereas a limited number of cells (~1% of total cells) expressed neuronal marker, ß-tubulin III. These results as a whole demonstrate that the EGF-immobilized substrate allows for iPS cell-derived NPCs to efficiently proliferate while maintaining the undifferentiated state.

Protective effects of cilengitide on inflammation in chondrocytes under excessive mechanical stress.

Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as matrix metalloproteinases (MMPs). Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well-known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVß3 and αVß5) and the expression of inflammatory factors under mechanical loading in chondrocytes by using an integrin receptor antagonist (cilengitide). Cilengitide suppressed the gene expression of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3), and MMP-13 induced by excessive mechanical stress. In addition, the protein expression of IL1-ß and MMP-13 was also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress-induced integrin signaling in chondrocytes by using western blotting. The levels of p-FAK, p-ERK, p-JNK, and p-p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrins αVß3 and αVß5 on the surface of chondrocytes and thereby induce an inflammatory reaction by upregulating the expression of IL-1ß, TNF-α, MMP-3, and MMP-13 through phosphorylation of FAK and MAPKs.

Stem cell-derived conditioned media from human exfoliated deciduous teeth promote bone regeneration.

OBJECTIVES: Cleft lip and palate (CL/P) are common congenital orofacial anomalies. Autogenous iliac bone grafting closes alveolar cleft defects but requires surgical intervention. Mesenchymal stem cell culture supernatant can regenerate tissues via paracrine activity. However, little is known about the bone-regenerative effects of stem cells from human exfoliated deciduous teeth (SHED) and conditioned media (CM). Our aim was to address this. MATERIALS AND METHODS: Stem cells were isolated from primary tooth pulp and cultured. Defects were made in calvariae of immunodeficient mice and implanted with stem cell- or CM-containing atelocollagen. Regenerated bone was analysed by microcomputed tomography, haematoxylin-eosin and Masson's trichrome staining. Vascular endothelial growth factor, CD31 and CD34 expression were confirmed by immunohistochemistry, and the presence of several proteins and growth factors was verified in SHED-CM. RESULTS: Bone regeneration was enhanced in defects treated with stem cells and CM compared to that in controls 8 weeks after transplantation. Mature bone formation and angiogenesis were confirmed with CM but not with stem cells or in controls. Secretome analysis using multiple cytokine assays revealed that SHED-CM contained tissue-regenerating factors with roles in angiogenesis and osteogenesis. CONCLUSION: CM non-invasively regenerate bone and might be effective to reconstruct alveolar clefts in CL/P patients.

FAK inhibition protects condylar cartilage under excessive mechanical stress.

OBJECTIVES: Excessive mechanical stress is assumed to be a major cause of temporomandibular joint (TMJ) osteoarthritis (OA). +Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase involved in a variety of signaling pathways. Little has been reported on the function of FAK in TMJ-OA. In the present study, we investigated the effect of FAK inhibition on TMJ cartilage under excessive mechanical loading stress. MATERIALS AND METHODS: Articular cartilage explants were harvested from the TMJ of rats and subjected to mechanical loading in the presence of an FAK inhibitor in organ culture. The gene expression of inflammatory cytokines was examined after the application of mechanical loading with or without FAK inhibitor. Paraffin-embedded sections of articular cartilage were stained with hematoxylin and eosin, safranin O and fast Green, toluidine blue, TUNEL staining, and immunohistochemical staining and was performed to investigate the protein expression of IL-1ß and MMP-13. RESULTS: Treatment with FAK inhibitor reduced the gene expression of inflammatory cytokines and inhibited the degradation of articular cartilage, as determined histologically. FAK inhibitor treatment also suppressed the protein expression of IL-1ß and MMP-13 in the hypertrophic zone, as determined immunohistologically. CONCLUSION: Treatment with FAK inhibitor suppresses inflammation and protects condylar cartilage under excessive mechanical loading.

Examination of the effect of combined use of Er:YAG laser irradiation and mechanical force loading on bone metabolism using primary human gingival fibroblasts.

Prolonged treatment and painful tooth movement are major problems for patients undergoing orthodontic treatment. Accelerating the movement of teeth leads to shortening of the treatment period, so various studies on the movement of teeth have been conducted in the field of orthodontics. In previous studies, we performed a fiber incision-like fiberotomy using an Er:YAG laser in rats and confirmed acceleration of tooth movement. Therefore, in this study, the effect of Er:YAG laser irradiation on human gingival fibroblasts was investigated in vitro. Human gingival fibroblasts (2.0 × 105 cells) were seeded in a 6-well plate and reached 80% confluence 24 h later. A control group not undergoing any irradiation and 3 groups undergoing laser irradiation at 0.6 W, 1.0 W, and 1.2 W were investigated. Laser irradiation was performed 24 h after cell seeding. The cells were then recovered 24 h later, and the cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), bone morphogenetic protein-2 (BMP-2), and BMP-4 genes were confirmed by PCR. In addition, a control group not undergoing any procedures, a group undergoing only Er:YAG laser irradiation, a group undergoing only centrifugal loading, and a group undergoing both Er:YAG laser irradiation and centrifugal force loading were investigated. After 24 h, cells were collected and PCR was performed. Twenty-four hours after laser irradiation, gene expressions were examined by quantitative RT-PCR, which showed that the gene expressions of COX-2, IL-1ß, TNF-α, BMP-2, and BMP-4 increased depending on the amount of irradiation energy, with the largest value at 1.2 W. Gene expressions of COX-2, IL-1ß, TNF-α, BMP-2, and BMP-4 were significantly higher in the laser with centrifugal load group than in the load group. These results suggest that genes related to bone metabolism are activated in human gingival fibroblasts when mechanical stimulation and laser irradiation are combined. This helps to elucidate the effects of Er:YAG laser irradiation during tooth movement.