Loss-of-function OGFRL1 variants identified in autosomal recessive cherubism families.

Cherubism (OMIM 118400) is a rare craniofacial disorder in children characterized by destructive jawbone expansion due to the growth of inflammatory fibrous lesions. Our previous studies have shown that gain-of-function mutations in SH3 domain-binding protein 2 (SH3BP2) are responsible for cherubism and that a knock-in mouse model for cherubism recapitulates the features of cherubism, such as increased osteoclast formation and jawbone destruction. To date, SH3BP2 is the only gene identified to be responsible for cherubism. Since not all patients clinically diagnosed with cherubism had mutations in SH3BP2, we hypothesized that there may be novel cherubism genes and that these genes may play a role in jawbone homeostasis. Here, using whole exome sequencing, we identified homozygous loss-of-function variants in the opioid growth factor receptor like 1 (OGFRL1) gene in 2 independent autosomal recessive cherubism families from Syria and India. The newly identified pathogenic homozygous variants were not reported in any variant databases, suggesting that OGFRL1 is a novel gene responsible for cherubism. Single cell analysis of mouse jawbone tissue revealed that Ogfrl1 is highly expressed in myeloid lineage cells. We generated OGFRL1 knockout mice and mice carrying the Syrian frameshift mutation to understand the in vivo role of OGFRL1. However, neither mouse model recapitulated human cherubism or the phenotypes exhibited by SH3BP2 cherubism mice under physiological and periodontitis conditions. Unlike bone marrow-derived M-CSF-dependent macrophages (BMMs) carrying the SH3BP2 cherubism mutation, BMMs lacking OGFRL1 or carrying the Syrian mutation showed no difference in TNF-ɑ mRNA induction by LPS or TNF-ɑ compared to WT BMMs. Osteoclast formation induced by RANKL was also comparable. These results suggest that the loss-of-function effects of OGFRL1 in humans differ from those in mice and highlight the fact that mice are not always an ideal model for studying rare craniofacial bone disorders.

Dental, skeletal and soft tissue changes after bimaxillary protrusion treatment with temporary anchorage devices using different retraction mechanics.

BACKGROUND: Temporary anchorage devices (TADs), which are absolute anchorage, are used for retraction of the anterior teeth in cases of severe bimaxillary protrusion. There have been a number of studies regarding anterior tooth movement using TADs performed by simulation systems and actual treated materials with sliding mechanics. However, there are few studies regarding anterior tooth movement using TADs treated by loop mechanics The purpose of this study was to investigate the effect of TADs in anterior tooth movement using loop mechanics performed in actual cases of bimaxillary protrusion. METHODS: This study was performed in 20 adult patients with severe bimaxillary protrusion treated with four bicuspid extraction with sliding or loop mechanics (n = 10 in each mechanics) using TADs. The skeletal and denture patterns, as well as the soft tissue profile from pre-treatment (T0) and post-treatment (T1) lateral cephalograms, were compared between sliding and closing loop mechanics. RESULTS: The use of TADs is useful for retraction of anterior teeth without molar anchorage loss. in sliding and loop mechanics. The upper anterior teeth were less lingual tipped and lower anterior teeth were more upright resulting in less clockwise rotation of the occlusal plane in loop mechanics compared to sliding mechanics. CONCLUSION: An oblique retraction force vector with a lower point of application causes less intrusion and more lingual tipping of upper anterior teeth as well as more clockwise rotation of the occlusal plane compared to a parallel retraction force vector.

Insulin potentiates inhibitory synaptic currents between fast-spiking and pyramidal neurons in the rat insular cortex.

Insulin plays roles in brain functions such as neural development and plasticity and is reported to be involved in dementia and depression. However, little information is available on the insulin-mediated modulation of electrophysiological activities, especially in the cerebral cortex. This study examined how insulin modulates the neural activities of inhibitory neurons and inhibitory postsynaptic currents (IPSCs) in rat insular cortex (IC; either sex) by multiple whole-cell patch-clamp recordings. We demonstrated that insulin increased the repetitive spike firing rate with a decrease in the threshold potential without changing the resting membrane potentials and input resistance of fast-spiking GABAergic neurons (FSNs). Next, we found a dose-dependent enhancement of unitary IPSCs (uIPSCs) by insulin in the connections from FSNs to pyramidal neurons (PNs). The insulin-induced enhancement of uIPSCs accompanied decreases in the paired-pulse ratio, suggesting that insulin increases GABA release from presynaptic terminals. The finding of miniature IPSC recordings of the increased frequency without changing the amplitude supports this hypothesis. Insulin had little effect on uIPSCs under the coapplication of S961, an insulin receptor antagonist, or lavendustin A, an inhibitor of tyrosine kinase. The PI3-K inhibitor wortmannin or the PKB/Akt inhibitors, deguelin and Akt inhibitor VIII, blocked the insulin-induced enhancement of uIPSCs. Intracellular application of Akt inhibitor VIII to presynaptic FSNs also blocked insulin-induced enhancement of uIPSCs. In contrast, uIPSCs were enhanced by insulin in combination with the MAPK inhibitor PD98059. These results suggest that insulin facilitates the inhibition of PNs by increases in FSN firing frequency and IPSCs from FSNs to PNs. (250 words).

Tension force causes cell cycle arrest at G2/M phase in osteocyte-like cell line MLO-Y4.

Bone remodelling is the process of bone resorption and formation, necessary to maintain bone structure or for adaptation to new conditions. Mechanical loadings, such as exercise, weight bearing and orthodontic force, play important roles in bone remodelling. During the remodelling process, osteocytes play crucial roles as mechanosensors to regulate osteoblasts and osteoclasts. However, the precise molecular mechanisms by which the mechanical stimuli affect the function of osteocytes remain unclear. In the present study, we analysed viability, cell cycle distribution and gene expression pattern of murine osteocyte-like MLO-Y4 cells exposed to tension force (TF). Cells were subjected to TF with 18% elongation at 6 cycles/min for 24 h using Flexcer Strain Unit (FX-3000). We found that TF stimulation induced cell cycle arrest at G2/M phase but not cell death in MLO-Y4 cells. Differentially expressed genes (DEGs) between TF-stimulated and unstimulated cells were identified by microarray analysis, and a marked increase in glutathione-S-transferase α (GSTA) family gene expression was observed in TF-stimulated cells. Enrichment analysis for the DEGs revealed that Gene Ontology (GO) terms and Kyoto Encyclopedia Genes and Genomes (KEGG) pathways related to the stress response were significantly enriched among the upregulated genes following TF. Consistent with these results, the production of reactive oxygen species (ROS) was elevated in TF-stimulated cells. Activation of the tumour suppressor p53, and upregulation of its downstream target GADD45A, were also observed in the stimulated cells. As GADD45A has been implicated in the promotion of G2/M cell cycle arrest, these observations may suggest that TF stress leads to G2/M arrest at least in part in a p53-dependent manner.

The changes in oral volume and hyoid bone position after maxillomandibular advancement and genioglossus advancement for patients with obstructive sleep apnea.

INTRODUCTION: Maxillomandibular advancement (MMA) and genioglossus advancement (GA) are surgeries for patients with obstructive sleep apnea (OSA). Postoperative evaluation is primarily based on the apnea-hypopnea index (AHI) measured by polysomnography. The purpose of this study was to identify the timing of hyoid bone relocation after MMA and GA surgery and to investigate whether or not hyoid bone relocation can be an indicator of postoperative evaluation of OSA. METHODS: Patients with OSA underwent MMA and GA surgery. Changes in hyoid bone position and tongue-to-oral volume ratio were analyzed on lateral radiographs before, immediately after, and 1 year after surgery. Then, a correlation was verified between these changes and postoperative AHI. RESULTS: In 18 patients studied, the position of the hyoid bone did not show a constant tendency immediately after surgery. One year after surgery, the bone had moved anteriorly and toward the oral cavity in all patients compared to its preoperative position. And AHI correlated with the movement of the hyoid bone to the oral side. DISCUSSION: One year after surgery, the tongue was adapted to the newly enlarged oral space, and as a result, the low position of the hyoid bone before the operation was improved. The findings suggest that the degree of lowering of the hyoid bone may be an indicator of the improvement of AHI.

Application of oxytocin with low-level laser irradiation suppresses the facilitation of cortical excitability by partial ligation of the infraorbital nerve in rats: An optical imaging study.

The effects of current treatments for neuropathic pain are limited. Oxytocin is a novel candidate substance to relieve neuropathic pain, as demonstrated in various animal models with nerve injury. Low-level laser therapy (LLLT) is another option for the treatment of neuropathic pain. In this study, we quantified the effects of oxytocin or LLLT alone and the combination of oxytocin and LLLT on cortical excitation induced by electrical stimulation of the dental pulp using optical imaging with a voltage-sensitive dye in the neuropathic pain model with partial ligation of the infraorbital nerve (pl-ION). We applied oxytocin (OXT, 0.5 µmol) to the rat once on the day of pl-ION locally to the injured nerve. LLLT using a diode laser (810 nm, 0.1 W, 500 s, continuous mode) was performed daily via the skin to the injured nerve from the day of pl-ION to 2 days after pl-ION. Cortical responses to electrical stimulation of the mandibular molar pulp under urethane anesthesia were recorded 3 days after pl-ION. Both the amplitude and area of excitation in the primary and secondary somatosensory and insular cortices in pl-ION rats were larger than those in sham rats. The larger amplitude of cortical excitation caused by pl-ION was suppressed by OXT or LLLT. The expanded area of cortical excitation caused by pl-ION was suppressed by OXT with LLLT but not by OXT or LLLT alone. These results suggest that the combined application of OXT and LLLT is effective in relieving the neuropathic pain induced by trigeminal nerve injury.

Continuous Compressive Force Induces Differentiation of Osteoclasts with High Levels of Inorganic Dissolution.

BACKGROUND Osteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor kappaB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force. MATERIAL AND METHODS Cells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm² compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates. RESULTS Multinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm² force than 0.3 g/cm². Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force. CONCLUSIONS Our study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.

Effect of mechanical strain-induced PGE2 production on bone nodule formation by rat calvarial progenitor cells.

High-magnitude mechanical strain inhibits bone nodule formation by reducing expression of bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (Runx2), and muscle segment homeobox 2 (Msx2). Mechanical strain also induces production of proinflammatory factor prostaglandin E2 (PGE2) by osteoblasts. We measured the effect of mechanical strain-induced PGE2 production on bone nodule formation and expression levels of bone formation-related factors. Osteoblast-like cells isolated from fetal rat calvariae were loaded with 18% cyclic tension force (TF) for 48 h in the presence or absence of NS-398, a selective inhibitor of cyclooxygenase-2. To investigate the effect of TF-induced PGE2 on bone formation, bone nodule area on day 21 was measured by von Kossa staining. BMP-2, Runx2, and Msx2 expression levels were examined at 1 day after TF loading. Bone nodule formation was significantly inhibited by TF but was restored to control level by PGE2 inhibition. Furthermore, TF loading-induced reductions in expressions of these factors were restored to control level by PGE2 suppression. These results indicate that PGE2 production induced by high-magnitude mechanical strain inhibits bone nodule formation by reducing expression levels of bone formation-related factors.

Treatment of bimaxillary protrusion with temporary anchorage devices.

For treatment of severe bimaxillary protrusion in adults, a condition known to be among the most difficult to manage, both the maxillary and mandibular anterior teeth must be fully retracted using all the extraction space available. This article reports the treatment of an adult with severe high-angle bimaxillary protrusion. To correct the protrusion of the anterior teeth, orthodontic anchor screws (OASs) were used to provide absolute anchorage during anterior retraction. Acceptable occlusion, facial profile, and balance were achieved. OASs appear to be very useful for treatment of severe bimaxillary protrusion in adults.

Continuous application of compressive force induces fusion of osteoclast-like RAW264.7 cells via upregulation of RANK and downregulation of LGR4.

AIMS: During orthodontic treatment, facilitating osteoclastic bone resorption in the alveolar bone exposed to the compressive force (CF) is an important factor for tooth movement. The present study investigated the effect of CF stimulation on the differentiation of RAW264.7 cells from precursors to mature osteoclasts. MAIN METHODS: The cells were continuously stimulated with 0.3, 0.6, or 1.1 g/cm2 CF-which was generated by increasing the volume of culture medium in the wells of a 96-well plate-in the presence or absence of receptor activator of nuclear factor κB (RANK) ligand (RANKL) for 4 days. KEY FINDINGS: In the presence of RANKL, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and the mRNA levels of dendritic cell-specific transmembrane protein (DC-STAMP) and osteoclast-stimulatory transmembrane protein (OC-STAMP) were increased by application of 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF. The mRNA level of RANK was upregulated whereas that of leucine-rich repeat-containing G-protein-coupled receptor (LGR)4-another RANKL receptor was downregulated by 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF in the absence of RANKL. The proportion of cells with nuclear translocation of the nuclear translocation of nuclear factor of activated T cells (NFAT)c1 was increased by 0.6 and 1.1 g/cm2 CF in the presence of RANKL. SIGNIFICANCE: Continuous application of CF induced the differentiation of RAW264.7 cells into TRAP-positive multinuclear cells by enhancing the expression of DC- and OC-STAMP and the nuclear translocation of NFATc1. This may result from the CF-induced increase in RANK and decrease in LGR4 expression.

Cherubism Mice Also Deficient in c-Fos Exhibit Inflammatory Bone Destruction Executed by Macrophages That Express MMP14 Despite the Absence of TRAP+ Osteoclasts.

Currently, it is believed that osteoclasts positive for tartrate-resistant acid phosphatase (TRAP+) are the exclusive bone-resorbing cells responsible for focal bone destruction in inflammatory arthritis. Recently, a mouse model of cherubism (Sh3bp2KI/KI ) with a homozygous gain-of-function mutation in the SH3-domain binding protein 2 (SH3BP2) was shown to develop auto-inflammatory joint destruction. Here, we demonstrate that Sh3bp2KI/KI mice also deficient in the FBJ osteosarcoma oncogene (c-Fos) still exhibit noticeable bone erosion at the distal tibia even in the absence of osteoclasts at 12 weeks old. Levels of serum collagen I C-terminal telopeptide (ICTP), a marker of bone resorption generated by matrix metalloproteinases (MMPs), were elevated, whereas levels of serum cross-linked C-telopeptide (CTX), another resorption marker produced by cathepsin K, were not increased. Collagenolytic MMP levels were increased in the inflamed joints of the Sh3bp2KI/KI mice deficient in c-Fos. Resorption pits contained a large number of F4/80+ macrophages and genetic depletion of macrophages rescued these erosive changes. Importantly, administration of NSC405020, an MMP14 inhibitor targeted to the hemopexin (PEX) domain, suppressed bone erosion in c-Fos-deficient Sh3bp2KI/KI mice. After activation of the NF-κB pathway, macrophage colony-stimulating factor (M-CSF)-dependent macrophages from c-Fos-deficient Sh3bp2KI/KI mice expressed increased amounts of MMP14 compared with wild-type macrophages. Interestingly, receptor activator of NF-κB ligand (RANKL)-deficient Sh3bp2KI/KI mice failed to show notable bone erosion, whereas c-Fos deletion did restore bone erosion to the RANKL-deficient Sh3bp2KI/KI mice, suggesting that osteolytic transformation of macrophages requires both loss-of-function of c-Fos and gain-of-function of SH3BP2 in this model. These data provide the first genetic evidence that cells other than osteoclasts can cause focal bone destruction in inflammatory bone disease and suggest that MMP14 is a key mediator conferring pathological bone-resorbing capacity on c-Fos-deficient Sh3bp2KI/KI macrophages. In summary, the paradigm that osteoclasts are the exclusive cells executing inflammatory bone destruction may need to be reevaluated based on our findings with c-Fos-deficient cherubism mice lacking osteoclasts. © 2017 American Society for Bone and Mineral Research.

Satisfaction with orthognathic surgery of skeletal Class III patients.

INTRODUCTION: Class III relationships can be corrected with single-jaw or bimaxillary surgery. The purpose of this research was to assess patient satisfaction after bimaxillary surgery, compared with setback surgery alone, for Class III corrections. Identifying patients' relative levels of satisfaction will provide guidance for the selection of surgical options. METHODS: The cephalometric outcomes for 25 patients who underwent 2-jaw surgery were compared with the outcomes in 40 patients who had mandibular setback. Soft and hard tissue changes were evaluated using initial and postsurgical lateral cephalograms. The patients were asked to complete self-administered questionnaires after orthognathic treatment. Correlations between cephalometric improvement and patient satisfaction were evaluated. RESULTS: The patients in the 2-jaw group reported significantly higher satisfaction in the appearance of the mouth (P <0.05), smile (P <0.05), and treatment outcome (P <0.001). These item scores and the changes in ANB, ANS-M, and nasolabial angle showed strong correlations in the 2-jaw group and moderate correlations in the 1-jaw group. CONCLUSIONS: ANS-M and nasolabial angle should be considered in the conventional diagnosis of skeletal Class III orthognathic surgery to obtain adequate correction of facial esthetics and patient satisfaction. Esthetic needs contribute to surgical decisions when treating patients with skeletal Class III malocclusions and dentofacial deformities such as maxillary deficiency and long facial height that causes a turned-up upper lip.

High-magnitude mechanical strain inhibits the differentiation of bone-forming rat calvarial progenitor cells.

PURPOSE: Orthodontic tooth movement occurs during the bone remodeling induced by therapeutic mechanical strain. It is important to investigate the relation between the strength of mechanical stress and bone formation activity. The aim of this study was to determine the effect of high-magnitude mechanical strain on bone formation in detail. MATERIALS AND METHODS: Osteoblast-like cells isolated from fetal rat calvariae were loaded with 18% cyclic tension force (TF) for 48 h. To phenotypically investigate the effect of TF, we measured the number and the size of bone nodules stained by von Kossa technique on day 21 after cell seeding and determined the calcium content of bone nodules on day 14. Furthermore, we examined the gene expression of BMP-2, Runx2 and Msx2, which are important factors for bone nodule formation, on days 1, 4 and 7 after TF loading. RESULTS: The maximum bone nodule size in the control group was 1620 and 719 µm in the TF group. Furthermore, the mean number of bone nodules sized over 360 µm in the TF group was significantly decreased compared to the control group. The calcium content was also significantly decreased to 42% by TF loading. The mRNA expression of BMP-2, Runx2 and Msx2 was decreased 1 and 4 days after TF loading. CONCLUSION: The differentiation of bone forming progenitor cells into bone nodule forming cells was inhibited by TF due to the decreased expression of bone formation related factors such as BMP-2, Runx2 and Msx2.

Longitudinal quantitative evaluation of the mid-palatal suture after rapid expansion using in vivo micro-CT.

New bone formation is known to occur between the opened palatal bones after rapid mid-palatal expansion (RME), although the time-dependent changes in the mid-palatal suture after RME have not been fully examined. Thus, we investigated time-dependent morphological changes in the mid-palatal suture using in vivo micro-computed tomography (mCT) and the expression of bone morphogenetic factors. RME was performed by inserting a 1.5-mm-thick circular metal ring between the maxillary incisors of rats, and morphological changes in the mid-palatal suture were investigated using in vivo mCT imaging after RME. Bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor-I (IGF-I) expression in the suture were also examined using reverse-transcription polymerase chain reaction and immunohistochemistry. The bone volume of the mid-palatal suture decreased after RME to a minimum of -0.34mm(3) on day 12, then increased with bone formation over time and reached -0.13mm(3) on day 24. Significant increases in BMP-2 and IGF-I mRNA expression after RME were found on day 3 compared with day 0. By immunohistochemistry, BMP-2 and IGF-I were detected in osteoblasts on days 5 and 7, in endothelial cells of blood vessels, and fibroblasts on day 7. Expansion of the mid-palatal suture continues for 12 days after a single RME, and restoration requires more than 30 days. Additionally, BMP-2 and IGF-I may play important roles in the restoration process.

Analysis of a force system for upper molar distalization using a trans-palatal arch and mini-implant: a finite element analysis study.

The purpose of this study was to analyse distal movements of molars in a force system using a trans-palatal arch (TPA), fixed to the maxillary first molar, and mini-implants placed at the palatal midline, considering the diagnostic standard for placement site in association with variation in upper molar locations, using finite element (FE) analysis. Three-dimensional FE models, divided by the differing direction of traction force, mesiodistal locations of the left and right molars, and the lateral location of the mini-implant were constructed. (1) When a traction force was fixed from the height of alveolar crest to the mini-implant placed at the middle of palate, the molars underwent bodily movement. (2) When the location of the mini-implant was moved to the left of the midline, the amount of distal movement of the left molar increased. When the mesiodistal locations of the left and right molars differed, the amount of distal movement of the molar located mesially was larger than that of the contralateral molar, even when the mini-implant was located on the midline.

Osteoblasts stimulate osteoclastogenesis via RANKL expression more strongly than periodontal ligament cells do in response to PGE(2).

OBJECTIVE: Periodontal ligament cells (PDLs) produce prostaglandin E(2) (PGE(2)) in response to orthodontic force. PGE(2) is a potent osteoclast-inducing factor that induces the receptor activator of nuclear factor-κB ligand (RANKL). Some studies reported that PDLs express RANKL in response to mechanical stress, whereas another study reported that they do not. Based on an immunohistochemical study, RANKL expression is localized around the alveolar bone surface 3 days after tooth movement. However, ankylosed teeth cannot be moved by therapeutic mechanical stress, suggesting that PDLs play a major role in alveolar bone resorption. In this study, we compared the functional difference in osteoclastogenesis between human PDLs (HPDLs) and normal human osteoblasts (HOBs) as a direct effect of PGE(2) exposure. DESIGN: We examined the expression of RANKL, osteoprotegerin, and macrophage colony-stimulating factor after 48-h culture with or without PGE(2) (10(-11) to 10(-5)M) in HPDLs and HOBs. Then to confirm whether RANKL produced by PGE(2) treatment induces osteoclastogenesis or not, RAW264.7 cells were co-cultured on HPDLs or HOBs pretreated with 10(-6)M of PGE(2). RESULT: PGE(2) exposure increased significantly RANKL expression in HOBs compared with HPDLs. PGE(2) exposure significantly decreased osteoprotegerin expression in HPDLs compared with HOBs. The number of tartrate-resistant acid phosphatase staining osteoclast-like cells from RAW264.7 cells increased significantly by PGE(2) pretreatment in HOBs and was reduced by small interfering RNA knockdown of RANKL. CONCLUSION: These results suggest that osteoblasts strongly influence the stimulation of osteoclastogenesis via RANKL, induced by PGE(2) in periodontal tissues, compared with PDLs.

Effect of Ga-Al-As laser irradiation on COX-2 and cPLA2-alpha expression in compressed human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Prostaglandin E(2) (PGE(2)), which has bone-resorptive activity, is produced by human periodontal ligament (PDL) cells in response to mechanical stress. We previously reported that low level laser (LLL) irradiation inhibited PGE(2) production in PDL cells in response to mechanical stress; however, the mechanism underlying this inhibitory effect was unclear. Thus, we sought to determine the mechanism underlying the inhibitory effect of LLL on PGE(2) production in compressed PDL cells. STUDY DESIGN/MATERIALS AND METHODS: A compressive force of 2.0 g/cm(2) was applied for 24 hours to human PDL cells obtained from premolars extracted for orthodontic treatment. LLL irradiation (Ga-Al-As laser, 830 nm, 3.82 J/cm(2)) was applied 6 hours before, 1 hour before, and immediately after the application of compressive force. The mRNA expression of COX-2 and cPLA(2)-alpha was then examined by real-time PCR. RESULTS: LLL irradiation significantly inhibited COX-2 and cPLA(2)-alpha mRNA expression, which was increased in response to the application of a compressive force. Moreover, LLL irradiation immediately after compression had the strongest inhibitory effect on the expression of these genes. CONCLUSIONS: The inhibition of PGE(2) production by LLL irradiation in compressed PDL cells may be due to the inhibition of COX-2 and cPLA(2)-alpha expression and is most pronounced immediately after the application of a compressive force.

Low-intensity laser irradiation stimulates mineralization via increased BMPs in MC3T3-E1 cells.

BACKGROUND: Previously, we reported that low-intensity laser irradiation accelerated bone formation, and that this mechanism deeply involved insulin-like growth factor I expression. However, as bone formation is supported by many local factors, the mechanism involved in laser irradiation remains incompletely understood. Therefore, the purpose of this study was to determine the effects of laser irradiation on the osteogenic response in vitro. METHODS: Mouse osteoblast-like cells, MC3T3-E1, were cultured and were irradiated for 5-20 minutes (0.96-3.82 J/cm(2)) at the subconfluent stage using a low-intensity Ga-Al-As diode laser apparatus. After laser irradiation, expression of bone morphogenetic proteins (BMPs), transcription factors (Runx2, Osterix, Dlx5, Msx2), and phosphorylation of Smad1 were determined, and calcium content of cell cultures was also determined. RESULTS: Irradiation at 1.91 J/cm(2) significantly increased the expression of BMPs and Runx2, Osterix, Dlx5, Msx2, and the phosphorylation of Smad1. Noggin, a BMP receptor blocker, inhibited the laser-induced Runx2 expression and phosphorylation of Smad1. Moreover, laser irradiation significantly increased the calcium content of cell cultures, and noggin inhibited this increase. CONCLUSION: These results suggest that low-intensity laser irradiation stimulates in vitro mineralization via increased expression of BMPs and transcription factors associated with osteoblast differentiation.

Compressive force stimulates the gene expression of IL-17s and their receptors in MC3T3-E1 cells.

During orthodontic tooth movement, cytokines released from periodontal ligament fibroblasts and alveolar bone osteoblasts can alter the process of bone remodeling. Recently, interleukin-17 (IL-17) was found to stimulate osteoclastic resorption through osteoblasts by inducing receptor activator of nuclear factor κB ligand (RANKL) expression. However, the relationship between mechanical stress and IL-17 production by osteoblasts is not clear. Therefore, we examined the effect of compressive force on the expressions of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, and their receptors (IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE) using MC3T3-E1 cells as osteoblast-like cells. We also examined the effect of IL-17A on the expression of IL-17Rs, RANKL, macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG). The cells were cultured with or without continuous compressive force (1.0 and 2.0 g/cm(2)) for up to 24 hr. The cells were also cultured with or without IL-17A (0.1, 1.0, or 10 ng/ml) for up to 72 hr. The mRNA expressions of IL-17s and their receptors were estimated by real-time polymerase chain reaction. The expression levels of IL-17s and their receptors increased depending on the compressive force. The addition of IL-17A increased the expression of IL-17RA, IL-17RB, IL-17RC, IL-17RE, RANKL, and M-CSF, whereas it decreased OPG expression. These results indicate that compressive force induces the expression of IL-17s and their receptors in osteoblast-like cells and that IL-17s and their receptors produced in response to compressive force may affect osteoclastogenesis through the expression of RANKL, M-CSF, and OPG.

Low-intensity laser irradiation stimulates bone nodule formation via insulin-like growth factor-I expression in rat calvarial cells.

BACKGROUND AND OBJECTIVE: We previously reported that low-intensity laser irradiation stimulated bone nodule formation through enhanced cellular proliferation and differentiation. However, the mechanisms of irradiation are unclear. Thus, we attempted to determine the responsibility of insulin-like growth factor (IGF)-I for the action observed. STUDY DESIGN/MATERIALS AND METHODS: Osteoblast-like cells were isolated from fetal rat calvariae and cultured with rat recombinant (r) IGF-I, IGF-I-antibody (Ab), and/or the cells were irradiated once (3.75 J/cm(2)) with a low-intensity Ga-Al-As laser (830 nm). The number and area of bone nodules formed in the culture were analyzed, and IGF-I expression was also examined. RESULTS: Treatment with rIGF-I significantly stimulated the number and area of bone nodules. This stimulatory effect was quite similar to those by laser irradiation, and this stimulation was abrogated dose-dependently by treatment with IGF-I-Ab. Moreover, laser irradiation significantly increased IGF-I protein and gene expression. CONCLUSION: The stimulatory effect of bone nodule formation by low-intensity laser irradiation will be at least partly mediated by IGF-I expression.