Effects of continuous and released compressive force on osteoclastogenesis invitro.

Objective: Compressive force has been found to be catabolic to alveolar bone during orthodontic tooth movement. This study quantified the fusion of mononuclear RAW 264.7 cells (a murine osteoclastic-like cell line) into multinucleated osteoclasts under a hydrostatic pressure-generated mechanical compression-the new model of various magnitudes and durations. Methods: RAW 264.7 cells were subjected to 0.3, 0.6 or 0.9 g/cm2 of compressive force by an acrylic cylinder custom-made by laser cutting or no compressive force for 4 days during osteoclastogenic induction. TRAP-positive multinucleated cells were quantified. For the release from force experiment, osteoclastogenesis was induced by 0.6 g/cm2 mechanical stimuli for 0, 1, 2, 3 or 4 days. Cell viability, TRAP-positive multinucleated cells, DCSTAMP and Cathepsin K (CTSK) gene expression were evaluated 4 days after release from force. Results: Compressive force at 0.6 and 0.9 g/cm2 significantly increase the number of TRAP-positive multinucleated cells (P < 0.05). Release from continuous mechanical compression after 4 days significantly elevated the number of TRAP-positive multinucleated cells and DCSTAMP and CTSK mRNA expression, with no adverse effects on cell viability (P < 0.05). Conclusions: Continuous stimulation with compressive force induced osteoclastogenesis in RAW 264.7 cells by enhancing DCSTAMP and CTSK expression, which provides new understanding of bone remodeling during orthodontic treatment.

Effects of compressive stress combined with mechanical vibration on osteoclastogenesis in RAW 264.7 cells.

OBJECTIVES: To investigate the effects of compressive force and/or mechanical vibration on NFATc1, DCSTAMP, and CTSK (cathepsin K) gene expression and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in RAW 264.7 cells, a murine osteoclastic-like cell line. MATERIALS AND METHODS: RAW 264.7 cells were subjected to mechanical vibration, compressive force, or compressive force combined with vibration. Cell viability and the numbers of TRAP-positive multinucleated cells were evaluated. NFATc1, DCSTAMP, and CTSK gene expressions were analyzed using real-time quantitative reverse transcription polymerase chain reaction. RESULTS: Compressive force combined with mechanical vibration significantly increased the numbers of TRAP-positive multinucleated cells but did not significantly affect cell viability. In addition, compressive force combined with mechanical vibration significantly increased NFATc1, DCSTAMP, and CTSK mRNA expression compared with compressive force or vibration alone. CONCLUSIONS: Compressive force combined with mechanical vibration induces osteoclastogenesis and upregulates NFATc1, DCSTAMP, and CTSK gene expression in RAW 264.7 cells. These results provide more insight into the mechanisms by which vibratory force accelerates orthodontic tooth movement.

Vibration activates the actin/NF-κB axis and upregulates IL-6 and IL-8 expression in human periodontal ligament cells.

We previously reported that mechanical vibration-induced proinflammatory cytokines, interleukin-6 (IL-6) and IL-8, expression in human periodontal ligament (hPDL) cells, however, the underlying mechanism remained unclear. Mechanical stimuli are able to activate cellular responses by inducing the activation of several signaling pathways including cytoskeletal changes and inflammation. The actin cytoskeleton is a highly dynamic network and plays many important roles in intracellular events. Here, we aimed to investigate the involvement of a pivotal mediator of inflammatory responses, nuclear factor-κB (NF-κB), and actin polymerization in vibration-induced upregulation of IL-6 and IL-8 expression in hPDL cells. hPDL cells were pretreated with the NF-κB inhibitor BAY 11-7082 or cytochalasin D, respectively, before exposure to vibration. IL-6 and IL-8 messenger RNA (mRNA) and protein expression were quantified by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. Subcellular localization of the NF-κB p65 subunit was visualized by immunofluorescent staining. We found an increase in NF-κB nuclear translocation in vibrated cells compared with control cells. Pretreatment with BAY 11-7082 significantly inhibited vibration-induced IL-6 and IL-8 mRNA and protein expression in hPDL cells. Moreover, pretreatment with cytochalasin D inhibited NF-κB nuclear translocation and attenuated upregulation of IL-6 and IL-8 mRNA and protein in vibrated cells. Therefore, modulation of actin cytoskeletal polymerization in response to vibration may activate the NF-κB signaling pathway and subsequently upregulate IL-6 and IL-8 expression in hPDL cells.

Low magnitude high frequency vibration induces RANKL via cyclooxygenase pathway in human periodontal ligament cells in vitro.

OBJECTIVE: This study aimed to examine the effects of PGE2 on RANKL expression in response to vibration and vibration in combination with compressive stress and characterise this transduction pathway in periodontal ligament (PDL) cells. METHODS: Cultured human PDL cells obtained from extracted premolar teeth (from six individuals) were subjected to three cycles of vibration (0.3 g, 30 Hz for 20 min every 24 h; V), compressive stress (1.5 g/cm2, 48 h; C) or vibration in combination with compressive stress (VC). To investigate whether the expression of RANKL and PGE2 was COX-dependent, PDL cells were treated with indomethacin prior to the onset of mechanical stimulation. RANKL and OPG expressions were examined by quantitative real-time polymerase chain reaction (qPCR). Quantification of PGE2, soluble RANKL (sRANKL) and OPG productions were measured using enzyme-linked immunosorbent assay (ELISAs). RESULTS: All mechanical stresses (V, C and VC) significantly increased PGE2 and RANKL. OPG was not affected by vibration, but was downregulated in compressed cells (C and VC). Indomethacin abolished induction of RANKL and downregulated OPG in response to all mechanical stresses. CONCLUSION: These results suggest that vibration, compressive stress and vibration in combination with compressive stress induce RANKL expression in human PDL cells by activating the cyclooxygenase pathway.

Vibration enhances PGE2 , IL-6, and IL-8 expression in compressed hPDL cells via cyclooxygenase pathway.

BACKGROUND: Although vibration combined with orthodontic force may accelerate orthodontic tooth movement, little is known about the mechanisms that regulate the associated cellular responses. The goal of this study was to investigate whether mechanical vibration combined with compressive force regulates cyclooxygenase (COX)-2/prostaglandin E2 (PGE2 ) and interleukin (IL)-6 and IL-8 messenger RNA (mRNA) and protein expression in human periodontal ligament (hPDL) cells via the COX pathway. METHODS: The primary cultured hPDL cells were exposed to mechanical vibration, compressive force or a combination of both mechanical vibration and compressive force at 24, 48, and 72 hours. The COX-2, IL-6, IL-8, receptor activator of nuclear factor kappa-Β ligand (RANKL), and osteoprotegrin (OPG) mRNA expressions were determined using quantitative real-time polymerase chain reaction (qPCR). The PGE2 , IL-6, and IL-8 protein expressions were quantified by enzyme-linked immunosorbent assay (ELISA). To demonstrate whether the expression of PGE2 , IL-6, and IL-8 was in the COX-dependent pathway, the hPDL cells were treated with indomethacin. To determine whether PGE2 stimulated the hPDL cells to express IL-6 and IL-8, exogenous PGE2 was added to the culture media. RESULTS: The combination of mechanical vibration and compressive force synergistically upregulated RANKL/OPG, COX-2/PGE2 , IL-6 and IL-8 mRNA, and protein expression. The indomethacin significantly attenuated the increases of PGE2 , IL-6, and IL-8 expression in cells stimulated with compressive force or mechanical vibration combined with compressive force. In addition, exogenous PGE2 increased IL-6 and IL-8 mRNA and protein expressions in hPDL cells. CONCLUSION: Mechanical vibration may enhance alveolar bone resorption at the compression side during orthodontic tooth movement via a mechanism involving the cyclooxygenase pathway.

Effects of low magnitude high frequency mechanical vibration combined with compressive force on human periodontal ligament cells in vitro.

Objective: Vibration can be used to accelerate tooth movement, though the exact mechanisms remain unclear. This study aimed to investigate the effects of low magnitude high frequency (LMHF) vibration combined with compressive force on periodontal ligament (PDL) cells in vitro. Materials and methods: Human PDL cells were isolated from extracted premolar teeth of four individuals. To determine the optimal frequency for later used in combination with compressive force, three cycles of low-magnitude (0.3 g) vibrations at various frequencies (30, 60, or 90 Hz) were applied to PDL cells for 20 min every 24 h. To investigate the effects of vibration combined with compressive force, PDL cells were subjected to three cycles of optimal vibration frequency (V) or 1.5 g/cm2 compressive force for 48 h (C) or vibration combined with compressive force (VC). Cell viability was assessed using MTT assay. PGE2, soluble RANKL (sRANKL), and OPG production were quantified by ELISA. RANKL, OPG, and Runx2 expression were determined using real-time PCR. Results: Cell viability was decreased in groups C and VC. PGE2 and RANKL, but not OPG, were increased in groups V, C, and VC, thus increasing the RANKL/OPG ratio. The highest level was observed in group VC. sRANKL was increased in groups V, C, and VC; however, no significant different between the experimental groups. Runx2 expression was reduced in groups C and VC. Conclusions: Vibration increased PGE2, RANKL, and sRANKL, but not OPG and Runx2. Vibration had the additive effects on PGE2 and RANKL, but not sRANKL in compressed PDL cells.

Effects of tannin-fluoride and milk-fluoride mixture on human enamel erosion from inappropriately chlorinated pool water.

This in vitro study aimed to investigate the efficacy of tannin-fluoride and milk-fluoride mixtures on human enamel erosion after exposure to inappropriately chlorinated pool water. Enamel specimens were immersed in swimming pool water (pH 2.7) for 30 min and in each test reagent for 4 min once a day for 60 consecutive days (group I: control, group II: tannin-fluoride, group III: milk-fluoride, group IV: tannin-fluoride before and milk-fluoride after erosive challenge, and group V: milk containing tannin-fluoride before and after erosive exposure). Surface microhardness was assessed on days 0, 30, and 60. Scanning electron microscopy (SEM) and electron probe microanalysis (EPMA) were performed after treatment of samples for 60 days. Surface microhardness of experimental groups was ranked as follows: group III > group IV-group V > group II > group I (P < 0.05). Moreover, SEM images revealed deposition of substances on erosive enamel surface after treatment with tannin-fluoride and milk-fluoride mixtures. Furthermore, EPMA profiles showed decrease of phosphorus and increase of fluoride content in groups II and IV. In conclusion, we demonstrated that treatment with fluoridated milk with or without tannin-fluoride has protective effects against enamel erosion caused by low-pH swimming pool water.

Vibratory stimulation increases interleukin-1 beta secretion during orthodontic tooth movement.

OBJECTIVES: To investigate the effects of application of vibratory stimuli on interleukin (IL)-1ß secretion during maxillary canine distalization. MATERIALS AND METHODS: Split-mouth design study in 15 subjects (mean age, 22.9 years; range 19-25 years) whose bilateral maxillary first premolars were extracted with subsequent canine distalization. On the experimental side, light force (60 g) was applied to the canine for 3 months in combination with vibratory stimuli provided using an electric toothbrush 15 minutes a day for 2 months; only orthodontic force was applied to the contralateral control canine. Gingival crevicular fluid (GCF) was collected from the mesial and distal sides of each canine at each monthly appointment. IL-1ß levels were analyzed using an enzyme-linked immunosorbent assay. Canine movement was measured monthly. RESULTS: Overall, enhanced IL-1ß secretion was observed at the pressure sites of experimental canines compared to control canines (mean, 0.64 ± 0.33 pg/µL vs 0.10 ± 0.11 pg/µL, respectively, P < .001). The accumulative amount of tooth movement was greater for the experimental canine than for the control canine (mean, 2.85 ± 0.17 mm vs 1.77 ± 0.11 mm, respectively, P < .001). CONCLUSIONS: This study demonstrates that, in combination with light orthodontic force, application of vibratory stimuli using an electric toothbrush enhanced the secretion of IL-1ß in GCF and accelerated orthodontic tooth movement.

Effects of 1.23% acidulated phosphate fluoride gel and drinkable yogurt on human enamel erosion, in vitro.

OBJECTIVES: In this in vitro study, the authors sought to determine the effects of 1.23% acidulated phosphate fluoride gel (APF) or drinkable yogurt on human enamel after exposure to an acidic drink. METHOD AND MATERIALS: Sixteen surgically removed, caries-free, human third molars were cut into four portions: mesial, distal, buccal, and lingual. Each portion was distributed into one of four groups, with each specimen embedded in acrylic resin and its enamel center lightly ground and polished. Three groups of specimens were immersed in orange juice for 2 minutes. One group received no other treatment (OR), one group was subsequently immersed in drinkable yogurt for 5 minutes (OR:YO), while another group had 1.23% APF applied for 4 minutes (OR:APF). The final group was immersed in only drinkable yogurt for 5 minutes (YO). Each exposure was performed twice daily for 60 days; between exposures, the samples were stored in artificial saliva. The enamel surfaces were monitored by three criteria: erosion depth, surface hardness, and SEM. RESULTS: Erosion depth increased progressively in all groups. Surface microhardness progressively decreased in all groups except the OR:APF group, where hardness was significantly higher than other groups at 60 days (P < .05). SEM inspection revealed preferential loss of rod crystallites with retention of interrod crystallites in all specimens exposed to orange juice. The enamel exposed to only drinkable yogurt revealed modest and uniform etching. CONCLUSION: Drinkable yogurt alone or posttreatment of enamel after exposure to an acidic drink with either 1.23% APF or drinkable yogurt leads to enamel dissolution and does not reduce enamel erosion, in vitro.

Bovine serum albumin release from novel chitosan-fluoro-aluminosilicate glass ionomer cement: stability and cytotoxicity studies.

OBJECTIVE: This study aimed to evaluate the effect of adding chitosan (CS) to conventional glass ionomer cement (GIC) on protein release and its cytotoxicity. METHODS: Bovine serum albumin (BSA) was used as the released protein from two glass ionomer formulations. One (GIC+BSA) contained fluoro-aluminosilicate glass mixed with BSA, and another (GIC:CS+BSA) used a similar glass and BSA with 20% chitosan. Six disc specimens per group (10mm in diameter, 2mm in height) were prepared and placed in phosphate buffer saline, which was replaced at various times over 2 weeks. The released protein was determined by a BCA assay. Cytotoxicity of the extracts from these materials for 1, 2 and 7 days to dental pulp cells was evaluated using MTT assay. RESULTS: The GIC:CS+BSA released a burst of BSA in the first 6h, and slowly released at different rates over the 2 weeks. GIC+BSA showed a similar result, but protein could not be detected at the 12h. The protein release rate of GIC:CS+BSA was significantly greater than GIC+BSA (P<0.01); nearly three times higher. The released BSA had the same molecular weight as evaluated by SDS-PAGE. From the MTT assay, the percentages of viable cells were significantly different and can be arranged as: GIC:CS+BSA>GIC:CS>GI+BSA>GI and the cytotoxicity was increased by time of extraction. CONCLUSION: Chitosan added in glass ionomer cement can prolong release of BSA as well as not increasing the toxicity to pulp cells. This material may be useful for protein delivery.

Effect of xylitol and fluoride on enamel erosion in vitro.

This study aimed to determine the anti-erosive effects of xylitol, fluoride and a xylitol/fluoride combination used as an additive in an acidic drink or as mouthrinse after enamel was exposed to an acidic drink, in vitro. Human third molars were divided into 7 groups (A-G). Samples from groups A to D were immersed for 5 min in orange juice only (A), orange juice plus either 25% xylitol (B), F(-) 1 ppm (C) or a 25% xylitol/F(-) 1 ppm combination (D), respectively. Samples from groups E to G were immersed in orange juice for 5 min and then in either 40% xylitol (E), F(-) 227 ppm (F) or a 40% xylitol/F(-) 227 ppm combination (G), for 1 min respectively. This process was performed four times daily for 14 days. Mineral loss was determined from the lesion depth and surface hardness. Erosion depth progressively increased in all groups, except E, where erosion depth was significantly lower than group A. Surface microhardness progressively decreased in all groups, except E, where hardness was significantly higher than group A. This study demonstrated that addition of xylitol, fluoride or a xylitol/fluoride combination to an acidic drink or post-treatment with fluoride or a xylitol/fluoride combination could reduce, but not prevent, enamel erosion.

Developmental patterning of the circumvallate papilla.

Organogenesis is regulated by the sequential and reciprocal interactions between epithelial and mesenchymal tissues. Many molecules, including growth factors, transcription factors, extracellular matrices, cell surface receptors, and matrix degrading enzymes, have been found to be involved in this process. To investigate the molecular mechanism responsible for morphogenesis of the circumvallate papilla/von Ebners' gland complex, we examined the expression patterns of selected cell adhesion molecules, extracellular matrix molecules, innervation and cell division in the circumvallate papilla of mouse embryos from embryonic day 11.5 (E11.5) to E14. At E11.5-E13.5, the lingual epithelium, the site where the circumvallate papilla will develop, is negative for BrdU labeling. At E14-E15, we detected cell division in the papillary area, especially in the epithelial invagination where von Ebners' minor salivary gland will form. The basement membrane component, laminin, is expressed as a continuous thin line separating the epithelia from the underlying mesenchyme, but it is broadly and strongly expressed in the area wherethe nervefibers penetrate into the circumvallate papilla. At the E12-E12.5 stage of development, the epithelial thickening shows intense E-cadherin staining in the superficial and basal layers, but weak E-cadherin staining in the suprabasal layer. E-cadherin is strongly expressed, but appears dispersed among the basal layer of lingual epithelium, the site where nerve fibers will innervate. At E13, nerve fibers reach the circumvallate papilla. These nerve fibers penetrate into and split the epithelial cell mass into two stalks which will later differentiate to form the von Ebners' gland. These results suggest that 1) the formation of the circumvallate papilla does not initially depend on cell division, 2) cell migration likely plays a major role during circumvallate placode formation, 3) E-cadherin and laminin may play a role in nerve guidance and 4) innervation impacts the final morphogenesis of the circumvallate papilla.