Four-point Bending System Research Articles

An investigation into the tension lap splices

The purpose of this study is to investigate the effect of lap joint length on structural performance under ultimate load using both nonlinear numerical analysis and laboratory experiments. Three-dimensional (3D) finite element models were employed for the numerical simulations, which were carried out using LUSAS software. A typical four-point bending system was modelled in order to analyze the impact of lap length. In total, 24 cases of lap length were studied across three concrete grades: C30, C45, and C60. The study also examined the influence of beam depth, shear link spacing, and concrete grade on lap joint performance. In order to validate the reliability of the 3D model, experimental data from RC beam test specimens were used.

Dkk-1-TNF-α crosstalk regulates MC3T3E1 pre-osteoblast proliferation and differentiation under mechanical stress through the ERK signaling pathway.

The study aims to explore the role of the ERK signaling pathway in the crosstalk between Dkk-1 and TNF-α in MC3T3E1 pre-osteoblasts under cyclic tensile/compressive stress. A forced four-point bending system was used to apply cyclic uniaxial tensile/compressive strain (2000 μ, 0.5Hz) to MC3T3E1 cells. Dkk-1 and TNF-α expression were upregulated in MC3T3E1 cells under compressive strain. Cell proliferation, the cell cycle, osteogenesis-related gene (Wnt5a, Runx2, Osterix) expression, β-catenin expression, and the p-ERK/ERK ratio were significantly enhanced, whereas apoptosis, the RANKL/OPG ratio, and TNF-α expression were significantly attenuated, by Dkk-1 silencing. Dkk-1 expression increased and the effects of Dkk-1 silencing were reversed when exogenous TNF-α was added. Mechanically, TNF-α crosstalked with Dkk-1 through ERK signaling in MC3T3E1 cells. ERK signaling blockade impaired Dkk-1-induced TNF-α expression and TNF-α-mediated Dkk-1 expression. Dkk-1 and TNF-α crosstalked, partially through ERK signaling, in MC3T3E1 cells under compressive/tensile strain, synergistically modulating various biological behaviors of the cells. These findings not only provide mechanical insight into the cellular events and molecular regulation of orthodontic tooth movement (OTM), but also aid the development of novel strategies to accelerate OTM.

Antiferromagnetic chromium thin films as piezoresistive sensor materials

Sputter-deposited thin films of pure chromium and of chromium with small amounts of nitrogen are characterized regarding their electrical resistivity and strain dependence, i.e., piezoresistivity. They show a temperature dependent piezoresistive effect with gauge factors ranging approximately from 10 to 20. Related to this effect, they exhibit signs of a paramagnetic–antiferromagnetic transition at temperatures of 420 K and higher. For characterization, resistivity is measured at different strain levels: in a bending setup with a fixed radius and in a four-point bending system with reference strain gauges. Several parameter series of the sputter deposition of pure Cr films show that the higher gauge factor is correlated to a higher temperature coefficient of resistivity (TCR). The addition of nitrogen extends the range of TCR toward negative values, with gauge factors still in the same range as pure Cr. A Cr–N strain gauge is characterized and shows a linear, low-hysteresis strain–resistivity effect as well as a relatively large transverse sensitivity. Resistivity and gauge factor of one Cr and one Cr–N sample are measured from room temperature up to 600 K. These films have a resistivity anomaly indicating an antiferromagnetic ordering temperature TN that is much higher than in bulk Cr. The gauge factor has a maximum near TN and falls to small values at higher temperatures. The results indicate that the piezoresistivity of Cr and Cr-rich films is coupled to their spin-density wave (SDW) antiferromagnetism. Since the SDW state is known to be tunable through alloying, internal stress, and crystallinity, it appears that piezoresistivity can be influenced by these parameters as well.

Flexure behavior of hollow steel beams strengthening with ferrocement

Experimental and numerical studies are carried out to investigate the flexural behavior of precast lightweight composite beams composed of a hollow steel beam strengthened with a ferrocement layer. This composite beam comprises of a slender cold-formed hollow section of dimension (140 × 180 mm) of different thicknesses surrounded by a ferrocement layer. This composite beam is named a precast ferrocement cold-formed hollow section (FCH). Nine beams with a span of 1500 mm were tested under a four-point bending system to evaluate their flexural strength. Four different parameters have been examined, which are the spacing of shear connectors, ferrocement thickness, hollow steel section thickness, and the effect of surface friction. The test results showed that the presence of 30 mm ferrocement layer increased the capacity by 18% compared with the control specimen. The presence of shear connectors increases the capacity by 30%. Increasing the steel cross-section thicknesses increase section capacity by 16–33% and finally, increasing the surface friction has a very small effect on the section capacity, which can be neglected. The finite element models were developed and validated by the test results; results show a close agreement between the experimental and finite element results. Extensive parameters were investigated, which were mortar compressive strength (f'c) and the position of the shear connectors. The results indicated that increasing mortar strength increases section capacity ranging between 11% and 18%. The presence of a shear connector in the web and the lower flange of the hollow section have an insignificant effect.

Restoration of NAD+ homeostasis protects C2C12 myoblasts and mouse levator ani muscle from mechanical stress-induced damage

ABSTRACT Excessive mechanical traction damages the levator ani muscle (LAM), increasing the incidence of pelvic floor dysfunction (PFD). In this study, we explored the effects of oxidized nicotinamide adenine dinucleotide (NAD+) on the damage to both muscle cells and LAM tissue induced by mechanical stress (MS) at the cellular and animal levels. The cell damage model was established using a four-point bending system. The LAM damage model was established using vaginal distention and traction. Exogenous addition of PJ34, an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1), and the nicotinamide mononucleotide (NMN) precursor of NAD+ increased NAD+ levels. ATP content and mitochondrial membrane potential were measured to assess mitochondrial function. NAD+ levels, cell viability, and PARP-1 activity were detected using commercial kits. DNA damage in cells was detected with immunofluorescence staining, and LAM damage was detected with tissue TUNEL staining. PARP-1 activity and DNA damage of LAM were detected by immunohistochemistry. A small amount of DNA damage and PARP-1 activation did not affect NAD+ levels, while excessive DNA damage and PARP-1 activation led to an imbalance of NAD+ homeostasis. Furthermore, increasing NAD+ levels in vivo and in vitro could rescue mitochondrial dysfunction and damage to both muscle cells and LAM tissue induced by MS. In conclusion, MS can induce damage to both C2C12 cells and LAM tissue. Restoring NAD+ homeostasis can rescue this damage by improving mitochondrial function.

Protective effect and potential mechanism of Schwann cell‐derived exosomes on mechanical damage of rat dorsal root ganglion cells

Pudendal nerve (PN) injury was one of the most important pathogenesis of stress urinary incontinence (SUI). Schwann cell (SC)-derived exosomes could promote axonal regeneration. Wnt protein could significantly promote axonal regeneration and participate in the regulation of proliferation and differentiation of neural stem cells. Therefore, we sought to determine whether SCs-derived exosomes might also protect against damaged dorsal root ganglion cells (DRGs) through the Wnt/β-catenin pathway. The DRGs injury model was fabricated using a four-point bending system. The exosomes were separated from the SCs supernatant. XAV939, which was a small molecule inhibitor, was used to inhibit the Wnt/β-catenin pathway. Next, Cell Counting Kit-8 (CCK8) kit was used to detect cell activity. We evaluated the proliferative activity of DRG cells using the cell cycle and apoptosis detection kit. We assessed the cell apoptotic rates through the Annexin V/PE double staining. Finally, we detect the expression of downstream proteins of Wnt/β-catenin pathway in DRG cells using western blotting. SC-derived exosomes had protective effects on DRGs after mechanical damage, which could promote cell proliferation, transition of the cell cycle to the G2 phase, and inhibit cell apoptosis. Exogenous administration of XAV939 suppressed the promoting effect of SCs -derived exosomes on DRG cells and the expression of downstream proteins of Wnt/β-catenin pathway in DRG cells was also suppressed. These results suggested that SC-derived exosomes have a repairing effect on DRG cells injury caused by cyclic mechanical stretching (CMS) and the Wnt/β-catenin pathway is potentially involved in the process.

U-type mechanical clamping to anchor the CFRP composites in strengthening of RC beams subjected to bending

The present study investigates the performance of U-type clamp anchorage on external strengthening of reinforced concrete beams using carbon fibre-reinforced polymer composites. Unidirectional carbon fibre was used to strengthen the beam having a cross-section of 150 × 200 mm2 and the fabricated U-type clamp was used as a clamping device to enhance the fibre-reinforced polymer (FRP) bonding. To test the beam, four-point bending system was adopted and the experimental parameters were the number of FRP layers. The failure mode of strengthened beams with U-type clamp was very similar (rupture of FRP composites) to the beams without U-type clamp. However, the delamination of FRP was evaded by counteracting the peeling stress development at the edge of fibre. Since the beams strengthened with U-type clamp exhibited more linear behaviour than that of the beams strengthened without U-type clamp, there may be a higher possibility for abrupt/rapid/brittle mode of failure and the ductility index of the beam with U-type clamp confirmed the above behaviour. However, the introduction of U-type clamp enhanced the flexural stiffness and ultimate strength by counteracting the debonding, and the beam with U-type clamp exhibited a maximum of 58.33% and 20.37% enhancement in flexural stiffness and ultimate strength than that of the beam without U-type clamp, respectively. The theoretical strength value of all strengthened beams was evaluated using models proposed in the previous research and compared with experimental results. It is inferred that the use of U-type clamp in FRP strengthening provides economic and structural benefits compared to a beam without U-type clamp.

Effect of depth of epoxy coated lapped splices on bond efficiency

This paper gives a report of research investigation carried out to study the effect of depth of epoxy coated lapped spliced bars on bond strength and efficiency. Thirty-six full size beams of varying lengths and sectional dimensions with lap spliced bars in constant moment region were cast and tested in a four-point bending system. Bond strength and efficiency of epoxy coated top reinforcing bars were investigated using beams with 300 mm and 500 mm depths. Three beam sizes (300 × 180 mm, 300 × 200 mm and 300 × 230 mm) and high yield reinforcing bars (16 mm, 20 mm and 28 mm) were coated with epoxy over the lap length and used with each depth. The ultimate moments from the tests were used to determine the stress developed in the reinforcing bars. Coated top reinforcing bars were found to have lower bond strength and efficiency than coated bottom reinforcing bars in the depths investigated. The location factor, α, which is a reduction factor determined using an average of three beams, for epoxy coated top bars were found to be 0.86, 0.84 and 0.89 respectively for 16 mm, 20 mm and 28 mm diameter bars at 300 mm depth and 0.62, 0.80 and 0.86 respectively for 16 mm, 20 mm and 28 mm diameter bars at 500 mm depth.

Strength and ductility behaviour of FRC beams strengthened with externally bonded GFRP laminates

The repair and rehabilitation of structural members are perhaps one of the most crucial problems in civil engineering applications. One of the advanced techniques of strengthening the reinforced concrete members is done by fiber-reinforced polymer composites. FRP is very effective to repair and strengthen the structural members that have become structurally weak over their life span. FRP repair system provides an economically viable alternative to traditional repair systems and materials. This experimental study focuses on the flexural strengthening of fiber reinforced concrete beams externally bonded with FRP laminates of different thicknesses. Six beams were cast for the study and tested under a four-point bending system. Out of which two beams were served as a control beam, one beam was considered as a reinforced concrete beam and the other was fiber reinforced concrete beam. The fibers used in this investigation were steel fiber. The beams were strengthened with GFRP of 3 mm and 5 mm of woven roving type. The study parameters of this investigation included yield load, ultimate load, deflection, yield load deflection, ultimate load deflection, deflection ductility, energy ductility, and the beam was found to be very effective in the load-carrying capacity, deflection, and ductility when compared to the control specimen. The fiber-reinforced concrete beam exhibit an increase in ultimate deflection by 79.3% when compared to the control specimen. GFRP strengthened beams showed an increase in ultimate deflection by 18.75% to 94.06%. GFRP strengthened fiber reinforced concrete beams showed an increase in ultimate deflection by 7.8 to 13.125%. GFRP strengthened beams showed an increase in ultimate deflection by 54.7% to 81.88%. GFRP strengthened fiber reinforced concrete beams showed an increase in ultimate load-carrying capacity by 36.9% to 48.7%. The ductility for the specimens increases by 1.27% to 1.34%, compared to the controlled specimen.

Effect of constant compressive stress induced by imitating Tuina stimulation with various durations on the cell cycle, cellular secretion, apoptosis, and expression of myogenic differentiation and myogenic factor 5 of rat skeletal muscle cells in vitro.

To investigate the effect of constant compressive stress induced by imitating Tuina stimulation with various durations on the cell cycle, cellular secretion, apoptosis, and expression of myogenic regulatory factors (MRFs), myogenic factor 5(Myf5) and myogenic differentiation (MyoD) of rat skeletal muscle cells (RSkMCs) in vitro. Third passage RSkMCs were subjected to constant compressive stresses with various durations at 2000 strain for 15, 30, 60, 90, and 120 min via a four-point bending system. The control group (CG) was cultured in the absence of mechanical loading. Alterations of the cell cycle and apoptosis rate were detected by flow cytometry (FCM). The concentrations of interleukin 6 (IL-6) / prostaglandin E2 (PGE2) and nitric oxide (NO) in supernatants were determined by enzyme-linked immunosorbent assays and the nitrate reductase method, respectively. Expression of Myf5 and MyoD was detected by immunohistochemistry. Compared with the CG, a significant alteration was observed in the synthesis phase fraction (SPF) (P < 0.01). The SPF and proliferation index (PI) were reduced from 15 to 90 min, but reached levels similar to those at 120 min. Apoptosis was increased significantly at 30 min (P < 0.05) and especially at 90 and 120 min (P < 0.01). Expression of MyoD and Myf5 was increased significantly at 15, 30, and 90 min (P < 0.01). Compared with 15 and 30 min, MyoD and Myf5 expression at 60 and 120 min was decreased significantly (P < 0.01). Compared with 60 min, MyoD expression at 90 min was increased significantly (P < 0.05), whereas MyoD and Myf5 expression at 120 min was significantly lower (P < 0.05). The IL-6 concentration was increased at 60 min compared with the CG and 15 min (P < 0.05), whereas the concentrations of PGE2 and NO were the highest at 15 and 30 min, respectively, compared with the CG and other time points (P < 0.05). The cell cycle, secretion, apoptosis, and Myf5 and MyoD expression of RSkMCs were regulated by compressive stress in a time-dependent manner. SPF and PI were inhibited at short durations (< 90 min), but NO and PGE2 secretion was the highest at shorter durations (< 30 min). With the prolongation of stimulation time, SPF, PI, and apoptosis were increased, but Myf5 and MyoD expression was decreased gradually at 15-30 min.

EXPERIMENTAL STUDY ON REINFORCED CARBON STEEL FIBERS BEAMS WITH GGBS AS PARTIAL REPLACEMENT OF CEMENT

The present paper gives detail study about effect of different percentages of carbon steel fibres and partial replacement of GGBS on deflection of reinforced concrete beams. The cement has been replaced by GGBS accordingly in the range of 30% by weight of cement for M30 grade concrete. It helps to reduce the heat of hydration. The beams were casted using two different percentages of Carbon steel fibres namely 1% and 2% according to the weight of concrete. The Carbon steel fibers used in the project are made of cold raddled steel wire. The deflection of reinforced concrete beams is tested at 28 days after casting using load frame. The dimensions of concrete specimens are 1500 mm × 230 mm × 230 mm. The experimental results are compared. It is concluded that 2% carbon steel fibres mix gives better strength compared to nominal mix of concrete. Objective of the study the objective of the paper was to study flexural behavior of carbon steel fiber reinforced concrete. Laboratory scale beams were cast and tested under four-point bending system (two loading point plus two simple supports) to study the ultimate load and deflection

Adhesion Properties of Electroplating Process Between Polyimide and Metal Layer for Polymer/Metal Hybrid Bonding

This article investigates the adhesion properties between the polyimide layers and the Ti/Cu layer after the electroplating process. The adhesion strength was evaluated by the four-point bending system, and the influence factors were confirmed by using different electroplating times and currents. The fracture interface after the four-point bending measurement was characterized, and the interface spectrum between the polyimide and the Ti/Cu layer was analyzed. The results showed that the modified polyimide-like material has better adhesion performance than the conventional polyimide. Moreover, the mechanism of degradation was proposed. As a result, the modified polyimide-like material can be applied in electroplating, a redistribution layer (RDL) formation, and packaging at the back-end-of-line processes.

Comparison of bond efficiency of various coating materials

The paper reports an experimental research investigation carried out to compare the bond resistance and efficiency of various coating materials, including epoxy, which were sourced locally. Thirty-four full size beams of varying lengths and sectional dimensions, with lap spliced bars in constant moment region were cast and tested in a four-point bending system. Three varying high yield diameter bars 16 mm, 20 mm and 28 mm were coated with various coating materials such as epoxy, chlorinated rubber, tyrolin, vinyl chloride and zinc ethyl silicate in an attempt to find a cheaper but effective coating material than epoxy. The ultimate moment from the tests were used to determine the stress developed in the steel rods. The bond efficiency, i.e. the ratio of test bond stresses and the theoretical bond stresses was used for the comparison of the various coating materials. The bond resistance and efficiency of vinyl chloride coated bars were found to be higher than that of epoxy and other materials, in all the beams tested. Statistically, there appear not to be significant difference between the bond efficiency of epoxy and vinyl chloride.

Flexural capacity of bi-directional GFRP strengthened RC beams with end anchorages

Purpose The purpose of this paper is to present the results of experimental and theoretical studies on the flexural capacity of reinforced concrete (RC) beams strengthened using externally bonded bi-directional glass fibre reinforced polymer (GFRP) composites and different end anchorage systems. Design/methodology/approach A series of nine RC beams with a length of 1,600 mm and a cross-section of 200 mm depth and 100 mm width were prepared and externally strengthened in flexure with bi-directional GFRP composites. These strengthened beams were anchored with three different end anchorage systems namely closed GFRP wraps, GFRP U-wraps and mechanical anchors. All these beams were tested with four-point bending system up to failure. The experimental results are compared with the theoretical results obtained using the relevant design guidelines. Findings The experimental results demonstrate a significant increase in the flexural performance of the GFRP strengthened beams with regard to the ultimate load carrying capacity and stiffness. The results also show that GFRP strengthened beams without end anchorages experienced intermediate concrete debonding failure at the GFRP plate end, whereas all the GFRP strengthened beams with different end anchorage systems failed in rupture of GFRP with concrete crushing. The theoretical results revealed no significant difference among the relevant design guidelines with regard to the predicted ultimate moment capacities of the bi-directional GFRP strengthened RC beams. However, the results show that ACI Committee 440 Report (2008) design recommendation provides reasonably acceptable predictions for the ultimate moment capacities of the tested beams strengthened externally with bi-directional GFRP reinforcement followed by FIB Bulletin 14 (2001) and eventually by JSCE (1997). Originality/value The research work presented in this manuscript is authentic and could contribute to the understanding of the overall behaviour of RC beams strengthened with FRP and different end anchorage systems under flexural loading.

Lithium chloride attenuates suppressed differentiation induced by mechanical strain in cementoblasts

ABSTRACTAim: The purpose of this study was to investigate the influence of mechanical strain on OCCM-30 cementoblast differentiation and Wnt/β-catenin pathway activity.Materials and Methods: Mechanical tension in the form of 2500-µ strain was applied to the cells using the Forcel four-point bending system, with or without the Wnt signaling activator, lithium chloride. Changes in cell differentiation and the expression of Wnt/β-catenin pathway components in response to strain and lithium chloride were assessed by real-time PCR, immunofluorescence, and western blotting.Results: The mRNA expression levels of the cementoblastogenesis-related genes alkaline phosphatase, runt-related transcription factor 2, and collagen 1, were decreased with mechanical strain. Similarly, the Wnt signaling pathway component genes LRP5, AXIN2, and LEF1 were decreased. The immunofluorescence assay demonstrated that scant β-catenin underwent nuclear translocation after the cells were subjected to mechanical strain. Moreover, western blotting showed that the protein levels of both β-catenin and phosphorylated β-catenin were increased after mechanical strain. In the presence of lithium chloride, the differentiation that was suppressed by mechanical strain was attenuated.Conclusions: 2500-µ strain mechanical strain inhibited cementoblast differentiation activity in vitro, which could be alleviated by actviating Wnt/β-catenin signaling using lithium chloride.

Primary cilia and autophagy interaction is involved in mechanical stress mediated cartilage development via ERK/mTOR axis

AimsBiomechanical reactivity is a special property of chondrocytes and mechanical stress can affect the development of cartilage. Primary cilia have been proved a cellular sensory which can detect physical and chemical stimuli extracellular and initiate multiple signaling transduction. Autophagy is an important environmental adaptive mechanism for cells maintenance of homeostasis. The aims of this study were to detect whether there is an interaction between primary cilia and autophagy in the regulation of mechanical stress-mediated cartilage development and to explore the underlying mechanism. Main methodsIn this study, chondrocytes were treated with cyclic tensile strain (CTS) by the four-point bending system. Chondrocytes viability, proliferation and differentiation capacities were analyzed by western blot and live/dead assays after CTS of different intensities. Meanwhile, primary cilia incidence and length changes, and autophagy expression were detected by immunofluorescence staining. The primary cilia and autophagy interaction regulation and the underlying mechanism were detected by immunofluorescence double staining and western blot. Key findingsMechanical stress could affect chondrocytes proliferation, phenotype and viability in an intensity dependent manner. The incidence and length of primary cilia as well as autophagy expression could be regulated by CTS. The integrity of primary cilia structure is vital for mechanical stress regulated ERK/mTOR signaling transduction and autophagy expression in chondrocyte. SignificanceThese findings indicate that mechanical stress could affect the interaction between primary cilia and autophagy and help to reveal the underlying mechanism of stress regulated cartilage development.

The effect of cyclic tensile force on the actin cytoskeleton organization and morphology of human periodontal ligament cells

To explore Girdin/Akt pathway protein expression and morphology change by cyclic tension in the periodontal ligament cells. Human periodontal ligament cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h though a four-point bending system. Cyclic tension force upregulated F-actin, Girdin and Akt expression in hPDL. In transmission electron microscope assay showed that there are more and bigger mitochondria, more and longer cynapses, more cellular organisms after tension force stimulation than control. The actin filament was changed to be regular lines and pointed to poles of cells. However, we found that the Girdin-depleted cells are small and there are more micro-organisms including more lysosomes and matrix vesicles than control. These finding suggest that the STAT3/Girdin/Akt pathway in PDL to response to mechanical stimulation as well, and Girdin may play a significant role in triggering cell proliferation and migration during orthodontic treatment. It provided an insight into the molecular basis for development of a vitro cell model in studying orthodontic treatment.

The effect of mechanical stretching stress on the proliferation, apoptosis of degenerative disc nucleus pulposus cells and expression of extracellular matrix

Objective To observe in vitro the effect of mechanical stress at different intensities on the proliferation, apoptosis and extracellular matrix of degenerative human nucleus pulposus cells. Methods The cells were isolated and cultured in vitro, and divided into a control group, a low-intensity group, a medium-intensity group and a high-intensity group. The low-, medium- and high-intensity groups were stretched mechanically by 1000 μ, 2000 μ or 4000 μ respectively for 6 hours using a four-point bending system, while the control group was not stressed. Flow cytometry was used to explore any changes in the cell cycle and the proliferation index (PI). The expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (BCL-2)/Bax, collagen II and aggrecan were measured using real-time quantitative polymerase chain reactions. Results The mechanical stretching significantly influenced proliferation, apoptosis and the extracellular matrices compared with the control group. The PI and PCNA expression increased at first and then decreased gradually with the exercise intensity. Compared with the control group, the mRNA expression level of Bcl-2/Bax increased significantly to 1.53 times that of the control group after 1000 μ stretching, but to only 0.71 times that of the control group at 2000 μ and 0.43 times at 4000 μ. The gene expression of collagen II increased significantly by 1.1 times and that of aggrecan by 1.3 times after 1000 μ stress stimulation compared with the control group (P≤0.05). However, the expression of collagen II and aggrecan was inhibited significantly at 2000 and 4000 μ , with the lowest levels at 4000 μ (P≤0.05). Conclusion Stretching at different intensities has different effects on the proliferation, apoptosis and extracellular matrix of human pulposus cells with degenerate nuclei. Key words: Mechanical stress; Stretching; Degenerate nuclei; Pulposus cells; Proliferation; Apoptosis; Extracellular matrices

PTH1R signalling regulates the mechanotransduction process of cementoblasts under cyclic tensile stress.

To investigate the regulatory role of type I parathyroid hormone receptor (PTH1R) signalling in the mechanotransduction process of cementoblasts under cyclic tensile stress (CTS). Immortalized cementoblast cell line OCCM-30 were employed and subjected to cyclic tensile strain applied by a four-point bending system. The expression of PTHrP and PTH1R, as well as cementoblastic transcription factor Runx-2, Osterix, and extracellular matrix protein COL-1 and OPN were assessed by quantitative real-time polymerase chain reaction and western blot analysis. PTH1R expression was knocked down by siPTH1R transfection, and the alteration of cementoblastic biomarkers expression was examined to evaluate the function of PTH1R. Furthermore, to investigate possible downstream molecules, expression of signal molecule ERK1/2 with or without siPTH1R transfection, and the effect of ERK inhibitor PD98059 on the expression of cementoblastic biomarkers was also examined. Cyclic tensile strain elevated the expression of PTHrP and PTH1R, as well as cementoblastic biomarkers Runx-2, Osterix, COL-1, and OPN in a time-dependent manner, which was inhibited by siPTH1R transfection. The expression of phosphorylated ERK1/2 was upregulated time-dependently under cyclic stretch, which was also inhibited by siPTH1R transfection, and pretreatment of p-ERK1/2 inhibitor PD98059 undermined the increase of Runx-2, Osterix, COL-1, and OPN prominently. The findings of the present study indicate that PTH1R signalling plays a regulatory role in the CTS induced cementoblastic differentiation in mature cementoblasts, and ERK1/2 is essentially involved as a downstream intracellular signal molecule in this mechanotransduction process.

The p38 MAPK and NF-κB Pathways are Involved in Cyclic Compressive Force-induced IL-6 Secretion in MLO-Y4 Cells

ABSTRACT We previously revealed the involvement of extracellular regulated protein kinases 1/2 (ERK1/2) in interleukin-6 (IL-6) secretion induced by cyclic compressive force (CCF) in MLO-Y4 cells. In this study, we investigated the contributions of the p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways to IL-6 secretion by stimulating MLO-Y4 cells with CCF. At 80% confluence, different magnitudes (1000μstrain, 2000 μstrain and 4000 μstrain), frequencies (0.5 Hz, 1.0 Hz and 2.0 Hz) and durations (10 min, 30 min, 1 h, 3 h and 6 h) of CCF were loaded onto cells using a four-point bending system. Flow Cytometry (FCM) analysis was used to analyze cell mortality rates after CCF loading. p38 and p65 phosphorylation as well as IκBα degradation in MLO-Y4 cells were detected by Western blotting (WB). Changes in IL-6 secretion after inhibitor treatment were assessed by enzyme-linked immunosorbent assays (ELISAs). Cellular viability was over 90 percent after CCF. p38 and p65 phosphorylation increased under all conditions, whereas IκBα protein levels decreased. However, phosphorylation and degradation were not completely dependent on the loading magnitude, frequency or duration. Furthermore, p38 inhibition using the specific inhibitor SB203580 reduced both p38 phosphorylation and IL-6 secretion. Similarly, NF-κB inhibition using BAY 11-7082 decreased p65 phosphorylation and IL-6 secretion but increased the concentration of IκBα. These findings reveal significant roles for the p38 and NF-κB signaling pathways in IL-6 secretion induced by CCF in MLO-Y4 cells.