Cyclic Tension Force Research Articles

Chemokine C-C motif ligand 8 in periodontal ligament during orthodontic tooth movement

ObjectivesTo investigate the roles of chemokine (C-C motif) ligand 8 (CCL8) in periodontal ligament during orthodontic tooth movement (OTM). MethodsBioinformatics analyzed 100 genes in human periodontal ligament cells that were most upregulated after 48 hours of mechanical stress, and these genes were classified through GO and KEGG databases. Nickel-titanium closed-coil springs were placed between right first molar and incisors to produce 20 cN of orthodontic force in eight-week-old male SD rats for 1 and 2 days, followed by immunohistochemical staining of CCL8. Human periodontal ligament fibroblasts (hPDLFs) were stimulated by 14% cyclic tension force (Flexcell FX-5000 T Tension System) or hypoxia conditions to mimic OTM for 1 and 2 days, then the resulting CCL8 were examined through ELISA. Scratching assay was performed by treating hPDLFs with different concentrations of CCL8 (1 ng/ml, 10 ng/ml, 100 ng/ml). The migration, proliferation, and adhesion abilities of 100 ng/ml CCL8-treated hPDLFs were also examined. qRT-PCR and western blot detected matrix metalloproteinase 3, periostin, and osteoprotegrin expressions of hPDLFs under 100 ng/ml CCL8. ResultsBioinformatic analysis demonstrated that CCL8 was upregulated after applying mechanical stress for 48 hours. CCL8 secretion showed upregulation after 24 hours of OTM applicationsin vivo and in vitro. CCL8-treated hPDLFs showed significant positive effects on cell proliferation and matrix metalloproteinase 3. It also inhibited periostin and osteoprotegrin expressions. ConclusionsCCL8 was upregulated in periodontal ligament during initial stage of OTM. Although CCL8 in human periodontal ligaments showed no significant effects on cell migration ability, it did enhance cell proliferation and osteoclastogenesis.

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.

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.

PYK2 mediates BzATP-induced extracellular matrix proteins synthesis

Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2’(3)-Ο-(4-Benzoylbenzoyl) adenosine-5’-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts.BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression.These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation.

The effect of biomechanical stimulation on osteoblast differentiation of human jaw periosteum-derived stem cells

BackgroundThis study was to investigate the effect of biomechanical stimulation on osteoblast differentiation of human periosteal-derived stem cell using the newly developed bioreactor.MethodsHuman periosteal-derived stem cells were harvested from the mandible during the extraction of an impacted third molar. Using the new bioreactor, 4% cyclic equibiaxial tension force (0.5 Hz) was applied for 2 and 8 h on the stem cells and cultured for 3, 7, and 14 days on the osteogenic medium. Biochemical changes of the osteoblasts after the biomechanical stimulation were investigated. No treatment group was referred to as control group.ResultsAlkaline phosphatase (ALP) activity and ALP messenger RNA (mRNA) expression level were higher in the strain group than those in the control group. The osteocalcin and osteonectin mRNA expressions were higher in the strain group compared to those in the control group on days 7 and 14. The vascular endothelial growth factor (VEGF) mRNA expression was higher in the strain group in comparison to that in the control group. Concentration of alizarin red S corresponding to calcium content was higher in the strain group than in the control group.ConclusionsThe study suggests that cyclic tension force could influence the osteoblast differentiation of periosteal-derived stem cells under optimal stimulation condition and the force could be applicable for tissue engineering.

Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment.

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.

Tension force-induced ATP promotes osteogenesis through P2X7 receptor in osteoblasts.

Orthodontic tooth movement induces alveolar bone resorption and formation by mechanical stimuli. Force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators that respond to bone cells by mechanical stimuli. However, the effect of tension force (TF)-induced ATP on osteogenesis is inadequately understood. Accordingly, we investigated the effect of TF on ATP production and osteogenesis in MC3T3-E1 cells. Cells were incubated in the presence or absence of P2X7 receptor antagonist A438079, and then stimulated with or without cyclic TF (6% or 18%) for a maximum of 24 h using Flexercell Strain Unit 3000. TF significantly increased extracellular ATP release compared to control. Six percent TF had maximum effect on ATP release compared to 18% TF and control. Six percent TF induced the expression of Runx2 and Osterix. Six percent TF also increased the expression of extracellular matrix proteins (ECMPs), ALP activity, and the calcium content in ECM. A438079 blocked the stimulatory effect of 6% TF on the expression of Runx2, Osterix and ECMPs, ALP activity, and calcium content in ECM. This study indicated that TF-induced extracellular ATP is released in osteoblasts, suggesting that TF-induced ATP promotes osteogenesis by autocrine action through P2X7 receptor in osteoblasts. J. Cell. Biochem. 116: 12–21, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.

Role of mechanical-stress inducible protein Hcs24/CTGF/CCN2 in cartilage growth and regeneration: Mechanical stress induces expression of Hcs24/CTGF/CCN2 in a human chondrocytic cell line HCS-2/8, rabbit costal chondrocytes and meniscus tissue cells

Mechanical stress plays an important role in the cartilage metabolism. The aim of this study is to determine the influence of mechanical load magnitude and frequency on cartilage metabolism in terms of the expression of hypertrophic chondrocyte-specific gene product 24/connective tissue growth factor/CCN family 2 (Hcs24/CTGF/CCN2), as an essential mediator of extracellular matrix (ECM) production. When a human chondrocytic cell line, HCS-2/8 was exposed to uni-axial cyclic mechanical force (6% elongation, 10 times/min) only for 30 min, the expression level of Hcs24/CTGF/CCN2 (CCN2) increased, and c-Jun N-terminal protein kinase (JNK) was activated. These findings suggest that stretch-induced CCN2 may be mediated by the JNK pathway. When HCS-2/8 cells were subjected to cyclic tension force at 15 kPa, 30 cycles/min, which has been reported to be a degradation force for HCS-2/8 cells, the expressions of CCN2 and aggrecan were inhibited, and such expressions remained unchanged in rabbit hyaline costal cartilage cells. However, these expressions increased in rabbit meniscus tissue cells. These findings suggest that the sensitivity of mechanical stretch may be different depending on the type of cells. Furthermore, CCN2 was co-localized with aggrecan in this meniscus tissue region exposed to mechanical stress in vivo. These findings suggest that CCN2 induced by mechanical stress may therefore play some role in meniscus growth and regeneration.

Immuno-localization of COX-1 and COX-2 in the rat molar periodontal tissue after topical application of lipopolysaccharide

Up-regulation of prostaglandin E 2 (PGE 2) production in the periodontal tissue is considered to be important for periodontal tissue destruction. The purpose of the study was to demonstrate the dynamic changes of immuno-localization of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS: 5 mg/ml in physiological saline) from Escherichia coli into the rat molar gingival sulcus. In the normal periodontal tissue, small numbers of junctional epithelium (JE) cells and numerous osteocytes embedded in alveolar bone constitutively expressed COX-1. The COX-1 expression was not effected by LPS application. JE cells, especially in the coronal portion of JE also expressed COX-2. LPS application induced the JE cells with consequent transient expression of COX-2 with a peak at day 1. These findings suggest that JE cells may play a critical role in first defense line against LPS challenge and PGE 2 from JE cells may be responsible for the initiation of periodontal inflammation. In the deep periodontal tissue, cementoblasts and osteoblasts showed constitutive expression of COX-2, which may be induced by continuous cyclic tension force due to occlusal pressure. LPS application caused a transient up-regulation of COX-2 expression in periodontal ligament fibroblasts, cementoblasts and osteoblasts. It is suggested that the inducible production of PGE 2 via COX-2 by these cells may be associated with connective tissue destruction and alveolar bone resorption.

The induction of c- fos mRNA expression by mechanical stress in human periodontal ligament cells

The periodontal ligament is subjected to mechanical loading during occlusion and mastication. Although internuclear transcription factors are associated with the regulatory pathway that converts these extracellular mechanical stimuli into a cellular response, there are no reports on these in human periodontal ligament fibroblasts. In this study, the amounts of c- fos mRNA in human periodontal ligament fibroblasts were investigated shortly after subjecting them to a cyclic tension force in vitro. The mRNA of alkaline phosphatase and the matrix proteins type I collagen, type III collagen, matrix Gla-protein, osteonectin, osteopontin, and osteocalcin were also examined. A significant, rapid, transient increase in c- fos mRNA was detected, which peaked 30 min after the application of mechanical force. However, there was no significant change in the mRNA for alkaline phosphatase or the matrix proteins. These results provide evidence that periodontal ligament fibroblasts and c- fos may play a critical part in the response of periodontal tissue to mechanical stimulation.

Cyclic tension force activates nitric oxide production in cultured human periodontal ligament cells.

Nitric oxide (NO) is involved in a number of physiological and pathophysiological processes. The aim of this study was to examine the ability of human periodontal ligament (PDL) cells to produce NO and whether mechanical forces could induce NO production in the PDL cells. Human PDL cells were seeded onto flexible bottoms of a culture plate and subjected to cyclic tension forces. NO production was evaluated by measuring concentration of NO2- and NO3- (NO2-/NO3-), the oxidized products of NO, in the culture medium. We employed reverse transcription polymerase chain reaction (RT-PCR) methods to detect NO synthase mRNA in the PDL cells. NO synthase immunoreactivity was also evaluated in both stimulated and unstimulated PDL cells. In unstimulated PDL cell culture, NO2-/NO3- increased to 140% of the initial value in 12 hours. In contrast, NO2-/NO3- showed a 3-fold increase when the cells had been subjected to cyclic tension forces for 12 hours. The increase in NO production was blocked by NG-monomethyl-L-arginine (5 x 10(-4) M), an inhibitor of NO synthase. Endothelial NO synthase (ecNOS) mRNA was expressed in both stimulated and unstimulated PDL cells, whereas inducible NO synthase (iNOS) mRNA was detected in neither culturing condition. We found strong ecNOS but not iNOS immunoreactivity in the stimulated PDL cells. These results suggest that human PDL cells produce NO by ecNOS and that the production is enhanced by stimulating the cells with cyclic tension forces. Mechanically stimulated PDL cells may modulate the function of periodontium by the upregulated NO production.

Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1.

To clarify the mechanism of cartilage degradation induced by mechanical stress, we investigated the influence of cyclic tension force (CTF) on the metabolism of cultured chondrocytes. The chondrocytes were exposed to CTF using a Flexercell strain unit. Five or 15 kPa of high frequency CTF significantly inhibited the syntheses of DNA, proteoglycan, collagen, and protein. Fifteen kPa of high frequency CTF induced the expression of interleukin-1 (IL-1), matrix metalloproteinase (MMP)-2 and -9 mRNA, and increased the production of pro- and active-MMP-9. The degradation of proteoglycan was inhibited by and MMP inhibitor, indicating that MMPs are involved in the degradation of proteoglycans induced by high frequency CTF. Moreover, reducing the frequency of CTF from high to low decreased the inhibition of proteoglycan synthesis. These findings suggest that the CTF frequency is one of the key determinants of chondrocyte metabolism. Low magnitude CTF, whether high or low frequency, did not cause the gene expression of cartilage degradation factors, suggesting that this CTF magnitude causes only minor changes in the cartilage matrix. High magnitude and frequency CTF caused the gene expression of IL-1 and MMP-9, followed by increases in the production of MMP-2 and -9 proteins, suggesting that excessive and continuous cyclic mechanical stress induces the production of IL-1 and MMP-9, resulting in cartilage degradation.

Effect of cellular aging on the induction of cyclooxygenase-2 by mechanical stress in human periodontal ligament cells

The production of prostaglandin (PGE 2) in human periodontal ligament fibroblast (hPLF) cells is increased by mechanical stress, however, the age-related changes in the susceptibility of hPLF cells in response to mechanical stress remain unclear. The purpose of this study was to examine the influence of in vitro cellular aging on PGE 2 production and the gene expression of cyclooxygenase (COX) in mechanically stressed hPLF cells. In vitro cellular aged hPLF cells were prepared by sequential subcultivations of hPLF cells from young healthy periodontal ligaments. In vitro cellular aged hPLF cells produced a significantly higher amount of PGE 2, as compared with young hPLF cells, when the cells were exposed to cyclic tension force in a time- and magnitude-dependent manner. The COX-2 mRNA level in aged cells was higher than that in young cells, whereas COX-1 mRNA remained unchanged. Since PGE 2 from hPLF cells was stimulated by in vitro aging as presented here, aging of hPLF cells may affect the severity of inflammation and bone resorption in the aged through the production of a large amount of PGE 2 in response to an excessive force such as a traumatic occlusion.

Effect of aging on functional changes of periodontal tissue cells.

Although the severity of periodontal disease is known to be affected by age, functional changes of periodontal tissue cells during the aging process are not well characterized. It is important to define how cellular aging affects the progression of periodontal diseases associated with the aging process. In vitro aging of human gingival fibroblast (HGF) and periodontal ligament fibroblast (HPLF) cells was prepared by sequential subcultivations (5 to 6 passages as young, 18 to 20 passages as old). GFs were also prepared from gingiva of Down's syndrome patients and 60-week-old rats. Fetal rat calvarial osteoblasts were prepared by sequential digestion with collagenase. HGF and HPLF cells were treated with lipopolysaccharide (LPS) and cyclic tension force, respectively. Amounts of PGE2, interleukin (IL)-1 beta, IL-6, and plasminogen activator (PA) in conditioned media were measured. Total RNA was extracted, and mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). LPS-stimulated PGE2, IL-1 beta, IL-6, and PA production was increased in "old" HGF compared to younger cells. According to RT-PCR analysis, gene expression of COX-2, IL-1 beta, IL-6, and tissue type (t) PA was higher in old cells than in young cells. Cyclic tension force to HPLF also stimulated phenotypic and gene expression of IL-1 beta, PGE2 (COX-2 gene) and tPA. These findings suggest that aging in both HGF and HPLF may be an important factor in the severity of periodontal disease through higher production of inflammatory mediators in response to both LPS and mechanical stress. In addition, oxygen radical-treated fibronectin (FN) as substratum diminished bone nodule formation by osteoblasts when compared with intact FN. This finding suggests that FN plays an important role in Osteoblast activity and that FN damaged by oxygen radicals during the aging process may be related to less bone formation.

Identification of factors mediating the decrease of alkaline phosphatase activity caused by tension-force in periodontal ligament cells

1. We examined the factors which mediate the decrease of alkaline phosphatase (ALP) activity in human peridontal ligament (PDL) cells in response to cyclic tension-force. 2. ALP activity in human PDL cells obtained from three donors in response to cyclic tension-force (24% elongation) was 43% lower than that of the corresponding control. 3. ALP activity was decreased by the addition of conditioned medium obtained from the culture of the cells exposed to tension-force. 4. The inhibitory effect of the conditioned medium on ALP activity was partially abolished in the presence of indomethacin (10 −6 M) and IL-1β antibody (10 ng/well). Moreover it was almost completely abolished in the presence of both indomethacin and IL-1β antibody. 5. Treatment of PDL cells with exogenous PGE 2 or IL-1β for 24 hr caused a dose-dependent decrease in ALP activity. Treatment with both PGE 2 (10 −8 M) and IL-1β (1.25 × 10 −10 M) together decreased ALP activity by 47% compared with the non-treated control. 6. These findings suggest that ALP activity in PDL cells was decreased in response to the cyclic tension-force and that the decrease in ALP activity was mainly mediated by PGE 2 and IL-1β produced by PDL cells in response to cyclic tension-force.