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Objective: To study the effects of long non-coding RNA linc-01135 on the osteogenic differentiation of inflammatory PDLSCs(P-PDLSCs) under 12％ static mechanical strain loading. Methods: Cells were isolated and cultured from the healthy and periodontitis samples respectively to obtain healthy PDLSCs(H-PDLSCs) and P-PDLSCs. RT-PCR were used to identify the expression level of linc-01135. Lentiviruses were used to upregulate and downregulate the expression of linc-01135, and the osteogenesis gene expression were analyzed by RT-PCR and the osteogenesis differentiation capability were evaluated by alizarin red staining. Results: linc-01135 expression in P-PDLSCs was lower than that in H-PDLSCs. When the expression of linc-01135 was upregulated in P-PDLSCs before 12％ SMS loading, the expression level of RUNX2, ALP, OPG was significantly increased. In contrast, the expression of RUNX2, ALP, OPG was significantly decreased when the expression of linc-01135 was suppressed. Alizarin red staining proved the same trend. Conclusion: linc-01135 can promote the osteogenic differentiation capability of P-PDLSCs under 12％SMS loading.
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Objective To investigate the effect and molecular mechanism of the combination of mechanical strain stimulation and icariin (ICA) on inhibiting the differentiation of osteoclasts induced by fatigue load stimulation. Methods The mouse mononuclear macrophage cell line RAW264.7 was cultured in vitro, and the blank control group was α-MEM complete medium. In the fatigue load group, RAW264.7 cells were treated with 5000 μεmechanical stretch strain, and then cultured in an osteoclast culture medium that was an α-MEM complete medium containing 40 ng/ml macrophage colony-stimulating factor and 40 ng/ml osteoclast differentiation factor. In the mechanical stimulation + ICA group, RAW264.7 cells were treated as the same procedure in the fatigue load group, and then cultured in an α-MEM complete medium containing 1 ×10 -5 mol/L ICA simultaneously with a 1000 μεtensile strain on the substrate. The activity of tartrate-resistant acid phosphatase (TRAP) was detected using a TRAP assay kit. The mRNA expression of the osteoclast marker genes, i.e. TRAP, cathepsin K(CTSK) and matrix metalloproteinase 9 (MMP-9) was detected by real-time RT-PCR. The nuclear translocation of nuclear factor kappa B (NF-κB) was analyzed by Western Blot. Results Compared with the fatigue load group, the combination of mechanical stimulation (1000 με substrate stretching) and ICA (1×10-5 mol/L) could significantly inhibit the activity of TRAP in osteoclasts (P<0.01) and reduce osteoclastosis. Moreover, that combination not only could down-regulate the mRNA expression of TRAP, CTSK and MMP-9 and the differences were statistically significant (all P<0.01), but also could inhibit the formation of osteoclasts by inhibiting the phosphorylation of P65, P50 and IκB-α in NF-κB signaling pathway. Conclusions The coupling of mechanical stimulation and ICA can effectively inhibit the osteoclast differentiation and the bone resorption induced by fatigue load, and the mechanism may involve regulating NF-κB signaling pathway.
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Objective To investigate the effect of 1,25-(OH)2-vitamin D3 (VD3) or mechanical strain alone and their combined treatment on proliferation and differentiation of pre-osteoblast MC3T3-E1 cells in vitro, as well as gene and protein expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-кB ligand (RANKL) in those cells. Methods MC3T3-E1 cells were treated with 10 nmol/L VD3, intermitted mechanical strain or with a combination of these two factors. Cell proliferation was assessed with flow cytometry, and alkaline phosphatase (ALP) activity was measured using a fluorometric detection kit. The mRNA expression of ALP, runt-related transcriptional factor 2 (Runx2), OPG, and RANKL genes was determined by real-time PCR. The proteins expression of Runx2, OPG, and RANKL was determined by Western blotting. ResultsVD3 inhibited the proliferation of MC3T3-E1 cells, but the mechanical strain had no effect on cell proliferation. Mechanical strain, VD3, and the combined treatment enhanced the ALP activity of MC3T3-E1 cells as well as the protein expression of Runx2. The effect of combined treatment was less pronounced than the effect of VD3 or mechanical strain alone. Mechanical strain promoted the gene and protein expression of osteoprotegerin (OPG) and increased the ratio of OPG/RANKL. However, the combination of VD3 and mechanical strain led to a decrease in ratio of OPG/RANKL. Conclusions Mechanical strain might be effective in inducing osteogenic differentiation and increasing bone formation. A joint stimulation with VD3 and strain can decrease proliferation and osteogenic differentiation and increase RANKL expression, which might affect bone remodeling. This study supplies some new data, which might be important in theoretical and clinical research of osteoporosis (OP) and other related bone diseases.
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Objective To evaluate differences in genes expression of rat bone marrow stromal cells (rBMSCs) under continuous mechanical strain by gene microarray technology．Methods rBMSCs were isolated and cultured in vitro. Continuous stresses with amplitude of 10% and frequency of 1 Hz were applied on rBMSCs for 6 hours by Flexercell mechanical loading system to investigate rBMSC gene expression profiles, and quantitative PCR was used to verify gene expression changes related to osteoblastic differentiation. Results Compared with the control group, 1 244 differentially expressed genes were found in mechanical loading group, among which 793 genes were up-regulated, while 451 genes were down-regulated．GO (gene ontology) analysis suggested that differentially expressed genes were mainly involved in multicellular organismal development, cell differentiation, chemotaxis, cell adhesion and so on. Four signaling pathways as Notch, Wnt, FGF and IGF might participate in the regulation of stress-induced osteoblastic differentiation. PCR validation results were consistent with the gene chip results. Conclusions Mechanical stress could induce osteoblastic differentiation of the BMSCs, while several differentially expressed genes screened by gene microarray may attribute to this process.
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Objective To study the effect of continuous strain on the proliferation and osteogenic differentiation of rat bone marrow stromal cells(BMSCs) in vitro. Methods Rat BMSCs were obtained from adult female Sprague-Dawley rats (3-month old), and purified by full-blood attachment culture. BMSCs between passage 3—5 were seeded on Flexercell mechanical loading system(10%, 1 Hz), and divided into 1 h group, 6 h group, 12 h group, 24 h group, 48 h group, respectively, according to the time subjected to strain. Effects of continuous strain on the morphology, proliferation and osteogenic differentiation of BMSCs were observed and analyzed. Results (1) Compared with the control group, cells subjected to 10% strain showed the particular orientation. Their alignment elongated mostly in the direction perpendicular to the strain axis in a time-dependent manner. (2)10% continuous strain could significantly decrease the proliferation of BMSCs. (3) Continuous strain could increase mRNA expression of ALP, COLⅠand Runx2 in a time-dependent manner. Compared with the control group, mRNA expression of ALP was increased significantly at 24 h, COLⅠat 24 h and 48 h, and Runx2 at 6 h. mRNA expression of osteocalcin (OC) ascended greatly in the beginning, but went down gradually and was significantly lower than that of control at 48 h（P＜0.05）. (4) Continuous strain could induce an increase in Runx2 protein level. A sharp increase in Runx2 protein was observed at 6 h(P＜0.05) , then Runx2 protein level decreased slowly with its mRNA expression being significantly lower than that of control at 24 h（P＜0.05） Conclusions Continuous strain could induce rat BMSCs to orient in an orderly manner, suppress its proliferation activity, but stimulate the osteoblastic differentiation at the early stage.
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Objective To investigate the effect from mechanical stimulation and osteogenic chemical inductor on osteoblastic differentiation markers and formation of calcified nodules in rat bone mesenchymal stem cells (rBMSCs). Method The rBMSC were cultured in medium contained with or without osteogenic chemical inductor. The cyclic biaxial mechanical strain (2%), at a frequency of 1 Hz, was applied to the rBMSCs for periods of 2 hours each time, at intervals of 2 hours, 3 times every day, lasting 3 days and 6 days, respectively. The mRNA expression of alkaline phosphatase (ALP), collagen type I (COL I) and osteocalcin (OCN) were analyzed with real time fluorescent quantitation reverse transcription polymerase chain reaction(qRT-PCR) and formation of calcified nodules were detected with alizarin red staining method. Results The mRNA expression of ALP, COL I and OCN were significantly increased in induced group compared with that in the corresponding uninduced group and calcified nodule was observed in the osteogenic chemical inductor group after 6 days with mechanical stimulation. Conclusions Osteogenic chemical inductor and mechanical stimulation can promote the osteoblastic differentiation of rBMSCs.
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Objective To investigate the effects of different magnitudes of mechanical strain on the expression of MMP-13/TIMP-1 and try to determine the signal transduction pathways in response to mechanical strain in human periodontal ligament cells (HPDLCs) in vitro. MethodHPDLCs were subjected to 0%，6%，12% or 18% elongation for 24 h by using cell stress loading system simultaneously. Then the MMP-13/TIMP-1 mRNA and protein expression in cells were tested by reverse transcription polymerase chain reaction (RT-PCR) and western blotting respectively. Furthermore, specific inhibitors were employed to examine the role of different signal transduction pathway on the expression of strain induced MMP-13 /TIMP-1 in HPDLCs. ResultsThe expression of MMP-13 /TIMP-1 in HPDLCs significantly increased in groups of 6%，12%, 18% elongation in a magnitude-dependent manner compared with the control group (0%) after the mechanical strain treatment for 24 h. PD098059 and cycloheximide could inhibit the increase in MMP-13 and TIMP-1 mRNA expression in response to mechanical strain respectively. Conclusions Different magnitudes of mechanical strain can affect the expression of MMP-13/TIMP-1 in HPDLCs in a magnitude dependent manner, and further affect the periodontium remodeling with the characteristics of degradation and synthesis of extracellular matrix in response to mechanical strain. The ERK-MAPK pathway is involved in strain induced MMP-13 expression while the strain-induced TIMP-1 expression depends on de novo protein synthesis.
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Objective To identify the differentially expressed proteins and clarify the major proteins involved in the molecular mechanism of osteoblasts under mechanical strain loading. Method Saos 2 osteoblastic cells were subjected to 12% elongation for 24 hours by using Flexcell strain loading system. Proteins extracted from Saos 2 cells were separated by two dimensional electrophoresis (2 DE). Differential expressed protein spots among groups were submitted to matrix assisted laser desorption/ionization time of flight mass spectrometer (MALDI TOF MS) assay and peptide mass fingerprinting (PMF) identification. The Swiss Prot and NCBI databases were used to obtain further information about proteins identified. Results Saos 2 stimulated by mechanical strain showed a significant difference in 2 DE system compared with the control group. A total of (1031±41) or (928±25) protein spots were resolved by 2 DE of controls or experimental groups extractions respectively. 17 significant up regulated proteins were identified. These associated proteins fell into 6 groups, including stress reaction, energy metabolism, cell proliferation, reconstruction of cytoskeleton, signaling and osteogenesis. Conclusions The Saos 2 can express differential proteins stimulated by mechanical strains and these proteins may play an important role in molecular mechanism of osteoblasts under mechanical strain loading.
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Objective To evaluate the role of angiotensin Ⅱ(AngⅡ) signal passway on the expression of Rho GDP dissociation inhibitor alpha (RhoGDIα) in hypertensive rats. Methods Protein and mRNA expressions of RhoGDIα in aortae of 4, 12 and 18 week-old spontaneously hypertensive rats (SHR, n = 4) and Wistar Kyoto rats (WKY, n= 4) were examined by Western blotting and real-time PCR. Aortas from SHR and WKY were analyzed using immonuchemical staining to locate the RhoGDIα in the aorta. The RhoGDIα expression in aorta of hypertensive rat model of aorta coarctation (ACR, n = 6) was also analyzed using Western blotting. Furthermore, The effect of mechanical strain at 10 % elongation on expression of RhoGDIα in vascular smoothmuscle cells (VSMCs) in the presence or absence of L-158809, an antagonist for AngⅡ type 1 receptor, was also evaluated by Western blotting. Results No significant difference of RhoGDIα expression was found between SHR and WKY at 4-week-old and 12-week-old. However, in 18-week-old group, RhoGDIα was significantly highly expressed in SHR than that of WKY at both mRNA and protein levels. RhoGDIα was located in the media of the aorta. Expression of RhoGDIα protein was upregulated in aortas of ACR at 2 and 4 weeks as compared with the controls. The expression of RhoGDIα in VSMCs was inhibited by mechanicalstrain at 10 % elongation, and further decreased by treatment of L-158809. Conclusion RhoGDIα is upregulated in aortae of the hypertensive rats. AngⅡ signal passway may be involved in the process of regulating expression of RhoGDIα.
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Objective To study the effects of early mechanical strain magnitude on formation and differentiation of osteoclasts. Methods RAW 264.7 cells induced by macrophage colony-stimulating factors and osteoclast differentiation factors were subjected to 0, 1 000, 1 500, 2 000, 2 500 and 5 000 με mechanical straining for three days. The morphological changes, number of osteoclasts and proliferation of precursor cells were determined at day 7. The activity of the tartrate-reaistant acid phosphatase (TRAP) in the culture medium was detected at days 4 and 7. Results The number of osteoclasts was decreased in 2 500 με group, while it was increased in 5 000 με group. The proliferation of precursor cells was increased in 2 000 and 2 500 με group, while it was decreased significantly in 5 000 με group. There was no significant difference in the number of osteoclasts and proliferation of precursor cells among 1 000 με group, 1 500 με group and 0 με group. The activity of TRAP was decreased in 1 000, 1 500, 2 000, 2500 and 5 000 με groups at days 4 and 7 when compared with με group. Conclusions Early mechanical straining plays a direct role in formation and differentiation of osteoclasts. The high strain magnitude within physiological load inhibits osteoclast formation, while high strain magnitude beyond physiological load stimulates osteoclast formation. Low strain magnitude has nearly no impact on formation of osteoclasts. Early mechanical straining may inhibit differentiation of osteoclasts.
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Tooth movement is a result of mutual physiologic responses between the periodontal ligament and alveolar bone stimulated by mechanical strain. The PDL cell and osteoblast are known to have an influence on bone formation by controlling collagen synthesis and alkaline phosphatase activation. Moreover, recent studies have shown that the PDL cell and osteoblast release osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa B ligand (RANKL) to control the level of osteoclast differentiation and activation which in turn influences bone resorption. In this study, progressively increased, continuous tensional force was applied to PDL cells. The objective was to find out which kind of biochemical reactions occur after tensional force application and to illuminate the alveolar bone resorption and apposition mechanism. Continuous and progressively increased tensile force was applied to PDL cells cultured on a petriperm dish with a flexible membrane. The amount of PGE2 and ALP synthesis were measured after 1, 3, 6 and 12 hours of force application. Secondly, RT-PCR analysis was carried out for OPG and RANKL which control osteoclast differentiation and MMP-1, -8, -9, -13 and TIMP-1 which regulate the resolution of collagen and resorption of the osteoid layer. According to the results, we concluded that progressively increased, continuous force application to human PDL cells reduces PGE2 synthesis, and increases OPG mRNA expression.
Subject(s)
Humans , Alkaline Phosphatase , Bone Resorption , Collagen , Dinoprostone , Membranes , Osteoblasts , Osteoclasts , Osteogenesis , Osteoprotegerin , Periodontal Ligament , RANK Ligand , Receptor Activator of Nuclear Factor-kappa B , RNA, Messenger , Tissue Inhibitor of Metalloproteinase-1 , Tooth Movement TechniquesABSTRACT
Objective To screen out the PI3K/Akt pathway related genes in vascular smooth muscle cells (VSMCs) subjected to cyclic strain. Method VSMCs of rat aorta were subjected to cyclic strain (10 %, 1 Hz) by using a FX-4000T system. Phosphorylation of Akt was examined by Western blotting. Suppression subtractive hybridization (SSH) method was employed to analyze the differently expressed cDNA sequence between the strained VSMCs pretreated with and without wortmannin, a specific inhibitor of PI3K. These fragments were ligated with T vectors, screened through the blue-white screening system to establish cDNA library, and was sequenced and analyzed in GenBank with BLASTN search.Result Level of Akt phosphorylation of VSMCs was significantly enhanced by the mechanical strain compared with the control. Ten different expressed sequence tags (EST) were gained after sequencing 30 clones randomly selected from 54 white clones. There may be six genes related with the mechanical strain and cell signal PI3K/Akt, such as High mobility group box 1 (HMGB1), Neural precursor cells expressed (Nedd4a), Cyclin-dependent kinase 5 (CDK5), Histone deacetylase 3 (HDAC3), Ubiquitin-like 1 (Uble1a) and Heterogeneous nuclear ribonucleoprotein H1 (hnRNP). Conclusion SSH is an effective and reliable approach to screen out the genes related with the mechanical strain and PI3K/Akt pathway in VSMCs.
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Objective:To investigate the effects of strain force on t he expression of osteoclast differentiation factor(ODF) and osteoclasto-genesis i nhibitory factor(OCIF) in human periodontal ligament cells (HPDLCs). Met hods: HPDLCs were subjected to cyclic strain force for 0, 6, 12 and 24 h ours, mRNA expression of ODF and OCIF were determined by RT-PCR. Result s:After treatment of the cells for 0,6,12 and 24 hours the ODF/?-actio n values were 0.7280?0.0261,0.6831?0.0411,0.5801?0.2230 and 0.4572?0.0373( P0.05) respectively.Conclusion:Strain force may decrease the expression of ODF and increase the expression of OCIF.
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Objective: To study the effects of mechanical strain on the proliferation of human osteoblasts. Methods: Cloned MG 63 human osteoblasts were cultured and seeded at 10 5/well in 6 well Bioflex cell cuture plates. Subsequently, the cells were exposed to mechanical strain stimulation (at 6%, 12% and 24% elongation rate respectively) for 24 hours using Flexercell 3000 Cell Stretching Unit. MTT colorimetric method was used to assess the proliferation of the cells. Results: The proliferation of MG 63 cells was significantly increased 24 h after mechanical strain treatment( P
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Objective To investigate the effects of different magnitudes of mechanical strain on proliferation and alkaline phosphatase (ALP) activity in mouse osteoblast-like cells (MC3T3-E1). Methods MC3T3-E1 cells were subjected to 0%, 6%, 12%, 18% elongation for 24h and 48h by using multi-passage cell stress loading system respectively. MTT colorimetric method was used to assess the proliferation of the cell, ALP activity was detected by ALP assay kit. Results The proliferation of MC3T3-E1 cells was increased significantly 24h and 48h after mechanical strain treatment concomitant with increasing stretching force (P