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The Initial Responses to Mechanical Strain and the Mechanotransduction in Osteoblastic Cells in Vitro

Author: ZhengYi
Tutor: LuoSongJiao
School: Sichuan University
Course: Clinical Stomatology
Keywords: osteoblast mechanotransduction cytoskeleton G protein tyrosine protein kinase Cbfa1/Osf2 NF-κB ERK1/2
CLC: R783
Type: PhD thesis
Year: 2004
Downloads: 354
Quote: 5
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Abstract


Orthodontic treatment involves the use and control of forces acting on the teeth and associated structures. The principle changes resulting from such forces are seen within the dentoalveolar system, but other structure can also be influenced, such as sutures and the temporomandibular joint area. Both modeling and remodeling are controlled by and interaction of metabolic and mechanical signals. These changes in mechanical stimulus modulate bone mass and architecture mainly through the osteoblast lineage, so it is important to study the biological response and mechanism of the osteoblast to the mechanical strain. However, recent studies have been limited to the late biological responses of osteoblast to various stress stimuli, and the results are different. In order to better understand what responses, especially the initial responses, are involved in mechano- transduction, we have examined the effect of mechanical strain lonely and coupled with the inhibitors of the relative signaling pathways on the expression of Cbfal, NF-κB, ERK1/2 and the proliferation and function of osteoblast-like cells.Osteoblast-like cells were cultured in DMEM with 10% FBS for 48 hours and starved in DMEM with no FBS for 12 hours, then subjected to mechanical strain by four-point bending system at 0.5Hz frequency for 5min, 15min, 30min, 1h, 3h, 6h, respectively. In each time-phase, the cells were loaded with tension and compression stress at 1000μstrain and 4000μstrain respectively. mRNA were analyzed by Real- time PCR and protein by Western Blotting. Changes in the experiments were expressed asratio (±standard error of the mean) to the controls. Statistical significance was determined for each comparison using the one-way ANOVA and multiple comparisons using the SNK, and p<0.05 was statistically considered significant. The ratios quoted were average changes from three independent experiments. The flow cytometry and MTT method were used to examine the number of osteoblast-like cells and their proliferation and metabolic enzymic activity 6h, 12h, 18h, 24h, 3 Oh and 36h after the top-bottom axial stress was exerted on the cells. Treatment of cultures with agonists: Before loading, the cells were treated with Genistein, Cytochalasin D, Pertussis Toxin and Indomethacin for 30min, lh, 24h, 24h, respectively. Then the cells were subjected to mechanical strain at 4000ustrain for 5min. The phosphorylation levels of ERK1/2 were measured by Western Blotting.In this study, we identify that the Four-Point Bending System by self-made could supply an accu- rate and effective cyclic uniaxial strain to the cultured adhesion cells in vitro. And the top-bottom axial stress at physiologic magnitude has a remarkable effect on the proliferation and function of osteoblast-like cells without pathological damage occurred. Whether lOOOustrain or 4000ustrain, the compress strain could induce a stronger and longer expression of Cbfal mRNA compared with the tension strain. The expression of NF-kB could be induced quickly and strongly by the stimulus of lOOOustrain tension strain, while repressed by compress strain and vice versa by the 4000ustrain stimulus. Both 4000ustrain tension stimulus and compress stimulus could induce the expression of ERK in a few minutes and the effect of tension strain is more significant. It may be due to the distinct mechanotransductive mechanism of the different directional mechanical strain. The microfilament cytoskeleton is the core of mechanotransduction. Both the cortical cytoskeleton and the internal cytoskeletal lattice are important to the mechanical signal transduction. Thereception and transduction of osteoblasts to the mechanical signal of tension or compression strain are relative to the microfilament cytoskeleton, while the activation of tyrosine protein kinases is mainly connected to response to the tension strain. G-protein coupled receptors and COX-2 pathways also contribute to the initial transduction of the mechanical strain. In summary, this study shows that the phosphorylation of proteins plays an important role in the initial responses of osteoblast to mechanical strain and multiple signaling pathways are involved in mechanotrans-duction. Our results provide a biological evidence for the principle of using light forces during the orthodontic treatment.

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