The Study Of The Effects And Mechanism Of Cyclic Dynamic Pressure On CLC-3 Chloride Channel In Osteoblasts

Bone tissue is very sensitive to mechanical stimulation,it can feel the conduction of various chemical and mechanical signals.The morphology,differentiation and proliferation of osteoblasts are also related to mechanical stimulation.When the mechanical forces act on bone tissue and the osteoblasts are under the stress environment,mechanical stimulation will active the cell membrane and transfer the signals to the nucleus by microfilaments and microtubules,series of biochemical reactions are caused during the process of activing varies of osteogenesis-related gene in osteoblasts,promote formation and remodeling of bone tissue.In this process,osteoblasts are the main function cells of bone formation and taking charge in synthesis,secretion and mineralization of bone matrix.Up to now,there were many reserches on the osteogenic effects of various stress conditions including tension and fluid shear stress,and there were aslo several static compressive stress reserches,but only a few reserches were concerned about the effects of dynamic compression stimulation on bone tissue and osteoblasts.Anion channels play essential roles in cellular processes such as excitation,contraction,salt and water transport,volume regulation,p H regulation,proliferation,migration,apoptosis,necrosis and ischemic injury.Studies have shown that there are many ion channels associated with the osteogenic differentiation,such as Ca2+,K+ and Cl–etc.CLC-3 chloride channel,a member of the CLC super-family of chloride channels,plays an important role in cell volume regulation and is also involved in cell proliferation,differentiation,apoptosis,and intracellular acidification of osteoclasts.Recent studies indicated that CLC-3 was related with the expression of osteogenic markers during osteogenesis through Runx2.Runx2(Runt-related transcription 2 factor,Runx2)is a transcription factor involved in osteoblast differentiation and acts as an important regulator ofbone formation at multiple stages.BMP-2-Smads-Runx2 signaling pathway plays an important role in the mechanical-stimulated differentiation of osteoblasts.In the osteogenic differentiation process,a variety of osteoblast specific genes,such as bone morphogenetic protein 2(Bone morphogenetic protein-2,BMP-2),Runx2 and Osteopontin(Osteopontin,OPN)will continuously express in different periods,and produce the corresponding protein secreted into the cell the extracellular matrix,so as to realize the mineralization of extracellular matrix and form mineralized nodules and bone.A recent study shows that persistent static compression stimulation can upregulate the expression of CLC-3 in MC3T3-E1 cells,and promotes the differentiation of osteoblasts.However,the effect of dynamic compression on the CLC-3 chloride channel and its mechanism by Flexcell Compression System has not been reported.Real-time PCR and Western Blot were used in this study to detect the effect and mechanism of cyclic dynamic compression on the CLC-3 chloride channel of osteoblasts in three parts.Part I: The effects of cyclic dynamic compression on the CLC-3,BMP-2,Runx2 and OPN in MC3T3-E1 osteoblasts;Part II: The changes of CLC-3,BMP-2,Runx2 and OPN after Chlorotoxin,the chloride channel specific blocker,were added in MC3T3-E1 osteoblasts;Part III: The effects of cyclic dynamic compression on CLC-3 chloride channel and osteoblast proliferation related genes Ki67 and PCNA.Part one The effects of cyclic dynamic compression on the CLC-3,BMP-2,Runx2 and OPN in MC3T3-E1 osteoblastsObjective: In this part,the effects of cyclic dynamic compression on the ultrastructure of MC3T3-E1 cells was observed by transmission electron microscope;Real-time PCR and Western blot were used to study the expression of CLC-3,BMP-2,Runx2 and OPN in osteoblasts after cyclic dynamic compression.And investigated the effects and correlation of cyclic dynamic compression on the CLC-3,BMP-2,Runx2 and OPN in MC3T3-E1 osteoblasts.Methods: MC3T3-E1 cells(Cell bank of Chinese Academy of Sciences,Shanghai,CHN)were maintained at 37℃ in a humidified atmosphere of 5%CO2 in α-modified Eagle’s minimum essential medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution.After reaching 90%confluence,the cells were detached by treatment with 10% trypsin-EDTA and cultured in 12-wells multiplates with a density of 1×105 cells/well in 1 ml of media.The osteoblasts were then cultured for 28 days in α-MEM supplemented with 10% FBS and 50 μg/ml ascorbic acid.During the last week,2 m M beta-glycerophosphate was added.After 28 days of culture,the osteoblasts were embedded in an abundant 3D extracellular matrix produced by the osteoblasts themselves,and forming a 1-2 mm deep 3D membrane.Two of these membranes,called 3D osteoblasts,were collected and placed into a well of a Biopress 6-wells plate containing 1.5 ml α-MEM supplemented with 5% FBS,100 U/ml penicillin,100 μg/ml streptomycin.All of the experiments were performed at 37℃ in an air atmosphere.Dynamic compression was applied individually to each pool of two membranes by the Biopress system with a magnitude of 30 Kpa at 1 Hz frequency(sinusoidal wave)for 2,4 and 8 h.In parallel,as a control,3D osteoblasts were collected and placed into the Biopress plate,but were unloaded.After the application of the mechanical regimen,the osteoblasts were then isolated from the extracellular matrix by an enzymatic digestion with 3 mg/ml collagenase D for30 min at 37 ℃ under agitation.After centrifugation,the effects of cyclic dynamic compression on the ultrastructure of MC3T3-E1 cells were observed by transmission electron microscope;Real-time PCR and Western blot were used to detect the expression of CLC-3,BMP-2,Runx2 and OPN in osteoblasts after cyclic dynamic compression.Results: Under transmission electron microscopy,before dynamic compression,there were little protrusions on the cell surface and little cytoplasmendoplasmic reticulum,Golgi apparatus and mitochondria in the cytoplasm before dynamic compression After dynamic compression for 2h,the cell surface protrusions increased slightly,the cytoplasm of the Golgi apparatus,endoplasmic reticulum and mitochondria were slightly increased inMC3T3-E1 cells.After dynamic compression for 4h,there were more cell surface protrusions in MC3T3-E1 cells,the nuclear was located in the side of the cell,there were more mitochondria,Golgi apparatus,themedullarystructure and expanded endoplasmic reticulum in MC3T3-E1 cells.After dynamic compression for 8h,there were more cell surface protrusions than those of 4h,and the amounts of Golgi apparatus,expanded endoplasmic reticulum and mitochondria further increased.Compared with the control group,cyclic dynamic compression loading MC3T3-E1 cells for 2,4and 8h,the expression of CLC-3,BMP-2,Runx2 and OPN genes were significantly up-regulated at m RNA and protein levels,the difference was statistically significant(P<0.05).There was no significant change in the expression of CLC-3,BMP-2,Runx2 and OPN after 2,4,8h in the control group.Part two The changes of CLC-3,BMP-2,Runx2 and OPN afterChlorotoxin,the chloride channel specific blocker,were addedin MC3T3-E1 osteoblastsObjective: For the further study of the role of CLC-3 chloride channel in the differentiation of osteoblasts after dynamic compression,in this part,Real-time PCR and Western blot were used to observe the expression of CLC-3,BMP-2,Runx2 and OPN after cyclic dynamic compression with Chlorotoxin added(Cltx,CLC-3 chloride channel specific inhibitor).Methods:Preparation of 3D cell membrane and cell extraction was the same as those of the first part.We divided the cells into two groups according to the experimental design.1 Group I: Compression without Cltx added30Kpa,1Hz,1/2 sinusoidal wave-form dynamic compression was performed in MC3T3-E1 cells for 8h,Real-time PCR and Western blot were used to detect the expression of CLC-3,BMP-2,Runx2 and OPN m RNA and protein in osteoblasts after cyclic dynamic compression.2 Group II: Compression with Cltx addedUsing the Cltx to block the CLC-3 channel 30 min bfore compression,then 30 Kpa,1Hz,1/2 sinusoidal wave-form dynamic compression was performed on MC3T3-E1 cells for 8h.Real-time PCR and Western blot were used to detect the expression of CLC-3,BMP-2,Runx2 and OPN in osteoblasts after cyclic dynamic compression.Results: Compared with the group of compression without Cltx added,application of Cltx blocking CLC-3 chloride channel before compression,CLC-3 m RNA and protein expression in MC3T3-E1 cells was not significantly decreased,while the expression of BMP-2,Runx2 and OPN gene m RNA and protein were significantly decreased,the result were statistically significant(P<0.05).Part three The effects and correlation of cyclic dynamic compression onCLC-3 chloride channel and osteoblast proliferation relatedgenes Ki67 and PCNA.Objective: In this part,Real-time PCR and Western blot were used to study the expression of CLC-3,PCNA and Ki67 in osteoblasts after cyclic dynamic compression,and investigate the effects and correlation of cyclic dynamic compression on the CLC-3,PCNA and Ki67 in MC3T3-E1 osteoblasts cells.Methods: Preparation of 3D cell membrane and extraction were the same as those of the first and second part.Membranes were collected and placed into a well of a Biopress 6-wells plate containing 1.5 ml α-MEM supplemented with 5% FBS,100 U/ml penicillin,100 μg/ml streptomycin.All of the experiments were performed at 37℃ in an air atmosphere.Dynamic compression was applied individually to each pool of two membranes by the Biopress system with a magnitude of 30 Kpa at 1 Hz frequency(sinusoidal wave)for 2,4 and 8 h.In parallel,as a control,3D osteoblasts were collected and placed into the Biopress plate,but were unloaded.After the application of the mechanical regimen,the osteoblasts were then isolated from the extracellular matrix by an enzymatic digestion with 3 mg/ml collagenase D for30 min at 37℃ under agitation.Real-time PCR and Western blot were used to detect the expression of CLC-3,BMP-2,Runx2 and OPN in osteoblasts after cyclic dynamic compression.Control group: the cells were not loaded with dynamic compression,and the residual treatment was the same as the experimental group.Results: The CLC-3 expression was significantly increased after loaded by cyclic dynamic compression for 2,4 and 8h(P<0.05).While there was no significant change in the m RNA and protein levels of PCNA and Ki67 in the osteoblast after the compression(P>0.05).There was no significant change in the expression of CLC-3,Ki67 and PCNA after 2,4,8h in the control group.Conclusions:1 The cyclic dynamic compression can promote the expression of CLC-3and increase the number of CLC-3 chloride channels on the surface of cell membrane.2 With the increased expression of CLC-3 gene,the expression of BMP-2,Runx2 and OPN were also increased,osteogenic differentiation may be related with the expression of CLC-3 chloride channels.3 Cyclic dynamic compression can up regulate the expression of BMP-2,Runx2 and OPN in ostoblasts,promote osteoblast differentiation.4 Cltx does not affect the expression of CLC-3 gene,but it can reduce the number of functional chloride channels in MC3T3-E1 cells.5 The expression of osteogenic differentiation related genes BMP-2,Runx2 and OPN may be related to the number of functional chloride channels.6 CLC-3 gene promotes osteoblast differentiation through functional CLC-3 chloride channels.7 The proliferation of osteoblasts were not significantly affected after30 Kpa,1Hz,1/2 sinusoidal wave-form dynamic compression for 2,4 and 8h.8 There was no significant correlation between the expression of CLC-3gene and the proliferation related genes Ki67 and PCNA after 30 Kpa,1Hz,1/2sinusoidal wave-form dynamic compression for 2,4 and 8h.