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Objective To explore the role of miR-199a-3p in osteoblast proliferation induced by fluid shear stress (FSS) and the potential molecular mechanism. Methods Osteoblast MC3T3-E1 was treated with 1. 2 Pa FSS with time gradients of 0, 15, 30, 45, 60, 75 and 90 min, respectively. MC3T3-E1 cells were transfected with miR-199a-3p mimic or miR-199a-3p inhibitor. MC3T3-E1 cells were transfected with miR-199a-3p mimic and itsnegative control and then treated with 1. 2 Pa FSS for 45 min. The pc DNA NC, pc DNA-CABLES -1, si RNA NC and si RNA CABLES-1 were transfected into MC3T3-E1 cells. The pc DNA-CABLES-1 and mir-199a-3p mimic and SI NA-cables-1 and miR-199a-3p inhibitor were co-transfected, respectively. Cell activity was detected by CCK-8 assay. Real-time quantitative PCR (RT-qPCR) was used to detect expression levels of CABLES-1, miR-199a-3p, CDK 6, Cyclin D1 and PCNA. Luciferase reporting assay was used to detect targeting relationship between CABLES-1 and miR-199a-3p. Immunofluorescence was used to detect protein expression of CABLES-1.Western blot was used to detect protein expression of CABLES-1, CDK 6, PCNA and Cyclin D1. Results Mir- 199a-3p in MC3T3-E1 cells was significantly down-regulated by FSS. Over-expressed miR-199a-3p inhibitedosteoblast proliferation, and down-regulated miR-199a-3p expression promoted osteoblast proliferation. miR-199a- 3p could reverse the FSS-induced proliferation in osteoblasts. Dual luciferase assay showed that miR-199a-3p targeted to CABLES-1 and over-expressed miR-199a-3p inhibited expression of CBALES-1 protein. CABLES-1 could promote proliferation of osteoblasts. miR-199a-3p inhibited osteoblast proliferation induced by FSS through CABLES-1. Conclusions FSS-induced osteoblast proliferation can be realized by down-regulated miR-199a-3p expression via targeting CABLES-1. The findings in this study provide new direction for researches on mechanism of FSS-induced osteoblast proliferation, as well as new ideas for future research on clinical application of mechanical loading in the treatment of bone and joint diseases.
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Bone defects have always been an important cause of threat to human health, and artificial biomimetic bone repair replacement materials are currently one of the most effective and feasible solution approaches to treat bone damage. To develop artificial bone biomimetic materials, an in vitro biomimetic mineralization system must be constructed first to study in vitro biomimetic mineralization mechanism of natural bone matrix. Collagen is a template for mineralization, and its properties such as crosslinking degree, diameter, osmotic pressure, and surface charge can all directly affect mineralization progress. The biochemical and mechanical environments in which mineralization occurs are also quite distinct in their effects on mineralization process, particularly noncollagenous proteins and fluid shear stress (FSS). FSS is considered to be the main mechanical stimulation of bone tissues in micro-environment, which is of great significance to bone growth, repair and health maintenance. FSS at different levels and loading regimes has significant effects on transformation of amorphous calcium phosphate to bone apatite, self-assembly and directional alignment of collagen fibrils, and formation of hierarchical intrafibrillar mineralization. In this paper, the factors affecting collagen mineralization and their mechanism were summarized, with focus on regulation of FSS on collagen mineralization, and development direction in future was also prospected.
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Objective To study fluid flow within alveolar bone under orthodontic and occlusal loading, so as to provide references for understanding the regulatory mechanism of bone remodeling during orthodontics. Methods An animal model for orthodontic tooth movement on rats was first constructed. The finite element model of tooth-periodontal ligament-alveolar bone was established based on micro-CT images and the strain field in alveolar bone under orthodontic or constant occlusal loading was analyzed. Then finite element model of alveolar bone was constructed from the bone near the cervical margin or apical root of mesial root. The fluid flow in this model under orthodontic and cyclic occlusal loading was further predicted by using fluid-solid coupling numerical simulation. Results The fluid velocity within alveolar bone cavity mainly distributed at 0-10 μm/s, and the fluid shear stress (FSS) was mainly distributed at 0-10 Pa. FSS on the surface of alveolar bone near the apical root was higher than that close to the cervical margin. Conclusions FSS at different levels could be produced at different location within alveolar bone cavity under orthodontic and cyclic occlusal loading, which might further activate biological response of bone cells on the surface of trabeculae and finally regulate the remodeling of alveolar bone and orthodontic movement of tooth. The results provide theoretical guidance for the clinical treatment of orthodontics.
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Objective To investigate the mechanism of mechano-chemical coregulation in chemokine-induced calcium response of Jurkat T cells under fluid shear stress (FSS). Methods By using parallel-plate flow chamber combined with fluorescence microscope, the calcium response of Jurkat T cells on CXCL12 was observed to extract the corresponding characteristic parameters under static or flow state, with or without extracellular Ca2+, respectively. Results Immobilized CXCL12 could induce firm adhesion of the circulating Jurkat T cells, and the arrested cells increased with the increase of CXCL12 concentration. Force could trigger the calcium response of Jurkat T cells and sharply raised the activation ratio from 4% up to 75% when the FSS increased from 0 to 20 mPa. Under 20 mPa FSS, extracellular Ca2+ could stimulate quickly the calcium response by shortening the delay time (about 23 s), and enhance calcium intensity by prolonging the climbing time (about 7 s) and half time (about 20 s). Conclusions The cooperation between FSS and extracellular Ca2+ would accelerate and enhance CXCL12-mediated-calcium response of Jurkat T cells, which indicated a fast mechanosensitive pathway through ‘extracellular calcium influx-intracellular calcium store release’. The research results would contribute to understanding the process of T cells activation and providing the clue for relevant pathological and drug research.
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Objective To investigate the effect of different fluid shear stress (FSS) on the regulation of planar cell polarity (PCP) signaling, and further to explore the relationship among FSS, PCP signaling pathway and ciliogenesis. Methods The hydrodynamic cell model of adjustable FSS was established. qPCR and immunofluorescence were used to detect the mRNA expression of PCP signaling pathway core protein Dvl2 and cilia assembly protein IFT88, cell targeting and co-localization under different FSS. Western blot (WB) was used to detect the protein expression of Dvl2 at 18 h under different FSS. Results The qPCR result showed that compared with 1.5 Pa FSS, under 0.1 Pa FSS, the mRNA expression of Dvl2 was higher at 6 h and 18 h (P＜0.05), significantly higher at 12 h (P＜0.01); the mRNA expression of IFT88 was significantly higher at 18 h (P＜0.01). The WB result showed that compared with 0 h, under 0.1 Pa FSS, the protein expression of Dvl2 was higher at 18 h (P＜0.05), significantly lower under 1.5 Pa FSS (P＜0.01); compared with 1.5 Pa FSS, the protein expression of Dvl2 was higher at 18 h under 0.1 Pa FSS (P＜0.05). The immunofluorescence result showed that the positive expression of Dvl2 increased with the loading time on FSS increasing, and gradually aggregated at a point around the nucleus; the positive expression of IFT88 was gradually transferred from the nucleus to the cytoplasm and aggregated at a point under 0.1 Pa FSS, and gradually decreased and depolymerized under 1.5 Pa FSS. Protein Dvl2 and IFT88 were located in the same position in cells under 0.1 Pa FSS and before 18 h under 1.5 Pa FSS, and colocalization of proteins Dvl2 and IFT88 was not observed after 18 h under 1.5 Pa FSS due to IFT88 depolymerization. Conclusions Laminar FSS played an inhibition on the transduction of PCP signaling pathway and a hindrance on the process of ciliogenesis, while low FSS played a promotion on the transduction. PCP signaling pathway might regulate FSS-induced ciliogenesis by Dvl2.
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Vascular endothelial cell glycocalyx is a layer of glycoprotein complex located on the surface of endothelial cells, forming a selective permeation barrier on the surface of endothelial cells. In the present review, after a brief introduction of glycocalyx, the relationship between glycocalyx and mass transport under fluid sheer stress (FSS), especially the relationship between glycocalyx and macromolecules such as low density lipoprotein (LDL) has been discussed. This relationship was reflected as following: on the one hand, the thickness and integrity of the glycocalyx affects the concentration polarization of LDL and its transendothelial transport and heparan sulfate proteoglycan (HSPG) participates in the whole process of residual lipoproteins metabolism. On the other hand, ox-LDL, an oxidized product of LDL, destroys heparan sulfate (HS) which is a major component of the endothelial cell glycocalyx. The study on relationship between vascular endothelial glycocalyx and lipoproteins will provide a new clue to elucidate the pathogenesis of atherosclerosis and provide more evidence to view the glycocalyx as a new control target.
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Objective To investigate the E-selectin mediated-calcium response of neutrophils under flow fields. Methods A parallel-plate flow chamber combined with a fluorescence microscope was used to observe the adhesion and subsequent calcium response of neutrophils on E-selectin at different concentrations under fluid shear stress (FSS) of 0-600 mPa. Results E-selectin could capture neutrophils from the flow to the chamber substrate and induce further intracellular calcium flux of firmly adhered cells. The arrested cell and activation ratio increased gradually as the concentration of E-selectin increased. Only immobilized E-selectin could conduct the external force-signal to trigger the calcium response of neutrophils effectively. By increasing FSS, not only was the activation ratio increased from 23% to 70%, but the calcium response intensity also increased from 0.92 to 1.45, while the delay time from cell adhesion to calcium response was greatly reduced from 70 s to 27 s. Conclusions FSS can modulate the calcium response of neutrophils in cooperation with E-selectin, and it positively regulates the activation rate and level of calcium response. This study may deepen the understanding of the immune response of leukocytes under a hemodynamic environment.
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Objective To investigate the gene expression of Piezo1 in four types of bone cells at different stages of osteogenic differentiation under fluid shear stress (FSS). Methods The mouse-derived mesenchymal stem cells (MSC), osteoblast-like cells MC3T3-E1, post-osteoblasts MLO-A5 and osteocytes MLO-Y4 were exposed to FSS at different magnitude (0.1, 1.1 Pa) with a custom-made cone-plate flow chamber for 0.5, 1, 3, 6, 12 h, respectively. The expression of Piezo1 mRNA was assessed by quantitative real-time polymerase chain reaction. Results Both Piezo1 and Piezo2 were expressed in four types of bone cells. The expression of Piezo1 was significantly up-regulated in all cells under FSS stimulation, and the expression level under 1.1 Pa FSS was significantly higher than that under 0.1 Pa FSS. In addition, the expression of Piezo1 in MSC, MC3T3-E1 and MLO-A5 cells increased to the highest level at 1 h under FSS stimulation. The expression of Piezo1 in MC3T3-E1 cells was much higher than that in the other three types of cells. Conclusions The expression of Piezo1 was related to the process of osteogenic differentiation, FSS level and loading time, and this research finding is of great significance to reveal the mechanism of mechanotransduction in bone tissues and to establish clinical treatment for bone diseases.
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Cells are exposed to mechanical stress, such as fluid shear stress (FSS), mechanical strain, hydrostatic pressure in vivo. FSS is considered to be the most important stress during bone homeostasis and remodeling. At present, most studies are mainly about the FSS effect on osteocytes and osteoblasts. However, the effects of FSS on bone mesenchymal stem cell (BMSCs) are not fully understood. BMSCs are of great significance in bone reconstruction and clinical treatment, so researchers increasingly focus on the response of BMSCs to FSS. The response of BMSCs to FSS depends on the alteration of cytoskeleton, matrix stiffness and elasticity, osteogenic signaling pathways and so on. In this review paper, the recent researches about the mechanotransduction mechanism of FSS, and its effect on differentiation and function of BMSCs are summarized, so as to provide new insights for studying construction of tissue engineered bone and treatment of bone diseases.
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Objective To investigate the effect of fluid shear stress (FSS) on the expression of B lymphoma MoMLV insertion region 1 (Bmi-1) in bone mesenchymal stem cells (BMSCs) and possible signal transduction mechanism.Methods BMSCs were isolated from SD rats and FSS at different magnitude (0.5,1.5,3.0 Pa)and under different time phase (1,2,6,24 h) were loaded by parallel-plate flow chamber system.The expression of Bmi-1 was measured by real-time RT-PCR at mRNA level and the levels of phosphorylated Akt (p-Akt)and extracellular signalregulated kinase 1/2 (p-ERK1/2) were detected by Western blotting.The signaling inhibitors,wortmannin (PI3K specific inhabitor) and PD98059 (ERK1/2 specific inhabitor),were used to investigate possible mechanical signal transduction pathway.Results Bmi-1mRNA expression increased when BMSCs were exposed to 1.5 Pa FSS for 1 h and reached the peak at 24 h.All FSS with different magnitude could increase Bmi-1 expression,especial at high FSS (3.0 Pa).Meanwhile,FSS resulted in a significant activation of p-Akt and p-ERK1/2 in BMSCs.After treated with wortmannin,the expression of Bmi-1 was inhibited prominently,however,PD98059,the expression of Bmi-1 did not change.Conclusions FSS can activate the expression of Bmi-1,the amount of Bmi-1 expression was closely related to the stimulating time and the magnitude of FSS,and Akt signal molecule plays an important role during the process.These findings provide significant references for studying the mechanical biological mechanisms of stem cell differentiation.
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Objective To investigate the effect of fluid shear stress (FSS) on the expression of B lymphoma MoMLV insertion region 1 (Bmi-1) in bone mesenchymal stem cells (BMSCs) and possible signal transduction mechanism.Methods BMSCs were isolated from SD rats and FSS at different magnitude (0.5,1.5,3.0 Pa)and under different time phase (1,2,6,24 h) were loaded by parallel-plate flow chamber system.The expression of Bmi-1 was measured by real-time RT-PCR at mRNA level and the levels of phosphorylated Akt (p-Akt)and extracellular signalregulated kinase 1/2 (p-ERK1/2) were detected by Western blotting.The signaling inhibitors,wortmannin (PI3K specific inhabitor) and PD98059 (ERK1/2 specific inhabitor),were used to investigate possible mechanical signal transduction pathway.Results Bmi-1mRNA expression increased when BMSCs were exposed to 1.5 Pa FSS for 1 h and reached the peak at 24 h.All FSS with different magnitude could increase Bmi-1 expression,especial at high FSS (3.0 Pa).Meanwhile,FSS resulted in a significant activation of p-Akt and p-ERK1/2 in BMSCs.After treated with wortmannin,the expression of Bmi-1 was inhibited prominently,however,PD98059,the expression of Bmi-1 did not change.Conclusions FSS can activate the expression of Bmi-1,the amount of Bmi-1 expression was closely related to the stimulating time and the magnitude of FSS,and Akt signal molecule plays an important role during the process.These findings provide significant references for studying the mechanical biological mechanisms of stem cell differentiation.
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Objective To investigate the effect of fluid shear stress (FSS) on the expression of B lymphoma MoMLV insertion region 1 (Bmi-1) in bone mesenchymal stem cells (BMSCs) and possible signal transduction mechanism.Methods BMSCs were isolated from SD rats and FSS at different magnitude (0.5,1.5,3.0 Pa)and under different time phase (1,2,6,24 h) were loaded by parallel-plate flow chamber system.The expression of Bmi-1 was measured by real-time RT-PCR at mRNA level and the levels of phosphorylated Akt (p-Akt)and extracellular signalregulated kinase 1/2 (p-ERK1/2) were detected by Western blotting.The signaling inhibitors,wortmannin (PI3K specific inhabitor) and PD98059 (ERK1/2 specific inhabitor),were used to investigate possible mechanical signal transduction pathway.Results Bmi-1mRNA expression increased when BMSCs were exposed to 1.5 Pa FSS for 1 h and reached the peak at 24 h.All FSS with different magnitude could increase Bmi-1 expression,especial at high FSS (3.0 Pa).Meanwhile,FSS resulted in a significant activation of p-Akt and p-ERK1/2 in BMSCs.After treated with wortmannin,the expression of Bmi-1 was inhibited prominently,however,PD98059,the expression of Bmi-1 did not change.Conclusions FSS can activate the expression of Bmi-1,the amount of Bmi-1 expression was closely related to the stimulating time and the magnitude of FSS,and Akt signal molecule plays an important role during the process.These findings provide significant references for studying the mechanical biological mechanisms of stem cell differentiation.
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Objective To examine the effects of fluid shear stress (FSS) on epithelial-mesenchymal transition (EMT) in Hep2 cells. Methods Hep2 cells were exposed to 140 mPa FSS. The morphologic changes of Hep2 cells exposed to FSS at different durations were observed using inverted microscope. The migration ability of Hep2 cells after FSS loading was investigated using scratch wound assay. The distribution and expression of cytoskeleton protein F-actin were examined by confocal microscope. The expression of the EMT marker proteins were detected by Western blotting. Results Most of Hep2 cells changed their morphology from polygon to elongated spindle with well-organized F-actin under FSS. After removing FSS, Hep2 cells recovered their initial morphology with flat polygon. FSS regulated Hep2 cells to enhance their migration capacity in a time-dependent manner. FSS promoted the rearrangement of cytoskeletal protein F-actin，which enhanced the migration behavior of Hep2 cells. In addition, FSS induced a time regularity of expression of the EMT marker proteins in Hep2 cells. Conclusions FSS as an important physical factor can induce EMT in Hep2 cells.
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Objective To construct the three-dimensional (3D) fluid model at the physiological level of shear stresses and study the effects of fluid shear stress (FSS) on adhesion, differentiation and mechanical sensitivity of osteoblasts. Methods The MC3T3-E1 osteoblasts cultured on β-tricalcium phosphate (β-TCP) scaffolds were subjected to various FSSs in the perfusion flow chamber for 6 hours to compare cell adhesion in FSS-loading groups and control group. Nitric oxide (NO) and alkaline phosphatase (ALP) were detected to compare mechanical sensitivity and cell differentiation. The FSS magnitude and distributions corresponding to various fluid rates were calculated with nonlinear fluid-structure coupling analysis. Results Cell adhesion rate was up to 74%~81% when the average FSS magnitude was lower than 0.4 Pa, but reduced to 60.22% when the average FSS was 0.41 Pa. The NO production rate reached the maximal concentration after loading for 5 min, then significantly reduced at 15 min, and gradually diminished to none at 30 min. ALP level significantly increased (P0.05) with the increase of shear stress. Conclusions Majority of the cells kept a normal adherence to the scaffold at the physiological level of shear stresses. The mechanical sensitivity of the cells under 3D condition was dependent on the FSS rate, which was consistent with two-dimensional (2D) condition. When the average FSS was lower than 0.304 Pa in the scaffold, FSS could significantly promote cell differentiation, but no significant change in cell differentiation could be found when FSS was higher than 0.304 Pa. The present study is expected to accelerate the realization of bone tissue engineering.