Effect of Fluid Shear Stress on Apoptosis and Osteogenesis of Mesenchymal Stem Cells

Effect of Fluid Shear Stress on Apoptosis and Osteogenesis of Mesenchymal Stem Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effect of Fluid Shear Stress on Apoptosis and Osteogenesis of Mesenchymal Stem Cells Fei Jiao, Xiao Zhang, Chongyang Ye, Qing Sun, Yan Gao, Chenglin Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1535410/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Mechanical cues, including fluid shear stress (FSS) of interstitial flow within bone cavities, induce osteogenic differentiation of mesenchymal stem cells (MSCs). However, whether FSS leads to the apoptosis of MSCs and its relation with osteogenic differentiation are still unclear. In this study, the effect of FSS on apoptosis and osteogenesis of MSCs is investigated. FSS is applied intermittently to MSCs through a cone-plate flow chamber system for 3 d. And results show that FSS inhibits the apoptosis of MSCs. Compared with static culture group, FSS promotes osteogenesis, as shown by the expression of three osteogenic differentiation markers, namely, alkaline phosphatase (ALP), osteocalcin (OCN), and collagen I (COL I). Double staining of individual cells shows that the relationship of apoptosis and osteogenic differentiation of MSCs is mutually exclusive. YAP may mediate the FSS-inhibiting apoptosis of MSCs. The results will further elucidate the mechanism of mechanical stimulation-induced bone remodeling. fluid shear stress mesenchymal stem cells osteogenesis apoptosis bone remodeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Mesenchymal stem cells (MSCs) are a self-renewing stem cells derived from the mesoderm and ectoderm during early embryonic development. MSCs are widely distributed in bone marrow and other tissues in animals. Under specific conditions in vitro, MSCs can differentiate into many cells, such as adipocytes, chondrocytes, osteocytes, myocytes, neurocytes, and endothelial cells 1 . Mechanical loading, especially fluid shear stress (FSS) in bone marrow and periosteum, can affect MSCs’ growth, apoptosis, and differentiation 2 . Interstitial fluid flowing through lacunar or canalicular spaces in bones generates FSS on MSCs, which may further translate into biochemical signals to regulate the biological behavior of cells 3 – 5 . FSS improves the osteogenic differentiation of MSCs 6 . The osteogenic differentiation of adipose tissue-derived mesenchymal stem cells is also observed in vitro in response to pulsating fluid flow 7 . Low-frequency (0.015, 0.044, or 0.074 Hz) pulsatile flow increases the expression of osteogenic differentiation factors, including collagen I (Col I), osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and bone morphogenetic proteins (BMP)-2, -4, and − 7 8 . Similarly, steady flow with a very low FSS of 10 − 3 to 10 − 5 Pa for 3 days significantly increases the osteogenic differentiation of MSCs, as shown by the expressions of alkaline phosphatase (ALP) and OPN 9 . The expressions of osteogenic genes, such as ALP, Runx2, COL Iα, and OCN, show that intermittent flow has greater effects on the osteogenic differentiation of hMSCs than continuous flow through the regulation of ERK1/2 and FAK activity 10 . Cell apoptosis can be significantly influenced by mechanical cues 11 – 13 . For example, FSS can regulate the fate of stem cells. The proliferation of MSCs exposed to 2 Pa FSS increases by 26.1% compared with static control 14 , 15 . FSS inhibits TNF-α-induced apoptosis of MC3T3-E1 cells by activating extracellular kinase 5 16 , in addition, the adhesion morphology of MSCs also influence their survival through their involvement with the cytoskeleton. In our previous study, the MSCs adhered to a small area of 314 µm 2 tend to undergo apoptosis, whereas those adhered to a large area of more than 1256 µm 2 tend to survive 17 . The disrupture of cytoskeleton by cytochalasin B is found to be related to the apoptosis of MSCs 18 . And the mechanical forces on MSCs are transduced into the nucleus to influence gene expression 19 . However, whether a correlation exists between osteogenesis and apoptosis under FSS exposure is still unknown and needs to be studied. In this study, we used a custom-made cone-plate flow chamber to apply constant wall FSS on MSCs, and to investigate how FSS at a physiological level regulates the apoptosis and osteogenic differentiation of MSCs. Results A cone-plate flow chamber provides controllable FSS distribution The numerical simulation results indicated that uniform FSS can be obtained in the annular region (Figs. 1D and 1F). At the center of the circle plate, the value of FSS was nearly zero. The fluctuation of FSS distribution was increased in the outer region of the circular plate. As shown in Figs. 1E and 1G, the wall FSS on the bottom increased along with the distance to the center (in the range of 0-5 mm) and displayed a relatively stable value of 1 or 2 Pa. The actual spreading area and circularity were measured based on the light-field images of single cell. Statistical analysis showed that no significant difference existed between the nuclear areas of MSCs upon exposure to FSS (1 and 2 Pa) compared with the control group. In addition, the differences of spreading area and circularity of cells were not statistically significant between static mode and FSS stimulation. The experimental results showed that FSS did not affect the spreading area, shape, or nuclear area of MSCs. MSCs apoptosis is inhibited by FSS After FSS exposure for 3 days, the apoptosis level of MSCs was detected by TUNEL assay. Typical photographs of each group are shown in Fig. 2A. The statistical results showed that compared with the control group, the average level of apoptosis decreased significantly in the FSS loading group (Fig. 2B). Compared with 2 Pa-FSS group, the average apoptosis level of 1 Pa-FSS group decreased. Osteogenesis of MSCs is enhanced by FSS To investigate the effect of FSS on osteogenic differentiation in MSCs, three types of osteogenic differentiation marker, namely, ALP, OCN and COL I, were detected after FSS exposure for 3 days. The total fluorescence intensity in a single cell was represented as the osteogenesis level of MSCs. Typical fluorescence photos of ALP, OCN and COL I are shown in Figs. 3A, 4A, and 5A. Statistical analysis showed that compared with static culture, MSCs expressed a significantly higher level of the protein of osteogenic marker ALP under FSS exposure (Fig. 3B). A similar trend was also observed for OCN (Fig. 4B) and COL I (Fig. 5B). Compared with the 1 Pa-FSS group, the expression levels of OCN and COL I were significantly higher in the 2 Pa-FSS group, whereas no significant difference was found in the expression of ALP between the two groups. The average fluorescence intensity of markers in each group was used to calculate the ratio of osteogenic differentiation. Differentiation ratios of the three markers in the loading group were significantly higher than those in the control group, and those in the 2 Pa-FSS group were significantly higher than those in the 1 Pa-FSS group (Figs. 3C, 4C, and 5C). These results demonstrated that FSS induced osteogenic differentiation in MSCs. Apoptosis and osteogenesis of MSCs are mutually exclusive Both TUNEL and ALP immunofluorescence were stained in a single cell to investigate the relationship between MSCs apoptosis and osteogenesis under the FSS exposure. Cells with high apoptotic levels had low osteogenic differentiation (Fig. 6A). The mean values of the relative fluorescence intensity of ALP and TUNEL in each group are shown in Fig. 6B. Compared with the control group, the FSS loading groups had higher osteogenesis level and lower apoptosis level. The osteogenesis level was higher and the apoptosis level was lower in the 1 Pa-FSS group than in the 2 Pa-FSS group. YAP nuclear translocation is regulated by FSS The nuclear transfer of YAP occurred when the mean YAP intensity in the nucleus was greater than that in the cytoplasm 20 . Typical fluorescence photographs are shown in Fig. 7A. The percentage of cells with YAP nuclear localization showed that FSS reduced the YAP nuclear localization. Compared with the control group, the percentage of the cells with nuclear localization was significantly reduced in the FSS loading group, whereas that in 1 Pa-FSS group was lower than in the 2 Pa-FSS group (Fig. 7B). Discussion FSS was applied intermittently to MSCs through a cone-plate flow chamber system. After 3 days of FSS exposure, the spreading state, apoptosis, osteogenic differentiation, and nuclear transfer of YAP of MSCs were detected and compared with those under static culture condition. FSS does not affect the spreading area, shape, or nuclear area but upregulates the osteogenesis level, downregulates the apoptosis level, and reduces the YAP nuclear localization of MSCs. A previous study demonstrated that the physiological FSS on osteocytes is between 0.8 and 3 Pa 21 . In the present study, the FSS of physiological levels at 1 Pa and 2 Pa decrease the apoptosis of MSCs, and the 1 Pa-FSS group has the lowest apoptosis level. This finding is consistent with the previous result that the exposure of hMSCs to 2 Pa FSS induces 126.1% proliferation compared with static controls 14 . Another study demonstrated that FSS inhibits TNF-α-induced apoptosis of MC3T3-E1 by activating extracellular kinase 5 16 . Qi et al. found that increasing FSS from 0.3 to 1.5 Pa for over 6 h significantly enhances the proliferation rate of PDL cells by 50% relative to the control group (0 Pa) 22 . Oscillating fluid flow (1 Pa, 1 Hz) can protect cells from apoptosis with or without TNF-α, and the reduction of FSS cannot inhibit the apoptosis of osteoblast-like MLO-Y4 cells 23 . All the above findings support our results that FSS inhibits MSC apoptosis. The second finding of this study is that physiological level FSS promotes the osteogenic differentiation of MSCs, which is consistent with the results of some previous studies. Kim et al. found that FSS induces the osteogenic differentiation of MSCs 24 . Yourek et al. showed that MSCs’ ALP activity is significantly increased in the osteogenic medium after 4 or 8 days of FSS exposure, and osteopontin expression is also higher than that in control groups 25 . Previous studies have shown that FSS can enhance ALP activity and upregulate osteogenic genes, such as OCN, COL I, and runt-associated transcription factor 2 (Runx2) 26 . The relationship between apoptosis and osteogenic differentiation was investigated by double staining single cells. The results showed that the apoptosis and osteogenic differentiation of MSCs are mutually exclusive. In our previous study, individual MSCs revealed a reciprocal relationship between osteogenesis and apoptosis during culture in different adhesive micropatterned islands 27 . The MSCs in large areas and low circularity prefer to differentiate into osteoblasts, whereas those in small areas may undergo apoptosis. Some previous studies showed that YAP/TAZ is an important signaling mediator of the substrate stiffness-induced fate of stem cells, and the force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction on molecular transport in nuclear pores 28 , 29 . However, our experimental results showed that FSS inhibits the nuclear localization of YAP in MSCs. The spread shape, area, and nuclear area of the cells are not changed after exposure to a physiological level of FSS for 3 days. Thus, we assume that the cytoskeleton tension and the forces applied to the nucleus are not changed, and the decrease of YAP nuclear transfer induced by FSS is mediated by other mechanisms. YAP1 coactivator is an essential regulator of cell proliferation 30 , and many studies showed that YAP is a pre-apoptotic regulator of mammalian cells 31 . When YAP is translocated to the nucleus, it binds to P73 in the nucleus, thereby promoting the transcription of downstream pro-apoptotic genes and inducing apoptosis 32 . A recent study found that FSS upregulates the expression of Piezo1 to promote the translocation of YAP to the nucleus, thereby inducing the apoptosis of suspending tumor cells. In contrast, FSS promotes YAP translocation to the cytoplasm in adherent tumor cells 33 . This finding is consistent with our present results, i.e., FSS promotes the translocation of YAP to the cytoplasm from the nucleus and decreases the apoptosis level. Further studies should be performed to clarify the mechanism underlying FSS-regulated MSC apoptosis. Methods Cell culture Mouse MSCs (Invitrogen, US) within passage 10 were cultured in α-MEM medium (Hyclone, UT) supplemented with 10% (v/v) fetal bovine serum (Gibco, NY) and 1% (v/v) penicillin and streptomycin (Invitrogen, CA) under humidified conditions at 37 °C and 5% CO 2 . After reaching 70% confluence, the cells were treated with 2% trypsin/EDTA solution (Invitrogen, CA). The suspended cells were seeded on the bottom of 6-well plates at a density of 5×10 3 cells/cm 2 . After seeding for 1 h, the culture medium was gently removed and changed every 3 days. All methods were carried out in accordance with relevant guidelines and regulations. Application of fluid shear stress In our previous study, we designed a cone-plate flow chamber based on 6-well plates, in which FSS was uniformly distributed on the annular region of the well bottom 34 . In the present study, the cone-plate flow chamber was further modified to apply FSS independently to each well (Fig. 1A). The device consisted of a stationary plate underneath a rotating cone (Fig. 1B). The distance between the cone’s tip and plate surface was controlled by placing a silicon membrane (Fig. 1C). Wall FSS exerted on the cells was controlled by specifying the cone’s rotation speed. Cone was fabricated according to hydrodynamic calculation 34 , 35 , in which a uniform wall FSS field was provided on the cell surface. MSCs were cultured on the bottom of a 6-well plate. MSCs were treated with a specific FSS thrice a day for 1 h each time. Numerical simulation Using COMSOL Multiphysics software, FSS was simulated by setting the parameters of device and the angular rate of the cone in accordance with the method used in the previous study 34 . For the cone-plate flow chamber model, the cone’s generatrix was machined as polyline to establish a uniform wall FSS field on the plate surface. The maximum radius \({R_c}\) of the cone was 15 mm, and its vertical distance \({h_c}\) to the tip was 1.3 mm. The gap \({h_0}\) between the cone’s tip and the plate surface was 0.2 mm. The radius of a well for the 6-well culture plate was 17 mm. A no-slip boundary condition was assumed for all rigid surfaces in the model except for the cone. A free surface boundary condition was used for the upper fluid surface within the well. Navier–Stokes equations were used to define the flow behavior of viscous fluids. Incompressible viscous fluid was assumed to have a density of 1×10 3 kg/m 3 and a viscosity of 1×10 − 3 Pa∙s. An iterative method was used to solve the equations for steady flow, and convergence was identified when the relative tolerance was less than 0.001. During numerical simulation, Reynolds number was computed as defined by Sdougos et al. 36 . According to the calculated Reynolds number, fluid flow in the cone-plate should be assumed as steady laminar flow, and FSS is calculated according to the following equation: \(\tau {\text{=}}\mu \frac{{\partial \nu }}{{\partial z}}\) We first calculated the shear strain rate \(\gamma {\text{=}}{{d\nu } \mathord{\left/ {\vphantom {{d\nu } {dz}}} \right. \kern-0pt} {dz}}\) along the direction close to the plate surface based on numerical simulation results and then obtained FSS by multiplying the shear strain rate with the viscosity coefficient \(\mu\) . All rigid surfaces in the model were assumed to be non-slip boundary conditions. Upper flow surface in the well was adopted as an open boundary condition, and the rotating cone was adopted as a sliding boundary condition. MATLAB and Origin software were used for data processing. Immunofluorescence staining After incubation in a 6-well plate for 3 days, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, rinsed with phosphate buffer saline twice, permeabilized with 0.2% Triton-X100 for 10 min, and blocked with 1% bovine serum albumin for 60 min at room temperature. In addition, the cells were labeled with primary antibody against alkaline phosphatase (ALP, Santa Cruz Biotechnology, Inc., USA), type I collagen (COL I, Santa Cruz Biotechnology, Inc., USA), osteocalcin (OCN, Santa Cruz Biotechnology, Inc., USA), or YAP (ABclonal, CN) at a 1:200 dilution overnight at 4 °C. Second antibody labeling was performed in the dark for 60 min at 37 °C. The nuclei were stained with 0.1% Hoechst (Invitrogen, USA) for 10 min. The cells were double-stained with an apoptosis assay kit (One Step TUNEL Apoptosis Assay Kit, Beyotime, CA) and ALP-conjugated TRITC. After fixation and permeabilization, the cells were incubated in the dark with a TUNEL reaction mixture for 60 min at 37 °C. Image analysis For mechanical experiments, MSCs were chosen from annular regions with inner and outer diameters of 5 and 12 mm, respectively, where the wall FSS appears uniform. The spreading area and shape of a single cell were obtained from bright-field image. Its nuclear area was acquired from Hoechst-staining image. Bright-field or fluorescence images were analyzed using ImageJ software 27 . Statistical analysis Statistical data were expressed as mean ± standard error of mean. Three independent experiments were performed with at least 50 cells for each group. One-way analysis of variance (ANOVA) with Turkey’s post hoc test for multiple comparisons was performed for statistical analysis by using Origin software. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1535410","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":98904565,"identity":"441f1670-ec68-4f3e-bd0b-27886dd0bcf5","order_by":0,"name":"Fei Jiao","email":"","orcid":"","institution":"Beijing Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Jiao","suffix":""},{"id":98904566,"identity":"c062d4d4-e7d1-481d-b4c5-30a34ddccd93","order_by":1,"name":"Xiao Zhang","email":"","orcid":"","institution":"Beijing Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Zhang","suffix":""},{"id":98904567,"identity":"05032c9c-384c-48c1-93b4-6007febcaebd","order_by":2,"name":"Chongyang Ye","email":"","orcid":"","institution":"Beijing Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chongyang","middleName":"","lastName":"Ye","suffix":""},{"id":98904568,"identity":"857c33d1-039d-4320-8982-c559eb8c3140","order_by":3,"name":"Qing Sun","email":"","orcid":"","institution":"Beijing Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Sun","suffix":""},{"id":98904569,"identity":"2ac6821d-ead0-4cf8-a8d9-2468e44f8b78","order_by":4,"name":"Yan Gao","email":"","orcid":"","institution":"Beijing Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Gao","suffix":""},{"id":98904570,"identity":"feb72192-40a7-4ec9-b371-a925eb7d61f0","order_by":5,"name":"Chenglin Liu","email":"","orcid":"","institution":"Capital University of Physical Education and Sports","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenglin","middleName":"","lastName":"Liu","suffix":""},{"id":98904571,"identity":"3785a395-f8ec-4644-8b1e-7b770c47225d","order_by":6,"name":"Bo Huo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYBACAwYGNgaGigM8II4ECVrOHODhIU0LY9sBBuK1mEskH3vwcd4dGXsG5oO3eRjs8ghqsZyRlm44c9szoMPYkq15GJKLCTvsRo6ZNO+2w0AtPGbSPAwHEhuI0zIHpIX/GylaGsC2sBGp5cyzdMMZx4B+OcxmbDnHIJkILceBIfah5o49e3vzwxtvKuwIa0EAZrAJxKsfBaNgFIyCUYAHAADxpjXpfGGWBQAAAABJRU5ErkJggg==","orcid":"","institution":"Beijing Institute of Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Huo","suffix":""}],"badges":[],"createdAt":"2022-04-08 03:29:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1535410/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1535410/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20501403,"identity":"296a46ce-63c4-42f5-baa7-2ffbe3e01535","added_by":"auto","created_at":"2022-04-19 14:05:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5099866,"visible":true,"origin":"","legend":"\u003cp\u003eCone-plate device for applying FSS to cells. (A) The photograph of cone-plate flow chamber system. (B) The side-view photo of the flow chamber. (C) The schematic graph of the flow chamber. Finite element analysis of wall FSS in cone-plate flow chamber. (D, F) Wall FSS distribution on the bottom plate for 36 and 80 rad/s angular velocity of the cone, respectively. (E, G) Radial distribution of wall FSS for 36 and 80 rad/s angular velocity of the cone, respectively.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1535410/v1/51cc69958ed80a87f0aa40f5.jpg"},{"id":20501981,"identity":"ae11caa7-bd79-4fe8-8b98-89068a56403f","added_by":"auto","created_at":"2022-04-19 14:10:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5476680,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis of individual MSCs under static condition or FSS exposure for 3 days. (A) Typical photographs of TUNEL staining. Scale bar, 50 μm. (B) Fluorescence intensity of TUNEL staining under static condition and FSS exposure. *, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1535410/v1/1b840c89cefeed8ec5267629.jpg"},{"id":20501401,"identity":"f25751bd-2140-48e7-8e85-f9085e8fb1e1","added_by":"auto","created_at":"2022-04-19 14:05:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3310072,"visible":true,"origin":"","legend":"\u003cp\u003eALP expression of individual MSCs cultured under static condition or FSS exposure for 3 days. (A) Typical fluorescent photographs of ALP. Scale bar, 50 μm. (B) Fluorescence intensity of ALP. (C) Osteogenesis ratio. *, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1535410/v1/f58536ca1dd7b96be9c0134c.jpg"},{"id":20501980,"identity":"c85ccaad-b5c6-45af-b7db-06d00fb4de1e","added_by":"auto","created_at":"2022-04-19 14:10:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1984689,"visible":true,"origin":"","legend":"\u003cp\u003eOCN expression in individual MSCs cultured under static condition or FSS exposure for 3 days. (A) Typical fluorescent photos of OCN. Scale bar, 50 μm. (B) Fluorescence intensity of OCN. (C) Osteogenesis ratio. *, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1535410/v1/462f2888b52a2d3770fb46d3.jpg"},{"id":20501402,"identity":"76621598-0902-44f2-9afa-2a6a8f299e93","added_by":"auto","created_at":"2022-04-19 14:05:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1875774,"visible":true,"origin":"","legend":"\u003cp\u003eCOL I expression in individual MSCs cultured under static condition or FSS exposure for 3 days. (A) Typical fluorescent photos of COL I. Scale bar, 50 μm. (B) Fluorescence intensity of COL I. (C) Osteogenesis ratio. *, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1535410/v1/0616d48231d15f78efbb2b5c.jpg"},{"id":20501405,"identity":"2bdc563f-e9d9-4ac4-ab62-fc5feb6117ff","added_by":"auto","created_at":"2022-04-19 14:05:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3497396,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between ALP expression and apoptosis in MSCs cultured under static condition or FSS exposure for 3 days. (A) Scatter plots of ALP and apoptosis. A dot represents the fluorescent intensity of ALP and the level of apoptosis of a single cell. (B) Mean fluorescence intensity of ALP and apoptosis of each group. Four quadrants in B were divided according to the values of the cells under static condition.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1535410/v1/095becf07673516622d67ca0.jpg"},{"id":20501406,"identity":"13b4a33c-e424-454c-b3f3-4103bc068a8d","added_by":"auto","created_at":"2022-04-19 14:05:27","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1916114,"visible":true,"origin":"","legend":"\u003cp\u003eNuclear localization of YAP in MSCs under static condition or FSS exposure for 3 days. (A) Typical fluorescence photos of YAP. Scale bar, 50 μm. (B) Percentage of YAP nuclear translocation under static condition and FSS exposure. *, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1535410/v1/472153f61146a0863743edb2.jpg"},{"id":31008411,"identity":"7506fd25-93ed-4bff-81b7-98ae5436e908","added_by":"auto","created_at":"2023-01-03 09:44:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":796316,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1535410/v1/6c0fa79e-edb8-4d9e-abe4-137c66ac0c46.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Fluid Shear Stress on Apoptosis and Osteogenesis of Mesenchymal Stem Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMesenchymal stem cells (MSCs) are a self-renewing stem cells derived from the mesoderm and ectoderm during early embryonic development. MSCs are widely distributed in bone marrow and other tissues in animals. Under specific conditions in vitro, MSCs can differentiate into many cells, such as adipocytes, chondrocytes, osteocytes, myocytes, neurocytes, and endothelial cells \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Mechanical loading, especially fluid shear stress (FSS) in bone marrow and periosteum, can affect MSCs\u0026rsquo; growth, apoptosis, and differentiation \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterstitial fluid flowing through lacunar or canalicular spaces in bones generates FSS on MSCs, which may further translate into biochemical signals to regulate the biological behavior of cells \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. FSS improves the osteogenic differentiation of MSCs \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The osteogenic differentiation of adipose tissue-derived mesenchymal stem cells is also observed in vitro in response to pulsating fluid flow \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Low-frequency (0.015, 0.044, or 0.074 Hz) pulsatile flow increases the expression of osteogenic differentiation factors, including collagen I (Col I), osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and bone morphogenetic proteins (BMP)-2, -4, and \u0026minus;\u0026thinsp;7 \u003csup\u003e8\u003c/sup\u003e. Similarly, steady flow with a very low FSS of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e Pa for 3 days significantly increases the osteogenic differentiation of MSCs, as shown by the expressions of alkaline phosphatase (ALP) and OPN \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The expressions of osteogenic genes, such as ALP, Runx2, COL Iα, and OCN, show that intermittent flow has greater effects on the osteogenic differentiation of hMSCs than continuous flow through the regulation of ERK1/2 and FAK activity \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCell apoptosis can be significantly influenced by mechanical cues \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. For example, FSS can regulate the fate of stem cells. The proliferation of MSCs exposed to 2 Pa FSS increases by 26.1% compared with static control \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. FSS inhibits TNF-α-induced apoptosis of MC3T3-E1 cells by activating extracellular kinase 5 \u003csup\u003e16\u003c/sup\u003e, in addition, the adhesion morphology of MSCs also influence their survival through their involvement with the cytoskeleton. In our previous study, the MSCs adhered to a small area of 314 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e tend to undergo apoptosis, whereas those adhered to a large area of more than 1256 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e tend to survive \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The disrupture of cytoskeleton by cytochalasin B is found to be related to the apoptosis of MSCs \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. And the mechanical forces on MSCs are transduced into the nucleus to influence gene expression \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, whether a correlation exists between osteogenesis and apoptosis under FSS exposure is still unknown and needs to be studied.\u003c/p\u003e \u003cp\u003eIn this study, we used a custom-made cone-plate flow chamber to apply constant wall FSS on MSCs, and to investigate how FSS at a physiological level regulates the apoptosis and osteogenic differentiation of MSCs.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eA cone-plate flow chamber provides controllable FSS distribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe numerical simulation results indicated that uniform FSS can be obtained in the annular region (Figs. 1D and 1F). At the center of the circle plate, the value of FSS was nearly zero. The fluctuation of FSS distribution was increased in the outer region of the circular plate. As shown in Figs. 1E and 1G, the wall FSS on the bottom increased along with the distance to the center (in the range of 0-5 mm) and displayed a relatively stable value of 1 or 2 Pa.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe actual spreading area and circularity were measured based on the light-field images of single cell. Statistical analysis showed that no significant difference existed between the nuclear areas of MSCs upon exposure to FSS (1 and 2 Pa) compared with the control group. In addition, the differences of spreading area and circularity of cells were not statistically significant between static mode and FSS stimulation. The experimental results showed that FSS did not affect the spreading area, shape, or nuclear area of MSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMSCs apoptosis is inhibited by FSS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter FSS exposure for 3 days, the apoptosis level of MSCs was detected by TUNEL assay. Typical photographs of each group are shown in Fig. 2A. The statistical results showed that compared with the control group, the average level of apoptosis decreased significantly in the FSS loading group (Fig. 2B). Compared with 2 Pa-FSS group, the average apoptosis level of 1 Pa-FSS group decreased.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsteogenesis of MSCs is enhanced by FSS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of FSS on osteogenic differentiation in MSCs, three types of osteogenic differentiation marker, namely, ALP, OCN and COL I, were detected after FSS exposure for 3 days. The total fluorescence intensity in a single cell was represented as the osteogenesis level of MSCs. Typical fluorescence photos of ALP, OCN and COL I are shown in Figs. 3A, 4A, and 5A. Statistical analysis showed that compared with static culture, MSCs expressed a significantly higher level of the protein of osteogenic marker ALP under FSS exposure (Fig. 3B).\u0026nbsp;A similar trend was also observed for OCN (Fig. 4B) and COL I (Fig. 5B). Compared with the 1 Pa-FSS group, the expression levels of OCN and COL I were significantly higher in the 2 Pa-FSS group, whereas no significant difference was found in the expression of ALP between the two groups. The average fluorescence intensity of markers in each group was used to calculate the ratio of osteogenic differentiation. Differentiation ratios of the three markers in the loading group were significantly higher than those in the control group, and those in the 2 Pa-FSS group were significantly higher than those in the 1 Pa-FSS group (Figs. 3C, 4C, and 5C). These results demonstrated that FSS induced osteogenic differentiation in MSCs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis and osteogenesis of MSCs are mutually exclusive\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth TUNEL and ALP immunofluorescence were stained in a single cell to investigate the relationship between MSCs apoptosis and osteogenesis under the FSS exposure. Cells with high apoptotic levels had low osteogenic differentiation (Fig. 6A). The mean values of the relative fluorescence intensity of ALP and TUNEL in each group are shown in Fig. 6B. Compared with the control group, the FSS loading groups had higher osteogenesis level and lower apoptosis level. The osteogenesis level was higher and the apoptosis level was lower in the 1 Pa-FSS group than in the 2 Pa-FSS group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYAP nuclear translocation is regulated by FSS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nuclear transfer of YAP occurred when the mean YAP intensity in the nucleus was greater than that in the cytoplasm\u0026nbsp;\u003ca href=\"#_ENREF_20\" title=\"Zhang, 2016 #10230\"\u003e\u003csup\u003e20\u003c/sup\u003e\u003c/a\u003e. Typical fluorescence photographs are shown in Fig. 7A. The percentage of cells with YAP nuclear localization showed that FSS reduced the YAP nuclear localization. Compared with the control group, the percentage of the cells with nuclear localization was significantly reduced in the FSS loading group, whereas that in 1 Pa-FSS group was lower than in the 2 Pa-FSS group (Fig. 7B). \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFSS was applied intermittently to MSCs through a cone-plate flow chamber system. After 3 days of FSS exposure, the spreading state, apoptosis, osteogenic differentiation, and nuclear transfer of YAP of MSCs were detected and compared with those under static culture condition. FSS does not affect the spreading area, shape, or nuclear area but upregulates the osteogenesis level, downregulates the apoptosis level, and reduces the YAP nuclear localization of MSCs.\u003c/p\u003e \u003cp\u003eA previous study demonstrated that the physiological FSS on osteocytes is between 0.8 and 3 Pa \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In the present study, the FSS of physiological levels at 1 Pa and 2 Pa decrease the apoptosis of MSCs, and the 1 Pa-FSS group has the lowest apoptosis level. This finding is consistent with the previous result that the exposure of hMSCs to 2 Pa FSS induces 126.1% proliferation compared with static controls \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Another study demonstrated that FSS inhibits TNF-α-induced apoptosis of MC3T3-E1 by activating extracellular kinase 5 \u003csup\u003e16\u003c/sup\u003e. Qi et al. found that increasing FSS from 0.3 to 1.5 Pa for over 6 h significantly enhances the proliferation rate of PDL cells by 50% relative to the control group (0 Pa) \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Oscillating fluid flow (1 Pa, 1 Hz) can protect cells from apoptosis with or without TNF-α, and the reduction of FSS cannot inhibit the apoptosis of osteoblast-like MLO-Y4 cells \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. All the above findings support our results that FSS inhibits MSC apoptosis.\u003c/p\u003e \u003cp\u003eThe second finding of this study is that physiological level FSS promotes the osteogenic differentiation of MSCs, which is consistent with the results of some previous studies. Kim et al. found that FSS induces the osteogenic differentiation of MSCs \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Yourek et al. showed that MSCs\u0026rsquo; ALP activity is significantly increased in the osteogenic medium after 4 or 8 days of FSS exposure, and osteopontin expression is also higher than that in control groups \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that FSS can enhance ALP activity and upregulate osteogenic genes, such as OCN, COL I, and runt-associated transcription factor 2 (Runx2) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe relationship between apoptosis and osteogenic differentiation was investigated by double staining single cells. The results showed that the apoptosis and osteogenic differentiation of MSCs are mutually exclusive. In our previous study, individual MSCs revealed a reciprocal relationship between osteogenesis and apoptosis during culture in different adhesive micropatterned islands \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The MSCs in large areas and low circularity prefer to differentiate into osteoblasts, whereas those in small areas may undergo apoptosis.\u003c/p\u003e \u003cp\u003eSome previous studies showed that YAP/TAZ is an important signaling mediator of the substrate stiffness-induced fate of stem cells, and the force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction on molecular transport in nuclear pores \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, our experimental results showed that FSS inhibits the nuclear localization of YAP in MSCs. The spread shape, area, and nuclear area of the cells are not changed after exposure to a physiological level of FSS for 3 days. Thus, we assume that the cytoskeleton tension and the forces applied to the nucleus are not changed, and the decrease of YAP nuclear transfer induced by FSS is mediated by other mechanisms. YAP1 coactivator is an essential regulator of cell proliferation \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and many studies showed that YAP is a pre-apoptotic regulator of mammalian cells \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. When YAP is translocated to the nucleus, it binds to P73 in the nucleus, thereby promoting the transcription of downstream pro-apoptotic genes and inducing apoptosis \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. A recent study found that FSS upregulates the expression of Piezo1 to promote the translocation of YAP to the nucleus, thereby inducing the apoptosis of suspending tumor cells. In contrast, FSS promotes YAP translocation to the cytoplasm in adherent tumor cells \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. This finding is consistent with our present results, i.e., FSS promotes the translocation of YAP to the cytoplasm from the nucleus and decreases the apoptosis level. Further studies should be performed to clarify the mechanism underlying FSS-regulated MSC apoptosis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse MSCs (Invitrogen, US) within passage 10 were cultured in\u0026nbsp;α-MEM medium (Hyclone, UT) supplemented with 10% (v/v) fetal bovine serum (Gibco, NY) and 1% (v/v) penicillin and streptomycin (Invitrogen, CA) under humidified conditions at 37\u0026nbsp;°C\u0026nbsp;and 5% CO\u003csub\u003e2\u003c/sub\u003e. After reaching 70% confluence, the cells were treated with 2% trypsin/EDTA solution (Invitrogen, CA). The suspended cells were seeded on the bottom of 6-well plates at a density of 5×10\u003csup\u003e3\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e. After seeding for 1 h, the culture medium was gently removed and changed every 3 days. All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApplication of fluid shear stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn our previous study, we designed a cone-plate flow chamber based on 6-well plates, in which FSS was uniformly distributed on the annular region of the well bottom\u0026nbsp;\u003ca href=\"#_ENREF_34\" title=\"Ye, 2019 #10350\"\u003e\u003csup\u003e34\u003c/sup\u003e\u003c/a\u003e. In the present study, the cone-plate flow chamber was further modified to apply FSS independently to each well (Fig. 1A). The device consisted of a stationary plate underneath a rotating cone (Fig. 1B). The distance between the cone’s tip and plate surface was controlled by placing a silicon membrane (Fig. 1C). Wall FSS exerted on the cells was controlled by specifying the cone’s rotation speed. Cone was fabricated according to hydrodynamic calculation\u0026nbsp;\u003ca href=\"#_ENREF_34\" title=\"Ye, 2019 #10350\"\u003e\u003csup\u003e34\u003c/sup\u003e\u003c/a\u003e\u003csup\u003e,\u003c/sup\u003e\u003ca href=\"#_ENREF_35\" title=\"Gao, 2019 #10877\"\u003e\u003csup\u003e35\u003c/sup\u003e\u003c/a\u003e, in which a uniform wall FSS field was provided on the cell surface. MSCs were cultured on the bottom of a 6-well plate. MSCs were treated with a specific FSS thrice a day for 1 h each time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNumerical simulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing COMSOL Multiphysics software, FSS was simulated by setting the parameters of device and the angular rate of the cone in accordance with the method used in the previous study \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. For the cone-plate flow chamber model, the cone\u0026rsquo;s generatrix was machined as polyline to establish a uniform wall FSS field on the plate surface. The maximum radius \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({R_c}\\)\u003c/span\u003e\u003c/span\u003e of the cone was 15 mm, and its vertical distance \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({h_c}\\)\u003c/span\u003e\u003c/span\u003e to the tip was 1.3 mm. The gap \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({h_0}\\)\u003c/span\u003e\u003c/span\u003e between the cone\u0026rsquo;s tip and the plate surface was 0.2 mm. The radius \u003cspan class=\"InlineEquation\"\u003e\u003c/span\u003e of a well for the 6-well culture plate was 17 mm. A no-slip boundary condition was assumed for all rigid surfaces in the model except for the cone. A free surface boundary condition was used for the upper fluid surface within the well.\u003c/p\u003e \u003cp\u003eNavier\u0026ndash;Stokes equations were used to define the flow behavior of viscous fluids. Incompressible viscous fluid was assumed to have a density of 1\u0026times;10\u003csup\u003e3\u003c/sup\u003e kg/m\u003csup\u003e3\u003c/sup\u003e and a viscosity of 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e Pa∙s. An iterative method was used to solve the equations for steady flow, and convergence was identified when the relative tolerance was less than 0.001.\u003c/p\u003e \u003cp\u003eDuring numerical simulation, Reynolds number was computed as defined by Sdougos et al. \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. According to the calculated Reynolds number, fluid flow in the cone-plate should be assumed as steady laminar flow, and FSS is calculated according to the following equation:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\tau {\\text{=}}\\mu \\frac{{\\partial \\nu }}{{\\partial z}}\\)\u003c/span\u003e \u003c/span\u003e \u003c/p\u003e \u003cp\u003eWe first calculated the shear strain rate\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\gamma {\\text{=}}{{d\\nu } \\mathord{\\left/ {\\vphantom {{d\\nu } {dz}}} \\right. \\kern-0pt} {dz}}\\)\u003c/span\u003e\u003c/span\u003ealong the\u003cspan class=\"InlineEquation\"\u003e\u003c/span\u003edirection close to the plate surface based on numerical simulation results and then obtained FSS by multiplying the shear strain rate with the viscosity coefficient \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu\\)\u003c/span\u003e\u003c/span\u003e. All rigid surfaces in the model were assumed to be non-slip boundary conditions. Upper flow surface in the well was adopted as an open boundary condition, and the rotating cone was adopted as a sliding boundary condition. MATLAB and Origin software were used for data processing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter incubation in a 6-well plate for 3 days, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, rinsed with phosphate buffer saline twice, permeabilized with 0.2% Triton-X100 for 10 min, and blocked with 1% bovine serum albumin for 60 min at room temperature. In addition, the cells were labeled with primary antibody against alkaline phosphatase (ALP, Santa Cruz Biotechnology, Inc., USA), type I collagen (COL I, Santa Cruz Biotechnology, Inc., USA), osteocalcin (OCN, Santa Cruz Biotechnology, Inc., USA), or YAP (ABclonal, CN) at a 1:200 dilution overnight at 4 °C. Second antibody labeling was performed in the dark for 60 min at 37 °C. The nuclei were stained with 0.1% Hoechst (Invitrogen, USA) for 10 min. The cells were double-stained with an apoptosis assay kit (One Step TUNEL Apoptosis Assay Kit, Beyotime, CA) and ALP-conjugated TRITC. After fixation and permeabilization, the cells were incubated in the dark with a TUNEL reaction mixture for 60 min at 37 °C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor mechanical experiments, MSCs were chosen from annular regions with inner and outer diameters of 5 and 12 mm, respectively, where the wall FSS appears uniform.\u0026nbsp;The spreading area and shape of a single cell were obtained from bright-field image. Its nuclear area was acquired from Hoechst-staining image.\u0026nbsp;Bright-field or fluorescence images were analyzed using ImageJ software\u0026nbsp;\u003ca href=\"#_ENREF_27\" title=\"Jiao, 2020 #10869\"\u003e\u003csup\u003e27\u003c/sup\u003e\u003c/a\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical data were expressed as mean ± standard error of mean. Three independent experiments were performed with at least 50 cells for each group. One-way analysis of variance (ANOVA) with Turkey’s post hoc test for multiple comparisons was performed for statistical analysis by using Origin software. The osteogenesis ratio of three osteogenic markers was calculated by chi-square test. The mean values of different groups were regarded as significantly different when p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China [12072034, 11572043 (BH)].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003ePittenger, M. F. \u003cem\u003eet al.\u003c/em\u003e Multilineage potential of adult human mesenchymal stem cells. 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Journal of Fluid Mechanics. \u003cstrong\u003e138\u003c/strong\u003e, 379\u0026ndash;404, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1017/s0022112084000161\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"fluid shear stress, mesenchymal stem cells, osteogenesis, apoptosis, bone remodeling","lastPublishedDoi":"10.21203/rs.3.rs-1535410/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1535410/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMechanical cues, including fluid shear stress (FSS) of interstitial flow within bone cavities, induce osteogenic differentiation of mesenchymal stem cells (MSCs). However, whether FSS leads to the apoptosis of MSCs and its relation with osteogenic differentiation are still unclear. In this study, the effect of FSS on apoptosis and osteogenesis of MSCs is investigated. FSS is applied intermittently to MSCs through a cone-plate flow chamber system for 3 d. And results show that FSS inhibits the apoptosis of MSCs. Compared with static culture group, FSS promotes osteogenesis, as shown by the expression of three osteogenic differentiation markers, namely, alkaline phosphatase (ALP), osteocalcin (OCN), and collagen I (COL I). Double staining of individual cells shows that the relationship of apoptosis and osteogenic differentiation of MSCs is mutually exclusive. YAP may mediate the FSS-inhibiting apoptosis of MSCs. The results will further elucidate the mechanism of mechanical stimulation-induced bone remodeling.\u003c/p\u003e","manuscriptTitle":"Effect of Fluid Shear Stress on Apoptosis and Osteogenesis of Mesenchymal Stem Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-19 14:05:25","doi":"10.21203/rs.3.rs-1535410/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8e8409cd-0605-40e2-b850-18ce42da3c80","owner":[],"postedDate":"April 19th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-03T09:44:30+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-19 14:05:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1535410","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1535410","identity":"rs-1535410","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}