The Effect And Mechanism Of Hypergravity On Osteoblasts And Bone Remodeling

Background and objective:Along with biochemical stimuli,mechanical stimuli are currently known to trigger essential intracellular signals in various cell species.Mechanical stimuli such as hydrostatic pressure,compressive stress and tensile stress are widely utilized in the field of mechanobiology.Mechanical stimuli play important roles in bone remodeling.Bone cells differentiate,proliferate,mature under mechanical stress in vivo,and are extremely sensitive to mechanical cues.Engineered bone tissue cultured in vitro always fails to provide adequate tissue structure and mechanical properties.This is largely attributed to the lack of mechanical stimuli during cell culture.Among mechanical stimuli,altered gravity involving microgravity and hypergravity has attracted interest for studying the effects of space flight.Since the cytosol and intracellular micro-organelles such as the nucleus,mitochondria,the cytoskeletons including actin fibers,intermediate filaments,and microtubules have different densities;changes in their relative position could result from altered gravity.Several reports have shown that altered gravity exerts various effects on mammalian cell models.Generally,it is known that microgravity strongly suppresses bone mass after a long period of exposure.While the hypergravity has the advantage of being easily realized in vitro by centrifugation,only limited studies are currently available with regard to hypergravity and osteogenesis.These recent findings suggest that hypergravity triggers complex mechanotransduction pathways regulated at both transcriptional and post-transcriptional levels.However,the precise molecular mechanisms underlying the effects of hypergravity on osteoblasts have yet to be clearly defined.The aim of this study is to investigate the effect and mechanism of hypergravity on osteoblasts and bone remodeling.It provides a theoretical basis for the study of growth,differentiation,reconstruction and adaptive changes of bone tissue cells under extreme mechanical conditions.This study is an extension of the research work on the growth,differentiation,reconstruction and adaptive changes of bone tissue/cell under physiological load environment,thus developing new theories,methods and techniques of biomechanics and mechanical biology,and expanding the new research field of biomechanics.Methods:1 Osteoblast-like MC3T3-E1 cells were cultured in?-MEM supplemented with10%fetal bovine serum,100 U/ml penicillin and 100?g/ml streptomycin at 37°C in a humidified incubator containing 5%CO₂.Unless otherwise specified,10⁵ cells per flask were exposed to 5 g,10 g,20 g,and 40 g hypergravity for 30 min once a day,last 3days,after seeding and spreading for 12 h.The control cells were subjected to the same conditions as the experimental cells in terms of timing,incubation media,and other procedures,with the exception of the gravity condition;for this,control cells were incubated at 1 g for 30 min in the 37°C incubator as that used for the experimental cells.2 We used HE staining,transmission electron microscopy and phalloidin staining to observe the morphology,ultrastructure and microfilament skeleton of MC3T3-E1cells after hypergravity exposure.3 Edu cell proliferation assay and Annexin V-FITC/PI flow cytometry were used to detect the effect of hypergravity on proliferation and apoptosis of MC3T3-E1 cells;qPCR and western blot were used to detect the expression and activity of osteogenic related genes and proteins in MC3T3-E1 cells under hypergravity environment.4 The differential coding and noncoding transcript expression profiling of MC3T3-E1 cells during exposure to hypergravity were established by mouse whole transcriptome microarray.Microarray data were validated using quantitative real-time polymerase chain reaction?Q-PCR?.Bioinformatic analyses were applied for further study of these differentially expressed lncRNAs and mRNA,the function of candidate genes and their regulatory mechanisms;5 We applied HPLC-MS/MS using an isobaric TMTs proteomics technology to analyse the differentially expressed proteins of MC3T3-E1 cells before and after hypergravity.In order to study of the role and mechanism of hypergravity on bone remodeling,multi-omics were used to analysis the information of transcription,translation,expression and other aspects.Results:1 Osteoblast-like MC3T3-E1 cells were cultured in the earth environment?1 g?.In the early stage of cell culture,the cells were mainly the fibroblast cell types,which are spindle-shaped and connected with others through the protrusions.In the late stage of cultivation,the cells were mainly epithelial cell types,which are cubic,connected into pieces and arranged cobblestone shapes.With the increasing of gravity acceleration?5 g,10 g,20 g,40 g?and culture time?24 h,48 h,72 h?,the contour of mouse MC3T3-E1cells in hypergravity were gradually rounded,the microfilament actin fibers were thickened and the cell volume were increased,which were consistent with the results of light microscope.The ultrastructure of cells,such as cell membrane,cytoplasm,nucleus and mitochondria,endoplasmic reticulum,ribosomes were all changed.2 The biological function of MC3T3-E1 cells were changed under hypergravity environment.The proliferation activity of MC3T3-E1 cells began to increase at 5 g exposure and significantly increase at 10 g and 20 g exposure.But when expose to 40 g,the proliferation activity of osteoblast was significantly inhibited.Low levels?5 g?of hypergravity stimulation had no significant effect on apoptosis of osteoblast.Moderate levels?10 g and 20 g?of hypergravity stimulation caused mild apoptosis of osteoblasts.High levels?40 g?of hypergravity stimulation cause massive apoptosis of osteoblasts.We observed that hypergravity at low and medium levels?5 g,10 g,and 20 g?promoted an increased gene and protein expression of the common genetic markers of osteoblasts differentiation.However,when expose to 40 g,the gene and protein expressions of mouse osteogenic genes?Runx2,ALP,and OCN?were decreased.3 A total of 58,809 mouse lncRNAs and 39,027 mouse mRNAs were detected by the gene chip.A large number of lncRNA and mRNA was changed in the hypergravity environment,and with the increasing of gravity acceleration,differentially expressed lncRNA and mRNA are also coming.A total of 35 coding transcripts with 6 genes significantly upregulated and 29genes downregulated in hypergravity-exposed?5 g?10g?20 g and 40 g?cells compared to the control cells,and 193 lncRNAs with 28 lncRNAs significantly upregulated and 165 lncRNAs downregulated were detected along with osteogenic differentiation under conditions of hypergravity.The potential regulatory mechanisms were analyzed by bioinformatics analysis and the interaction between differentially expressed lncRNAs and mRNAs was further predicted.mRNAs-lncRNAs co-expression network analysis also revealed 10 core regulatory genes including seven mRNAs and three lncRNAs.Based on the analysis,we identified one lncRNA,NONMMUT012703,which is likely to play important roles in bone remodeling process under hypergravity.4 We applied HPLC-MS/MS using an isobaric TMTs proteomics technology to analyse the differentially expressed proteins of MC3T3-E1 cells under hypergravity environment.It was shown that there were 24 differentially expressed proteins,of which22 were up-regulated and 2 were down-regulated after hypergravity exposure.The potential regulatory mechanisms were analyzed by bioinformatics analysis and the interaction between differentially expressed proteins was predicted.We further studied the function of key proteins.5 In order to study of the role and the mechanism on bone remodeling in hypergravity,multi-omics[non-coding RNA?lncRNAs?expression profiles,encoding RNA?mRNAs?expression profiles and protein expression profiles]were used to analysis the information of transcription,translation,expression and other aspects.Based on the bioinformatics analysis and some related research results,we identified one lncRNA,NONMMUT01270,as a candidate for hypergravity sensitive and bone remodeling related lncRNA.NONMMUT012703,which may interact with target genes?such as CFTR,NFATC2,SYCN,SOCS3,GIP,SNHG7OS,and VMN2R90?,or bind with transcription factors?c-Rel,HNF-3beta,E47,HLF,RFX1,and COMP1?through multiple signaling pathways?eg,MAPK,Jak-STAT,cGMP-PKG,Wnt,PI3K-Ak,and VEGF signaling pathways?participate in the bone remodeling process in hypergravity environment.Conclusions:1 The high-acceleration centrifugal loading device,which is independently developed by our research group,can simulate the hypergravity environment and is suitable for the study of the mechanical and biological effects of cells in hypergravity environment.2 Osteoblast-like MC3T3-E1 cells have the biological characteristics of osteoblasts and can respond to mechanical stimulation well.It is a good model for studying the biological effects of osteoblasts under stress.3 In order to resist mechanical stimulation,MC3T3-E1 cells morphology,microfilament skeleton and ultrastructure were changed under hypergravity.4 Under the extreme mechanical environment of hypergravity,the biological function of mouse MC3T3-E1 cells were changed.The effect of hypergravity on osteoblast proliferation,apoptosis and differentiation depends on the magnitude of gravity acceleration.A gravity acceleration of 20 g or less is a safe loading range and does not cause a large amount of apoptosis of osteoblasts.At medium levels of hypergravity?10 g and 20 g?stimulation,the biological behavior of mouse osteoblasts significantly changed.5 The non-coding RNA?lncRNAs?,coding RNA?mRNAs?and protein expression profiles of MC3T3-E1 cells were changed when exposed to the extreme hypergravity environment.Moreover,as the gravity acceleration increases,the differentially altered genes and proteins were gradually increased.6 The lncRNA obtained in the process of osteogenic differentiation during exposure to hypergravity may be a potential target in the process of bone tissue damage,repair and reconstruction.7 Differentially expressed proteins of osteoblasts in hypergravity may be the biomarker proteins which are d sensitive and bone remodeling-associated.