The Research Of Titanium Elastic Deformation On The Bone Marrow Mesenchymal Stem Cells Biological Behaviors

Objective:Titanium is one of most commonly used materials in the orthopedics.Traditional titanium implant research mainly focused on the alloy composition improvement,porosity and pore size optimization,metal surface modification,etc.There are few studies that focus on the relatationship between the physical deformation of the titanium and the bone tissue regeneration.Early research has shown that titanium under certain stress can occur elastic deformation,but it is unkonwn whether the deformation will affect the biology behavior of bone marrow mesenchymal stem cells.Therefore,in this research we will study the effect of titanium elastic deformation on the bone marrow mesenchymal stem cells biological behaviors,we will control the change of the cytoskeleton through the nano-modification on the metal surface and explore the cell adhesion,proliferation,apoptosis,and osteogenic differentiation and molecular mechanism of the changes.We will finally to improve the integration of metal-bone interface and bone tissue regeneration.Methods:1.The research of a mechanical loading system based on the elastic deformation of titaniumAccording to the experimental requirements for the mechanical loading of titanium specimens,it is necessary to develop a device capable of applying cyclic mechanical stress to the titanium specimen,and the parameters such as the magnitude,frequency and period of the output force of the device are precisely controllable.The device needs to meet the conditions of convenient operation,stable working conditions and reliable performance.2.The preparation and characterization of nanotube morphology on the titanium surfaceWe adopted organic solution to anodize titanium specimen and use scanning electron microscope,atomic force microscope and contact angle test to represent the nanotube morphology.3.The influence of titanium elastic deformation on BMSCs biological behaviorsFirst,the relative optimal variables such as size,frequency and period for promoting osteogenic differentiation of BMSCs were explored.We set the different magnitude(0.3%,0.6%,0.9% and 1.2%),different frequency(0.5Hz,1Hz,2.5Hz and 5Hz),different period(5min/d,15min/d,30min/d and 60min/d)of the mechanical device.ALP staining was used to evaluate the osteogenesis of BMSCs.Then studying the proliferation,apoptosis,adhesion,cell morphology,osteogenic marker genes and protein expression of BMSCs through CCK8,flow cytometry,scanning electron microscopy,immunofluorescence,real-time quantitative PCR,Western Blot and other methods.The research of molecular mechanism of titanium elastic deformation on BMSCs osteogenic differentiation are needed.Results:1.We developed an experimental device for mechanical loading of titanium specimens successfully.The device is mainly composed of drive system,execution system and control system.It mainly includes motor,motor driver,motor encoder,lever type pressing force structure,experimental device base,titanium specimen supporting structure and titanium specimen.The device can apply dynamic mechanical loading of different magnitude,frequency and period to the titanium specimen,and has the advantages of stable working condition,convenient operation,high precision of mechanical loading control,simple and durable structure and reliable performance.2.The titanium dioxide nanotube distributed homogenously.The diameter was about 80 nm,the height was about 2?m,the contact angle was 93.38 ± 1.387°,and the nanotubes were composed of titanium(Ti)and oxygen(O).3.According to the results of ALP staining,the best mechanical parameters are: magnitude: 0.9%,frequency: 5Hz,period: 30min/d.This optimal mechanical parameter will be used in subsequent experiments to investigate the effects of titanium elastic deformation on BMSCs proliferation,apoptosis,adhesion,cell morphology,osteogenic gene expression,and molecular signaling pathways.The result of CCK-8 showed that there was no significant difference in cell proliferation on day 1 and day 2 after mechanical loading,and cell proliferation was promoted on the third day(P<0.05).The result of flow cytometry showed that the apotosis of BMSCs was no signifant difference between mechanical strain and control groups.The result of scanning electron microscopy and cytoskeleton staining showed that mechanical strain faciliated cell spreading and cytoskeleton arrangement.The result of immunofluorescence staining showed that mechanical strain accerated the expression of vinculin;The results of real-time quantitative PCR showed that mechanical strain promoted the expression of osteogenic genes such as Runx2,ALP,BSP,Col-1,OCN and OPN(P<0.05);Western Blot results showed that titanium metal elastic deformation promoted the phosphorylation level of FAK and Erk1/2,and enhanced the expression of Runx2.Our results preliminarily demonstrated that mechanical strain might enhance the osteogenic differentiation of MSCs through the FAK-Erk1/2-Runx2 pathway.Conclusion: Titanium elastic deformation promoted osteogenic differentiation of BMSCs.The relative optimal mechanical parameters in this study are 0.9%,5Hz,30min/d.Titanium elastic deformation enhanced cell adhesion and spreading,facilitated cell proliferation,and promotes the expression of vinculin.Titanium elastic deformation could promote the expression of the osteogenic genes such as Runx2,ALP,BSP,Col-1,OPC and OCN.Our results preliminarily demonstrated that mechanical strain might enhance the osteogenic differentiation of MSCs through the FAK-Erk1/2-Runx2 pathway.