Preparation Of Graphene Coating And Biological Studies On The Surface Of Graphene-modified Biomedical Titanium Alloy（Ti₆Al₄V）

BackgroundDesign and thermo mechanical processing control of titanium alloys,thereby providing titanium alloys with many excellent mechanical propertys,such as lower modulus of elasticity,corrosion resistance and higher yield strength.These alloys are widely used in orthopaedics and dentistry such as artificial bones,artificial joints,bone plates and screws,dental implants and as substitute materials for other hard tissues.Up to today the most widely used titanium based implant materials are α-β Ti₆Al₄V,which accounts for approximately 80% of all titanium alloy products.The high elastic modulus of titanium alloy（Ti₆Al₄V,110GPa）as compared to bone（10 – 30GPa）.But,as biomedical materials for the implantation into the human body,corrosion resistance is of prime importance other than its mechanical properties.On the other hand,titanium alloys are very readily oxidized,the surface forms a layer of dense and strong Ti O2 film,thereby providing the underlying substrate with strong corrosion resistance.This property makes titanium and titanium alloys attractive for use in implants.However,the success or failure of implants,except for the mechanical and biological properties of the implant materials,are primarily dependent on surface characteristics of materials such as biocompatibility and corrosion resistance.Indirectly these surface factors also effect mechanical behavior such as stress shielding,wear debris or fatigue failure.The biocompatibility and bioactivity of the implant surface play a crucial role as the surface is directly exposed to the living organism.Goal of implant surface engineering is not only to fit the demands of avoiding negative effects of implanted materials on the surrounding tissue but even more to enhance the interplay between the designed technical material and the living matter.In best case the physical and chemical properties of the chosen implant material should be in accord with the replaced tissue.Living tissue has the ability to renew itself continuously,whereas implant materials typically lack this ability.Therefore major attention must be paid to the surface of a material system as its reaction with the host tissue is often decisive on success or failure of implantation.The oxidized layer of titanium alloys is a biologically inert film and hinders the direct combination of the implant with bone tissues,resulting in poor osteointegration and poor long-term implant outcomes.To address this issue while retaining the mechanical properties of the implant material,the surface of the implant must be able to fully induce bone tissue regeneration so that osteointegration can be promoted.In recent years,the modification of implant surfaces to improve their biological activity and to promote osteointegration has become a popular area in biological materials engineering.The coating of implant surfaces and loading of bioactive substances or drugs on an implant can effectively improve the compatibility and bioactivity of implants,enhance osteoblast function,promote bone tissue regeneration,and improve bonding strength and osteointegration at the implant/bone interface bonding strength.Graphene is isolated from graphite by mechanical exfoliation and could exist stably in 2004 by A K Geim and his co-workers.As an allotropes of elemental carbon,graphene is a planar monolayer of carbon atoms,and arranged into a two-dimensional honeycomb lattice.Graphene has a carbon–carbon covalent bond length of 0.142 nm.Graphene as an elementary building hunk of graphitic materials with different geometries,graphene can be wrapped into zero-dimensional fullerenes,rolled into one-dimensional carbon nanotubes,fastened into three-dimensional layered structures graphite.Graphene has demonstrated a variety of unique physicochemical properties including high electron mobility at room temperature（15,000 cm-1/V?s）,exceptional thermal conductivity（5000 W/m?K）,the highest theoretical specific surface area（2630 m Z/g）,and superior mechanical properties with Young’s modulus of 1 TPa,the highest strangth（130 GPa）.These physicochemical property allow graphene to be deliberated as a perfect material for a wide-ranging applications include quantum physics,single molecule gas detection,transparent conducting electrodes,engineering of nanocomposites and energy storage devices such as supercapacitors and lithium ion batteries.Beyond all doubt,graphene has risen as an outstanding star materials by scientists’ searching for future electronic and composite industry.Since 2008,numerous interesting studies have been carried out to discover the use of graphene and its derivatives for extensive biomedical applications and tissue engineerings.A large number of studies have shown that graphene and derivatives can induce the adhesion,proliferation and differentiation of bone marrow stromal cells（BMSCs）on implants and biological scaffolds.Graphene has also shown good performance in medical applications,such as for cancer treatments,disease diagnosis,microbiological detection and drug/gene delivery,antibacterial materials,biosensor and biological imaging.Hence,the graphene can be suggested as an excellent biocompatible material for surface engineering and other medical applications.In this study,we prepared graphene coatings on the surface of Ti₆Al₄V and performed in vitro and in vivo animal experiments.The results suggest that graphene and its derivatives may be new types of nano-coating materials that can improve cells adhesion,proliferation and osteogenic differentiation and can also promote osteointegration at implant-bone interfacesObjective 1.To fabricate the surface graphene coating of titanium alloy（Ti₆Al₄V）and analysis the characterization of these scaffolds.2.In vitro,To analysis the biological activity of G-Ti₆Al₄V and Ti₆Al₄V in following aspects: Adhesion,proliferation and differentiation of bone marrow stromal cells（BMSCs）on scaffolds.3.For in vivo experiments,a New Zealand white rabbit femoral condyle defects model was established.To evaluate the osteogenesis and osteointegration of these scaffolds via implant these scaffolds into rabbit body.Methods 1.Through the chemical etching and Physisorption method to transfer the graphene growth on copper foil（Coverage rate of graphene is 100%）to the surface of titanium alloy Ti₆Al₄V for preparation graphene coating.The titanium alloy Ti₆Al₄V specifications are as follows: Ti₆Al₄V sheets（Φ=10 mm,δ=5 mm）were used for cell experiments.Ti₆Al₄V（Φ=5 mm,L=10 mm）cylinders were used for animal experiments.Scanning electron microscopy（SEM）was used to observe changes in the surface morphology of the materials.The Raman spectroscopy was used to characterize the peak of these materials（G-Cu,G-Ti₆Al₄V and Ti₆Al₄V）.Optical contact angle measuring instrument and optical profilometer was used to determine the surface hydrophilicity and roughness.The influence of graphene coating on the mechanical properties of the titanium alloy was observed using a material testing machine.Mechanical stability of graphene coating was evaluated using ultrasonication test.2.In vitro,Bone Marrow Stromal Cells（BMSCs）were extracted from Sprague–Dawley（SD）rats.The BMSCs were isolated and cultured using a whole bone marrow adherent culture method.After the BMSCs cultures were passed to the P3 generation,the cells were inoculated on the different scaffolds（G-Ti₆Al₄V,Ti₆Al₄V）.After cells were inoculated 1 d,3 d,5 d and 7 d,a Cell Counting Kit-8（CCK-8）was used to evaluate the proliferation of BMSCs on the different scaffolds.After Inoculated 1 d and 5 d,using SEM to observe the cell growth states and adhesion statuses of the different scaffold.After cells were inoculated 1 d,3 d,5 d,the adhesion ability of BMSCs on two scaffolds were evaluated through cell immunofluorescent staining and using a confocal laser scanning microscopy（CLSM）to observe and analyses.A live/dead cell kit was used for live/dead cell staining and quantitative analysis to evaluate the cytoactive of the different scaffolds after 1 d,3 d,5 d and 7 d of BMSCs culture.After osteogenesis induction of BMSCs on different scaffolds for 21 d,an optical microscopy was used to analysis the alizarin red S-dyed red bone nodules.At 7 d and 14 d of BMSCs osteogenesis induction on different scaffolds,alkaline phosphatase（ALP）activity was measured using an ALP activity assay kit,and using real-time polymerase chain reaction（RT-PCR）and Western Blot to detect the relevant osteogenic gene expression.3.For in vivo experiments,a New Zealand white rabbit femoral condyle defects model was established and implant these scaffolds into rabbit body.The effect of osteogenesis and osseointegration of these scaffolds were evaluated through Micro-CT,Tetracycline-Calcein fluorescence label and Van-Gieson staining at 4w,12 w,and 24 w after surgery.Results 1.The SEM images and Raman spectroscopy results are showed that graphene coating was successfully immobilized on the surfaces of titanium alloy Ti₆Al₄V and that the coating was very stable.The average water contact angle results indicated a significant increase of hydrophily by the graphene coating on the G-Ti₆Al₄V scaffolds（P<0.05）.The surface roughness results showed that the G-Ti₆Al₄V group was significantly higher than that of the Ti₆Al₄V group（P<0.05）.The compression test results showed that there was no difference between the two groups in terms of the compressive modulus（P=0.9695）,which indicated that the mechanical properties of the overall titanium alloy were not significantly affected.2.In vitro,the cell proliferation was evaluated by the Cell Counting Kit-8（CCK-8 assay）,cell proliferation was significantly greater in the G-Ti₆Al₄V group compared to Ti₆Al₄V group at 1 d,3 d,5d and 7 d（P<0.05）.Using SEM to observe the cell growth states.SEM images showed,the morphology of the BMSCs was normal and showed a typical elongated-spindle shape,cells pseudopodia and nuclei were observable,the cell density of the G-Ti₆Al₄V group was high and showed a completely enveloped cell matrix.Using CLSM to observe BMSCs were fluorescently stained for F-actin,vinculin and nuclei to investigate cell adhesion behavior.The results showed,cells from the G-Ti₆Al₄V group showed significantly higher mean integrated optical density（IOD）of vinculin staining compared with those from the Ti₆Al₄V group at inoculated 1 d,3 d and 5d（P< 0.05）.Live/dead cell staining show that over time,the number of living cells in the two groups gradually increased,but the number of living cells at each time point,compared with the Ti₆Al₄V group,the G-Ti₆Al₄V group was significantly higher.At each time point,the number of dead cells in the Ti₆Al₄V group increased more than that in the G-Ti₆Al₄V group.BMSCs on the scaffolds after 21 d of osteogenic induction were stained with alizarin red S and quantified.The results are shown compared with the Ti₆Al₄V group,the G-Ti₆Al₄V group showed darker and larger alizarin red S staining with more calcium nodules.The OD value（570 nm）of the alizarin red S staining was measured to quantify the formation of calcium nodules.Compared with Ti₆Al₄V group,the generation of calcium nodules in the G-Ti₆Al₄V group was significantly higher（P<0.05）.ALP staining and quantitative analyses results showed that the G-Ti₆Al₄V group demonstrated a deeper color of ALP staining compared with the Ti₆Al₄V group at 7 d and 14 d.At each time point,compared with the Ti₆Al₄V group,the OD value in the G-Ti₆Al₄V group was significantly higher（P<0.05）.Cell differentiation was assessed at 7 days and 14 days with osteogenic markers including Runx2,ALP,OCN,BMP-2 and Col-1 by quantitative RT-PCR tests.The levels of Runx2,ALP,BMP-2 and Col-1 gene expressions from the G-Ti₆Al₄V group were significantly higher compared with the Ti₆Al₄V group at 7 days and 14 days（P<0.05）.But at 7 days,there were no significant difference for both OCN gene expressions between the G-Ti₆Al₄V and Ti₆Al₄V groups（P>0.05）,while their gene expressions were significantly higher in the G-Ti₆Al₄V group at 14 days（P<0.05）.Western Blot also showed the same trend as RT-PCR 3.In vivo,at 4 w 12 w and 24 w the biomechanical testing（push-out test）results show the maximum failure load of both groups increased over time.However,at each time point,the maximum failure load of the G-Ti₆Al₄V group was significantly greater than that of the Ti₆Al₄V group（P<0.05）,indicating that the graphene-coated group required a greater compressive strength to detach the specimen from the femoral condyle.Micro-CT results showed,the BV/TV,Tb Th and Tb N of the two groups increased,especially from 4 weeks to 12 weeks.However,at each time point,the BV/TV,Tb Th and Tb N in the G-Ti₆Al₄V group was higher than that in the Ti₆Al₄V group（P<0.05）.At over time,Tb.Sp gradually reduced in the two groups.At each time point,compared with the Ti₆Al₄V group,the Tb.Sp of the G-Ti₆Al₄V group was significantly lower（P<0.05）.There was no significant difference for BS/BV at 4 weeks between the G-Ti₆Al₄V and Ti₆Al₄V groups（P>0.05）,while BS/BV was significantly higher in the Ti₆Al₄V group at 12 weeks and 24 weeks（P<0.05）.To detect the bone mineral apposition rate（MAR）,double immunofluorescence staining was used.The result showed the MAR of the G-Ti₆Al₄V group was significantly higher than that the Ti₆Al₄V group（P<0.05）.Over time,the bone volumes of the two groups increased,but compared with the Ti₆Al₄V group,the density and volume of new bone in the G-Ti₆Al₄V group were significantly higher（P<0.05）.Additionally,compared with the G-Ti₆Al₄V group at each time point,the Ti₆Al₄V group showed more blue-dyed fibrous tissues between the implant and the bone tissue.Especially at 24 weeks,the scaffolds of G-Ti₆Al₄V group and bone tissues were closely attached with almost no gaps or fibrous tissues present.This affects the attachment between the new bone and the material and thereby influences the outcome of osteointegration.These results confirmed the Micro-CT and biomechanical push-out test results.Conclusions 1.Graphene coating was successfully immobilized on the surfaces of titanium alloy Ti₆Al₄V and that the coating was very stable.And graphene coating significantly improved the hydrophilic and roughness of Ti₆Al₄V scaffolds surface but did not change the mechanical and physical properties of titanium alloy Ti₆Al₄V.2.The graphene coating enhanced the bioactivity of Ti₆Al₄V scaffolds and strongly promoted the adhesion,proliferation and osteogenic differentiation of BMSCs on scaffolds.3.The osteogenesis and osteointegration of the implant-bone interface were promoted when using the graphene-coated Ti₆Al₄V scaffolds.These scaffolds accelerated bone defect repair.