Construction Of Bio-Active Interface Of Rgd Peptide-Grafted Graphene Oxide And Its Biological Properties

Graphene oxide(GO)is an oxidized derivative of graphene(GN),which is easy to be surface modified by its rich oxygen functional groups.Functionalized GO is widely used for electrochemical biosensor manufacturing or bone tissue engineering.However,the current used RGD peptide functionalized graphene oxide requires the introduction of a third linkage,which would not only increase the complexity of the experimental procedure,but also may change the physical and chemical properties of GO.Therefore,it is necessary to provide a more direct and simple method to realize the surface biological functionalization of graphene oxide and to study its biological properties.RGD peptide is also known as the cell adhesion peptide that can promote cell adhesion,proliferation and differentiation.However,Whether the linkage of RGD peptides to GO can enhance cell viability,be served as a cell biosensor interface in vitro cytological analysis and be applied in bone tissue engineering,are worthy of our study to explore.Objective: 1.RGD covalently modified GO nanomaterials via the EDC/NHS amidation reaction to form a novel RGD-GO biofilm and characterize it.The ITO electrode was modified by RGD-GO biofilms as the sensing electrode to study the application of electrochemical biosensors.2.Study the ability of RGD-GO biofilms to promote the osteogenic differentiation of C3H10T1/2 stem cells in vitro.3.Investigate the biocompatibility of RGD-GO biofilms in vivo.4.Observe the ability of subcutaneous ectopic osteogenesis of RGD-GO biofilms composited C3H10T1/2 stem cells in nude nice,and explore the application of RGD-GO biofilms in bone tissue engineering.Methods: 1.The GO films were firstly formed by natural drying of aqueous solution of GO.Then RGD peptides with amine functional groups were immobilized onto the GO films surface via the EDC/NHS coupling amidation reaction to form RGD-GO biofilms.The morphologies of the GO film before and after modification were characterized by AFM.The chemical compositions of the GO films before and after modification were detected by FTIR measurements.The hydrophobicity of synthetic materials was measured by the contact angle measuring instrument.Meanwhile,the electrochemical properties of RGD-GO biofilms were investigated by electrochemical impedance spectroscopy.2.Cell viability of HPDLFS cultured on RGD-GO biofilms and GO films was evaluated using the well-established CCK-8 assay.The cytoskeleton images were captured using a laser scanning confocal microscope and the data were used to analysis and calculate the available adhesive area of each substrate by image J software.The RGD-GO biofilms were modified on the surface of ITO electrode,and the growth of HPDLFS on RGD-GO-modified ITO electrode was observed under microscope.The proliferation and adhesion of HPDLFS cells on the surface of RGD-GO biofilms /GO films modified electrode were detected by EIS.The effect of lipopolysaccharide on HPDLFs cells viability was detected by EIS.In parallel,it was also analyzed using CCK-8 assay.3.Using C3H10T1/2 as the stem cell model,the growth morphology of cells on the RGD-GO biofilms surface was observed by microscopy.The cytoskeleton was observed under CLSM.By detecting of alkaline phosphatase activity,alkaline phosphatase staining experiments,alizarin red staining experiments and the expression of osteogenesis-related genes(OPN,OCN and Runx2)by fluorescence quantitative PCR,the ability of osteogenic differentiation of C3H10T1/2 cells cultured on RGD-GO biofilms without osteogenic induction was investigated.4.RGD-GO biofilms were implanted into SD rats subcutaneously and the biocompatibility in vivo was studied by gross observation and morphological analysis,Bio-Oss as control.5.RGD-GO biofilms composited C3H10T1/2 stem cells were implanted subcutaneously in nude mice.Bio-Oss was used as control.The ectopic osteogenic ability was investigated by HE staining,Masson staining and immunohistochemical staining.Results: 1.Compared with Go films,the line roughness and the surface roughness of the RGD-GO biofilms increased,hydrophilicity improved,the FTIR spectrum of RGD-GO biofilms clearly displayed two new peaks at 1533 and 1290 cm-1,corresponding to the characteristic peaks of amide I and amide II of the RGD peptides.It was also noticed that the absorption peak at 1732cm-1 assigned to carboxyl groups(C-OH)became weak.EIS measurement showed that the Rct value increased from 2.1E4? to 4.7E4?,the CPE-T value further increased to 5.3E-6C.2.Cell viability gradually increased with the culture time both on GO films and RGD-GO biofilms,but HPDLFs on RGD-GO biofilms showed better cell viability than that of GO films with significant difference(P<0.05).The results of CLSM showed that quantitative analysis of cell number per unit area and cell spreading area on RGD-GO biofilms were significantly higher than GO films(P<0.05).It was observed that HPDLFs can be cultured on the surface of RGD-GO-modified ITO electrode under microscope.With the adhesion and proliferation of cells on the RGD-GO-modified ITO electrode surface,the EIS showed that the values of Ccell rapidly decreased within the incubation time of 2-24 h.This value reached to a stable range of 8.2 to 8.5nF when cells completely spread out to form cell monolayer at 72 h.The final value was just slightly higher than that of the GO-modified ITO electrode,which was stable at the range of 7.6-8.1nF.The measured Rcell values of the RGD-GO-modified ITO electrode were higher than that of the GO-modified ITO electrode,especially at the incubation time of 48 h.At the formation of cells monolayer at 72 h,Rcell reached to the maximum value of 1382? for the RGD-GO-modified ITO electrode.The measured R?S value of the RGD-GO-modified ITO electrode surface was higher than that of the GO-modified ITO electrode.In addition,the value of R?S sustained increasing and reached to a maximum value around 338 ? after 72 h incubation.All the results were in accordance with the previous data of cell counting.The inhibition effect of the LPS on the HPDLFs proliferation was studied using RGD-GO biofilms based EIS sensor,it could be seen that the obtained EIS results showed similar trend with those from traditional colorimetric CCK-8 assay.3.RGD-GO biofilms could promote C3H10T1/2 cells adhesion and proliferation by cell morphology observation.Compared with GO films,the activity of alkaline phosphatase of cells on RGD-GO biofilms was increased at 3 days,5 days,7 days and 9 days(P <0.05).Alkaline phosphatase staining showed positive for 9 days.Alizarin red staining was positive after 14 days.The ostegenic gene expression of OPN,OCN and Runx2 were significantly higher than those of GO group and control group for 9 days(P <0.05).4.RGD-GO biofilms were implanted into SD rats subcutaneously for 2 weeks,4 weeks,8 weeks,12 weeks later,the results showed that no inflammatory cells and foreign macrophages were found like Bio-Oss.The boundaries of implant materials and tissue gradually cleared from the initial thin layer of fiber to the formation of fiber membrane samples.At the same time,the surrounding fibrous tissue gradually thicken and the range of materials implanted subcutaneous gradually reduced.5.When RGD-GO biofilms composited C3H10T1/2 stem cells were implanted subcutaneously in nude mice for 8 weeks,HE staining and Masson staining showed that Bio-Oss/RGD-GO were positive.Immunohistochemical analysis showed osteogenic matrix or bone-like tissue.Conclusion: 1.RGD-modified GO films were successfully synthesized and characterized.RGD-GO biofilms possess good stability and special electrochemical property.It can be used as a novel cell sensor interface to realize the electrochemical detection and analysis of cells in vitro.2.RGD-GO biofilms have good biocompatibility in vitro and can enhance cell adhesion and proliferation.Besides,RGD-GO biofilms can induce C3H10T1/2 stem cell osteogenic differentiation in the absence of osteogenic induction fluid.3.RGD-GO biofilms have good biocompatibility in vivo.Ectopic osteogenesis experiments in vivo show that the RGD-GO biofilms composited C3H10T1/2 stem cells can be osteogenic.In this study,a direct and simple method was used to realize the biological functionalized GO.Through a series of characterization and EIS analysis,this experiment provides basis for RGD-GO biofilms as the sensor interface of cell biosensor.Through biocompatibility studies,osteogenic differentiation studies and ectopic osteogenesis research in vivo,it provides a reference basis for the late experimental study of electrical stimulation to promote osteogenic differentiation,the possibility of periodontal regeneration and being used as biofilms covered on the surface of biomimetic bone scaffolds to strengthen osteoblast differentiation.