Feasibility Study Of Graphene Oxide As A Scaffold For Tooth Regeneration

Objectives The purpose of this study was to analyze the cell abilities of adhesion,growth and calcification on the graphene oxide(GO)scaffold,and to explore the biocompatibility and feasibility as a new scaffold for tooth regeneration.Methods 1 Rat dental pulp stem cells(DPSCs)were cultured by modified tissue block method and were identified by immunofluorescence staining with CD34,CD44 and STRO-1 antibodies.The rat dental epithelial cells(DECs)which was obtained from mandibular process of fetal rats were cultured by enzyme digestion method and were identified by immunofluorescence staining with CK14 antibiody.2 GO scaffold was prepared by Hummers method and gradient freeze-drying method.The surface morphology of the scaffold was observed by scanning electron microscope and the composition of the scaffold was determined by Raman spectrometer.3 DPSCs were inoculated respectively into ?-MEM medium(control group)and medium containing GO dispersion at diffewas detected by MTT method after 24 hours,and then the cell viability was calculated to rent concentrations(experimental group).The absorbance of each group evaluate the cytotoxicity of GO.4 The DPSCs were co-cultured with GO scaffold for 3days,and the immunofluorescence staining was used to detect the tubulin-? on GO scaffold in order to evaluting the adhesion staus of DPSCs on it.5 The DECs and DPSCs were taken as seed cells,and implanted on GO scaffolds(DPCSs/DECs/GO group)and gelatin sponge scaffolds(DPCSs/DECs/GS group)at 1:1 ratio according to the stratified inoculation method.Then the three-dimensional culture models were transplanted into omentum majus of rats.After 4 weeks,the graft tissues were detected by histological observation and immunohistochemical staining.The content of calcium and phosphorus in the graft was measured by energy disperse pectroscopy,the tissue structure of the graft was determined by X-ray diffraction,which were used to detect the degree of mineralization.The animal studies was used to detect the differentiation ability of cells on GO scaffold in vivo.Results 1 The cultured dental pulp cells were negative for CD34,and positive for CD44 and STRO-1,which conformed that these cells were DPSCs.The cultured mandibular process cells were positive for CK14,which conformed that these cells were DECs.2 The results of characterization of GO scaffolds showed that the pore size was 50-100 ?m,the porosity was as high as 95%,and there were many wrinkle structures on the surface of the scaffold.Raman spectra showed that D peak appeared at 1355 cm-1 and G peak appeared at 1582 cm-1,which was consistent with the identification of graphene oxide.3 MTT results showed that the cell viability had no significant difference between the experimental group and the control group(P>0.05).This confirmed that GO has no toxicity of DPSCs proliferation.4 The results of immunofluorescence showed the GO scaffold wad positive for tubulin-?,which indicating that the cells grew well on GO scaffolds and had good cell adhesion,indicating that GO had good biocompatibility.5 HE staining showed that new tissue grew into the scaffolds in every group,and the cells grew well.Immunohistochemical staining showed that the dentin sialprotein(DSP)was positive in DPCSs/DECs/GO group,negative in DPCSs/DECs/GS group.Scanning electron microscopy showed that there are collagen and angiogenesis in DPCSs/DECs/GO group.The content of calcium and phosphorus in the graft was significantly lower than that in the normal teeth,X-ray diffraction showed that the implant had not reached the mineralized level of normal teeth.These indicated that the GO scaffold is more conducive to DPSCs differentiation than GS.Conclusions 1 Rat DPSCs could be obtained by modified tissue block method.Fetal rat DECs could be cultured by enzyme digestion.2 The GO scaffold has good biocompatibility,which is beneficial to the growth and attachment of cells.3 GO,as a new scaffold for tissue engineering tooth regeneration,can promote tooth differentiation of DPSCs.Figure 14;Table 1;Reference 110...