Preparation And Osteogenesis In Vitro Of Sulfonated Carbon Fiber Reinforced Polyether Ether Ketone Composites Modified With Graphene Oxide

Objective:The aim of this study was to prepare carbon fiber reinforced polyether ether ketone(CFR-PEEK)composite as a new type of loaded bone substitute material,and to treat its surface with sulfonation and modification of graphene oxide(GO).The cell response and osteogenic ability of modified CFR-PEEK composites were studied in vitro.Methods:1.Polyether ether ketone(PEEK)powder and carbon fiber(CF)powder were extruded and molded by high temperature vertical injection molding machine to obtain CFR-PEEK composites with 40%weight ratio of CF.Sulfonated CFR-PEEK composites(S-CFR-PEEK)were obtained by sulfonation of concentrated sulfuric acid.On this basis,GO modified S-CFR-PEEK(GO-S-CFR-PEEK)was obtained by soaking and coating with GO.Three groups of sample materials were obtained:CFR-PEEK、S-CFR-PEEK、GO-S-CFR-PEEK.The surface morphology of the materials were observed by scanning electron microscope(SEM).2.CCK-8 method was used to evaluate the cytotoxicity of each group of materials in vitro.L929 mouse fibroblasts were cultured with the extracted solution of the prepared materials.Cell morphology was observed at 1d,4d and 7d,respectively.and the absorbance(OD)value was measured by enzyme labeling instrument.The biological safety of each group was evaluated according to the relative growth rate(RGR).3.Primary rat bone marrow mesenchymal stem cells(BMSC_S)were cultured and identified by flow cytometry.The cell adhesion,cell proliferation and osteogenic differentiation of BMSC_S were tested by direct contact with each group of materials to evaluate their cytocompatibility and osteogenic ability in vitro.Cell adhesion test:BMSC_S was inoculated on the surface of the material for 48 hours.After immobilization and permeation,immunofluorescence staining was performed with TRITC Phalloidin Rhodamine labeled phalloidin working fluid and DAPI dye solution,and the photographs were taken under laser confocal microscope.Cell proliferation test:BMSC_S was inoculated on the surface of the material for 1、4、7 d,and the proliferation activity of the cells on the material was detected by CCK-8.Cell osteogenic differentiation test:BMSC_S was inoculated on the surface of material to induce osteogenesis for 7 d and 14 d.ALP activity was calculated by ALP alkaline phosphatase kit.Results:1.CFR-PEEK was prepared,and S-CFR-PEEK、GO-S-CFR-PEEK was obtained by surface modification.CFR-PEEK was flat and S-CFR-PEEK was three-dimensional porous structure observed by SEM.The surface of GO-S-CFR-PEEK shows that the porous structure was completely covered by the lamellar GO structure.2.Cells cultured in three groups of leach liquor of material changed from oval to mostly fusiform on 1、4 and 7 d.There was no difference in the cell morphology between three groups of materialand the negative control group.Most of the toxicity levels are level 1according to the RGR,CFR-PEEK and GO-S-CFR-PEEK materials are non-toxic.The RGR of S-CFR-PEEK was lower(64.47%),and the level of toxicity was grade 2,however,combined with the observation of cell morphology,S-CFR-PEEK was also non-toxic.3.After primary culture of BMSC_S to the third generation(P3),the cells showed homogeneous long fibrous structure and concentrated swirl growth.The results of flow cytometry showed that CD 29(98.3%),CD 90(97.65%)was positive,CD 34(1.90%).CD45(3.07%)was negative,indicating that the primary cells were BMSC_S;Cell adhesion test:the adhesion density and morphology of bmscs on the surface of three groups were observed by laser confocal microscope.The adhesion density of bmscs cells on GO-S-CFR-PEEK was significantly higher than that on S-CFR-PEEK and CFR-PEEK,and the cytoskeleton was more obvious;Cell proliferation test:the cells proliferation of CFR-PEEK was not obvious at three time points,almost no difference.S-CFR-PEEK did not increase at 1 and 4d,but at the 7th day,the proliferation of S-CFR-PEEK was significantly larger than that of CFR-PEEK group.The proliferation of GO-S-CFR-PEEK cells continued to proliferate rapidly on day 1,4 and 7,although the cells proliferation of GO-S-CFR-PEEK was lower than that of the other two groups at 1 d,but the cell activity was good on 4 d and 7 d;Cell osteogenic differentiation test:the activity of ALP in CFR-PEEK was not significantly increased on day 7 and day 14,but the activity of ALP in S-CFR-PEEK and GO-S-CFR-PEEK was significantly increased.At each time point,the activity of ALP in GO-S-CFR-PEEK was higher than that in CFR-PEEK and S-CFR-PEEK,and the ability of cell osteogenic induction was greater.Conclusion:The three-dimensional porous structure of S-CFR-PEEK was prepared by CFR-PEEK sulfonation,GO-S-CFR-PEEK was prepared by modifying S-CFR-PEEK with go simple immersion coating method.In vitro cytotoxicity test the each group of materials of are within the range of normal biological safety.The results of cell adhesion and cell proliferation showed that GO-S-CFR-PEEK had better cytocompatibility than the other two groups.In addition,cell osteogenic differentiation also reflects that GO-S-CFR-PEEK has better osteogenic ability in vitro.In conclusion,GO improves the cytocompatibility and bioactivity of composites,and has great application prospect in orthopedic and dental implants.