Preparation Of Magnetically Responsive Carbon Composite Scaffolds For Treatment Of Tumorous Bone Defects In Vitro

With respect to the problem of tumorous bone defects,it is of great significance to provide a bifunctional biomaterial for both postoperative adjuvant treatment of tumor and bone defect repair.Carbon materials such as graphene and carbon nanotubes have potential application in bone tissue engineering due to their great specific surface area,good mechanical properties and certain bone induced properties.However,compared with traditional bone repair materials,there is still a gap in biocompatibility and bone induced properties.Therefore,it is particularly important to improve the comprehensive properties of carbon scaffold materials by modifying or combining with other functional particles.In addition,as a non-invasive external physical stimulus,magnetic field has been proved to have a certain influence on cell behavior.Drug delivery can control the movement of magnetic drug carriers and concentrate in the lesion area to control drug release.Based on above-mentioned studies,we constructed a three-dimensional carbon scaffold with layered-columnar microstructure,in-situ precipited composites of hydroxyapatite and Fe₃O₄,endowing the scaffold with certain biocompatibility and magnetism,and investigated its drug-loading and drug-released behaviors,and evaluated its osteogenic activity and anti-tumor effect with cell co-culture experiments in vitro.The conclusions are as follows:1.Owing to the agglomerate and poor dispersion of the graphene and carbon nanotubes,the reduced graphene oxide(RGO)/carbon nanotube(CNT)scaffold with three-dimensional layered-columnar microstructure is constructed by electrostatic self-assembled method to improve the specific surface area and promenting the agglomerate of the graphene and carbon nanotubes.On this basis,hydroxyapatite and Fe₃O₄ nanoparticles were in-situ precipeted in the scaffold to endow the biocompatibility and magnetismthe.Results showed that The RGO/CNT scaffold prepared at the 180?for 3 hours has a larger specific surface area(216.29 m²/g)and pore volume(0.46 cm³/g),whilethe RGO/CNT/HA/Fe₃O₄ scaffold prepared under the same condition has a smaller specific surface area(67.06 m²/g)and pore volume(0.23 cm³/g).Moreover,the RGO/CNT/HA/Fe₃O₄ scaffold had been found to be ferrimagnetism with the coercivity of 64.1 Oe and residual magnetizationof 0.3 emu/g,which is convenient for magnetization and demagnetization.2.The drug-loaded and drug-released capacities of the carbon-based scaffolds under the static magnetic field(SMF)with different intensity were studied.The results showed that the drug-loaded content of RGO/CNT scaffold was 255.1?g/mg without SMF stimulation.The drug-loaded content of RGO/CNT/HA/Fe₃O₄ scaffold was 310.8?g/mg with SMF stimulation of 130 m T.It indicated that a certain intensity of magnetic field stimulation was beneficial to drug adsorption.The drug-released experiment results showed that RGO/CNT/HA/Fe₃O₄ scaffold has the slower drug-released behavior with SMF stimulation.3.The results of cell co-cultured with BMSCs in vitro showed that the RGO/CNT/HA/Fe₃O₄composite has better cell proliferation with a certain extent of SMF stimulation due to the existence of HA and Fe₃O₄ functional particles in the composite scaffold.RT-q PCR results showed that the RGO/CNT/HA/Fe₃O₄ composite combined with 60m T of SMF stimulation could promote the expression of multiple osteogenic related genes(ALP?COL-1?Runx2?OPN?ROX?BMP-2),which is beneficial to the osteogenic differentiation of BMS cells.4.In order to investigate the anti-tumor effect of RGO/CNT/HA/Fe₃O₄composite,Berberamine Hydrochloride(BBM)was used as the model drug to be loaded in this composite with the different SMF.The results of cell co-cultured with UMR-106 in vitro showed that the north direction of the magnetic field is beneficial to inhibit tumor cell proliferation.Compared with other SMF stimulation,RGO/CNT/HA/Fe₃O₄composite with 130m T stimulation has the best inhibitory effect on tumor cells proliferation.Co-culturated with UMR-106 in vitro for 48hours,the BBM@RGO/CNT/HA/Fe₃O₄ group has the lowest cell survival rate of 39%.It indicated that the combination of magnetic material(RGO/CNT/HA/Fe₃O₄),SMF stimulation and drugs strengthened the inhibition effect of tumor cells proliferation.RT-q PCR results showed that the BBM@RGO/CNT/HA/Fe₃O₄ system could promote cell apoptosis by inhibiting the expression of Bcl-2 with SMF stimulation.