A Biphasic Injectable Bone Cement With Fe₃O₄/GO Nanocomposites For The Treatment Of Tumor-induced Bone Defect

Part I Preparation and characterization of injectable magnetic bone cement samplesObjective:In order to prepareα-TCP/CS（αC）,α-TCP/CS/GO（αCG）andα-TCP/CS/Fe₃O₄@GO（αCFG）injectable bone cement samples,and to characterize the three groups（αC,αCG andαCFG）of bone cement samples.Methods:Fe₃O₄ modified Graphene oxide（Fe₃O₄/GO）was prepared by hydrothermal reaction without further purification.Then the Fe₃O₄/GO was sufficiently mixed withα-TCP/CS dual-phase bone cement to obtain the solid phase with 5%,10%and 15%of mass fraction（Fe₃O₄/GO）,respectively.After that,the solid phase was fully mixed with the liquid phase（2.5%Na H₂PO₄）,followed by solidification,molding,fixation and dehydration to obtain the cylindrical composite material samples.The characteristics of the bone cement samples were studied by scanning electron microscope（SEM）,X-ray diffraction（XRD）and the test of compressive strength,coagulation time and injectability.In vitro ion release and degradation experiments were carried out by soaking the samples in Tris-HCl buffer solution.The hysteresis curve of the bone cement samples was measured by magnetic property measurement system（MPMS）.The temperature-time curve of magnetic bone cement with different Fe₃O₄ content was measured under various alternative magnetic field and dry/wet environment.Results:The SEM images clearly showed the microcrystals of calcium-deficient hydroxyapatite（CDHA）and calcium sulfate in each group of bone cement.The elemental map distribution showed that Fe₃O₄/GO was well distributed inαCFG bone cement.The XRD results figured that the Fe₃O₄/GO had little effect on the phase transition of cement samples during the solidification.Regarding the ion-release experiments,in each group of bone cement,the level of Ca²⁺ions increased rapidly in the first 7 days and then declined and stabilized.With the increase of Fe₃O₄/GO content,the degradation rate and the level of iron ions was also higher at the same time.αC-15FG group showed the most significant mass loss compared with other groups.αC,αCG andαCFG cements all showed similar injection property and initial solidification time.The compressive strength of theαC cement group（14.73±0.75 MPa）was higher than the other groups（p<0.001）,and there was significant difference between the compressive strength ofαC-5FG（12.91±0.38 MPa）,αC-10FG（10.33±0.5 MPa）andαC-15FG（6.25±0.28 MPa）,which suggested that the compressive strength of bone cement decreased with the increasing of Fe₃O₄/GO content.The hysteresis curve of bone cement doped with Fe₃O₄/GO was similar to"S",and as the Fe₃O₄/GO content increased,the magnetic properties of bone cement also increased.Under the same magnetic field,the magnetocaloric temperature ofαCFG cement increased with the increasing of Fe₃O₄/GO content,demonstrated that the magnetocaloric properties ofαCFG cement can be flexibly controlled by controlling the Fe₃O₄/GO content and magnetic field strength.Conclusion:Fe₃O₄/GO has rare effect on the phase change of cement during the setting process.αC,αCG andαCFG cement all performed well in injection property test.The magnetic bone cementαCFG containing Fe₃O₄/GO exhibits excellent magnetocaloric effect.Adding GO or Fe₃O₄/GO toαC cement has a special effect on its mechanical properties,but it can still maintain excellent mechanical properties and meet the support of new bone repair.αCFG is an excellent degradable magnetic bone cement.Part II Tumoricidal and osteogenesis activity ofαCFG bone cement in vitroPurpose:To explore the osteogenesis and selectively tumoricidal ability ofαCFG scaffold through magnetothermal effect in vitro.Method:In order to evaluate the tumoricidal effect of high temperature and its effect on the viability of r BMSCs in vitro,the osteosarcoma cells 143b and primary r BMSCs were seeded in a 12-well plate,and theαC-10FG bone cement sheet was gently placed on the plate,and by adjusting the strength of AMF,the sample was heated at predetermined temperature（41℃,43℃,45℃）for 10 minutes monitored by thermal infrared imager.After the heating,the sample was removed immediately,and the culture medium was added and the cells were cultured for 1,2 and 3 days before the live-dead cell staining,respectively.Following the procedures in ISO 10993,the extracts of bone cement samples were prepared and added into r BMSCs.Then the r BMSCs’viability was assessed by CCK-8 kit and spectrophotometry following the manufacturer’s introductions.To evaluate the osteogenic differentiation of r BMSCs,the expression level of osteogenic genes（RUNX2,OPN,OCN and BMP-2）was measured by q RT-PCR,and the cell mineralization of r BMSCs was evaluated via alizarin red（AR）staining.After r BMSCs were seeded onαC,αCG andαCFG bone cement sheets and cultured for 7 days,the cytoskeleton and cell nucleus were stained by FITC and DAPI,respectively,and then the confocal laser scanning microscope（CLSM）was used to observe the cell adhesion and growth on the cement sample.Results:When the temperature ofαCFG bone cement was maintained at 43-45°C via controlling AMF,the viability of 143b cells was significantly lower than normal control.The CCK-8 results showed that compared with the control group,αC andαCG group with high concentration（200 mg/m L）of extract figured a certain level of cytotoxicity than normal control.However,all concentration of the extract ofαCFG bone cement showed rare cytotoxicity.The alizarin red（AR）staining figured thatαCFG group induced more mineralized nodules than other groups.The expression of bone-related genes in r BMSCs withαCFG was significantly higher than that ofαC andαCG.Moreover,the cell adhesion and growth of r BMSCs onαCFG cement sheet were better than other groups according to CLSM images.Conclusion:αCFG bone cement can effectively ablate tumor cells by high temperature induced by AMF.At the same time,αCFG bone cement can promote the adhesion,proliferation and osteogenic differentiation of r BMSCs.Part III Tumoricidal and osteogenesis activity ofαCFG bone cement in vivoPurpose:To explore the Tumoricidal and osteogenesis ability ofαCFG scaffold through high temperature in vivo by subcutaneous tumor-bearing and skull defect animal model in vivo.Method:To evaluate the Tumoricidal effect ofαCFG bone cement in vivo,143b osteosarcoma cells were injected into the back of nude mice.After the subcutaneous tumor reached a diameter of about 8 mm,each group of cement samples were implanted in the center of the tumor.All nude mice received an AMF of same intensity and were monitored in real time by thermal infrared imager.The tumor volume was measured every other day.On day 14,all mice were sacrificed and the tumor and lung tissue were isolated and collected for H&E staining.To evaluate the osteogenesis effect ofαCFG in vivo,24 SD rats were used as a skull defect model by drilling in the skull to make a full-thickness 4 mm round defect.The rats were divided into four groups:αC,αCG,αCFG andαCFG+AMF according to the different implanted materials and whether the magnetic field was interfered.The rats inαCFG+AMF group were interfered by AMF for 10 minutes every other day until day 14.All rats were sacrificed and the skull tissues were collected for micro-CT assay and three-dimensional reconstruction analysis at week 8.After that,H&E and Masson staining were performed for pathological analysis,and the percentage of new bone formation was quantitatively analyzed by software.Result:The temperature of tumor issues implanted withαCFG bone cement could rapidly rise to about 50℃under the AMF with a density of 250 Gs.The tumor volume ofαCFG+AMF group was remarkably smaller than that of theαCFG group without AMF interference.The H&E staining results showed significantly large areas of tumor necrosis and a significant reduction in the number of pulmonary metastatic nodules.The micro-CT results of the rats’skull figured that the ratio of bone volume to tissue volume（BV/TV）inαCFG andαCFG+AMF group was 18.1%and 17.6%,respectively,which is significantly higher than control groups（αC,αCG）.The H&E and Masson staining images suggested that there were more new bone tissues and collagen fiber deposition onαCFG,which means that the area of new bone tissue onαCFG bone cement was apparently higher than that on the exposedαC andαCG group.Conclusion:αCFG bone cement can effectively ablate tumor cells,inhibit tumor growth and lung metastasis through AMF-induced high temperature,and induce bone regeneration in vivo.