Novel Magnetic Bioglass Combined With PMMA Bone Cement For The Treatment Of Tumor-induced Bone Defect

Part I Synthesis and property characterization study of magnetic bioglassesObjective:To synthesize a new type of magnetic bioglass by improved gel method,and to test the characterization and biocompatibility of magnetic bioglass with different Fe₂O₃ loading.Methods:Using sodium metasilicate pentahydrate as raw material,Magnetic biological glass（MBG）was prepared by modified gel method under alkaline condition.MBG powder was photographed with a digital camera.Scanning electron microscopy was used to observe the morphology of MBG.The types and distribution of elements on the surface of magnetic active glass were observed and detected by EDS.The phase composition of magnetic bioglass was analyzed by infrared diffractometer（IR）.X-ray diffractometry（XRD）and magnetic measurement system（MPMS）were used to analyze the phase composition and magnetic properties of the magnetic bioglyphes with different Fe₂O₃ loads,and the alternating magnetic field and temperature change curves with time under dry/wet environment were measured.The toxicity of magnetic bioglass was tested by CCK-8 assay.Results:SEM showed that the new type of magnetic bioglass was micrometer,porous and the pore size was about 200 nm.The distribution of the element map shows that the elements are evenly distributed,and iron is protected within other elements.IR,XRD and other results show that there areα-Fe₂O₃ andγ-Fe₂O₃ ferrites in the magnetic bioglass.Theγ-Fe₂O₃ content in the magnetic bioglass with 40%Fe₂O₃ loading is the highest.The hysteresis curve and magneto-thermal curve also show that the temperature of the magnetic bioglass with 40%Fe₂O₃ load rises the fastest and the highest temperature is the largest.The results of CCK-8 experiment show that magnetic bioglass has no biological toxicity.Conclusion:A kind of magnetic bioglass was prepared by the modified gel method.The magnetic bioglass with 40%Fe₂O₃ load has the best magneto-thermal properties and biocompatibility.Part II Synthesis and property characterization of injectable magnetic bone cementsObjective:To obtain magnetic bone cement,which is referred to as PBG（Polymethyl methacrylate/magnetic Biological Glass）,traditional Polymethyl methacrylate（PMMA）is modified by magnetic biofilm Glass,and composite Different ratios of magnetic bioglass and PMMA bone cement will be mixed to prepare and synthesize different ratios of PBG bone cement,and the characterization test will be conducted to select the most appropriate ratio.Methods:PMMA cement was mixed with solid phase at 10%,20%and 30%mass percentage（wt%）,and then mixed with MMA liquid phase to solidify,mold,fix and dry to obtain cylindrical composite material samples with different proportions of magnetic biogas.The most suitable proportion of magnetic bioglass was screened by measuring the temperature change with time,compressive strength test and injectable property test of magnetic bone cement with different ratio of magnetic bioglass under different alternating magnetic field and dry/wet environment.The in vitro simulated mineralization experiment was conducted to verify the in vitro osteogenic properties of PMMA and PBG.The surface morphology of bone cement before and after mineralization was observed by SEM,and the surface element composition of bone cement was analyzed by EDS+mapping.The curing time,curing temperature and water inclination Angle were used to detect the characterization characteristics of bone cement.Tris-HCl buffer solution was used to soak cement materials for ion release and degradation experiments in vitro.Results:The magneto-thermal curves showed that the temperature of bone cement in the magnetic field rose faster and reached the highest temperature with the increase of the proportion of biological glass.The results of compressive strength test and injectability showed that the mechanical strength and injectability of PBG decreased with the increase of the proportion of magnetic bioglass,but between 20%and 30%,the mechanical strength and injectability showed a fault decline.The SEM results clearly show that the magnetic bioglory in PBG is evenly mixed in the surface and gap of circular PMMA particles.After soaking in simulated body fluid（SBF）for 14 days,PBG has more obvious mineralization layer than PMMA surface.The distribution and analysis of the element map after mineralization also show that the mineralization degree of PBG is significantly higher than that of PMMA.The experimental results of curing time and curing temperature show that the curing time of PBG is about 30%longer than that of PMMA,and the curing temperature decreases by about 20℃.The results of mass degradation experiment showed that only 2%of PMMA and 4%of PBG were degraded,and the solution of PBG degradation was alkaline.The results of ion release experiment showed that Ca and Fe ions in PBG bone cement were released rapidly in the first 7 days,and then tended to be stable.Conclusion:magnetic bioglass proportion of 20%PBG various properties of bone cement most conforms to our expectations,in the proportion,the advantage of PMMA bone cement itself properties such as mechanical strength,injectable sex has no effect,but the disadvantage of PMMA bone cement performance is improved,reduces the curing temperature,increased the ability of magnetic thermal performance and mineralization in vitro.Part III Cellular experiments to study the tumor cell killing ability and in vitro osteogenesis of PBG bone cementObjective:To explore the different effects of different temperatures on stem cells and tumor cells at the cellular level,as well as to investigate the biocompatibility and in vitro osteogenic effects of PBG bone cement.Methods:143B osteosarcoma cells and primary bone marrow mesenchymal stem cells（r BMSCs）were inoculated in 96-well plates,briefly sealed and then maintained for 10min using a water bath heated to 43°C and 46°C,after which the viability of the cells was determined by CCK-8 after removal of the sealed culture for 1 d.Bone cement extracts were prepared according to international standards for interventional culture of BMSCs,and cell viability was determined by CCK-8.After inoculation of BMSCs onto PMMA and PBG bone cement sheets for 7 days,the cytoskeleton and nuclei were stained using FITC-labeled ghost pen cyclic peptide and DAPI,respectively,and cell adhesion and growth status on the cement samples were observed using confocal laser scanning microscopy.The effects of different bone cement extracts on the cell mineralization of BMSCs were compared with alkaline phosphatase staining and alizarin red staining,and the expression of relevant osteogenic genes（OPN,OCN and BMP-2）was detected by real-time quantitative PCR（q RT-PCR）.Results:The survival rate of 143B cells was significantly lower than that of normal controls at temperatures of 43-46°C,without much difference in BMSCs.CCK-8analysis showed that the PMMA and PBG extracts did not show significant cytotoxicity at 1,3 and 5 d.The distribution and adhesion of BMSCs on PBG bone cement samples were observed by confocal than PMMA group.the results of ALP staining showed that the PBG group had darker alkaline phosphatase staining than the blank and PMMA groups,and the results of alizarin red staining showed that the PBG group had more mineralized nodules than the blank and PMMA groups.The results of osteogenic gene expression showed that at 7 days,the osteogenic-related gene expression of BMSCs in the PBG bone cement samples was significantly higher than that of the blank group and PMMA.Conclusion:43-46°C is the temperature interval that can selectively kill tumor cells,while PBG bone cement can promote the attachment,proliferation and osteogenic differentiation of BMSCs.Part IV Study of the tumor killing ability and osteogenic effect of PBG bone cement on tumor tissues at animal levelObjective:To investigate the ability of PBG bone cement to repair tumorigenic bone defects under alternating magnetic fields by the New Zealand rabbit tibial plateau tumorigenic bone defect model,and to investigate the osteogenic effect of PBG scaffold in vivo by the New Zealand rabbit femoral condyle defect model.Methods:A deep hole was turned in the tibial plateau of New Zealand rabbits,and the VX2 tumor tissue was filled into the hole.In the PBG-heated group,New Zealand rabbits were placed in an alternating magnetic field for 10 minutes under inhalation anesthesia,first once every two days and then once a week.Tumor changes were detected using CT once a week for 4 weeks.Twelve New Zealand rabbits were individually molded with femoral condylar defects of 6 mm in diameter and 1 cm in depth,and divided into two groups according to the implant material:PMMA and PBG groups.6 weeks later,the rats were executed and the femurs were collected.Micro-CT scanning of the femoral specimens was performed and 3D reconstruction analysis was performed to quantify the percentage of bone volume around the bone cement.Pathological analysis was then performed by H&E staining.Results:Weekly CT showed uniformly varying degrees of bone destruction in New Zealand rabbits in the blank and PBG unheated groups,with the PBG heated group having a significantly better postoperative bone tumor profile than the other two groups.The results of Micro-CT analysis of New Zealand rabbits’femur showed that the thickness and density of bone around PBG was significantly higher than PMMA.the results of H&E showed that the bone formation in the ring around PBG bone cement was more dense and continuous.Conclusion:PBG bone cement can effectively inhibit tumor recurrence and growth in vivo by alternating magnetic field-induced hyperthermia,and the osseointegration effect in vivo is significantly due to PMMA.