PMMA-Fe₃O₄ For Internal Mechanical Support And Magnetic Thermal Ablation Of Bone Tumors

PART? The preparation and mechanical properties of PMMA-Fe₃O₄ magnetic bone cementObjective A new magnetic PMMA-Fe₃O₄ was prepared to test its basic properties include setting time,polymerization temperature,injectability and mechanical properties.Method Four types of PMMA based magnetic bone cement containing 0%,3%,6% and 9% Fe₃O₄ nanoparticles was developed.The microstructure of solidified bone cement were observed by electron microscope.Standard samples were prepared according to ISO5833:2002?E?standard.Compressive strength,bending strength and bending modulus of different magnetic bone cement were measured by "compressive test" and "three-point bending test".The basic properties of bone cement are determined by measuring its polymerization temperature,setting time and injectability.Result PMMA-Fe₃O₄ magnetic bone cement was successfully developed.The diameter of Fe₃O₄ nanoparticles in PMMA-6%Fe₃O₄ was measured under electron microscopy.The nanoparticles were 50-100 nm and uniform distributed around the matrix of polymethyl methacrylate?PMMA?.With the increase of the proportion of Fe₃O₄ in magnetic bone cement,the compressive strength,bending strength and polymerization temperature were decrease.With the increase of the proportion of Fe₃O₄ in magnetic bone cement,bending modulus,setting time and injectability of magnetic bone cement were increase.PMMA-9%Fe₃O₄ was excluded from the subsequent experiments in vivo because the compressive strength was lower than 70 MPa.Conclusion After mixing different Fe₃O₄,the basic properties and mechanical properties of the prepared magnetic bone cement were different,and some specific properties were significantly different?P < 0.05?.The mechanical properties of magnetic bone cement containing 0%,3% and 6% Fe₃O₄ are up to the standard and can be used for subsequent experimental study.PART ? Evaluation of induction heating efficiency of PMMA-Fe₃O₄ magnetic bone cement in vitroObjective The induction heating efficiency of PMMA-Fe₃O₄ magnetic bone cement in alternating magnetic field were studied.Method According to the preparation method of the first part,0.15 m L magnetic bone cement containing 0,3,6,9% Fe₃O₄ was made into small balls during the working period and placed at room temperature for 24 hours.The small balls were put into EP tubes containing 1.5 m L saline?2 m L specification?;EP tubes were placed in coils of magnetic induction heater and exposed to alternating magnetic field for 180 seconds.Temperature changes were recorded every 10 seconds by infrared thermal imager,and time-temperature curves were obtained.After screening,PMMA-Fe₃O₄ with optimum Fe₃O₄ content was obtained.The 0.1 m L and 0.2 m L optimum concentration of PMMA-Fe₃O₄ was developed and then evaluated its induction heating efficiency in saline water.The time-temperature curves of PMMA-Fe₃O₄ with different volumes at the same concentration were made.The best concentration of 0.15 m L bone cement sphere was cut into two hemispheres at the same size.The sphere of hemisphere was embedded in bovine liver?4 cm × 2 cm × 2 cm?,while the section of the hemisphere was kept parallel to the surface of bovine liver.After exposed in the AMF,the temperature changes of bovine liver around the spherical section were recorded every 10 seconds by infrared thermography.Then using Analyz IR 7.1 software to analyze the temperature changes of bovine liver at different distances from the surface of the globule at different times,the temperature-distance-time curve was obtained.The bone cement small ball was embedded into the bovine liver mass?2 cm × 2 cm × 2 cm?and exposed in AMF for 180 s,the temperature changes of bovine liver were recorded every 10 seconds by infrared thermography.The bovine liver was cut into half after exposure AFM for 120 s,150 s and 180 s,and the range of coagulative necrosis of bovine was measured.Result For the proportion of magnetic bone cement,the larger the proportion of Fe₃O₄,the stronger the magnetic heat properties;the larger the volume of magnetic bone cement,the better the magnetic heat properties.The longer the time of magnetic induction heating,the larger the area of coagulation necrosis was achieved.The temperature decreased from 82.7 ±13.8 °C?1 mm away from the surface of the embedded PMMA-6%Fe₃O₄?to 71.2±5.5 °C,61.4±2.8 °C,53.8±0.4 °C,47.5±0.2 °C,44.5±0.3 °C,40.8±0.2 °C,38.4±0.5 °C,and 36.9±0.9 °C?1.5,2,2.5,3,3.5,4,4.5,and 5 mm,respectively,away from the surface of the embedded PMMA-6%Fe₃O₄?after 150 s of heating.The ablated tissue distance was 1.38±0.12 mm,3.06±0.24 mm,and 4.82±0.23 mm for 120 s,150 s and 180 s.Conclusion The PMMA-6%Fe₃O₄ magnetic bone cement exhibit excellent magnetic heating property and controllability.Appropriate magnetic heating time can be selected to achieve better ablation effect for different sizes of bone tumors.It not only ensures that the effective temperature range of thermal ablation covers all bone tumors,but also avoids the damage of normal tissues caused by excessive heating.To provide comprehensive data to support the following experiments in vivo.PART ? The biosafety of PMMA-Fe₃O₄ magnetic bone cementObjective To investigate the biosafety of PMMA-Fe₃O₄ magnetic bone cement in vivo,and providing experimental basis for clinical translational of magnetic bone cement.Method A total of 150 ?L of PMMA-6%Fe₃O₄ was injected into the lateral thigh muscle of 9 New Zealand white rabbits?2 months old,and weight of 2.0-2.5 kg,any sex?.Six rabbits were randomly selected for blood collection.Three milliliters of blood specimens were collected through the ear-vein of rabbits at pre-injection,the 1st day,the 1st week,the 2nd week,the 3rd week and the 4th week,respectively.The blood tests included white blood cell?WBC?,red blood cell?RBC?,hemoglobin?HB?and platelet?PLT?counts.The serum tests included measurements of alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,creatinine?CR?,blood urea nitrogen?BUN?,creatine kinase?CK?and lactate dehydrogenase levels?LDH-L?.For the rest three rabbits,After 4 weeks,the other three rabbits were sacrificed to collect the major organs,include heart,liver,spleen,lung,kidney and the muscle tissue around the PMMA-6%Fe₃O₄,for pathological examination.Result There was no significant difference in the results of all hematological and serological examinations before and 1,7,14,21 and 28 days after injection.On the 28 th day,there were no abnormalities in the visceral sections compared with those of normal rabbits.There was no white blood cell aggregation and no obvious necrosis of muscle cells near PMMA-Fe₃O₄ magnetic bone cement.Conclusion These results clearly demonstrate the excellent biocompatibility and biosafety of magnetic bone cement.PART ? Preparation of a tumor model in the rabbit tibial plateau with a VX2 tumor mass and evaluation of the mechanical properties of tumor planted leg containing PMMA-Fe₃O₄ magnetic bone cement.Objective Develop a rabbit tibial plateau tumor model with VX2 tumor mass.To investigate the effect of PMMA-Fe₃O₄ magnetic bone cement on the bone support ability of rabbits with tumors in vitro.Method Tumor tissue were harvested from tumor planted rabbit,fresh white fish-like tumors were cut and separated into tumors about 1 mm³ in size.After anesthesia,two tumors mass were injected into the tibial plateau of rabbits through bone marrow puncture needle and closed by gelatin sponge.On the 14 th day after implantation,CT scan was performed and measured.When the destruction volume of tibial plateau reached 180.0 mm³,the model was successfully established.Twelve tumor-bearing and four healthy rabbits were chosen for this experiment.The twelve tumor-bearing rabbits were randomly divided into 3 groups.For the PMMA-6%Fe₃O₄-Heating?PMMA-6%Fe₃O₄-H?and PMMA-6%Fe₃O₄ groups,four rabbits were randomly selected and anesthetized,and then150 ?L of PMMA-6%Fe₃O₄ cement paste was injected into the tumor under CT guidance.he tumor-bearing legs in the PMMA-6%Fe₃O₄-H group were exposed to an AMF as above for 150 s,while the other tumors in the PMMA-6%Fe₃O₄ group were not exposed.For the Tumor group,no materials injected into the tumors.For the Normal group,4 legs without tumors were harvested from the four healthy rabbits.The compressive properties of each rabbit calf bone in vitro were tested by dynamic and static fatigue testing machine..Result The bilateral tibial plateau of 7 New Zealand white rabbits was modeled by puncture modeling method.Thirteen models were successfully established,with a success rate of 93%.The compressive forces were 179.0±32.0 N,310.3±70.5 N,328.0±23.5 N,353.0±73.1 N for the Tumor,PMMA-6%Fe₃O₄,PMMA-6%Fe₃O₄-H and Normal groups.The stiffness was 231.7±73.4 N/mm,485.7±145.1 N/mm,501.3±117.0 N/mm,562.7±83.0 N/mm for the Tumor,PMMA-6%Fe₃O₄,PMMA-6%Fe₃O₄-H and Normal groups.Conclusion It is a convenient and efficient method for establishing in situ bone tumors model of tibial plateau using this puncture approach.This magnetic bone cement can repair bone defect fast and strength the mechanical properties of rabbit legs,and achieve nearly the compressive properties of normal rabbit leg.After injection of PMMA-6%Fe₃O₄ and its transformation to a solid,regardless of whether heating was used,the mechanical properties recovered and showed no statistically significant difference from the normal tibial plateau in ex vivo measurements.PART ? PMMA-Fe₃O₄ for internal mechanical support and magnetic thermal ablation of bone tumorsObjective Minimally invasive modalities are of great interest in the field of treating bone tumors.Providing reliable mechanical support and fast killing tumor cells to achieve fast physical function recovery for rapid repair of bone defects is still challenging in clinical works.The PMMA-Fe₃O₄ magnetic bone cement with reliable mechanical support and high magnetic induce heating efficiency.The effect of thermal ablation of bone tumors under alternating magnetic field was also investigated.Method According to the results of the above four parts,the PMMA-6%Fe₃O₄ bone cement is the optimum ration of Fe₃O₄ containing.Sixty unilateral New Zealand rabbits were randomly divided into three groups: bone cement group?PMMA-6%Fe₃O₄?,hyperthermia group?PMMA-6%Fe₃O₄-H?and control group?Tumor?.For the PMMA-6%Fe₃O₄-H group,rabbits were randomly selected,and 0.15 m L of PMMA-6%Fe₃O₄ was injected into the tumors under CT guidance.After injection,the tumor was heated simply by placing the tumor-bearing part of the leg into a water-cooled magnetic induction coil for 150 s,and the surface skin temperature of the tibial plateaus was continuously measured by the same far-infrared thermometer as above.For the PMMA-6%Fe₃O₄ group,0.15 m L of PMMA-6%Fe₃O₄ was injected into the tumors as described above,and for the untreated Tumor group,0.15 m L of saline was injected into the tumor instead of PMMA-6%Fe₃O₄.On the first day after injection,three rabbits in each group were randomly selected and euthanized for tumor pathological examination by H&E straining.On the 4th day after injection,3 rabbits in each group were randomly selected and euthanized,and terminal deoxynucleotidyl transferase d UTP nick end labeling?TUNEL?and proliferating cell nuclear antigen?PCNA?assay for tumor cell apoptosis and proliferation,respectively,were detected through immunohistochemistry.The proliferating on index?PI?and apoptotic index?AI?were calculated.All the remaining 14 rabbits in each group were fed for further observation of the tumor growth trend through CT examination and the knee perimeter on the 1st d,4th d,7th d,14 th d,21 st d,28 th d,35 th d,42 nd d,49 thd and 56 th day after treatment.The destroyed bone volume of the tibial plateau in all rabbits was calculated from CT images and comparison with the pre-injection image,and the knee perimeter was measured using a soft ruler.For the PMMA-6%Fe₃O₄ group,the thickness of the upper tibial plateau cortical bone was measured by CT images,and the CT value of the upper tibial plateau cortical bone was also measured by CT images.During the follow-up,any rabbits that had died were dissected to check metastasis to viscera.At the end of the experiment,all rabbits were euthanized using appropriate approved methods.The mortality and the percentage of visceral metastasis were calculated.Result In the hyperthermia group,11 rabbits?78.57%?survived for 56 days,and 2 of them had visceral metastasis.The average volume of bone destruction did not increase significantly,the average circumference of leg did not increase significantly,the average weight increased significantly,and the average thickness of tibial plateau epithelial bone and CT value increased significantly.In bone cement group,the longest survival time was 30 days.Visceral metastasis occurred in 10 rabbits,the average volume of bone destruction increased significantly,the average circumference of leg increased significantly,and the average weight decreased significantly.In the control group,the longest survival time was 28 days,visceral metastasis occurred in 8 rabbits,the average volume of bone destruction increased significantly,the average circumference of leg increased significantly,and the average weight decreased significantly.Pathological examination showed that: On the first day after intervention,a large number of coagulative necrotic tumor tissues were observed around the material of rabbit leg bone in hyperthermia group,while a large number of tumor cells were observed in bone cement group and control group.On the fourth day after intervention,apoptotic index was the highest and proliferative index was the lowest in the hyperthermia group.The apoptotic index of the control group was the lowest and the proliferation index was the highest.The apoptotic index of the bone cement group was low,and there was no significant difference between the two groups.The proliferation index was significantly lower than that of the control group and significantly higher than that of the hyperthermia group.Conclusion The PMMA-6% Fe₃O₄ magnetic bone cement can be accurately and controllably injected into the bone defect caused by tibial plateau tumors in rabbits under CT guidance.After 150 seconds of continuous exposure to alternating magnetic field,the magnetic bone cement can cause obvious necrosis of tumor tissue,promote apoptosis of tumor cells and inhibit proliferation of tumor cells,and has no obvious damage to normal surrounding tissues.The survival time of VX2 model rabbits with tibial plateau tumors in situ after magnetotherapy was significantly longer than that of pure bone cement filling group and control group.We have presented a novel,highly efficient and minimally invasive approach for complete bone tumor regression and bone defect repair by magnetic hyperthermia based on PMMA containing Fe₃O₄ NPs;this approach shows excellent heating ability for rabbit VX2 tibial plateau tumor ablation upon exposure to an AMF and provides mechanical support for bone repair.The new and powerful dual-function implant is a promising minimally invasive agent for the treatment of bone tumors and has good clinical translation potential.