| Background The repair of bone defects caused by trauma,bone infection,and bone tumor has always been one of the difficulties in clinical treatment.Autogenous bone transplantation combined with rigid internal fixation technology is recognized as the "gold standard" for repairing mandibular defects.Titanium alloy is the most commonly used internal fixed materials.It has the advantages of stable nature,corrosion resistance,good tissue compatibility,and light quality.However,due to the high elastic modulus of titanium alloy,there is a stress shielding effect between it and the bone cortex.If the internal fixation is not removed after the bone healing,it may lead to osteoporosis for a long time.In recent years,biodegradable polymer materials have been increasingly used for internal fixation of fractures in the maxillofacial region.However,its insufficient strength,the degradation product is acidic,leading to aseptic inflammation,affecting the repair and healing of bone and other problems,which limit its application.In order to develop an internal fixation material with good biocompatibility,mechanical properties and biodegradable properties,biodegradable metallic materials have become a hot research topic in medical and material science in recent years.Biodegradable magnesium alloys were introduced into the field of orthopedics as early as last century,with the advantages of biodegradability,low stress-shielding effect on fixed bones,and the release of magnesium ions to promote bone growth.The limitation is that the degradation is too fast,the corrosion mode is mostly local corrosion,which is easy to cause stress concentration and fracture.Degradation will produce excessive hydrogen,resulting in high local p H value;In severe cases,inflammatory reaction and osteolysis may even occur.Another biodegradable iron-based alloy is characterized by uniform degradation and excellent mechanical properties.The main problem is that the degradation rate is very slow and the degradation products are difficult to metabolize.In recent years biodegradable zinc-based alloys have become a new research hotspot.Zinc is the second most abundant micronutrient in the human body.It is involved in hundreds of enzymatic reactions that affect development,maturation,immune function,and multiple disease states.Zinc plays an important role in bone formation and mineralization,stimulating osteoblasts and inhibiting osteoclasts.The standard electrode potential of Zn（-0.76 V/SCE）is between that of Mg（-2.37 V/SCE）and Fe（-0.44 V/SCE）.The degradation of Zn is slower than that of Mg,but faster than that of Fe,and no hydrogen is released during the biodegradation process.The mechanical properties of pure zinc are poor.Pure zinc has a tensile strength of about 28-120 MPa and its poor ductility at room temperature（2-2.5%）limits the use of pure zinc for internal fixing.The group developed a biodegradable,high strength Zn-Fe-X zinc alloy,which was prepared as a maxillofacial internal fixation system with a yield strength of 171 MPa and a tensile strength of 180 MPa.In the completed preliminary in vitro experiments and animal experiments on mandibular fractures,it was demonstrated that the biodegradable zinc alloy internal fixation system has sufficient strength to provide good support for simple linear fractures of the canine mandible,good histocompatibility,plasticity,uniform degradation,and about 20% degradation in 1 year.After approval,the project has successfully entered into clinical multicenter trials and has completed dozens of clinical trial cases,achieving good clinical results.The biodegradable zinc alloy internal fixation system developed by our group is the only zinc alloy medical bone implant that is currently in clinical trials internationally and has great clinical application prospects.With the progressive study of degradable Zn-Fe-X alloys,the group found deficiencies such as insufficient strength,single shape,and narrow indications,but also found advantages such as better bone healing in the zinc alloy group than in the control group.In order to further explore the potential of zinc-based alloys in maxillofacial internal fixation,the following questions need to be further explored: First,the previous experiment was only a simple single linear fracture of the mandible,but clinically we often encounter complex fractures of the mandible,or mandibular defects caused by trauma,tumors,etc.Can the mechanical properties of zinc alloy be further optimized for the fixation of mandibular defect repair? The second question is that because the repair of mandibular defects is more difficult,traditional splints need to be bent by the physician intraoperatively,which not only increases the operating time,but also the repeated bending of the internal fixation plate can easily lead to fracture.Digitally designed personalized splints are widely used in clinical practice because of their speed,accuracy and individualized shape.Can resorbable zinc alloy be made into personalized plates to produce complex shapes and achieve better facial reconstruction results? Third,in the previous experiment,we found that the concentration of zinc ions in the bone tissue around the zinc alloy internal fixation was significantly increased,and the bone healing effect of the other zinc alloy group was better than that of the control group.So what role does the degradation release of zinc ions play in bone healing? Fourth,zinc alloy is degradable.After degradation,its mechanical strength changes.What effect will it have on the bone healing process?In view of the above problems,the aim of this project is to explore a new generation of biodegradable zinc alloy internal fixation system.The strength of the zinc alloy was improved by adjusting the alloy ratio and optimizing the post-treatment process,and personalized biodegradable zinc alloy plates and screws for mandibular defects were produced by digital design,finite element analysis and 5-axis cutting.In vitro and animal studies were conducted to observe the feasibility of personalized resorbable metal internal fixation systems for the treatment of maxillofacial bone defects.Comparison with the titanium personalized internal fixation system currently in clinical use to confirm the safety and effectiveness of the product.Comparison with the titanium personalized internal fixation system currently in clinical use to confirm the safety and effectiveness of the product.To explore the degradability,tissue compatibility and tissue response of the biodegradable zinc alloy fracture fixation system in animals,and to investigate the molecular and biomechanical mechanisms of biodegradable zinc alloy fixation in bone healing.Objective 1)To create digital models of different models of zinc alloy reconstruction plates and screws used for fixation of repaired bone defects.The stress-strain conditions of different models of digitized personalized zinc alloy splinters were evaluated through finite element simulations to investigate the best reconstruction plate model suitable for fixing segmental mandibles in dogs.2)Enhancement of zinc alloy material properties through alloying and post-treatment processes.Processing of personalized degradable Zn-Mg-Fe alloy splints and screws by CAD/CAM technology.Characterization of the material to evaluate the mechanical properties,in vitro biosafety,and degradability of the material.3)To verify the possibility of fixation of canine mandibular segmental defect model with degradable Zn-Mg-Fe alloy through animal experiments.To observe the safety and efficacy of the new biodegradable Zn-Mg-Fe alloy internal fixation system for the treatment of segmental mandibular defects in dogs and the degradability of the Zn-MgFe alloy internal fixation system in vivo.4)To explore the bone-promoting mechanism of Zn-Mg-Fe alloy: evaluate the biocompatibility and bone-promoting performance of degradable Zn-Mg-Fe alloy through cell experiments in vitro.Create a digital model after degradation of Zn-MgFe alloy.Through finite element biomechanical analysis,observe the biomechanical and stress shielding changes of bone tissue after degradation of zinc alloy to repair mandibular defects,and explore the relationship between the mechanical changes of internal fixation degradation and bone healing.5)Preclinical exploration: To evaluate the feasibility of personalized biodegradable ZnMg-Fe alloy internal fixation material for application in human mandibular defects by finite element simulation of human mandibular segmental defects,to observe whether the maximum stress of plates and nails exceeds the limit of the material,to analyze the stress concentration site of the internal fixation system,and to provide a basis for the next personalized modification and final application in humans.Methods Part Ⅰ 1)The canine mandibular cortical bone,cancellous bone,and teeth were extracted from the CT by Mimics and Geomagic software,and three-dimensional models were created separately.Model the segmental bone defect of canine mandible by 3-matic software.Digital models of different models of personalized reconstruction plates and screws were established.2)Based on the model of a 3-cm segmental bone defect in the mandible of a Beagle dog and nine different types of personalized bone plate models,the maximum Von-Mises stresses and maximum Von-Mises strains of the nine personalized bone plate models for the repair of mandibular defects in dogs with anterior and posterior teeth were analyzed using finite element analysis software in conjunction with the mechanical properties of zinc alloy.The pattern of stress distribution was analyzed to find the model with the best mechanical properties of the individualized splints.The stress concentrations in the internal fixation were analyzed to facilitate optimization in the subsequent digital processing.Part Ⅱ3)The weight ratio of Zn-Mg-Fe alloy was adjusted,and the Zn-Mg-Fe alloy was cast by vacuum method,and the casting ingots were further processed by post-treatment methods such as rolling and extrusion to explore the Zn-Mg-Fe alloy material with better strength.The stress concentration area of the reconstructed plate obtained from the finite element analysis was modeled and optimized by UG NX 12.0 software.The Zn-Mg-Fe alloy personalized joint plate was manufactured using 5-axis simultaneous machining center.4)The prepared Zn-Mg-Fe alloy materials were subjected to mechanical tests,scanning electron microscopy observations,X-ray diffraction observations,electrochemical tests,in vitro degradation experiments and cytotoxicity experiments.The mechanical strength,microstructure,physical phase,in vitro degradation properties and safety of the prepared Zn-Mg-Fe alloy materials were observed.Part Ⅲ5)An animal model of mandibular defect in Beagle was established.24 Beagles were randomly divided into 2 groups,including 12 in the experimental group（personalized Zn-Mg-Fe alloy internal fixation system）and 12 in the control group（personalized titanium alloy internal fixation system）.A 3-cm segmental bone defect was made in the right mandible of each Beagle,and a set of 12-hole personalized fixation products was used for each bone defect model.6)To evaluate the safety,efficacy and degradability of a personalized degradable Zn-MgFe alloy internal fixation system for the treatment of mandibular defects in dogs.After the animal model was established,X-rays were taken and blood was drawn regularly for routine blood tests,blood biochemistry and serum trace elements.The animals were euthanized in batches at 1,3 and 6 months after surgery,and the mandibular bone blocks were examined by gross observation,Micro-CT scanning,SEM scanning electron microscopy,hard tissue sectioning,and triple fluorescence labeled bone deposition.The important organs and soft tissues around the implants were taken for pathological sections.Part Ⅳ7)MC3T3-E1 cells were used as the study subjects.After co-culture of different concentrations of zinc alloy extracted liquid with MC3T3-E1 cells.The adhesion and proliferation of MC3T3-E1 cells were observed by CCK8 assay and cytoskeleton immunofluorescence staining.The induction of osteogenic differentiation of MC3T3-E1 cells by the materials was observed by alkaline phosphatase staining and activity assay,alizarin red staining and activity assay,and RT-qPCR.8)The degradation rate of the Zn-Mg-Fe alloy in vivo obtained from animal experiments was simulated to create CAD models of the Zn-Mg-Fe internal fixation plates at different time points after surgery.Through finite element simulation of the healing process of canine mandibular defect model,the biomechanical and stress shielding changes of bone tissue after the repair of mandibular defect with degradable zinc alloy were explored,and the relationship between internal fixation degradation and bone healing was analyzed from the biomechanical point of view.Part Ⅴ9)A complete finite element model of the human mandible is created,solved and computed,and then compared with relevant literature to evaluate the reliability of the established finite element model.10)A finite element model of the personalized Zn-Mg-Fe alloy internal fixation system combined with fibular flap to reconstruct RBS-type bone defects in the mandible was constructed.Simulate the mechanical loading of occlusion.Analyze the stress-strain of Zn-Mg-Fe alloy reconstructed plates and nails,and compare them with titanium alloy reconstructed plates and nails to evaluate the feasibility of personalized Zn-Mg-Fe alloy biodegradable internal fixation material in the application of human mandibular defects.Observe whether the maximum stress of the plates and nails exceeds the limits of the material.Analyze the stress concentration areas of the internal fixation system to provide a basis for the next personalized modification and final application in human.ResultsPart Ⅰ.Design and biomechanical finite element analysis of a personalized biodegradable zinc alloy internal fixation system1)Cortical bone,cancellous bone,and teeth were separated and extracted in Mimics software depending on the gray scale.The model was repaired using Geomagic Studio 2014 software.A segmental bone defect of the canine mandible 3 cm long from the third premolar to the first molar was created using 3-matic software.Personalized bone splints were designed according to the requirements of the experimental subgroup,and CAD models of nine different models of personalized reconstruction plates and matching screws were obtained.2)After the completion of finite element analysis,the maximum equivalent stress and maximum equivalent strain of 9 groups of reconstruction plates,bone screws and the whole bone defect model were considered as the best model design of this experiment.The stress concentration areas of the personalized zinc alloy reconstructed plates were at the edges of the screw holes and the turning areas of the outer edges of the reconstructed plates.Further optimization of the above locations is needed to reduce the risk of fracture due to stress concentration in the plate.Part Ⅱ Preparation and characterization of personalized degradable Zn-Mg-Fe alloy internal fixation system3)The Zn-Mg-Fe alloy plate used for the successful preparation of the bone plate and the bar used for the preparation of the bone screw.The shape optimization of the reconstruction board CAD model was successfully carried out through UG NX12.0 software.The personalized Zn-Mg-Fe alloy reconstructed plate was successfully processed by forging and forming with 5-axis simultaneous cutting.The overall dimensions of the prepared personalized biodegradable zinc alloy reconstructed plate were kept intact,and all the designed features were effectively formed with smooth surface and no roughness by hand.The screw holes are well constructed with smooth and complete countersunk recesses and good contact relations with the screws.4)The yield strength of the prepared Zn-Mg-Fe alloy plates was 172.4±1.7 MPa and the ultimate tensile strength was 353±6.4 MPa;The yield strength of the Zn-Mg-Fe alloy screws was 341.7±20.3 MPa and the ultimate tensile strength was 354.4±6.6 MPa.The XRD scan detection could detect the Zn,Mg and Mg2Zn11 compounds diffraction peaks.Scanning electron microscopy revealed that the surfaces of the internal fixing plate and screws were smooth and uniform.The electrochemical polarization curve showed a corrosion potential（Ecorr）of-1.076±0.03 V and a corrosion current density（Icorr）of 0.664±0.26 μA/cm2 for the internal fixation plate and a corrosion potential（Ecorr）of-1.091±0.04 V and a corrosion current density（Icorr）of 1.064±0.39 μA/ cm2 for the screw.Electrochemical impedance spectroscopy tests revealed that the corrosion performance of the screws was stronger than that of the internal fixation plates.In vitro degradation experiments revealed that the degradation rate of Zn alloy plates was 0.152±0.002 mm/year after 70 days of immersion;the in vitro degradation rate of Zn alloy nails was 0.216±0.055 mm/year.Part Ⅲ Animal experimental study of personalized biodegradable Zn-Mg-Fe alloy internal fixation system for implant fixation of mandibular defects5)A 3-cm segmental bone defect in the mandible of Beagle dogs was successfully established,and the defect in the mandible of Beagle dogs was repaired using a personalized degradable Zn-Mg-Fe alloy（experimental group）and a personalized titanium alloy（control group）internal fixation system combined with autologous bone.6)The personalized biodegradable Zn-Mg-Fe alloy internal fixation system provided stable fixation for the repair of mandibular defects in this animal experiment,and healing was obtained in all bone graft blocks.At 1 month postoperatively,the internal fixation effect of the zinc alloy group was found to be better than that of the titanium alloy group by X-ray observation,Micro-CT quantitative analysis,hard tissue section observation,triple fluorescence labeled bone deposition and quantitative analysis.At 3 months postoperatively,the difference between the zinc alloy group and the titanium alloy group became smaller.At 6 months postoperatively,the bone healing of the zinc alloy group was still better than that of the titanium alloy group by combining the Xray,Micro-CT quantitative analysis,and bone mechanics test results.Some granular degradation products were found on the surface of the zinc alloy internal fixation plates and nails,and the amount of degradation particles increased with time.The degradation rates of personalized zinc alloy degradable implants were 0.087±0.019 mm/year at 1 month postoperatively,0.1337±0.02 mm/year at 3 months postoperatively,and0.1554±0.023 mm/year at 6 months postoperatively.A variety of degradation product morphologies were observed by scanning electron microscopy.The process of degradation advanced slowly and uniformly from the zinc alloy surface to the basal layer,and there was no destruction,fragmentation or disintegration of the material with degradation.The EDS results showed that the content of zinc elements in the transition from the zinc alloy material layer to the bone tissue layer decreased when entering the bone tissue,and the zinc elements released from the material were not deposited in the bone tissue,showing a better degradation safety of the material.The zinc alloy showed good biocompatibility in vivo.Blood routine,blood biochemistry,serum zinc,magnesium and iron ion concentrations fluctuated slightly within the normal range.The pathological results of heart,liver,kidney,lung,spleen and gonad were normal.Part Ⅳ Study on the bio-biomechanical mechanism of degradable Zn-Mg-Fe alloy to promote bone healing7)After co-culture of different concentrations of Zn-Mg-Fe alloy extract and control group culture solution with MC3T3-E1 cells,it was found that 100% Zn-Mg-Fe alloy extract had obvious inhibitory effects on MC3T3-E1 cell proliferation,and 50%,20% and 10% Zn-Mg-Fe alloy extract had the ability to promote MC3T3-E1 cell proliferation.The zinc alloy extracts were diluted 1-fold,5-fold and 10-fold,and there was no significant cytotoxicity to MC3T3-E1 cells in each group.50%,20% and 10% of zinc alloy extracts had pro-proliferative ability of MC3T3-E1 cells.The greater osteogenic differentiation ability of cells in 20% zinc alloy extract was observed by alkaline phosphatase staining and activity assay,alizarin red staining and activity assay,and RT-qPCR assay.The classical Wnt signaling pathway was activated by 20% concentration and 10% concentration of zinc alloy infusion fluid and significantly upregulated the expression of Wnt1,LRP6,and β-catenin.8)Biomechanical analysis found that,with the degradation of Zn-Mg-Fe alloy at 1,3 and 6 months after operation,the implanted bone mass and proximal and distal callus of zinc alloy group received higher stress stimulation than that of titanium alloy group.The stress shielding rate of Zn-Mg-Fe alloy group is lower than that of titanium alloy group,and the downward trend is more obvious than that of titanium alloy group.Part Ⅴ.Biomechanical finite element study of personalized biodegradable Zn-Mg-Fe alloy internal fixation system for bone graft fixation of human mandibular defects9)A digital finite element model of the human mandible was successfully created,and the measured occlusal forces were in general agreement with the literature,and the pattern of stress distribution was consistent with that described in the references.It is verified that the created finite element model is reliable and can be used for further analysis.10)The finite element model of personalized Zn-Mg-Fe alloy internal fixation system combined with fibula flap reconstruction for repairing RBS-type bone defect in mandible was successfully constructed.Through finite element simulation,the analysis results show that: 1)the maximum equivalent force of zinc alloy internal fixation reconstruction plate was 167.98 MPa,which was lower than the yield strength of zinc alloy plate;the maximum equivalent force of zinc alloy nail was 87.771 MPa,which was lower than the yield strength of zinc alloy nail material.（2）The equivalent force and equivalent variation of zinc alloy screws and reconstruction plates were smaller than those of titanium alloy,and the stress distribution was more uniform;（3）The equivalent force and equivalent variation of bone were found to be slightly higher in the zinc alloy group than in the titanium alloy;（4）The chin turn and condylar process are the areas where stresses are easily concentrated,and the maximum equivalent force here is very close to the yield strength of zinc alloy,and there is a risk of fracture.ConclusionIn this project,the mechanical strength of zinc alloy was further enhanced by changing the alloy formula and optimizing the post-treatment process to address the shortcomings of the first generation of zinc alloy internal fixation system,such as insufficient strength,single type and narrow indications.A new generation of biodegradable Zn-Mg-Fe alloy internal fixation system with individualized shape and excellent mechanical properties was explored by CAD/CAE/CAM technology and applied to the mandibular defect for the first time.The physical and chemical properties,degradable properties,biocompatibility and bone joining effectiveness of the personalized degradable Zn-Mg-Fe alloy internal fixation system were systematically investigated by material preparation and characterization,in vitro degradation experiments,cell safety experiments,animal experiments and human finite element simulations.Following this,the mechanism of degradable Zn-Mg-Fe alloys for bone healing was further investigated at both the cellular molecular level and biomechanical dimensions.The following conclusions were drawn from this study.1)Through CAD design and CAE finite element biomechanical analysis,we can find the best reconstruction plate model for mandibular defect and find the stress concentration area,which lays the foundation for the follow-up research.2)The mechanical and biological properties of the alloy can be optimized by changing the elemental ratio of Zn-Mg-Fe and using different post-treatment methods.Customized zinc alloy reconstructed plates can be produced by forging and forming with 5-axis simultaneous cutting.The reconstructed plate is suitable as an internal fixation material for mandibular defects because of its significantly improved mechanical properties and individualized shape compared to the first generation of internal fixation plates.3)The personalized biodegradable Zn-Mg-Fe alloy internal fixation system could provide solid fixation of a 3-cm segmental bone defect in the canine mandible,and the bone healing effect was better than that of the titanium alloy group,especially the difference was significant at 1 month after surgery.The degradable Zn-Mg-Fe alloy showed degradation properties in the canine mandibular site,with a degradation rate of 0.1554±0.023mm/year and a volume decrease of 15.61±0.9521% at 6 months after implantation.In the long-term embedding experiment of Zn-Mg-Fe alloy conducted by our group at the same time,it was found that the degradation rate was 0.063±0.003 mm/year at 1 year after surgery,and the percentage of volume loss was 19.2±1.25%.The degradable Zn-Mg-Fe alloy had degradation properties at the canine mandibular site,with relatively slow and uniform degradation rates,no significant abnormal elevation of zinc ions in the blood,and no deposition of zinc elements in the bone tissue.The degradable Zn-Mg-Fe alloy had no toxic effects on the heart,liver,spleen,lung,kidney,testis and soft tissues around the implants,and had good biocompatibility.4)The mechanism of degradable Zn-Mg-Fe alloy for promoting bone healing: On the one hand,Zn-Mg-Fe alloy extract has a concentration-dependent phenomenon,in which low concentrations of metal ions are beneficial to cells,while high concentrations are harmful to cells.In the appropriate concentration range,Zn-Mg-Fe alloy extract has the ability to promote proliferation and osteogenic differentiation of MC3T3-E1 cells.On the other hand,the gradual degradation of the Zn-Mg-Fe alloy internal fixation system with implantation reduces the stress shading of the implanted bone,and the implanted bone mass can be stimulated by more stress and higher bone volume in the osteogenic strain range,thus promoting bone remodeling.However,the degradation rate of the ZnMg-Fe alloy fixation system is still relatively slow,resulting in a stress masking effect after bone healing,and continuous improvement and optimization are needed to further explore the balance between the degradation rate and the biosafety and bone healing properties.5)Finite element simulations revealed that the mechanical properties of the Zn-Mg-Fe alloy could meet the mechanical requirements for reconstructing RBS-type bone defects in the mandible.The chin turn and condyle are stress concentration sites with fracture risk.Our research pioneered a new generation of biodegradable Zn-Mg-Fe alloy internal fixation system with individualized shape and excellent mechanical properties,and investigated a working method for the design-fabrication of a Zn-Mg-Fe alloy internal fixation system suitable for mandibular defect reconstruction.Our study is the first to apply degradable Zn-Mg-Fe alloy to mandibular defect fixation,which further opens up the application of degradable Zn-Mg-Fe alloy and explores the bio-biomechanical mechanism of degradable Zn-Mg-Fe alloy to promote bone healing from both cellular molecular level and biomechanical dimension.Meanwhile,our research has provided experimental data for further research and development of personalized degradable metal internal fixation materials with better comprehensive performance,and accumulated rich theoretical basis and technical experience for the research and development of digital personalized medical Zn-Mg-Fe alloy maxillofacial internal fixation and its biomechanical research,it provides reference data for personalized degradable metal internal fixation materials to enter clinical trials in the future. |
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