| Bone defects caused by trauma,tumor,infection and other diseases are common clinical problems.More than 2 million bone transplants are performed globally each year,and it is the second most common tissue transplant after blood transfusion.Autologous bone is the most commonly used material in bone graft surgery and is the gold standard for bone grafting.However,there are limited donors and risks of trauma,deformity,and infection at the donor site.Allografts are also commonly used bone grafts,but carry risks such as immune rejection and disease transmission.In order to solve these problems,polymers,ceramics,composite materials and metal implants have become the commonly used materials for bone graft substitutes.Among them,metal implants have become a good substitute for bone defects in load-bearing sites due to their high strength,good ductility and good biocompatibility.The clinically most common used titanium alloys like Ti-6Al-4V and Ti-6Al-7Nb contain toxic elements Al and V,which may adversely affect tissues and organs;also the biologically inert characteristics of titanium require further surface modification to obtain good osseointegration.Tantalum is known as a "biophilic" metal with good biocompatibility,corrosion resistance and mechanical properties.It has been widely used in Class II and Class III medical devices,especially porous tantalum,which has been widely used in total joint replacement prosthesis,spinal fusion cage,bone scaffold and so on.Biological high-entropy alloy is a new type of biological alloy that has emerged in recent years.It has more than five main biological elements,and the content of each component is between 5% and 35% to increase the entropy of mixing and stabilize the solid solution.Compared with pure tantalum implants,biological high-entropy alloy implants can reduce implants density and weight,and improve mechanical properties and biocompatibility through element selection and ratio,and have a higher degree of design freedom.Biological high-entropy alloys have good biocompatibility,corrosion resistance,and tunable mechanical properties,making it a promising candidate for bone implants.As bone defect implants,tantalum scaffold and biological high-entropy alloy scaffolds need to have macroscopic porous structures to provide channels for bone ingrowth;moreover,the mechanical properties of the scaffolds can be adjusted by the size of the pores,porosity,and solid content of the scaffolds to obtain suitable elastic modulus and strength.In addition,since tantalum and some biological high-entropy alloy elements are biologically inert elements and cannot get further osseointegration,tantalum and biological high-entropy alloy scaffolds also need to have microscopic surface chemistry and morphology that are conducive to osteogenesis and biocompatibility,thereby promoting the regeneration of bone tissue.3D printed metal can form complex three-dimensional structures in a layer-by-layer manner through computer-aided design,which is especially suitable for the preparation of personalized bone implants and functional graded macroporous and microporous structures.3D printed sample can optimize the lattice structure to have more suitable mechanical properties and functions.The net shape manufacturing simplifies production and assembly steps and greatly reduces manufacturing time and cost,and reduces material waste and environmental impact.At present,selective laser melting,laser engineered net shaping and electron beam melting are the main methods of metal 3D printing.However,since tantalum and some biological high-entropy alloy elements are high-melting-point metals,which are easy to be oxidized,they have extremely high requirements on the power and air tightness of the equipment,and the raw material powder needs to meet a certain degree of sphericity,particle size and fluidity.These methods for 3D printing tantalum and biological high-entropy alloy scaffolds still face the problems of high difficulty and high costs.In this study,we prepared porous tantalum scaffold and porous biological high-entropy alloy bone implant scaffolds with hierarchical macro/micro/nano structure by direct ink writing and sintering,and constructed a convenient,fast,green,and low-cost 3D printing method for the porous metal bone implant scaffold.The scaffold has a hierarchical macro/micro/nano structure: the macropores provide channels for bone ingrowth,and the microscopic surface chemistry and structure improve the osseointegration of the porous implant.In addition,by adjusting the scaffold pore size,porosity,scaffold rod diameter,solid solution content,biological element types and proportions,we can reduce the density and weight of the scaffolds,and adjust the mechanical properties and biological properties of the scaffolds.We comprehensively evaluated the scaffolds through in vitro cell experiments and in vivo bone defect implantation experiments,and the results showed good biocompatibility and osteogenic properties.The porous tantalum scaffold and porous biological high-entropy alloy scaffolds have tunable mechanical properties and biological properties,and can effectively repair bone defects without loading cells,factors and drugs,and are expected to be used as a new generation of porous metal scaffolds for load-bearing sites and large bone defects.Objective: 1.The hierarchical macro/micro/nano-porous tantalum scaffolds were prepared by direct ink writing and sintering.Explore the way to prepare hierarchical macro/micro/nano-porous metal scaffolds by direct ink writing and sintering.2.Evaluate the biocompatibility and osteogenic properties of porous tantalum scaffold.Explore the scaffold osteogenesis mechanism,and provide a basis for more reasonable surface design of bone implant scaffolds.3.The hierarchical macro/micro/nano-porous titanium-tantalum-niobium-zirconium-molybdenum biological high-entropy alloy scaffold was prepared by direct ink writing and sintering.Based on the concept of biological high-entropy alloys,the direct ink writing and sintering method was used to develop biological high-entropy alloy scaffolds,which provided new ideas for the preparation of hierarchical macro/micro/nano-porous highentropy alloy for bone implant scaffolds.4.The biocompatibility and osteogenic properties of the porous biological highentropy alloy scaffolds were evaluated by in vitro experiments to screen better scaffolds.5.Use animal experiments to verify the bone defect repair ability of porous metal scaffolds,and lay an experimental foundation for further clinical applications of the scaffolds.Method: 1.Mix F127 with tantalum powder to print a porous tantalum scaffold,then degreasing and sintering.Scanning electron microscopy,X-ray diffraction,compressive strength and elastic modulus tests were performed to observe the microstructure,phase and mechanical properties of the prepared porous tantalum scaffolds.2.Rat bone marrow mesenchymal stem cells were extracted and seeded on the surface of 3D printed tantalum scaffold(3D＿Ta),and smooth tantalum(S＿Ta)was used as a control.The adhesion and proliferation of rat bone marrow mesenchymal stem cells were observed by scanning electron microscopy,cytoskeleton immunofluorescence staining,and integrin immunofluorescence staining;alkaline phosphatase staining and activity assay,alizarin red staining and activity assay,and RT-q PCR were used to observe osteogenic differentiation of rat bone marrow mesenchymal stem cells.3.The 3D＿Ta rods and S＿Ta rods were implanted into the distal end of the rat femur,and the osteogenesis of the materials was observed and compared by VG staining and scanning electron microscope Surrounding bone was observed by immunohistochemical staining for osteoblasts markers,stem cells markers,and macrophage polarization-related markers.4.RAW264.7 cell line was seeded on the surface of 3D＿Ta and S＿Ta.M1/M2 macrophage markers were detected by immunofluorescence staining and flow cytometry.The expression of integrins was observed by immunofluorescence staining.The expressions of growth factor-related genes,inflammation-related genes,and integrin-related genes were detected by RT-q PCR.The level of secreted inflammation related cytokines was detected by enzyme-linked immunosorbent assay;the expression of cell polarization related pathway proteins was detected by Western blot.5.Collect the supernatant of RAW264.7 cells cultured on the surface of 3D＿Ta and S＿Ta,and mix with medium or osteogenic induction medium to prepare conditioned medium,and observe cell migration and osteogenic differentiation by co-culture.The effect of conditioned medium on the migration of rat bone marrow mesenchymal stem cells was assayed by Transwell and monolayer wound repair.Alkaline phosphatase staining and activity assay,alizarin red staining and activity assay,RT-q PCR,osteogenesis Immunofluorescence detection and Western blot detection of related proteins were used to observe the regulation of macrophage polarization and secretion function on the osteogenic differentiation of rat bone marrow mesenchymal stem cells.6.Mix F127 with different proportions of titanium,tantalum,niobium,zirconium,and molybdenum powders to print porous biological high-entropy alloy scaffolds.The control was pure titanium scaffold.Ti0.6Zr0.6Nb1.4Ta1.4Mo1.4 group,Ti Zr Nb Ta Mo group,Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 group,and Ti group were degreasing and sintered.Scanning electron microscopy,X-ray diffraction,compressive strength and elastic modulus tests were performed on the scaffolds to observe the microstructure,phase and mechanical properties of the prepared porous tantalum scaffolds.7.Rat bone marrow mesenchymal stem cells were extracted and seeded on the surface of the Ti0.6Zr0.6Nb1.4Ta1.4Mo1.4 group,Ti Zr Nb Ta Mo group,Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 group,and Ti group.The adhesion and proliferation of rat bone marrow mesenchymal stem cells were observed by CCK8 assay,live and dead cell staining,cytoskeleton immunofluorescence staining,scanning electron microscopy,and RT-q PCR staining;alkaline phosphatase staining and activity assay,alizarin red staining and activity assay,RTq PCR and osteogenesis-related protein immunofluorescence were used to observe the osteogenic differentiation of rat bone marrow mesenchymal stem cells.8.A rabbit cranium critical defect model(four full-thickness bone defects with a diameter of 8 mm)was established.The groups were Ti0.6Zr0.6Nb1.4Ta1.4Mo1.4 group,Ti Zr Nb Ta Mo group,Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 group,Ti group,Ta group,and blank group.The observation time points were 4,8 and 16 w.The general condition and osteogenesis of the scaffolds were observed by gross observation and VG staining.9.A rabbit femur condyle defect model(bone defects of φ4mm*h6mm were prepared on the bilateral condyles of each rabbit).The groups were Ti0.6Zr0.6Nb1.4Ta1.4Mo1.4 group,Ti Zr Nb Ta Mo group,Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 group,Ti group,Ta group and blank group.The observation time points were 4 and 8 weeks.The general condition,osteogenesis,and in vivo biocompatibility of the scaffolds were observed through gross observation,VG staining,sequential fluorescence labeling,blood routine,blood biochemistry,and visceral histology.Results: 1.The hierarchical macro/micro/nano-porous tantalum scaffolds were successfully prepared.The surface morphology of the scaffold was rough,caused by crystal growth and fusion,particles melting and bonding.It was found by XRD analysis that the scaffolds surface is Ta B2.The compressive strength of porous tantalum scaffolds tested by mechanical testing machine was 153.40±8.02 MPa,and the elastic modulus was 2.55±0.07 GPa.2.Rat bone marrow mesenchymal stem cells were seeded on the surface of 3D＿Ta and S＿Ta,and it was found that the cell adhesion,proliferation and osteogenic differentiation ability of the S＿Ta group was better.3.By observing the effect of 3D＿Ta and S＿Ta group in osseointegration in rats,it was found that the results were different from the in vitro experiment.The 3D＿Ta group had a better ability to induce osseointegration in rats than the S＿Ta group.By immunohistochemical staining,it was observed that there were more M2 macrophages in the bone tissue around the 3D＿Ta group,and more M1 macrophages in the bone tissue around the S＿Ta group.4.The RAW264.7 cell line was cultured on the surface of the 3D＿Ta and S＿Ta.By immunofluorescence staining and flow cytometry,it was found that the cells of the 3D＿Ta group highly expressed M2 related macrophage markers,and cells on the S＿Ta group highly expressed M1 related macrophage markers.Cells in 3D＿Ta group highly expressed Integrin β1 and cells in S＿Ta high expressed Integrin αM.By RT-q PCR,it was found that the 3D＿Ta group cells expressed more growth factor related genes,anti-inflammatory related genes,Integrin α5 and Integrin β1,while the S＿Ta group cells expressed lower growth factor related genes,and more pro-inflammatory related genes,Integrin αM and Integrin β2 genes.The enzyme-linked immunosorbent assay showed that the cells in 3D＿Ta group secreted more anti-inflammatory factors,while the cells in the S＿Ta group secreted more proinflammatory factors.Western blot showed that the phosphorylation levels of PI3 K and AKT were higher in the 3D＿Ta group,while the phosphorylation level of p65 in the S＿Ta group was higher than that in the 3D＿Ta group.5.Co-culture of rat bone marrow mesenchymal stem cells in conditioned medium,it was found that the migration ability of rat bone marrow mesenchymal stem cells in the 3D＿Ta group was better;Phosphatase staining and activity assay,alizarin red staining and activity assay,RT-q PCR,immunofluorescence detection of osteogenesis-related proteins and Western blot detection showed that the osteogenic differentiation ability of cells in 3D＿Ta group was better.6.The Ti0.6Zr0.6Nb1.4Ta1.4Mo1.4 group,Ti Zr Nb Ta Mo group,Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 group biological high-entropy alloy scaffolds were successfully prepared.The surface morphology of the scaffold was rough,and the micro-nano morphology and pores formed by the melting and bonding of metal particles.With the increase of the ratio of titanium and zirconium and the decrease of the ratio of other elements,the metal particles of the scaffold were more fully fused,and the micropores were reduced.The XRD of the three groups of scaffolds were consistent with BCC phase,and the disordered solid solution structure of BCC phase can be formed between various elements.With the increase of Ti and Zr content,the compressive strength and elastic modulus of the scaffolds increased.The scaffold strengths of Ti0.6Zr0.6Nb1.4Ta1.4Mo1.4 group,Ti Zr Nb Ta Mo group,Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 group and Ti group were 72.69±4.65 MPa,120.80±6.28 MPa,163.00±6.25 MPa and 98.80±4.10 MPa;the elastic modulus were 0.19±0.02 GPa,0.34±0.01 GPa,0.64±0.03 GPa and 1.60±0.03 GPa,respectively.7.Rat bone marrow mesenchymal stem cells were seeded on the Ti0.6Zr0.6Nb1.4Ta1.4Mo1.4 group,Ti Zr Nb Ta Mo group,Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 group,Ti group.All of them have good ability to promote cell adhesion,proliferation and osteogenic differentiation,among which Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 group was the best.8.In rabbit cranium critical defect model,no swelling,no hyperplasia,no exudation,and no granulation formation were observed at the implantation site of each rabbit.VG staining showed that the new bone gradually grew into the scaffold from the edge of the defect and the dura mater.At 16 weeks,the new bone grew into the center of the defect and filled the pores inside the scaffold,and the new bone formed osseointegration with scaffolds.9.In rabbit femoral condyle defect model,it was observed that there was no swelling,no hyperplasia,no exudation,and no granulation formation at the implantation site of each rabbit.There was no abnormality in the morphology,color and texture of the animal heart,liver,spleen,lung and kidney.VG staining observed that the amount of new bone tissue in the scaffolds increased with time.At 8 weeks,the area of new bone formation outside the scaffolds was more than that at 4 weeks.At the same time,the internal scaffold pores were also filled with bone tissue.The new trabecular bone was interwoven and integrated with the host bone,and the scaffold and the bone form an osseointegration.The results of sequential fluorescence labeling of bone deposition showed that the new bone mineralization deposition rate in Ta group and Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 group was significantly higher than that of other groups.The blood routine and blood biochemical indexes of the experimental rabbits in each group fluctuated within the normal range.The heart,liver,spleen,lung,and kidney tissues showed no abnormality.Conclusion: Our research group prepared porous tantalum and porous biological high-entropy alloy scaffolds by direct ink writing and sintering for the first time.The physicochemical properties,biocompatibility and osteogenic properties were systematically studied.The direct ink writing and sintering can effectively repair bone defects,without additional steps to prepare a surface structure that promotes osseointegration,and without loading cells,factors and drugs.By adjusting the scaffold printing parameters such as size,porosity,rod diameter,solid solution content,and biological element types and ratios,we can change the mechanical properties and biological activity of scaffolds with better design freedom.The following conclusions can be drawn from this study: 1.Porous tantalum scaffolds can be prepared by direct ink writing and sintering.The scaffold has hierarchical macro/micro/nano – structure and good mechanical properties,and the deposition of Ta B2 crystals on the surface of the scaffold is beneficial to osseointegration.The scaffold is suitable as bone implant scaffold.2.The porous tantalum scaffold has a benefit effect on the adhesion,proliferation,and osteogenic differentiation of rat bone marrow mesenchymal stem cells.By regulating the polarization and secretory function of macrophages around the material,the osteogenic microenvironment of the implantation site can be changed,thereby regulating the osseointegration of the material in vivo.3.Ti0.6Zr0.6Nb1.4Ta1.4Mo1.4,Ti Zr Nb Ta Mo,Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 biological highentropy alloy scaffolds can be prepared by direct ink writing and sintering.The scaffolds have hierarchical macro/micro/nano-structure,good mechanical properties,and are suitable as bone implant scaffolds.By adjusting the pore size,porosity,rod diameter,solid solution content,biological element types and proportions,we can reduce the density and weight of the scaffolds and optimize the mechanical properties and biological properties of the scaffolds,and have a higher degree of design freedom.4.Ti0.6Zr0.6Nb1.4Ta1.4Mo1.4,Ti Zr Nb Ta Mo,Ti1.4Zr1.4Nb0.6Ta0.6Mo0.6 biological highentropy alloy scaffolds can promote the adhesion,proliferation,and osteogenic differentiation of rat bone marrow mesenchymal stem cells.5.The prepared porous tantalum and biological high-entropy alloy scaffolds can effectively repair the critical defect of the rabbit cranium and condyle defect of the rabbit femur,and have good biosafety,which is expected to be used as a new generation of bone defect implants.Our research provides new ideas for 3D printing metal scaffolds,and provides experimental data for further research and development of 3D printed scaffolds with better comprehensive properties.It also provides data for the future clinical trials of porous tantalum scaffold and biological high-entropy alloy scaffolds prepared by direct ink writing and sintering. |
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