Preparation Of 3D Printed Ti-6Al-4V Porous Implants Based On Bionic Design And Study Of Bone Integration Performance

Background:The treatment of bone defects that are not capable of self-healing due to trauma,tumor,osteoarthritis,and other bone diseases remains a major concern in modern orthopedics.Currently,autogenous bone transplant(mostly autogenous iliac bone)and inactivated bone allograft are the most common treatments for critical size bone defects.Meanwhile,in clinical practice,induced membrane procedures(such as the Masquelet technique)and bone transport techniques(such as the Ilizarov technique)are also prevalent.However,these treatments still have certain drawbacks,such as iliac autograft’s potential to induce iliac defect and increase blood loss;bone allografts’ possibility to cause immunological rejection and raise the risk of infection;the Masquelet technique and the Ilizarov technique have lengthy treatment cycles,demanding technical specifications for surgeons,and costly surgery expenditures.Bone tissue engineering(BTE)was developed to more effectively address this clinical issue.By combining the benefits of autologous and allograft bone grafts,porous scaffolds with high biocompatibility and similar elastic modulus to bone tissue may be produced as bone graft replacements to enhance the present treatment technology for critical size bone defects and lower the occurrence of potential problems such as secondary trauma and immune rejection.Scaffolds constructed by BTE are designed to simulate artificial mechanical structures that are conducive to bone tissue formation.The primary objective is to create an osteogenic environment in which bone remodeling can be accomplished across the limitations of critical bone defects.The optimal bone defect filling scaffold should be biocompatible,have the appropriate pore size and porosity,and contain the mechanical strength of genuine bone.Materials now accessible for BTE scaffolds include metals,ceramics,polymers,etc.Metals and alloys have a long history of usage as orthopedic implants,with Ti-6Al-4V being a preferred alloy in recent years due to its high biocompatibility and lack of toxic side effects following insertion.However,Ti-6A1-4V orthopedic implants are not immune to stress shielding—their elastic modulus is substantially higher than that of natural bone.This significant disparity in mechanical strength might result in osteoporosis of the bone around the implant,which can lead to bone resorption and eventual implant failure.Therefore,it is of great significance to optimize the mechanical strength of Ti-6A1-4V implant and reduce its modulus difference with human bone.In recent years,microstructural materials have shown significant advantages,that refining implants into porous structures with adjustable porosity and pore size.The improved implant’s porous structure not only decreases stress shielding,but also directs new bone growth into the porous structure,resulting in a tight bond between the implant and the host bone,i.e.,bone integration.Traditional scaffolds for filling bone defects contain regular and repetitive porous structures made up of simple cell repeating filling structures.However,the mechanical properties and biocompatibility of porous scaffolds may be influenced by the invariable pore size,consistent cell lattice and uniformly distributed porosity.Natural cancellous bone’s trabecular structure does not have the same pore size and cell lattice,and its uneven porous structure exhibits random variation in pore geometry and size.The resulting wide pore size distribution range,variable pore structure and porosity may be additional potential factors for improving the biocompatibility of porous scaffolds in BTE.In addition,it is tough to precisely manage pore size,porosity and shape through traditional manufacturing technologies,and the porous titanium alloy scaffolds constructed by them cannot guarantee pore connectivity and the microstructure is uncontrollable.In latest days,the rapid development of 3D printing technology,including selective laser melting(SLM),selective laser sintering(SLS),electron beam melting(EBM),and other technologies,has made it feasible to create programmable metal porous structures.Among them,SLM is a powder bed fusion 3D printing technology that uses high-energy lasers to melt metal particles into complex geometric shapes layer by layer.SLM technology is not only superior to SLS and EBM in terms of precision,but also offers significant benefits in terms of the toughness and fatigue strength of the samples it produces.This enables the fabrication of Ti-6A1-4V porous scaffolds with an interporous structure designed by computer-aided design(CAD)based on voronoi tessellation(VT).Purpose:This study aims to design and prepare a high-precision porous titanium alloy scaffold with imitation of bone trabecular structure(ITS)using CAD and SLM metal 3D printing technology,which could be used as a bone graft substitute for the treatment of clinically critical size bone defects.Through physical and chemical characterization and in vitro and in vivo biological experiments,the relationship between bionic structure,pore size and pore distribution of porous titanium scaffolds with ITS and bone defect repair was analyzed,and the mechanism promoting bone tissue repair and enhancing bone integration performance was further explored,in order to provide scientific reference for the design and manufacture of porous bionic micro-structure scaffolds.In addition,it offers novel concepts and techniques for enhancing orthopedic implants.Methods:1.Using the VT design concept,a series of ITS porous scaffolds with varied pore size distributions were designed and constructed through CAD,and the regular structure porous scaffolds(RS)with the corresponding same average pore size were established as the control group.According to the different average aperture designed(300 μm,400 μm,500 μm),two types of scaffolds with distinct structure were divided into 6 groups,namely ITS300,ITS400,ITS500 and RS300,RS400,RS500.2.Using SLM metal 3D printing technology,porous titanium alloy scaffolds were manufactured,and their element composition,morphology and structure,mechanical characteristics,hydrophilic properties,and in vitro mineralization capability were examined.3.In vitro co-cultivation of porous titanium alloy scaffolds with rat bone marrow mesenchymal stem cells(BMSCs).Using CCK-8 cell proliferation assay and live/dead cell staining,the toxicity and biocompatibility of scaffolds were evaluated.By utilizing a laser confocal microscope and scanning electron microscope,the effects of scaffolds on the morphology of BMSCs were observed and evaluated.Alkaline phosphatase staining,alkaline phosphatase activity quantification and alizarin red staining were utilized to detect osteogenic differentiation and calcium deposition of BMSCs on scaffolds.Using RT-PCR and Western blotting,the expression of osteogenic genes and proteins in BMSCs was analyzed.4.The rabbit tibial bone defect model was established,and each group of porous titanium alloy scaffolds was implanted into the defect.Micro-CT scanning,the implant ejection stress test,and bone tissue section staining were used to detect the samples 4 and 8 weeks following surgery.In vivo animal experiments were conducted to further evaluate and verify the bone defect repair ability and osseointegration performance from each group of porous titanium alloy scaffolds.Results:1.In this work,using CAD design and SLM metal 3D printing technology,a bionic design-based Ti-6Al-4V porous scaffold with ITS was successfully manufactured.The pores are interconnected,and the pores of varying pore sizes are combined with each other,which has the same bone trabecular structure as human bone.According to the in vitro characterization data,the compression modulus of the scaffold falls as the aperture size increases.The water contact angle of the scaffold decreases as the aperture size increases.Compared to the two groups with the same average pore diameter but varied pore structures,the ITS group had a lower compression modulus and greater hydrophilicity.This study’s Ti-6A1-4V porous scaffold has high in vitro mineralization capability,and the ITS400 group has good hydrophilicity while preserving compressive performance comparable to that of genuine bone.2.The CCK-8 cell proliferation assay and live/dead cell staining were used to verify that the porous titanium alloy scaffolded in each group had no cytotoxicity.The cell inoculation effectiveness was inversely proportional to the pore size,and the porous titanium alloy scaffold with irregular pore structure and multiple pore sizes was more favourable to cell proliferation.Laser confocal imaging and scanning electron microscopy indicated that the porous titanium alloy scaffold with ITS structure facilitated the cells’ adhesion,extension,and pseudopod dilatation.Alkaline phosphatase activity quantification and alizarin red or alkaline phosphatase staining demonstrated that the porous titanium alloy scaffold with ITS structure was more conducive to osteogenic differentiation of BMSCs and calcium deposition.RT-PCR and Western Blot further verified that the porous titanium alloy scaffold with ITS structure could up-regulate the expression of osteogenic genes in BMSCs co-cultured with the scaffold,hence promoting osteogenic differentiation.3.For in vivo animal investigations,the porous titanium alloy scaffold was implanted into the rabbit tibial bone defect model.Analysised by micro-CT scanning and Masson staining of tissue sections shown that the porous titanium alloy scaffold with ITS structure had a greater capacity to stimulate osteogenic differentiation and bone formation.The implant ejection stress test demonstrated that the ITS400 group had the highest mechanical locking strength between the scaffold and the new bone,indicating superior bone integration.Conclusion:In this work,SLM 3D printing technology was utilized to effectively design and fabricate a variety of porous titanium alloy scaffolders with varying average pore sizes and distributions that resemble bone trabeculae.Using regular structural scaffolds corresponding to the same average pore size as controls,the bone integration ability of porous titanium alloy scaffolds with imitation of bone trabeculae and regular structural porous titanium alloy scaffolds was evaluated by in vitro characterization,in vitro cell experiment and in vivo animal experiment.In vitro and in vivo investigations have demonstrated that porous titanium alloy scaffolds with ITS have the approximate compression modulus as natural bone,have improved hydrotropy and histocompatibility,and promote the formation of new bone.By comparing ITS porous titanium alloy scaffolds with the same structure but different average pore sizes revealed that the ITS400 group had the greatest potential for bone integration while maintaining sufficient mechanical strength.This study investigated the effect of irregular distribution of bone trabecular pore structure and variation in pore size on the osseointegration capability of titanium alloy porous scaffolds.It serves as a reference for the design of porous implants that facilitate biological binding.