| Infective bone defect is a common problem in orthopaedics.The pathogenesis mainly includes two aspects,namely,bone trauma defect and infection.A large part of the reason is the increasing number of traffic accidents and construction accidents.Usually the above causes will lead to a wide range of high-energy fractures and bone defects,especially open fractures,pollution is more serious.The infective bone defect often leads to osteonecrosis,delayed union of fracture,nonunion and failure of internal implantation due to infection.In severe cases,it may cause disability or induce sepsis,which would lead to death of patients.Even if the infection is limited to the local,because of the bacterial biofilm and local microenvironment and other factors,it will be difficult to completely remove the infected focus.In that case,the bone defect is difficult to repair.Therefore,in the treatment of infective bone defect,anti-infection treatment can be said to be the most important.The principle of clinical treatment of infective bone defect is different from acute osteomyelitis.It can not only rely on the systemic use of antibiotics.Antimicrobial treatment must be combined with surgery to remove the infected sections.In addition to the surgical removal of dead bone and clearance of infected tissues,the defect also needs to be repaired.The shape of infective bone defect is often complex,so the implanted repair material should be customized to reconstruct the normal anatomic shape and function of bone.While offering the individualized customization of macroscopic morphology,the microstructure of implants should provide enough space for cell growth.Microporous structure can be used as scaffolds to induce vascular growth and osteogenesis,so that implants can be closely combined with bone.However,the microscopic porous structure also provides an excellent place for bacteria to reproduce.In order to prevent implantation failure caused by infection,the built-in should have antibacterial properties to some extent.3D printing technology,is also known as additive manufacturing or rapid prototyping technology.Compared with traditional subtraction and formation,3D printing has many advantages,such as creating complex design of complex structures,while making use of various materials,Includes plastics,metals,waxes,rubber,wood,cloth,food and biomaterials.Titanium alloy(Ti-6Al-4V)is generally used for metal3D printing because titanium alloy has good biocompatibility with human bone.For bone defects with complex macroscopic morphology,3D printing can be individualized designed and manufactured according to the requirements of its original anatomic structure and implants,in order to achieve the purpose of reconstructing anatomic morphology and restoring function.At the same time,at the microscopic level,3D printing of metal porous structure is very important.In the physical mechanical aspect,the design of metal porous structure can change its mechanical properties and elastic modulus and other mechanical parameters.So that the mechanical parameters are closer to the bone tissue in order to bear the corresponding stress,thus improving the healing of the fracture and the reconstruction of the bone.In biological aspect,the suitable microporous structure can be used as a scaffold to induce the cell adhesion and proliferation,so that the implant could be closely integrated with the bone.Although 3D printed metal implants can meet the requirements of macroscopic morphology and microstructure of infective bone defects,they cannot solve the problem of infection.Therefore,it is urgent to provide antibacterial properties for 3D printed metal mesh structures.Nowadays,for infective bone defects treatment,PMMA bone cement containing vancomycin is mainly used.However,this kind of material has poor biocompatibility and can not be degraded,so it requires to be removed by another operation.Biodegradable materials will replace PMMA bone cement as the drug carrier for the future clinical treatment of infective bone defects.Among biodegradable materials,hydrogels are excellent drug carriers.Hydrogel,as a three-dimensional crosslinked polymer network system,can control drug release,expand on a large scale in liquid environment such as body fluid to maintain structural stability,and gradually degrade while release drugs.It can meet the requirement of antibacterial biomaterials.The antibacterial hydrogel loaded with vancomycin can effectively increase the local drug concentration.At the same time,with the degradation of the hydrogel,vancomycin is released gradually,which can ensure prolonged antibacterial effect,which means antibacterial hydrogel could meet the requirements of anti-infective treatment of infective bone defects.At present,there are many cases of bone defect reconstruction using 3D printed metal microporous scaffolds universally,but antibacterial modification is rarely reported.The application of antibacterial hydrogel can also be seen in literature and practical clinical application.However,it is rarely reported that hydrogel has been composited with 3D printed metal microporous scaffold.In this study,antibacterial hydrogels were loaded on 3D printed metal microporous scaffolds as a composite system,and their antibacterial and microporous structural characteristics were complementary to each other to treat infective bone defects.This research is mainly divided into the following two major experimental parts:Part Ⅰ The preparation,physical and chemical characterization and in vitro antibacterial experiment of vancomycin loaded hydrogels and 3D printed metal microporous scaffolds.Preparation and characterization of antibacterial hydrogels:biodegradable hydrogels were synthesized through liquid A and B from N-carboxymethyl chitosan and sodium alginate oxide and mixed by chemical crosslinking.Vancomycin was added to liquid A as antibacterial component,and then mixed and evenly added to liquid B to form gel.The antibacterial hydrogel was placed in PBS solution and elastase solution to degrade in vitro.The results showed that the material had good degradation performance in vitro.The release curve of vancomycin from antibacterial hydrogel in vitro was analyzed by high performance liquid chromatography.It was proved that the material could effectively release vancomycin and achieve prolonged antibacterial effect.500μl hydrogel was injected subcutaneously into the back of SD rats(250±50 g).The degradation experiment in vivo showed that the material could be degraded in vivo and finished in about 30 days.Antibacterial experiment:S.aureus(representing Gram-positive bacteria)and E.coli(representing Gram-negative bacteria)were selected as standard strains,and the size of bacteriostatic ring was observed at 24 h.The gel was added to the liquid medium containing S.aureus and E.coli for bacterial culture,and the effect of hydrogel on bacterial proliferation was observed.The hydrogels cultured in S.aureus and E.coli liquid medium for 2 h were stained with live&dead to observe the survival of bacteria.Finally,the hydrogels cultured in S.aureus and E.coli liquid medium for 2h were observed by electron microscope after fixation,and the distribution and morphological characteristics of bacteria were recorded.The antibacterial test showed that the hydrogel had excellent antibacterial ability.Preparation of 3D printed metal microporous scaffolds:according to the preset CAD model,the STL file was imported into 3D printing machine(EBM-S12,Arcam).The titanium alloy powder is solidified layer by layer using electron beam melting technology(ARCAM EBM?),The cylindrical titanium alloy microporous scaffold with a height of 5 mm and a diameter of 6 mm was manufactured.The pore diameter was 400 mm and the porosity was 70%.At the same time,the microstructure of the porous structure was observed by scanning electron microscope(SEM).Part Ⅱ Animal model of treatment of infective bone defect with vancomycin hydrogel and 3D printed metal microporous scaffold.Animal model experiment:using New Zealand white(NZw)rabbits(3.0±0.5 kg)as model,a circular critical size bone defect with diameter of 6 mm and depth of 10mm was established at 1/3 lateral flat of upper tibia,and 500μl*10~4 colony-forming unit(CFU)/ml S.aureus liquid culture medium was injected into the experimental animal model of infective bone defect.One week later,the tibia was taken to verify the success of the model.Formal animal experiment:osteomyelitis model was established in 4 groups of rabbits.one week later,three groups of materials(blank stent,antibacterial hydrogel and antibacterial hydrogel stent)and a group of blank control were implanted after debridement.At the 2nd and 4th month,MicroCT examination,gross external phase score,HE section of femur,hard tissue section,HE section of important organs,biomechanical push-out test were performed respectively.The number of leukocytes and the severity of inflammation in the antibacterial hydrogel group were significantly lower than those in the non-antibacterial hydrogel group(P<0.05).The results were consistent with the gross external phase score,imaging score and pathological results.MicroCT,HE sections and biomechanical experiments showed that the effect of the group containing antibacterial hydrogel scaffolds was better than that of other bone components,and there was significant difference between the two groups(P<0.05).The sections of peripheral muscles and important organs showed that the material was not biotoxic.The above experiments show that the antibacterial hydrogel 3D printed titanium alloy microporous composite system has a good antibacterial effect and osteogenic effect in the treatment of infective bone defects.In summary,the 3D printed metal microporous composite system carrying vancomycin loaded hydrogel was effectively constructed,and the release rate of vancomycin from the system was reasonably prolonged.To elucidate the mechanism of antibacterial hydrogel and 3D printed metal microporous structure in the treatment of infective bone defects would greatly improve the transformation speed of the antibacterial osteogenic composite system in clinical application and bring novel therapy to this kind of patients.At the same time,this scheme might provide a new idea for the anti-infection design of orthopaedic joint prosthesis(especially 3D printed prosthesis)in the future. |
