| Traditional metal implant materials(such as stainless steel,titanium,titanium alloys etc),because of its excellent biocompatibility,excellent machinability,corrosion resistance,as well as its modulus of elasticity close to the human bones close,which has been widely used in orthopedic,dental orthopaedic surgery and biomedical fields.But some new problems have also emerged during the application process,such as stress shielding,implant loosening,implant-related infection,and implant-related infections may lead to the worse results,It is very easy to form a biofilm on the implantation material surface after infection,Bacterial biofilm has isolated the attack from the body's immune system,as well as resistance to the antibiotic effect,which has brought great pain and burden for the patient and also increased the difficulty of clinical treatment.Clinical treatment of implant-associated infections is surgical debridement again renovated,high doses of antibiotics applied,and the treatment has its obvious limitations,reoperation increase patients suffering and the burden of money,high doses of antibiotics can lead to antibiotics resistance.So how to reduce or avoid the occurrence of infections after such implants have become a major and urgent medical problem.Metal ions has a broad spectrum of antimicrobial,good dispersibility in vivo,and has no advantages of drug-resistant bacteria,developed new metal ion implanted biomaterial with antimicrobial characteristics,has become a hot topic in new biomaterials development.Metal ions are mainly silver,zinc and copper ions,which is most widely applied to biological materials research.In this work,Zn-incorporated pure Ti was prepared by thealkali-and heat-treatment technique.Studies on the antibacterial properties and biocompatibility in vitro and in vivo will be performed to deeply evaluate the biosafety of the biomaterial,which will provide the scientific evidences for the research,development and clinical application of the novel antibacterial titanium biomaterials.Research objectives:The objective of this project is to prepare a new biomaterial with porous structure on the surface and Zn ions by alkali-heat treatment.The novel Ti-Zn as the experimental group and pure titanium for the control group to study its antibacterial by co-culture of bacteria and materials,as well as cell compatibility and blood compatibility by co-culture of cells and materials,A implant-associated infection animal model was built to study the antimicrobial activity and toxicity of Ti-Zn biomaterials in vivo,which will provide the scientific evidences for the research,development and clinical application of the novel antibacterial titanium biomaterials.Methods:The main subject of the study is the new development of zinc-containing antibacterial titanium materials.Using the most widely of clinical application of the titanium pure Ti as the negative control group.The Staphylococcus aureus was used as the experimental bacteria.We tested the antibacterial activity of the Ti-Zn biomaterials through the co-culture method of the materials and the bacteria.The human bone marrow mesenchymal stem cells(hBMSC)were used as the experimental cells.Through the co-culture method of the materials and the cells,and then test the toxicity of the co-cultured cells,materials and cells adhesion,materials and cells proliferation toxicity,cells on the surface of materials were observed by Live/Dead staining and SEM observation.Acute hemolytic test was done to test the blood compatibility of the new antibacterial biomaterials Through the material culture of Staphylococcus aureus and determined antibacterial properties of biological materials of the new Ti-Zn biomaterials and observed the bacterial morphological changes after co-culture under scanning electron microscope.Using New Zealand White rabbits as the experimental animal to build an animal models which is susceptible implant infection because of immune suppression by hormone and study the antimicrobial activity and toxicity of the new Ti-Zn biomaterial in vivo.Results:(1)We have prepared porous structure of titanium surface through alkali-and heat-treatment technique.And using chemical method to make the zinc ions attached to titanium surface,and then make it in simulation of biological fluids to reach the purpose of sustained-release.(2)It is confirmed that the new Ti-Zn biomaterial has good biocompatibility.By MTT test,surface cell staining test,acute hemolysis test and electron microscopic observation of cell morphology on the material surface.(3)By co-culture of the material and the bacteria,it is proved that Ti-Zn biomaterials has excellent sterilization rate,which is above 90%,statistically significant(p<0.05)(4)Using New Zealand White Rabbit implants-infection susceptibility model for model,make the Ti-Zn and pure Ti biomaterials which should be coated with certain amount of bacteria implanted in rabbit femoral condyle.it is observed that the animal's body temperature is increased,and lower temperature was in the experimental group than in the control group and have statistically significant.It is more serious wound infection on control group,White blood cell count from venous blood is lower than pure titanium group,It is lower that the bacterial mount contented around implanted per unit weight bone tissue in Ti-Zn material group than control group.(p<0.05),And Zn in the blood test group and control group has no statistically significant difference,so the new Ti-Zn material has certain antibacterial activity in vivo.Conclusions:(1)From the results of the antibacterial test,we found that the sterilization rate of Ti-Zn was above 90%,which was demonstrated excellent antibacterial activity.Test the zinc ion precipitated from Ti-Zn materials to make a curve,we can find that the zinc ion released quickly in the beginning,then slowly released.And by means of SEM,we further found that bacterial membrane rupture after co-culture with Ti-Zn material,so we speculate that the sterilization mechanism of metal ions is to destroy bacteria membrane.(2)The materials and the blood were co-cultured,and then we found that the hemolysis rate of Ti-Zn was 0.36%,which was much lower than that of the 5%.It is proved that the new Ti-Zn biomaterial has good blood compatibility.Through the method of hBMSC and materials co-cultured,proliferation activity is obtained.Through the live/dead dyeing methods and the SEM scanning,we intuitive observation the cells adhesion in the material surface,and the cells spreading,grow well.From all above we confirmed that the growth of the hBMSC cells in the new Ti-Zn materials were good.That is to say,the Ti-Zn biomaterials has good cell compatibility.(3)The Ti-Zn and pure Ti biomaterials which should be coated with certain amount of bacteria has been implanted in rabbit femoral condyle,Then we tested that the animal's body temperature,white blood cell and observed surgical incisions.Lower infection in the experimental group than in the control group has been observed which was statistically significant.So the new Ti-Zn material has certain antibacterial activity in vivo,and there is no statistical different about the Zn ions in blood.So the new Ti-Zn material has no toxicity and no effect of zinc metabolism in vivo.In summary,the new Zn-incorporated coatings antibacterial biological material has good cell compatibility,blood compatibility and good antibacterial activity.On the one hand,the sterilization mechanism of metal ions is to destroy bacteria membrane.Due to its good compatibility which can promote the cells adhered,proliferation.So it can increase the adaptability of the implants between the hosts,and reduce the formation of bacteria biofilm on the surface of implants.Though the animal experiment we can find that the new Ti-Zn material has certain antibacterial activity in vivo and no toxicity and no effect of zinc metabolism in vivo.It has good prospects of clinical application. |
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