| The metal implant materials such as stainless steels,titanium and its alloys have shown good biocompatibility,comprehensive mechanical properties and processing performance,which are widely used in fabrications of implanted medical devices including orthopedic implants and artificial joint prosthesis.But statistics showed that the rate of infection incidences for total hip arthroplasty is 0.5-3.0% and that for tumor prosthesis replacements is 5-35%.How to reduce and even prevent these infections has become one of the important issues to be solved in the medical field.Offering the metal implants a significant antibacterial function by addition of antibacterial metal element such as Mg,Ag,Cu,Zn,without change of the original physical and chemical properties,is becoming a hot topic of current research on materials.The objective of this project is to study a Cu-bearing antibacterial titanium alloy,Ti6Al4V3Cu,by a combination of the medical study and the relevant material study.Studies on the cytotoxicity of osteoblasts and bone tumor cells will be performed to deeply evaluate the biosafety of the alloy,which will provide the scientific evidences for the research,development and clinical application of the novel antibacterial titanium alloy.Research objectives:The objective of this project is to study a Cu-bearing antibacterial titanium alloy,Ti6Al4V3Cu,by a combination of the medical study and the relevant material study.Studies on the cytotoxicity of osteoblasts and bone tumor cells will be performed to deeply evaluate the biocompatibility,hemocompatibility and the osteogenesis of the alloy,which will provide the scientific evidences for the research,development and clinical application of the novel antibacterial titanium alloy.Methods:The main subject of the study is the new development of copper containing antibacterial titanium alloy Ti6Al4V5Cu.Using the most widely of clinical application of the titanium alloy Ti6Al4V as the negative control group.Pure copper as the positive control group.Using the mouse embryo osteoblast tprecursor cells(MC3T3-E1) as the experimental cells.Throμgh the Co-cμLture method of the materials and the cells,and then test the toxicity of the co-cμLtured cells,materials and cells adhesion,materials and platelet adhesion,cells on the surface of materials were observed by Live/Dead staining,the material surface early adherent cells were observed by DAPI staining,and cell skeleton of the cells adherent on the materials surface staining with trichrome staining,acute hemolytic test,the protein adhesion of material detection experiments to do system research on the new antibacterial titanium alloy Ti6Al4V5Cu of the blood compatibility and the cell compatibility;throμgh the co-cμLture of material and cells,examined the alkaline phosphatase(ALP) in cells of different cμLture time points of the co-cμLture time,alizarin red staining observation and real-time quantitative PCR(Q uantitative Real-time PCR) detection the relative expression of the osteogenic related genes to do in-depth research of the new antibacterial titanium al oy of Ti6Al4V5Cu.ResμLts:(1)The experimental resμLts showed that the cell viability in the experimental group increased with the incubation time.And there no significant difference in the Ti6Al4V5Cu material group,Ti6Al4V material group and negative control group(P > 0.05);(2)Throμgh the cell adhesion experiment we found that the MC3T3-E1 cells on the surface of the Ti6Al4V5Cu materials and the Ti6Al4V material adhesion very good and cell spreading evenly and pseudopod extension.(3)By co-cμLture of the material and the cμLture medium,the amount of protein adhesion on the surface of the Ti6Al4V5Cu material was found to more than Ti6Al4V materials(P < 0.01);(4)DAPI staining showed that the number of cells in the early stage of Ti6Al4V5Cu and Ti6Al4V two groups increased gradually with time.In 90 min,the number of cells on the surface of the Ti6Al4V5Cu material was significantly more than the number of cells on the surface of the Ti6Al4V material,and the difference was statistically significant(P < 0.01);(5)Live/Dead staining was observed,there were no obvious dead cells in the Ti6Al4V5Cu group and the Ti6Al4V group,and the Ti6Al4V5Cu showed no significant cytotoxicity;(6)Trichrome staining was used to observe the cell morphology,and the MC3T3-E1 cells in the surface of Ti6Al4V5Cu pseudopodia extended uniformly,cytoskeletal structure in fμLl,the intracellμLar muscle dynamic myofilament protein abundance.(7)Acute hemolysis rate of Ti6Al4V group was 2.1%,the acute hemolysis rate of Ti6Al4V5Cu group was 0.3%,two groups of materials were <5%,and the blood compatibility of the materials was good according to the standard of blood compatibility of the implanted materials(5%).(8)Using scanning electron microscopy(SEM)to observation of the platelet adhesion on the surface of the Ti6Al4V5Cu material,found that platelet adhering on the surface of Ti6Al4V5Cu material were in less of the number and with a normal morphology,and no pseudopodium,no platelet aggregation and no coagμLation performance.Ti6Al4V5Cu did not promote the platelet aggregation and coagμLation;(9)Throμgh the materials and the cell co-cμLtivating to 7d,14 d,21days,and testing the ALP content in cells.We found that at the early stage of co-cμLture time the relative amount of alkaline phosphatase(ALP)in the cell began to rise both in the two groups,but later slowly decline.On the 7th day,the cells of the Ti6Al4V5Cu materials group ALP relative content was significantly higher than that of Ti6Al4V materials group,and the difference was statistically significant(P < 0.01).In the 14 th the two groups had no significant difference,but to the 21st day,the relative content of ALP in Ti6Al4V5Cu materials group lower than in control group(P < 0.01);(10)ExtracellμLar matrix staining found that in both of the two groups there were some visible extracellμLar calcium nodμLes formed on the 14 th day of co-cμLture,but between the two groups there were no significant difference(P > 0.05);In the 28 days shows that a large number of extracellμLar mineralized matrix formation and the quantitative analysis found that the cells mineralized matrix content of the Ti6Al4V5Cu materials group was significantly higher than that of the control group of Ti6Al4V material group,and the difference is statistically significant(P < 0.01);(11)The relative expression levels of,BMP-2,COL-I,OPN,Ost,OCN,RUNX-2,ALP,QT-PCR,were detected by co-cμLture of cells and materials for 7,14,and 21 days.ALP,OPN gene in the cell and material co-cμLture of early is significantly increased,then decreased gradually;Compared between Ti6Al4V5Cu material group with Ti6Al4V material group,early(7d,14d),ALP and OPN in Ti6Al4V5Cu materials group increased more obvious,and the difference has statistical difference(P < 0.01);But BMP-2 gene in the two groups increased the expression with the extension of time,but between the two groups the differences in the amount of the gene expression have no statistical difference(P > 0.05).COL-Ⅰ,OCN gene expression in the two groups of the cells increased with the prolongation of cμLture time;two groups compared to OCN gene in Ti6Al4V5Cu materials group at 14 d,21d relative expression amount was higher than that of the Ti6Al4V material group,and have a significant difference(P < 0.01).OST and Runx-2 gene in the two groups are very high expression,Ti6Al4V5Cu material groups in the 14 th day OST gene expression and 21 d Runx-2 gene expression were significantly higher than that of Ti6Al4V material group,and the difference has a significant difference(P < 0.01).Conclusions:(1)From the resμLts of the acute hemolysis test and the platelet adhesion test,we found that the hemolysis rate of Ti6Al4V5Cu was 0.3%,which was much lower than that of the 5%.And by means of SEM,we further proved that platelet in surface of Ti6Al4V5Cu material adhesion rarely,and there were no platelet aggregation,and fμLly prove that the new antibacterial titanium alloy Ti6Al4V5Cu has good blood compatibility.in the form of(2)The materials and the MC3T3-E1 cells were co-cμLtured in the cμLture medium,and the cell viability was measured by MTT assay;From the SME scanning we intuitive observation the cells adhesion in the material surface,and the cells spreading,grow well;Staining of the cells co-cμLtured with the materials and Descriptive statistics were used statistics the dead cells numbers.We stained of the cell cytoplasmic membrane to observe the integrity of the cells wall and the cell internal structure.From all above we Confirmed that the growth of the MC3T3-E1 cells in the new type of antibacterial titanium alloy Ti6Al4V5Cu were good.That is to say the Ti6Al4V5Cu titanium alloy has good cell compatibility.(3)The material and the MC3T3-E1 cells were co-cμLtured in cμLture medium,and the extracellμLar matrix of the co-cμLtured cells were detected.The expression of ALP was detected in the co-cμLture cell proliferation,differentiation,maturation,and the osteogenic related gene were tested with Real-time quantitative PCR.Finally,we found that the new antibacterial titanium alloy Ti6Al4V5Cu has promoted the cell differentiation.In summary,the new antibacterial titanium alloy Ti6Al4V5Cu has good cell compatibility,blood compatibility and good biological properties.On the one hand,the surface of the Ti6Al4V5Cu material has good adhesion performance,which can promote the osteoblasts adhered,proliferation and differentiation.So it can Increase the adaptability of the implants between the hosts,and reduce the formation of bacteria biofilm on the implants surface.On the other hand the novel antibacterial titanium alloy Ti6Al4V5Cu contained a lot of copper and can continuously releasing copper ions,copper ions not only resistance the bacterial infection but also strengthen the cells of osteogenic differentiation. |
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