Biocompatibility In Vivo Of New Kinds Of Medical Mg Alloy

BackgroundWith the development of biomedicine,biomaterials are applied more and more widely.Biomaterials not only have important social effects in bettering the living quality of human beings,improving their health and saving their lives,but also become a new growth point in economic and scientific-technological areas.There have been a variety of metallic biomaterials widely used in clinic,especially in orthopedics(such as spinal orthopedic,re-conjunction of fractures,restoration of cranium and so on),metals have been the most popular bearing-force implant materials.However,none of them could meet our needs completely.Therefore,it is necessary and urgent to develop new kinds of metallic biomaterials.With the excellent performance of abundant resources,low cost,low density, high strength and stiffness,Magnesium and its alloys are called of "Green Project Materials in 21st century".Thus,they have been widely used in aerospace, electronic communications and other fields.As applicated in orthopedics,the density of magnesium and its alloy is so close to human bone,especially the dense bone,that they do not increase the patient's weight and discomfort,so they are extremely beneficial to their rehabilitation.Young's modulus of elasticity of magnesium and its alloys is almost equal to the human skeleton.Consequently,they are advantageous to reduce or eliminate "stress shielding" between the implant and bone,and to promote growth and healing of fractures.Recently,researchers both in and abroad found that magnesium has the promotion of osteogenesis,considering that magnesium alloy was suitable to be artificial bone or implant.Orthopedics and materials experts are taking wide attention to magnesium alloy because of these unique advantages. However,the chemical properties of magnesium alloy are lively,corrosion resistance is poor.If the implant degraded before the fracture healing,it will not achieve a fixed effect.Thus,scholars both at home and abroad improved corrosion resistance of magnesium alloy through a variety of ways.And Chinese scholars increased the corrosion resistance and friction resistance of magnesium alloy effectively through step anodization technology,slowed down the degradation of it. As a result,it is expected to become a new type of biomedical materials.However, after deal with the magnesium alloys with this technology,how about their biocompatibility,is unclear.ObjectiveAnimal experiments were applied in this study.The magnesium alloys were implanted in animals to observe local inflammation,bone-induced;the leaching liquor were injected into animals to evaluate stimulus response,allergic reaction, acute systemic toxicity action.To evaluate biocompatibility in vivo and bone-induced of step anodizational magnesium and its alloys initially,titanium alloy Ti-6Al-7Nb,which has so good biocompatibility,was compared with.And,it is supposed to explore how different types of membrane parameters and materials influence the biocompatibility of materials through the experiments,as well as correlation and essence between them.Thus,it could provide the of biomedical feasibility evidence for the realization of magnesium and its alloys to be biomedical materials.Methods1.Materials in experimentsMg alloy AZ91D(AZ₀),ME20M(ME₀) and their step anodizational materials under three different conditions(AZ₁,AZ₂,AZ₃,ME₁,ME₂,ME₃),pure magnesium(H),medical titanium alloy(Ti-6Al-7Nb),were made into block samples with the size of 6mm×5mm×2mm and column samples with 8mm diameter and 20mm length separately.The samples were cleaned,disinfected,and prepared to use.2.Preparation of extractsThe above massive samples prepared extracts,extraction medium using 0.9% sterile saline,the ratio of the sample surface area(cm²) and medium volume(ml) was 3:1.The extracts were placed in 37℃incubator within 72h to make metal ions separated as much as possible,sterilization by 0.22μm millipore filter,prepared of material extracts.3.Animals in experiments24 New Zealand white rabbits,88 Kunming mice,80 guinea pigs from animal testing center,Nanfang Hospital,Southern Medical University,open systems environment for animal husbandry.4.Implantation experimentSelected three implant points each on both sides of New Zealand white rabbit's spine.The above-mentioned cylindrical samples were implanted into muscles.Each rabbit was implanted Ti,H,one for each,and 4 magnesium alloys AZ(AZ₀-AZ3) or 4 magnesium alloys ME(ME₀-ME3).Tested concentration of magnesium ions in blood regularly,X-ray films were taken to know the approximate information of material degradation.8 animals were executed at 1 month,3 months and 6 months each,observed of wound healing,removed the material and the surrounding muscle tissue to detect the material compositions on the surface of samples,observed the fibrous capsule thickness and tissue inflammatory response around the samples under the microscope.5.Acute systemic toxicity testMice were randomly divided into experimental group(magnesium alloy), negative control group(Ti),positive control group(H),blank control group(saline), 8 in each group.Mice in each group were injected corresponding material extracts and saline via the tail vein.Immediately after injection,4,24,48 and 72h,mice general performance status,toxicity and deaths were observed.Monitor changes in body weight at 24,48,72h after injection.Observe the liver inflammatory response under the microscope.6.Intradermal stimulation experimentSelected two points each on both sides of the guinea pig spine in shaved district, the interval of each two points was 2cm at least.Injected 0.1ml extracts of test group, the positive control group(H),negative control group(Ti) as well as normal saline (blank control group) at these four points randomly.Inspected the injection site at 24,48 and 72h after injection,observed skin tissue reaction.Types of inflammatory cells and graded response were observed under the microscope.7.Sensitization experimentThe above extracts,saline and the formaldehyde solution(volume fraction of 0.05),respectively,mixed with complete Freund's adjuvant of the same volume as to emulsification.The method of combining injection and illinition was used.48 guinea pigs were randomly divided into experimental group(AZ Group,ME group,H group,4 in each and 36 in all),blank control group(saline,4 in it),negative control group(Ti,4 in it) and the positive control group(formaldehyde,4 in it).After shaved the back,each guinea pig was intradermal injected symmetrically at the six points on both sides of spine.1 weeks after intradermal injection,2cm×4cm saturated filter paper,which have been soaked in the standard material extracts, negative and positive control solution,was fixed on the injection sites for 48h for the 2nd allergens.14d later,shaved the abdomen of guinea pigs,sticked with the previous filter paper and fixed for 24h for stimulating.Observed the reaction or rash on stimulated abdominal skin at the time of 24h after the filter paper removed.Types of inflammatory cells and graded response were observed under the microscope.8.Statistical analysisSPSS13.0 software,using paired t test to deal with the data of Magnesium ion concentration before and after surgery;the concentration of magnesium ions increased between groups useing factorial analysis;tissue inflammatory response surrounding implanted sample using rank sum test;fibrous capsule thickness around the implant specimen using factorial analysis;body weight of mice before and after the test using paired t test,between each group using one-way ANOVA.while P＜0.05 there is statistical significance.Results1.Implantation experimentThe general situation:the general situation is good after implantation of the samples.Blood magnesium ion concentration:gradually increased,and were significantly different at different time points,AZ group has no difference between the ME group(P＞0.05),the concentration of magnesium ions have a positive increase after 1 month,3 months,6 months between AZ,ME group. X-ray film:the samples nearly intact,there were the density-reduced areas surround all samples of the magnesium alloy at each time point(bubbles).Visual observation:at each time point,the capsules and air bubbles were existing around of the magnesium alloy samples implanted,no swelling,no abnormal secretions.Histological observation:inflammatory response of AZ₁,AZ₃,H,Ti groups were classâ… ,other magnesium alloy groups' were less.The capsule reaction of AZ₃,Ti groups were classâ…¡,AZ₁ group's was less at 6 month,other groups' were serious.Thichness of capsule:AZ₁＜AZ₃＜Ti＜other magnesium alloy. Osteogenesis:the cartilage tissue formed around some samples of AZ₃ and ME₀ group,osteoblasts were active.2.Acute systemic toxicity testThe general situation:the general situation after the experiment in each group was OK.Body weight of mice in AZ₁,AZ₃ group did not change significantly after injected magnesium alloy extracts.Body weight of mice in other groups reduced more significantly after injected the magnesium alloy extracts.Histological observation:in Ti,AZ₁,AZ₃ groups,liver tissue had no significant changes in morphology,no significant congestion and edema,or necrosis.Mice in H group had morphologically normal liver tissue,mild congestion and edema.There were no significant inflammatory responses.In other groups,the livers had mild to moderate occurrence of morphological changes and liver cell degeneration.3.Intradermal stimulation experimentVisual observation:no erythema and edema exist at the injection site of normal saline.Ti,AZ₁,AZ₃ groups,the skin stimulate reaction score 0 points;the rest of the sample injection site exist sporadic mild skin erythema and edema,skin stimulate recation score 1 point.Histological observation:normal saline,Ti,AZ₁,AZ₃ groups have no inflammation cell at the injection site,no hyperemia,edema,necrosis in tissue, inflammatory cell response is gradeâ… .The rest of sample injection sites exist a small number of lymphocytes,mild congestion and edema,no necrosis, inflammatory cell reaction is gradeâ…¡.4.Sensitization experimentVisual observation:necrosis of stimulated abdominal skin is obvious in formaldehyde group,allergic reaction is serious,graded as 4.No erythema and edema exist in Saline group,graded as 0.Very slight edema and erythema exist in Ti, AZ₁,AZ₃ group(grade 1).Limitative erythema and mild edema exist in other samples,allergic reaction graded as 2.Histological observation:cell necrosis,lymphocytic gathered significantly at the surrounding parts of necrosis in formaldehyde group,inflammatory response to gradeâ…£.Saline group without inflammatory cell accumulation,no congestion and edema,inflammation graded as gradeâ… .there are a small number of lymphocytes gathered,no significant congestion and edema in Ti,AZ₁,AZ₃(gradeâ…¡),middle volume of inflammatory cells,a little obvious congestion and edema in other groups, inflammatory response graded as gradeâ…¢.ConclusionsSamples of AZ₁,AZ₃,H groups had good performance of the local inflammatory response in implantation experiment,AZ₃ with better capsule reaction. AZ₁,AZ₃ gave better performance in acute systemic toxicity test,intradermal stimulation experiment and sensitization experiment.To sum up,AZ₃ has the best General biocompatibility in vivo,AZ₁ can.