The Biocompatibility Study Of Step-up Micro-arc Oxidation Magnesium Alloy In Vitro

BackgroundAs we know, biomedical material plays an important role in the therapy of orthopaedic disease. In the orthopaedics research, metal material is better than any other material especially in the mechanical capacity. Magnesium alloy as a novel biomedical material displayed more advantages than others, such as abundant resource, better specific strength and rigidity, more similar to the density and elastic modulus of human normal bone tissue, resolved stress dodge effect feasibly, promoted the bony growth and concrescence. In addition, magnesium as one of the macrometabolic elements takes part in the metabolic process.However, magnesium and magnesium alloy can not tolerate the corrosion in body liquid stably, and degrade persistently, which is considered as the fatal shortcoming. Investigators have altered the thermochemistry property in the surface of magnesium alloy and enhance the resistance of corrosion through many approaches such as alkali treatment, thermal treatment, alkali steeping combined thermal oxidation treatment, thermal oxidation combined organism coating treatment and ion plating titanium. The suppliers of material in this experiment enhance the resistance to corrosion of two kinds of magnesium alloy (AZ91D and ME20M) through step anodic oxidation surface reshaping under three different preparation conditions. Nevertheless, the biocompatibility of surface ceramic membrane in magnesium alloy after reshaping which is very important in the application of medical implantation material is unknown. Therefore, in vitro experiment was performed to estimate the biocompatibility of the previous mentioned material.Objective and significanceThrough in vitro hemolysis test and cellular compatibility test, we expect to evaluate the biocompatibility of magnesium alloy treated by step anodic oxidation surface reshaping, screen suitable magnesium alloy ceramal membrane material for clinical application and give evidence to support the in vivo experiment. Moreover, we could explore the internal relationship between different preparation and biocompatibility of magnesium alloy ceramal membrane material, in order to prepare the material better in biocompatibility feasibly, meanwhile, it may be a novel approach for implantation material in clinical application.Methods1. experimental materialsTwo kinds of magnesium alloy (AZ91D and ME20M ) were prepared under three different conditions, and became six kinds of magnesium alloy ceramal membrane (AZ₁,AZ₂,AZ₃,ME₁,ME₂ and ME₃). As control groups in the experiment, untreated AZ91D and ME20M, high purified magnesium and Ti₆Al₇Nb were used. All material above we mentioned supplied by South China University of Technology. We performed hemolysis test previously, in terms of good compatibility, subsequently performed cellular compatibility test.2. Preparation of material leaching liquorWe used physiologic saline and DMEM contained 10% fetal bovine serum in hemolysis test and cellular compatibility test, respectively. In the two tests, the material above mentioned were soaked into the medium at a ratio of surface area and medium volume (3:1), and cultured at 37℃in 5%CO2 atmosphere for 72h, and then the material leaching liquor was filtered by 0.22μm micropore filter in order to achieve sterilization.3. hemolysis testIn accordance with the latest national standard (GB/T16886.4-2003), the fresh anticoagulation diluted rabbit blood was added to 10mL physiologic saline material leaching liquor, sterilized distilled water (positive control) and physiologic saline (negative control), respectively, then aqueous bath at 37℃for 60 minutes, subsequently centrifuged at 1000 rpm per min for 5 minutes. The supernatant was aspirated to a clear tube, and determined the absorbance value at wavelength of 540 nm by microplate spectrophotometer. Each group contained 5 samples in parallel. The formula for calculating the haemolysis rate as below: haemolysis rate (%) = (absorbance value of sample - absorbance value of negative control) / (absorbance value of positive control - absorbance value of negative control)×100%.4. Isolation, culture and identification of rat osteoblastWe operated on the tissue in cranium from SD rat which born in 24 hours, then isolated, cultured and identified the osteoblasts in according with the protocol of cell primary culture5. cell cytotoxicity testAccording to the latest national standard (GB/T16886.5-2003), the osteoblasts were seeded in 96-well plates, cultured 24 hours for cell adherent, and then the medium was discarded. Blank control was added into simple medium, the experimental groups were added into different concentration of leaching liquor (100%,50%,10%,l%), incubated at at 37℃in 5% CO2 atmosphere for 3 days. MTT assay was performed to determine the OD value which should be reformed into proliferation rate.6. direct cell contact testMaterial AZ₁ and AZ₃ proved in good biocompatibility by hemolysis test lay in 12-well tissue culture plate, respectively, and then the osteoblasts were inoculated on the surface of material previously mentioned in a density of 1×10⁵/ml, subsequently co-cultured at 37℃in 5% CO2 atmosphere. We observed the growth status through scanning electron microscope on day 3 and 5 after co-cultured.7. cellular compatibility testThe osteoblasts were seeded in 96-well plates, cultured 24 hours for cell adherent, and then the medium was discarded. Blank control was added into simple medium, the experimental groups were added into different material leaching liquor (AZ₁, AZ₃ and Ti₆Al₇Nb), incubated at at 37℃in 5% CO2 atmosphere incubator. MTT assay and the quantitation detection of alkaline phosphatase (ALP) were performed to determine the proliferation and cell activity, respectively, on day 1, 3 and 5 after co-cultured.8. Statistical analysisTwo-way classification ANOVA of SPSS13.0 were used to assess differences between different groups of absorbance value from MTT assay and ALP activity. P values less than 0.05 were considered statistically significance.Results1. hemolysis testThe hemolysis rates of AZ₁, AZ₃ and Ti₆Al₇Nb were 2.19%,2.77% and 1.33 %,respectively, less than the national prescript standard (5%), consistent with the required haemolyticus of biomedical material, therefore, it was considered that the material we mentioned above could not induce hemolytic reaction. In contrast, the other material used in our experiment did not suitable for biomedical material as medical implantation application.2. cytotoxicity testAccording to the correspondence between cytotoxicity and cell proliferation rate, we considered the cytotoxictiy of AZ₁, AZ₃ and Ti₆Al₇Nb as grade 0 or 1, which were consistent with the national prescript requirement of cytotoxicity for biomaterial.3. Observation by scanning electron microscopeWe found that the osteoblasts adherent on the surface of the material displayed a fine growth status and eumorphism. Also, we could find the cell body of osteoblasts elongate, look like fusiform, polygon or heteromorphosis. There were many reductus and microvillus on the cell surface. The cells elongated veil to each other, the fibriform or multiwire contacted and reticulate. The osteoblasts were well adherent on the surface of material.4. MTT assay and the quantitation detection of ALPAs the culture time prolonged, the counts of cells from all groups were increased, however, at the same time point, there was no significance between control and experiment group, nor was between different experiment groups (P< 0.05).ConclusionsAZ₁ and AZ₃, the magnesium alloy ceramal membrane treated by step anodic oxidation surface reshaping, displayed wonderful biocompatibility, well adherence, fine cell compatibility and lower cytotoxicity. Importantly, they did not induce hemolytic reaction. Compared to Ti-6Al-7Nb frequently used in clinical, there was no significance in statistically. They are expected to further development of a new type of implant material in orthopedics...