The Evaluation Of The Biocompatibility Of Nickel-free Austenitic Stainless Steel Using Annexin V/PI And Hoechst33342/PI Double Staining Method

ObjectiveIn this study, L929 mouse fibroblasts were cultured with the extracts from a new type of nickel-free austenitic stainless steel, BIOSSN4, which was developed by the Metal Institute of Chinese Academy of Sciences. Apoptotic cells were seen under fluorescence microscope for each experimental group after Annexin V-FITC/PI and Hoechst33342/PI double staining and apoptosis rate was examined by the method of flow cytometry Annexin V/PI double staining in order to evaluate the biocompatibility of BIOSSN4 stainless steel from the aspect of apoptosis so that the biological basis for its clinical use can be verified.Materials and Methods1. Experimental grouping and the preparation of the tested metalsThe tested material was divided into 6 study groups which included:Group A: BIOSSN4 stainless steel; Group B:317L stainless steel; Group C:cobalt-chromium alloy; Group D:nickel-chromium alloy; Group E:Gold alloy; and Group F:the negative control which contained RPMI1640 culture medium with 10% fetal calf serum. Based on the length of culture time, each study group was further divided into 24 and 48 hours groups.A round wax sheet with 10 mm diameter and 1 mm thickness was first prepared for each group. The circular metal wafers were then casted with corresponding metals, polished, cleaned with ultrasound for 15min, washed three times with deionized water, kept in the glass vials, and finally autoclaved under 121℃and were ready to use.2. Preparation of the extracts from the metal wafersThe metal wafers were placed in the 24-well plate under sterile condition. Two milliliters of RPMI1640 medium containing 10% fetal calf serum were added to each well. The wafers were extracted continuously for 72 hours in the 5% CO2 incubator at 37℃and were ready for the next step.3. Cell cultureL929 cells were cultured and passaged in the RPMI1640 cell culture medium containing 10% fetal calf serum in the incubator at 37℃with 5% CO2.4. Detection of apoptotic cellsExtracts from the negative control and the tested alloy metals were cultured with L929 cell. At 24 and 48 hours, respectively, the cultured cells were then stained with FITC/PI double staining. The apoptotic cells were observed under the fluorescence microscope. Apoptotic rate for each group was evaluated by passing through the flow cytometry. Hoechst33342/PI double staining was also performed and the apoptotic cells were observed under fluorescence microscope to calculate the apoptotic rate.Results1. Apoptotic cells were seen under fluorescence microscope for each experimental group after Annexin V-FITC/PI and Hoechst33342/PI double staining. Apoptosis rate from low to high was in the following order:Gold alloy