Construction Of ZnO/nHa Bone-like Structural Activity Enhanced Hydroxyapatite Bone Repair Material And Study On Its Biological Activity In Vitro

Objective: According to the biological evaluation standard of medical devices(ISO10993-5),this paper will evaluate the in vitro biological performance of ZnO/nHA materials with different mass concentration ratios of Zn and their composite materials with RBMSCs,and screen out the Zn content.The best ZnO/nHA composite.Methods: In this study,ZnO/nHA composites were divided into 6 groups: blank control,0%,1%,3%,5%,and 7% ZnO/nHA composites with Zn content.The experiments in this paper are mainly divided into the following parts:(1)For the hemolysis rate of the material itself: prepare the extracts of 6 groups of materials,and calculate the hemolysis rate of the rabbit blood by reading the OD value of the spectrophotometer;(2)detection of the antibacterial effect of the material: Six material groups were selected to qualitatively and quantitatively determine the antibacterial effect of different material groups on Staphylococcus aureus and calculate the antibacterial rate.(3)Culture of rabbit primary bone marrow mesenchymal stem cells: take young rabbits as the test object,Take the bone marrow for primary cell culture,and verify the purity by flow cytometry;(4)Co-cultivate RBMSCs with bone-like materials and measure the adherence rate of RBMSCs on the materials: select 0%,1%,3%,5%,and 7% ZnO/nHA groups Five material groups directly adhered to rabbit bone marrow derived mesenchymal stem cells(RBMSCs).The adherence rate of RBMSCs was calculated 24 hours later,and the scanning electron microscope(SEM)was used to observe the effect of 5 materials on RBMSCs24 hours later.The effect of adhesion;(5)The effect of materials on the proliferation of RBMSCs: In order to avoid the influence of the surface properties of materials on the number of RBMSCs adhesion,six groups of materials were prepared and the CCK-8 method was used to analyze the effects of different materials at different time points.Impact of RBMSCs proliferation.Results:(1)The hemolysis rate of the 6 groups of materials was less than 5%,and no hemolysis occurred in each group of materials,which can be used as a reliable basis for the next stage of the test.(2)In the qualitative antibacterial experiment,compared with the control group and 0% ZnO/nHA,the zinc-doped hydroxyapatite material has obvious antibacterial effect.1%,3%,5% and 7% Zn content ZnO/nHA composite The diameter of the bacteriostatic circle in the material plate was 1.3cm,1.5cm,1.6cm,1.7cm,indicating that the antibacterial effect was more significant with the increase of zinc content;quantitative antibacterial experiments further confirmed this view,and the zinc content was 3% and Above content,the antibacterial rate is above 90%.(3)CD90 was used as the positive marker,and CD45 was used as the negative marker.It was confirmed by flow cytometry that the cultured cells were bone marrow mesenchymal stem cells,which had adherent growth characteristics.(4)Through SEM observation,RBMSCs can adhere to the surface of the material and grow healthily;after calculating the adherence rate,it is found that compared with the0% ZnO/nHA group,the 1% ZnO/nHA group has a significant difference,and the adherence rate reaches 80%.The best effect is adherence.(5)The proliferation of RBMSCs cultured for 1d,2d,3d,4d,and 5d was determined.Compared with the control group,the proliferation curves of ZnO/nHA materials with 0%,1%,and 3%Zn content were roughly the same,and the 1% group Compared with the 3% group,the proliferation effect of the 1% group is better;and the proliferation curve of Zn O /n HA materials with 5% and 7% Zn content is smooth and even has a downward trend.Conclusion: Based on the experimental results of hemolysis,antibacterial,adhesion and proliferation of RBMSCs,it was found that 1% ZnO/nHA composites have better cell viability in this experimental study.