The Toxicity And Antibacterial Activity Of Graphene And Graphene Oxide: In Vitro And In Vivo Evaluation

BackgroundSkeleton is a hard organ which provides support protection for the human beings, and it is extremely easy to be defected with infection due to various traumas. Artificial bone repair materials have become one of the implants with the largest demand, with complicated and diversified types. However, at present, no material can replace human bone completely in performance. It is one of the hot issues in the current orthopaedic research to continuously find out and explore a high-quality orthopaedic implant material. In addition to good biological compatibility, an ideal orthopaetic implant material should have antibacterial function. With the development of bone tissue engineering, more and more new biological materials have been applied in the field of orthopaetic repair and treatment.As a special two-dimension structure of carbon, grapheme and grapheme oxide are characterized by great hardness and good extensibility, which have been greatly concerned in the field of biomedicine. However, the research on the interaction between grapheme and its derivatives and living body is still at the initial stage. Due to the diversification and complexity of biological system, a comprehensive conclusion still cannot be made in the issue of biological safety, some researches have shown that grapheme has a good biological compatibility and antibacterial role, but it still has certain potential biological damage to cells and tissues, and is closely related with the dosage used. At the same time, there is still a lack of simple and effective detection and evaluation to the antibacterial capacity of grapheme and grapheme oxide. Therefore, we make clear the biological safety and antibacterial capacity of grapheme and grapheme oxide, and this will be beneficial for their application in nano-medicine. Objectives1. To discuss the influence of grapheme and grapheme oxide nano particles on biological compatibility of BMSCs.2. To discuss the influence of grapheme and grapheme oxide nano particles on the surrounding tissues after being implanted in the animal body.3. To discuss the antibacterial capacity of grapheme and grapheme oxide nano particles to staphylococcus aureus. Methods1. The toxicity of graphene and graphene oxide: in vitro evaluationMarrow culture method was used to separate the SD rat BMSCs, and osteogenic induction and adipogenic induction were used for identification. P2-P3 BMSCs were taken for co-culture with G and GO. Alama Blue colorimetric method was used to detect the cell reproductive capacity, scanning electron microscope was used to observe the change of cell status, transmission electron microscope was used to observe the internal change of cell, and Western Blot was used to detect the expression level of conglutination and autophagy associated protein in the cells.2. The toxicity of graphene and graphene oxide: in vivo evaluationG and Go were injected into Balb/c mouse medial vastus muscle bag and the material was taken after 7 d, HE staining was used to observe the influence of nano materials on the surrounding tissues, and the expression level of autophagy associated protein in the tissues.3. The antibacterial activity of graphene and graphene oxide: in vitro and in vivo evaluationThe bioluminescence staphylococcus aureus was sued to co-culture with various groups of nano materials in vivo and in vitro respectively, and the antibacterial capacity of G and GO was evaluated by counting the biological lighting strength of the staphylococcus aureus. Results1. The rat BMSCs were successfully separated and identified, the result of Alama Blue colorimetric method showed that under the same concentration, when the concentration of GO was higher than 500 μg/m L, there will be a serious restriction role to the cells(P < 0.01), and various G groups had a small influence on the cell proliferation; the transmission electron microscope result showed that when the G concentration was less than 50 μg/m L, there was no significant change in cell status; when it was more than 50 μg/m L, the cell was stimulated; when GO concentration was more than 50 μg/m L, cell death was observed; the transmission electron microscope result showed that both G and GO could enter the cell and influence the cell; the Western Blot detection result showed that in the GO group, with the increasing of concentration, the cell Vinculin protein expression decreased(P < 0.01), while the expressions of autophagy associated protein Beclin1, ATG5 and LC3 B protein were up-regulated(P < 0.01).2. The in vivo experiment result showed that compared with the blank control group, there was necrosis in the mouse muscle tissue in GO group, and the autophagy associated protein Beclin1, ATG5 and LC3 B protein in the tissue were up-regulated(P < 0.01).3. The antibacterial experiment result showed that when the concentration was more than 100 μg/m L, the GO group presented a strong antibacterial capacity, while the antibacterial in G group was weak. ConclusionIn conclusion, G and GO have some biological toxins with concentration-dependent manners. Before their application to tissue engineering, their mechanisms of toxicity and modified ways to reduce toxicity need to be clarified, which will contribute to their applications in nanomedicine.