The Biocompatibility Evaluation Of Nano-carbon Particles

Diamond-Like Carbon(DLC)films present a remarkable excellent wear resistance,chemical inertness and biocompatibility,and can significantly reduce the generation of debris.Nonetheless,during the long-term service of DLC coating,DLC wear debris is still generated in vivo and in vitro.These wear debris are carbon particles and have a circular or irregular shape with size range from nano to micro-meter scales.The wear carbon particles have different sp~2/sp~3 ratios,which is mainly depended on the graphitization of DLC film during wear process.The generation and release of DLC debris have attracted a lot of attentions and raised concerns.Whether they will cause the adverse reactions around the DLC modified joint prosthesis and will pose a risk of aseptic loosening or not.In order to improve the application of DLC film on artificial joint modification,it is an urgent mission to evaluate the cytotoxicity of DLC wear debris with different structure.But because of the excellent wear resistance and the circulation of body fluid,it is difficult to collect DLC debris and estimate the biosafety of them,so in this paper,nano-carbon particles(NCs)with different microstructure(sp~2/sp~3 ratios)have been prepared to simulate DLC debris produced in vivo,and the biocompatibility of NCs are also researched in this article to promote the clinical application of DLC film.In this paper,NCs with different sp~2/sp~3 ratios are produced by nano-diamond vacuum annealing treatment in 300℃,500℃ and 700℃.The microstructure of nano-carbon particles(NCs)are investigated via Raman spectrum,X-ray photoelectron spectroscopy(XPS)and Transmission electron microscope(TEM),Malvern Zetasizer Nano-ZS90 apparatus is used to evaluate the zeta potential of particles.Macrophages are cultured to evaluate the inflammation reaction,and osteoblasts adhesion and growth are also evaluated.The results show that the structure(sp~2/sp~3 ratios)of NCs have great impact on its surface charge,which further influence the behavior of macrophages and osteoblasts.In the meantime,the particles concentration has a significant influence on particles biocompatibility.NCs with different structure are successfully prepared by vacuum annealing treatment.NCs are round in shape and have a mean size around 10nm,which have the similar size and shape with DLC debris produced in vivo.Meanwhile,with higher annealing temperature,the graphite content and the sp~2/sp~3 ratios of NCs drastically increased.The presence of graphite component in NCs gives the negatively property in NCs surface and show a negative zeta potential.Negatively charged NCs can’t enter into the macrophages and osteoblasts due to the electrostatic repulsion.Thus those NCs with different spS/sp3 ratios show better osteogenesis ability,and induce lower inflammatory reaction with little amount IL-6 and TNF-a.No significant difference is observed among the zeta potentials of NCs with different sp~2/sp~3 ratios.Thus the cytocompatibility of NCs have also no significant difference.The effect of concentration on the biocompatibility of debris shows that the cytotoxicity of NCs present dose-dependent.Higher particles concentration induces severe macrophages inflammatory response and lower osteogenesis ability.Noteworthy,when particles concentration reach up to 50μg/mL,particles toxicity to macrophages and osteoblasts is significantly increased.In a word,NCs with different structure present different electrical properties,which further influence the interaction between NCs particles and cells.The sp~2/sp~3 ratios of NCs change within a certain range have no significant toxicity on cells.And NCs still present good biocompatibility with macrophages and osteoblasts.At the same time,the toxicity of NCs is dose-dependent,with higher particles concentration,the severe macrophages and osteoblasts toxicity.