Biocompatibility Of Diamond-like-carbon Film And Wear Debris

Diamond-like-carbon(DLC)film is considered as an excellent candidate for the artificial joint surface modification due to its good mechanical property and biocompatability.From in vitro and clinical studies,DLC can significantly reduce wear and corrosion of the metal substrates.Nevertheless,wear debris can still be generated through wear processes and enter the synovial fluid,leading to periprosthetic osteolysis.In order to promote clinical application of DLC coated joint prosthesis,this thesis had investigated both the toxicity and the biocompatibility effect on different doses,structures and sizes of DLC wear debris and the inner related mechanism.Aimed at the theme of biocompatibility evaluation of DLC film and wear debris,the following work had been carried out.(1)The biocompatibility of DLC films with different structures was investigated using macrophages,osteoblasts and fibroblasts.The results showed that DLC films with a low ratio of sp2/sp3,which tend to have a structure similar to that of diamond,led to less inflammatory,excellent osteogenic and fibroblastic reactions.The results also demonstratedthat the high-density diamond structure(low ratio of sp1/sp3)of DLC films was beneficial for cell adhesion and growth because of better protein adsorption without electrostatic repulsion,while higher graphite structure(high ratio of sp2/sp3)of DLC film inhibit cell adhesion and protein adsorption because of electrostatic repulsion.(2)The cytotoxicity of graphite nanoparticles with different doses had been evaluated.The macrophages and osteoblasts of mouse were incubated with graphite nanoparticles(<30nm diameter)respectively,followed by cytotoxicity evaluations on index of inflammatory cytokines,alkaline phosphatase(ALP)assays and related signal protein expressions.Cytotoxity evaluation showed that cell viability had a dose-dependent(10-100 ?g/mL)decrease,and has the threshold of 30 ?g/mL that caused a steep decline when crossing.Noticeable cytotoxicity was observed as the dose exceeded this threshold,owing to the upregulated RANKL expression and downregulated OPG expression.Meanwhile,activated macrophage morphosis was noticed as a result of the intense inflammatory response caused by high doses(>30 ?g/mL),characterized by the increased release of TNF-a and IL-6.(3)The cytotoxicity of DLC wear debris with different structures had been evaluated on index of cell viability,morphology,ELISA(enzyme-linked immuno sorbent assay)kits and quantitative real-time PCR,through four representative types of cells in vitro,including Raw 264.7 macrophages,primary mouse osteoblast,national institute of health 3T3 mouse fibroblast(NIH-3T3)and primary mouse mesenchymal stem cell,.Related clinicopathologic analysis of rat joint after induced by DLC wear debris had been executed in vivo.In general,DLC wear debris with higher ratio of sp2/sp3(tend to graphite phase)exhibited higher cytotoxicity,presenting significant inhibition of osteoblastogenesis.Due to the similarity to graphite structure,inflammatory response was increased by upregulated TNF-?,IL-6,MMP-2 and MMP-9 expression and downregulated IL-10 and TIMP-1 expression,while osteoblastogenesis was decreased owing to upregulated RANKL expression and downregulated OPG expression.For lack of electrostatic repulsion,the structure with higher ratio of sp2/sp3 enhanced BSA adsorption and promoted cell activities.(4)The cytotoxicity of DLC wear debris in different size ranges(0-0.22 ?m,0.22-0.65 ?m,0.65-1.0 ?m,1.0-5.0 ?m)had been evaluated.Microstructure and physical characteristics of DLC wear debris were investigated by Raman spectroscopy,transmission electron microscopy(TEM),scanning electron microscope(SEM)and dynamic light scattering(DLS).Furthermore,the macrophages,osteoblasts and fibroblast of mouse were incubated with DLC wear debris in different dimensions respectively,followed by cytotoxicity evaluations on inflammatory cytokines,alkaline phosphatase(ALP)assays and related signal protein expressions.The results showed that when comparing with the DLC wear debris samples of 0.22-0.65 ?m and 0.65-1.0 ?m,DLC wear debris samples of 0-0.22?m and 1.0-5.0 ?m showed a lower cytotoxicity.As BSA has a negative potential in physiological conditions,DLC wear debris in dimensions of 0-0.22 ?m and 1.0-5.0 ?m would attract BSA through electrostatic adsorption.Generally,BSA could enhanced the surface affinity to cells,so it also could improve the biocompatibility of particles.As a results,the DLC wear debris in dimensions of 0-0.22 ?m and 1.0-5.0 ?m exhibited less cytotoxicity benefited from the BSA-encapsulated effect.On the other hand,DLC wear debris in dimensions of 0.22-0.65?m and 0.65-1.0 ?m has a negative potential,it would reject the BSA through electrostatic repulsion and exhibited higher cytotoxicity.Therefore,DLC wear debris samples in dimensions of 0-0.22 ?m and 1.0-5.0 ?m showed a lower cytotoxicity than the samples in dimensions of 0.22-0.65 ?m and 0.65-1.0 ?m.