| Background and Objective:With the rapid development of economy in our society, Great changes have taken place in every field. In the field of dental prosthesis, the repairing methods of dentition defect and teeth missing, also obtained the effective development. The dental implant have many advantages such as favorable stability, comfortable, does not damage the healthy tissue in the mouth compared with the traditional fixed and removable dentures. So far, titanium as implantable biomaterial has been widely used because it has good biocompatibility, corrosion resistance and mechanical properties.But titanium still has some disadvantages, such as biological inert, does not have bone induction ability; would release some metal ions to the surrounding fluid, etc. It is a hot spots to modificate its biological activity in current implant materials. The method to biologically modificate the implant material is mainly to add the bioactive coating surface, to improve its elasticity modulus, surface wettability and surface energy. Nanocrystalline diamond film receives much concern in recent years, as its have good abrasion resistance, biocompatibility, importantly it can reach high bonding strength with all kinds of substrates, and has high surface activity due to its size effect of nanometer particles.In the study, we investigated the surface characteristics and its biocompatibility of pure titanium whose surface was modified with nanocrystalline diamond( NCD) by Microwave Plasma Chemical Vapor Deposition(MPCVD). Scanning electron microscope(SEM), Spectruman analysis ray diffraction and X-Ray photoelectron spectroscopy(XPS) were used to analyze the chemical composition and surface topography of NCD film.Also we used Human Osteoblast-like cells MG63 to investigate the bioactivity of NCD film.The attachment, proliferation and differentiation of the cells were tested by NAPI, CCK-8 and ALP.Method:1. The preparation of NCD filmThe NCD film was prepared on pure titanium by MPCVD. In the process, the dangling bonds of the surface of NCD were hydrogen terminated(NCD-h). By thermal processing at 400 ℃for 4h with 21 % oxygen, the NCD-h turned oxygen terminated(NCD-o), whose dangling bonds were carbonyls, carboxyl and aether ect.2. The physical and chemical properties of NCD film(1) Observe the surface morphology of the material by SEM(2) The chemical constitution of the surface of material by XPS(3) The evaluation of particle size of material by XRD(4) The wettability of material by contact angle mater3. The biological performance of NCD(1) The cytotoxicity of material(2) The attachment of the cells by DAPI(3) The attachment morphology of the cells by SEM(4) Cells proliferation assays(5) Alkaline phosphatase(ALP)Result:1. The physical and chemical properties of NCD film(1) SEM and spectroscopyAfter 30 min of deposition, nucleation of diamond particles was observed, and the film which was formed by diamond particles nucleation was uniformly coated on the substrate, the average diameter of diamond was determined to be about 20-250 nm. When the deposition time was 10 min, particle size of the diamond was uniform, but there were gaps between the cores, and the integrity of the film was imperfect. However, at longer times, there was a trend that the diamond grain size was increasing; although diamond particle size could be uniformly dense coverage on the substrate surface, the particle size had been a dozen oreven several microns(E, F). Spectroscopy measurements for NCD-h revealed that, chemical elements of the surface of the diamond film synthesized was almost all carbon, and the(Atom percent)AT% was 95.27%, containing only a small amount of surface oxygen.(2) XPSCarbon and Oxygen 1s(C1s and O1s) signals were respectively showed in NCD-o, and the peak of NCD-h C1 s was 285 ev, closer with the peak of diamond, and then we can guess that there is diamond or diamond-like in the samples.(3) XRDWe calculate the size of the diamond grain is about 13 nm by Scherrer formula, so the diamond film is nanometer level.(4) Contact angleThe result of NCD-h is hydrophobic, and the contact angle is 125°, while the NCD-o is very hydrophilic whose angle is 38°.2. The biological performance of NCD(1) The cytotoxicity of materialAfter cell seeding for 6 hours and 12 hours, the RGR of single cells groups, NCD-o groups and titanium discs groups had no significant difference(p>0.05). However after 24 h, 48 h and 72 h, the number of NCD-o was obviously larger than the rest of the groups(p<0.001),especially NCD-h groups. According tobiocompatibility evaluation criteria, the results prove that each groupwere not cytotoxic and has good biocompatibility.(2) The attachment of the cells by DAPIAfter seeding 6h, the number of cells attached on NCD-o groups and titanium discs groups had no distinctly difference, and was slightly larger than NCD-h groups. In the 24 hours group, there was a significant growth on each group, particularly the NCD-o groups and titanium discs groups, and the number of cell on the surface of NCD-o was obviously more than the NCD-h groups, suggesting NCD-o groups had a significant role in promoting the adhesion and proliferation of MG63 cells.(3) The attachment morphology of the cells by SEMThe cells on the titanium discs group were elongated very much and we adhered to only a matrix like a seat at high magnification. Whereas compared with the titanium discs groups, the filopodia of the cells extended well and diverged radically from the cell membrane in NCD-o and NCD-h groups. The NCD-o groups compared to NCD-h, the filopodia were more numerous and longer as well as interconnected with each other, all of which were strongly in favor of cell adhesion to the substrate.(5) Cells proliferation assaysThe result showed no significantly difference among the groups at day1. With the extension of the cell culture time, at 4 days, there was a significant increase in the number of cells. The cells proliferateddistinctly in NCD-o groups compared with NCD-h(P<0.05). After 7 days, the proliferation of NCD-o groups still was still better than NCD-h groups(p<0.001) and was statistically significant compared to the titanium discs groups(P<0.05).(6) ALPIn the third day, the ALPactivities of the cells in NCD-o is the more compared with the pure cells, titanium, and NCD-h. With time goes on, in day 7 and 14, the activities of ALP were enhanced in experimental group and control group, particularly in NCD-o group.Conclusion:1. The NCD films in 30 min group which is consistent, high purity, and without other impurity elements introduced is nanoscale size.2. After thermally treatment in the air, the NCD-h turned NCD-o which is strongly hydrophilic, and good for the attachment, proliferation and differentiation of cells. |
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