Processing Optimization And Properties Evaluation Of Tb-Doped Nano-Hydroxyapatite

Objective:This study explored the role of process parameters on the particle size of hydroxyapatite nanoparticles (HAnp) by hydrothermal synthesis method, investigated the effect of different Terbium (Tb) dropping amount on the properties of HAnp, and evaluated the biocompatibility and cellular endocytosis of Tb-HAnp, in order to lay a foundation for building a novel porous titanium implant by gene activation.Materials and methods:1)HAnp preparation process optimization: HAnp was prepared by hydrothermal synthesis method using Ca(NO3)2.4H2O and (NH4)2HPO4for precursor reactants under170℃3h. Regulating the size of HAnp by controlling the (NH4)2HPO4dropping speed. The products’structure and phase were analysised by X-ray diffraction (XRD), and the particle size morphology and dispersion were characterized using transmission electron microscopy (TEM).2)Preparation of fluorescent HAnp:Different Tb doping amount of HAnp was prepared by hydrothermal synthesis method under170℃3h controlling the dropping speed of (NH4)2HPO4for0.12ml/min. The products’structure and phase were analysised by X-ray diffraction (XRD) and the particle size, morphology and dispersion were characterized using transmission electron microscopy (TEM), the Zeta potential were detected by Zeta potential analyzer, the fluorescence properties of particle was observed by inverted fluorescence microscope.3)Cytotoxicity of Tb-HAnp:MTT assay method was used to evaluate the cytotoxicity of different Tb doped amount of HAnp concentration ranged from100μg/ml～2000μg/ml on L929.4) Tb-HAnp and MG63cell co culture:Different amont of Tb-HAnp co cultured with MG63cells for48h, using inverted microscope and fluorescence microscopet to observe the endocytosis effect.Results:1) HAnp was prepared by hydrothermal synthesis method. Regulating the size of HAnp by controlling the (NH4)2HPO4dropping speed, the0.12ml/min group of HAnp’s average particle size were controlled in30nm with good dispersion.2) New diffraction peaks appear in Tb-doped HAnp which corresponding structure is TbPO4.H2O, Indicates that the Tb3+has been mixed with HAnp. The images of TEM shows that size of HAnp rising with Tb addition amount increasing from0mol%to5mol%, and dropping with Tb addition amount further increasing from5mol%to10mol%, which suggests it is possibly that the dominant growth orientation of HAnp could be regulated by Tb. The results of Zeta potential indicates that in the range of Tb amount from0mol%to5mol%the potential absolute rising from-7.60mV to-18.3mV, while in the range of Tb amount from5mol%to10mol%the potential of HAnp absolute dropping from-18.3mV to-6.73mV, which corresponding to the variation trend of particle size. Fluorescence microscopy reveals that green fluorescents can be seen under ultraviolet excitation. The number of fluorescents increase with Tb addition rate rising.3) Inverted microscope shows that the cells are normal and strech well in both experimental groups and negative control group while the positive control group completely cracked. Irregular aggregates are visible in the experimental groups. Aggregates increase with Tb-HAnp concentration rising which have no significant effect on cells. Statistical analysis reveals that RGR of HAnp with three differents Tb addition amount range from100ug/ml to2000ug/ml show significant difference (P <0.05) compared with positive control group. Cell toxicity were within1grade.4) Inverted microscope shows that the cell are normal and strech well in both experimental group and negative control group. High density and irregular aggregates are visible in the cells of the experimental groups which increase with Tb-HAnp concentration rising. Fluorescence microscopy reveals that blank control group are completely scotopic vision while the experimental group shows green fluorescents. The position and morphology of the fluorescents are corresponding to the high density aggregates. Conclusions:1) The size of HAnp can be regulated by (NH4)2HPO4dropping speed. Spherical or rod-like HAnp which in average of30nm with good dispersion can be prepared under the (NH4)2HPO4dropping speed for0.12ml/min by hydrothermal synthesis method.2) HAnp obtain remarkable fluorescence by the means of Tb doping.10mol%Tb-HAnp show the best of the fluorescence.3) Tb-HAnp show no cytotoxicity. Tb-HAnp can enter MG63through endocytosis.