Research On Biological Characteristics Of Nanotubes Surface Of Pure Titanium And Nanomicelle

Background To make the implant functional load as soon as possible, shorten healing time as much as possible, get long-term resistance to oral bacterial infection, and maintain the functional stability of the implant, large number of studies attempt to further improve the surface properties of titanium implants in order to establish and maintain soft tissue-implant biologic seal and implant-bone osseointegration in good condition. Research on the nanometer-scale to improve implant property is a hot topic because of excellent property of nano-structure. The standard surface with same structures has not be made at the nanoscale level. The preparation process of Titania nanotube arrays by electrochemical anodic oxidation is relatively simple, and the tube diameter, tube length and morphology can be controlled. Thus help to further in-depth study of the interfacial interaction between nano-surface and peri-implant in the cellular and molecular levels. It has been demonstrated that Titania nanotube arrays are not only conducive to bone formation, but also conducive to activation through dopant fill. The interface action between implant and surrounding tissue before and after bioactive substances doped in Titania nanotubes is still to be studied in depth. Objection (1) To study on the preparation process and biological characteristics of Titania nanotubes, and study on the possible effect in promoting titanium osseointegration and titanium-fiber combination.(2) To study on the preparation process and biological characteristics of Simvastatin(SV)-loaded micelles, and study on the ideal dosage form of SV to improve the bone biological activity.(3) To study on nanotubes-micelles combined effect in the release of SV and in bone cytology, and provide a new idea and a new activation modified method for titanium implants developed with the biological activity.Methods and materials (1) Preparation of Large-diameter Titania nanotube arrays by anodic oxidation, and load bovine serum albumin (BSA) in the nanotubes and determination of load rate; Observation on the surface morphology and structure of the sample by environmental scanning electron microscope (SEM), analysis of the elements of the sample by surface X-ray photoelectron spectroscopy (XPS), measurement of the surface contact angle and calculation of the surface free energy (SFE), using surface profiler to observe the surface of the sample profile and measure the roughness; observation on human gingival fibroblasts (HGF) morphological changes on the specimen surface by environmental SEM; Using confocal laser scanning microscopy (CLSM) to observe the immunofluorescence staining of HGF and calculation of early adhesion rate; MTS assay for cell proliferation activity on the specimen surface; real-time quantitative PCR (QT-PCR) detection of type I collagen (COL-1) gene expression of HGF before and after BSA loaded in Titania nanotubes; enzyme-linked immunosorbent assay (ELISA) for COL-1of HGF in the extracellular matrix secretion.(2) Preparation of SV-loaded PECL micelles by dialysis method, using dynamic light scattering (DLS) for determination of particle size and distribution of the micelles, observation on the micelles morphology by atomic force microscopy (AFM), ultraviolet determination of drug-loaded micelle drug loading (DL) and the encapsulation rate (ER), in vitro release assay for release function of the SV-loaded micelles; Using CCK-8assay, ALP activity determination by colorimetric method, alizarin red staining, QT-PCR, and Western blot to detect differently cell proliferation, ALP activity, calcification deposition, BMP-2gene expression, and BMP-2protein expression activity of the human osteoblast-like MG63cells affected by SV-loaded micelles.(3) Loading micelles in Titania nanotubes, in vitro release assay for nanotubes-micelles combined effect in the release of SV; Observation on MG63cells morphological changes on the specimen surface by field emission SEM, Using CLSM to observe the immunofluorescence staining MG63cells on the specimen surface and calculation of early adhesion rate; Using MTS assay, ALP activity determined by colorimetric method and ELIS A to detect differently the changes of cell proliferation activity, ALP activity, osteocalcin content of MG63cells on the specimen surface before and after SV-loaded micelles doped in Titania nanotubes.(4) SPSS17.0software for data analysis, using One-Way ANOVA and ANOVA of analyzing of factorial designed data; Levene test for homogeneity of variance of separate effects; Using Bonferroni method for pairwise comparisons test in the case of homogeneity of variance, while using Tamhane’T2method for pairwise comparisons test in the case of unhomogeneity of variance; Test level (a) is0.05.Results (1) Titania nanotube arrays surface of pure titanium could be successfully prepared, with anatase crystal of main structure and80-100nm of tube diameter. BSA could be successfully loaded in the nanotubes, and the loading rates of200μg,400μg,600μg BSA in Titania nanotubes were up to99%. XPS analysis showed Titania nanotube surface was rich in-OH group. Among the three kinds of smooth surface, respectively being simply polishing titanium surface, Titania nanotubes surface, and BSA-loaded nanotubes surface, the hydrophilicity, the SFE and Ra were in turn increased, accordingly the early adhesion rate of HGF was gradually increased. The nanotubes surface could increase early adhesion rate and late proliferative activity, and vigorously increase the COL-1gene expression in HGF but also at some time point for extracellular COL-1secretion. The BSA-loaded nanotubes smooth surface was able to increase early adhesion rate, decrease late proliferative activity, lower early COL-1gene expression but increase its late expression, and reduce the secretion of extracellular COL-1at any time point.(2) In AFM images it could be seen that the SV-loaded PECL micelles were spherical, and sizes were not very uniform, about80nm of diameter, which were consistent with DLS. SV-loaded PECL micelles used in In vitro release assay and cytological experiments, made by the content ratio of PECL:SV in100:10, with ER78.94±11.51%and DR7.89±1.15%. The release profile of SV-loaded PECL micelles extended than the same content of SV. SV-loaded micelles at2.5×10"7M could reduce early proliferative activity of MG63cells less than SV at the same concentration, and increase the calcification area, and also show the largest early calcified nodules. SV-loaded micelles at2.5×10-7-2.5×10-10M could increase the ALP activity more than SV at the same concentration, and increase BMP2gene protein expression.(3) The profile of nanotubes-micelles joint release of SV was slightly extended than the same SV content of drug-loaded micelles, and the highest accumulative release rate was the same. The specimen surface could increase early adhesion rate of MG63cells, ALP activity before and after SV-loaded micelles doped in Titania nanotubes, and the promotion would be more powerful on the micelles-nanotubes surface. It was not a significant impact on proliferative activity that SV-loaded micelles loaded in nanotubes or not. Titania nanotubes surface could increase OC content inside of MG63cells but lower OC content outside, micelles-nanotubes surface could increase OC content both inside and outside of MG63cells.Conclusion (1) Titania nanotubes smooth surface could promote the early adhesion of HGF, and was expected to form a closed loop of dense fibrous connective tissue; So the smooth surface after nanotubes modified could become one of the choice for implant collar surface; Loading the serum protein affected the formation of fibrous connective tissue closed loop or not, it was needed for further study.(2) SV-loaded PECL micelles could have a slow release profile of SV, and show better biological property than SV; So PECL micelles were expected to become a drug delivery system in order to improve the bioavailability of SV (3) Titania nanotubes could contribute to contact osteogenesis on implant surface, thus contributing to osseointegration before and after SV-loaded PECL micelles doped, and the osteogenesis performance of micelles-nanotubes surface would be more powerful.(4) Nanotubes could be a good carrier for the micelles with sustained release effect, and the combination of the two could better reflect the sustained release of drug effects, so that the bone bioactive substances loaded could be slowly released to focus on the implant-bone interface. Thus provided a new viable idea for the formation of the bioactive implant surface.