電化學沉積膠原蛋白/氫氧基磷灰石及萬古黴素/幾丁聚醣複材於Ti6Al4V之研究

Abstract

Previously in our laboratory, electrolytic hydroxyapatite (HA)/TiO2 composite coatings have been successfully developed to improve the bioactivity and the bonding strength between Ti alloy and bone tissue. In this study, the top layer composed of collagen/HA composite was further deposited on post bio-ceramics (HA/TiO2) coated Ti alloy in order to improve bioactivity, such as the initial cell proliferation and the finally osteo-integration. Through XRD, FTIR, SEM/EDS, ICP, electrochemical polarization tests, immersion tests, and cell culture including MTT and ALP assays, it was found that only the collagen could not be solely deposited by electrochemical method, but it has been done with the co-deposited HA, unstable octa-calcium phosphate (OCP) was not found since the previous bio-ceramic coating preferred HA to OCP. The further deposition of composite revealed the enhancing effects on bioactivity, proliferation, differentiation, and mineralization since the collagen contained in the composite not only retarded the ion release from the substrate, but also provided amino group to attract biological protein for the former two, and the stable HA offered the excellent environment for the latter two. In order to reduce or prevent the probability of osteomylitis, a novel method of vancomycin/chitosan composite coating on the Ti alloy implant has been carried out in the vancomycin/chitosan mixed aqueous solution by the electrochemical deposition. Though the deposition of vancomycin could not be carried out independently, it has been codeposited with chitosan by the hydrogen bonds between them. The coatings resulting from the deprotonation of chitosan by electrochemical reactions, were characterized by x-ray diffraction (XRD) for crystal phase, transmission electron microscopy (TEM) for microstructure, field emission scanning electron microscopy (FESEM) for the morphological observation, Fourier transform infrared (FTIR) spectroscopy for the specified chemical bonds, and ultraviolet/visible (UV/Vis) spectroscopy for the loading and releasing of vancomycin. It was found that the as-received chitosan powder was form Ⅱ, the as-deposited film was the mixture of form Ⅰ and form Ⅱ, and form Ⅰ became the major after immersed phosphate buffer solution (PBS). The chitosan film was composed of 2.0 nm diameter fibers which aligned in the direction of fiber axis, and further reduced to 1.6 nm by adding vancomycin due to the lowered pH. The vancomycin loading in the composite coating could be tuned from 150 to 550 μg/cm2 by the deposition time and potential. From antibacterial assay, no bacteria colonies were found in tubes containing the vancomycin/chitosan composite coating. In other words, the vancomycin was not denaturalized during the electrochemical deposition. However, the post porous hydroxyapatite coated Ti4Al4V was prepared for the subsequent electrolytic deposition of vancomycin-chitosan composite to control the drug release. As expected, the initial burst was reduced to 55 %, followed by a steady release about 20 % from day 1 to day 5 and a slower release of the retained 25 % after day 6, resulting in bacterial inhibition zone diameter 30 mm which could last for more than a month in antibacterial tests, compared with the coated specimen without hydroxyapatite gradually loosing inhibition zone after 21 days. Besides, the cell culture indicated that the vancomycin-chitosan/HA composite coated has enhanced the proliferation, the differentiation and the mineralization of the osteoblast-like cell. In general, it was helpful for the osteointegration on permanent implants. Consistently, it effectively provided the prophylaxis and therapy of osteomyelitis according to the results of the rabbit infection animal model.