Osseintegrasyonu Arttıracak Hidroksiapatit/Siklodekstrin Kompozit Taşıyıcıların Geliştirilmesi

Abstract

ABSTRACT

DEVELOPMENT OF HYDROXYAPATITE/CYCLODEXTRIN COMPOSITE CARRIERS TO ENHANCE OSSEOINTEGRATION

Meryem Sümeyye AKDEMİR Master of Science, Bioengineering Division Supervisor: Prof. Dr. Menemşe GÜMÜŞDERELİOĞLU September 2015, 150 pages

This thesis was prepared with support of FHD-2015-7226 Hacettepe University funding project and TÜBİTAK 2210-C Primary Subject National Scholarship Program for MSc Students. The aim of this thesis was to prepare hydroxyapatite (HA)/hydroxypropyl-βcyclodextrin (HPβCD) composite carriers that will allow the formation of HPβCD/Melatonin inclusion complex to improve osseointegration of titanium (Ti) which is commonly used in hard tissue implants, and to investigate the effects of it on osseointegration in vitro. In the first stage of this study, formation of a titanium oxide (TiO2) layer on the surface was aimed to improve the speed of HA formation and to enhance the stability of the coating. In this context, Ti of 99% purity was used and the surface area of Ti was increased by grinding with silicone carbide (SiC) sandpapers of different grit sizes. After creating a micro roughness on Ti surface by grinding, alkaline and anodic oxidation treatments were applied to achieve the desired oxide layer. Following alkaline oxidation, the samples were heat treated at 600oC to improve the biocompatibility and stability of the oxide layer. In anodic oxidation method, an oxide layer with nano-sized roughness was generated on the titanium plates. Optimum anodic oxidation conditions were determined to obtain a TiO2 layer with the most suitable surface morphology. By applying the optimum parameters in anodic oxidation (20 V, 1 cm distance between anode and cathode, 45 min processing time) TiO2 layers consisting of hills of nanotubules, which are approximately 75 nm in diameter, were formed. According to atomic force microscopy (AFM) results, surface roughness (Ra) was increased by 150% and 400% after alkaline and anodic oxidation treatments, respectively. In addition, contact angle measurements showed that both oxidation methods increased the wettability of the samples. Energy dispersive X-ray (EDX) analysis demonstrated that surface coating was consisting of 100% TiO2. iv

In the second stage of the thesis, the Ti plates that were modified with mechanical, chemical and electrochemical processes were coated with HA in room conditions via biomimetic methods using 10xSynthetic Body Fluid (SBF). While the chemical structure of the HA coating was analysed with Fourier Transform Infrared Spectroscopy (FTIR) and EDX analyses, its thickness and morphological properties were visualized with Scanning Electron Microscopy (SEM). The binding stability of HA coating on modified Ti surfaces was determined via micro scratching analysis. It was found that HA coating exhibited better physical stability when Ti was modified with alkaline oxidation method. In the third stage of the thesis, water-soluble or insoluble (crosslinked) HPβCD based carrier surfaces that would enable controlled release of melatonin were formed on HA layer. Water insoluble HPβCD carrier was obtained via crosslinking with 1,2,3,4butanetetracarboxylic acid (BTCA). While the properties of this structure were obtained via FTIR, SEM and thermogravimetric (TGA) analysis. To obtain stable HPβCD layers on HA coated Ti plates, 2 different pH values, pH 1 and pH 4, were adjusted during crosslinking process, and it was seen that pH 4 resulted to the formation of better stability. To prepare water-soluble HPβCD/Melatonin complexes, microwave irradiation method, which was developed and optimized by our research group, was used. In the next stage of the study, HPβCD was complexed with melatonin that has effects on bone metabolism, depending on inclusion complex forming property of HPβCD. FTIR analysis was conducted to determine the formation of HPβCD/Melatonin inclusion complexes and it was found that the melatonin and HPβCD, which were solved in an ethanol/water solution (pH 1 and 4), had formed a complex. Two different loading methods were used while forming the HPβCD/Melatonin complexes, which were pipetting and immersing. In vitro release studies were performed to determine the melatonin release kinetics of the HA/HPβCD composite carrier, and it was found that cross-linked HPβCD membranes had extended release profiles, while water-soluble membranes had burst release profiles. Osseointegration potential of Ti/HA/HPβCD/Melatonin composite systems was determined with in vitro culture studies conducted with preosteoblastic MC3T3-El cell line. Cell viability analysis had shown that HA inclusion had supported cell adhesion. Furthermore, it was determined that cross-linked HPβCD membranes had not supported cell adhesion, and this had shown that they are suitable surfaces that support controlled release of biosignals when cell adhesion is not desired. It was also found in the studies conducted with water-soluble complexes that the existence of HPβCD hinders cell adhesion and suppresses cell proliferation. While HPβCD has many pharmaceutical applications, cell surface interactions of crosslinked HPβCD have not been investigated in the related literature. For this reason, the findings of this study carry importance to seek new applications of HPβCD systems