Surface functionalization of titanium for biomedical applications

SURFACE FUNCTIONALIZATION OF TITANIUM FOR BIOMEDICAL APPLICATIONS Zhang Fan NATIONAL UNIVERSITY OF SINGAPORE 2009 SURFACE FUNCTIONALIZATION OF TITANIUM FOR BIOMEDICAL APPLICATIONS ZHANG FAN (B.Eng.(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS My time at NUS has been an incredible experience. For that I owe thanks to a great many people for their guidance, advice, support, and friendship. First of all I would like to express my deepest thanks and appreciation to my supervisors: Professor Kang En Tang and Professor Neoh Koon Gee. They believed in me during my most difficult time with their great patience, continuous encouragement and invaluable advice, and offered me this amazing and enlightening opportunity to work and learn in their labs. Their vision and enthusiasm have been most inspiring to me. There are many Kang and Neoh Lab members who have been a source of great support along the way and I thank them all because it has been a great time working with them. Special thanks go to Dr. Xu Fujian, Dr. Shi Zhilong and Dr. Fu Guodong for their help from their great experience and skills. I am also grateful to Mr. Chua Poh Hui, Dr Lim Siew Lay and Dr. Wuang Shy Chyi for their useful discussions on my research work. In addition, thanks are also due to all technical staffs of Department of Chemical and Biomolecular Engineering, especially Dr. Yuan Zeliang and Ms Samantha, for their assistance in the project. Last, but certainly not least, I cannot express enough thanks and appreciation to my parents. Their consistent support and unconditional love has truly enabled me to get through this entire journey. TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS SUMMARY . NOMENCLATURE LIST OF FIGURES LIST OF TABLES CHAPTER PROJECT SCOPE 14 . CHAPTER LITERATURE SURVEY 15 . 2.1 Properties of Titanium and its Alloys 20 21 2.1.1 General Physical Properties 21 2.1.2 Reactivity and Surface Properties 22 2.1.3 Corrosion Properties 26 2.1.4 Mechanical Properties 28 2.1.5 Biocompatibility 32 2.2 Applications of Titanium and its Alloys 35 2.2.1 Hard Tissue Replacements 35 2.2.2 Cardiovascular Applications 40 2.2.3 Other applications 42 2.3 Surface Modification of Titanium 45 2.3.1 Mechanical Methods 46 2.3.2 Chemical Methods 46 2.3.3 Biochemical Modification 51 CHAPTER MODIFICATION OF TITANIUM VIA SURFACE-INITIATED ATOM TRANSFER RADICAL POLYMERIZATION 3.1 Introduction 55 56 3.2 Experimental Section 59 3.3 Results and discussion 64 3.4 Conclusions 78 CHAPTER FUNCTIONALIZATION OF TITANIUM SURFACES FROM ANTIBACTERIAL SURFACE TO SURFACE FOR OSTEOBLAST ADHESION 79 4.1 Introduction 80 4.2 Experimental Section 82 4.3 Results and Discussion 94 4.4 Conclusions 118 CHAPTER BACTERIAL ADHESION AND OSTEOBLAST FUNCTIONS ON HEPARIN-FUNCTIONALIZED TITANIUM SURFACES 119 5.1 Introduction 120 5.2 Materials and Methods 121 5.3 Results and Discussion 128 5.4 Conclusions 146 CHAPTER SILK-FUNCTIONALIZED TITANIUM SURFACES FOR ENHANCING OSTEOBLAST FUNCTIONS AND REDUCING BACTERIAL ADHESION 147 6.1 Introduction 148 6.2 Materials and Methods 150 6.3 Results and Discussion 155 6.4 Conclusions 177 CHAPTER CONCLUSIONS . 178 CHAPTER RECOMMENDATIONS FOR FUTURE RESEARCH 183 REFERENCES 188 SUMMARY . Titanium and its alloys have been widely used in biomedical devices and implants. Surface modifications of titanium and its alloys are usually employed to further enhance their biocompatibility and biological functions, while retaining their intrinsic bulk properties. In this work, titanium surfaces were modified via surface-initiated atom transfer radical polymerization (ATRP) and bioconjugation to tailor their functionalities. Further functionalization of the grafted surfaces via biomolecular immobilization or post derivatization was carried out and the biological performance of the resulting substrates was assayed. Brushes of poly(poly(ethylene poly((2-dimethylamino)ethyl glycol)methacrylate) methacrylate) or or P(PEGMA), P(DMAEMA), and poly(2,3,4,5,6-pentafluorostyrene) or P(PFS), as well as their block copolymers, were tethered on the silane-coupled titanium surfaces via ATRP. Diblock copolymer brushes consisting of PEGMA and DMAEMA blocks were obtained by using the initial homopolymer brushes as the macroinitiators for the ATRP of the second monomer. The compositions of functionalized surfaces were analyzed by X-ray photoelectron spectroscopy (XPS). The wettability of the titanium surfaces could be modified by surface initiated ATRP of different monomers. The functional polymer-metal hybrids were found to be stable to hydrolysis. Both antibacterial effects and enhancement in mammalian cell adhesion were achieved separately on different titanium surfaces via controlled surface graft polymerizations and post functionalization. Surface-initiated ATRP of 2-hydroxyethyl methacrylate (HEMA) was carried out on titanium surface. The pendant hydroxyl end groups of the grafted HEMA chains were then converted into carboxyl or amine groups to allow the coupling of gentamicin, penicillin, or collagen via the carbodiimide chemistry. The covalently immobilized antibiotics retain the antibacterial properties, as indicated by a significant reduction in the viability of contacting Staphylococcus aureus. The collagen-immobilized surfaces, on the other hand, promote fibroblast and osteoblast adhesion and proliferation. Thus, the present surface-initiated living radical graft polymerization technique allows the tailoring of Ti surface with vastly different functions and is potentially useful to the design or improvement of Ti-based biomedical implants. In an attempt to prepare the desirable implants which can simultaneously inhibit bacterial adhesion and promote osteoblast functions, titanium was functionalized with a biomimic anchor molecule, dopamine. The dopamine-modifed titanium surfaces conjugate with heparin via the carbodiimide chemistry. The covalently immobilized heparin significantly reduces the adhesion of the two bacteria strains (Staphylococcus aureus and Staphylococcus epidermidis) tested. At the same time, osteoblast cells adhesion, proliferation, and alkaline phosphatase activity can be enhanced, depending on the dopamine and heparin concentration. Thus, the technique of using dopamine together with heparin to functionalize Ti surfaces is a potentially useful mean to combat biomaterial-centered infection and enhance osseointegration. The possility of preparing ideal biomedical implants, which can simultaneously inhibit bacterial adhesion and promote osteoblast functions, have also been explored with the silk-functionalized titanium. Titanium surfaces were modified with poly(methacrylic acid) (P(MAA)) followed by immobilization of silk sericin. With the coupling of ATRP initiator, titanium was modified via surface-initiated ATRP of methacrylic acid sodium salt (MAAS). The pendant carboxyl end groups of the grafted and partially protonated MAA chains were subsequently coupled with silk sericin via the carbodiimide chemistry. The covalently immobilized MAA brushes significantly reduce the adhesion of the two bacteria strains tested. The silk sericin immobilized surfaces, at the same time, promote osteoblast cells adhesion, proliferation, and alkaline phosphatase activity. Thus, the P(MAA) and silk sericin functionalized Ti surfaces have potential applications for combating biomaterial-centered infection and promoting osseointegration. NOMENCLATURE AA AAm AFM acrylic acid acrylamide atomic force microscopy ALP ANOVA APS Ar alkaline phosphatase one-way analysis of variance (3-Aminopropyl)trimethoxysilane Argon ATCC ATRP American Type Culture Collection atom transfer radical polymerization B. mori BE Bombyx mori binding energy BMP Bpy bone morphogenetic protein 2,2'-Bipyridine –COOH cp CuCl CuCl2 carboxyl group commercially pure Copper(I) Chloride Copper(II) Chloride CVD DMAEMA DMAP DMEM DMF ECM EDAC EDTA eV FTIR chemical vapor deposition (2-dimethylamino)ethyl methacrylate 4-(dimethylamino)pyridine; Dulbecco's modified Eagle’s medium dimethyl formamide extracellular matrix 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ethylenediaminetetraacetic acid electronvolt, a unit of energy Fourier transform infrared FWHM -g- full width at half-maximum graft GE GMA h HA HEMA HF HRB HRC gentamicin sulfate glycidyl methacrylate hour hydroxyapatite 2-hydroxyethyl methacrylate hydrofluoric acid hardness Rockwell B scale hardness Rockwell C scale IR MAA MAAS MMA MW NaSS –NH2 NHS OCPC –OH P(ABCD) PBS PE PEG PEGMA infrared methacrylic acid methacrylic acid sodium salt minute methyl methacrylate molecular weight sodium styrene sulfonate amine group N-hydroxysuccinimde; ortho-cresolphthalein complexone hydroxyl group polymer of ABCD phosphate buffered saline penicillin G sodium salt poly(ethylene glycol) poly(ethylene glycol) monomethacrylate PFS PI PLL PMDETA PNPP PVD RGD RPM S. aureus S. epidermidis SA SAM 2,3,4,5,6-pentafluorostyrene propidium iodide poly(L-lysine) N,N,N',N'',N''-pentamethyldiethylenetriamine p-nitrophenylphosphate physical vapor deposition Arg-Gly-Asp peptides revolutions per minute Staphylococcus aureus Staphylococcus epidermidis succinic anhydride self-assembled monolayer SBF SEM simulated body fluid scanning electron microscopy TEA Chlorosilane THF Ti UTS UV triethylamine trichloro(4-(chloromethyl)-phenyl)silane tetrahydrofuran titanium ultimate strength ultraviolet v VBC volume 4-vinylbenzyl chloride XPS YS X-ray photoelectron spectroscopy yield strength References to Orthopedic Devices and Issues of Antibiotic Resistance, Biomaterials, 27, pp. 2331-2339. 2006. 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Biotechnol., 81, pp. 136-145. 2006. 205 [...]... serve as a good coupling agent for the immobilization of ATRP initiator on titanium substrates This PhD project aims to modify titanium surfaces with a series of biologically active molecules to improve the performance of titanium substrates for biomedical applications In Chapter 2, the properties of titanium and its applications in biomedical field are summarized Various surface modification techniques... illustrating the two routes for the immobilization of collagen on the Ti-g-P(HEMA) surface 90 Figure 4-5 XPS wide scan spectra of (a) the pristine or Ti-OH surface, and (b) Ti-Cl surface 95 Figure 4-6 XPS wide scan spectra of (a) the Ti-g-P(HEMA) surface from 1 h of surface- initiated ATRP of HEMA, (b) the Ti-g-P(HEMA-NH2) surface, and (c) the Ti-g-P(HEMA-PE) surface 97 Figure 4-7... Ti-Cl surface, and immobilization of silk sericin on the Ti-polymer hybrid 151 Figure 6-2 XPS wide scan and C 1s core-level spectra of (a, b) the pristine or Ti-OH surface, (c, d) the Ti-Cl surface, (e, f) the Ti-g-P(MAA) surface after 3 h of surface- initiated ATRP of MAAS and (g, h) the corresponding Ti-g-P(MAA-Silk) surface 156 Figure 6-3 Comparison of osteoblast attachment on surfaces of. .. silanization of the Ti-OH surface to give rise to the Ti-Cl surface, surface- initiated ATRP of PEGMA, DMAEMA, PFS, and PEGMA/DMAEMA block copolymer brushes from the Ti-Cl surface 58 Figure 3-2 Wide scan spectra of the (a) Ti-OH surface and (b) Ti-Cl surface, and Si 2p and Cl 2p core-level spectra of the (c,d) Ti-Cl surface 66 Figure 3-3 XPS core-level and wide scan spectra of (a,b) the Ti-g-P(PEGMA) surface. .. spectra of (a) the Ti-g-P(PEGMA) surface, (b) the Ti-g-P(DMAEMA) surface, (c) the Ti-g-P(PFS) surface, prepared under the ATRP conditions as described in Table 3-1 Test conditions: in water:THF (1:1, v:v) solution of pH =2 at 37 °C for 3 weeks 77 Figure 4-1 Schematic diagram illustrating the processes of silanization of the Ti-OH surface to give rise to the Ti-Cl surface, surface- initiated 9 ATRP of HEMA... 1s core-level spectra of the Ti-g-P(HEMA) surface from 1 h of surface- initiated ATRP of HEMA, and the corresponding Ti-g-P(HEMA-COOH) surface, and (c,d) C 1s and N 1s core-level spectra of the Ti-g-P(HEMA-SA-GE) surface 98 Figure 4-8 (a, b) C 1s and N 1s core-level spectra of the Ti-g-P(DMAEMA) surface, and (c, d) Br 3d and N 1s core-level spectra of the Ti-g-P(DMAEMA-Q) surface 99 Figure... surface obtained at the ATRP time of 3 h, (c,d) the Ti-g-P(DMAEMA) surface obtained at the ATRP time of 5 h, and (e,f) the Ti-g-P(PFS) surface obtained at the ATRP time of 3 h 72 Figure 3-4 Wide scan and C 1s core-level spectra of (a,b) Ti-g-P(PEGMA)-bP(DMAEMA) surface and (c,d) the Ti-g-P(DMAEMA)-b-P(PEGMA) surface (The ATRP conditions for the preparation of surface- grafted block copolymers... which can adversely affects biological function of surrounding tissues and can lead to mechanical failure of the device It is also this corrosion resistance in saline environments that forms the basis for the use of titanium in biomedical application Fortunately the corrosion resistance of the pure titanium is largely carried with it into the alloys as well Titanium- based alloys, such as Ti-6Al-4V has... properties of titanium and some of its alloys 29 Table 2-3 Typical hardness of titanium and some of its alloys 31 Table 3-1 Surface composition bonding ratio and static water contact angle of the polymer-functionalized titanium surfaces 67 14 CHAPTER 1 PROJECT SCOPE 15 Chapter 1 Over the last two decades, titanium and its alloys have been used extensively in biomedical devices and components... (d)Ti-g-P(HEMA-SA-Col) surfaces after 2 days of 3T3 fibroblast cell culturing at an initial seeding concentration of 104 cells/mL 112 10 Figure 4-14 SEM images of (a, c) Ti-g-P(HEMA-SA-Col) surfaces and (b, d) pristine Ti surfaces after 4 days of 3T3 osteoblast cell culturing at initial seeding concentrations of 2 × 104 cells/mL (for a and b) and 5 × 103 cells/mL (for c and d) 114 . SURFACE FUNCTIONALIZATION OF TITANIUM FOR BIOMEDICAL APPLICATIONS Zhang Fan NATIONAL UNIVERSITY OF SINGAPORE 2009 SURFACE FUNCTIONALIZATION. on titanium substrates. This PhD project aims to modify titanium surfaces with a series of biologically active molecules to improve the performance of titanium substrates for biomedical applications. . FUNCTIONALIZATION OF TITANIUM FOR BIOMEDICAL APPLICATIONS ZHANG FAN (B.Eng.(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF