In vitro study of surface functionalization of titanium substrates for potential enhancement of osseointegration and reduction of bacterial infection

IN VITRO STUDY OF SURFACE FUNCTIONALIZATION OF TITANIUM SUBSTRATES FOR POTENTIAL ENHANCEMENT OF OSSEOINTEGRATION AND REDUCTION OF BACTERIAL INFECTION HU XUEFENG NATIONAL UNIVERSITY OF SINGAPORE 2013 IN VITRO STUDY OF SURFACE FUNCTIONALIZATION OF TITANIUM SUBSTRATES FOR POTENTIAL ENHANCEMENT OF OSSEOINTEGRATION AND REDUCTION OF BACTERIAL INFECTION HU XUEFENG (B.Eng., M.Sci., BUCT) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Hu Xuefeng 11 June 2014  ACKNOWLEGEMENT Firstly, I would like to express my sincere gratitude to my supervisor, Prof. Neoh Koon Gee, for her thorough guidance and continuous support throughout this work. Her critical way of thinking and enthusiastic attitude towards work has been of great value for me. This thesis would not have been completed without her invaluable suggestions and profound discussion. I owe my deep gratitude to my co-supervisor, Assoc. Prof. Wilson Wang, for his constructive comments and important support throughout this project. I am also grateful to Prof. Kang En-Tang for his permission to access the equipment in his lab. I would like to thank Dr. Yuan Ze Liang for his help in XPS and SEM training and operation. I appreciate all my colleagues, Dr. Shi Zhilong, Dr. Wang Liang, Rusdianto Budiraharjo, Tan Lihan, Yang Wenjing, Huang Chao, Wang Rong, Lu Shengjie, Zheng Dong, Li Min and Xu Liqun, for their warm encouragement and kind help. I am also grateful to the lab officers Ms. Li Fengmei, Ms. Li Xiang, and Dr. Yang Liming for their assistance in my study. Last but not least, I would like to thank my deeply beloved wife, Zhang Jieyu, for her understanding and support. I would also like to show my gratitude to my family for their unconditional support and love. i  TABLE OF CONTENTS ACKNOWLEGEMENT TABLE OF CONTENTS i ii SUMMARY vi LIST OF TABLES viii LIST OF FIGURES ix NOMENCLATURE xvi CHAPTER INTRODUCTION 1.1 Background 1.2 Research objective and scope CHAPTER LITERATURE REVIEW 2.1 Introduction 2.2 Bone healing processes at bone-implant interface 2.2.1 Human bodys initial responses to an implant 2.2.2 Woven bone formation 10 2.2.3 Bone remodeling 11 2.3 Ti and its alloys as implant materials 12 2.3.1 Requirements for implant materials 12 2.3.2 Ti and its alloys 13 2.4 Surface modification of Ti to enhance osseointegration 14 2.4.1 Enhancement of osseointegration by surface topography 15 2.4.2 Enhancement of osseointegration by surface chemistry 16 2.5 Surface modification of Ti to reduce infections 22 2.5.1 Surface topographical modification 23 2.5.2 Surface modification with bactericidal agents 24 2.5.3 Surface modification with anti-adhesive agents 27 CHAPTER BACTERIAL AND OSTEOBLAST BEHAVIOR ON Ti, Co-Cr AND SS TREATED WITH ALKALI AND HEAT: A COMPARATIVE STUDY FOR POTENTIAL ORTHOPEDIC APPLICATIONS 29 3.1 Introduction 30 3.2 Materials and methods 30 ii 3.3 3.2.1 Materials 30 3.2.2 Substrate preparation 31 3.2.3 Characterization 31 3.2.4 Measurement of surface ROS density 32 3.2.5 Bacterial culture and adhesion assay 32 3.2.6 Cell culture and cytotoxic assay 33 3.2.7 Statistical analysis 34 Results and discussion 34 3.3.1 Surface characterization of the pristine and treated Ti substrates 34 3.3.2 ROS generation on the pristine and treated Ti substrates 37 3.3.3 Bacterial adhesion on the pristine and treated Ti substrates 38 3.3.4 Mammalian cell behavior on the pristine and treated Ti 41 substrates 3.3.5 Comparison with alkali and heat-treated Co-Cr and SS 44 substrates 3.4 Conclusion 50 CHAPTER AN IN VITRO ASSESSMENT OF Ti FUNCTIONALIZED WITH POLYSACCHARIDES CONJUGATED WITH VEGF FOR ENHANCED OSSEOINTEGRATION AND INHIBITION OF BACTERIAL ADHESION 51 4.1 Introduction 51 4.2 Materials and methods 52 4.2.1 Materials 52 4.2.2 Synthesis of CMCS and HAC 53 4.2.3 Preparation of substrates 54 4.2.4 Characterization 54 4.2.5 Bacterial adhesion assay 55 4.2.6 Cell culture 55 4.2.7 Cell attachment and proliferation 55 4.2.8 ALP activity and calcium deposition (mineralization) assay 56 4.2.9 Statistical analysis 56 Results and discussion 57 4.3.1 Surface characterization 57 4.3.2 Antibacterial properties 59 4.3 iii 4.4 4.3.3 Cell attachment and proliferation 64 4.3.4 ALP activity and calcium deposition 66 4.3.5 Stability of immobilized VEGF 69 Conclusion 71 CHAPTER STRATEGY FOR IMMOBILIZING VEGF ON IMPLANT SURFACES TO OPTIMIZE ITS CONCURRENT BIOACTIVITY TOWARDS ENDOTHELIAL CELLS AND OSTEOBLASTS 72 5.1 Introduction 73 5.2 Materials and methods 73 5.2.1 Materials 73 5.2.2 Synthesis of HAC and HepC 74 5.2.3 Preparation of substrates 74 5.2.4 Characterization 74 5.2.5 Cell culture 75 5.2.6 Endothelial cell metabolic activity 75 5.2.7 CD31 and vWF expression 75 5.2.8 In vitro angiogenesis assay 76 5.2.9 Calcium deposition assay 77 5.2.10 Bacterial culture and adhesion assay 77 5.2.11 Statistical analysis 77 Results and discussion 78 5.3.1 HepC synthesis and substrate surface characterization 78 5.3.2 Bioactivity of the immobilized VEGF 82 5.3.3 Osteoblast mineralization 88 5.3.4 Antibacterial properties 93 Conclusion 97 5.3 5.4 CHAPTER AN IN VITRO ASSESSMENT OF FIBROBLAST AND OSTEOBLAST RESPONSE TO ALENDRONATE-MODIFIED Ti AND THE POTENTIAL FOR DECREASING FIBROUS ENCAPSULATION 98 6.1 Introduction 99 6.2 Materials and methods 99 6.2.1 Materials 99 6.2.2 Substrate preparation 99 6.2.3 Surface characterization and alendronate release test 100 iv 6.3 6.2.4 Cell culture 101 6.2.5 Cell attachment and proliferation 101 6.2.6 ALP activity assay 101 6.2.7 Apoptosis assay 101 6.2.8 Co-culture of fibroblasts and osteoblasts 102 6.2.9 Statistical analysis 102 Results and discussion 102 6.3.1 Surface characterization and alendronate release test 102 6.3.2 Fibroblast attachment, proliferation and apoptosis 106 6.3.3 Osteoblast attachment, proliferation, differentiation and 111 apoptosis 6.4 6.3.4 Co-culture of fibroblasts and osteoblasts 117 Conclusion 120 CHAPTER CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER STUDY 121 7.1 Conclusions 122 7.2 Recommendations for further study 124 REFERENCES 127 LIST OF PUBLICATION 158 v SUMMARY The main reasons for implant failure are defective osseointegration and bacterial infections. Surface modification is a promising strategy to overcome these problems since it can endow the implant surface with the desired functions while simultaneously retaining the implants intrinsic mechanical properties. Since titanium (Ti) and its alloys are the most commonly used biomaterials for implants, different strategies for Ti surface modification to enhance osseointegration and reduce bacterial infection have been investigated, and are reported in this thesis. Firstly, Ti was treated with alkali and heat to convert the amorphous titanium dioxide into anatase since anatase has been shown to exhibit antibacterial effect. The anatase-functionalized Ti significantly reduced bacterial adhesion due to reactive oxygen species (ROS) generated by the anatase. Unfortunately, the ROS exhibited cytotoxicity towards osteoblasts. Cobalt-chrome (Co-Cr) alloys and stainless steel (SS) treated in a similar fashion did not generate ROS, and exhibited no cytotoxicity towards osteoblasts. The treated Co-Cr and SS reduced bacterial adhesion due to their hydrophilic surfaces, which is a different mechanism from that of the alkali and heat-treated Ti. Thus, while this strategy for Ti surface modification may be useful for antibacterial applications, it is not deemed suitable for orthopedic applications. A second strategy was then developed, involving covalent immobilization of a growth factor on Ti via a pre-coated antibacterial polysaccharide layer. Vascular endothelial growth factor (VEGF) was chosen as the target growth factor with the aim of investigating its direct effect on osteoblasts. Antibacterial assay showed that the polysaccharide-modified substrates significantly decreased bacterial adhesion. Osteoblast behavior on the different substrates was also assessed, and the results showed that osteoblast functions were enhanced by the immobilized VEGF on the polysaccharide-grafted Ti. Since the bioactivity of covalently immobilized VEGF may be compromised due to adverse conformational changes and possible interference with the functional region in the immobilization process, the possibility of bioactivity changes upon vi immobilization was investigated. VEGF was immobilized on Ti surfaces via either covalent binding or heparin-VEGF interaction. The bioactivity of the covalently immobilized VEGF on endothelial cell functions was found to be significantly lower than that of the heparin-bound VEGF. The heparin-bound VEGF also enhanced mineralization in an osteoblast/endothelial cell co-culture to a much greater extent than in an osteoblast monoculture, illustrating the importance of crosstalk between osteoblasts and endothelial cells. In addition, the surfaces of the heparin-modified substrates are highly hydrophilic and negatively charged, which significantly inhibit bacterial adhesion. Lastly to address the issue of fibrous encapsulation which can impede osseointegration, alendronate, a drug that can induce fibroblast apoptosis, was loaded on Ti surfaces via a hydroxyapatite coating. With a surface density of loaded alendronate of 0.05 mg/cm2 or higher, fibroblast proliferation was suppressed due to increased apoptosis, while osteoblast functions increased with minimal apoptosis. In a co-culture of fibroblasts and osteoblasts in a 1:1 ratio, ~75% of the cells on these alendronate-loaded substrates were osteoblasts four days after cell seeding. These results suggest that the strategy of loading alendronate on Ti can potentially be capitalized to reduce fibrous encapsulation. vii References Liu, C. X., Zhang, D. R., He, Y., Zhao, X. S., & Bai, R. 2010. Modification of membrane surface for anti-biofouling performance: Effect of anti-adhesion and antibacteria approaches. J Membr Sci, 346(1): 121-130. Liu, S., Yu, J., & Jaroniec, M. 2011. 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Biomaterials, 25(14): 2695-2711. 157 Appendix LIST OF PUBLICATIONS Hu XF, Neoh KG, Shi ZL, Kang ET, Poh C, Wang W. An in vitro assessment of titanium functionalized with polysaccharides conjugated with vascular endothelial growth factor for enhanced osseointegration and inhibition of bacterial adhesion. Biomaterials, 2010, 31(34), 8854-63. Neoh KG, Hu XF, Zheng D, Kang ET. Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces. Biomaterials, 2012, 33(10), 2813-22. Hu XF, Neoh KG, Zhang JY, Kang ET, Wang W. Immobilization strategy for optimizing VEGF's concurrent bioactivity towards endothelial cells and osteoblasts on implant surfaces. Biomaterials, 2012, 33(32), 8082-93. Hu XF, Neoh KG, Shi ZL, Kang ET, Wang W. An in vitro assessment of fibroblast and osteoblast response to alendronate-modified titanium and the potential for decreasing fibrous encapsulation. Tissue Engineering Part A, 19(17-18), 1919-1930. Hu XF, Neoh KG, Zhang JY, Kang ET. Bacterial and osteoblast behavior on titanium, cobalt-chromium alloy and stainless steel treated with alkali and heat: a comparative study for potential orthopedic applications. Journal of Colloid and Interface Science, 2014, 417, 410-419. Zhang JY, Neoh KG, Hu XF, Kang ET, Wang W. Combined effects of direct current stimulation and immobilized BMP-2 for enhancement of osteogenesis. Biotechnology and Bioengineering, 2013, 110(5), 1466-75. 158 [...]...LIST OF TABLES Table 3.1 Surface elemental compositions as determined by XPS, contact angle and surface roughness of the pristine and treated Ti substrates Table 4.1 Elemental composition as determined by XPS and contact angle at the surface of pristine and functionalized Ti substrates Table 4.2 Elemental composition as determined by XPS at the surface of the Ti-CMCS-VEGF and Ti-HAC-VEGF substrates before... wide-scan spectra of the Ti-CMCS-VEGF and Ti-HAC-VEGF substrates before and after aging in PBS Figure 5.1 Scheme showing the conversion of heparin to HepC Figure 5.2 FT-IR spectra of heparin and HepC Figure 5.3 XPS wide-scan spectra of the pristine Ti, Ti-HAC, Ti-HAC-VEGF, Ti-HepC, and Ti-HepC-VEGF substrates The concentration of VEGF in the loading solution was 1 g/ml Figure 5.4 Surface density of immobilized... objective of this thesis is to formulate surface modification strategies to enhance osseointegration and reduce bacterial infections for Ti substrates This thesis consists of seven Chapters Chapter 1 presents a general introduction and the research objective and scope, while Chapter 2 provides a detailed literature review In Chapter 3, a strategy of alkali and heat treatment for forming anatase on Ti, and. .. topography and chemical composition (Ratner, 1993) The adsorbed proteins are important for successful bone healing For example, fibronectin and vitronectin can interact with the integrins on mesenchymal stem cells (MSCs) and enhance their attachment on the implant surface, and fibrinogen, von Willebrand factor (vWF) and immunoglobulin G are important for platelet activation, coagulation, and inflammation... failure are: defective osseointegration at the bone-implant interface and bacterial infections For ideal orthopedic implants, the materials must be habitable by bone-forming cells (favoring adhesion of osteoblasts), and be anti-infective (discouraging bacterial adhesion) Orthopedic implants can be integrated in bone by mechanical fit such as using screws to fix the device, or by osseointegration (i.e bone... functional coatings on implant surfaces to enhance osseointegration or inhibit bacterial infections (Chen et al., 2012a; Liu et al., 2004; Zhao et al., 2009), few studies have focused on achieving these dual functions simultaneously Enhancement of osseointegration and prevention of infection are sometimes contradictory For example, a surface that can prevent bacterial adhesion may be unfavorable for the attachment... 2.3 Chemical structure of BPs R1 and R2 indicate the different side chains Figure 2.4 Interaction of cells with soluble and immobilized growth factors Figure 3.1 Surface characterization of the pristine and treated Ti substrates a-b: XPS wide-scan spectra (a) and XRD spectra (b) of the pristine Ti, TiS, TiH, TiSH, TiH-10 and TiSH-10 substrates and indicate the presence of anatase and rutile, respectively... substrates before and after aging in PBS Table 5.1 Elemental composition as determined by XPS, contact angle, and zeta potential at the surface of pristine and functionalized Ti substrates Table 5.2 Elemental composition as determined by XPS at the surface of the Ti-HepC-VEGF substrate before and after immersion in PBS for 7 days Table 6.1 Elemental composition as determined by XPS, surface density of loaded... Biocompatibility and osseointegration The implantation of artificial implants induces a cascade of reactions in biological 12 Chapter 2 micro-environment due to the interaction of the device with body fluid, proteins, and cells, which often result in the formation of fibrous tissue on the implant surface as a result of wound healing Therefore, biocompatibility which reflects host response to a foreign material and. .. 1999) Therefore, an implant with osteoactive surface would be helpful in shortening the healing process since it can encourage bone growth from the implant surface 2.2.3 Bone remodeling Bone remodeling refers to the processes of pre-existing bone removal and new bone formation, which occurs throughout the healing process and continues during the lifetime of the implant It includes five sequences of events: . UNIVERSITY OF SINGAPORE 2013 IN VITRO STUDY OF SURFACE FUNCTIONALIZATION OF TITANIUM SUBSTRATES FOR POTENTIAL ENHANCEMENT OF OSSEOINTEGRATION AND REDUCTION OF BACTERIAL INFECTION. IN VITRO STUDY OF SURFACE FUNCTIONALIZATION OF TITANIUM SUBSTRATES FOR POTENTIAL ENHANCEMENT OF OSSEOINTEGRATION AND REDUCTION OF BACTERIAL INFECTION . Ti and its alloys 13 2.4 Surface modification of Ti to enhance osseointegration 14 2.4.1 Enhancement of osseointegration by surface topography 15 2.4.2 Enhancement of osseointegration by surface