CHEMICALLY-MODIFIED CO-SUBSTITUTED HYDROXYAPATITE NANOMATERIAL FOR BIOMEDICAL APPLICATIONS LIM POON NIAN NATIONAL UNIVERSITY OF SINGAPORE 2013 CHEMICALLY-MODIFIED CO-SUBSTITUTED HYDROXYAPATITE NANOMATERIAL FOR BIOMEDICAL APPLICATIONS LIM POON NIAN B.Eng.(Hons), Nanyang Technological University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MECHANICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 Declaration I hereby declare that this 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. LIM POON NIAN 18 December 2013 i Abstract Bioactivity of a biomaterial plays an integral role for bone regeneration. During this process, optimal healing of the defect region is heavily reliant upon the prevention of bacterial infection after implant placement. Hydroxyapatite (HA) is commonly used as a bone replacement material, but has slow osseointegration rate and tends to represent a site of weakness for the host defence, making it susceptible to implant-related infections. The rate of osseointegration in HA can be enhanced by the substitution of silicon whilst antibacterial property can be created by the substitution of silver. Given the beneficial properties of both elements, it is thus desirable to incorporate both silver and silicon ions into HA to achieve antibacterial and enhanced osseointegration properties, respectively. Current literature reported on the use of silver-substituted HA and silicon-substituted HA. However, the cosubstitution of these two elements into HA has not been explored yet. Therefore, this motivates the development of silver, silicon co-substituted hydroxyapatite (Ag,Si-HA). In this study, Ag,Si-HA is a novel bi-functional bone graft material that possesses antibacterial and enhanced biological properties, to facilitate bone healing. A phase-pure Ag,Si-HA with a bonemimicking morphology (60 nm x 10 nm) was successfully synthesised using an aqueous precipitation technique. Phosphate, hydroxyl and silicate functional groups were observed in the FTIR spectra. Silver and silicon ions were structurally incorporated in the HA structure as reflected by the increment of the lattice parameters and unit cell volume. A silver content of 0.5 wt.% was demonstrated to produce the optimum antibacterial effect with a ii 7-log reduction in the growth of adherent Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) over a period of days. Subsequently, the antibacterial action and biological properties of Ag,Si-HA containing optimised content of 0.5 wt.% of silver and 0.7 wt.% silicon were investigated. Silver ions of Ag,Si-HA was first reported to diffuse towards the crystal surface, damaging the cell wall and thereby inducing potassium ions leakage from the adherent S. aureus. Furthermore, the substitution of silicon promoted the proliferation of human adipose-derived mesenchymal stem cells on Ag,SiHA, which in turn, permitted greater bone differentiation (alkaline phosphatase, type I collagen and osteocalcin) as compared to HA and AgHA. On the whole, this study shows that Ag,Si-HA functions by firstly allowing the incorporated silver in Ag,Si-HA to exert its antibacterial action against adherent bacteria and subsequently, the incorporated silicon promotes the bone differentiation process. All these results demonstrated that the approach of cosubstituting silver and silicon could complement their functions in the apatite by facilitating bone regeneration, to perform as an ideal bone graft material. iii Acknowledgements The following are my heartfelt appreciations to those people who gave me the support, knowledge and insight to begin, conduct and complete this thesis. First and foremost, I would like to thank my supervisors Dr. Thian Eng San and Dr. Tay Bee Yen, both of whom provided excellent support in terms of their guidance, scientific input, encouragement and opportunities. I would like to thank Prof. Ho Bow for his expert advice and help in the antibacterial work, and also to Mr. Ng Han Chong and the members of Prof Ho Bow's lab for their assistance and help when conducting the antibacterial experiments. Special thanks also go to Dr. Shi Zhilong and Dr. Teo Yiling, Erin. It is my great pleasure to work with all of them. I am grateful for their help of Dr. Li Tao, Dr. Florencia Wira, Ms. Ma Cho Cho and Ms. Xie Hong from SIMTech. Thanks to Mr. Lucas Lu for his assistance in the TEM preparation work. On a personal level, I would like to thank Dr. Thian Eng San and Dr. Tay Bee Yen for their help and time in reading the first drafts of this thesis and making valuables suggestions for its improvement. My greatest appreciations would also go to Jing Wen for reading parts of this thesis, checking and giving improvement for the grammatical errors. I would also like to say a big thank you to the members of BIOMAT lab. In particular, Ruey Na, Yi Min, Chang Lei, Zu Yong, Qinyuan, Jason, Jin Lan, iv Wu Yang and Yiling, without them the past years wouldn't have been half as much fun. I would also like to thank Mr. Thomas Tan, Mr. Khalim and Mr. Ng Hong Wei of Materials lab for their constant kind assistance. I would like to acknowledge the National University of Singapore for the financial support. Finally, I would like thank my family and friends especially Jianyi, Abriena, Jiahui, Jialing, Xiu Ning, Chew Yen, Beverly and Fan Yan for their constant friendship, patience, encouragement and support during good and bad times. v Publications, Conferences and Awards Journals: 1) Lim PN, Shi ZL, Neoh KG, Ho B, Tay BY, Thian ES. The Effects of Silver and Silicon in Co-Substituted Apatite Towards Bacteria and Cell Responses, Biomedical Materials, 2014, 9: 015010. 2) Lim PN, Teo YL E, Ho B, Tay BY, Thian ES. Effect of Silver Content on the Antibacterial and Bioactive Properties of Silver-Substituted Hydroxyapatite. Journal of Biomedical Materials Research: Part A, 2013, 101A: 2456-2464. 3) Thian ES, Chang L, Lim PN, Gurucharan B, Sun J, Fuh JYH, Ho B, Tay BY, Teo EY, Wang W. Chemically-Modified Calcium Phosphate Coatings via Drop-On-Demand Micro-Dispensing Technique. Surface & Coatings Technology, 2013, 231: 29-33. 4) Thian ES, Konishi T, Kawanobe Y, Lim PN, Choong C, Ho B, Aizawa M. Zinc-Substituted Hydroxyapatite: A Biomaterial with Enhanced Bioactivity and Antibacterial Property. Journal of Materials Science: Materials in Medicine, 2013, 24:437-445. 5) Lim PN, Tay BY, Chan ML C, Thian ES. Synthesis and Characterisation of Silver/Silicon Co-Substituted Nanohydroxyapatite Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2012, 100B: 285–291. Conferences (Oral): 1) Lim PN, Chang L, Ho B, Tay BY, Choong C, Thian ES. Development of New Generation Bone Graft Material: Silicon and Silver Co-substituted Apatite with Bi-Functional Properties. Material Research Society Fall Meeting and Exhibit (MRS), Boston, Massachusetts, 1st-6th December 2013. 2) Lim PN, Teo YL E, Ho B, Tay BY, Thian ES. Silver-Substituted Hydroxyapatite: A Potential Bone Substitute Material for Postoperative Infection Treatment. International Conference of Young Researchers on Advanced Materials (ICYRAM), Singapore, 1st July- 6th July 2012. 3) Lim PN, Shi ZL, Tay BY, Neoh KG, Thian E. Silver/Silicon CoSubstituted Hydroxyapatite: A Potential Biomaterial with Antibacterial and Bioactive Properties. 9th World Biomaterials Congress (WBC), Chengdu, China, 1st June – 5th June 2012. 4) Thian ES, Lim PN, Shi Z, Tay BY, Neoh KG. Silver-Doped Apatite as a Bioactive and an Antimicrobial Bone Material. 23rd International Symposium on Ceramics in Medicine (ISCM), Istanbul, Turkey, 6th November – 9th November 2011. 5) Lim PN; Yu ET J; Thian ES, Tay BY; Chan ML C. Physiochemical Stability of Chemically-Modified Nanoapatites Sintered at Different Temperatures. Defence Science Research Conference and Expo (DSR) Singapore, 3rd August-5th August 2011. vi 6) Lim PN, Tay BY, Chan ML C, Thian ES. Silver/Silicon Co-Substituted Nanohydroxyapatite: A Novel Bone Material for Biomedical Applications. International Conference on Materials for Advanced Technologies, Singapore (ICMAT), 26th June – 1st July 2011. Conferences (Poster): 1) Lim PN, Ho B, Tay BY, Choong C, Thian ES. Development of Next Generation Bone Substitute Material: Silicon and Silver Co-Substituted Apatite with Bi-Functional Properties, SIMTech Post-Graduate Research Posters Exhibition, 16th August 2013. 2) Thian ES, Lim PN, Ho B, Teo EY, Tay BY. The Effect of Silver Concentration on Antimicrobial Property of Silver-Substituted Apatite. 9th World Biomaterials Congress, Chengdu, China, 1st June – 5th June 2012. 3) Lim PN, Ho B, Tay BY, Thian ES. Evaluation on the Antibacterial Capability of Silver-Substituted Hydroxyapatite for Implant-Associated Infection, SIMTech Post-Graduate Research Poster Exhibition, 7th September 2012. 4) Lim PN, Ho B, Chan ML C, Tay BY, Thian ES. Silver/Silicon CoSubstituted Hydroxyapatite: A New Generation of Functional Biomaterial, SIMTech Post-Graduate Research Posters Exhibition, 23 September 2011. 5) Thian ES , Lim PN, Lee LY, Zhang ZY, Tay BY, Chan J, Teoh SH Nanostructured Substituted Apatites for Bone Tissue Engineering. MRS-S Trilateral Conference, Singapore 11th August- 13 August 2010. Conference Proceedings: 1) Lim PN, Chang L, Ho B, Tay BY, Choong C, Thian ES. Development of New Generation Bone Graft Material: Silicon and Silver Co-Substituted Apatite with Bi-Functional Properties. MRS Online Proceedings Library, 2013, accepted. 2) Thian ES, Lim PN, Shi Z, Tay BY, Neoh KG. Silver-Doped Apatite as a Bioactive and an Antimicrobial Bone Material. Key Engineering Materials, 2012 (493-494), 27-30. 3) Lim PN; Yu ET J; Thian ES, Tay BY; Chan ML C. Physiochemical Stability of Chemically-Modified Nanoapatites Sintered at Different Temperatures. Defense Science Research Conference and Expo (DSR), 3rd-5th August. 2011, pp.1-4, doi: 10.1109/DSR.2011.6026836. Awards 1) SIMTech Best Ph.D Student of the Year, 15 March 2013 2) Best Poster Presenter Award (First Runner-Up) at the SIMTech PostGraduate Research Posters Exhibition, 23 September 2011 vii Table of Contents Declaration . i Abstract . ii Acknowledgements iv Publications, Conferences and Awards vi Table of Contents . viii Lists of Figures . xii Lists of Tables . xvii Lists of Symbols xviii CHAPTER INTRODUCTION 1.1 Background . 1.2 Objectives 1.3 Scope . CHAPTER LITERATURE REVIEW 2.1 Bone Physiology . 2.1.1 Structure of Bone . 2.1.2 Bone Cells 12 2.1.3 Bone Matrix . 13 2.2 Bone Repair . 17 2.2.1 Bone Graft . 18 2.2.2 Synthetic Bone Graft . 21 2.2.3 Factors Affecting Bone Healing and Implant-Related Infections . 26 2.3 Calcium Phosphate: Hydroxyapatite (HA) . 31 2.3.1 Synthesis of HA . 34 2.3.2 Crystal Structure of HA . 37 2.3.3 Substituted Apatite 39 2.3.4 Biological Properties of HA 43 2.4 Silver-Substituted Hydroxyapatite (AgHA) 48 2.4.1 Silver as an Antibacterial Agent 49 2.4.2 Synthesis of AgHA 51 2.4.3 Chemical and Physical Characterisation of AgHA . 53 2.4.4 Antibacterial Properties of AgHA . 56 viii References 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. Vaughan, J.M., The physiology of bone 1975: Clarendon Pres. Kielty, C.M., Hopkinson, I., Grant, M. E. , Connective Tissue and Its Heritable Disorders. 1993: Willey-Liss, Inc. 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Neo, M., Nakamura, T., Ohtsuki, C., Kokubo, T., and Yamamuro, T., Apatite Formation on Three Kinds of Bioactive Material at an Early Stage In Vivo: A Comparative Study by Transmission Electron Microscopy. Journal of Biomedical Materials Research, 1993. 27(8): p. 999-1006. 211 [...]... Although studies concerning substituted apatites are conventionally performed by the addition of single cation or anion, co- substitution has been considered in recent years [43-48], and is certainly becoming a considerable field of research for apatites Co- substitution combines the functions of various ions into a single apatite compound, which can eliminate problems encountered in the composite system... AgHA Silver -Substituted Hydroxyapatite Ag,Si-HA Silver, Silicon Co- substituted Hydroxyapatite ALP Alkaline Phosphatase ANOVA Analysis of Variance ATP Adenosine-5'-Triphosphate BEs Binding Energies C albicans Candida albicans Ca Calcium COL I Type I Collagen ECM Extracellular Matrix E coli Escherichia coli FESEM Field Emission Scanning Electron Microscope FTIR Fourier Transform Infrared HA Hydroxyapatite. .. 60 2.5 Silicon -Substituted Hydroxyapatite (SiHA) 62 2.5.1 Silicon as Bone Promoter 63 2.5.2 Synthesis of SiHA 65 2.5.3 Chemical and Physical Characterisation of SiHA 66 2.5.4 Biological Characterisation of SiHA 74 2.6 Co- Substituted Hydroxyapatite 80 2.6.1 Various Types of Co- Substituted Hydroxyapatite 81 2.6.2 Synthesis of Co- Substituted Hydroxyapatite ... Hydroxyapatite 81 2.6.2 Synthesis of Co- Substituted Hydroxyapatite 81 2.6.3 Chemical Characterisation of Co- Substituted Hydroxyapatite 82 2.6.4 Biological Characterisation of Co- Substituted Hydroxyapatite 85 2.7 Summary 86 CHAPTER 3 SILVER, SILICON CO- SUBSTITUTED HYDROXYAPATITE: SYNTHESIS AND CHARACTERISATION 3.1 Introduction 88 3.2 Materials and Methods ... Procollagen molecules consist of 3 pro-α chains, which undergo processing to α-chains, and subsequently assemble to form collagen fibrils (tropocollagen), which are coiled to form a helical structure Each tropocollagen molecule is longitudinally displaced by approximately one quarter of its length relative to its nearest neighbour when they are aggregated together This staggering effect of tropocollagen... tissue metabolism, particularly for bone and cartilage To mimic the mineral component of bone better, silicon -substituted HA (SiHA) has been extensively investigated by many researchers [13-26] The incorporation of silicon into HA has been shown to have the potential to increase the rate of bone apposition on HA implants significantly [19, 21, 27, 28] Therefore, SiHA is considered as state-of-the-art... Properties of Ag,Si-HA 136 5.3.2 Silver Composition on the Surface of Ag,Si-HA 141 5.3.3 Antibacterial Assessment of Ag,Si-HA 142 5.3.4 Surface Silver Ions of Ag,Si-HA for Antibacterial Action 147 5.3.5 Effect on S aureus treated by Ag,Si-HA 149 5.4 Conclusions 158 x CHAPTER 6 IN-VITRO BIOCOMPATIBILITY OF SILVER, SILICON CO- SUBSTITUTED HYDROXYAPATITE 6.1 Introduction ... The specific objectives of this research project are as follows:  To determine the feasibility of co- substituting silver and silicon into hydroxyapatite to form Ag,Si-HA via a wet precipitation method, and characterise its physicochemical properties,  To optimise the silver content in hydroxyapatite for effective antibacterial properties,  To determine the mechanism of the antibacterial action in... effects of silver and silicon on the biological properties of Ag,Si-HA 1.3 Scope Chapter 1 established the background, illustrating the need for synthetic bone substitute materials Particularly, HA and improvements to its property were discussed The co- substitution of silver and silicon was also proposed to incorporate antibacterial property in conjunction with enhanced bone formation in HA Subsequently,... matrix is composed of two major phases namely the organic phase (~ 30-40 wt.% protein) and the inorganic phase (~ 50-60 wt.% minerals), with the remaining making up of biological compounds [58] Collagen is the most abundant protein found in the human body, which comprises approximately 95 % of the non-mineralised component of bone matrix Osteoblasts synthesise collagen in the form of long procollagen . CHEMICALLY- MODIFIED CO- SUBSTITUTED HYDROXYAPATITE NANOMATERIAL FOR BIOMEDICAL APPLICATIONS LIM POON NIAN NATIONAL UNIVERSITY OF SINGAPORE 2013 CHEMICALLY- MODIFIED CO- SUBSTITUTED. 2.6 Co- Substituted Hydroxyapatite 80 2.6.1 Various Types of Co- Substituted Hydroxyapatite 81 2.6.2 Synthesis of Co- Substituted Hydroxyapatite 81 2.6.3 Chemical Characterisation of Co- Substituted. ML C, Thian ES. Silver/Silicon Co- Substituted Nanohydroxyapatite: A Novel Bone Material for Biomedical Applications. International Conference on Materials for Advanced Technologies, Singapore