Content Of Thesis Working 08 03 [307638]

[anonimizat]-medical devices

Tran Thi Thanh

February, 2020

Supervised by

Prof. Dr. M. TÂRCOLEA

Bucharest – 02/2020

[anonimizat]-medical devices

by

Tran Thi Thanh

This dissertation is submitted for the degree of Doctor of Material Sciences

Faculty of Materials Science and Engineering

University Politehnica of Bucharest

Approved by

Prof. Dr. M. TÂRCOLEA

Advisor

Bucharest – 02/2020

[anonimizat]-[anonimizat]. The research reported herein was approved by the committee of Thesis Appraisal

Bucharest – 02/2020

Thesis Review Committee

Chairman: (signature)

Member: (signature)

Member: (signature)

Member: (signature)

Member: (signature)

Declaration

This thesis is submitted for the degree of Doctor of Material Sciences at the Politehnica University of Bucharest. The research was conducted under the supervision of Professor Dr. [anonimizat] 2015 – 2020.

[anonimizat]. [anonimizat] a degree, diploma or other qualification at any other university.

Part of this work has been submitted to appear in the following publications:

[anonimizat]6Al4V for medical purposes, D.M. Vraceanu, T. Tran., E. Ungureanu, V. Negoiescu, M. Tarcolea, M. Dinu, A. Vladescu, C.M. Cotrut, Scientific Bulletin U.P.B 2018, Series B – [anonimizat]-active and antibacterial coatings for medical applications, D.M., Vranceanu, A. Vladescu, M. Dinu, T. Tran, C.M Cotrut, EMRS 2017 – Spring Meeting, 2017, 22-26 May, 2017, Strasbourg, [anonimizat], M.Tarcolea, D.M.Vranceanu, A.Vladescu, M.Dinu, T.Tran, C.M.Cotrut, 7th International Conference “Biomaterials, Tissue Engineering & Medical Devices”-BIOMMEDD’2016, 15-17 September 2016, Constanta, Romania – [anonimizat], [anonimizat], [anonimizat], [anonimizat] U.P.B 2020, Series B – [anonimizat] 2020

Acknowledgements

At the very beginning, I would like to acknowledge and express my sincere gratitude to my supervisor Professor Dr. [anonimizat], [anonimizat]. I also would like to consider Prof. Dr. M. O [anonimizat] I spent one year under his supervision at the beginning of study and a portion of the current study was carried out.

Further, I would like to thank the members of The Electrochemical Deposition Laboratory for their help, suggestions and support in progressing this work. A special thanks to Dr. Cosmin COTRUȚ and Dr. Diana VRÂNCEANU who did not hesitate to offer valuable time, assistance and understanding into this area of research.

I am also thankful to all the staffs of The National Institute of Research and Development for Biological Sciences especially to Prof. Norica NICHITA, Olivia DOBRICĂ for their support and helping me in carrying out different experiments in their respective laboratories.

Many thanks to all! I am greatly indebted to my parents and family for their support, devotion and understanding.

.

Abstract

This research aims to investigate the micro-structure development of HAp coating on Ti and its alloys. The electrochemical deposition technique was applied to obtained pure HAp coatings, HAp-Ag coatings, HAp-Zn coatings with the requirements of micro-structure's properties, biocompatibility, bioactivity, etc.

The theories of all the major surface modification of Ti and its alloy via electrochemical deposition technique, composite coating of HAp formed by electrochemical deposition, silver doped hydroxyapatite coating on titanium and its alloy, zinc doped hydroxyapatite coating on titanium and its alloy, characterization method for hydroxyapatite deposited layers, biocompatibility test for medical devices, involved are reviewed in Chapter 2.

Experimental work is presented in chapters: Characterization of the calcium phosphate coating on Ti6Al4V substrates by electrochemical process, Pulsed electrochemical deposition of Ag doped hydroxyapatite bioactive coatings on Ti6Al4V for medical purposes, Studies of micro-structure and composition of the modified HAp coating via SEM investigations, In vitro biocompatibility investigation of silver and zinc modified hydroxyapatite on metallic implant materials.

Chapter 4 summarizes the whole research work, and attention is drawn to new areas of interest for future research.

Content

Preface/ Declaration

Acknowledgments

Abstract

Contents

Statement of objectives

Nomenclature

List of Figures

List of Tables

Surface bio-functionalization for bio-medical devices

Chapter 1: Introduction

Chapter 2: Literature review

2.1 Introduction

2.2 Surface modification of Ti and its alloy via electrochemical deposition technique.

2.2.1 Substrate

2.2.1.1 Mechanical polishing

2.2.1.2 Cleaning

2.2.1.3 Another method

2.2.2 Formation of HAp coating on Ti and its alloy

2.2.2.1 Hydroxyapatite

2.2.2.2 Electrolyte solution

2.2.3 Applied voltage

2.2.4 Time

2.2.5 Post-treatment

2.3 Composite coating of HAp formed by electrochemical deposition

2.4 Silver doped hydroxyapatite coating on titanium and its alloy

2.5 Zinc doped hydroxyapatite coating on titanium and its alloy

2.6 Characterization method for hydroxyapatite deposited layers

2.6.1 Photon-based techniques

2.6.1.1 Infrared spectroscopy

2.6.1.2 Raman spectroscopy

2.6.1.3 X-ray Photoelectron Spectroscopy

2.6.1.4 X-ray diffraction

2.6.2 Microscopy techniques

2.6.2.1 Scanning electron microscopy

2.6.2.2 Energy Dispersive X-ray Spectroscopy

2.6.2.3 Transmission electron microscopy

2.6.3 Mechanical characterization techniques

2.6.3.1 Adhesion Testing

2.6.3.2 Roughness Testing

2.6.4 Another Testing

Biocompatibility test for medical devices: review

Introduction

In Vitro Tests for Biocompatibility

2.7.2.1 Cytotoxicity tests

2.7.2.2 Cell adhesion

Cell activation

In Vivo Tests for Biocompatibility

2.7.4 The laboratory equipment for biocompatibility evaluation

2.8 Summary of literature review

Chapter 3: Experimental details/ Experimental Methods/ Experiment Procedures

Characterization of the calcium phosphate coating on Ti6Al4V substrates obtained by electrochemical process

.1 Abstract

3.1.2 Material and methods

3.1.2.1 Preparation of Ti6Al4V specimen

3.1.2.2 Deposition procedure

3.1.2.3 Electrochemical measurement

Structural characterizations

3.1.3 Results and discussion

3.1.3.1 Surface and morphological studies via SEM investigations

Composition of the deposited specimen

3.1.4 Conclusions and Further research

3.2 Pulsed electrochemical deposition of Ag doped hydroxyapatite bio-active coatings on Ti6Al4V for medical purposes.

.1 Abstract

3.2.2 Material and methods

3.2.3 Deposition procedure

3.2.3.1 Preparation of electrolyte solution

3.2.3.2 Electrochemical measurement

3.2.3.3 Coatings characterization

3.2.4 Results and discussion

3.2.4.1 Micro-structure and composition of coatings studies via SEM investigations

3.2.4.2 The phase composition of the coatings

3.2.5 Conclusions

3.3 Studies of micro-structure and composition of the modified HAp coating via SEM investigations

3.3.1 Materials and Methods

3.3.1.1 Preparation of titanium samples

3.3.1.2 Electrochemical deposition process

3.3.1.3 Post-treatment: Annealing heat treatment of HAp coating

3.3.1.4 Characterization and composition analysis of coatings

3.3.2 Results and discussions

3.3.2.1 Morphological investigation

3.3.2.2 Elemental composition

3.3.3 Conclusions

3.4 In vitro biocompatibility investigation of silver and zinc modified hydroxyapatite deposited on metallic implant materials

3.4.1 Materials and Methods

3.4.1.1 Pretreatments of Ti substrate

3.4.1.2 Electrochemical deposition of HAp, HAp-Ag, HAp-Zn coatings on titanium

3.4.1.3 Characterization and composition analysis of coatings

3.4.1.4 In vitro biological activity analysis

3.4.1.5 Evaluation of biocompatibility

– Cell culture

– Immunofluorescence assay

+ Sample preparation

+ Staining procedure

Staining for Actin and Ki67

Staining for Tubulin and PDI

Microscopic immunofluorescence analysis.

– Cell proliferation assay

3.4.2 Results and discussions

3.4.2.1 Morphological investigation

3.4.2.2 Evaluation of Bioactivity

3.4.2.3 Evaluation of biocompatibility

– Cell morphology

– Cell proliferation

3.4.3 Conclusions

Chapter 4: Conclusions and future work

4.1 Conclusions

4.2 Future work

Bibliography references

Statement of objectives

This dissertation is submitted for the degree of Doctor of Materials Science at the Faculty of Materials Science and Engineering, University Politehnica of Bucharest. The research described herein was conducted under the major advisor of Prof. Dr. M. TÂRCOLEA and co-advisor of Prof. Dr. M. O COJOCARU in the Department of Materials Science, Faculty of Natural Sciences and Engineering, University Politehnica of Bucharest, between October 2015 and February 2020.

Nomenclature

List of Figures

Figure 2.1 – The crystal structure of HAp (hexagonal)

Figure 2.2 – FTIR spectra of as-used graphene oxide

Figure 2.3 – FTIR spectra of the CSHAp-Ag composite coating on TNs

Figure 2.4 – Raman spectra of samples HAp1/60, HAp2/60, HAp3/60 and ceramics HAp-COM

Figure 2.5 – XPS general spectrum of the prepared CSHAp-Ag coating (a) and deconvolution of Ag (3d) XPS peak (b)

Figure 2.6 – XPS profile of SWNTs/HAp composite coatings and pure HAp coating

Figure 2.7 – The XRD patterns of Ca–P coating deposited on 316L SS at various current densities without the addition of H2O2 into the electrolyte (a) 0.5 mA/cm2, (b) 1 mA/cm2, (c) 2 mA/cm2 and (d) 3 mA/cm2

Figure 2.8 – The XRD patterns of Ca–P coating deposited on 316L SS at various current densities with the addition of 1000 ppm H2O2 into the electrolyte (a) 0.5 mA/cm2, (b) 1 mA/cm2, (c) 2 mA/cm2 and (d) 3 mA/cm2

Figure 2.9a – Cone-like structure of HAp coating

Figure 2.9b – Sharp-angle tip structure of HAp coating

Figure 2.9c – Flower-like structure of HAp coating

Figure 2.10 – Ribbon-like structure of HAp coating

Figure 2.11a – TEM images of HAp crystals

Figure 2.11b – The wrinkled-paper-like graphene oxide (GO) sheets in GO/HAp composite coating

Figure 2.12 – The tensile test

Figure 3.1.1 – PHOENIX BETA (Grinder/ Polisher and Power head, BUEHLER)

Figure 3.1.2 – Ultrasonic machine (BANDELIN SONOREX DIGITEX, Germany)

Figure 3.1.3 – HI 4221-02 RESEARCH GRADE pH/ORP/°C – 1 Channel-Meter

Figure 3.1.4 – PARSTAT MC, Princeton Applied Research, USA

Figure 3.1.5 – Schematic illustration of voltage –time profile used for electrochemical deposition of calcium phosphate coating by voltammetry

Figure 3.1.6 – Phenom ProX system

Figure 3.1.7a – The surface of the deposited coating

Figure 3.1.7b – SEM images of HAp coating on Ti6Al4V from low magnification to high magnification.

Figure 3.1.8 – SEM image of CaP coating on Ti6Al4V

Figure 3.1.9 – The EDX spectra (elemental composition) by SEM

Figure 3.2.1 – Schematic illustration of the a) applied pulsed electrochemical deposition cycles

Figure 3.2.2 – The HAp/Ti6Al4V (2a) and HAp-Ag/Ti6Al4V (2b) coatings

Figure 3.2.3 – SEM images of HAp/Ti6Al4V and HAp-Ag/Ti6Al4V coatings

Figure 3.2.4 – EDS elemental spectrum of the HAp/Ti6Al4V (a) and HAp-Ag/Ti6Al4V (b) coatings obtained by pulsed electrochemical deposition

Figure 3.2.5 – SEM images of elements distribution for the obtained coatings HAp/Ti6Al4V (a, b) and HAp-Ag/Ti6Al4V (c, d)

Figure 3.2.6 – XRD pattern of the obtained coatings a) HAp/Ti6Al4V, b) HAp-Ag/Ti6Al4V, c) their overlay

Figure 3.3.1 – SEM micrograph of the HAp, HAp-Ag, HAp-Zn coatings at magnifications ×500 (A1, B1 C1), ×1000 (A2, B2, C2), × 3000 (A3, B3, C3), ×5000 (A4, B4, C4), ×10000 (A5, B5, C5), ×15000 (A6, B6, C6), ×25000 (A7, B7, C7), ×30000 (B8, C8) obtained using electro-deposition at 0.6 m/cm2 applied current density

Figure 3.3.2 – Elemental composition for HAp/Ti (a), HAp-Ag/Ti (b) and HAp-Zn/Ti (c) coatings were examined by EDS elemental mapping

Figure 3.4.1 – The sample before Biocompatibility test

Figure 3.4.2 -The sample before observation by Microcopy

Figure 3.4.3 – The SEM/EDS measurements on substrate material: HAp (a, d), HAp-Ag (b, e), HAp-Zn coatings (c, f).

Figure 3.4.4 – The chart shows the increase in the mass of the apatite layer on Ti.

Figure. 3.4.5 – The results of staining for Actin and Ki67 of the HEK 293T cells (Actin-red, Ki67-green)

Figure. 3.4.6 – The results of staining for Tubulin and PDI of the HEK 293T cells (PDI-green, Tubulin-red)

Figure. 3.4.7A – Staining for Actin of the coating at Magnification X63 (a1, b1, c1, d1)

Figure. 3.4.7B – Staining for Tubulin of the coating at Magnification X63 (a2, b2, c2, d2)

Figure. 3.4.8A – Staining for PDI of the coating at Magnification X63 (a1, b1, c1, d1)

Figure. 3.4.8A – Staining for PDI of the coating at Magnification X63 (a1, b1, c1, d1)

Figure 3.4.9 – The number of cells on the coating after 1, 3 and 4 days.

Figure 3.4.10 – Cell Viability of the coatings after 1, 3 and 4 days

List of Tables

Table 2.1 – Types of biomaterials, characteristics, and their common application

Table 2.2 – Different techniques to deposit HAp coatings

Table 2.3 – Treatment of Ti and its alloy substrate

Table 2.4 – The parameter of anodic oxidation process for Ti

Table 2.5 – Alkaline treatment

Table 2.6 – Ca/P with different Ca/P ratio

Table 2.7 – The requirements for HAp coatings

Table 2.8 – Electrolyte solution for HAp coating on Ti and its alloy

Table 2.9 – The capacity to incorporate various ions into the HAp coating

Table 2.10 – The incorporation of reinforcing materials into the HAp coating

Table 2.11 – Treatment of substrate for HAp-Ag coating on Ti and its alloy

Table 2.12 – The conditions of the electrolyte solution before electrodeposition process of co-substituted HAp.

Table 2.13 – The conditions of the electrolyte solution before electrodeposition process of co-substituted HAp

Table 2.14 – The parameter of electrochemical deposition

Table 2.15 – CFUs of antibacterial tests and bactericidal ratios of the coatings (n = 3)

Table 2.16 – ISO 10993: Biological evaluation of medical devices

Table 2.17 – ASTM standards and tests for biocompatibility

Table 2.18 – Advantages and Disadvantages between vitro test and vitro test

Table 2.19 – Cell culture

Table 2.20 – Cytotoxicity tests

Table 2.21 – Ion concentrations of the human blood plasma and the SBF solution.

Table 2.22 – Laboratory equipment

Table 3.1.1 – Condition for electrochemical hydroxyapatite deposition by cyclic voltammetry.

Table 3.1.2 – Composition of the elements

Table 3.1.3 – Composition of the elements

Table 3.2.1 – Samples codification and chemical composition of the electrolyte

Table 3.2.2 – Condition for electrochemical hydroxyapatite deposition by cyclic voltammetry

Table 3.2.3 – Elemental composition of the obtained coatings were examined by EDS

Table 3.3.1 – Samples codification and chemical composition of the electrolyte

Table 3.3.2 – Elemental composition of the obtained coatings by electrochemical deposition

Table 3.4.1 – The electrolyte used for fabrication of the coatings

Table 3.4.2 – The concentrations of SBF (1000 mL)

Table 3.4.3 – The increase of apatite layer on substrate at every period of 1, 3, 7, 14, 21 days in SBF

Chapter 1: Introduction

Nowadays, when science and technology have been develop rapidly, it has been associated with human life, present in all fields, with the aim of improving the quality and longevity of human life and materials for bio-medical applications are no exception.

If physics was the central science in the 20th century, then into the 21st century a good deal of research will be directed to life science as the focus. This means that bio-medical science is central to all other sciences. Bio-medical material is the science of searching for new types of materials that can adapt to the biological – physiological mechanisms of the human body. In recent years, materials for bio-medical applications have attracted a great deal of interest in the scientific community. Over the decades, numerous studies related to this topic have been published, contributing significantly to the development of this field. Currently, the research continues to be carried out based on previous research by the special features and practical applicability that it brings. Typically, bio-medical material will be found in cosmetic surgeries (silicone chin implants, silicone breast implant surgery, etc.), dentistry (porcelain teeth, dental restorations, etc.), orthopedic surgery (fixed screw for broken bones, joint replacement implants such as knees, hips, elbows, etc.), artificial valves replacement in the heart (pulmonic valve, tricuspid valve, mitral valve and aortic valve), stents in blood vessels (stents are made of metal or plastic), etc. Hence, the development of new biomaterial with high longevity, have high-level corrosion resistance, excellent mechanical property (it means combination of high strength and low Young´s modulus, high fatigue and wear resistance, high ductility), great biocompatibility and non-cytotoxicity is highly all-important and necessary. These purposes still contain many challenges for the scientific community.

Titanium (Ti) and its alloy are traditionally metallic biomaterials, which have been the most widely used material in medical applications because of its main load-bearing properties, such as dental applications (orthopedic, dental implants), joint replacement and bone fixation. In addition, Ti and it alloys contain outstanding typical advantages such as excellent corrosion resistance, low density, non-toxic [1, 2]. However, their biggest drawback is their biological incompatibility and lack of antibacterial properties. Due to the interactions will take place between cells and tissues with biomaterials at the tissue implant interface are almost exclusively surface, consequently surface properties of biomaterials are of great importance. For many years, surface modification methods for Ti and its alloy have been extensively studied with various considerations. There are various approaches possible to modify the surface of Ti and its alloy to enhance adhesion, anti-bacterial properties, biocompatibility and corrosion resistance of coating materials are still challenging. The contact surface is the interface where bio-medical materials interact with the biological environment (such as bone, soft tissue, blood) so the properties of the surface are crucial factors for the existence of bio-medical materials in the body. Modification of the surface of Ti and its alloy in order to provide enhanced cell attachment at interactions, growth and tissue formation can be achieved without detrimental effect to the mechanical properties of them have been continuing to receive the attention of the scientific community.

A material has been found in contrast to Ti and its alloy is Hydroxyapatite (HAp), which belongs to the calcium phosphate (Ca/P) family, brings all the characteristic features of biomaterials: bio-compatible, bio-active, osteo-conductive, osteo-integration, osteo-induction, non-toxic, non-inflammatory and non-immunogenic properties, … but it displays low mechanical and fracture toughness hence HAp is an unsuitable candidate for load-bearing in implant applications [3].

Recent years have seen an increased interest in the modification of Ti and its alloy by HAp coating. There are many researchers focus on the combination between of the excellent mechanical properties of Ti and its alloy to compensate for the poor mechanical properties of HAp for load-bearing applications. There are a lot of technologies have been presented on the previous research such as plasma spraying process, sputter coating, pulsed laser deposition, sol-gel, electrophoretic deposition, electrochemical deposition … The choice of a suitable technology is dependent on many factors, including the substrate material, component design and geometry, cost, coating thickness and process temperature, etc. Among all of the techniques listed, the electrochemical deposition had attracted much more interest due to it is inexpensive and simple process, it could be carried out quickly and operated under mild experimental condition such as relatively low temperature [4]. This technology is seen as the method of choice for modifying the surfaces of Ti and its alloy by HAp coating for bio-medical and tissue engineering applications. In additional, the thickness, chemical composition and the micro-structure of the calcium phosphate coating could also be controlled by the adjusting the electrochemical deposition condition. Hence, electrochemical deposition of HAp coating has focused considerably in recent years and presented at a lot of published research.

On other hand, the surface modification of Ti and its alloy is also concerned with the antibacterial and biocompatibility properties, one of the very important requirements of bio-medical materials. HAp does not show any antibacterial activity so antibacterial materials were added into the HAp coating as a solution to effectively inhibit infection. The key advantages of inorganic antibacterial materials bring convincingly such as improved safety and stability. Moreover, the calcium ion in HAp can be easily replaced by various metal ions, which results in enhanced osseointegration, biological activity, and antibacterial properties. The ion exchange of HAp with metal ions is promising to improve the properties of HAp coating in various applications. Hence, surface modification of Ti and its alloy for improved biocompatibility can be achieved by this way. In the recent years, the selection of inorganic antibacterial materials has attracted interest of science community. To improve the antibacterial property of HAp, various metallic nanoparticles were introduced such as zinc (Zn), copper (Cu) and silver (Ag), strontium (Sr), etc. Compared with other heavy metal ions, silver antibacterial metal ions have shown outstanding advantages, attracting the attention of scientists due to broad spectrum antibacterial activity, strong inhibition, and high efficiency and low toxicity on long-term use. Moreover, the antibacterial properties of silver are strongly expressed in their ability to fight against the most common bacteria which are Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Staphylococcus aureus and Bacillus subtilis).

With the already mentioned aspects above, the primary aim of the work detailed in this thesis focuses on the surface modification topic for Ti and its alloys based on electrochemical deposition techniques to create HAp coatings. This thesis begins, in chapter 2, with an introductory review of the literature that explores our current understanding of surface modification of Ti and its alloy via electrochemical deposition technique, composite coating of HAp formed by electrochemical deposition, silver doped hydroxyapatite coating on titanium and its alloy, zinc doped hydroxyapatite coating on titanium and its alloy, characterization method for hydroxyapatite deposited layers, biocompatibility test for medical devices.

Chapter 3 is devoted to the detail experiment. This section of the thesis draws together many of the topics addressed in previous chapters. The results obtain that HAp coating adapted the requirements of homogeneity, biocompatibility and bioactivity. In addition, the author also studied the adding of Ag+, Zn2+ ions into the HAp coating to explore the non-cytotoxic and biocompatible properties. The samples were investigated by SEM for morphological features and EDS analysis. Moreover, the deposited coatings were required to further perceive whether this material in good biocompatibility with host. In this regard, the cellular behavior (human embryonic kidney 293 cells, HEK 293T cells) on HAp/Ti, HAp-Ag/Ti, HAp-Zn/Ti coatings was evaluated in vitro. Bioactivity behavior of the coatings also were assessed in simulated body fluid (SBF).

Finally, concluding remarks and the scope for future work are given in chapter 4.

Chapter 2: Literature review

2.1 Introduction about the biomaterials

There are many types of materials with different applications. The biomaterials are currently developing very fast, is one kind of materials have been attracting a great deal of attention in the scientific community due to wide application range such as Replacement of damaged part (joint replacement on hip, shoulder, knee, etc.), assist in healing (bone plates, screws, etc.), aid to treatment (catheters, drains, etc.), correct cosmetic (silicone chin implants, silicone breast implant surgery, etc.), improve function (contact lens, etc.), etc. In this aspect, biomaterials stand out because of their ability to remain in contact with tissues of the human body. The biomaterial is one of the most important concepts ever formed by the application of materials science to the medical field.

Biomaterial has a long history of use in medicine application. At the eighteenth and nineteenth centuries marked the outstanding developments in biomaterials field. The word ‘biomaterial’ was formed in the mid twentieth century. The concept of a biomaterial was first suggested by Williams in 1987: "nonviable material used in a medical device, intended to interact with biological systems". Since that time, the definitions have been seen differently at different times to accommodate the new developments.

The design and selection of biomaterials depend on the specific application. Development of new biomaterials is a challenge for the scientific community. Biomaterials comes in many types, generally they are divided into five main categories including metals, ceramics, composites, polymers and natural materials. Each type has different advantages and disadvantages, were demonstrated at [Table 2.1] [3]. The selection of biological materials depends on the purpose of specific applications. In many cases, the combination of materials aims to make optimal use of the advantages and significantly reduce the disadvantages of biological materials. Moreover, the selection must strictly adhere to the basic requirements of biomaterials, which are materials that must be compatible with the surrounding physiological environment when in contact with the body without causing any effect.

Metallic materials, including metals and alloys, have found many important medical applications. They are attractive as biomaterial of choice for applications that require about the high-level mechanical property. Especially, they have been focusing a great attention for load-bearing applications because of favorable mechanical properties (especially fracture toughness and fatigue strength), such as orthopedic implants (wires, plates, screws, total or partial joint replacements) and dental applications. The mechanical properties include hardness, tensile strength, elastic modulus and elongation. The material replaced for bone will expect to have a modulus equivalent to that type of bone (from 4 to 30 GPa). Thus, a material with excellent combination of high strength and low elastic modulus closer to bone has been used for implantation application. The most common metals and alloys that are seen for medical application are stainless steels (Iron-base alloys of the 316L stainless steel), pure titanium and titanium and titanium-base alloys, such as commercially pure (98.9% Ti) and Ti6Al4V (Ti, 6% Al, 4%V), and Ti-Ni (55% Ni and 45% Ti), and cobalt-based of four types Cr (27-30%), Mo (5-7%), Ni (2-5%); Cr (19-21%), Ni (9-11%), W (14-16%); Cr (18-22%), Fe (4-6%), Ni (15-25%), W (3-4%); Cr (19-20%), Mo (9-10%), Ni (33-37%). The main attention in selecting metals and alloys for bio-medical applications are biocompatibility, excellent mechanical properties, corrosion resistance, and reasonable cost. Moreover, the mechanical properties of metallic biomaterials depend not only on the type of metal but also on the fabrication process of the material and device. For example, thermal and mechanical process can change the micro-structure of metallic biomaterials. On another hand, the physiological environment of human body is typically aqueous solution at 37oC, pH value at 7.3, with dissolved gases (such as oxygen), electrolytes, cells, and proteins. During the electrochemical process of corrosion, biomaterials can release ions, which may reduce the biocompatibility of materials and their influence may affect the longevity of the material. Compare with other biomaterials, metallic biomaterials have more favorable corrosion resistance for long-term implant applications such as joint and dental prostheses, especially Ti and its alloys as well as cobalt-chromium alloys. However, there are some disadvantages of these metal materials involving the possible release of harmful metal ions through corrosion processes when they are in contact to body environment. The release of toxic ions will cause effects on the surrounding tissues.

Biopolymers are used mainly for no-load-bearing applications, such as vascular prosthesis, catheters, drug delivery aids, facial prosthesis, skin/cartilage, intraocular prosthesis, and in conjunction with metals in orthopedics and dentistry. It includes acrylics, polyamides, polyesters, polyethylene, polysiloxanes, polyurethane.

Bio-ceramics (aluminum oxide, zirconium oxide, calcium phosphates, apatite, graphite) are generally used in dental restorations and for certain orthopedic applications (such as part of artificial joints, articulating surfaces in joints and in teeth) due to their hardness, the high compression and wear resistance. Composite materials are specifically engineered for particular use of medical applications.

Natural biomaterials are divided into three types of bio-polymers: proteins, polysaccharides, polynucleotides.

Table 2.1 – Types of biomaterials, characteristics, and their common application [3]

2.2 Surface modification of Ti and its alloy via electrochemical deposition technique.

Calcium phosphate (Ca-P) ceramics are bio-active material, they could form directly bond with bone tissue [4]. However, the mechanical properties of Ca-P ceramics are very weak, and these materials could not be used under load bearing applications like are used titanium implants or titanium alloy implants.

There are many researchers focus on the development of new biomaterials that combine the osteo-conductive characteristics of bio-active ceramics with enough strength and toughness for load-bearing applications. Hence, several coating methods have been developed for combination the high strength of titanium or its alloy with the bioactivity and osteo-conductivity of calcium phosphate such as plasma spraying process [5], sputter coating [6], pulsed laser deposition [7, 8], sol-gel [9], electrophoretic deposition [10], electrochemical deposition. Each technique has different advantages and disadvantages, depend on reality condition to choose suitable method, which are demonstrate at [Table 2.2] [97].

Among the techniques listed, the electrochemical deposition had exhibited much more interest due to it is inexpensive and simple process, it could be carried out quickly and operated under mild experimental condition such as relatively low temperature [11], at atmospheric pressure. The thickness, chemical composition and micro-structure of the calcium phosphate coating could also be controlled by the adjusting the electro-deposition condition. Hence, electrochemical deposition of calcium phosphate coating has attracted considerable attention in recent years.

Table 2.2 – Different techniques to deposit HAp coatings [97]

2.2.1 Substrate

Titanium (Ti) and its alloys are the most commonly used metallic materials for medical implants in orthopedic and dental applications, due to their low density, non-toxicity, excellent corrosion resistance [2], great biocompatibility and mechanical properties for load-bearing orthopedic application [1]. For many years, surface modification methods for Ti and its alloys have been extensively investigated. The various surface treatments utilize technologies to enhance the adhesion strength, anti-bacterial property, biocompatibility and corrosion resistance of implant coating materials remain challenging.

Among all titanium and its alloys, the widely applied materials in medical application are the commercially pure titanium (cp Ti, grade 2) and Ti-6Al-4V (grade 5) alloy, they are attractive currently gaining increasing attention in science community.

The development on titanium alloys contain of non-toxic elements were suggested and are under development with the increasing continuing. As a result, niobium (Nb), tantalum (Ta), zirconium (Zr), molybdenum (Mo) and tin (Sn) elements are the favorable non-toxic alloying elements for titanium alloys for bio-medical applications. Recently, the titanium alloys suggested in this research and development contain fairly a large amount of Nb , Ta, Zr, Mo and/or Sn such as systems including two elements (Ti–Zr system, Ti–Mo system, Ti–Ta system alloys), systems including three elements (Ti–Ta–Zr system, Ti–Nb–Hf system, Ti–Nb–Zr system, Ti–Nb–Sn system, Ti–Fe–Ta system, Ti–Mo–Ga system, Ti–Mo–Ge system, Ti–Mo–Al system alloys), and systems including four elements (Ti–Nb–Ta–Zr system, Ti– Mo–Zr–Sn system, Ti–Sn–Nb–Ta system, Ti–Mo–Zr– Fe system, Ti–Mo–Nb–Si system alloys) [12, 13, 14, 15, 16, 17, 18]. Some type of Ti-based alloys, as Ti-12.5Mo, Ti-8Al-7Nb, Ti-13Nb-13Zr, Ti-29Nb-13Ta-4.6Zr and Ti-12Mo-6Zr-2Fe, has also been considered for medical purposes. Among the research, Ti6Al4V is the most popular Ti-based alloy, containing Al (5.5–6.5 %wt.) and V (3.5–4.5 %wt.). As the same with the all metallic biomaterials, the mechanical properties of Ti and its alloys depend on %wt. of elements, and the processing technology.

Physically, commercially pure Ti has some characteristic features such as the ultimate tensile strength values of 240 to 550 MPa, yield strength of 170 to 485 MPa, and elongation of 15 to 24%. While, the tensile strength of Ti-6Al-4V alloys is approximately 860 MPa, with yield strengths of 758 (cast alloy) and 795 MPa (wrought alloy), minimum elongation of 8% (cast alloy) and 10% (wrought alloy). Chemically, titanium is in group IV and period 4 of Mendeleev’s periodic table.

The preparation of Ti and its alloy as a substrate before electrolyte deposition process plays an important role, its effect to the obtained HAp coating result was showed in many studies. There is a variety of techniques were presented in the previous research such as mechanical polishing, electro-polishing, chemical etching, plasma treatment, sonication (ultrasonic bath), etc. To enhance the effectiveness and efficiency, several of the techniques are combined. Generally, the substrate surface preparation before the deposition consists of three major steps: mechanical polishing (grinding/abrading and polishing), cleaning and testing the surface cleanliness. Detail of steps for the substrate treatment were illustrated in [Table 2.3].

Table 2.3 – Treatment of Ti and its alloy substrate

2.2.1.1 Mechanical polishing

Mechanical method is first steps in series procedure of the substrate preparing for the electrochemical deposition. Purpose of mechanical polishing is descaling, removal a part of a material layer on the surface and the smooth surface finish by using an abrasive. Mechanical polishing is used to smooth rough surface, clean off the contaminants, and obtain a mirror finish for hard materials as bio-metallic. Rough or smooth surfaces are achieved in the range of μm to mm scale depend on the requirement of experiment. Typically, the mechanical polishing process consists of grinding and polishing. Grinding is usually performed to remove substantial amounts of metal. Polishing is the final process to generate a smooth, highly reflective and mirror-like finish. Mechanical polishing method were used by sandpaper, emery paper, with various grit size, or the kind of powders such as silicon carbide (SiC), aluminum oxide (Al2O3), diamond, cerium oxide (CeO2), glass beads, silica sand, with different particle size. It depends on kind of material and the experiment condition to choose suitable techniques. In fact, the most widely used process were wet-grinded and with SiC paper from 400 to 1200 grit, then they were wet-polished by aluminum oxide particle.

Additional, grit blasting (also called abrasive blasting) also was used as a mechanical method for the preparation of the substrate specimen. It leads to the formation of a porous/wrought layer on the surface through the collision with microscopic particles. Grit blasting uses grit such as aluminum oxide (Al2O3), titanium dioxide (TiO2), or calcium carbonate (CaCO3) are projected by a fluid carrier (compressed air or liquid) onto the surface. During the erosion processes it has an effect to the micrometer scale, depending on the size of particles used for blasting, and allow to obtain the surfaces have an average roughness index of less than 1 mm. In some case, Ca/P ceramic particles with various sizes are used to blast (for Ti, Ti6Al4V, etc.) [39, 40]. According to some reports [41, 42, 43], surface roughness of the titanium obtains in the range 0.5 – 1.5 μm when using alumina particles in the size 25 – 75 μm to grit blasting, while roughness of titanium in range 2 – 6 μm are reported for surfaces blasted with particles size of 200 – 600 μm [44, 45].

2.2.1.2 Cleaning

The substrate surface may be contaminated by naturally occurring process results such as oxides, carbides on metals or plasticized particles on polymers; adsorbed layers form by the absorption or diffusion of material from the surrounding; contaminants particles such as dust, grease on the surface, etc. In some cases, surface contamination may result from the cleaning techniques used.

The cleaning of the substrate surface is an important step to HAp deposition. It is necessary to remove the contaminants that would otherwise affect the properties of the coating. The presence of even tiny amounts of contaminants on the surface such as oil, grease, oxides, etc. cause the reduction of coating adhesion to the substrate. The choice of the cleaning procedure depends on the composition, physical properties and chemistry of the substrate, and the type of contaminants on the surface. There are some methods for cleaning the sample surface: cleaning by solvents, ultrasonic cleaning, cleaning by heating, cleaning by etching, etc. In fact, by the combination between two or more cleaning methods would be obtained the desire result.

Cleaning by solvents: The specimens were gently rinsed by solvents to be sure they always clean. Some common solvents were used such as acetone, ethanol, distilled water, deionized water, ultra-pure water, etc. After finishing of some procedure of the specimen preparation as mechanical polishing, alkaline etching process, anodization, etc. the substrate specimens always were rinsed with distilled water or deionized water. To enhance cleaning, acetone or ethanol were used in the ultra-sonic bath.

Ultrasonic bath: Simple cleaning methods such as wiping, soaking and rinsing with solvents can remove particles and loose contaminants. In order to remove strongly adhered particles on the substrate those methods are not perfectly adequate. Ultrasonic bath (involving the use of ultrasound) has proved to be a more effective technique. Cleaning by this technique is performed with an ultrasonic bath and a suitable liquid. Choice of liquid play an importance role since the use of a suitable solvent or solvent mixture in which the contaminants are soluble will significantly improve the efficiency of cleaning. The common liquids were used as acetone and ethanol for the substrate treatment before HAp deposition. Inside the bath, a transducer generates ultrasonic waves within the liquid with the ultrasonic frequency usually 15–400 kHz. Particles and contaminants on a sample immersed in the bath are removed through the agitation caused by the movement of the voids and bubbles within the liquid. Rinsing with fresh liquid is used to ensure cleaning after sonication. For the experiment of HAp deposition, the specimen was usually cleaned by ultrasonic technique in distilled water, or deionized water, or acetone, or ethanol for about 15 min to 30 min and then dried.

Cleaning by etching (also called pickling): Etching is the technical term when used for removing oxide scales order, Ti and its alloy have an oxide layer (titanium oxide, TiO2) on the surface with a thickness of few nanometers layer on its surface. To obtain clean and uniform surface finishes, acid etching generally leads to a thin (< 10 nm) surface oxide layer. The specimens were immersed in the common acid solution such as HCl, HF, HNO3, with different duration. In the experiment, the samples were treated by in mixture of acids with different proportions. The most commonly used and recommended standard solution for acid pickling of titanium and titanium alloys consists of 10-30 volume-% of nitric acid HNO3 (69 mass-%) and 1-3 volume-% of hydrofluoric acid HF (60 mass-%) in distilled water [46]. The specimens were etched with acid before HAP deposition in order to remove natural oxide layers and increase roughness of surfaces and it was proved in a bulk of reports.

Cleaning by heating: Heating the substrate may remove the volatile impurities. The temperature should be chosen by the melting point and/or surface reactivity of the substrate. Heating is not useful if it causes stresses and cracking due to non-uniform heating or oxidation of the surface.

After cleaning, the samples are dried by in different ways: by the oven or in the air.

2.2.1.3 Another method

Beside the methods mentioned above, in some cases can use other methods such as anodic oxidation (also called anodizing), alkaline etching, etc. also applied to treat Ti surface bring the effect for HAp deposition process.

Anodic oxidation (also referred to as anodizing) was applied for the pre-treatment of the metal substrate, which is a powerful technique that can produce surfaces with improved oxide layer or increased oxide thickness of up to micron size. It is a good established method for producing difference of protective oxide film on metal. In anodic oxidation, electrode reaction in combination with electrical-field driven in metal and oxygen ion diffusion lead to the formation of an oxide film at the anode surface. This method aims to improve the adhesive bonding, increased oxide thickness, porous coating, etc. Some acids are used as electrolytes for anodic oxidation of titanium: solution of H3PO4, NaF, H2SO4, NH4H2PO4, NH4F, CH3COOH, etc. In recent years, many studies have mentioned to this content as well as a method aim to improve the mechanical property of HAp coating by reinforcing materials by nanotubes on surface [19, 21, 22, 24, 25, 26, 31, 35, 47, 48, 49]. An ordered oxide layer can be tailored when the type of electrolyte, applied current density, electrolyte concentration, electrolyte temperature, agitation speed and cathode-to-anode surface area ratios are controlled. In some papers reported about HAp deposition, the substrates were prepared by anodic oxidation with a series of the experimental parameters [Table 2.4].

Table 2.4 – The parameter of anodic oxidation process for Ti

Titanium dioxide (TiO2) nanotube arrays were grown on the surface of titanium with thickness of about 350 nm by the anodization process. With this TiO2 structure enhance the formation of apatite and results indicated that the morphology of gel graphene oxide HAp coating on TiO2 nanotube is more porous and thicker, with better bond strength [19]. Another TiO2 nanotube arrays with diameter of 100 nm induced the formation of apatite and enhances the bond strength of magnesium doped HAp coating [22].

Prior to electrochemical deposition, pre-treating process of Ti by anodization can improve the strength of the HAp on titanium substrate. This result was showed in [48], the average strength of coating deposition on Ti without anodic oxidation is about 5.0 MPa, while the average strength of Ti with anodic oxidation is about 7.3 MPa. In another case, the anodization process is potential in forming oxide layer between the surface substrate and HAp coating, leads to obtain the pure and homogeneous HAP coating [50].

Alkaline etching treatment aims to improve apatite formation and surface roughness [51, 52]. Alkaline solution typically used was sodium hydroxide (NaOH) with different concentration at various temperatures depending on experiment condition, [Table 1.5] illustrates the parameters about alkaline treatment of Ti prior to electrochemical deposition technique.

Table 2.5 – Alkaline treatment

Alkaline etching enhanced the bonding strength of HAp onto nano-porous Ti. The result revealed that the best for surface structure (a ring-like morphology was formed around the neck of the nano-pores) lead to enhance the formation of the HAp coating and improved the bond strength of the coating with the Ti substrate [56].

2.2.2 Electrolyte

In recent years, hydroxyapatite (HAp, (Ca10(PO4)6(OH)2) has been widely used to coat load-bearing metallic implants. The main reason of using HAp coating on metallic substrates is to keep the mechanical properties of the metal such as load-bearing ability, to take advantage of the coating is chemical similarity and biocompatibility with the bone [71]. It has been studied as a coating of dental and orthopedic implants and as a component of bio-polymer composite material. Several techniques have been used to create the HAp coating on metallic implants, such as plasma spraying [5, 72, 73, 74, 75, 76, 77], thermal spraying [78], sputter coating [6, 79], pulsed laser ablation [7, 80, 81], dynamic mixing [82], dip coating [83], sol–gel [9, 84, 85], electrophoretic deposition [10, 86], biomimetic coating [87], ion-beam-assisted deposition [88], hot iso-static pressing [89], and electrochemical deposition [90, 91, 92].

Different coating technologies have been developed over the years as mentioned above, among which electrochemical deposition is the most frequently used coating technique. The use of electrochemical deposition has specific advantages, such as low cost, simplicity of performance, and controllability of the phase, which make this method more popular than other techniques. The electrochemical method allows crystalline HAp to be deposited rapidly from aqueous solution under mild operating conditions. In addition, this method is applied to the medical devices with complex shapes (curved surfaces such as dental implant screws and orthopedic fixation pins) may be evenly coated electrochemically in a single step. This method becomes a popular coating method owing to its ease of processing control, variability of the coating composition and suitability for complex implant geometries.

2.2.2.1 Hydroxyapatite

Hydroxyapatite belongs to the Ca/P family [Table 1.6] and was found in a wide variety of applications in bio-medical such as bio-medical devices, the implants, the repair and reconstruction of bone tissue defects, etc. It brings all the characteristic features of biomaterials: biocompatible (1), bioactive (2), osteoconductive (3), osteointegration (4), osteoinduction (5), non-toxic, non-inflammatory and non-immunogenic properties, etc. About chemical composition, pure HAp has formula Ca5(PO4)3OH but is usually written Ca10(PO4)6(OH)2, it is a naturally mineral made up of calcium and phosphate and is found in human bones. It consists principally of Ca2+ (39.6 wt. %) and PO43- (18.5 wt. % as P) [3], show only the O-H (for OH group) and P-O (for PO4 group) absorption band in the FT-IR β spectra.

(1) Biocompatible: The term refers to the ability of a material to perform with an appropriate host response in a specific situation.

(2) Bioactive: is the ability of the material to directly ‘bond’ to bone through chemical interaction and not physical or mechanical attachment.

(3) Osteo-conductive: This means bone grows on a surface. An osteo-conductive surface is one that permits bone growth on its surface.

(4) Osteo-integration: Direct anchorage of an implant by the formation of bony tissue around the implant without the growth of fibrous tissue at the bone–implant interface.

(5) Osteo-induction: This term means that primitive, undifferentiated and pluripotent cells are somehow stimulated to develop into the bone-forming cell lineage. One proposed definition is the process by which osteo-genesis is induced.

The Ca/P molar ratio of HAp is 1.67, it has the most stable status at normal temperatures and pH value from 4 to 12 [57]. The crystal structure of HAp most frequently displays a hexagonal symmetry P63/m [Figure 2.1] with preferred orientation along the c axis and lattice parameters a = b = 9.432 Å, c = 6.881 Å, γ = 120o. HAp crystals typically display a needle-like morphology. Another crystal structure of HAp is monoclinic P21/b with a = 9.842 Å, b = 2a, c = 6.881 Å, γ = 120o [58]. The hexagonal structure of HAp is typically obtained by the precipitation process from supersaturated solutions at temperature range of 25–100oC, while the monoclinic structure is primarily formed by heating the hexagonal form at 850°C in air and then cooling to room temperature. HAp can appear to have brown, yellow, or green color. In its powder form is typically white. Molecular weight of HAp is 1004.6 g/mol, its theoretical density is 3.156 g/cm3, elastic modulus of HAP is 10 GPa, tensile strength is about 100 MPa.

Table 2.6 – Ca/P with different Ca/P ratio [59]

Figure 2.1 – The crystal structure of HAp (hexagonal) [46]

As mentioned above, HAp is material mainly known for its ability to contact bone tissue. Despite of the HAp contain strength properties; it displays low mechanical and fracture toughness hence HAp is an unsuitable candidate for load-bearing in implant applications. Thus, there are many researchers focus on the combination between of the osteo-conductive characteristics of HAp with enough strength and toughness of Ti and its alloy for load-bearing applications. HAp is applied as a thin coating on metallic orthopedic and dental implants.

The requirements for HAp coatings have been declared in the Food and Drug Administration guidelines as well as in the ISO standards [Table 2.7] [98]

Table 2.7 – The requirements for HAp coatings

The HAp structure allows the substitution of their Ca, PO4 and OH groups by many other ions (both anionic and cationic) lead to change in various structural properties such as crystallizing, lattice parameters, and stability and morphology. For example, the substitution of F for OH, the carbonate (CO3) can substitute for the OH or the PO4 groups. While Ca can be substituted by varied metal cations, e.g. Na, K, Mg, Sr, Ba, Mn and Pb.

2.2.2.2 Electrolyte solution

Components and concentration

Electrochemical deposition process of HAp is a wet chemical method that allow the coatings to be synthesized on metallic substrates under mild conditions. HAp coating grows from electrolyte solution onto the surface of a metal cathode when an electrical current is applied. The coating composition and morphology can be adjusted by series of condition such as the applied electrical current, pH of the solution, temperature, and additives. The electrolyte solution contains dissolved calcium and phosphate ions. The pH was controlled depending on each experiment condition.

The process of electrochemical deposition involves the decomposition of an aqueous electrolyte which, for the formation of the apatite coating on the substrate, must contain calcium and phosphorus ions with the suitable concentration. Depending on experimental condition, the precipitation of difference types of Ca/P such as di-Ca/P dehydrate (brushite, DCPD), HAp, octa-Ca/P (OCP) and calcium-deficient HAp (Ca-def HAp) on the cathode is possible. Typically, HAp coating is obtained thought two steps, beginning process with obtained coating of Ca/P and then transforming to HAp coating. Some studies tried to use di-Ca/P di-hydrate coating as an initial step to obtain HAp coating. Formation of bio-active HAp coatings were performed by the immersion of specimen with di-Ca/P di-hydrate (brushite) coating in Ringer’s solution with
pH = 7.1 and pH = 8.91 and in Hank’s Balanced Salt (HBS) solution of pH = 7.4 for 24h and 48h, the thickness of obtained coatings is in the range 10 – 20μ [60]. On another study, the obtained result of cathodic polarization process were Ca/P di-hydrate (brushite) coating, and this coating conversion was made via the immersion in one of the aqueous solution: NaOH (pH = 11), Ca(OH)2 (pH = 11.5), Mg(OH)2 (pH = 10.2) at 80oC for 2h with a surface/volume ratio of 4ml/cm2 [61]. Additionally, the transformation was carried out at temperature in deionized water, deionized water with added calcium ions, and modified Hank’s type solution without calcium and magnesium ions, modified Hank’s type solution with calcium and magnesium ions, and modified Hank’s type solution with added calcium ions [62]. Similar results were obtained, in another study, after treatment of deposited brushite coating by the immersion in NaOH with 600 ppm H2O2 for 1 hour; SEM image presented the crystallized coating of single-phase HAp of needle-like crystals, and EDX analysis showed that the Ca/P ratio of electrodeposited HAp is 1.69 [63]. In addition, in report [64] is indicated that initial the as-deposited coating mainly consisted of di-Ca/P dehydrate (DCPD, CaHPO4.2H2O) and β-tri-Ca/P (β-TCP, Ca3(PO4)2), and after immersion in 1M NaOH solution for 2h they transformed into HAp with uniform flake-like morphology. Conversion of the Ca/P coating to HAp upon soaking in Simulate body fluid (SBF) is also a method that was applied [65], the brushite coating after deposition was immersed in SBF for 7 days, and the obtained HAp coating had sphere-like morphology.

On the contrary, HAp coating on the substrate also could directly obtain through deposition process. This is achieved either by increasing the temperature during deposition, using electrolytes of higher pH (and consequently lower Ca and phosphate ion concentrations) [66] or combinations of these conditions.

The formation of HAp coating on the substrate is affected by various factors such as electrolytic solution composition or concentration, electric current density, deposition time, and pH value. These factors can be changed to control the purity, crystalline, stoichiometry, morphology and mechanical strength of the resulting coatings. On the study where was performed by electrochemical deposition of HAp [67] was shown that the coating consists of mixed phases of di-Ca/P dehydrate (brushite) – DCPD) and octa-Ca/P (whitlockite) – OCP) and was formed at various current densities without the addition of hydrogen peroxide (H2O2). In contrast, the addition of H2O2 lead to the apparition of HAp phase. However, the concentration of H2O2 effects the morphology of Hap: with the concentration ranging from 600 to 2000 ppm was obtained the pure HAp layer, on the contrary increasing it to 3000 ppm the coating is not uniform.

The main HAp phases produced by the electrodeposition process depend on the electrochemical conditions such as electrolyte concentration and pH, applied voltage, ionic strength of the electrolyte, electrolytic temperature, cathodic surface state, solution agitation and ionic species in the electrolyte and several other factors. Among of them, the electrolyte composition plays an important role to affect the obtained HAp coating. Additional, when referring to the electrolyte solution the emphasis was on the following factors: composition of the electrolytes, concentration, pH, temperature, and additives.

The electrolyte solution provides the ions to be electrodeposited. Electrolyte solutions used for deposition of HAp were salt solutions containing Ca2+ and PO43- ions. Typically, situation for ultrafine-grained nano-phase HAp coating obtained through electrochemical deposition was the use of salts dilution of the calcium nitrate Ca(NO3)2 and ammonium dihydrogen phosphate NH4H2PO4 in distilled water or deionized water. In many cases peroxide H2O2 was used to substitute water; its effects onto the result of the obtained coating will be mentioned below. Note that ensuring the quality of electrolyte solution relates to completely dissolving of the salts in the aqueous solution. Magnetic stirring is used to control heat for electrolyte solution. On the other hand, stirring with a constant speed allow to keep uniform concentration of the electrolyte. In many cases, electrolyte solution was stirred during the deposition.

Although the precursors used for the electrodeposition of pure HAp coatings were Ca(NO3)2 and NH4H2PO4, in fact Ca(NO3)2 can be substituted by calcium nitrate tetra-hydrate Ca(NO3)2·4H2O or calcium chloride CaCl2 and NH4H2PO4 can be substituted by dipotassium hydrogen phosphate K2HPO4. Concentration of electrolyte solution must keep a constant ratio of Ca/P always at 1.67 similar to Ca/P ratio of HAp. Typically, electrolyte and its concentration were used for deposition of HAP were 0.042 M Ca(NO3)2 and 0.025 M NH4H2PO4. Besides that, in some reports was also used electrolyte solution with another concentration [Table 2.8].

Table 2.8 – Electrolyte solution for HAp coating on Ti and its alloy

Additives

Deposition process of HAp requires an enough volume of electrolyte to surface ratio; approximately 20 ml of electrolyte is needed to coat 1 cm2 of the specimen. In addition, various electrochemical reactions occurring with the ions in the electrolyte solution near electrodes during the deposition. The OH– ions are created at the surface of cathode (substrate) due to the chemical reaction which is always the reduction reaction of water H2O lead to formation of large amount hydrogen (H2) gaseous bubbles.

2H2O + 2e‒ = H2 ↑ + 2OH‒

These H2 gas adheres onto the surface of the substrate and causes poor adhesion of the HAp coating on the substrate; result can be seen on the coating: cracks, craters and gas pores. To overcome the withdraw, some ways are reported in the studies.

To obtain the effective coating on the surface of the substrate some additives were added in the electrolyte solution during the electrochemical deposition process such as the enhancement of the adhesion of the HAp coating on the substrate. The addition of peroxide (H2O2) into the electrolyte solution were widely used. Indeed, H2O2 is a strong oxidative reagent and this is a method that could improve the adhesiveness between coating and substrate in the electrodeposition process which have showed in many studies. There by, amount of OH– at the substrate surface is increased and leads to improvement of the quality of electrodeposition.

H2O2 + 2e- → 2 OH –

Additionally, some studies showed that to improve the ionic strength and conductivity of electrolyte solution sodium nitrate (NaNO3) was mainly used due to the chemical reduction of NO3‒ ions that also generate OH‒ such as in reports [23]. Nitrogen gas (N2) was used as a method aim to reduce the amount of dissolved carbon dioxide to prevent the formation of calcium carbonate (CaCO3) deposit; electrolyte was de-aerated before and during the experiments. Besides the role of the improvement of the ionic strength, NaNO3 solution also benefited to refine the crystal grain of HAp. At 110 V of the voltage value, the addition of NaNO3 changed the morphology of HAP coating from rod-like to lotus-like with 0.33 μm particle size and improved the density of HAp particles. Other salts also dissolved in the electrolyte solution for ionic conductivity was Ag NO3 [21], KNO3 [25], NaCl [27].

Nitrogen gas (N2) was used as a method aim to reduce the amount of dissolved carbon dioxide to prevent the formation of calcium carbonate (CaCO3) deposit; electrolyte was de-aerated before and during the experiments [34, 37].

pH value

In fact, the deposition process of the HAp coating can take place at ambient temperature and neutral pH value. It is also possible to change the pH value of the electrolyte solution for HAp deposition from 4.0 to 6.5 and adjust the electrolyte composition by using dilute solution with HNO3, NH4OH, NaOH, HCl, NH3·H2O [Table 2.7]. Effect of pH value on coating thickness and weight increasing were revealed by [63], according to two sets of experiments which were performed with pH value range from 3.8 to 10. Otherwise, the thickness and weight of HAp coating reduces when increasing pH value more than 8.8. In other words, one can produce the precipitation of HAp on the bottom of the electrolytic cell rather than its coating on the cathode (substrate).

Temperature

The temperature of electrolyte for HAp deposition produce effect on mass of HAp coating. An experiment was performed to prove that the mass of HAp coating increases when the temperature reaches to 70oC, nevertheless HAp coating mass reduces with continuous increasing of the temperature to 85oC [68].

2.2.3 Applied voltage

There are two conventional electrochemical techniques used for HAP coating on the substrate, which are galvanostatic (constant current) and potentiostatic (constant potential) coating techniques. Additional, pulse-electrodeposition techniques were also used, in this techniques potential or current is applied as a pulse instead of a constant mode.

Galvanostatic/potentiostatic HAP coating

In galvanostatic method, a constant current is applied for a period, whereas a constant potential is applied for potentiostatic method. The potentiostatic method is a potential technique to overcome the hydrogen evolution problem performing a more homogeneous coating [69]. The influence of current density on the HAp coating composition, micro-structure and bonding strength between coating and substrate were investigated in [33], the current density values applied were of 1.25, 1.87, 2.50, 3.12 and 3.61 mA/cm2. The increase of current density could enhance the ion migration and develops the reaction speed in the electrolyte leading to increase of the hydrogen gas. The escaping hydrogen gas would lead to the HAp coating to be broken away from the substrate due to the thickness of the reducing of HAp coating. The HAp coating thickness was about 21.8 μm with current density at 2.5 mA/cm2 and the bonding strength between the substrate and HAp coating was 18 MPa. Additionally, the crystals structure change from porous to dense and then back to porous when increasing current density.

On other study, the influence of the applied current density on the phase composition, crystalline domain size and morphology were investigated [65]. The increasing of deposition time up to 25 min and current density reached 7 mA/cm2 do not affect significantly the mass of the coatings. At 30 min and at 7 mA/cm2 the mass of the coatings was the greatest, and over those values would not influence the coating mass. With the increasing of the current density would lead to the increase in porosity due to increased generation of hydrogen bubbles that conduct to smaller crystalline forming.

Pulse-electrodeposition techniques

In pulse-electrodeposition method, potential or current is applied as a pulse instead of a constant mode; it involves on–off cycles. In principle, off cycle discharges the negatively charged layer and allows the ions to diffuse towards the substrate and results in a more evenly distribution of ions for coating.

Current research show that researchers more interested on the pulsed electrochemical deposition technique aim to give the HAp coatings with much advantages including simple use, high purity of coatings, easy adjust of the experiment conditions and low cost [93, 94, 95, 96]. The pulse technique allows the properties and structures of coating to be controlled and improved by adjusting pulse parameters such as pulse on time, pulse off time and current density. [75].

The effect of pulsed voltage on the morphology by the electrochemical deposition techniques were reported at [69]. The voltage values were applied in range of 90 – 120 V. With the increasing of voltage up to in range of 100 – 110 V, the obtained HAp coating had lower porosity and is more homogeneous, and the size of HAp particles were reduced from 0.35 to 0.25 μm. When voltage value was reached to 120 V, the surface of HAp coating appeared to be much heterogeneous with the size particle of 0.3 μm. On another study [70], the obtained result showed that the pulse-electrodeposition techniques could be improved the degradation behavior of AZ31 magnesium alloy. In addition, the study also compared the obtained effect when applied both direct and pulsed voltage. The XRD indicated that the peak intensity of HAp coating prepared by pulsed voltage is stronger.

Pulse-electrodeposition is a potential technique to overcome the hydrogen evolution problem observed in some reports. The influence of using pulsed current on the crystallinity and adhesion of HAp films deposited via electrochemical deposition on titanium substrate has been investigated at [27]. The results showed that at low current densities pulsed deposition increased the thickness, the crystallinity and the adhesion of the HAp coating. On the contrary, at the high current densities also increased thickness, however due to the generation of the hydrogen bubbles lead to, at both high constant current densities and high pulsed current densities, low crystalline and poor adhesion of the HAp coating.

2.2.4 Time

To investigate the effect of the deposition time on HAp coating, different time was applied in range of 20 – 60 min [69]. The results showed that in the range of 20 – 40 min the porosity of HAp coating decreases, the obtained HAp coating was denser, and the thickness was increased. However, the time value overcoming 40 min point and up to 60 min conducts to the porosity decreasing continually, and the thickness decreasing too. Hence, the investigation concluded that the optimum deposition time range was of 30 – 40 min. The study showed that the obtained coating with the smallest crystalline domain size of 15.6 nm and the greatest porosity was obtained at higher current density of 9 mA/cm2. On the contrary, the less porous coating with the largest crystalline domain size were obtained at lower current density with range of 5 – 7 mA/cm2.

2.2.5 Post-treatment

After the coating process, the specimens were removed from the electrolyte and subsequently washed with distilled water to remove residual electrolyte and then dried at room temperature. After the chemical deposition process, the obtained coating could be a mixture of different types of Ca/P such as di-Ca/P dehydrate (brushite, DCPD), HAP, octa-Ca/P (OCP) and calcium-deficient HAp (Ca-def HAp). The almost the deposited samples would be treated to obtain a pure HAp coating and this content was mentioned above. However, in this part, it was mentioned as one of the steps to treat deposited specimen in some studies.

With the alkaline method (NaOH solution with concentration about 0.1 – 0.25 M, at temperature about 60 – 80oC), the cleaning (with distilled water, ultrapure water) and drying (at 60 – 450oC) were applied much. Thereby, the Ca/P would be transformed to HAp.

Heat treatments promoted as post-treatment technique for HAp coating to investigate. The other effect of heat treatments is the increase in the adhesive strength of the coatings.

2.5 Composite coating of HAP formed by electrochemical deposition

Composite coating of HAp with inorganic or organic additives were attended by many studies. As studied previously, the capacity to incorporate various ions into the HAp coating has been exploited with electrochemical deposition by some reports [Table 2.9]. Additionally, one of the most common approach to provide better corrosion resistance, incorporation of reinforcing materials like CNTs, TiO2, etc. is followed in some studies [Table 2.10]. In addition, recently some studies have been performed to prove that the composite coatings are promising materials for the fabrication of implantable biomaterials with advanced functional properties.

Table 1.9 – The capacity to incorporate various ions into the HAp coatings

Table 2.10 – The incorporation of reinforcing materials into the HAp coatings

2.6 Silver doped hydroxyapatite coating on titanium and its alloys

Hydroxyapatites do not show any antibacterial activity. In order to overcome these problems, a possible solution that could prevent bacterial infections is to modify the implant surfaces with antibacterial coatings while maintaining good biocompatibility. Antibacterial materials were added into the HAp coating to effectively inhibit infection. These substitutions may alter the crystal structure and induce some changes in the materials properties like phase stability and reactivity. With respect to its biological usage, this may also change the bioactivity, biocompatibility along with some material’s surface characteristics. In the recent years, the use of inorganic antibacterial agents has attracted interest in control of microbes. The calcium in hydroxyapatite can be easily replaced by various metal ions, which results in enhanced osseointegration, biological activity, and antibacterial properties. To improve the antibacterial property of HAp, various metallic nanoparticles such as zinc (Zn), copper (Cu) and silver (Ag), strontium (Sr), etc. exhibit antibacterial properties are incorporated into HAp through substitution Ca2+ ion. Compared with other heavy metal ions, the silver-based an antibacterial type of metal ions have captured much attention and are used in different bio-medical fields because of its broad spectrum antimicrobial activity, has strong inhibitory effect, have high efficiency and low toxicity for long-term use [107]. The cationic change rate of HAp is very high with silver ions, thus silver ions substituted HAp was the most commonly used to create antibacterial coating [108, 109, 110]. In addition, silver was chosen into HAp coating due to Ag-doped HAp is active against the most common bacterial are Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Staphylococcus aureus and Bacillus subtilis) [111, 112, 113].

Silver nanoparticles or silver atoms incorporated into HAp coatings provide antimicrobial properties while maintaining the bioactivity of HAp. Several methods have been used to create silver-containing HAp coatings, including electrochemical deposition [114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124], plasma spray [125, 126, 127, 128, 129, 130, 131], magnetron sputter coating method [132, 133, 134], sol-gel synthesis [135, 136, 137, 138], hydrothermal method [139, 140], wet chemical method [141], precipitation [142, 143], dipping [144, 145], ball milling [146], ion exchange method [147], etc. and other methods. These methods are line-of-sight technologies, each method contains advantages and disadvantages. Depend on the aims, the condition of the experiment chooses the effective method. However, the uniform distribution of Ag in coatings is still a challenge.

Different coating technologies have been developed over the years as mentioned above, among which electrochemical deposition is the most frequently used coating technique. The use of electrochemical deposition has specific advantages, such as low cost, simplicity of performance, and controllability of the phases, which make this method more popular than other techniques. In recent years, various approaches have been examined for the introduction of silver into HAp coating on Ti and its alloy substrate through electrochemical deposition process. Nevertheless, electrochemical deposition on HAp coatings also has numerous disadvantages, including the weak bonding strength at the coating metal interface. However, the success of an orthopedic implantable biomaterial depends not only on the bone implant integration, but also on having a sterile environment around the implant, which prevents bacterial-related infection.

The electrochemical method allows crystalline HAp to be deposited rapidly from aqueous solution under mild operating conditions. In addition, this method is applied to the medical devices with complex shapes: curved surfaces such as dental implant screws and orthopedic fixation pins may be evenly coated electrochemically in a single step. This method becomes a popular coating method owing to its ease of processing control, variability of the coating composition and suitability for complex implant geometries. The HAp/Ag composite coatings were produced using electrochemical deposition by adding silver powder in the electrolyte.

As the preparation of substrate before deposition for HAp coating were reported by previous paper, with Ag-containing HAp coating, it also plays an important role, its effect to the obtained HAp-Ag coating results was showed in many studies. There are a variety of techniques for preparing biomaterial surface before HAp-Ag deposition such as mechanical polishing, electro-polishing, chemical etching, plasma treatment, sonication (ultrasonic bath) etc. To enhance the effectiveness and efficiency, usually several of the techniques have combined each other. Typically, the substrate surface preparation before the deposition consists of three major steps: mechanical polishing (grinding/abrading and polishing), cleaning and testing the surface cleanliness. Several technologies for treatment of the substrate before deposit HAp-Ag coating are available, above on them some are presented in [Table 2.11].

Typical technique to enhance adhesive strength of HAp-Ag coating on the substrate is anodization pre-treatment which was mentioned in various previous research [114, 121, 122].

Table 2.11 – Treatment of substrate for HAp-Ag coating on Ti and its alloy

The electrolyte solution provides the ions to be electrodeposited. Electrolyte solutions used for deposition of HAp were salt solutions containing Ca2+ and PO43‒ ions. Typically, situation for ultrafine-grained nano-phase HAP coating obtained through electrochemical deposition was the use of salts dilution of the calcium nitrate Ca(NO3)2 and ammonium di-hydrogen phosphate NH4H2PO4 in distilled water or deionized water with Ca/P molar ration 1.67 the same molar ratio of Ca/P in HAp. To obtain HAp-Ag coating, as the resource of silver AgNO3 was added into the electrolyte [Table 2.12].

Table 2.12 The conditions of the electrolyte solution before electrodeposition process of

co-substituted HAp.

The uniform distribution of nano-sized Ag in the HAp coatings is one of the critical factors determining the successful application of HAp-Ag coatings. The key to success is to carefully adjust the composition of the electrolyte and the processing parameters to control the deposition rate of HAp and Ag. The reequipments of coating on surface is the uniform distribution therefore the deposition rates of two phases in the composite coatings should match each other. To solve this problem, cysteine (Cys, HSCH2(NH2)COOH) is put into the electrolyte as the coordination agent to stabilize Ag ions [117]. Cysteine is an amino acid, inexpensive, simple and environmentally friendly, and has a strong affinity to metal ions to form metal–ligand complexes, which have been used to prepare inorganic nano-crystals.

In other ways, the information of HAp-Ag coating on the substrate were took place through two steps. In the first step, a Ti substrate was coated with HAp through electrochemical crystallization from mixed aqueous solution (138 mM NaCl, 1.3 mM CaCl2, 50 mM tris amino-methane, 0.84 mM K2HPO4) with pH = 7.2 at T = 95oC and I = 12.5 mA/cm2 in t = 2 min. In the second step, silver nanoparticles were deposited onto the HAp layer through electrochemical reduction of aqueous Ag+ to Ago with mixed aqueous solution (125 ml NaCl and 125 ml AgNO3) at T = 95oC. The obtained result was silver nanoparticles distributed uniformly on the HA coating. Additionally, the quantity and size of silver nanoparticles were controlled by electrochemical deposition time under applied current density [115]. Similarly, Liu et al [124] also tried to deposit Ca-P coatings on Ti surfaces and subsequently electroplate silver on the surfaces of Ca-P coatings in nicotinic acid bath.

In other case with the multi-ionic substitution, not only Ag was doped into HAp, there are some cations were added as a second binary element to offset the potential cytotoxicity of Ag. Co-substituted HAp has attracted attention in recent years. One of the secondary chemicals appropriate for Ag-doped HAp in bone-associated implants is strontium (Sr). Results showed that Sr2+ and Ag+ could be evenly incorporated into the HAp lattice to form Sr-HAp-Ag coatings [121]. The role of zinc (Zn) as a cationic substitution within hydroxyapatite is to counteract the effects of osteoporosis. The zinc ions can affect the body and the skeleton. In a previous research introduced to co-substitution of Ag and Zn into HAp coating [119]. Considering the importance of Chitosan (CS), which is a widely used bio-polymer, because it exhibits non toxicity, biodegradability, antibacterial and hemostasis properties. CS also exhibits a broad-spectrum of antimicrobial activities against Gram-negative and Gram-positive bacteria. Due to these advantages, CS also was mentioned to prepare the chitosan-silver/hydroxyapatite (CSAgHAp) composite antibacterial coatings on titanium (Ti) in aqueous solutions by electrochemical deposition [122]. The electrolyte solutions used for electrochemical deposition process of co-substituted HAp was demonstrated in [Table 2.13].

Table 2.13 – The conditions of the electrolyte solution before electrodeposition process of co-substituted HAp.

Based on the above advantages of pulsed electro-deposition, previous research used this technique to obtain HAp-Ag coating on the substrate. Liu et al [124] demonstrated that HAp-Ag coating was performed through pulse electro-deposition by adding Ag silver into the electrolyte solution. A previous research demonstrated the possibility of co-depositing HAp and Ag simultaneously by pulsed electrochemical deposition, which generated a uniform distribution of Ag particles in the coating [123]. The experiment was employed by pulsed electrochemical deposition to co-deposit HAp and Ag simultaneously, which realized the uniform distribution of Ag particles in the coatings. The results indicated the as-prepared coatings had good antibacterial properties and biocompatibility [117].

Hydroxyapatite doped with Ag+ ions (HAp-Ag) was synthesized via electrochemical deposition method on anodized titanium to improve antibacterial activity and cell-material interactions was performed by supplying a constant current density of 0.85 mA/cm2 for 2100 s. The obtained HAp-Ag coatings with 2.03 wt.% silver, did not only show highly significant antibacterial properties but also considerable biocompatibility and low toxicity in vivo. The corrosion tests also indicated the HAp-Ag coatings have efficient corrosion resistance [114]

To deposit HAp-Ag coating on the substrate, these experiments were carried out on electrochemical workstation. A conventional three-electrode electrochemical cell was used: the specimen as the working electrode (WE), a platinum foil as a count electrode (CE) and saturated calomel (SCE) as the reference electrode. The parameters of electrochemical deposition were demonstrated via [Table 2.14].

Table 2.14 – The parameter of electrochemical deposition

After the chemical deposition process, the obtained coating could be a mixture of different types of Ca/P such as di-Ca/P dehydrate (brushite, DCPD), HAp, octa-Ca/P (OCP) and calcium-deficient HAp (Ca-def HAp). With the alkaline method (NaOH solution with concentration about 0.1 – 0.25 M, at temperature about 60 – 80oC), the cleaning (with distilled water, ultrapure water) and drying (at 60 – 450oC) were applied much. Thereby, the Ca/P would be transformed to pure HAp coating [114, 121].

Heat treatments promoted as post-treatment technique for HAp-Ag coating to investigate the effect of it on the antibacterial properties was showed in a recent research [117]. The specimens went through heat treatments at one of designated temperatures (700°C, 800°C and 900°C) for 2 h in a furnace at a ramp speed in 21 min and then cooled in the furnace. By the comparison of the antibacterial effects of the heat-treated and untreated HAp-Ag coating author indicated that the heat-treated coatings nearly exhibited the same bactericidal ability as the coatings without heat treatments, which suggested that heat treatments were not detrimental to the antibacterial properties of the coatings [Table 2.15]. Moreover, pure HAp is stable until
1300°C but it decomposes at 700°C with the existence of Ag nanoparticle. In addition, the effect of heat treatments on the biocompatibility of HAp-Ag coatings was investigated. From the result [117] showed that it enhances the biocompatibility of Ag-HAp coatings. Moreover, the other effect of heat treatments is an increase in the adhesive strength of the coatings. The adhesive strength of HAp-Ag coatings was detected of 9.7 ± 0.6 MPa before heat treatments and is raised to 14.1 ± 0.9 MPa after heat treatments process, which show that silver is useful for the enhancement of the adhesion between the coatings and Ti substrates.

Table 2.15 -CFUs of antibacterial tests and bactericidal ratios of the coatings (n = 3) [117]

2.7 Zinc doped hydroxyapatite coating on titanium and its alloys

Zinc (Zn) is one of the most abundant nutritionally essential elements in the human body and has basic safety for bio-medical applications. Zn is found in all body tissues with 85% of whole the body zinc in muscle and bone, 11% in the skin and the liver and the remaining in all the other tissues. In multicellular organisms, virtually all zinc is intracellular, 30–40% is in the nucleus, 50% in the cytoplasm, organelles and specialized vesicles (for digestive enzymes or hormone storage) and the remainder in the cell membrane. The daily requirement of Zn in the human body is found to be 15 mg [148].

Zn substitution in HAp has been the attention of widely interest because of its presence in all the tissues and its wide roles in biological functions, such as enzyme activity, nucleic acid metabolism, etc. Many efforts are being a try to develop modified calcium phosphate coating, especially HAp coating on metallic biomaterials in order to enhance their biocompatibility, the corrosion and degradability properties, etc. Previous studies have shown that the structure of HAp can substitute readily cationic and anionic. The incorporation of Zn in HAp has a considerable influence on the mechanical and physicochemical properties of HAp-Zn coating. Many approaches, the main methods of preparation of HAp-Zn coating, which have been reported, were sol-gel [149, 150, 151, 157, 158], chemical precipitation [152, 153, 154, 160, 161, 162, 163, 164, 165], hydrothermal [155, 156], ion-exchange [159], wet chemical method [166], plasma electrolytic oxidation [167], electrochemical deposition [119], etc.

The role of Zn as a cationic substitution for Ca in apatite structures of HAp, in most studies, HAp-Zn coating were obtained using zinc nitrate (Zn(NO3)2) [166], zinc chloride (ZnCl2) [162] and zinc acetate (Zn(CH3COO)2.2H2O) solutions [167].

Due to its antibacterial properties, some previous research has presented studies on hydroxyapatites doped with zinc ions to confirm effective bioactivity and antibacterial properties [160, 161]. Besides to their biological properties, HAp-Zn coating confirmed its inhibitory effect on the development of bacteria including E. coli, S. aureus, C. albicans, and Streptococcus mutans [158].

2.8 Characterization method for hydroxyapatite deposited layers

The surface HAp coating has a variety of characteristic properties. Among various characterizations, the most important ones related to biomaterials are chemical, physical, mechanical, and biological in relation to their surface and bulk properties. Chemical properties relate to the materials include composition, bonding and structures. Physical properties consist of micro-structures, phases, density and porosity. Mechanical properties deal with strength and toughness. Biological properties show how materials behave in a body environment. [168].

There are several advanced techniques available for characterization of HAp surface coatings and some excellent reviews have already been published in this area. Generally, techniques include as follows:

(1) Photon-based: Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray photo-electron spectroscopy, X-ray diffraction;

(2) Microscopy techniques: scanning electron microscopy, Energy Dispersive X-ray Spectroscopy and transmission electron microscopy;

(3) Mechanical characterization techniques: Adhesion Testing (The tensile test (pull-off) method, Scratch Test method), Roughness Testing (Atomic force microscopy);

(4) Another Testing: Contact angle method.

However, the choice of technique is dependent on a lot of factors. Consideration is required of factors such as availability or cost, the destructive or non-destructive nature of the technique, resolution, the requirement of data acquisition etc. Previously, for the thick of hydroxyapatite coatings as well as the characterization of the hydroxyapatite coatings, traditionally infrared spectroscopy, X-ray diffraction, electron microscopy and mechanical testing were used. Currently, these techniques are still valuable for analyzing hydroxyapatite coatings, specifically many other techniques have also proven to be powerful in the characterization of hydroxyapatite coatings.

2.8.1 Photon-based techniques

2.8.1.1 Infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a typically used for analytical technique, that show the characterization of biomaterials. Infrared (IR) spectroscopy is technique based on the interaction of electromagnetic radiation with molecules, it is use fast and convenient. This method reveals for the variations of structural characteristic groups and vibrations bonds. To chemical analysis of HAp coating, two important methods are used, complementing each other: Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). XRD method is useful to use for characterization, providing data about the crystal structure of material and its phase composition, however, it has disadvantages is not convenient to examine the amount of hydroxyl (OH-) or carbonate (CO32-) groups in HAp.

FTIR method, in many cases is more sensitive than XRD when determining presence of new phases. In a general way, FTIR provides useful information about location of peaks, their intensity, width and the shape in the required wave number range and determining phase composition. Most molecules have functional groups that absorb radiation in the mid-FTIR range, which is found between 4000 and 400 cm-1. This technique is sensitive to the presence of chemical functional groups in a sample, thereby allowing identification of structural fragments of the molecules. Using FTIR could be examined HAp base on considering three spectrum parameters [169]:

(1) Location of absorption maximum indicates the composition of HAp, even slight variations of that composition influence energy of bonding lead to influence frequency of variations;

(2) Peak width shows degree of the atoms’ order in the apatite elementary cell;

(3) Considering the absorption maximum of (OH‒) vibrations, presence of HAp and its thermal stability can be determined, as well as hydroxyl group concentration in the sample.

A biological HAp usually has a calcium deficiency; it is always substituted with a carbonate. Two types of carbonate substitution are possible: direct substitution of (OH‒) with (CO32‒) or substitution of (PO43‒) with (CO32‒). The most characteristic chemical groups in the FTIR spectrum of synthesized HAp are (PO43‒), (OH‒), (CO3‒) as well as (HPO42‒) [170].

Theoretically, there are four vibrational modes present for (PO43‒) band v1 (symmetrical stretching vibration), v2, v3 (anti-symmetrical stretching vibration) and v4 (bending vibration). The v2 band is a weak band, not as strong as the v3 and v4 bands. The v3 was calculated and used to determine phosphate/carbonate ions ratio and the v3 domain appears to be the most affected by (CO32‒) substitution [171]. Additional, (CO32‒) band have four vibrational modes, three of which are observed in the infrared spectrum and two of which are observed in the Raman spectrum [172]. Essentially, the v2 and v3 vibrational modes are observed in the infrared spectra and the v2 has only one site, the v3 has three sites. The carbonate v4 bands have very low intensity and are seldom seen in the infrared spectrum [173] so it was usually observed in the Raman spectrum. The (OH-) group was considered to vibration mode of adsorption water.

In many other cases, the HAp composite coating were observed by FTIR beside the chemical groups in the formation of phase pure HAp also detected other chemical groups such as C-O stretching vibration, C-OH stretching vibration, C-O-H deformation vibration, C=C stretching vibration, C=O stretching vibration of COOH groups at 1055 cm-1, 1222 cm-1, 1392 cm-1, 1623 cm-1 and 1730 cm-1, respectively [20] [Figure 2.2]. Additionally, a peak noted at 1610 cm-1 of NH2 absorption band in chitosan, and peak of C=O at 1635 cm-1 due to the coordinate interaction between Ag ions and the NH2 groups of chitosan [21] [Figure 2.3].

2.8.1.2 Raman spectroscopy

Raman spectroscopy is a convenient technique for qualitative analysis, especially the identification for functional group and molecular structure. Raman vibrational spectra show all aspects of the molecular structure such as frequencies, intensities, etc. The complementary information can be found from IR spectroscopy. As Raman and IR spectra examine many bands at practically the same frequency with different intensities.

For HAp coating investigated by Raman spectroscopy technology would complement information about the internal (PO43‒) band and essentially, the v3 vibrational modes are observed [175] [Figure 2.4].

Figure 2.4 – Raman spectra of samples HA1/60, HA2/60, HA3/60 and ceramics HA-COM (Reprinted from [175])

2.8.1.3 X-ray Photoelectron Spectroscopy

X-ray photoelectron spectroscopy (XPS) operate based on the phenomenon of photoelectric effect for chemical analysis, is a powerful technique for detecting the chemical elements in the near surface region of a sample. The technique can be used for analyzing all elements, except hydrogen and helium, in the near surface region of 1 to 10 nm depth [174]. The relative atomic concentrations were determined from the C 1s, O 1s, Ca 2p, and P 2p peaks by using the software. The amounts of elements on the surface were quantified by molar percentages. XPS narrow survey scan spectra for Ca, P, O, and C were obtained.

Additionally, the XPS was used to quantitatively determine surface components and compositions of the sample. For example, the chitosan-silver/hydroxyapatite composite coatings on TiO2 nanotube was investigated by XPS [21], the binding energies of Ca (2p, 347.3 eV), P (2p, 133.09 eV), O (1s, 532.1 eV), Ag (3d5/2, 367.5 eV) and Ag (3d3/2, 373.5 eV) [Figure 2.5a]. Binding energies of Ag (3d5/2) core level for Ag, Ag2O, and Ag-O are 368.6, 368.2, and 367.8 eV, respectively [Figure 2.5a].

Figure 2.5 (Reprinted from [21])

XPS general spectrum of the prepared CSAgHAp coating (a) and deconvolution of Ag (3d) XPS peak (b)

On another study [23], C 1s, O 1s,Ca 2p, P 2p peaks were detected in the XPS spectra of pure HAp coating and single-walled carbon nanotubes/HAp composite coatings [Figure 1.6]. Moreover, the elemental contents of Ca and P in the coatings were determined by the ratio of the area under their respective elemental peak in the XPS narrow scan spectrum. The Ca/P ratios calculated directly from the XPS data were 1.42 for pure HAp coating. With the increase of the single-walled carbon nanotubes concentration to 0.5 and 1.0 mg/mL, the measured Ca/P ratio reached to nearly 1.64, which was closed to 1.67 of stoichiometric HAp.

Figure 2.6 – XPS profile of SWNTs/HAp composite coatings and pure HAp coating (Reprinted from [23])

2.8.1.4 X-ray diffraction

X-ray diffraction is an analytical technique which uses the diffracted intensity of a monochromatic X-ray beam but do not destroy the surface. This technique provides the information about the crystallographic structure, chemical composition, and physical properties of materials and thin films. In addition, it can be used to calculate precise lattice parameters, crystallite size, preferred orientation, and lattice stress.

X-rays are produced by bombarding a metal target (Cu, Mo, Cr usually) with a beam of electrons emitted from a hot filament (often tungsten). The incident beam will ionize electrons from the K-shell (1s) of the target atom and X-rays are emitted as the resultant vacancies are filled by electrons dropping down from the L (2p) or M (3p) levels. These gives rise to Ka and Kb lines.

X-ray diffraction methods have long been widely applied to characterize HAp coating. On the study [67], the phase and composition of the HAp coating were analyzed with Cu Kα radiation with λ=1.5406 Å generated at 35 kV and 25 mA. Data collected over the 2θ range 20 – 60° and also at the low angle (< 20°) with a step size of 0.010° and a count time of 0.2 s. [Figure 2.7] displays the X-ray diffraction patterns of the as-deposited coatings on 316L SS without H2O2 into the electrolyte at various current densities; the main composition of the coating layer was di-calcium phosphate di-hydrate with octa-calcium phosphate on the 316L SS substrates. On the contrary, addition of H2O2 with concentration of 1000 ppm into the electrolyte lead to change composition of the coating phase [Figure 2.8].

2.8.2 Microscopy techniques

2.8.2.1 Scanning electron microscopy

A scanning electron microscope (SEM) is typically applied to examine about the topography and composition of sample's surface. It is an electron microscope, works based on scanning with a focused beam of electrons, the electrons interact with atoms in the sample to obtain various signals, finally result give images of the sample's surface.

Although SEM can’t have good resolution like transmission electron microscopy (TEM) although SEM don’t destroy the sample and can operate at the low vacuum. Another strong point of SEM is simple controls more than TEM, SEM costs are much lower compared with TEM, and SEM is much more popular than TEM.

The surface morphology of the hydroxyapatite coatings was examined by scanning electron microscopy. The morphology of HAp coating is very diverse and obtained under different experimental conditions. For example, cone-like structure [Figure 2.9a] obtained at 2 and 5 min, however increasing of time up 20 and 60 min obtained HAp coating transform to hexagonal prism with sharp-angle tip [Figure 2.9b], continually increasing up to 90 min the obtained morphology of HAp coating turned to rod-like with regular hexagonal cross section and finally transform to flower-like structure at 120 min [Figure 2.9c] [91].

Figure 2.9a – Cone-like structure of HAp coating (Reprinted from [91])

Figure 2.9b – Sharp-angle tip structure of HAp coating (Reprinted from [91])

Figure 2.9c – Flower-like structure of HAp coating (Reprinted from [91])

The similar result on the other study, revealed a ribbon-like HAp crystals deposited on titanium surface by electrochemical deposition with higher concentration of 4×10-2 M Ca2+ [176] [Figure 2.10].

Figure 2.10 – Ribbon-like structure of HAp coating (Reprinted from [176])

2.8.2.2 Energy Dispersive X-ray Spectroscopy

Energy Dispersive X-Ray Spectroscopy (EDS or EDX) is typically use with SEM. It is a chemical analysis technique, work base on detects x-rays emitted from the sample by an electron beam, result give the elemental composition of sample's surface.

2.8.2.3 Transmission electron microscopy

As mentioned above, the TEM has several drawbacks and it is as not popular used as SEM. However, in some case it still used. On the study [20], TEM images showed that HAp crystals in pure HAp coating were easy to accumulate even after ultrasonic treatment [Figure 2.11a] and they were uniformly dispersed and attached onto the wrinkled-paper-like graphene oxide (GO) sheets in GO/HAp composite coatings [Figure 2.11b].

2.8.3 Mechanical characterization techniques

2.8.3.1 Adhesion Testing

The evaluation of adhesion of HAp coating to the substrate plays an importance role. There are several methods have been developed to evaluate adhesion. Tensile test (pull-off) and the scratch test have been widely used, they bring the outstanding advantages compare to other technique.

The tensile test (pull-off) method

The tensile test can be done as shown in [Figure 2.12]. The adhesion strength of the HAp was assessed by pull-out test. For example, the mechanical characterization of HAp coatings were tested by Universal Instron Mechanical Testing System (Instron 5569, Instron Co.) according to ASTM F1044-05 standard and the measured bonding strength values was 13.7 ± 1.25 MPa, and adhesion strength of 16.2 ± 2.5 MPa, 17.1 ± 1.6 MPa, 23.9 ± 2.1 MPa, 25.4 ± 1.4 MPa were achieved for 25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL graphene oxide/HAP composite coatings, respectively [20].

Figure 2.12 – The tensile test

Scratch Test method

The scratch test has been typically used as a technique for the evaluation of the adhesion of coatings, this technique was established in 1950 by Heavens. It works base on a stylus that is drawn over the sample surface under a force; the force continuously increasing until the coating is detached. The coating detachment can be observed in practice by optical microscopy.

The adhesion strength of the HAp coatings on the surface were examined by high energy low current DC electron beam (HELCDEB) via the standard scratch tester using a Universal Instron Mechanical Testing system (Instron 5565, Instron Co.) according to ASTM F 1044-05 standard. Adhesive strength depends on the surface roughness and coating properties. On the study [34], the adhesion strength values of the HAp coating were examined by HELCDEB, the result showed good bond strength with the Ti6Al4V substrate.

2.8.3.2 Roughness Testing

Atomic force microscopy

The atomic force microscope (AFM) was used in 1985 by Binnig, Quate and Gerber. As the same other scanning probe microscopes, the AFM works base on scanning process by a sharp probe over the surface of a sample and then measures the changes of force between the probe tip and the sample. A cantilever with a sharp tip is positioned above a surface. Depending on the distance between them, the forces will determine the interaction, which form the bending of the cantilever; therefore this force is measured by an optical lever technique (it means a laser beam is focused on the back of a cantilever and reflected into a photodetector). By scanning the tip across the surface and recording the change of force show a map of surface topography and other properties obtain.

In addition, the AFM was widely used to investigate the roughness of HAp coating. For example, on the study [23] show that the surface roughness (Ra) of the single-walled carbon nanotubes/hydroxyapatite (SWNTs/HA) composite coatings was measured by an Atomic force microscope (AFM, SPM-9600, Japan) at five different areas of the coatings. The measurements by AFM were controlled in ambient air with a scan rate of 0.7016 Hz and a scan size of 20 × 20 µm. The result showed that the Ra of the SWNTs/HA composite coatings was 600.19 ± 57.12, 565.63 ± 49.53, 551.44 ± 66.09 and 593.13 ± 53.37, respectively.

2.8.4 Another Testing

Contact angle method

Contact angle (CA) measurement is a popular and inexpensive methods to investigate surface structure. It brings the useful information about the surface. Its operating principle is based on producing a drop of liquid on a solid, the angle formed between the solid/liquid interface is the contact angle.

Biocompatibility test for medical devices: review

2.9.1 Introduction

Biocompatibility is a word that is used extensively in biomaterial science. It is a key concept in understanding the host response to implants and biomaterials. In 1987, a book was published offering a definition of biocompatibility: ‘the ability of a material to perform with an appropriate host response in a specific application” [177]. This definition showed that though it was accurate and historically important in the design, development and application of biomaterial in medicine, although it does not have insights in the mechanism of biocompatibility, how to test the biocompatibility of material or how to optimize or enhance the biocompatibility of the material [178].

There is a good deal of regulatory guidance in place to guide one through biocompatibility evaluation of a medical device and depending on which country a device is to be registered in, some variations exist. Recently, extensive efforts have been made by government agencies such as US Food and Drug Administration (FDA) and regulatory bodies, American Society for Testing and Materials (ASTM), International Standards Organization (ISO), and the United States Pharmacopeia (USP), to provide procedures, protocols, guidelines, and standards that may be used in the in vivo assessment of the tissue compatibility of medical devices. For example, the US FDA and European Union commonly accept the ISO 10993 standards for biocompatibility which is provided by a list of types of tests to be considered for biological response evaluation under ISO 1099312 [Table 2.16] [179, 180, 181]. This standard gives the basic guidelines of biocompatibility. In that, Part 1 of the standard is the Guidance on Selection of Tests, Part 2 covers animal welfare requirements, and Parts 3 through 20 are guidelines for specific test procedures or other testing related issues. [Table 2.17] provides a list of ASTM standards that can be used for biocompatibility evaluation [182].

Table 2.16 – ISO 10993: Biological evaluation of medical devices (Reprinted from [179, 180, 181])

Table 2.17 – ASTM standards and tests for biocompatibility

The biocompatibility could be taken place in vitro test or in vivo test. Both of type have the difference advantages and disadvantage [Table 2.18] [183].

Table 2.18 – Advantages and Disadvantages between vitro test and vitro test (Reprinted from [183].)

2.9.2 In Vitro Tests for Biocompatibility

In vitro tests for biocompatibility take place outside a living organism, it means the biomaterial contact with a cell, enzyme, etc. The contact can be direct or indirect. The most common type of biocompatibility assay is the use of cell culture systems to determine the cytotoxicity, cell adhesion, cell activation, or cell death. Cell culture assays are used extensively to study the biocompatibility characterization. There are three types of cell culture assays, which are extract dilution, direct contact, and indirect contact test. Among them, direct contact cell culture is most commonly used.

In addition, in tests on extracts including some test such as L929 elution test; Neutral red uptake test; Colony formation test; 3-(4,5-dimethylthiazol-2-yl)-2,5­; diphenyltetrazolium bromide; (MTT) and related tests. Indirect contact test includes Agar diffusion test and Filter diffusion [178].

Cell culture for biocompatibility tests were used a lot of kind [Table 2.19] [183]

Table 2.19 – Cell culture

2.9.2.1 Cytotoxicity tests

Cytotoxicity tests aim to examine the cell death status was caused by a material. This method works base on measuring cell number and the growth of their before and after interacting to that material. List of cytotoxicity tests and their relevant standards are mentioned in [Table 2.20].

Table 2.20 – Cytotoxicity tests

The cytotoxicity test is a biological evaluations method and screening tests, that mean using the tissue cells in vitro aim to observe the cell growth, the reproduction and the morphological effects of cells by medical devices. Among the biocompatibility tests, the cytotoxicity plays the most important role for biological evaluation because of it has a series of advantages. The cytotoxicity is preferred as an important indicator for the evaluation of medical devices because of it is simple, fast, has a high sensitivity and can save host from toxicity property [184]. There are three types of cytotoxicity test, which are extract, direct contact and indirect contact tests, it pointed in the International Organization for Standardization 109993-5.

Extract test: The method also known as the 3-(4, 5-dimethyl-2-thiazolyl) – 2, 5-diphenyl-2H-tetrazolium bromide (methyl thiazolyl tetrazolium; MTT) assay. This method works is based on the measures of the reduction of the yellow, water-soluble MTT by mitochondrial succinate dehydrogenase. It is currently the most widely used method to investigate the cell growth rate, it shows the result more accurate than other methods. Moreover, it is relatively simple and was published in previous research [37, 114, 116, 126, 127, 130, 133, 139, 140, 146, 185]. However, this method has some disadvantages such as there are numerous steps in this test, and it take a lot of time, repetitive process, etc. This method operates on the base of the following principle: the yellow, water-soluble MTT is reduced to form a purple crystalline formazan. This substance has soluble ability in dimethyl sulfoxide and other organic solvents but has insoluble ability in water. Several crystals formed, they had a positive relation to the number of cells and their activity. They are measured the absorbance (optical density) colorimetric value, which shows the number of surviving cells and their activity [186].

Direct contact is high sensitivity method. This test operates base on observing the changes of cell morphological and the changes in the number of cells. The direct contact biocompatibility test works based on the addition of a test sample directly on the top of a sub-confluent cell layer (without agar layer in between). The guidance for this test is followed by the international standards such as ISO 10993-5:2005 (Tests for cytotoxicity – In Vitro Methods) and ISO 7405:2008 (Dentistry – Evaluation of biocompatibility of medical devices used in dentistry) [184].

2.9.2.2 Cell adhesion

Adhesion ability of cell plays an important role in the development and maintenance of tissues. Cell adhesion is the ability of a single cell to stick to another cell, or cells interact with and attach to a surface, substrate. Cell adhesion occurs base on the action of transmembrane glycoproteins, is called cell adhesion molecules.

This method is the most widely uses of adhesion assays aim to investigate the ability of cell adhere to the substrate, and to examine the sensitivity ability of cell in the substrate interaction to inhibitors [187].

Standard adhesion assays measure cell binding either to immobilize ligands or to cell monolayers in flat-well microtiter plates under static conditions. Typically, these test systems require several washing steps to separate adherent from non-adherent cells.

In the previous research [188], it was described an adhesion assay which avoids these washing steps by employing V-bottom 96-well plates. In this assay, fluorescently labelled leukocytes can adhere to V-well plates coated with soluble ligand for a fixed time. Then, they are detached the adherent cells from non-adherent cells by the centrifugal force. Non-adherent cells with the V-shaped wells are quantified by using a fluorimeter with a narrow aperture.

There are various techniques to measure cell adhesion, they have been applied to many fields of study in order to gain understanding of cell adhesion. The review [189] discussed the overview of the available methods to study cell adhesion through attachment and detachment events.

2.9.2.3 Cell activation

Bioactivity was checked through tests of immersion in Simulated Body Fluid (SBF). Because of its similarity to the human blood plasma, SBF, with pH = 7.42, was applied as a solution for the in-vitro test [Table 2.21]. The SBF solution was prepared by dissolving reagent-grade chemicals of NaCl, NaHCO3, KCl, K2HPO4.3H2O, MgCl2.6H2O, CaCl2 and Na2SO4 in distilled water, respectively and buffered at pH 7.4 with tris-hydroxymethyl-aminomethane ((CH2OH)3CNH2) and 1 M HCl at 36.5 °C as described by Kokubo and Takadama. Moreover, the SBF used was according to the bioactivity evaluation of the implant materials through the formation of HAp surface layers (ISO 23317) [190, 191]. The solution temperature was maintained at body temperature 37°C. In order to maintain the ion concentration, the SBF solution was refreshed every two days. The samples were immersed in SBF for a pre-determined period. And then, they washed with distilled water and dried. There are much previous research apply in vitro bioactivity test [21, 121, 128, 185, 192]

Table 2.21 – Ion concentrations of the human blood plasma and the SBF solution.

2.10 In Vivo Tests for Biocompatibility

The goal of the in vivo assessment of biocompatibility is to detective whether a medical device cause harm to the host by the evaluation under conditions simulating clinical use such as toxicity, genotoxicity, hemocompatibility, biodegradation, etc. [178].

2.10.1 The laboratory equipment for biocompatibility evaluation

List of cytotoxicity tests and their relevant standards are mentioned in [Table 5] and minimum required laboratory equipment list is given in [Table 2.22] [193]

Table 2.22 – Laboratory equipment

2.11 Summary of literature review

Chapter 3: Experimental details

3.1 Characterization of the calcium phosphate coating on Ti6Al4V substrates by electrochemical process

3.1 1 Abstract

Electrochemical depositions of calcium phosphate film on Ti–6Al–4V in 5mM Ca(NO3)2 (calcium nitrate) and 3mM NH4H2PO4 (ammonium di-hydrogen phosphate) in distilled water , were carried out by the pulsed electro-deposition using cyclic voltammetry with scanning the potential ranging between -2V to 0V, using a scan rate of 100 mV/s. The results showed that the uniform calcium phosphate coatings layer was successfully deposited on Ti–6Al–4V at 50oC for 120 min. Aim of this study was to investigate the surface morphology, element composition identification of the coatings by field-emission scanning electron microscopy (Phenom ProX, Netherlands) at an electron beam energy of 15 keV with different magnifications.

Material and methods

3.1.2.1 Preparation of Ti6Al4V specimen

Commercially available Ti–6Al–4V alloy, with chemical composition contains 6% Al and 4% V as well as the balance is Ti (Bibus Metals AG, Germany), was used as a substrate in this present study. Some previous research suggested Ti6Al4V as the substrate for electrochemical deposition [34, 35, 36, 38].

The Ti6Al4V specimen bar was cut by wet cutting method on cutting machine (DELTA Abrasimet Cutter, Buehler, Germany) divide into the plate samples in 2 centimeters of the diameter and 0.05 centimeter of the thick. The samples were rinsed by distilled water and dry at room temperature.

The preparation of substrate before deposition for HAp plays an important role, its effect to the obtained calcium phosphate coating. There is a variety of techniques for preparing biomaterial surface before electrochemical deposition. The effectiveness and efficiency are greatly enhanced due to the combination between several of the techniques. Generally, the preparation of substrate surface before the deposition process consists of two major steps are the mechanical polishing (grinding/abrading and polishing) and cleaning process. Detail of steps for the substrate treatment were described as follows:

Mechanical method is first steps in series procedure of the substrate preparing for the electrochemical deposition that includes the grinding and polishing process. Purpose of mechanical polishing is descaling, removal a part of a material layer on the surface and the smooth surface finish by using an abrasive. Mechanical polishing is often used to smooth rough edges, remove contaminants, and provide a mirror finish for materials. In this study, both of two process were performed by PHOENIX BETA machine (Grinder/ Polisher and Power head, BUEHLER) [Figure 3.1.1].

Grinding process used the wet-abraded method, the liquid is water, two of the diamond sheets (Buehler Hercules, US) were used as grinding paper:

The diamond grinding disc was in size 74 micrometer (µm) with the time in 3 minutes and the speed of the grinding wheel in 300 RPM.

Type of diamond size 15 micrometer (µm) in 3 minutes and the speed of grinding wheel in 250 RPM. After every time the grinding, the samples were rinsed by distilled water.

Figure 3.1.1 – PHOENIX BETA (Grinder/ Polisher and Power head, BUEHLER)

And then the specimens were abraded with four kinds of the Silicon Carbide (SiC) Wet Grinding paper (ATM, Germany):

Type of the SiC paper with 320 and 600 grit for 3 minutes and the speed of the grinding wheel was 200 RPM.

Type of the SiC paper with 800 and 1000 grit for 4 minutes and the speed of the grinding wheel was 200 RPM. After finishing of the mechanical grinding with every type of SiC paper, the sample were rinsed with distilled water.

Finally the samples were polished with Micro-cloth type of 10” PSA 10/PK (Buehler Hercules, US) and Polycrystalline Diamond Polishing Suspension (Buehler Hercules, US) of
9 µm for 10 minutes and the speed of the polishing wheel was 100 RPM. The Ti6Al4V samples were rinsed by distilled water and dried at room temperature. The Titanium samples finished aspect was mirror-like.

The cleaning of the substrate surface is an important step to electrochemical deposition. It is necessary to remove the contaminants that would otherwise affect the properties of the coating. The appearance of even tiny amounts of surface contaminants such as oil, grease, oxides cause the reduction of coating adhesion to the substrate. Additionally, the choice of the cleaning procedure depends on the composition, physical properties and chemistry of the substrate, the type of contaminants on the surface. For this experiment of electrochemical deposition, the Ti6Al4V sample was cleaned by the ultrasonic machine (BANDELIN SONOREX DIGITEX, Germany) [Figure 3.1.2] in iso-propanol alcohol for 20 min with the ultrasonic frequency 15kHz and then dried at room temperature.

Figure 3.1.2 – Ultrasonic machine (BANDELIN SONOREX DIGITEX, Germany)

3.1.2.2 Deposition procedure

The electrolyte for deposition was prepared by dissolving the analytical grade include 5mM Ca(NO3)2 (calcium nitrate) and 3mM NH4H2PO4 (ammonium di-hydrogen phosphate) (both from Sigma Aldrich, Germany) in distilled water. With these concentrations, a previous study suggested to be used for electrochemical deposition [194]. Additional, some previous research also performed, however Ca(NO3)2 was be substituted by CaCl2·2H2O (calcium nitrate di-hydrate), NH4H2PO4 was be substituted by (NH4)2HPO4 (Di-ammonium phosphate) [34] and Ca(NO3)2 was be substituted by CaCl2 .4H2O (calcium nitrate tetra-hydrate) [195].

1000 ml total volumes of the used solutions were put into the cell while magnetic stirring. To ensuring the quality of electrolyte solution relates to completely dissolving of the salts in the aqueous solution, magnetic stirring stirs with a constant speed about 300 rpm to keep the concentration of the electrolyte uniform during the deposition process, a magnetic bar with 4 cm length was laid down in the electrolyte solution bath.

The initial pH of electrolyte (pH = 4.7) was adjusted to 3.990 at 23.1oC by solution of nitric acid 1M (HNO3). The pH and the temperature were determined by pH meter (HI 4221-02 RESEARCH GRADE pH/ORP/°C – 1 Channel-Meter) [Figure 3.1.3].

Figure 3.1.3 – HI 4221-02 RESEARCH GRADE pH/ORP/°C – 1 Channel-Meter

There are various electrochemical reactions occurring with the ions in the electrolyte solution near electrodes during the deposition. The OH– ions are created at the surface of cathode (substrate) due to the chemical reaction which is always the reduction reaction of water H2O lead to formation of large amount hydrogen (H2) gaseous bubbles (1). To overcome this withdraw there are suggested some ways, in previous research, such as addition of peroxide (H2O2) into the electrolyte solution [67], leading the nitrogen gas into the electrolyte solution before electrochemical deposition process [37, 34, 67].

In this study, nitrogen gas (N2) was led into the electrolyte bath before electrochemical deposition process in 20 min as a method aim to reduce the formation of large amount hydrogen (H2) gaseous bubbles which were obtained and raised from various electrochemical reactions occurring in the electrolyte solution. On another hand, this method also reduces the amount of dissolved carbon dioxide to prevent the formation of carbonate (CaCO3) deposit.

2H2O + 2e– = H2 ↑ + 2OH– (3.1)

These H2 gas bubbles adheres onto the surface of the substrate and causes poor adhesion of the HAp coating on the substrate; result can be seen on the coating: cracks, craters and gas pores.

3.1.2.3 Electrochemical measurement

Electrochemical deposition of HAp coating on Ti6Al4V surface was carried out in a regular three electrode cell arrangement using an electrochemical workstation (PARSTAT MC, Princeton Applied Research, USA) [Figure 3.1.4]; the sample as the working electrode was used for the electrochemical measurement. The counter electrode was made of platinum foil and the reference electrode was saturated calomel electrode (SCE) (Ag/KCl).

Figure 3.1.4 – PARSTAT MC, Princeton Applied Research, USA

The Potentiostatic method is a potential technique to overcome the hydrogen evolution problem performing a more homogeneous coating [69]. The pulsed electro-deposition was made with cyclic voltammetry by scanning the potential ranging between 0 V to -2 V, a break time of 100s, using a scan rate of 100 mV/s. Thirty six cycles were employed for the electrochemical deposition. The pulse cycle is schematically illustrated in [Figure 3.1.5] and condition of electrochemical deposition are showed in [Table 3.1.1].

Figure 3.1.5 – Schematic illustration of voltage–time profile used for electrochemical deposition of calcium phosphate coating by voltammetry

Table 3.1.1 – Condition for electrochemical hydroxyapatite deposition by cyclic voltammetry

Magnetic stirring was used to control heat for electrolyte solution. The experiment was carried out at 50oC. The electrolyte solution was stirred with the magnetic stirring at a speed of 100 rpm during the deposition process in order to degas hydrogen gas from the cathode and to improve the coating uniformity.

After the coating process, the specimens were removed from the electrolyte and subsequently washed with distilled water to remove residual electrolytes and then dried at room temperature.

3.1.2.4 Structural characterizations

The surface morphology and element composition of the deposited specimen were identified by field-emission scanning electron microscopy (Phenom ProX, Netherlands) [Figure 3.1.6] at an electron beam energy of 15 keV with difference magnification ×500, ×1000, ×2000 and ×30.000. This is accomplished in the Phenom ProX with a fully integrated and specifically designed EDS detector. EDS is an analyze technique, operate base on the generation of X-rays by the bombardment of the sample via an electron beam. The EDS elemental analysis is fully combined into the Phenom ProX system. The Element Identification (EID) software was provided to allows the user to program various point analysis.

Figure 3.1.6 – Phenom ProX system

3.1.3.1 Surface and morphological studies via SEM investigations

Although SEM can’t have good resolution like transmission electron microscopy (TEM) although SEM don’t destroy the sample and can operate at the low vacuum. Another strong point of SEM is simple controls more than TEM, SEM costs are much lower compared with TEM, and SEM is much more popular than TEM.

The pulsed electro-deposition using cyclic voltammetry by scanning the potential, the electrolyte composition and time deposition significantly affect the deposition kinetics as well as the morphology of calcium phosphate coating. During the electrochemical deposition process, some reaction occurs as the following equations:

O2 + H2O + 4e‒ → 4 OH‒ (3.2)

2 H2PO4 + 2e‒ → 2 HPO42‒ + H2 (3.3)

2 HPO42‒ + 2e‒ → 2 PO43‒ + H2 (3.4)

2 H2O + 2e‒ → H2 + 2OH‒ (3.5)

H2O + NO3‒ + 2e‒ → NO2‒ + 2 OH‒ (3.6)

Ca2+ + HPO42‒ + 2 H2O → CaHPO4·2H2O (3.7)

3 Ca2+ + PO43‒ → Ca3(PO4)2 (3.8)

10 CaHPO4·2H2O + 12 OH‒ → Ca10(PO4)6(OH)2 + 4 PO43‒ + 30 H2O (3.9)

Firstly, the reduction of hydrogen form H2PO4‒ and HPO42‒ (3.2), (3.3) and (3.4).

After that, Ca2+ ions react with HPO42‒ and PO43‒ to form CaHPO4.2H2O (brushite) and Tri-calcium phosphate (α-TCP, β-TCP) (3.7) and (3.8).

The generated hydroxide and phosphate ions will react with Ca2+ in electrolyte solution to form HAp particles on the specimen surface (3.9).

The result clearly showed that electrodeposited coating has consisted several kinds of calcium phosphates forming on specimen surface.

[Figure 3.1.7a] shows the image of the sample surface via SEM at an electron beam energy of 5 keV. [Figure 3.1.7b] illustrates the observation result from SEM images with magnification ×500 (A), ×1000 (B), ×2000 (C) and ×30.000 (D), that is micro-structure of deposited specimen surface. Coatings appeared relatively homogeneous and completely covered the substrate surface of the specimens, there is no significant difference in coating morphology at the regions.

The clusters of calcium phosphate formed on Ti6Al4V were distributed almost even [Figure 3.1.8a]; this SEM image showed that the deposited calcium phosphate coating have thickness comparatively uniform over the specimen surface.

As can be seen from [Figure 3.1.8b and c], morphology of the deposited coating exhibited clearly a flake-like structure which stacking to the surface of the titanium alloy substrate and forming clusters.

[Figure 3.1.8d and e] present the micro-structure of calcium phosphate flakes by SEM with higher magnification.

3.1.3.2 Composition of the deposited specimen

The composition of the deposited coating was investigated by SEM to find out the distribution of the elemental composition on the coating surface. Almost the marks on the surface region investigated indicate the presence of O, Ca, P, Ti, Al, Si and V elements. From SEM images, the distribution of Ca, O and P is comparatively homogeneous. High Ca/P ratio correspond to thickness and homogeneous calcium phosphate coatings. The homogeneity of calcium phosphate coating was assessed using the Ca/P ratio, which ranged from 0.952 to 1.699 at the examined region of the coating surface that was revealed by SEM image [Figure 3.1.8a].

It appeared that high Ca/P ratio are related to calcium-rich phases like hydroxyapatite (HAp). SEM image found out Ca/P molar ratio = 1.699 like Ca/P molar ratio of HAp at the fifth marked point on the surface region in [Figure 3.1.8a], composition of the elements was showed by [Table 3.1.2a] and schematic illustration of peaks of the elements by [Figure 3.1.9a]. Additional, at the marked points as (9), (10) and (12) the Ca/P ratio were identified with value of 1.054, 1.049, and 1.0, respectively that similar to Ca/P molar ratio of di-calcium phosphate dehydrate (DCPD – brushite) and di-calcium phosphate anhydrate (DCPA – monetite); these investigations were revealed by [Table 3.1.2b, c, d] and the peak of the elements were showed by [Figure 3.1.9b].

The Ca/P molar ratio = 1.32 – 1.34 at (7) in [Figure 3.1.8a] [Table 3.1.3a], at (1), (3) and (6) in [Figure 3.1.8d] [Table 3.1.3b, c, d], at (8) in [Figure 3.1.8c] [Table 3.1.3e]. Those results were lower than the Ca/P ratio of 1.67 which corresponds to the standard stoichiometry for HAp, they showed clear that like Ca/P molar ratio of octa-calcium phosphate (OCP – whitlockite) that were confirmed by SEM image.

Additionally, the Ca/P molar ratio = 1.490 – 1.511 at (7) in [Figure 3.1.8c] [Table 3.1.4a], at (5) in [Figure 3.1.6d] [Table 3.1.4] revealed that Ca/P molar ratio was like of Tricalcium phosphate (α-TCP, β-TCP).

Intense peaks for Ca and P also were displayed. Small peaks corresponding to Ti, Al and V present in the substrate were also detected. These peaks indicate the homogeneous coating structure of the coatings in [Figure 3.1.9a, c].

The intensity of the peaks of Ti, Ca and P obtained with SEM were used to evaluate in a qualitative way the thickness. The more homogeneous and thicker coatings were obtained at the marked point (1), (2), (3), (4), (5), (7) and the less homogeneous and thinner coatings were obtained at the point (6), (8) (9), (10), (11) and (12) on the region of coating surface by [Figure 3.1.8a].

On the other region of deposited coating was examined by SEM with the higher magnification such as [Figure 3.1.8b], showed clearly that the crystallized coating consists of flake-like crystals which stacking to the surface of the titanium alloy substrate and forming clusters. The ratio molar of Ca/P in range from 1.0 to 1.2 which is characteristic for the calcium phosphate coating and there was comparatively homogeneous. The coating at the marked region such as (5), (6), (7) and (8) was slightly thinner than the marked region such as (1), (2), (3) and (4), the ratio of Ca/P at the thinner coating region was about 1.0 and at the thicker region was about 1.2.

The molar ratio Ca/P was higher at the thicker coating, about from 1.0 to 1.4 [Figure 3.1.8c], from 1.1 to 1.5 [Figure 3.1.8d] and from 1.3 to 1.4 [Figure 3.1.8e].

Table 3.1.3 Elemental Composition

3.1.4 Conclusions and further research

The calcium phosphate deposit film on Ti6Al4V substrates by electrochemical process was successfully formed. The main results obtained are summarized as follows:

The uniform calcium phosphate coating cover comparatively over specimen substrate with micro-structure flake-like morphology, which stacking to the surface of the titanium alloy substrate and forming uniformly clusters.

The homogeneity of calcium phosphate coating was assessed using the Ca/Ti ratio, which ranged from 0.952 to 1.699 at the examined region of the coating surface that was revealed by SEM image.

The deposition aspect should be improved further through studies on the electrochemical deposition parameters influence on the general aspect of the deposit, its homogeneity, the desired Ca/P ratio, etc. Also, the deposited layer will be characterized further in terms of its crystallographic structure, depth, adhesion, etc. For such determinations will be necessary to cooperate with other laboratories and the help of researchers inside and outside University POLITEHNICA of Bucharest.

3.2 Pulsed electrochemical deposition of Ag doped hydroxyapatite bio-active coatings on Ti6Al4V for medical purposes.

3.2.1 Abstract

Based on the above advantages of pulsed electrodeposition, this study is focused on the pulsed electrodeposition of Ag substituted HAp on Ti6Al4V for improved and enhanced bioactivity, antibacterial properties of HAp coating.

3.2.2 Material and methods

The substrate: Commercially available Ti–6Al–4V alloy with chemical composition contains 6% Al and 4% V as well as the balance is Ti (Bibus Metals AG, Germany) that is promoted as a substrate in this experiment plan.

The Ti6Al4V specimens bar were cut by wet cutting method on cutting machine (DELTA Abrasimet Cutter, Buehler, Germany), divided into the plate samples in 2 centimeter of the diameter and 0.3 centimeter of the thick. The samples were rinsed by distilled water and dry at room temperature.

The mechanical polishing was performed by SiC abrasive paper (300-1200 grit), after which the samples have been cleaned in distilled water.

The Ti6Al4V samples were cleaned by the ultrasonic machine (BANDELIN SONOREX DIGITEX, Germany) in acetone and water for 20 min at 55oC with the ultrasonic frequency 15 kHz and then dried at room temperature.

3.2.3 Deposition procedure

3.2.3.1 Preparation of electrolyte solution

Concentration of electrolyte solution: Electrolyte solution will be suggested for keeping a constant ratio of Ca/P always is 1.67, equal to that in the stoichiometric composition of hydroxyapatite by dissolving Ca(NO3)2·4H2O, NH4H2PO4 and AgNO3 in ultra-pure water (ASTM I) at 75 °C, in different concentrations indicated in [Table 3.2.1].

Table 3.2.1 – Samples codification and chemical composition of the electrolyte

The content of silver: The appropriate silver content should be chosen to balance the biocompatibility and antibacterial properties of the coatings. Silver nitrate with concentration 0.5 mM/L was used to obtain the amount of silver in the coatings [Table 3.2.1].

For ensuring the quality of electrolyte solution related to completely dissolving of the salts in the aqueous solution, magnetic stirring with a constant speed about 100 rpm was used to keep the concentration of the electrolyte uniform during the deposition process, using a magnetic bar laid down in the electrolyte solution bath.

The pH value: The pH value was adjusted to 4.0 by 1M nitric acid (1M HNO3) and controlled via the pH meter (HI 4221-02 RESEARCH GRADE pH/ORP/°C – 1 Channel-Meter).

Reductions of the gaseous bubbles: In this experiment, nitrogen gas (N2) will be led into the electrolyte bath before electrochemical deposition process in 20 min as a method aim to reduce the formation of large amount hydrogen (H2) gaseous bubbles raised from various electrochemical reactions occurring in the electrolyte solution. On another hand, this method also reduces the amount of dissolved carbon dioxide to prevent the formation of carbonate (CaCO3) deposit.

3.2.3.2 Electrochemical measurement

Electrochemical workstation: Electrochemical deposition of HAp coating on Ti6Al4V surface was carried out in a regular three electrode cell arrangement using a potentiostat/ galvanostat (Parstat MC, PMC 2000, Princeton Applied Research, USA), the samples as the working electrode were used for the electrochemical measurement. The counter electrode was made of platinum foil and the reference electrode was saturated calomel electrode (SCE) (KCl).

Set up the voltage: The Potentiostatic method is a potential technique to overcome the hydrogen evolution problem performing a more homogeneous coating [69]. The pulsed electro-deposition using cyclic voltammetry by scanning the potential ranging between 0V to -2V, a break time of 100s, using a scan rate of 100 mV/s. Thirty six cycles (t = 7200s) were employed for the electrochemical deposition. The pulse cycle is schematically illustrated in Figure 1 and conditions of electrochemical deposition are summarized in [Table 3.2.2].

Magnetic stirring was used to control heat for electrolyte solution. The experiment was carried out at 75oC. The electrolyte solution was stirred with the magnetic stirring at a speed of 100 rpm during the deposition process to help degassing hydrogen gas from the cathode and to improve the coating uniformity. After the coating process, the specimens were removed from the electrolyte and subsequently washed with distilled water to remove residual electrolyte, and then they were dried at room temperature.

Figure 3.2.1 – Schematic illustration of the applied pulsed electrochemical deposition cycles

Table 3.2.2 – Condition for electrochemical hydroxyapatite deposition by cyclic voltammetry

3.2.3.3 Coatings characterization

The structure and elemental composition of Hap, Hap-Ag coatings were studied using a scanning electron microscope (SEM, TableTop 3030Plus, Hitachi, Japan) equipped with an energy dispersive X-Ray (EDX) system (Quantax70, Bruker, USA).

The phase composition of the HAp, HAp-Ag coatings were investigated by a X-Ray diffractometer (SmartLab XRD, Rigaku, Japan) with CuKα radiation (λ = 1.5405 Å). Grazing incidence measurements were performed in the range of 20-80° with a 0.01° step, at an incidence angle of 0.2°.

3.2.4 Results and discussion

After the coating process, the specimens were removed from the electrolyte and subsequently washed with distilled water to remove residual electrolyte, and then they were dried at room temperature.

[Figure 3.2.2] shows the HAp/Ti6Al4V (2a) and HAp-Ag/Ti6Al4V (2b) coatings obtained by pulsed electrochemical deposition method.

[Figure 3.2.3] presents the morphology of the obtained HAp/Ti6Al4V (3a-3c) and HAp-Ag/Ti6Al4V (3e-3g) coatings by pulse electrochemical deposition method via SEM investigations. It illustrates the observation results from SEM images with different magnification, that is micro-structure of deposited specimen surfaces. Coatings appeared relatively homogeneous and completely covered the substrate surface of the specimens, there is no significant difference in coating morphology at the regions on both of type un-doped coating and doped coating (3a, 3e).

SEM micrographs of the coatings deposited at a higher magnification are represented in Figure 3, indicating that HAp coatings [Figure 3.2.3c] have needle-like shape distributed comparatively homogeneity on Ti6Al4V. While the needle-like morphology characteristic for HAp is embedded with agglomeration of spherical white particles of Ag were found via SEM investigations [Figure 3.2.3f]. Indeed, silver addition has led to changes in the coating’s morphology of HAp on Ti6Al4V. However, it does not lead to major alterations in the crystalline structure of HAp, this is illustrated by X-Ray diffraction (XRD) analysis [Figure 3.2.6].

Table 3.2.3 – Elemental composition of the obtained coatings were examined by EDS

The composition of the deposited coating was investigated by SEM to find out the distribution of the elemental composition on the coating surface. Almost the marks on the surface regions indicated the presence of Ca, P, O, Ti, C [Figure 3.2.4a and Table 3.2.3] Ca, P, O, Ti, C and Ag elements [Figure 3.2.4b and Table 3.2.3].

The data obtained by the energy dispersive spectroscopy (EDS) [Table 3.2.3] revealed that Ti have registered small value, fact which can be attributed to a significant thickness of the obtained coatings.

From SEM images, revealed that the distribution of Ca, O and P is comparatively homogeneous [Figure 3.2.5]. The elemental composition evidenced the presence of the characteristic elements (Ca, P, O, Ti) of hydroxyapatite and Ti6Al4V substrate. High Ca/P ratio (1.86, 1.92, 1.97, 1.90) corresponded to thickness and homogeneous calcium phosphate coatings. The homogeneity of calcium phosphate coating was assessed using the Ca/P ratio at the examined region of the coating surface that was revealed by SEM [Figure 3.2.5].

3.2.4.2 The phase composition of the coatings

X-Ray diffraction (XRD) analysis was employed to determine the changes in the crystalline structure and phase composition of obtained HAp-Ag coating.

XRD diagrams of the obtained HAp coating on Ti6Al4V are shown in [Figure 3.2.6a]. All the diffraction peaks (002), (102), (211) could be assigned to the most intense crystalline planes of HAp (Power Diffraction File (PDF) no. 9-432), according to the presence of a polycrystalline apatite. No characteristic diffraction angles from other calcium phosphate phases have been identified. Among HAp peaks, the most pronounced HAp reflection is found at 2θ ~ 26°, which indicates a (002) preferred orientation of the deposited HAp crystals just as reported in other studies [68, 197, 198, 199] .

In the case of Ag doped HAp, the corresponding diffraction peaks of Ag where compared with those according to The International Centre for Diffraction Data (ICDD) no. 01-077-6577. However, there are no significant changes in coating morphology at the regions on doped coating [Figure 3.2.6b]. Specifically, shifts towards smaller angles after Ag addition were noted, and can be explained by the substitution of Ca ions with Ag [196].

3.2.5 Conclusions

In this study, electrochemical deposition process has been successfully used for achievement of un-doped HAp coatings and doped Ag/HAp coatings, which have antimicrobial properties. The analysis of XRD, SEM and EDX showed that this technique is very effective in making deposits of HAp. The obtained coating was dense, uniform and pure, corresponding to the data Power Diffraction File (PDF) no. 9-432 and The International Centre for Diffraction Data (ICDD) no. 01-077-6577.

The Ag/HAp phase micro-structure and the XRD results showed that there are no significant changes in coating morphology at the regions on doped coating. HAp morphology has changed after silver doping, revealing a surface with agglomeration of spherical particle of Ag. Phase composition had confirmed the hydroxyapatite formation and, by adding Ag, the peaks have slightly shifted towards left, indicating that Ag is substituting the Ca present in the HAp lattice.

3.3 Studies of microstructure and composition of the modified HAp coating via SEM investigations

Modification of the surface of a biomaterial in order to provide enhanced cell attachment, growth and tissue formation can be achieved via various processes. The most common involving the deposition of calcium phosphates onto the implant surface were mentioned in this study. Calcium phosphates such as hydroxyapatite (HAp) are the main structural component of natural bone. Also, HAp have received much attention and was used on orthopedic and dental implants due to its excellent biocompatibility and osseointegration [3, 200].

Thereby, composite coating of HAp with inorganic or organic additives were obtained via this technique and reported in many previously studies. Various ions may be incorporated into the HAp coating such as ions of Mg, Ag, Sr, Si, Zn, Cu. These substitutions may modify the crystalline structure and induce some changes in the materials properties like phase stability and reactivity. With respect to its biological usage, this may also change the bioactivity, biocompatibility along with some material’s surface characteristics.

In the present work, electrochemical depositions of HAp, HAp-Ag, HAp-Zn coating on commercially pure titanium (cp-Ti) were carried out by the same technique. The coatings were successfully deposited under 0.6 mA/cm2 current density for 20 min. Morphology and elemental composition of the obtained samples were investigated using a scanning electron microscope (SEM) and the elemental composition obtained by the energy dispersive spectroscopy (EDS). The incorporation of Ag, Zn substantially changed the morphology of HAp crystals. The morphology and composition of coatings were investigated by scanning electron microscopy (SEM) and image analyses. Result indicated that Ag, Zn were uniformly distributed into the coatings. The microstructures of the HAp coating have been transformed from a plate-like structure to the plate-like crystals combined with white flowering branches-like structure and interconnected network-type structure.

3.3.1 Materials and Methods

3.3.1.1 Preparation of titanium samples

Commercially pure titanium of 99.9% purity (cp Ti) – ELI bar (Bibus Metals AG, Germany) was used as a substrate material for coatings. The cp Ti bar was cut into disks of 14 mm diameter and 1 mm thickness by wet cutting method on Cutting Machine (DELTA Abrasimet Cutter, Buehler, Germany). The surface of substrates was incrementally grinded by utilizing Silicon Carbide paper (SiC) with 320, 600 and 800 grit. Thenceforth, the substrates were thoroughly washed with soap, ultrapure water. They were sonicated in 2-propanol by the Ultrasonic Machine (BANDELIN SONOREX DIGITEX, Germany) for 20 min at 55 °C with 15 kHz ultrasonic frequency, and subsequently dried in air.

3.3.1.2 Electrochemical deposition process

The electrolyte used for fabrication of the coatings was prepared by mixing Ca(NO3)2·4H2O, NH4H2PO4, AgNO3 and Zn(NO3)2·6H2O in ultra-pure water (ASTM I) in different concentrations indicated in [Table 3.3.1]. Each type of the electrolyte solution was kept at a Ca/P constant ratio of 1.67.

The electrolyte was de-aerated with N2 for 20 min prior to the tests. This procedure was adopted in order to reduce the amount of dissolved carbon dioxide and thus preventing the formation of CaCO3 deposits. The pH value of the electrolytes was 5.0. The coating process was carried out at 75 °C in a three-electrodes cell fitted with a platinum plate works as counter-electrode (anode), a titanium substrate works as the working electrode (cathode) and a saturated calomel electrode (SCE) works as reference electrode. The cathode current density is kept constant at a value of 0.6 mA/cm2 for 20 min using a potentiostat/galvanostat (Parstat MC, PMC 2000, Princeton Applied Research, USA) controlled by PC equipped with VersaStudio Software.

Magnetic stirring was used to control heat of the electrolyte solution. The magnetic stirring was performed at a speed of 50 rpm during the deposition process in order to keep the concentration to degas hydrogen from the cathode and to improve the coating uniformity.

After the coating process, the specimens were removed from the electrolyte, followed by generous washing with distilled water in order to remove residual electrolyte, and then they were dried at room temperature.

Table 3.3.1 – Samples codification and chemical composition of the electrolyte

3.3.1.3 Post-treatment: Annealing heat treatment of HAp coating

After the electrochemical deposition process, selected samples were annealed in a furnace at 80 °C for 1h in argon atmosphere and cooled back to room temperature within the furnace.

3.3.1.4 Characterization and composition analysis of coatings

A scanning electron microscope (Phenom ProX, Netherlands) was used to analyze the samples, operating at 10 kV. For comparison purposes, the surfaces were examined at magnifications ×500, ×1000, ×3000, ×5000, ×10000, ×15000, ×25000, ×30000.

3.3.2 Results and discussions

3.3.2.1 Morphological investigation

Figure 3.3.1 show the SEM micrographs of the HAp, HAp-Ag, HAp-Zn coatings at different magnifications, obtained using conventional electrodeposition at 0.6 m/cm2 applied current density. SEM images revealed the uniform and dense layers composed of numerous
Ca-P nanoparticles distributed overall Ti substrates exhibiting significant differences in the surface morphology of HAp, HAp-Ag and HAp-Zn coatings with plate-like crystals, plate-like crystals combined with white flowering branches-like crystals and interconnected network-type structure, respectively.

SEM images of crystallized HAp coating of the titanium substrate revealed that crystallized coating consists of a regular thin plate-like crystals having constant sizes, interweaving rigidly with each other on the surface of the titanium substrate [Figures 3.3.1 A2-A7]. Moreover, the magnified figure [A5, A6, A7] demonstrates that thin plate-like crystals grow outward, vertical onto the substrate surface and nearly perpendicular to the substrate.

Meanwhile, the deposition morphology remains thin plate-shaped with clusters of white flowering branches-like shape interleaved between the plates and relatively uniformly distributed. At higher magnifications, when comparing the micrograph images [A5-B5, A6, B6, A7-B7] then micrographs obtained by HAp-Ag coating shows a more compact but less uniform plate-like morphology than those observed from HAp coating. Interestingly, a high-magnification SEM image [B6, B7] shows clearly the formation of white flowering branches-like shape of Ag particles.

According to surface morphology and crystal structure analyses of the coatings, interconnected network-like hydroxyapatite crystals were observed on the surface of the Ti. Clearly visible in [Figures 3.3.1 C1-C5], the layer is a homogeneous fully covered deposit showing some cracks. The morphology of the coated surface has significantly changed from thin plate-like crystals to a porous and interconnected network-type structure seen on the HAp-Zn coating, and coating was denser [C6, C7, C8].

3.3.2.2 Elemental composition

EDS elemental mapping attested the uniformity of HAp coatings, the uniform distribution of Ag, Zn in the HAp-Ag and HAp-Zn, respectively. The data obtained by energy dispersive spectroscopy (EDS) were shown in [Table 3.3.2]. The Ca/P ratio corresponded to thick and homogeneous calcium phosphate coatings. The obtained result showed that the HAp-Ag/Ti coating is the thickest (with Ca/P ratio of 1.67 – 1.94) and HAp/Ti coating is the thinnest (with Ca/P ratio of 1.47). Indeed, zinc and silver addition has led to changes in the coating morphology of HAp on Ti although they have been performed in similar conditions by electrochemical deposition. The HAp-Ag/Ti, HAp-Zn/Ti coatings become thicker with a higher Ca/P ratio.

EDS result of HAp-coated surface indicated the presence of HAp components on the substrate consisting in Ca, P, O, Ti [Fig. 3.3.2a]. Additionally, Ca/P ratio was of about 1.47, which agreed with the mole ratios of Ca and P of Tricalcium phosphate (α-TCP, β-TCP). The EDS detected only Ca, P, Ag on the HAp-Ag coating [Fig. 3.3.2b] and Ca, P, Zn on the HAp-Zn coating [Fig. 3.3.2c]. SEM image showed a Ca/P molar ratio of 1.699 at the marked point on the surface region of HAp-Ag coating, like the Ca/P molar ratio of HAp. Additionally, the range of the Ca/P molar ratios of HAp-Zn coating is from 1.60 to 1.62, like Ca/P molar ratio of Calcium-deficient HAp (CDHA) [Table 3.3.2].

a b

c

Fig 3.3.2 – Elemental composition for HAp/Ti (a), HAp-Ag/Ti (b) and HAp-Zn/Ti (c) coatings examined by EDS elemental mapping

Table 3.3.2 – Elemental composition of the obtained coatings by electrochemical deposition

3.3.3 Conclusions

HAp, HAp-Ag and HAp-Zn coatings were deposited successfully on a titanium surface by an electrodeposition method at temperatures around 75°C, low current densities (0.6 mA/cm2) and 20 min time. Uniform and thick coatings were obtained. These electrodeposited coatings are expected to enhance the biocompatibility of the material. Next efforts will be addressed to study the coatings obtained by in vitro and in vivo in a new series of experiments in order to investigate the biocompatibility.

3.4 In vitro biocompatibility investigation of silver and zinc modified hydroxyapatite deposited on metallic implant materials.

One of the features of hydroxyapatite is the capacity for ion substitution. The ion exchange of HAp with metal ions is promising to improve the properties of HAp coating in various applications. Various ions such as Cu+2, Zn+2, Ag+, Mg+2 and Mn+2 are incorporated into HAp through substitution Ca+2 ion. Among these, two metal ions were studied in this work: Ag+ and Zn2+, the HAp-Ag and HAp-Zn coating obtained via electrochemical deposition process. The samples were investigated by SEM for morphological features and EDS analysis. Moreover, the deposited coatings were required to further perceive whether this material persists in good biocompatibility with the host. In this regard, the cellular behavior (human embryonic kidney 293 cells, HEK 293T cells) on HAp/Ti, HAp-Ag/Ti, HAp-Zn/Ti coatings was evaluated in vitro. Bioactivity behavior of the coatings also were assessed in simulated body fluid (SBF). The purpose of this in vitro study was to evaluate the biocompatibility, bioactivity characterization of silver and zinc modified hydroxyapatite on titanium. Preliminary cells culture investigations showed that HAp-Ag and HAp-Zn coating were non-cytotoxic and bio-compatible.

3.4.1 Materials and Methods

3.4.2.1 Pretreatments of Ti substrate

The coatings were deposited on commercially pure Ti (cp-Ti, purchased from Bibus Ag Metals, Germany) which acted as substrate. Samples in disc-shape with dimension of 14 mm in diameter and 2 mm in thickness were prepared. The substrates were metallographically prepared using different grades of silicon carbide paper (400 – 800 grit). After, the substrates were thoroughly cleaned with ultrapure water and ultrasonically cleaned in 2-propanol for 20 min and dried in air.

3.4.2.2 Electrochemical deposition of HAp, HAp-Ag, HAp-Zn coatings on titanium

The electrochemical deposition was carried out with a Potentiostat/Galvanostat Parstat MC, PMC 2000 (Princeton Applied Research, USA) employed to deposit the coatings on Ti, using a typical three cell system configured as following: the Ti substrate was set as working electrode (WE), the platinum foil was the counter electrode (CE) and the saturated calomel electrode (SCE) as reference electrode (RE).

In Table 3.4.1 is presented the chemical composition of the electrolytes used in the study. Briefly, the electrolytes were obtained by dissolving the salts (analytical grade, purchased form Sigma Aldrich) in ultra-pure water, ASTM I (Milli-Q). The pH value of electrolyte was adjusted to 5.0 by addition of 1M NaOH. The electrolyte was purged with nitrogen gas for 20 min prior the experiments in order to reduce the hydrogen evolution. During the deposition the electrolytes were continuously stirred. The experiments were performed at 75 °C by applying a constant current density of – 0.85 mA/cm2 for 1200 s. After the deposition, the samples were gently rinsed in distilled water and dried in air.

Table 3.4.1: The electrolyte used for fabrication of the coatings

3.4.2.3 Characterization and composition analysis of coatings

The morphology and elemental distribution of the coatings were studied using scanning electron microscopy equipped (SEM) with an energy dispersive spectroscopy (EDS) (Phenom ProX, Phenom World, Netherlands).

3.4.2.4 In vitro biological activity analysis

Bioactivity was evaluated by soaking the coated specimens in 40 mL of simulated blood fluid solution (SBF). The chemical composition of the media (Table 2) used in the present study resemblance to human blood plasma and uses the receipt proposed by Kokubo [74, 201]. The tests were achieved at 37 °C for 21 days and at different periods of 1, 3, 7, 14, 21 days samples were removed from the media and gently washed with distilled water and then dried in a desiccator. The SBF was refreshed every 3 days to preserve the ions concentration.

Table 3.4.2 – The concentrations of SBF (1000 mL)

The samples mass (substrate and coating) evolution was gravimetrically monitored using an analytical balance with an accuracy of 0.01 mg. The weight variation of the formed/lost mass on the surface was determined using the following equation:

where Δm represents the mass variation on the surface, mf and mi are sample weights before and after exposure to SBF media.

3.4.2.5 Evaluation of biocompatibility

– Cell culture:

Human embryonic kidney 293T cells (HEK293T) were used to assess the biocompatibility of HAp, HAp-Ag, HAp-Zn coatings. HEK293T were cultured in Dulbecco’s modified eagle medium 1X (DMEM, Life technologies), supplemented with Glutamax-I (Life technologies), 10% FBS (Life technologies) and 1% Non-essential amino acids (Life technologies).

Cells were seeded on the Ti samples that were placed in 24-well polystyrene cell culture plates (internal well diameter is 15 mm) and previously rinsed twice with ethanol 70%. Cells seeded on 24-well plates served as control.

Cells were seeded onto the samples at an initial density of 25.000 cells/well and were cultured for 1, 3, and 4 days at 37 °C under a humidified air atmosphere consisting of 5% CO2 and 95% air. The cells were seeded and further were used for cell proliferation assay or stained for immunofluorescence. Culture medium was renewed every 2 days.

Fig 3.4.1 – The samples before Biocompatibility test

– Immunofluorescence assay:

Sample preparation: After 24 h culture, the extracellular media was removed, the cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS (1X), Life technologies) and fixed with Paraformaldehyde 4% (PFA 4%) for 20 min at room temperature. Further, the cells were washed again with DPBS (1X), followed by permeabilization with detergent Triton X-100 0.2% in PBS for 4 min and washing with DPBS (1X). Subsequently, blocking was performed using Bovine serum albumin 1% (BSA) in phosphate buffer solution (PBS) 1X.

Staining procedure:

a) Staining for Actin and Ki67

Cells seeded on the Ti samples were stained with Alexa Fluor® 594 phalloidin (A594) 1/40 in BSA 1% for 60 min. Further, the same samples were incubated with anti-Ki67 antibody (rabbit, Thermo Scientific) 1/200 in BSA 1% for 30 min and further with anti-rabbit Alexa Fluor 488 secondary antibody, dilution 1/500 in BSA 1%, for 30 min. Cells were washed 5 times with PBS after each incubation.

After staining, samples were mounted on glass slides using Anti-fade solution with DAPI (Invitrogen).

b) Staining for Tubulin and PDI

Cells seeded on the samples were incubated with Anti-Tubulin antibody (rabbit, Abcam) 1/1000 in BSA 1% for 30 min and further with Anti-Rabbit Alexa Fluor-A594 secondary antibody, 1/500 in BSA 1% for 30 min.

Afterwards, samples were stained with Anti-PDI antibody (mouse, Abcam) 1/1000 in BSA 1% for 30 min, followed by incubation with anti-mouse Alexa Fluor 488 secondary antibody, 1/500 in BSA 1%, for 30 min. Cells were washed 5 times with PBS after each incubation.

After staining, samples were mounted on glass slides using Anti-fade solution with DAPI (Invitrogen).

c) Microscopic immunofluorescence analysis

The cells were visualized under microscope (ApoTome.2, AXIO) with the magnification ×20 (mode: dry) and X63 (mode: oil, Immersol 518F), respectively.

Fig 3.4.2 – The samples before microscopic observation

– Cell proliferation assay:

Cells were harvested at days 1, 3 and 4 counted using a hemocytometer. A dilution of the mixture with the number of cells to be counted should be used Trypan Blue Stain 0.4% (T10282, USA) was used to stain the dead cells and calculate cell viability (volumetric ratio 1:1).

The biocompatibility tests were performed in triplicate using different samples to ensure the validity of the results, means and standard deviations were calculated.

3.4.2 Results and discussions

3.4.2.1 Morphological investigation

[Fig. 3.4.3] shows the scanning electron microscope (SEM) image for simple and modified HAp coatings prepared by the electrochemical deposition method. The images are clearly visible in [Fig. 3.4.3 a] that the electrodeposited of simple HAp coating presents a morphology made of ribbon-like crystals, with a Ca/P ratio of 1.47. The addition of Ag into HAp hasn’t modified the plate like morphology of HAp but some silver particle agglomerations are visible on the HAp crystals [Fig. 3.4.3 b]. In this case the Ca/P ratio is about 1.67 which is like the stoichiometric HAp (Ca/P=1.67). The HAp-Zn coatings is characterized by an interconnected network made of very thin and small crystals [Fig. 3.4.3 c] suggesting that the morphology of HAp is visible modified by addition of zinc. The Ca/P ratio registered a value of 1.62 which is very close to the stoichiometric one.

According to the EDS analysis silver and zinc signals, respectively, appear on the spectra [Fig. 3.4.3 e, f].

Thus, it can be said that the addition of Ag and Zn has induced some small modifications in the HAp and the Ca/P ratio has increased from a value of 1.47 in the case of HAp coatings to 1.67 and 1.62 for HAp-Ag and HAp-Zn, respectively.

Fig 3.4.3. The SEM/EDS measurements on substrate material:

HAp (a, d), HAp-Ag (b, e), HAp-Zn coatings (c, f).

3.4.2.2 Evaluation of Bioactivity

Immersion in SBF media has commonly been used to study the bioactivity of materials as a quick, easy and low-cost method. The SBF used was according to the bioactivity evaluation of the implant materials via the formation of apatite layers on surface of substrates (ISO 23317:2014) and is an important feature in order to estimate the material ability to bond with leaving tissues. After every period of 1, 3, 7, 14, 21 days immersion in SBF, a thick and dense apatite layer was formed on the specimen surface, mineral crystals covered almost all the surface of the specimen. The growth of apatite layers was measured carefully and the obtained values are presented in [Table 3.4.3]. Each sample was measured five times.

A slight increase in the initial mass was observed on all the samples after one day exposure to SBF media, except Ti substrate without coating which didn’t present any mass changes. The apatite growth rate on HAp-Ag is the fastest at each period. For HAp-Zn samples, the growth rate of the new apatite layer was higher compared to HAp samples at 7 days and 14 day but smaller than at 21 days [Fig 3.4.4 a]. Hence, the bioactivity sequence of the coatings was
HAp-Ag > HAp-Zn > HAp > Ti. The presence of bone-like apatite layers on the substrate has been considered as a positive biological response to host.

Table 3.4.3 – The increase of apatite layer on substrate at every period of
1, 3, 7, 14, 21 days in SBF

Fig 3.4.4 – The chart shows the increase in the mass of the apatite layer on Ti

3.4.2.3 Evaluation of biocompatibility

– Cell morphology: Cell morphology was analyzed by staining the cells for cytoskeletal proteins as actin and tubulin. The actin filaments (polymerized actin) were visualized using phalloidin. No significant differences in the cell’s morphology was noted [Fig. 3.4.5 and Fig. 3.4.6]. A reduced polymerization of actin was observed for all the coatings (HAp, HAp-Zn and HAp-Ag) compared to the Ti samples [Fig. 3.4.7], suggesting the HAp coatings alter the organization of actin in filaments.

Tubulin staining revealed a better organization of the microtubules in the cells seeded onto HAp-Zn and HAp-Ag than those seeded onto cp-Ti or HAp [Fig. 3.4.7]. This suggest that Zn2+ and Ag+ ions could add a benefit for tubulin polymerization, which is an important dynamic process for cell intracellular transport and cell division.

The intracellular transport is also related to the function of reticulum endoplasmic, which was evaluated by staining for a specific protein, called protein-disulphide-isomerase (PDI). PDI expression was similar in cells grown on either material [Fig. 3.4.8]. To evaluate cell proliferation, HEK293T cells were also stained for Ki67 protein, which is associated with this process. It is expressed during G1 (growth phase), S (synthesis phase), G2 (gap phase) and mitosis phases of cell cycle, but not in G0 (metabolic state). As it is shown in [Fig. 3.4.8], Ki67 expression was identified for the majority cells seeded on all samples. A higher level of Ki67 protein was found in cells during mitosis phase, fact confirmed by visualizing the separation of chromosomes during mitosis (prophase, metaphase, telophase and anaphase) by staining with DAPI. Comparison between cell number (by DAPI staining) on the different samples indicates a higher proliferation rate for cells grown on HAp-Zn and HAp-Ag coatings and the lowest on the control sample, cp-Ti.

These results suggest a possible advantage of using HAp-Zn and HAp-Ag coatings to improve cell growing parameters, but further experiments should be carried out using other types of cells.

– Cell proliferation:

In [Fig 3.4.9] are presented the values obtained for the cell proliferation assay. It can be observed that on day 1 no significant differences were noted between all tested materials. On day 3, a slight increase of cells was observed on all materials. Compared to control sample, the lowest cell proliferation was registered for the uncoated Ti and sequence of the increase rate was Ti < HAp < HAp-Ag < HAp-Zn < control. Similarly, a constantly increase on day 4 was observed on all the samples. Thus, according to cell proliferation assay, it can be said that all coatings have enhanced the cell proliferation of Ti, but better results were noted for HAp coatings with addition of Ag and Zn.

In [Fig 3.4.10] are presented the cell viability results which demonstrated that all the samples have good cytocompatibility of HEK 293T cells. In vitro biocompatibility clearly indicated that all tested samples showed no cell cytotoxic activity. The cell viability was above 80% (non-cytotoxic), the highest values being obtained for HAp-Zn coatings (86.41 % at day 4). Moreover, on the 4th day, the cell viability was similar for all coatings as following: HAp (85.14 %), HAp-Ag (86.19 %) and HAp-Zn coating (86.41 %). There are no significant differences found between them at day 1 and day 3.

Fig 3.4.9 – The number of cells on the coating after 1, 3 and 4 days.

Fig 3.4.10 – Cell Viability of the coatings after 1, 3 and 4 days

3.4.3 Conclusions

In the present study, biocompatibility and bioactivity of simple and Ag and Zn modified HAp deposited by electrochemical deposition on pure titanium was evaluated. Based on the presented results the following conclusions were drawn:

the morphology of HAp was made of thin ribbon like crystals; the addition of Ag into HAp didn’t induce major modification of the ribbon like morphology, but some Ag particle agglomerations were noted; in the case of HAp-Zn coatings, the morphology was made of very thin crystals which formed an interconnected porous network, suggesting that Zn alters the nucleation kinetics;

the elemental composition indicated that the addition of Ag and Zn enhances the Ca/P ratio of HAp from a value of 1.49 for simple HAp to 1.62 for HAp-Zn and 1.67 for HAp-Ag, the latter ones being close to the stoichiometric HAp (1.67);

in terms of bioactivity, all coatings have enhanced the biomineralization ability of cp-Ti; after 21 days of immersion in SBF the highest values were registered for HAp-Ag coatings (7.11 mg), followed by HAp (4.58 mg) and HAp-Zn (4.38 mg), while the cp-Ti have registered the smallest value (2.11 mg);

the preliminary cell culture investigations showed that Ag-HAp and Zn-HAp coating were non-cytotoxic and biocompatible and overall addition of Ag and Zn into HAp enhanced the behavior of HAp.

As a conclusion, it can be said that HAp based coatings with small amounts of Ag and Zn can improve the bioactivity and biocompatibility of titanium and can be considered as potential materials for medical application usage.

3.4.4 Acknowledgements

This work was supported by grants of the Romanian Ministry of Research and Innovation, CCCDI – UEFISCDI, projects number PN-III-P1-1.2-PCCDI-2017-0239/60PCCDI 2018.

Chapter 4: Conclusions and future work

4.1 Conclusions

4.2 Future work

Chapter 5: Personal contributions

Bibliography references

[1] M. Long, H.J. Rack, Biomaterials, 19 (1998), p. 1621

[2] Brunette DM, Titanium in medicine: material science, surface science, engineering, biological responses, and medical applications, Springer Verlag Engineering, Materials; 2001

[3] D.Shi (2004), Biomaterial and Tissue Enginerring

[4] G. Ran, A. Pandit and D.P.Apatsidis, (2006), Biomaterials

[5] Feng C. F, Khor K. A, Liu E. J, Cheang P, (1996), Phase transformation in plasma-sprayed hydroxyapatite coating, Seripta Materialia 42 (1), 103-109

[6] Ding, S.J., Ju, C.P., Chern, L.J.H., (1999), Characterization of hydroxyapatite and titanium coatings sputtered on Ti-6Al-4V substrate, J. Biomedicine Mater. Res., 44 (3), 266-279

[7] C.K. Wang, J. H. Chern L, (1997), Pulse laser deposition of hydroxyapatite, Biomaterials 18, 1331-1338

[8] Wang, C.K., Chern, L.J.H., Ju, C.P., Ong, H.C., Chang, R.P.H., (1997), Structural characterization of pulsed laser-deposited hydroxyapatite film on titanium substrate, Biomaterials, 18 (20), 1331-1338

[9] Hsieh, M.F., Perng, L.H., Chin, T.S., (2002), Hydroxyapatite coating on Ti6Al4V alloy using a sol-gel derived precursor, Mater. Chem. Phys., 74 (3), 245-250

[10] Cong Wang, J. Mo, Wen Cheng, Ruifang Zhang, (2002), Thick hydroxyapatite coatings by electrophoretic deposition, materials Letters 57, 99-105

[11] Achariya Rakngarm, Yoshiharu Mutoh, (2009), Electrochemical depositions of calcium phosphate film on commercial pure titanium and Ti–6Al–4V in two types of electrolyte at room temperature, Materials Science and Engineering C 29, 275–283

[12] Achariya Rakngarm, Yoshiharu Mutoh, (2009), Electrochemical depositions of calcium phosphate film on commercial pure titanium and Ti–6Al–4V in two types of electrolyte at room temperature, Materials Science and Engineering C 29, 275–283

[13] K. Nitta, S. Watanabe, N. Masahashi, H. Hosoda, S. Hanawa, Ni-free Ti–Nb–Sn sHApe memory alloys, in: M. Niinomi, T. Okabe, E.M. Taleff, D.R. Lesuer, H.F. Lippard (Eds.), Structural Biomaterials for the 21st Century, TMS, 2001, pp. 25–34.

[14] Y. Daimatsu, A. Yamamoto, H. Hosoda, S. Miyazaki, SHApe memory characteristics of Ti–Mo–Ga for bio-medical applications, Proc. Fall Meet. Jpn Inst. Met., 2001, p. 401.

[15] H. Hosoda, A. Yamamoto, S. Miyazaki, Mechanical properties of Ti–Mo–Ge sHApe memory alloy for bio-medical applications, Proc. Fall Meet. Jpn Inst. Met., 2001, p. 401.

[16] H. Hiromoto, F. Mizuno, T. Hanawa, C. Kuroda, H. Hosoda, K. Wakashima, S. Miyazaki, Polarization behavior of Ti–Mo–Al sHApe memory alloy in simulated body liquid, Proc. Annu. Meet. Jpn Inst. Met., 2002, p. 443.

[17] M. Ikeda, Y. Nakamura, N. Takahama, Effect of Zr contents on heat treatment behaviors and phase constitution of Ti-50 mass% Ta–Zr alloy, Proc. Annu. Meet. JIM, 2003, p. 130.

[18] T. Maeshima, T. Eto, H. Uchiyama, K. Uchiyama, M. Nishida, Development of Ti–Sc–Mo sHApe memory alloy, Annu. Meet. JIM, 2003, p. 134.

[19] Yajing Yan, Xuejiao Zhang, Huanhuan Mao, Yong Huang, Qiongqiong Ding, Xiaofeng Pang, (2015), Hydroxyapatite-gelatin functionalized graphene oxide composite coatings deposited on TiO2 nanotube by electrochemical deposition for bio-medical applications, Applied Surface Science 329, 76–82

[20] Yongxiang Zeng, Xibo Pei, Shuying Yang, Han Qin, He Cai, Shanshan Hu, Lei Sui, Qianbing Wan, Jian Wang, (2015), Graphene oxide-hydroxyapatite composite coatings fabricated by electrochemical deposition, Surface & Coatings Technology

[21] Yajing Yan, Xuejiao Zhang, Caixia Li, Yong Huang, Qiongqiong Ding, Xiaofeng Pang, (2015), Preparation and characterization of chitosan-silver-hydroxyapatite composite coatings onTiO2 nanotube for bio-medical applications, Applied Surface Science 332, 62–69

[22] Yajing Yan, Qiongqiong Ding, Yong Huang, Shuguang Han, Xiaofeng Pang, (2014), Magnesium substituted hydroxyapatite coating on titanium with nanotublar TiO2 intermediate layer via electrochemical deposition, Applied Surface Science 305, 77–85

[23] Xibo Pei Yongxiang Zeng Rui He Zhongjie Li Lingyang Tian Jian Wang Qianbing Wan Xiaoyu Li Hong Bao, (2014), Single-walled carbon nanotubes-hydroxyapatite coatings on titanium obtained by electrochemical deposition, Applied Surface Science

[24] Kang Lee, Yong-Hoon Jeong, Yeong-Mu Ko, Han-Cheol Choe, William A. Brantley, (2013), Hydroxyapatite coating on micropore-formed titanium alloy utilizing electrochemical deposition, Thin Solid Film

[25] M.A. El Mhammedi, M. Achak, M. Bakasse, (2013), Evaluation of a platinum electrode modified with hydroxyapatite in the lead (II) determination in a square wave voltammetric procedure, Arabian Journal of Chemistry 6, 299–305

[26] D. Gopi, E. Shinyjoy, M. Sekar, M. Surendiran, L. Kavitha, T.S. Sampath Kumar, (2013), Development of carbon nanotubes reinforced hydroxyapatite composite coatings on titanium by electrodeposition method, Corrosion Science 73, 321–330

[27] Nik Norziehana Che Isa, Yusairie Mohd and Norjanah Yury, (2012), Electrochemical Deposition and Characterization of Hydroxyapatite (HAP) on Titanium Substrate, APCBEE Procedia 3, 46 – 52

[28] D.J. Blackwood, K.H.W. Seah, (2010), Galvanostatic pulse deposition of hydroxyapatite for adhesion to titanium for bio-medical purposes, Materials Science and Engineering C 30 561–565

[29] D.J. Blackwood, K.H.W. Seah, (2009), Electrochemical cathodic deposition of hydroxyapatite -Improvements in adhesion and crystallinity, Materials Science and Engineering C 29, 1233–1238

[30] Jian Wang, Yonglie Chao, Qianbing Wan, Zhimin Zhu, Haiyang Yu, (2009), Fluoridated hydroxyapatite coatings on titanium obtained by electrochemical deposition, Acta Biomaterialia 5, 1798–180

[31] Zhao Xuhui, Yang Lingfang, Zuo Yu and Xiong Jinping, (2009), Hydroxyapatite Coatings on Titanium Prepared by Electrodeposition in a Modified Simulated Body Fluid, Chinese Journal of Chemical Engineering, 17(4) 667- 671

[32] Ming-Jie Jiao, Xiao-Xiang Wang, (2009), Electrolytic deposition of magnesium-substituted hydroxyapatite crystals on titanium substrate, Materials Letters 63, 2286–2289

[33] Dai-Hua He, Pu Wang, Ping Liu, Xin-Kuan Liu, Feng-Cang Ma, Jun Zhao, (2015), HA coating fabricated by electrochemical deposition on modified Ti6Al4V alloy, Surface & Coatings Technology

[34] D. Gopi, El-Sayed M. Sherif, D. Rajeswari, L. Kavitha, R. Pramod, Jishnu Dwivedi, S.R. Polaki, (2014), Evaluation of the mechanical and corrosion protection performance of electrodeposited hydroxyapatite on the high energy electron beam treated titanium alloy, Journal of Alloys and Compounds 616, 498–504

[35] Lidia Benea, Eliza Mardare-Danaila, Jean-Pierre Celis, (2014), Increasing the tribological performances of Ti–6Al–4V alloy by forming a thin nanoporous TiO2 layer and hydroxyapatite electrodeposition under lubricated conditions, Tribology International 78, 168–175

[36] N. Metoki, L. Leifenberg-Kuznits, W. Kopelovich, L. Burstein, M. Gozin, N. Eliaz, (2014), Hydroxyapatite coatings electrodeposited at near-physiological conditions, Materials Letters 119, 24–27

[37] Dhanaraj Gopi, Arumugam Karthika, Subramani Nithiya, Louis Kavitha, (2014), In vitro biological performance of minerals substituted hydroxyapatite coating by pulsed electrodeposition method, Materials Chemistry and Physics 144, 75 – 85

[38] M. Saremi and B. Mottaghi Golshan, (2006), Electrodeposition of nano size hydroxyapatite coating on Ti alloy, Iranian Journal of Materials Science and Engineering, Vol. 3, Numbers 3 and 4, Summer and Autumn 2006

[39] Marco Antonio Lopez-Heredia · P. Weiss · P. Layrolle, (2007), An electrodeposition method of calcium phosphate coatings on titanium alloy, J Mater Sci: Mater Med 18:381–390

[40] Ishikawa K, Miyamoto Y, Nagayama M, Asaoka K (1997), Blast coating method: new method of coating titanium surface with hydroxyapatite at room temperature, J Biomed Mater Res 38(2):129-134

[41] Baleani M, Viceconti M, Toni A, (2000), The effect of sandblasting treatment on endurance properties of titanium alloy hip prostheses, Artif Organs 24(4):296-299

[42] Wennerberg A, Albrektsson T, Lausmaa J, (1996), Torque and histomorphometric evaluation of c.p. titanium screws blasted with 25- and 75-microns-sized particles of Al2O3, J Biome Mater Res 30(2):251-260

[43] Wennerberg A, Albrektsson T, Johansson C, Andersson B, (1996), Experimental study of turned and grit-blasted screw-sHAped implants with special emphasis on effects of blasting material and surface topography, Biomaterials 17(1):15-22

[44] Buser D, Nydegger T, Oxland T, Cochran DL, Schenk RK, Hirt HP, Snetivy D, Nolte LP, (1999), Interface shear strength of titanium implants with a sandblasted and acid-etched surface: a biomechanical study in the maxilla of miniature pigs, J Biomed Mater Res 45(2):75-83

[45] Hacking SA, Bobyn JD, Tanzer M, Krygier JJ, (1999), The osseous response to corundum blasted implant surfaces in a canine hip model. Clin Orthop 364:240-253

[46] ASTM standard B600, (1997), Standard guide for Descaling and cleaning titanium and titanium alloy surface. Annual Book of ASTM Standards, Vol.2.04, American Society for testing and Materials, Phiadelphia, PA, pp 6-8

[47] Wang Yue-qin, Tao Jie, Wang Ling, HE Ping-ting, Wang Tao, (2008), HA coating on titanium with nanotubular anodized TiO2 intermediate layer via electrochemical deposition, Trans. Nonferrous Met. Soc. China 18, 631 – 635

[48] Zhang Yuan-yuan, Tao Jie, Pang Ying-chun, Wang We, Wang Tao, (2006), Electrochemical deposition of hydroxyapatite coatings on titanium, Trans. Nonferrous Met. SOC. China 16, 633-637

[49] A. Kar, K.S. Raja, M. Misra, (2006), Electrodeposition of hydroxyapatite onto nanotubular TiO2 for implant applications, Surface & Coatings Technology 201, 3723–3731

[50] Cheng, X., Wang, J., Wang, Y., Wang, G., Zhao, L., (2002), Preparation of thin hydroxyapatite layers on cp titanium through anodic oxidation followed with hydrothermal treatment, Sheng wu yi xue gong cheng xue za zhi J. Biomed. Eng. Shengwu yixue gongchengxue zazhi vol. 19, 378–382.

[51] Nishiguchi S, Nakamura T, Kobayashi M, Kim HM, Miyaji F, Kokubo T, (1999), The effect of heat treatment on bone-bonding ability of alkali-treated titanium, Biomaterials 20(5):49-500

[52] Wen HB, Liu Q, de Wijn JR, de Groot K, Cui FZ, (1998), Preparation of bio-active microporous titanium surface by a new two-step chemical treatment. J Mater Sci: Mater Med 9(3):121-128

[53] Dai-Hua He, Pu Wang, Ping Liu, Xin-Kuan Liu, Feng-Cang Ma, Jun Zhao, (2015), HA coating fabricated by electrochemical deposition on modified Ti6Al4V alloy, Surface & Coatings Technology

[54] N. Metoki, L. Leifenberg-Kuznits, W. Kopelovich, L. Burstein, M. Gozin, N. Eliaz, (2014), Hydroxyapatite coatings electrodeposited at near-physiological conditions, Materials Letters 119, 24–27

[55] D.J. Blackwood, K.H.W. Seah, (2009), Electrochemical cathodic deposition of hydroxyapatite -Improvements in adhesion and crystallinity, Materials Science and Engineering C 29, 1233–1238

[56] A. Kar, K.S. Raja, M. Misra, (2006), Electrodeposition of hydroxyapatite onto nanotubular TiO2 for implant applications, Surface & Coatings Technology 201, 3723–3731

[57] S. Koutsopoulos (2002), Synthesis and characterization of hydroxyapatite crystals: A review study on the analytical methods, Journal of Bio-medical Material Research, Vol. 62, pp. 600-612

[58] J.C. Elliott (1994), Structure and Chemistry of the Apatite and other calcium orthophosphates,

[59] Dorozhkin, S.V, (2007), Calcium orthophosphates. J. Mater. Sci, 42, 1061-1095.

[60] Mihai V. Popa, Jose Maria Calderon Moreno, Monica Popa, Ecaterina Vasilescu, Paula Drob, Cora Vasilescu, Silviu I. Drob, (2011), Electrochemical deposition of bio-active coatings on Ti and Ti–6Al–4V surface, Surface & Coatings Technology 205, 4776–4783

[61] L. De Nardo, D. Pedretti, A. Cigada, R. Chiesa, Electrochemical Deposition of Calcium Phosphate for Implant Osseointegration and Drug Release

[62] Mukesh Kumar, Harani Dasarathy, Clyde Riley, (1998), Electrodeposition of brushite coatings and their transformation to hydroxyapatite in aqueous solutions, Materials Science Program, Department of Chemistry, University of Alabama in Huntsville, Alabama 35899

[63] E.A. Abdel-Aal D. El-Sayed M. Shoeib A.T. Kandil, (2013), Enhancing coating of brushite-hydroxyapatite layer on titanium alloy implant surface with additives, Applied Surface Science

[64] Y.W. Song, D.Y. Shan, E.H. Han, (2008), Electrodeposition of hydroxyapatite coating on AZ91D magnesium alloy for biomaterial application, Materials Letters 62, 3276–3279

[65] M.S. Djosi, V. Panic, J. Stojanovic, M. Mitric, V.B. Miskovi Stankovi, (2012), The effect of applied current density on the surface morphology of deposited calcium phosphate coatings on titanium, Colloids and Surfaces A: Physicochem. Eng. Aspects 400, 36–43

[66] M. Shirkhanzadeh, (1998), Direct formation of nanophase hydroxyapatite on cathodically polarized electrodes, Journal of materials science, Materials in medicine 9, 67-72

[67] D. Gopi, J. Indira, L. Kavitha, (2012), A comparative study on the direct and pulsed current electrodeposition of hydroxyapatite coatings on surgical grade stainless steel, Surface & Coatings Technology, 206, 2859–2869

[68] Dinh Thi Mai Thanh, Pham Thi Nam, Nguyen Thu Phuong, Le Xuan Que, Nguyen Van Anh,Thai Hoang, Tran Dai Lam, (2013), Controlling the electrodeposition, morphology and structure of hydroxyapatite coating on 316L stainless steel, Materials Science and Engineering C 33, 2037–2045

[69] Jia Li’nan, Liang Chenghao, Huang Naibao, Duan Feng, WPang Lixia, (2015), Formation and Characterization of Hydroxyapatite Coating Prepared by Pulsed Electrochemical Deposition, Rare Metal Materials and Engineering, Volume 44, Issue 3

[70] M. Saremi, Sh. Mohajernia n, S. Hejazi, (2014), Controlling the degradation rate of AZ31 Magnesium alloy and purity of nano-hydroxyapatite coating by pulse electrodeposition, Materials Letters

[71] Silva P, Santos J, Monteiro F, Knowles J, Adhesion and microstructural characterization of plasma-sprayed hydroxyapatite/glass ceramic coatings onto Ti–6A1–4V substrates, Surface and Coatings Technology 1998; 102:191–6.

[72] De Groot K, Geesink R, Klein C, Serekian P, Plasma sprayed coatings of hydroxyapatite, Journal of Bio-medical Materials Research 2004;21:1375–81.

[73] Y.P. Lu, Y. Jiao, J.H. Wang, W.H. Xu, G.Y. Xiao, R.F. Zhu, A further insight into pores in plasma sprayed hydroxyapatite coating, Surf. Coat. Technol., 206 (2012), p. 3550

[74] L. Sun, C.C. Berndt, K.A. Gross, A. Kucuk, Material fundamentals and clinical performance of plasma-sprayed hydroxyapatite coatings: a review, J. Biomed. Mater. Res. B Appl. Biomater, 58 (2001), p. 570

[75] V. Sergo, O. Sbaizero, D.R. Clarke, Mechanical and chemical consequences of the residual stresses in plasma sprayed hydroxyapatite coatings, Biomaterials, 18 (1997), p. 477

[76] M. Roy, A. Bandyopadhyay, S. Bose, Induction plasma sprayed nano hydroxyapatite coatings on titanium for orthopaedic and dental implants, Surf. Coat. Technol., 205 (2011), p. 2785

[77] Y. Yang, K.-H. Kim, J.L. Ong, A review on calcium phosphate coatings produced using a sputtering process—an alternative to plasma spraying, Biomaterials, 26 (2005), p. 327

[78] Gross KA, Berndt CC. Thermal processing of hydroxyapatite for coating production. Journal of Bio-medical Materials Research 1998; 39:580–7.

[79] Ding S-J, Properties and immersion behavior of magnetron-sputtered multilayered hydroxyapatite/titanium composite coatings, Biomaterials 2003; 24:4233–8

[80] Cleries L, Martınez E, Fernandez-Pradas J, Sardin G, Esteve J, Morenza J, Mechanical properties of calcium phosphate coatings deposited by laser ablation, Biomaterials 2000;21:967–71.

[81] K. Van Dijk, H.G. Schaeken, J.G.G. Wolke, J.A. Jansen, Pulsed laser deposition of hydroxyapatite thin films, Biomaterials, 17 (1998), p. 159

[82] Yoshinari M, Ohtsuka Y, Dérand T, Thin hydroxyapatite coating produced by the ion beam dynamic mixing method, Biomaterials 1994;15 : 529–35.

[83] Kaciulis S, Mattogno G, Napoli A, Bemporad E, Ferrari F, Montenero A, et al, Surface analysis of bio-compatible coatings on titanium, Journal of Electron Spectroscopy and Related Phenomena 1998; 95:61–9.

[84] Li P, Groot Kd, Kokubo T. Bio-active Ca10(PO4)6(OH)2/TiO2 composite coating prepared by sol–gel process. Journal of Sol–Gel Science and Technology 1996; 7:27–34.

[85] P. Choudhury, D.C. Agrawal. Sol–gel derived hydroxyapatite coatings on titanium substrates. Surf. Coat. Technol., 206 (2011), p. 360

[86] Han Y, Fu T, Lu J, Xu K. Characterization and stability of hydroxyapatite coatings prepared by an electro-deposition and alkaline‐treatment process. Journal of Bio-medical Materials Research 2000; 54 :96–101.

[87] Habibovic P, Barrere F, Blitterswijk CA, Groot K, Layrolle P. Biomimetic hydroxyapatite coating on metal implants. Journal of the American Ceramic Society 2002; 85:517–22.

[88] Choi J-M, Kim H-E, Lee I-S. Ion-beam-assisted deposition (IBAD) of hydroxyapatite coating layer on Ti-based metal substrate. Biomaterials 2000; 21:469–73.

[89] Wie H, Herø H, Solheim T. Hot isostatic pressing-processed hydroxyapatite coated titanium implants: light microscopic and scanning electron microscopy investigations. The International Journal of Oral & Maxillofacial Implants 1998; 13:837.

[90] M. Manso, C. Jimenez, C. Morant, P. Herrero, J.M. Martinez-Duart. Electrodeposition of hydroxyapatite coatings in basic conditions. Biomaterials, 21 (2000), p. 1755

[91] M.H. Ma, W. Ye, X.X. Wang. Effect of supersaturation on the morphology of hydroxyapatite crystals deposited by electrochemical deposition on titanium. Mater. Lett., 62 (2008), p. 3875

[92] Y.Y. Zhang, J. Tao, Y.C. Pang, W. Wang, T. Wang. Electrochemical deposition of hydroxyapatite coatings on titanium. Trans. Nonferrous Metals Soc. China, 16 (2006), p. 633

[93] R. Drevet, H. Benhayounea, L. Worthama, S. Potirona, J. Dougladeb, D.L. Maquina, Effects of pulsed current and H2O2 amount on the composition of electrodeposited calcium phosphate coatings, Mater. Charact. 61 (2010), 786-795.

[94] T. Frade, V. Bouzon, A. Gomes, M.I.S. Pereira, Pulsed-reverse current electro-deposition of Zn and Zn-TiO2 nanocomposite films, Surf. Coat. Technol. 204 (2010), 3592-3598.

[95] H. Adelkhani, M.R. Arshadi, J. Alloys Compd. Properties of Fe–Ni–Cr alloy coatings by using direct and pulse current electro-deposition, 476 (2009) 234-237.

[96] R. Drevet, H. Benhayoune, Pulsed electro-deposition for the synthesis of strontium-substituted calcium phosphate coatings with improved dissolution properties, Mater. Sci. Eng. C 33 (2013) 4260-4265

[97] R.I.M. Asri, W.S.W. Harun, M.A. Hassan, S.A.C. Ghani, Z. Buyong, (2016), A review of hydroxyapatite-based coating techniques: Sol–gel and electrochemical depositions on bio-compatible metals, Journal of the mechanical behavior of bio-medical materials 57, 9 5 – 1 0 8

[98] Yang, Y., Kim, K.-H., Ong, J.L., 2005. A review on calcium phosphate coatings produced using a sputtering process—an alternative to plasma spraying. Biomaterials 26, 327–337

[99] Ming-Jie Jiao, Xiao-Xiang Wang, (2009), Electrolytic deposition of magnesium-substituted hydroxyapatite crystals on titanium substrate, Materials Letters 63, 2286–2289

[100] Jian Wang, Yonglie Chao, Qianbing Wan, Zhimin Zhu, Haiyang Yu, (2009), Fluoridated hydroxyapatite coatings on titanium obtained by electrochemical deposition, Acta Biomaterialia 5, 1798–180

[101] Xiong Lu, Bailin Zhang, Yingbo Wang, Xianli Zhou, Jie Weng, Shuxin Qu, Bo Feng, Fumio Watari,, Yonghui Ding and Yang Leng, (2011), Nano-Ag-loaded hydroxyapatite coatings on titanium surfaces by electrochemical deposition, J. R. Soc. Interface 8, 529–539

[102] Yong Huang, Qiongqiong Ding, Xiaofeng Pang, Shuguang Han, Yajing, (2013), Corrosion behavior and biocompatibility of strontium and fluorine co-doped electrodeposited hydroxyapatite coatings, Applied Surface Science 282, 456–462

[103] Yong Huang, Shuguang Han, Xiaofeng Pang, Qionqion Ding, Yajing Yan, (2013), Electrodeposition of porous hydroxyapatitecalcium silicate composite coating on titanium for bio-medical applications, Applied Surface Science 271, 299–302

[104] D. Gopi, E. Shinyjoy, M. Sekar, M. Surendiran, L. Kavitha, T.S. Sampath Kumar, (2013), Development of carbon nanotubes reinforced hydroxyapatite composite coatings on titanium by electrodeposition method, Corrosion Science 73, 321–330

[105] Yongxiang Zeng, Xibo Pei, Shuying Yang, Han Qin, He Cai, Shanshan Hu, Lei Sui, Qianbing Wan, Jian Wang, (2015), Graphene oxide-hydroxyapatite composite coatings fabricated by electrochemical deposition, Surface & Coatings Technology.

[106] Yong Huang, Yajing Yan, Xiaofeng Pang, (2015), Electrolytic deposition of fluorine-doped hydroxyapatite-ZrO2 films on titanium for bio-medical applications, Ceramics International 39, 245–253

[107] O. Choi, K.K. Deng, N.-J. Kim, L. Ross, R.Y. Surampalli, Z. Hu. The inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth. Water Res., 42 (12) (2008), pp. 3066–3074

[108] Svitlana Chernousova, Prof. Dr. Matthias Epple, Silver as Antibacterial Agent: Ion, Nanoparticle, and Metal, Angewandte Chemie International Edition, Volume 52, Issue 6, February 4, 2013, Pages 1636–1653

[109] Mahendra Rai, Alka Yadav, Aniket Gade, Silver nanoparticles as a new generation of antimicrobials, Biotechnology Advances, Volume 27, Issue 1, January–February 2009, Pages 76–83

[110] Gianluigi Franci , Annarita Falanga, Stefania Galdiero, Luciana Palomba, Mahendra Rai, Giancarlo Morelli and Massimiliano Galdiero, Silver Nanoparticles as Potential Antibacterial Agents, Molecules 2015, 20, 8856-8874

[111] Nida Iqbala, Mohammed Rafiq Abdul Kadira, Nik Ahmad Nizam Nik Malekb, c, Nasrul Humaimi Mahmooda, Malliga Raman Muralid, Tunku Kamaruld, Rapid microwave assisted synthesis and characterization of nanosized silver-doped hydroxyapatite with antibacterial properties, Materials Letters, Volume 89, 15 December 2012, Pages 118–122

[112] U. Anjaneyulu, V.K. Swaroop, U. Vijayalakshmi. Preparation and characterization of novel Ag doped hydroxyapatite–Fe3O4–chitosan hybrid composites and in vitro biological evaluations for orthopaedic applications. RSC. Adv., 6 (2016), pp. 10997–11007

[113] H. Funao, S. Nagai, A. Sasaki, T. Hoshikawa, T. Tsuji, Y. Okada, et al, A novel hydroxyapatite film coated with ionic silver via inositol hexa-phosphate chelation prevents implant-associated infection. Sci. Rep., DOI: 10.1038/srep23238

[114] Yajing Yan, Xuejiao Zhang, Yong Huang, Qiongqiong Ding, Xiaofeng Pang, Antibacterial and bioactivity of silver substituted hydroxyapatite/TiO2 nanotube composite coatings on titanium, Applied Surface Science 314 (2014) 348–357

[115] Cong Fu, Xuefei Zhang, Keith Savino, Paul Gabrys, Yun Gao, Wanaruk Chaimayo, Benjamin L. Miller b, Matthew Z. Yates, Antimicrobial silver-hydroxyapatite composite coatings through two-stage electrochemical synthesis, Surface & Coatings Technology 301 (2016) 13–19

[116] Anish Shivaram, Susmita Bose, Amit Bandyopadhyay, Mechanical degradation of TiO2 nanotubes with and without nano-particulate silver coating, journal of the mechanical behavior of bio-medical materials 59 (2016) 508-518

[117] Xiong Lu, Bailin Zhang, Yingbo Wang, Xianli Zhou, Jie Weng, Shuxin Qu, Bo Feng, Fumio Watari, Yonghui Ding and Yang Leng, Nano-Ag-loaded hydroxyapatite coatings on titanium surfaces by electrochemical deposition J. R. Soc. Interface (2011) 8, 529–539

[118] Chao-Ming Xie, Xiong Lu, Ke-Feng Wang, Fan-Zhi Meng, Ou Jiang, Hong-Ping Zhang, Wei Zhi, and Li-Ming Fang, Silver Nanoparticles and Growth Factors Incorporated Hydroxyapatite Coatings on Metallic Implant Surfaces for Enhancement of Osteo-inductivity and Antibacterial Properties, Applied materials & Interfaces

[119] M. Furko, Y. Jiang, T.A. Wilkins. Balázsi, Electrochemical and morphological investigation of silver and zinc modified calcium phosphate bio-ceramic coatings on metallic implant materials, Materials Science and Engineering C 62 (2016) 249–259

[120] F. Bir, H. Khireddine, A. Touati, D. Sidane, S. Yala, H. Oudadesse, Electrochemical depositions of fluoro-hydroxyapatite doped by Cu2+, Zn2+, Ag+ on stainless steel substrates, Applied Surface Science 258 (2012) 7021–7030

[121] Yong Huang, Xuejiao Zhanga, Honglei Zhang, Haixia Qiaoa, Xiaoyun Zhanga, Tianjun Jiaa, Shuguang Hanb, Yuan Gaod, Hongyuan Xiaoe, Hejie Yang, Fabrication of silver- and strontium-doped hydroxyapatite/TiO2 nanotube bilayer coatings for enhancing bactericidal effect and osteo-inductivity, Ceramics International 43 (2017) 992–1007

[122] Yajing Yan, Xuejiao Zhang, Caixia Li, Yong Huang, Qiongqiong Ding, Xiaofeng Pang, Preparation and characterization of chitosan-silver/hydroxyapatite composite coatings onTiO2 nanotube for bio-medical applications, Applied Surface Science 332 (2015) 62–69

[123] Zhang, B. L., Wang, Y. B., Lu, X., Zhou, X. L., Qu, S. X., Feng, B. & Weng, J. In press. Accepted HA/Ag composite coatings prepared by pulse electrochemical deposition on titanium surfaces. Rare Metal Mater. Eng.

[124] Liu, R. F., Xiao, X. F., Zhen, L. Q. & Huang, J. M, HA/Ag composite coatings prepared by two-step electro-deposition. Chin. J. Appl. Chem, 2003, 20, 547–551.

[125] B.S. Necula, I. Apachitei, F.D. Tichelaar, L.E. Fratila-Apachitei, J. Duszczyk , An electron microscopical study on the growth of TiO2–Ag antibacterial coatings on Ti6Al7Nb bio-medical alloy, Acta Biomaterialia 7 (2011) 2751–2757

[126] Gary A. Fielding, Mangal Roy, Amit Bandyopadhyay, Susmita Bose, Antibacterial and biological characteristics of silver containing and strontium doped plasma sprayed hydroxyapatite coatings, Acta Biomaterial 8 (2012) 3144–3152

[127] Yikai Chen, Xuebin Zheng, Youtao Xie, Chuanxian Ding, Hongjiang Ruan, Cunyi Fan, Anti-bacterial and cytotoxic properties of plasma sprayed silver-containing HA coatings, J Mater Sci: Mater Med (2008) 19:3603–3609

[128] Jinhua Li, Xuanyong Liu, Yuqin Qiao, Hongqin Zhu, Chuanxian Ding, Antimicrobial activity and cytocompatibility of Ag plasma-modified hierarchical TiO2 film on titanium surface, Colloids and Surfaces B: Biointerfaces 113 (2014) 134–145

[129] Venkateswarlu Kotharu, Rameshbabu Nagumothua, Chandra Bose Arumugam, Muthupandi Veerappan, Subramanian Sankaran, MubarakAli Davoodbasha, Thajuddin Nooruddin, Fabrication of corrosion resistant, bio-active and antibacterial silver substituted hydroxyapatite/titania composite coating on Cp Ti, Ceramics International 38 (2012) 731–740

[130] Mangal Roy, Gary A. Fielding, Haluk Beyenal, Amit Bandyopadhyay, and Susmita Bose, Mechanical, In vitro Antimicrobial, and Biological Properties of Plasma-Sprayed Silver-Doped Hydroxyapatite Coating, Applied materials & Interfaces

[131] Baoe Li, Xuanyong Liu, Fanhao Meng, Jiang Chang, Chuanxian Ding, Preparation and antibacterial properties of plasma sprayed nano-titania/silver coatings, Materials Chemistry and Physics 118 (2009) 99–104

[132] S. Ciuca, M. Badea, E. Pozna, I. Pana, A. Kiss, L. Floroian, A. Semenescu, C.M. Cotrut, M. Moga, A. Vladescu, Evaluation of Ag containing hydroxyapatite coatings to the Candida albicans infection, Journal of Microbiological Methods 125 (2016) 12–18

[133] W. Chena, Y. Liua, H.S Courtney, M. Bettenga, C.M. Agrawal, J.D. Bumgardnere, J.L. Ong, In vitro anti-bacterial and biological properties of magnetron co-sputtered silver-containing hydroxyapatite coating, Biomaterials 27 (2006) 5512–5517

[134] M. Badea, M. Braic, A. Kiss, M. Moga, E. Pozna, I. Pana, A. Vladescu, Influence of Ag content on the antibacterial properties of SiC doped hydroxyapatite coatings, Ceramics International 42 (2016) 1801–1811

[135] A. Balamurugan, G. Balossier, D. Laurent-Maquin, S. Pina, A.H.S. Rebelo, J. Faure, J.M.F. Ferreir, An in vitro biological and anti-bacterial study on a sol–gel derived silver-incorporated bioglass system, dental materials 2 4 (2008) 1343–1351

[136] W. Chen, S. Oh, A.P. Ong, N. Oh, Y. Liu,4 H.S. Courtney, M. Appleford, J.L. Ong, Antibacterial and osteogenic properties of silver-containing hydroxyapatite coatings produced using a sol gel process, Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31197

[137] Swarna Jaiswal, Patrick McHale, Brendan Duffy, Preparation and rapid analysis of antibacterial silver, copper and zinc doped sol–gel surfaces, Colloids and Surfaces B: Biointerfaces 94 (2012) 170–176

[138] O. Akhavan, E. Ghaderi, Self-accumulated Ag nanoparticles on mesoporous TiO2 thin film with high bactericidal activities, Surface & Coatings Technology 204 (2010) 3676–3683

[139] Zhen Geng, Zhenduo Cui, Zhaoyang Li, Shengli Zhu, Yanqin Liang, Yunde Liu, Xue Li, Xin He, Xiaoxu Yu, Renfeng Wang, Xianjin Yang, Strontium incorporation to optimize the antibacterial and biological characteristics of silver-substituted hydroxyapatite coating, Materials Science and Engineering C 58 (2016) 467–477

[140] Chao Shi, Jianyong Gao, Ming Wang, Jingke Fu, Dalin Wang, Yingchun Zhu, Ultra-trace silver-doped hydroxyapatite with non-cytotoxicity and effective antibacterial activity, Materials Science and Engineering C 55 (2015) 497–505

[141] Brajendra Singh, Ashutosh Kumar Dubey, Samayendra Kumar, Naresh Saha, Bikramjit Basu, Rajeev Gupta, In vitro biocompatibility and antimicrobial activity of wet chemically prepared Ca10−xAgx(PO4)6(OH)2 (0.0≤x≤0.5) hydroxyapatites, Materials Science and Engineering C 31 (2011) 1320–1329

[142] Mustafa Turkoz, Aykan Onur Atilla, Zafer Evis, Silver and fluoride doped hydroxyapatites: Investigation by micro-structure, mechanical and antibacterial properties, Ceramics International 39 (2013) 8925–8931

[143] Carmen Steluta Ciobanu, Florian Massuyeau, Liliana Violeta Constantin and Daniela Predoi, Structural and physical properties of antibacterial Ag-doped nano hydroxyapatite synthesized at 100°C, Ciobanu et al. Nanoscale Research Letters 2011, 6:613

[144] A. Yanovsk, V. Kuznetsov, A. Stanislavov, S. Danilchenko, L. Sukhodub, Calcium–phosphate coatings obtained biomimetically on magnesium substrates under low magnetic field, Applied Surface Science 258 (2012) 8577–8584

[145] Anchun Mo, Juan Liao, Wei Xu, Suqin Xian, Yubao Li, Shi Bai, Preparation and antibacterial effect of silver–hydroxyapatite/titania nanocomposite thin film on titanium, Applied Surface Science 255 (2008) 435–438

[146] Andrea Ewald, Daniel Hösel, Sarika Patel, Liam M. Grover, Jake E. Barralet, Uwe Gbureck, Silver-doped calcium phosphate cements with antimicrobial activity, Acta Biomaterialia 7 (2011) 4064–4070

[147] Xiao Bai, Stefan Sandukas, Mark Appleford, Joo L. Ong, Afsaneh Rabiei, Antibacterial effect and cytotoxicity of Ag-doped functionally graded hydroxyapatite coatings, Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.31985

[148] Tapiero H, Tew KD. Trace elements in human physiology and pathology: zinc and metallothioneins. Biomed Pharmacother 2003;57(9):399–411

[149] Grandjean-Laquerriere A, Laquerriere P, Jallot E, Nedelec JM, Guenounou M, Laurent-Maquin D, Phillips TM, Influence of the zinc concentration of sol-gel derived zinc substituted hydroxyapatite on cytokine production by human monocytes in vitro, Biomaterials. 2006 Jun;27(17):3195-200. Epub 2006 Feb 17

[150] Jallot E, Nedelec JM, Grimault AS, Chassot E, Grandjean-Laquerriere A, Laquerriere P, Laurent-Maquin D, STEM and EDXS characterisation of physico-chemical reactions at the periphery of sol-gel derived Zn-substituted hydroxyapatites during interactions with biological fluids, Colloids Surf B Biointerfaces. 2005 May 25;42(3-4):205-10. Epub 2005 Apr 11.

[151] Kui Cheng, Jiabei Zhou, Wenjian Weng, Sam Zhang, Ge Shen, Piyi Du, Gaorong Han, Composite calcium phosphate coatings with sustained Zn release, ThinSoildFims(519), 2011, 4647-4651

[152] Fujii E, Ohkubo M, Tsuru K, Hayakawa S, Osaka A, Kawabata K, Bonhomme C, Babonneau F, Selective protein adsorption property and characterization of nano-crystalline zinc-containing hydroxyapatite, Acta Biomater. 2006 Jan;2(1):69-74. Epub 2005 Oct 24.

[153] Fumiaki Miyaji, Yoko Suyama, Yoshiteru Kono, Formation and Structure of Zinc-Substituted Calcium Hydroxyapatite, Materials Research Bulletin 40(2):209-220, February 2005

[154] LeGeros RZ, Bleiwas CB, Retino M, Rohanizadeh R, LeGeros JP, Zinc effect on the in vitro formation of calcium phosphates: relevance to clinical inhibition of calculus formation, Format: AbstractSend to Am J Dent. 1999 Apr;12(2):65-71.

[155] Von W. L. Jolly, The Synthesis and Characterization of Inorganic Compounds, Prentice Hall International, Hearts 1970. 1. Aufl., XI, 590 S., geb. ca. DM 74

[156] Ming’Ou Li, Xiufeng Xiao, Rongfang Liu, Cuiyu Chen, Lizhong Huang, Structural characterization of zinc-substituted hydroxyapatite prepared by hydrothermal method, J Mater Sci: Mater Med (2008) 19:797–803

[157] S.J. Kalita,H.A.Bhatt, Nano crystalline hydroxyapatite doped with magnesium and zinc: synthesis and characterization, Mater.Sci.Eng.C 27 (2007)837–848.

[158] R.J. Chung, M.F.Hsieh, C.W.Huang, L.H.Perng,H.W.Wen T.S. Chin, Antimicrobial effects and human gingival biocompatibility of hydroxyapatite sol–gel coatings, J.Biomed.Mater.Res.76B(2006) 169–178.

[159] S. Hayakawa, K.Ando, K.Tsuru, A.Osaka, E.Fujii, K.Kawabata, et al., Structural characterization and protein adsorption property of hydroxyapatite particles modified with zinc ions, J.Am. Ceram. Soc.90 (2007) 565–569.

[160] Y. Sogo, T.Sakurai, K.Onuma, etal., The most appropriate (Ca + Zn)/P molar ratio to minimize the zinc content of Zn TCP/HAP ceramic used in the promotion of bone formation, J.Biomed. Mater. Res. 62(2002) 457–463.

[161] A. Ito, K.Ojima, H.Naito, N.Ichinose, T.Tateishi, Preparation, solubility, and cytocompatibility of zinc-releasing calcium phosphate ceramics, J.Biomed. Mater. Res. 50(2000) 178–183.

[162] Y. Tang, H.F.CHAppell, M.T.Dove, R.J.Reeder, Y.J.Lee, Zinc incorporation into hydroxylapatite, Biomaterials 30(2009) 2864–2872.

[163] A. Ito, H.Kawamura, M.Otsuka, M.Ikeuchi, H.Ohgushi, K.Ishikawa, K. Onuma, N.Kanzaki, Y.Sogo, N.Ichinose, Zinc-releasing calcium phosphate for stimulating bone formation, Mater. Sci. Eng .C22 (2002) 21–25.

[164] A. Bigi ,E.Foresti, M.Gandolfi, M.Gazzano, N.Roveri, Inhibiting effect of zinc on hydroxylapatite crystallization, J.Inorg. Biochem. 58 (1995) 49–58.

[165] G. SureshKumar, A.Thamizhavel, Y.Yokogawa, etal., Synthesis, characterization and in vitro studies of zinc and carbonateco-substituted nano-hydroxyapatite for bio-medical applications, Mater. Chem. Phys. 134 (2012)1127–1135.

[166] F. Ren, R. Xin, X. Ge, Y. Leng, Characterization and structural analysis of zinc substituted hydroxyapatites, Acta Biomater. 5 (2009) 3141–3149

[167] H. Hu, W. Zhang, Y. Qiao, X. Jiang, X. Liu, C. Ding, Antibacterial activity and increased bone marrow stem cell functions of Zn-incorporated TiO2 coatings on titanium, Acta Biomaterialia 8 (2012) 904–915

[168] Amit Bandhyopadhya and Susmita Bose, (2013), 'Characterization of Biomaterials', ISBN: 978-0-12-415800-9

[169] Liga Berzina-Cimdina and Natalija Borodajenko, (2012), ‘Research of Calcium Phosphates Using Fourier Transform Infrared Spectroscopy’, Infrared Spectroscopy – Materials Science, Engineering and Technology, Editor Theophile Theophanides, ISBN 978-953-51-0537-4

[170] Shi D., (2006), ‘Introduction to biomaterials’, World Scientific Publishing, p. 253

[171] I. Rehman and W. Bonfield, (1997), ‘Characterization of hydroxyapatite and carbonated apatite by photo acoustic FTIR spectroscopy’, Journal of Materials science: Materials in medicine 8, 1-4

[172] D.G. Nelson and J. D. B. Featherstone, (1982), ‘Preparation, analysis, and characterization of carbonated apatite’, Calcif Tissue Int. 34, S69-81

[173] H. El Feki, Christian Rey, M. Vignoles, (1991), ‘Carbonate Ions in Apatite: Infrared Investigations in the v4 CO3 Domain’, Calcif Tissue Int 49:269-274

[174] D. Briggs, M.P. Seah, (1990), ‘Auger and X-ray Photoelectron Spectroscopy’, Practical Surface Analysis. Vol. 1 Wiley, New York

[175] C.C. Silva, A.S.B. Sombrab, (2004), 'Raman spectroscopy measurements of hydroxyapatite obtained by mechanical alloying', Journal of Physics and Chemistry of Solids 65, 1031–1033

[176] Wei Ye, Xiao-Xiang Wang, (2007), 'Ribbon-like and rod-like hydroxyapatite crystals deposited on titanium surface with electrochemical method', Materials Letters 61, 4062–4065

[177] Definitions in Biomaterials, Williams, ‘Progress in Bio-medical Engineering’, 4, Elsevier, Amsterdam (1987).

[178] Buddy D. Ratner, ‘The Biocompatibility of Implant Materials’, Host Response to Biomaterials, the Impact of Host Response on Biomaterial Selection 2015, Pages 37–51.

[179] FDA. Blue Book Memorandum G95-1: FDA-Modified Version of ISO 10993-Part 1, Biological Evaluation of Medical Devices – Part 1: Evaluation and Testing; Silver Spring, MD: FDA, 1995

[180] ISO 10993. Biological Evaluation of Medical Devices, www.iso.org

[181] K.L. SHARMA, Biomaterial & Biocompatibility Testing Laboratory, Government of India Ministry of Health & family Welfare Nirman Bhavan, New Delhi – 110011

[182] ASTM. 2008 Annual Book of ASTM Standards; ASTM International: West Conshohocken, PA, 2008; Vol. 13.01

[183] ‘Biocompatibility and Tissue Reaction to Biomaterials’

[184] De Melo WM, Maximiano WM, Antunes AA, Beloti MM, Rosa AL, 'Cytotoxicity testing of methyl and ethyl 2-cyanoacrylate using direct contact assay on osteoblast cell cultures', de Oliveira PT J Oral Maxillofac Surg. 2013 Jan; 71(1):35-41.

[185] Salim Levent Aktuğ, Salih Durdu, Emine Yalçın, Kültigin Çavușoğlu, Metin Usta, ‘Bioactivity and biocompatibility of hydroxyapatite-based bioceramic coatings on zirconium by plasma electrolytic oxidation’, Materials Science and Engineering C 71 (2017) 1020–1027.

[186] WEIJIA LI, JING ZHOU, and YUYIN XU, 'Study of the in vitro cytotoxicity testing of medical devices', Bio-medical Report. 2015 Sep; 3(5): 617–620.

[187] Humphries MJ, Cell adhesion assays, Methods Mol Biol. 2009; 522:203-10.

[188] Weitz-Schmidt G, Chreng S. Cell adhesion assays, Methods Mol Biol. 2012; 757: 15-30

[189] Amelia Ahmad Khalili, Mohd Ridzuan Ahmad, A Review of Cell Adhesion Studies for Bio-medical and Biological Applications, Int J Mol Sci. 2015 Aug; 16(8): 18149–18184.

[190] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi, T. Yamamwo, ‘Solutions able to reproduce in vivo surface structure changes in bio-active glass-ceramic’, A-W. J. Biomed. Mat. Res. 24 (1989) 721–734

[191] T. Kokubo, H. Takadama, ‘How useful is SBF in predicting in vivo bone bioactivity?’ Biomaterials 27 (2006) 2907–2915

[192] Rym Taktaka, Achwek Elghazel, Jamel Bouaziz, Slim Charfi, Hassib Keskes, 'Tricalcium phosphate-Fluorapatite as bone tissue engineering: Evaluation of bioactivity and biocompatibility' Materials Science & Engineering C

[193] K.L. SHARMA, Biomaterial & Biocompatibility Testing Laboratory, Government of India Ministry of Health & family Welfare Nirman Bhavan, New Delhi – 110011

[194] Sung Hwan Kim, Yong Hoon Jeong, Han Cheol Choe, William A. Brantley, (2014), “Morphology change of HAp films on highly ordered nanotubular Ti-Nb-Hf alloys as a function of electrochemical deposition cycle”, Surface & Coatings Technology.

[195] In-Seop Byeon, Kang Lee, Han-Cheol Choe, William A. Brantley, (2015), ‘Surface morphology of Zn-containing hydroxyapatite (Zn-HA) deposited electrochemically on Ti–xNb alloys’, Thin Solid Films

[196] P.N. Lim, B.Y. Tay, C.M. Chan, E.S. Thian, "Synthesis and characterization of silver/silicon-cosubstituted nanohydroxyapatite", J. Biomed. Mater. Res. – Part B Appl. Biomater., Vol. 100 B, 2012, pp. 285–291.

[197] N. Eliaz, O. Ritman-Hertz, D. Aronov, E. Weinberg, Y. Shenhar, G. Rosenman, M. Weinreb, E. Ron, "The effect of surface treatments on the adhesion of electrochemically deposited hydroxyapatite coating to titanium and on its interaction with cells and bacteria", J. Mater. Sci. Mater. Med., Vol. 22, 2011, pp. 1741–1752.

[198] N. Eliaz, T.M. Sridh, "Electrocrystallization of hydroxyapatite and its dependence on solution conditions", Cryst. Growth Des., Vol. 8, 2008, pp. 3965–3977.

[199] J. Huang, S.M. Best, W. Bonfield, T. Buckland, "Development and characterization of titanium-containing hydroxyapatite for medical applications", Acta Biomater., Vol. 6, 2010, pp. 241–249.

[200] S. Koutsopoulos, Synthesis and characterization of hydroxyapatite crystals: A review study on the analytical methods, Journal of Bio-medical Material Research, Vol. 62, pp. 600-612, 2002

[201] Vranceanu, D. M., Parau, A. C., Cotrut, C. M., Kiss, A. E., Constantin, L. R., Braic, V., & Vladescu, A. (2019). In vitro evaluation of Ag doped hydroxyapatite coatings in acellular media. Ceramics International, 45(8), 11050-11061.