Laser Surface Treatment of Bio-Implant Materials

Laser Surface Treatment of Bio-Implant Materials
Laser Surface Treatment of Bio-Implant Materials L. Hao and J. Lawrence 2005 John Wiley & Sons, Ltd ISBN: 0-470-01687-6
Laser Surface Treatment of Bio-Implant Materials
Liang Hao Loughborough University, UK Jonathan Lawrence Nanyang Technological University, Singapore
Copyright 2005
John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England Telephone (44) 1243 779777
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To Yan-jun and My Parents For the world they bring to me.
To Louise and Ethan, Always there; beyond compare.
Contents
Acknowledgements Introduction Bio-Implants and Surface Modication of Biomaterials Wettability in Biomaterials Science and Modication Techniques Lasers and Their Application for Modication of the Biomaterials 1 Bioactivity and Biointegration of Orthopaedic and Dental Implants 1.1 Introduction 1.1.1 Biocompatibility 1.1.2 Host Response to Biomaterials 1.1.3 In vitro Models of Biological Response to Implants 1.2 Bioactivity of Bone Implants 1.2.1 The Mechanism of Apatite Formation 1.2.2 Functional Group 1.3 Biointegration of Orthopaedic and Dental Implants 1.3.1 Osseointegration 1.3.2 Bone Cell Adhesion [44] 1.3.3 OsteoblastMaterial Interactions 1.4 Controlling the BoneImplant Interface 1.4.1 Physicochemical Methods 1.4.2 Biochemical Methods [9] Surface Modication of Biomaterials 2.1 Introduction 2.1.1 Orthopaedic and Dental Implants 2.1.2 Surface Properties of Biomaterials xiii xv xv xvi xvii
1 1 2 2 2 3 3 4 5 5 5 6 6 7 7 11 11 11 12
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2.2
2.3
2.4
2.5
2.1.3 Surface Analysis of Biomaterials Ceramic Implants [65] 2.2.1 Nearly Bioinert Ceramics [64, 69] 2.2.2 Alumina 2.2.3 Zirconia Ceramics Metallic Implants 2.3.1 Mechanical Properties 2.3.2 Corrosion Surface Modication of Biomaterials 2.4.1 Introduction 2.4.2 Radiation Grafting and Photografting [76] 2.4.3 Plasma Surface Modication of Biomaterials 2.4.4 Ion Beam Processing 2.4.5 Other Methods [65] Laser Surface Modication of Biomaterials 2.5.1 Introduction 2.5.2 Laser Patterning and Microfabrication 2.5.3 Pulsed Laser Deposition (PLD) of Biocompatible Ceramics 2.5.4 Matrix-Assisted Pulsed Laser Evaporation and MAPLE Direct Write 2.5.5 Other Laser Surface Treatments
12 13 13 14 14 15 16 16 17 17 17 18 18 19 19 19 20 20 21 21
Wettability in Biomaterials Science and Modication Techniques 3.1 Introduction 3.2 Wettability, Adhesion and Bonding: Theoretical Background 3.2.1 The Wetting Process 3.2.2 Contact Angle and Work of Adhesion 3.2.3 Surface Energy and the Dispersive/Polar Characteristics 3.2.4 Physical Bonding 3.2.5 Mechanical Bonding 3.2.6 Chemical Bonding 3.3 Wettability in Biomaterial Science 3.3.1 Biomaterial Interfaces [110] 3.3.2 Tensiometry 3.3.3 Interfacial Biophysics 3.3.4 Thermodynamic Concepts in Biomaterials Science 3.4 Current Methods of Wettability Modication 3.4.1 Chemical Reactions 3.4.2 Plasma Surface Modication
23 23 23 23 24 25 28 28 28 29 29 29 30 31 33 33 33
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3.4.3 3.4.4 3.4.5 3.4.6 3.4.7 3.5 Laser 3.5.1
Ion Beam Processing Radiation Grafting UV and Ozone Corona Discharge Electrowetting Wettability Characteristics Modication Laser Surface Modication of Ceramic Materials for Improved Wettability 3.5.2 Laser Surface Modication of Metallic Materials for Improved Wettability
34 34 34 35 35 35 35 36
CO2 Laser Modication of the Wettability Characteristics of Magnesia Partially Stabilised Zirconia 4.1 Introduction 4.2 Experimental Procedures 4.2.1 Material Specications 4.2.2 CO2 Laser Experimental Arrangement 4.2.3 Morphological, Chemical and Phase Analysis Procedures 4.2.4 Wettability Characteristics Analysis Procedure 4.3 The Effects of CO2 Laser Radiation on Wettability Characteristics 4.3.1 Contact Angle 4.3.2 The Effect of Surface Oxygen Content 4.3.3 The Effect of Surface Roughness 4.3.4 The Effects of Solidied Microstructures and Surface Melting on Wettability Characteristics 4.4 Surface Energy and Its Component Parts 4.5 Identication of the Predominant Mechanisms Active in Determining Wettability Characteristics 4.6 The Role Played by Microstructures in Terms of Crystal Size and Phase in Effecting Surface Energy Changes 4.6.1 The Role of Crystal Size on Surface Energy 4.6.2 The Role of Phase Change on Surface Energy 4.7 Investigation of Wettability and Work Adhesion Using Physiological Liquids 4.8 Summary In vitro Biocompatibility Evaluation of CO2 Laser Treated Magnesia Partially Stabilised Zirconia 5.1 Introduction 5.2 Sample Preparation 5.3 Bone-Like Apatite Formation
37 37 38 38 39 39 40 41 41 42 43 45 47 52 56 56 61 61 63
65 65 67 67
Contents
5.4
5.5
5.6 5.7
5.3.1 Experimental Procedures 5.3.2 Spectral Analysis and Hydroxyl Group 5.3.3 The Correlation between OH Groups and Wettability Characteristics 5.3.4 The Effects of CO2 Laser Treatment on the MgOPSZ in Simulated Body Fluids Protein Adsorption 5.4.1 Experimental Procedures 5.4.2 Albumin and Fibronectin Adsorption on CO2 Laser Treated MgOPSZ Osteoblast Cell Response 5.5.1 Experimental Procedures 5.5.2 Osteoblast Cell Response on the CO2 Laser Treated MgOPSZ 5.5.3 The Effect of CO2 Laser Treatment on the Osteoblast Cell Response Predictions for Implantation in an in vivo Clinical Situation Summary
68 69 71 73 75 76 77 80 81 83 89 95 98
The Effects of CO2 Laser Radiation on the Wettability Characteristics of a Titanium Alloy 6.1 Introduction 6.2 Experimental Procedures 6.2.1 Material Specications and Preparation 6.2.2 CO2 Laser Surface Treatment 6.2.3 Morphological, Chemical and Phase Analysis Procedures 6.2.4 Wettability Characteristics Analysis Procedure 6.3 The Effects of CO2 Laser Radiation on Wettability Characteristics 6.3.1 Contact Angle 6.3.2 Morphological Analysis and Its Effect on Wettability Characteristics 6.3.3 Phase and Chemical Analysis and Its Effects on Wettability Characteristics 6.4 Surface Energy and Its Component Analysis 6.5 Identication of the Predominant Mechanisms Active in Determining Wettability Characteristics 6.6 Investigation of Wettability and Work Adhesion Using Physiological Liquids 6.7 Summary
99 99 101 101 102 102 102 103 103 103 105 109 111 114 116
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In vitro Biocompatibility Evaluation of CO2 Laser Treated Titanium Alloy 7.1 Introduction 7.2 Sample Preparation 7.3 Bone-Like Apatite Formation on Titanium Alloys 7.3.1 Experimental Procedures 7.3.2 The Effects of CO2 Laser Treatment on the Ti6Al4V in Simulated Body Fluid 7.4 Protein Adsorption 7.4.1 Experimental Procedures 7.4.2 Albumin and Fibronectin Adsorption on CO2 Laser Treated Titanium Alloy 7.5 Osteoblast Cell Adhesion 7.5.1 Experimental Procedure 7.5.2 Osteoblast Cell Response on CO2 Laser Treated Titanium Alloy 7.5.3 The Effect of CO2 Laser Treatment on the Osteoblast Cell Response 7.6 Predictions for Implantation in an in vivo Clinical Situation 7.7 Summary Enquiry into Possible Generic Effects of the CO2 Laser Treatment on Bone Implant Biomaterials 8.1 Introduction 8.2 Ascertaining the Generic Effects of CO2 Laser Treatment on Bioinert Ceramics 8.2.1 Experimental Procedures 8.2.2 Modication of the Surfaces Properties and Wettability Characteristics of a YPSZ Bioinert Ceramic 8.2.3 Identication of the Predominant Mechanism Active in the Wettability Characteristics Modication of a YPSZ Bioinert Ceramic 8.2.4 Generic Effects of CO2 Laser Treatment on the Wettability Characteristics of Bioinert Ceramics 8.2.5 CO2 Laser Induced Effects on the Cell Response on a YPSZ Bioinert Ceramic 8.2.6 Generic Effects of CO2 Laser Treatment on the Cell Response on Bioinert Ceramics 8.3 Ascertaining the Generic Effects of CO2 Laser Treatment on Metal Implants 8.3.1 Experimental Procedures
117 117 119 120 121 121 123 123 124 127 127 128 131 135 138
141 141 142 143 144
149 150 153 157 157 158
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8.3.2 Modication of Surfaces Properties and Wettability Characteristics of a 316 LS Stainless Steel 8.3.3 Identication of the Predominant Mechanism Active in the Wettability Characteristics Modication of a 316 LS Stainless Steel 8.3.4 Generic Effects of CO2 Laser Treatment on the Wettability Characteristics of Biometals 8.3.5 CO2 Laser Induced Effects on Protein Adsorption and the Cell Response on a 316 LS Stainless Steel 8.3.6 Generic Effects of CO2 Laser Treatment on Protein Adsorption and the Cell Response on Biometals 8.4 Summary Conclusions References Index
159
166 168 170 175 176 179 185 209
Acknowledgements
First, we gratefully appreciate the understanding and encouragement of our families who live on opposite sides of the world in China and England. We are indebted to all the technicians in the Materials Laboratory at Nanyang Technological University for their advice and assistance on optical microscopy, XRD, SEM, EDX, contact angle analysis, XPS analysis and sample preparation. Many thanks also to the Doctoral Research students in the Materials Laboratory for sharing their knowledge on material treatment and analysis. We acknowledge the tremendous contribution made by the rst-rate work of the 20032004 Final Year Project students: Y.F. Phua, T.L. Tan, M.W. Koo and T.H. Wang. On numerous occasions we obtained superb instruction and assistance from Mr Ma Dong Rui on the subject of osteoblast cell culture, for which we are extremely grateful.
Introduction
Bio-Implants and Surface Modication of Biomaterials There is archaeological evidence that lost teeth were replaced by handcarved ivory or wood implants as long ago as ancient Egypt. In the early 1950s Swedish orthopaedic surgeon Per Ingvar Branemark began studying the healing process of titanium anchoring screws, which proved to be a seminal point for modern dental and orthopaedic implants [1]. His work showed that fusion between bone and the titanium implant could take place, a phenomenon he called osseointegration. Orthopaedic implants to treat joint degradation due primarily to osteoarthritis, osteoporosis or injury are now commonplace. These include hip (around 325 000 US implants in 2001) and shoulder, wrist and knee (around 300 000 US implants in 2001). Biomaterial applications make use of all classes of materials, metals, ceramics, polymers and composite. These are divided roughly into three user types [2]: (a) inert or relatively inert with minimal host response; (b) bioactive, which actually stimulates bonding to the surrounding tissue; and (c) biodegradable, which resorb in the body over a period of time. Events leading to integration of an implant into bone, which in turn determine the performance of the device, take place largely at the tissue implant interface. The main requirements for a biomaterial to function properly in an osseous site include good biocompatibility favouring bone apposition, adequate mechanical properties and the ability to ensure skeletal functions [35]. Bioactivity and biointegration are the two essential aspects of these interactions. Bioactivity and the maintenance of skeletal functions are usually attributed to the ability to induce an apatite layer on a materials surface in physiological conditions [68]. The close apposition between bone and an implant surface, or osseointegration, presented as the ability to promote bone cells anchorage, attachment, spreading, growth and differentiation [9, 10], is another key factor for successful implantation of a biomaterial for dental and orthopaedic applications [35].
xvi
Introduction
Biointegration is the ideal outcome expected of an articial implant. This implies that the phenomena that occur at the interface between the implant and host tissues do not induce any deleterious effects such as chronic inammatory response or formation of unusual tissues. It is, therefore, of paramount importance to design biomaterials used in implants with the best surface properties. Meanwhile, these biomaterials must possess bulk properties that meet other requirements, especially mechanical properties, in order to function properly in a bioenvironment. As it is quite difcult to design biomaterials fullling both needs, a common approach is to fabricate biomaterials with adequate bulk properties followed by a special treatment to enhance the surface properties. Hence surface modication of biomaterials is becoming an increasingly popular method to improve device multifunctionality, tribological and mechanical properties, as well as biocompatibility of articial devices while obviating the needs for large expenses and a long time to develop brand new materials [11]. Materials can be surface modied by using biological or physicochemical methods.
Wettability in Biomaterials Science and Modication Techniques The wetting of a surface by a liquid and the ultimate extent of spreading of that liquid are very important aspects of practical surface chemistry. Even with all the new information of the last 20 years, however, there still remains a great deal to learn about the mechanisms of movement of a liquid across a surface and the factors that govern such movement [12]. Biomaterial scientists have long sought a single, material-related parameter that effectively measures biocompatibility and might serve as a practical design guide. The theories of surface energy and wetting for such parameters present an attractive means to do this as surface properties are important determinants of a biomaterial function [12]. The ability to control the surface wettability of solid substrates is important in many situations. Various surface processes are used for modifying the surfaces of materials depending on the actual material and the application. A number of laser-based techniques for altering the wettability characteristics of engineering materials have been investigated [13]. Surface sensitivity is of critical importance in biomaterials surface science because only the uppermost layers are in direct physicochemical contact with the biological environment; consequently only the upper few molecular layers determine biocompatibility. Thus the interfacial chemistry of concern to biomaterial scientists is determined by material composition within the upper nanometre or so. Tensiometry encompasses a broad range of related wetting techniques that measure surface energy. These include the observation of contact angles, which is perhaps the most familiar and widely
Lasers and Their Application for Modication of the Biomaterials
xvii
applied method. Tensiometric methods have singular potential in biomaterials surface science based on the criteria of surface sensitivity, kind of analytical information obtained and relevance of that information to biomedical problems. First, with respect to surface sensitivity, wetting measurements are sensitive only to the upper 0.5 nm or so of a surface [14, 15] and are therefore among the most surface-sensitive techniques available. Second, tensiometry directly measures the fundamental energy at an interface that drives important processes such as adsorption and adhesion. This kind of information must be particularly pertinent to biomaterial problems because of the overwhelming importance of protein adsorption and cell/tissue adhesion. Third, wetting measurements can be made using proteinaceous saline solutions that are particularly relevant to biomedical applications. Special high-vacuum preparation techniques that might introduce experimental artefacts are not required. Various methods are used to improve the surface wettability of materials and their adhesion to other materials. Hydrophilicity is a characteristic of materials exhibiting an afnity for water. Hydrophilic literally means waterloving and such materials readily adsorb water. Hydrophobic describes materials possessing a characteristic that has the opposite response to water interaction compared to hydrophilic materials. Smaller water contact angles correspond to more hydrophilic surfaces and higher surface free energies. At present, the processes available to engineers for the modication of a materials wettability characteristics are invariably complex and consequently somewhat difcult to control. Lasers, on the other hand, can offer the user not only an exceedingly high degree of process controllability but also a great deal of process exibility. There is a growing amount of published work that testies to the potential of lasers for altering the surface properties of materials in order to improve their wettability characteristics. Laser radiation was found to effect signicant changes in the wettability characteristics of materials. Lawrence and Li [13] have amply demonstrated the practicability of employing different types of lasers to effect changes in the wettability characteristics of composites and ceramics, metals and plastics for improved adhesion and bonding.
Lasers and Their Application for Modication of the Biomaterials The laser has some unique properties for surface treatment. The electromagnetic radiation of a laser beam is absorbed within the rst atomic layers for opaque materials, such as metals, and there are no associated hot gas jets, eddy currents or even radiation spillage outside the optically dened beam area. In fact, the applied energy can be placed precisely on the surface only where it is needed. Thus it is a true surface heater and a unique tool for
xviii
Introduction
surface engineering. Common advantages of laser surfacing compared to alternatives are [16]: chemical cleanliness; controlled thermal penetration and, therefore, distortion; controlled thermal prole and, therefore, shape and location of the heat affected region; less after-machining, if any, is required; remote noncontact processing is usually possible; and relatively easy to automate. Surface treatment is a subject of considerable interest at present as it seems to offer the chance to save strategic materials or to allow improved components with idealised surfaces and bulk properties. At present, the lasers are being used in the following surface modications of the biomaterials: Laser patterning and microfabrication Pulsed laser deposition (PLD) of biocompatible ceramics Matrix-assisted pulsed laser evaporation (MAPLE) and MAPLE direct write (MDW) Laser surface treatment for improving corrosion Laser grafting Laser treatment of plasma sprayed hydroxyapatite coatings However, little work has been carried out to investigate employing lasers to modify the surface properties of biomaterials in order to improve their biocompatibility. Having said that, it is recognised within the currently published work that laser irradiation of material surfaces can affect changes in the cell adhesion on biomaterials. Lately, several publications have investigated the modication of biocompatibility of a biomaterials surface following laser irradiation. A CO2 pulsed laser was used to graft a polymer [17] and a rubber [18]. The results showed a marked reduction of the platelet adhesion and aggregation for the modied polymer surface and cell attachment, with a greater degree of spreading and attening on the unmodied rubber surface. L929 broblast cells attached and proliferated extensively on the CO2 and KrF laser treated lms [19] in comparison with unmodied PET (polyethylene teraphthalate), with surface morphology and wettability being found to affect cell adhesion and spreading. However, so far, no work has investigated the use of laser surface modication of bioinert ceramics and biograde metals for improved biocompatibility. With a view to improve the biocompatibility of the dental and orthopaedic implant, a CO2 laser was used to modify the surface properties of the widely used bioinert ceramics and biograde metals. By varying the CO2 laser power densities, the work investigated how the CO2 laser affected the surface
Lasers and Their Application for Modication of the Biomaterials
xix
properties of magnesiapartially stabilised zirconia (MgOPSZ), a bioinert ceramic, such as morphology, surface roughness, surface oxygen content and rapidly solidied microstructure. It particularly analysed the change in the wettability characteristics and basic mechanisms governing this modication [2022], since wettability is widely recognised as an important determinant of cell adhesion and biomaterials function. Then, the bioactivity evaluation of the MgOPSZ was conducted to nd whether the bone-like apatite could form on the MgOPSZ following CO2 laser irradiation in the stimulated body uid and what were the functional groups for apatite nucleation [23]. Furthermore, it investigated how the CO2 laser modied surface properties inuence the protein adsorption [24, 25] and osteoblast cell adhesion [26] that would manipulate the biointegration between the implant and tissue. Moreover, the effects of the CO2 laser on the modication of the wettability characteristics of a titanium alloy (Ti6Al4V) and thereof the adhesion with the simulated physiological liquids were analysed. Further, it observed the apatite formation on the CO2 laser treated Ti6Al 4V alloy after soaking in simulated body uid and the signicant difference of albumin and bronectin adsorption between the untreated sample and CO2 laser treated samples. More importantly, osteoblast cell adhesion and proliferation performed on the CO2 laser treated Ti6Al4V was compared with untreated titanium alloy. With the aim of establishing the laser as the innovative technique for the surface modication of the biomaterials, the generic effects of CO2 laser irradiation on the biocompatibility of a yttriapartially stabilised zirconia (YPSZ) and a biograde stainless steel are investigated. Similar effects of the CO2 laser treatment on the wettability characteristics, protein adsorption and osteoblast cell were observed on the YPSZ and stainless steel. The study therefore proved the generic and great potential application of the laser surface process of widely used bioinert ceramic and biograde metals.
1
Bioactivity and Biointegration of Orthopaedic and Dental Implants
1.1 Introduction Events leading to integration of an implant into bone, which in turn determine the performance of the device, take place largely at the tissue implant interface. The main requirements for a biomaterial to function properly in an osseous site include good biocompatibility favouring bone apposition, adequate mechanical properties and the ability to assure skeletal functions [35]. Bioactivity and biointegration are the two essential aspects of these interactions. Bioactivity and the maintenance of skeletal functions are usually attributed to the ability to induce an apatite layer on a materials surface in physiological conditions [68]. The close apposition between bone and an implant surface, or osseointegration, presented as the ability to promote bone cells anchorage, attachment, spreading, growth and differentiation [9, 10], is another key factor for successful implantation of a biomaterial for dental and orthopaedic applications [35]. Recent years have seen considerable interest in the systematic investigation of the relationship between surface chemistry [27, 28] and morphology [29] and biological interfacial reactions. Ultimately, such studies may lead to an enhanced understanding of the surface chemical cues that guide the combination of apatite induction ability with attachment, growth and differentiation of bone cells and to the design of improved synthetic materials for dental and orthopaedic applications.
Bioactivity and Biointegration
1.1.1 Biocompatibility A biocompatible material has been dened as a material that does not induce an acute or chronic inammatory response and does not prevent a proper differentiation of implant surrounding tissues [30]. It is recognised that some adverse tissue reaction around the implanted biomaterial is inevitable, owing to surgical trauma during insertion. This denition implies that biocompatibility depends on the purpose of the implant. Terms such as bioinert or bioactive, rather than biocompatibility, more accurately describe the features required of an ideal biomaterial or device. These terms better describe the action or nonaction required from surrounding tissue and are thus related to the choice of materials and material characteristics (hydrophilic/hydrophobic). 1.1.2 Host Response to Biomaterials The biocompatibility of the implant material is closely related to the reactions between the surface of the biomaterial and the inammatory host response [31]. The implantation response in bone differs in some ways from that taking place in soft tissue. There is an inammatory and a reparative response which occur one on the other. The reparative response starts 23 days after the implantation. The stem cells of bone develop into osteoblasts, which form a layer near the implant together with broblasts. Fibroblasts, osteoblasts and capillaries penetrate into the blood clot, replacing it, and ll the space between the implant and bone [32]. After the formation of a collagen-rich extracellular matrix (ECM), mineralization follows. Normally, there are vesicles in the ECM and some of them include calcication focuses. The presence of vesicles with biomaterial in the early period is a sign of good primary acceptance. When the membranes of these vesicles rupture, the erupted apatite crystals unite and form calcifying structures [33]. Early trabecles grow and continue to mineralise, and some of them reach the implant surface. In an optimal situation, the material is covered by bone tissue and not by brous capsule. The healing of bone tissue continues like fracture healing. Remodeling bone tissue begins after two weeks and continues for the lifetime. When the material is biocompatible, there is an abundance of the ECM and osteoblasts. This is conrmed by the close attachment and fast proliferation of these cells [34]. 1.1.3 In vitro Models of Biological Response to Implants In vitro models have been widely used to investigate biological responses to biomaterials, and the materials adopted for implantology have been no exception. Given their relative ease of use and lack of expense, in vitro models would appear to investigate the biological response to implant
Bioactivity of Bone Implants
materials [35]. Cell culture methods have been used to evaluate the biological compatibility of materials for more than two decades [36]. Bone cell culture models are increasingly employed to study bonebiomaterial interactions. Most of the cultures have utilized osteoblastic cells (reviewed by Cooper et al. [37]), with only a few using osteoclastic cells [38, 39]. In vitro models have the potential to help elucidate events at the boneimplant interface (reviewed by Davies [40]), by providing morphological, biochemical and molecular information regarding osteoblastic development and synthesis of the matrix at the interface with various biomaterials.
1.2 Bioactivity of Bone Implants For an articial material to bond to living bone, it is essential that the material has the ability to form a biologically active, bone-like, apatite layer on its surface in the human body. Under normal conditions, the body uid is already supersaturated with respect to apatite, and once apatite on its nuclei form on the surface of a material, they can spontaneously grow by consuming the calcium and phosphate ions from the body uid. The nucleation of apatite on the surface of a material is induced by the functional groups on its surface. Naturally, the development of bioactive materials that have improved and ultimately the bone-like mechanical properties is desirable [41]. 1.2.1 The Mechanism of Apatite Formation The biological activity of most orthopaedic and dental biomaterials is related to their ability to promote the formation of a neoformed layer of carbonate apatite crystals analogous to bone mineral. This layer also associates specic bone proteins and is the starting point of bone reconstruction [42]. Orthopaedic biomaterials may be classied as passive or active with regard to their propensity simply to allow the nucleation and growth of carbonate apatite crystals from body uids (hydroapatite, titanium oxide, neutral hydrogels, collagen) or to supply ions to build and develop this layer (bioglasses, alkaline hydroxides, CaP compounds, calcium carbonate). Bioactive ceramics have a common characteristic at the interface with bone after integration. In fact, bioactive ceramics, including bioglass and hydroxyapatite (HA) reveal a layer of apatite at the interface, mediating integration with bone. Histological examination in vivo shows that this apatite layer is formed on the ceramic surface early in the implantation period and thereafter the bone matrix integrates into the apatite. Detailed characterisation indicated that this apatite layer consists of nanocrystals of carbonateion-containing apatite that has a defective structure and low crystallinity. These features are, in fact, very similar to those of the mineral phase in bone;
hence bone-producing cells (osteoblasts) can preferentially proliferate on the apatite and differentiate to form an extracellular matrix composed of biological apatite and collagen. As a result, the surrounding bone comes into direct contact with the surface apatite layer. When this process occurs, a chemical bond is formed between the bone mineral and the surface apatite to decrease the interfacial energy between them. It can be concluded that an essential requirement for an articial material to bond to living bone is the formation of a layer of biologically active bone-like apatite on its surface in the body. 1.2.2 Functional Group Bioactivity can be induced on surfaces of nonbioactive materials either by the formation of the functional groups that are able to induce apatite formation or by forming thin ceramic phases that have the potential to form the functional groups on exposure to a body environment [41]. The catalytic effect of the SiOH groups and TiOH groups for apatite nucleation has been proven from the observation that silica and titania gels produced by the sol-gel method form apatite on their surfaces in simulated body uids (SBF), and these functional groups are abundant on their surfaces. Zirconia, niobium oxide and tantalum oxide gels have also been shown to form apatite on their surface in SBF, as shown in Figure 1.1. This indicates that ZrOH, NbOH and TaPH groups are effective for apatite nucleation. Other assessments using self-assembled monolayers (SAM) in SBF have indicated that COOH and PO4H2 groups are also effective for apatite nucleation [41].
Figure 1.1 Scanning electron microscopy (SEM) photographs of the surfaces of silica (A), titania (B), zirconia (C), niobium oxide (D) and tantalum oxide (E) gels after soaking in an SBF for 14 d [41]
Biointegration of Orthopaedic and Dental Implants
These groups have specic structures revealing negatively charge, and induce apatite formation via formations of an amorphous calcium compound, e.g. calcium silicate, calcium titanate and amorphous calcium phosphate. Moreover, the efcacy of apatite nucleation of the above functional groups is determined, not by their composition alone but in a complicated fashion that is dependent on their concentration and structural arrangement.
1.3 Biointegration of Orthopaedic and Dental Implants 1.3.1 Osseointegration Integration of the implant into the bone is a property of paramount importance in the proper functioning of the implant; consequently extensive studies have been carried out using different techniques to improve osseointegration. Osseointegration was dened by Branemark [43] as: A direct structural and functional connection between living bone and the surface of a load-carrying implant. The integration of a biomaterial to bone involves essentially two processes: interlocking with bone tissue and chemical interactions with bone constituents. It is essential for the efcacy of orthopaedic or dental implants to establish a mechanically solid interface with complete fusion between the materials surface and the bone tissue with no brous tissue interface. 1.3.2 Bone Cell Adhesion [44] The term adhesion in the biomaterial domain covers different phenomena: the attachment phase, which occurs rapidly and involves short-term events like physicochemical linkages between cells and materials involving ionic forces, van der Waals forces, etc., and the adhesion phase, occurring in the longer term and involving various biological molecules: extracellular matrix proteins, cell membrane proteins and cytoskeleton proteins which interact together to induce signal transduction, promoting the action of transcription factors and consequently regulating gene expression. It is widely acknowledged that a major determinant of the bonebiomaterial interfacial response is the initial attachment, spreading and growth of osteoblasts on the implant surface and that improvements in these processes may lead to faster and long-term stability [3, 45]. It has been possible to demonstrate cellular attachment to implant surfaces and there is good evidence for new bone formation being initiated at a distance from but towards the surface of an implant rather than on the surface itself [3, 45]. The biocompatibility of biomaterials is very closely related to cell behaviour on contact with them and particularly to cell adhesion to their surface.
1.3.3 OsteoblastMaterial Interactions Osteoblastmaterial interaction depends on the surface aspects of materials, which may be described according to their topography, chemistry or surface energy. These surface characteristics determine how biological molecules will adsorb to the surface and more particularly determine the orientation of adsorbed molecules [46]. They also determine the cell behaviour on contact. It is known that osteoblast cells initially respond in a differential manner to the material surface. The comparison of the behaviour of different cell types on materials shows that they react differently according to surface roughness [28, 47]. Scanning electron microscopic examination of bone cells on materials with various surface roughness generally demonstrated that cell spreading and continuous cell layer formation were better on smooth surfaces compared to rough surfaces [4850]; however, higher levels of cellular attachment have been found on rough surfaces of titanium with irregular morphologies [5153] in vitro. Similarly, recent studies have shown that alkaline phosphatase (ALP) specic activity is enhanced on rough Ti and Ti6Al4V [54, 55]. Cells grown on rougher surfaces exhibited increased production of collagen [52, 54] and transforming growth factor b [54]. These differences of cell response could be attributed to either the microstructure, crystalline or chemistry, as different methods were used to obtain different roughnesses. A contact guidance phenomenon has also been described on osteoblastic cells. On smooth surfaces, bone cells were randomly oriented although they were aligned parallel to the direction of the grooves in an end-to-end fashion in 5 mm deep grooves [47]. Osteoblast attachment is also affected by the chemistry of the biomaterials surface, but a conclusive picture has not yet emerged. Early in vitro cytocompatibility studies focused on the morphological aspect, growth capacity and the state of differentiation of cells on materials with various chemical compositions. The diversity of cell responses to the different materials tested highlighted the capacity of cells to distinguish the effects of subtle changes in substratum surface chemistry. For primary bovine osteoblasts, the wettability of the surface has been shown to be one of the important factors [56]. On the other hand, the functional groups present on the surface have been demonstrated to be of even greater importance than the wettability for the adhesion of MC3T3-E1 osteoblasts [57].
1.4 Controlling the BoneImplant Interface Different approaches are being used in an effort to obtain the desired bone implant interface. As Kasemo and Lausmaa [58], among others, have described, biological tissues interact mainly with the outermost atomic
Controlling the BoneImplant Interface
layers of an implant; consequently, much effort is being devoted to methods of modifying surfaces of existing biomaterials to achieve desired biological responses. The approaches can be classied as physicochemical and biochemical methods. 1.4.1 Physicochemical Methods Wettability characteristics are among the physicochemical characteristics that have been altered with the aim of improving the boneimplant interface [9]. Polymer surfaces were modied by glow discharge to study the effect of surface treatment on cell adhesion using polyethylene, polytetrauoroethylene, poly(ethylene terephthalate), polystyrene and polypropylene lms. The surface wettability of all the lms decreased with respect to the length of plasma treatment. For each of the polymers, a different dependence of cell adhesion on the length of plasma treatment was observed, but, in each case, the optimal water contact angle for cell adhesion was approximately 70 [59]. Alterations in surface morphology and roughness have been used to inuence cell and tissue responses to implants [9]. In addition to providing mechanical interlocking, surfaces with grooves can induce contact guidance, whereby the direction of cell movement is affected by the morphology of the substrate. For osteoblast-like cloned mouse cells (MC3T3-E1) cultured on titanium plates roughened by wire-type electric discharge machining or plasma coating, proliferation and ALP activity were enhanced on the roughened surfaces [60]. Considering the role of electrostatic interactions in many biological events, charged surfaces have been proposed as being conducive to tissue integration. Calcium phosphate coatings have been extensively investigated because of their chemical similarity to bone mineral [9]. The osteoblast-like cells cultured on RKKP- and AP40-bioglass layer coated zirconia showed a higher proliferation rate, leading to conuent cultures with higher cell density and a generally better expression of osteoblast ALP activity in comparison with zirconia substrate [61]. Each approach, however, has drawbacks. Contradictory results with charged materials in bone have been reported: indeed both positively and negatively charged surfaces were observed to promote bone formation. Although short-term clinical results have been encouraging, dissolution of coatings as well as cracking and their separation from metallic substrates remain concerns [9]. 1.4.2 Biochemical Methods [9] Biochemical methods of surface modication offer an alternative or adjunct to physicochemical and morphological methods. Biochemical surface modication endeavours to utilise current understanding of the biology and biochemistry of cellular function and differentiation. Much has been learned
about the mechanisms by which cells adhere to substrates, and major advances have been made in understanding the role of biomolecules in regulating differentiation and remodelling of cells and tissues, respectively. The goal of biochemical surface modication is to immobilise proteins, enzymes or peptides on biomaterials for the purpose of inducing specic cell and tissue responses or, in other words, to control the tissueimplant interface with molecules delivered directly to the interface. One approach to controlling cellbiomaterial interactions utilises cell adhesion molecules. Since identication of the arginineglycineaspartic acid (RGD) sequence as mediating attachment of cells to several plasma and extracellular matrix proteins, including bronectin, vitronectin, type I collagen, osteopontin and bone sialoprotein, researchers have been depositing RGD-containing peptides on biomaterials to promote cell attachment. A second approach to biochemical surface modication uses biomolecules having demonstrated osteotropic effects. A large amount of information has been obtained about biomolecules involved in bone development and fracture healing. By delivering one or more of these molecules, which normally play essential roles in osteogenesis, directly to the tissueimplant interface, bone formation may be promoted. To control exposure and concentration, retention and/or release of biomolecules from implant surfaces can be altered using different methods, including adsorption, covalent immobilisation and release from coatings (Figure 1.2). The simplest way to deliver biomolecules to the tissueimplant interface is by dipping the device in a solution of protein before inserting it. Studies using simple adsorption indicate that delivery of TGF-b to the tissueimplant interface can improve bone formation in the periprosthetic
Figure 1.2 Schematic illustration of methods for controlling retention and/or release of biomolecules at the tissueimplant interface [9]
Controlling the BoneImplant Interface
gap and can enhance bone ingrowth into porous coatings [62]. Using a similar approach, ALP adsorbed on titanium implants enhanced periprosthetic bone formation [63]. One drawback with the adsorption method, however, is that it provides little control over the delivery, including release/retention and orientation, of molecules. Proteins are initially retained on the surface by weak physisorption forces; then, depending on the implant microenvironment, which varies between anatomical sites and between patients, they desorb from the surface in an uncontrolled manner to initiate desired responses. Considering the necessity of specic receptor ligand interactions for activity of many relevant biomolecules, appropriate presentation of protein may also be needed. Although positive responses have been observed using this simple approach, there is no indication that they are optimal for clinical applications. Bonding biomolecules to implants is an alternate way of delivering them to the tissueimplant interface, albeit protein will not be released. This approach is more complicated than adsorption, because of the chemistry involved, but the activity of molecules immobilised on plastics has been shown to equal or exceed that of soluble protein [64]. For orthopaedic and dental applications, metal surfaces possess a relative paucity of functional groups needed for immobilising molecules. However, the passivating oxide lm on these materials does have surface hydroxyl groups that provide locations for bonding using silane chemistry. This approach has been used to immobilise peptides, enzymes and adhesive proteins on different biomaterials, including CoCrMo, Ti6Al4V, Ti and NiTi [9].
2
Surface Modication of Biomaterials
2.1 Introduction Biomaterials have been studied for many years and have been dened by Ratner [65] as being nonviable materials used in a medical device and intended to interact with a biological system. There is a big demand for biomaterials to assist or replace organ functions and to improve patients quality of life. Biomaterial applications make use of all classes of materials, metals, ceramics, polymers and composite. These are divided roughly into three user types [2]: (a) inert or relatively inert with minimal host response; (b) bioactive, which actually stimulates bonding to the surrounding tissue; and (c) biodegradable, which resorb in the body over a period of time. The biological responses to biomaterials and devices are largely controlled by their surface chemistry and structure. That is to say, the surface characteristics play a role in the functioning of biomaterial. The rationale for the surface modication of biomaterials is straightforward. Either biological or physicochemical methods are often employed to modify the material surface. Various physicochemical methods will be introduced in this chapter; among them, laser surface treatment has proved to have good potential for it is a unique feature. 2.1.1 Orthopaedic and Dental Implants There is archaeological evidence that lost teeth were replaced by handcarved ivory or wood implants as long ago as ancient Egypt. In the early 1950s Swedish orthopaedic surgeon Per Ingvar Branemark began studying
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the healing process of titanium anchoring screws, which proved to be a seminal point for modern dental and orthopaedic implants [1]. His work showed that fusion between bone and the titanium implant could take place, a phenomenon he called osseointegration. Orthopaedic implants to treat joint degradation due primarily to osteoarthritis, osteoporosis or injury are now commonplace. These include hip (around 325 000 US implants in 2001) and shoulder, wrist and knee (around 300 000 US implants in 2001). These types of implant have undergone continual advancement of their metal and plastic components in order to improve wear resistance and xation in the insertion site; nevertheless, research continues for more wear-resistant joint interface materials, for materials with improved mechanical properties, and to improve long-term reliability, especially as it relates to xation. The objective, of course, is to develop implants that can serve patients for an indenite period. 2.1.2 Surface Properties of Biomaterials In the case of medical implants the importance of surface science is quite obvious [66, 67]. It has been hypothesised that tissuebiomaterial interactions are governed by surface properties and that the important interactions occur within around 1 nm of the biomaterial surface [58]. Natural biological structures appear to be able to interact selectively with relevant biomolecules while resisting nonspecic interactions. Furthermore, biological interfaces are highly dynamic. As pointed out by Blawas and Reichert [68], simply trapping cells at a particular point on a surface is not enough. Cells must rst be encouraged to differentiate (i.e. change their behaviour to perform as required) and once their function is complete their activity must be turned off again. 2.1.3 Surface Analysis of Biomaterials Scanning electron microscopy (SEM) has been the traditional method for studying the microscopic surface structure of biomaterials. While allowing visualisation and chemical analysis of the specimen, SEM is not a surfacespecic technique in the strictest sense. Elemental microanalysis (EDAX) may also be carried out using SEM. This relies on the detection of X-rays of characteristic energy, which are emitted on interaction with the electron beam. With the recent, rapid growth of methods for preparing spatially welldened materials, the focus of biomedical surface science is now on high spatial resolution surface chemical state analysis. The driving forces for developing biomedical surface chemical state imaging techniques are addressed below. X-ray photoelectron spectroscopy (XPS), time-of-ight secondary ion mass spectrometry (ToF SIMS), scanning probe microscopy
Ceramic Implants [65]
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(SPM) and near-edge X-ray absorption ne structure (NEXAFS) each has its own strengths and weaknesses with respect to generating surface chemical state information at high spatial resolution, but together they provide a powerful set of complementary techniques. For example, XPS and ToF SIMS can be used to improve the level of chemical state information obtainable with SPM, while SPM can be used to improve the spatial resolution obtainable with XPS and ToF SIMS.
2.2 Ceramic Implants [65] It is essential to recognise that no one material is suitable for all biomaterial applications. As a class of biomaterials, ceramics, glasses and glassceramics are generally used to repair or replace skeletal hard connective tissues. Their success depends upon achieving a stable attachment to connective tissue. The mechanism of tissue attachment is directly related to the type of tissue response at the implanttissue interface (see Table 2.1). 2.2.1 Nearly Bioinert Ceramics [65, 69] Bioceramics are compatible because they are composed of ions commonly found in the physiological environment (calcium, potassium, magnesium, sodium, etc.) and of ions showing limited toxicity to body tissue (zirconium and titanium). Two nearly inert ceramics most used in surgical implants are
Table 2.1 Types of bioceramictissue attachment and their classication [65] Type of attachment 1. Dense, nonporous, nearly inert ceramics attach by bone growth into surface irregularities by cementing the device into the tissues or by press-tting into a defect (termed morphological xation) 2. For porous inert implants, bone ingrowth occurs that mechanically attaches the bone to the material (termed biological xation) 3. Dense, nonporous surface-reactive ceramics, glasses and glassceramics attach directly by chemical bonding with the bone (termed bioactive xation) 4. Dense, nonporous (or porous) resorbable ceramics are designed to be slowly replaced by bone Example Al2O3 (single crystal and polycrystalline) ZrO2 (partially stabilised zirconia)
Al2O3 (polycrystalline) Hydroxyapatite-coated porous metals Bioactive glass Bioactive glassceramics Hydroxyapatite Calcium sulfate (plaster of Paris) Tricalcium phosphate Calciumphosphate salts
14 Table 2.2 Property Chemical composition Density Bending strength Compression strength Young modulus Fracture toughness KIC Hardness g/cm3 MPa MPa GPa MPa/m HV 0.1 Properties of bioinert ceramics [70] Units Alumina 99.9 %MgO !3.97 >500 4100 380 4 2200
MgOPSZ Al2O3 ZrO2 MgO (810 mol %) 5.746 450700 2000 200 715 1200
YPSZ (TZP) ZrO2 Y2O3 (3 mol %) >6 9001200 2000 210 710 1200
alumina and zirconia. The characteristics of bioinert ceramics for biomedical application are shown in Table 2.2. High-strength ceramics used for implants are very inert in the body and exhibit minimal ion release. Inert bioceramics undergo little or no chemical change during long-term exposure to the physiological environment. 2.2.2 Alumina High-density, high-purity (>99.5%) alumina is used in load-bearing hip prostheses and dental implants because of its excellent corrosion resistance, good biocompatibility, high wear resistance and high strength [69, 71]. Although some dental implants are single-crystal sapphires most Al2O3 devices are very ne-grained polycrystalline a-Al2O3 produced by pressing and sintering at T 16001700 C. Alumina has been used in orthopaedic surgery for nearly 20 years. Its use has been motivated largely by two factors: its excellent biocompatibility and very thin capsule formation, which permits cementless xation of prostheses and its exceptionally low coefcients of friction and wear rates. 2.2.3 Zirconia Ceramics Zirconia is also exceptionally inert in the physiological environment and zirconia ceramics have an advantage over alumina ceramics of higher fracture toughness and higher exural strength and lower Youngs modulus [69]. Partially stabilised zirconia (PSZ) is a ceramic that has found wide usage in medical and dental surgery. During a heating process, zirconia will undergo a phase transformation process. The change in volume associated with this transformation makes the usage of pure zirconia in many applications impossible. Addition of some oxides, such as calcia (CaO), magnesia (MgO) and yttria (Y2O3), into the zirconia structure in a certain degree
Metallic Implants
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Figure 2.1 Medical-grade zirconia used as (a) femoral balls, (b) thumb and (c) dental implant [69]
results in a solid solution, which is a cubic form and has no phase transformation during heating and cooling. This solid solution material is termed stabilised zirconia. Within medicine it is commonly used to fabricate hip ball joints, knee, thumb, etc., while in dentistry it is used to manufacture dental implants, dental posts, brackets and inlays. Some zirconia implants for medical and dental applications are shown in Figure 2.1. The published results of in vitro wear tests demonstrated that zirconia has a superior wear resistance. Saikko [72] showed no wear of zirconia femoral heads on his hip simulator wear test against the 10.9 mm ultra high molecular weight polyethylene (UHMWPE) cup, and Oka et al. [73] demonstrated the high wear resistance of zirconia against UHMWPE and the superiority of zirconia ceramics even over alumina ceramics in terms of low wear and low friction.
2.3 Metallic Implants Metals have been the primary materials in the past for damaged human bones due to their superior mechanical properties [74], albeit dangerous ions that are released in vivo from these alloys. Although pure metals are sometimes used, alloys (metals containing two or more elements) frequently provide improvement in material properties, such as strength and corrosion resistance. Three material groups dominate biomedical metals: 316 L stainless steel, cobaltchromiummolybdenum alloy, and pure titanium and titanium alloys. The main considerations in selected metals and alloys for biomedical applications are biocompatibility, appropriate mechanical properties, corrosion resistance, and reasonable cost.
16 Table 2.3
Surface Modication of Biomaterials Selected properties of metallic biomaterials Youngs modulus, E (GPa) 190 210253 110 116 1530 Yield strength, sy (MPa) 2211213 4481606 485 8961034 3070 Tensile strength, sUTS (MPa) 5861351 6551896 760 9651103 70150 Fatigue limit, send (MPa) 241820 207950 300 620
Material Stainless steel Cobaltchromium (CoCr) alloys Titanium (Ti) Ti6Al4V Cortical bone
2.3.1 Mechanical Properties The mechanical properties of materials are of great importance when designing load-bearing orthopaedic and dental implants. Some mechanical properties of metallic biomaterials are listed in Table 2.3. With a few exceptions, the high tensile and fatigue strength of metals, compared with ceramics and polymers, make them the material of choice for implants that carry mechanical loads. The elastic moduli of the metals list in Table 2.3 are at least seven times greater than that of natural bone. This mismatch of mechanical properties can cause stress shielding, a condition characterised by bone resorption (loss of bone) in the vicinity of implants. The key problems associated with the use of these metallic femoral stems are thus the release of dangerous particles from wear debris, the detrimental effect on the bone remodelling process due to stress shielding and also loosening of the implant tissue interface. It has been shown that the degree of stress shielding is directly related to the difference in stiffness of bone and implant material [75]. Titanium alloys are favourable materials for orthopaedic implants due to their good mechanical properties. Titanium, however, does not bond directly to bone, resulting in loosening of the implant. Undesirable movements at the implanttissue interface results in failure cracks of the implant. 2.3.2 Corrosion The physiological environment is typically modelled as a 37 C aqueous solution, at pH 7.3, with dissolved gases (such as oxygen), electrolytes, cells and proteins. Immersion of metals in this environment can lead to corrosion, which is deterioration and removal of the metal by chemical reactions. During the electrochemical process of corrosion, the metallic biomaterial can release ions, which may reduce the biocompatibility of materials and jeopardise the fate of implants. For example, the type and concentration of released corrosion products can alter the functions of cells in the vicinity of implants as well as of cells as remote locations after transport of the
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corrosion by-products to distant sites inside the body. These circumstances become stronger possibilities in the bodies of sick and elderly patients, who are the largest group of recipients of prostheses. Titanium and its alloys, as well as cobaltchromium alloys, have more favourable corrosion resistance for long-term implant applications such as joint and dental prostheses. 2.4 Surface Modication of Biomaterials 2.4.1 Introduction Biointegration is the ideal outcome expected of an articial implant. This implies that the phenomena that occur at the interface between the implant and host tissues do not induce any deleterious effects such as chronic inammatory response or formation of unusual tissues. It is, therefore, of paramount importance to design biomaterials used in implants with the best surface properties. Meanwhile, these biomaterials must process bulk properties that meet other requirements, especially mechanical properties, in order to function properly in a bioenvironment. As it is quite difcult to design biomaterials fullling both needs, a common approach is to fabricate biomaterials with adequate bulk properties followed by a special treatment to enhance the surface properties. Hence surface modication of biomaterials is becoming an increasingly popular method to improve device multifunctionality, tribological and mechanical properties, as well as biocompatibility of articial devices while obviating the need for large expenses and a long time to develop brand new materials [11]. Materials can be surfacemodied by using biological or physicochemical methods. A few of the more widely used physicochemical methods are briey described here. 2.4.2 Radiation Grafting and Photografting [76] Radiation is widely used in biomaterials science for surface modication, sterilization and to improve bulk properties. The use of gamma, ultraviolet (UV) and electron beam radiation has enabled the biomaterial scientist to perform bulk and surface modications that improve the biological response of materials and, subsequently, the performance of many medical devices. Radiation grafting has proven to be a simple technique that enables control placement of bioactive molecules on a polymer surface. Radiation-induced cross-linking has allowed the tailoring of the composition and properties of hydrogels to meet numerous biomedical applications. In addition, photocross-linking can serve to enhance the tribological properties of load-bearing components of the total articial knee and hip. UV radiation appears to have the potential to facilitate in situ curing of adhesives, in situ production and modication of devices and the generation of smart biomedical devices such as biochips.
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2.4.3 Plasma Surface Modication of Biomaterials In the plasma surface modication process, glow discharge plasma is created by evacuating a vessel, usually quartz because of its inertness, and then relling it with a low-pressure gas. The gas is then energised using techniques such as radiofrequency energy, microwaves and alternating current or direct current. The energetic species in gas plasma include ions, electrons, radicals, metastables and photons in the short-wave ultraviolet (UV) range. These energy transfers are dissipated within the solid by a variety of chemical and physical processes, to result in surface modication. Plasma-based techniques combining the advantages of conventional plasma and ion beam technologies are effective methods for medical implants with complex shapes [77]. In particular, modication of the surface energetics of the materials can improve the adhesion strength, surface and coating properties, and biocompatibility, to name just a few [78]. However, the apparatus used to produce plasma depositions can be expensive [65] and the chemistry produced on a surface can be ill-dened.
2.4.4 Ion Beam Processing Biomaterial modication by ion beam processing is becoming popular for improving medical device function, biocompatibility and as a new mutation breeding method. Ion beam base processes, such as ion implantation and ion beam assisted deposition (IBAD), can provide benecial surface layers with desirable properties without detrimentally affecting the bulk properties. The ion beam method injects accelerated ions with energies ranging from 101 to 106 eV (1 eV 1.6 1019 J) into the surface zone of a material in order to alter its surface properties. Important potential applications for biomaterials include modication of hardness (wear), lubricity, toughness, corrosion, conductivity and bioreaction [79]. The primary advantage of ion implantation is selective surface modication without detrimentally affecting bulk properties. The drawbacks of this process are the high cost and the relatively shallow depth of modication. Ion beam assisted deposition (IBAD) is a vacuum deposition process that combines physical vapour deposition (PVD) with ion beam bombardment. The major feature of IBAD is bombardment with a certain energy (ranging from several hundred to several thousand eV) ion beam during the deposition of coating. IBAD is used in hydroxyapatite coating preparation, diamond-like carbon (DLC) lm, CN lm and other coatings. Another ion beam process is ion beam texturing (IBT). IBT has the ability to create desirable microfeatures and macrofeatures on the biomaterials to meet the requirement of biocompatibility in vivo.
Laser Surface Modication of Biomaterials
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2.4.5 Other Methods [65] Silanization Silane reactions can be used to modify hydroxylated or amine-rich surfaces. Since glass, silicon, germanium, alumina and quartz surfaces, as well as many metal oxide surfaces, are all rich in hydroxyl groups, silanes are particularly useful for modifying these materials. LangmuirBlodgett Deposition The LangmuirBlodgett (LB) deposition method covers a surface with a highly ordered layer. Each of the molecules that assemble into this layer contains a polar head group and a nonpolar region. Self-Assembled Monolayers Self-assembled monolayers (SAMs) are surface coating lms that spontaneously form highly ordered structures (two-dimensional crystals) on specic substrates. Surface-Modifying Additives Certain components can be added in low concentrations to a material during fabrication and will spontaneously rise to and dominate the surface. Conversion Coating Conversion coatings modify the surface of a metal into a dense oxide-rich layer that imparts corrosion protection, enhanced adhesivity and sometimes lubricity to the metal. Parylene Coating Parylene (para-xylylene) coatings occupy a unique niche in the surface modication literature because of their frequent application and the good quality of the thin-lm coatings formed.
2.5 Laser Surface Modication of Biomaterials 2.5.1 Introduction Lasers can rapidly and specically induce surface changes in organic and inorganic materials. The advantages of using lasers for such modication are the precise control of the frequency of the light, the wide range of frequencies available, the high energy density, the ability to focus and raster the
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light, the possibilities for using both heat and specic excitation to effect change and the ability to pulse the source and control reaction time. Treatments are pulsed (100 nanoseconds to picoseconds pulse times) and continuous wave (CW), with interaction times often less than 1 microsecond. Laser-induced surface alterations include annealing, etching, deposition and polymerisation. 2.5.2 Laser Patterning and Microfabrication Laser patterning is based on the possibility of focusing an intense laser beam at certain spots on a surface, where the high beam intensity causes evaporation of the material. By this approach, pits can be produced down to $1 mm, in the size range of interest to match cell sizes. By controlled motion of the beam (either by using clever optics or by sample motion), predesigned patterns can be made. With a kinoform, it is possible to laser-machine multiple pits in a surface at once [80]. A new method combines microphotolithographical techniques with laser excimer beam technology to create surfaces with well-dened three-dimensional microdomains in order to delineate critical microscopic surface features governing materialcell interaction [81]. Most laser-based patterning techniques use UV photoablation to micromachine biological substrates in order to generate mesoscopic patterns, and arrays of viable cells are required to fabricate next-generation tissue-based sensing devices to build three-dimensional cellular structures for advanced tissue engineering and to separate selectively and culture differentially microorganisms for a variety of basic and applied research applications [82]. Recently, laser etching (or laser ablation) and microlithography have been adopted to achieve micrometer dimensions with high precision in order to develop a large number of miniaturised systems for the analysis of biological tissues. Excimer laser etching was used to microtexture a biocompatible substratum for high-contrast microscope cell analysis [83]. 2.5.3 Pulsed Laser Deposition (PLD) of Biocompatible Ceramics Pulsed laser deposition (PLD) is a new technique for the deposition of thin lms of biocompatible ceramics [84]. Pulsed laser deposition is especially well suited to the deposition of bone-like ceramics (e.g. hydroxyapatite (HA) and calcium phosphates) on to metal, ceramic, semiconductor or polymer substrates for potential application in medical implants, prosthetic devices and biocompatible probes or sensors. The degree of control over lm characteristics offered by PLD exceeds that of other known deposition techniques presently applied to production of thin lms of biocompatible ceramics. It is anticipated that PLD will develop into the technique of choice
Laser Surface Modication of Biomaterials
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for the manufacture of implant or prosthetic devices comprising biocompatible lms on structurally robust substrates. Pulsed laser ablation is a new method for deposition of thin layers of HA on to biomaterial surfaces. Differences in cell spreading were apparent which were correlated with the uence used to deposit the HA [85]. Pulsed laser ablation and deposition of bioactive glass [86, 87] have been performed and the plume and lm compositions have been characterised. All the elements present in the target have been found in the gaseous phase. 2.5.4 Matrix-Assisted Pulsed Laser Evaporation and MAPLE Direct Write Two techniques, matrix-assisted pulsed-laser evaporation (MAPLE) and MAPLE direct write (MDW) were developed to deposit biomaterial thin lms [88]. MAPLE involves dissolving or suspending the biomaterial in a volatile solvent, freezing the mixture to create a solid target and using a low uence pulsed laser to evaporate the target for deposition of the solute inside a vacuum system. Using simple shadow masks, i.e. lines, dots and arrays, pattern features with length scales as small as 20 mm can be deposited using multiple materials on different types of substrates. MAPLE utilises a low uence pulsed UV laser and a frozen target consisting of a dilute mixture of the material to be deposited and a high vapour-pressure solvent. MDW uses pulsed laser radiation to directly transfer material from a ribbon to a substrate. Patterns with a spatial resolution of approximately 10 mm can be written directly. Biomaterials ranging from polyethylene glycol to eukaryotic cells (Chinese hamster ovaries) were deposited with no measurable damage to their structures or genotype. Deposits of immobilized horseradish peroxides (an enzyme) in the form of a polymer composite with a protective coating, i.e. (polyurethane) retained their enzymatic functions. A dopamine electrochemical sensor was fabricated by MDW using a natural tissues/graphite composite. The novelty of the MDW process is that the interaction of the incident laser pulse with the coating on the ribbon can transfer the micrometre-size powder, nanopowders and especially the chemical precursors to form a densely packed composite on the receiving substrate [89]. 2.5.5 Other Laser Surface Treatments Laser Surface Treatment for Improving Corrosion It was found that the corrosion behaviour of NiTi samples was improved by excimer laser surface melting [90]. Laser treatment improvement resistance is explained by a combination of the homogenisation of the surface by melting, the hardening due to N incorporation and the thickening of the
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oxide layer. Moreover, excimer laser surface treatment in air showed a remarkable improvement in pitting corrosion resistance for 316LS biograde stainless steel. The results show that excimer laser surface melting can effectively eliminate carbides and second phases alike, while also serving the function of homogenising the microstructure. N2 induced into the lasertreated surface could promote new precipitates and as a result lowered the corrosion resistance of 316LS stainless steel [91] and Ti6Al4V alloy [92]. Laser Grafting For the purpose of improved surface hydrophilicity and biocompatibility of ethylenepropylene rubber, 2-hydroxyethyl methacrylate (HEMA) and N-vinyl pyrrolidone (NVP) have been grafted on to the surface of this polymer using a CO2 pulsed laser at different uence (output power J/cm2) as the excitation source [17]. Moreover, ethylenepropylene rubber (EPR) based vulcanizates have been surface-grafted with acrylamide (AAm) and HEMA using a CO2 pulsed laser as the excitation source [18]. Surface hydrophilicity (measured by the water drop contact angle) increased for the grafted samples and comparative results indicate that the adhesion of macrophages to EPR samples modied with AAm and HEMA, with no respiratory burst and cellular damage, is signicantly lower than their adhesion on unmodied surfaces, which show an activated state of the attached macrophages. Laser Treatment of Plasma Sprayed Hydroxyapatite Coatings [93] The three requirements generally expected of biomaterials coating are: crystallinity, porosity and adhesion. Crystallinity is essential because amorphous coatings are more resorbable. The study found that laser treatment of plasma-sprayed coatings led to a wide range of microstructures. The porosity of the coatings was reduced signicantly. Nd:YAG (neodymiumdoped yttrium aluminium gainet) laser (pulsed) treatment signicantly changes the characteristics of the plasma-sprayed coating microstructure in several ways. It ranges from a at and smooth surface prole containing ne grains to an irregular surface comprising re-melted particles, spherical pores and tracks. Laser treatment of plasma-sprayed HA coatings basically generates a molten layer that rapidly solidies.
3
Wettability in Biomaterials Science and Modication Techniques
3.1 Introduction The wetting of a surface by a liquid and the ultimate extent of spreading that liquid are very important aspects of practical surface chemistry. Even with all the new information of the last 20 years, however, there still remains a great deal to learn about the mechanisms of movement of a liquid across a surface and the factors that govern such movement [12]. Biomaterial scientists have long sought a single, material-related parameter that effectively measures biocompatibility and might serve as a practical design guide. The theories of surface energy and wetting for such parameters present an attractive means to do this as surface properties are important determinants of a biomaterial function [12]. The ability to control the surface wettability of solid substrates is important in many situations. Various surface processes are used for modifying the surfaces of materials depending on the actual material and the application. A number of laser-based techniques for altering the wettability characteristics of engineering materials have been investigated [13]. 3.2 Wettability, Adhesion and Bonding: Theoretical Background 3.2.1 The Wetting Process The term wetting in its most general sense is used to denote the displacement of air from a liquid or solid surface by water or any aqueous or molten
24
Wettability in Biomaterials Science
solution [94]. Wetting is fundamentally a thermodynamic process and the changes in free energy that may occur determine whether or not wetting will happen, at what rate it will proceed and how far it will progress against the external forces. 3.2.2 Contact Angle and Work of Adhesion When a drop of liquid is in free space it is drawn into a spherical shape by the tensile forces of its surface tension, which results from the attractive and repulsive forces that exist between the molecules of the liquid. When such a drop of liquid is brought into contact with a at solid surface, the nal shape taken by the drop, and thus whether it will wet the surface or not, depends upon the relative magnitudes of the molecular forces that exist within the liquid (cohesive) and between the liquid and the solid (adhesive) [95]. The index of this effect is the contact angle, y, which the liquid subtends with the solid. In practice, for wetting to occur the contact angle should be less than 90 . If the contact angle is greater than 90 then the liquid does not wet the solid surface and no adhesion takes place [95]. Figure 3.1 shows a schematic view of a liquid droplet on a solid surface. The contact angle is related to the solid and liquid surface energies, gsv and glv , and the solidliquid interfacial energy, gsl , through the principal of virtual work expressed by Youngs equation: gsv glv cos y gsl 3:1
If an equilibrium for the droplet of liquid melt shown in Figure 3.1 is established, then the relation of y to gsv , glv and gsl is described by the rearranged Youngs equation: cos y gsv gsl glv 3:2
Clearly, to achieve wetting gsv should be large, while gsl and glv should be small. Hence liquids of a lower surface tension will always spread over a solid surface of higher surface tension in order to reduce the total free energy of the system [96, 97]. Whether the drop of liquid spreads across the solid
lv
sv
sl
Figure 3.1 Schematic of the wetting of a solid medium by a liquid melt [95]
Wettability, Adhesion and Bonding
25
surface to wet the surface and provide a coating or remains as a nite drop with an equilibrium angle is dependent upon the spreading coefcient S. For spreading to occur spontaneously S glv cos y 1 > 0 3:3
The adhesion intensity of a liquid to a solid surface is known as the work of adhesion Wad and is given by the YoungDupre equation Wad glv 1 cos y 3:4
Based on the nature of the attractive forces existing across the liquidsolid interface, wetting can be classied into the two broad categories of physical wetting and chemical wetting. In physical wetting the attractive energy required to wet a surface is provided by the reversible physical forces, such as the van der Waals and dispersion forces. In chemical wetting adhesion is achieved as a result of reactions occurring between the mating surfaces, giving rise to chemical bonds [98]. 3.2.3 Surface Energy and the Dispersive/Polar Characteristics The intermolecular attraction that is responsible for surface energy, g, results from a variety of intermolecular forces whose contribution to the total surface energy is additive [99]. The majority of these forces are functions of the particular chemical nature of a certain material, and as such the total surface energy comprises gp (polar or nondispersive interaction) and gd (dispersive component, since van der Waals forces are present in all systems regardless of their chemical nature); therefore, the surface energy of any system can be described by [100] g gd gp 3:5
Similarly, Wad can be expressed as the sum of the different intermolecular forces that act at the interface [101]: 1 p p 1 d Wad Wad Wad 2 gd gd 2 2 gp glv 2 sv lv sv 3:6
If a liquid that has both dispersive and polar forces is in contact with a solid surface where the surface energy is due to dispersion forces only, then the relationship between the contact angle and the surface energies of the liquid and solid are given by [101, 102] 1 2 gd gd 2 sv lv 1 cos y glv 3:7
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1
0.5
Cos
0.5
1 0 0.04
1 2
0.08
0.12
0.16
Figure 3.2 substrate
Plot of cos y against gd =glv for a theoretical liquid system on any solid lv
However, by equating Equation (3.7) with Equation (3.4), the contact angle for solidliquid systems where both dispersion forces and polar forces are present can be related to the surface energies of the respective liquid and solid by 1 p p 1 2 gd gd 2 2 gsv glv 2 sv lv 1 cos y glv 3:8
Therefore, from Equation (3.8), one can estimate the dispersive component of a solid substrate surface energy, gd , by plotting the graph of cos y against sv 1 (gd )2 =glv . This is shown in Figure 3.2 for a theoretical liquid system on any lv solid substrate. Thus, according to Fowkes [100], the value of gd is estimated by the sv 1 gradient ( 2 [(gd ) ]2 ) of the line (----) that connects the origin (cos y 1) sv 1 with the intercept point (cos y against (gd )2 =glv ) of the straight line () lv correlating the data point with the abscissa at cos y 1 In contrast, it is not possible to determine the value of the polar component of a solid substrate 1 p surface energy, gsv , directly from cos y against (gd )2 =glv . This is because the lv p p 1 intercept of the straight line (cos y against (gd )1=2 =glv ) is at 2(gsv glv )2 =glv , and lv thus only refers to individual control liquids and not the control liquid system as a whole. However, it has been established that the entire amount of the surface energies due to dispersion forces either of the solids or the liquids are active in the wettability performance [100, 103]. As such, it is d possible to calculate the dispersive component of the work of adhesion, Wad , by using only the relevant part of Equation (3.6). Thus 1 d Wad 2 gd gd 2 sv lv 3:9
Wettability, Adhesion and Bonding
27
d If one plots a graph of Wad against Wad for the solid substrate, then for each particular liquid in a given system in contact with the solid surface, Wad , which was determined from Equation (3.4), can often be correlated with d Wad , which was determined from Equation (3.9), by the straight line relationship d Wad aWad b
3:10
Therefore, for a solid substrate the constants a and b can be deduced respectively by calculating the gradient of the best-t straight line and by extrapolating the best-t straight line to nd the intercept point on the p axis. Also, if one plots a graph of (glv ) against (gd ), then for the liquids in a lv p given liquids system, (glv ) can often be correlated with (gd ) by the straight lv line relationship p 1 1 glv 2 c gd 2 d lv 3:11
Again, for a solid substrate the constants c and d can be deduced respectively by calculating the gradient of the best-t straight line and extrapolating the best-t straight line to nd the intercept point on the axis. By introducing Equation (3.10) into Equation (3.6) and rearranging, then
d Wad a 1Wad b p
3:12
or, alternatively, p 1 p 1 1 1 b gsv 2 glv 2 a 1 gd 2 gp 2 sv sv 2 3:13
By introducing Equation (3.11) into Equation (3.13) and differentiating 1 1 p 1 with respect to (gd )2 , considering that (gd )2 and (glv )2 are constant, then the sv lv following can be derived: gp sv 1 2 d 1 g 2 a 1 sv c 1:14
Since gd for the solid substrate can be determined previously directly from sv 1 the plot of cos y against (gd )2 =glv , then it is possible to calculate gd for the sv lv solid substrate Equation (3.14) directly. By employing this approach it is possible to determine, from y measurements and the control liquid surface energy properties, the changes in the wettability characteristics of the materials.
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3.2.4 Physical Bonding Physical bonding is essentially the effect that occurs when two perfectly at surfaces are brought together to atomic interaction distances, resulting in local atomic rearrangement and consequently adhesion. A typical example of physical bonding is that of van der Waals bonding. The energy difference between the specic surface energy of one material and that of the other is the work of adhesion. The work of adhesion can yield a theoretical breaking stress similar to the strength of either of the materials used. Physical bonding provides a useful guideline to the selection of materials that will bond well together. 3.2.5 Mechanical Bonding Mechanical bonding basically refers to the interlocking microstructure of rough surfaces to provide tensile strength and, in the case of shear, frictional strengthening. During the bonding process, the liquid or melt can ow with varying degrees of ease into cavities and asperities; a ductile metal or glass melt can conform to a rough solid substrate surface, or a vapour can deposit in surface asperities. The solid substrate surface may be roughed by means of acid or base chemical attack, grinding, grit or sand blasting, or laser treatment to enhance mechanical bonding. The effectiveness of these different surface-roughening techniques is entirely dependent upon their optimum application as well as on the specic methods and materials being used. In addition, chemical interaction between materials that are being bonded can lead to mechanical bonding. Further, the increase in the surface area of a mechanically roughened surface can affect an increase in the level of physical bonding. 3.2.6 Chemical Bonding Considerable research into the various chemical mechanisms that can be present during the bonding process is in process. Although most of the research is qualitative or semi-quantitative in nature, it is providing a useful background of chemical data that is contributing to a basic understanding of the principles of chemical bonding. A chemical bond is formed at an interface when a balance of bond energies and a continuous electronic structure are present across the interface for any two dissimilar phases. This structure occurs when a thermodynamically stable chemical equilibrium exists at the interface and is essentially achieved by chemical reactions at the interface. Generally, equilibrium compositions (which can be determined if an equilibrium phase diagram of the two phases being bonded is available) at the interface are attained at the reaction temperature very rapidly.
Wettability in Biomaterial Science
29
3.3 Wettability in Biomaterial Science From a historical perspective, Baiers proposal that critical surface energy can be directly linked to biocompatibility is perhaps the most penetrating concept among the few generalities offered to explain rules of biocompatibility. This theory, in its most general form, recognises that surface energy must control the way biologic uids interact with materials and that this interaction, in turn, must primarily inuence tissue and cell reactions. As examples, Baier pioneered the use of Zismans critical surface tension as an indicator of blood compatibility [104, 105] and bioadhesion [106, 107]. Neumann et al. employed their equation-of-state approach to calculate interfacial tensions from contact-angle measurements that, in turn, were used to predict cell adhesion [108] and thromboresistance [109]. Whereas concepts such as these have served as useful general guidelines or rules of thumb for biomaterials design, each has fallen far short of being the desired quantitative predictor of biocompatibility, particularly when applied to proteinaceous environments. 3.3.1 Biomaterial Interfaces [110] Surface sensitivity is of critical importance in biomaterials surface science because only the uppermost layers are in direct physicochemical contact with the biological environment; consequently, only the upper few molecular layers determine biocompatibility. Chemical events such as acidbase reactions, hydrogen bonding and ion exchange occur over atomic bondlength distances. Longer-range hydrophobic forces can extend up to about 10 nm and are responsible for nonspecic adsorption, adhesion and surfaceinduced water structure within this zone. Thus the interaction of a material with the biological environment occurs at or through the narrow region termed the interface. Therefore, the interfacial chemistry of concern to biomaterial scientists is determined by the materials composition within the uppermost few nanometres. 3.3.2 Tensiometry Tensiometry encompasses a broad range of related wetting techniques that measure surface energy. These include the observation of contact angles, which is perhaps the most familiar and widely applied method. Tensiometric methods have singular potential in biomaterials surface science based on the criteria of surface sensitivity, kind of analytical information obtained and relevance of that information to biomedical problems. First, with respect to surface sensitivity, wetting measurements are sensitive only to the upper 0.5 nm or so of a surface [14, 15] and are therefore among the most surface-sensitive techniques available. Second, tensiometry directly
30
measures the fundamental energy at an interface that drives important processes such as adsorption and adhesion. This kind of information must be particularly pertinent to biomaterial problems because of the overwhelming importance of protein adsorption and cell/tissue adhesion. Third, wetting measurements can be made using proteinaceous saline solutions that are particularly relevant to biomedical applications. Special highvacuum preparation techniques that might introduce experimental artefacts are not required. 3.3.3 Interfacial Biophysics The physicochemical nature of biomaterial interfaces was considered, leading to the conclusion that interfacial energy is a primary determinant of biocompatibility. A colloid science theory that quanties interactions at small distances and has biomaterial applications is the so-called Derjaguim Landau Verwey Overbek (DLVO) theory [111, 112], shown in the Figure 3.3. DLVO illustrates the relationship between particle (cell) distance from the surface and repulsive (electrostatic) and attractive (namely van der Waals) interaction energies. The basis of this theory is that attractive van der Waals potentials and repulsive electrostatic forces are additive. Formulation of these interaction potentials can be quite detailed for each case, but the qualitative predictive aspects for a macroscopic substrate are quite straightforward. From a physicochemical point of view, the kinetics of adhesion can be described as long-range interactions [100] and short-range interactions (acidbase, hydrogen bonds) [113]. Others describe these forces as dispersive and polar [114].
G GE 10 G 50 10 GVDW 100 D()
Figure 3.3 Interaction energies between a particle (cell) approaching a solid surface [65]
Wettability in Biomaterial Science
31
The total interaction energy (G, solid line) is composed of attractive van der Waals forces GVDW and repulsive electrostatic forces (GE ). A secondary minimum can be observed at approximately 100 A and a primary minimum at a distance <5 A. An energy barrier can be observed at approximately 40 A from the surface: G GE GVDW 3:15
where G is the sum of energy forces involved in Equation (3.15). GVDW is Ha=(6D) (which represents the van der Waals interaction between a particle with a radium a and a distance D to a at substratum), H is the Hamaker constant and GE (:) Zl Z2 is the electrostatic interaction between a particle and a solid plate (in which Z1 and Z2 are the zeta potentials of the particle and the solid substratum). Long-range interaction forces probably result in bringing a particle into the secondary minimum at approximately 100 A from the surface, but only a little energy is needed to remove the particle from the surface again. Shortrange interactions between a particle and a solid can only take place at distances <20 A. 3.3.4 Thermodynamic Concepts in Biomaterials Science An important conceptual tool in modelling of biological response is surface thermodynamics. A principal utility of thermodynamics is the ability to handle multicomponent systems in a phenomenological manner. Surface hydrophobicity, or wettability, is an important determinant of cell adhesion. It is related to surface free energy and is typically evaluated by the water contact angle. Smaller water contact angles correspond to more hydrophilic surfaces and higher surface free energies. In general, more hydrophilic substrates support cell adhesion and spreading to a greater extent than hydrophobic materials, which have low surface free energies. Substratum surface free energy is related to cell spreading, as illustrated in Figure 3.4. Poor spreading on hydrophobic substrata and good spreading on hydrophilic substrata can be observed in both the absence and presence of preadsorbed serum proteins. Specically, work with wettability gradient surfaces has shown that protein adsorption occurs to a greater extent on hydrophobic substrata than on hydrophilic substrata, that exchange of a pre-adsorbed protein by another protein occurs more readily on hydrophilic substrata than on hydrophobic substrates, that adsorption-induced conformational changes are greater on hydrophobic substrates than on hydrophilic substrates and that cell adhesion reaches a maximum on moderately hydrophilic substrates that have contact angles around 60 .
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1.0
Relatively Cell Spreading
0.5
0 0 10 50 sv (mJ/m2) 100
(Dotted line and solid line represents cell spreading in the absence of proteins and presence of proteins respectively)
Figure 3.4 Cell spreading as a function of substratum surface free energy [115]
Thermodynamically, the process of adhesion and spreading of cells from a liquid suspension on to a solid substrate can be described by [114] Fadh gcs gcl gsl 3:16
in which Fadh is the interfacial free energy of adhesion, gcs is the cellsolid interfacial free energy, gcl is the cellliquid interfacial free energy and gsl is the solidliquid interfacial free energy. If Fadh < 0, adhesion and spreading are energetically favourable, while if Fadh > 0, adhesion and spreading are unfavourable. Figure 3.5 illustrates the relationship between Fadh and the substratum surface free energy (or wettability). It should be noted that hydrophobic substrata (gs < 40 erg=cm2 ) do not promote adhesion of broblasts.
Fadh +10 UNFAVORABLE FOR ADHESION
50
100
FAVORABLE FOR ADHESION
s[erg.cm2]
10
Figure 3.5 Interfacial energy of adhesion Fadh as a function of the substratum surface energy gsv [114]
Current Methods of Wettability Modication
33
Extremely hydrophilic substrata do not promote adhesion either (e.g. highenergy methacrylates or hydrogels).
3.4 Current Methods of Wettability Modication Various methods are used to improve the surface wettability of materials and their adhesion to other materials. Hydrophilicity is a characteristic of materials exhibiting an afnity for water. Hydrophilic literally means waterloving and such materials readily adsorb water. Hydrophobic describes materials possessing characteristics that have the opposite response to water interaction compared to hydrophilic materials. Smaller water contact angles correspond to more hydrophilic surfaces and higher surface free energies. 3.4.1 Chemical Reactions A chemical reaction can be used to leave the functional groups and change the hydrophobic or hydrophilic behaviour of a materials surface. Chemical reactions were used to attach various hydrophilic functional groups and one hydrophobic group to cross-linked polystyrene resins. Those with hydrophilic groups had much better wettability by water [116]. Technically established treatments to modify the surface of silicone produced by plasma discharge and following hydrogel coating yield to a less hydrophobic surface [117, 118]. The wettability measurements of Si (111) surfaces treated in aqueous HF and H2SiF6 solutions indicated that the HF-etched surface is hydrophobic y $ 70 , while the H2SiF6-treated surface is, if anything, hydrophilic y $ 55 . 3.4.2 Plasma Surface Modication Plasma treatment can produce the extremes of both highly wettable and highly nonwettable (or hydrophobic) surfaces. Plasma surface modication can be used to tailor surface energies. Hydrophilic and hydrophobic surfaces can be created on polymers through interaction with gas plasma. Using oxygen to create hydroxyl functionality will increase the wettability of the surface. This has been used to enhance the performance of a catheter by the creation of a wettable surface on the polymer tubing. Most plasma cleaning operations will result in a surface that is more wettable (hydrophilic) than the starting surface. The increase of the surface wettability is mainly due to the grafting on to the NH3 plasma-treated polypropylene (PP) lms of nitrogen and oxygen polar groups [119]. The treatment of composite materials [120] by means of a cold plasma allows both the increase of their surface wetting properties and the improvement of their mechanical strength in terms of adhesion between bres and matrix.
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3.4.3 Ion Beam Processing Ion beam based processes, such as ion implantation and ion beam assisted deposition (IBAD) can provide benecial surface layers with desirable properties without detrimentally affecting the bulk properties. Wettability of organic materials was changed by ion implantation [118]. Doping effects can be obtained on electrical conductivity and wettability, induced by highdose metal implantation. To date, ion implantation has been successfully applied in metal and polymer biomaterials. For the polymer substrate, a specic functional chemical group is produced at the surface of the polymer to improve the surface wettability, anticalcic behaviour and biocompatibility of biomaterials. A new surface modication technique, ion assisted reaction (IAR) [121, 122], has been developed for improving wettability of materials and enhancing adhesion to other materials. The contact angles of water drops with modied polymers were reduced more by Ar ion irradiation with a owing oxygen gas environment than without owing oxygen gas. 3.4.4 Radiation Grafting Radiation grafting can be used, for instance, for introducing polar groups in the bulk or on the surface of nonpolar polymers, for increasing or reducing the wettability of a polymer, for imparting a better compatibility of a polymer to a specic coating and the like. Radiation grafting and related methods have been widely used for surface modication of biomaterials, and comprehensive review articles are available [123]. The radiation breaks chemical bonds in the material to be grafted, forming free radicals, peroxides, or other reactive species. These reactive surface groups are then exposed to a monomer. The monomer reacts with the free radicals at the surface and propagates at a free radical chain reaction, incorporating other monomers into a surface-grafted polymer. 3.4.5 UV and Ozone An ultraviolet (UV)ozone oxidation process [124] is shown to be an effective adhesion pre-treatment for polyethylene (PE) and polyetheretherketone (PEEK). The data obtained indicate that the treatment gives considerable oxidation and improved wettability for PE and PEEK surface types. It produces changes in surface oxygen chemistry and free energy which improves surface polarity and wetting. Exposing PP to ozone in the presence of UV light [125] is a simple and effective way of modifying its surface to improve its wettability and adhesion. Atomic force microscopy (AFM) showed a dramatic change in the morphology and a clear increase in the adhesion force resulting from the modication of a PP lm by UV/ozone exposure.
Laser Wettability Characteristics Modication
35
3.4.6 Corona Discharge The corona discharge is a commonly used procedure to modify surfaces of materials such as metals, glass or plastics. It is generally accepted that this treatment modies the substrate polymer surface properties like wettability and adhesion. Lee et al. [126] developed a method for preparing a wettability gradient on polymer surfaces using corona discharge treatment. The gradient was produced by treating the polymer sheets with the corona from a knife-type electrode whose power was changed gradually along the sample length. The polymer surfaces oxidised gradually with increasing corona power and the wettability gradient was created on the sample surfaces. 3.4.7 Electrowetting The wettability of poly(ethylene terephthalate) (PET) lms by water and aqueous solutions is increased by applying a voltage between the water and a rear electrode placed under the polymer lm [127]. This electrowetting effect can decrease contact angles by more than 30 under applied voltages of 200 Veff. The electrowetting effect can help the solutions to wet the solid surface.
3.5 Laser Wettability Characteristics Modication At present, the processes available to engineers for the modication of a materials wettability characteristics are invariably complex and consequently somewhat difcult to control. Lasers, on the other hand, can offer the user not only an exceedingly high degree of process controllability but also a great deal of process exibility. There is a growing amount of published work that testies to the potential of lasers for altering the surface properties of materials in order to improve their wettability characteristics. Laser radiation was found to effect signicant changes in the wettability characteristics of materials. 3.5.1 Laser Surface Modication of Ceramic Materials for Improved Wettability At present, very little published work exists regarding the effects of laser radiation on the wettability characteristics of ceramics materials. Indeed, the published work is predominantly concerned with the use of excimer laser radiation. Kappel [128] has shown that the texturing of ceramics (with an excimer laser of 248 nm) can improve the adhesion strength by up to 20 %. Such an improvement is said to be due to the formation of raised microscopic protrusions over the surface. The wettability characteristics of the
36
selected ceramic materials, ceramic tile (SiO2/Al2O3-based), clay quarry tile (SiO2/Al2O3/Fe2O3-based) and Al2O3 and SiO2TiO2 (crystalline), were improved after high-power diode laser (HPDL) treatment [129131]. The changes in surface roughness, surface oxygen content and surface energy resulted in the enhancement of the wettability characteristics. Recently, Lawrence [132] conducted work on ceramics and metals to isolate each of these mechanisms, which permitted the magnitude of their inuence to be qualitatively determined. Also, for ordinary Portland cement (OPC), surface energy, by way of microstructural changes, was seen to inuence a change in the wettability characteristics, while surface roughness was found to play a minor role in inducing changes in the wettability characteristics. 3.5.2 Laser Surface Modication of Metallic Materials for Improved Wettability It is recognised within the currently published work that laser irradiation of material surfaces can affect their wettability characteristics. Previously Heitz et al. [133], Henari and Blau [134] and Olfert et al. [135] have found that excimer laser treatment of metals results in improved coating adhesion. The improvements in adhesion were attributed to the fact that the excimer laser treatment resulted in a smoother surface and as such enhanced the action of wetting. It was demonstrated by using contact angles that CO2 laser treatment was sufcient to produce a fully wettable surface [136]. Selfuxing FeCrNiBSi alloy powders with various Ni contents were laser clad on medium carbon steel substrates [137]. The wettability increases with increasing Ni content in the cladding alloy. Good wettability of the cladding alloy on the substrate has a benecial effect on crack prevention in the cladding layer, because it reduces the formation of pores in the cladding layer, such pores usually being where stress concentrates and cracking initiates. Lawrence and Li [138] compared the interaction of CO2, Nd:YAG and the HPDL; excimer laser radiation with the surface of the mild steel studied was found to effect changes in the wettability characteristics of the material. It was observed that interaction of the mild steel with Nd:YAG and HPDL radiation brought about an improvement in the wettability characteristics of the steel. In contrast, interaction of the mild steel with CO2 and excimer laser radiation resulted in a depreciation of the wettability characteristics of the steel.
4
CO2 Laser Modication of the Wettability Characteristics of Magnesia Partially Stabilised Zirconia
In this chapter the modication of the wettability characteristics of an MgOPSZ bioceramic following CO2 laser irradiation is investigated. To study the wettability characteristics, contact angles between a set of control test liquids and the surface of the untreated and CO2 laser treated MgOPSZ were measured. The investigation revealed that CO2 laser treatment occasioned a marked increase in the wettability of the MgOPSZ. The inuential factors active in determining the wettability characteristics were analysed and the primary mechanism was deduced.
4.1 Introduction During recent decades a vast number of materials have been tested as potential biomaterials. Ceramic implants have aroused great interest because of their excellent compatibility with the physiological environment [71, 139]. Partially stabilised zirconia (PSZ) has found wide usage in medical and dental surgery. Within orthopaedics, PSZ is commonly used as femoral head, articial knee, bone screws and plates, while in dentistry it is used to manufacture dental implants, dental posts, crowns, brackets and inlays. Magnesiapartially stabilised zirconia (MgOPSZ) was the very rst zirconia implant approved by the US Food and Drug Administration (FDA).
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CO2 Laser Modication of the Wettability Characteristics
However, although bioinert ceramics, including MgOPSZ, have long been appreciated for their biocompatibility, they have often clinically failed due to lack of direct bonding with bone, i.e. insufcient osseointegration [140]. The wettability and interfacial interactions in a bioceramicbodyliquid system were investigated and low adherence was observed at the interface between the ZrO2 and the body liquids [141]. For an implant to be successful, close apposition of bone to the surface of the implant (osseointegration) is essential. From a historical perspective, Baiers proposal that critical surface energy can be directly linked to biocompatibility is perhaps the most penetrating concept among the few generalities offered to explain rules of biocompatibility [104107]. This theory, in its most general form, recognises that surface energy must control the way biologic uids interact with materials and that this interaction, in turn, must primarily inuence tissue and cell reactions [12]. Since the applied energy of lasers can be placed precisely on a surface, lasers provide the contemporary scientist and engineer with a controllable and exible tool for surface engineering. Kappel [128] has shown that the texturing of ceramic (with an excimer laser of 248 nm) can improve the adhesion strength by up to 20 %. Such an improvement was said to be due to the formation of raised microscopic protrusions over the surface. Lawrence and Li have demonstrated the practicability of employing different types of lasers to effect changes in the wettability characteristics of ceramics [130, 131, 142]. Yet, despite a growing amount of work conducted with engineering materials, no work has been conducted to assess laser processing as a means for enhancing the wettability characteristics of a bioinert ceramic.
4.2 Experimental Procedures 4.2.1 Material Specications The material investigated was a 4 % MgOPSZ obtained in sheet form with dimensions of 50 50 2:15 mm3 (Goodfellow, Ltd). For experimental purposes, the sheet was cut into 10 blocks each of 50 12 2:15 mm3 with a cutting machine (Miniton; Struers, GmbH) using a diamond-rimmed cutting blade and used as received prior to CO2 laser treatment. The 10 blocks were then divided into two groups of ve samples, with the groups being: untreated and CO2 laser treated. The main physical properties of the MgOPSZ were a density of 5.74 g/cm3, a specic heat at 25 C of 400 500 J/K kg and thermal conductivity at 20 C of 1.52.5 W/mK. The main mechanical properties were a compressive strength of 15002000 MPa, a tensile modulus of 200 GPa and a Vickers hardness of 1200 kgf/mm2.
Experimental Procedures
39
Figure 4.1 Schematic diagram of the set-up for the CO2 laser treatment experiments
4.2.2 CO2 Laser Experimental Arrangement A 3 kW CO2 laser emitting with a wavelength of 10.6 mm was used in this study. The laser produced a transverse electromagnetic multimode (TEM01) beam and was operated in the continuous wave (CW) mode. Figure 4.1 illustrates the full arrangement with a ve-axis workstation (TLC105; Trumpf, Ltd). It can be seen that a series of optical units was used to deliver the CO2 laser beam to the workpiece through the laser head, which was positioned by means of two linear axes (y and z axes) and two rotary axes (b and c axes). The defocused CO2 laser beam was traversed a single time across the surface of the MgOPSZ samples placed on the stage using the x axis. The fumes produced were removed with an extraction system, while O2 process gas with 2 bar pressure was used to shield the laser optics and assist the surface treatment. 4.2.3 Morphological, Chemical and Phase Analysis Procedures The surface roughness of the samples was measured by a surface prolometer (Surface Tester SV-600; Mitutoyo, Inc.). The surface and cross-section characteristics of the untreated and CO2 laser treated MgOPSZ samples were examined without etching using optical microscopy and scanning electron microscopy (SEM)(JSM 5600LV; JEOL, Ltd). The samples were sectioned with a cutting machine (Miniton; Struers, GmbH) using a diamond-rimmed cutting blade in order to analyse the cross-section. The sectioned samples were polished with 180, 400, 800 and 1000 grit SiC abrasive papers and then ne-polished using cloths and diamond pastes down to 3 mm. On account of the nonconductive nature of the MgOPSZ, it was necessary to coat the samples with Au in order to conduct the SEM examination. In order to determine any changes that may have occurred in the chemical make-up of the MgOPSZ following CO2 laser treatment, the samples were examined using energy dispersive X-ray (EDX) analysis to
40
obtain the chemical constituents and X-ray photoemission spectroscopy (XPS) to ascertain the elemental content. XPS (AXIS Ultra; Kratos, Inc.) analysis was performed on the sample surface using a spectrometer with monochromatic Al Ka (1486.71 eV) X-ray radiation (15 kV and 10 mA) and a hemispherical electron energy analyser. The changes in the phase of the MgOPSZ were detected by means of X-ray diffraction (XRD) (PW 1830; Philips Ltd) analysis with a Cu Ka source working at 30 kV and 20 mA across a range of 2080 2-theta. 4.2.4 Wettability Characteristics Analysis Procedure To investigate the effects of CO2 laser irradiation on the wetting and surface energy characteristics of the MgOPSZ, a set of sessile drop control experiments were carried out using glycerol, formamide, etheneglycol, polyglycol p E-200 and polyglycol 15-200 with known values of glv , gd and glv [143], lv detailed in Table 4.1. The contact angles, y, of the test liquids on the untreated and CO2 laser treated MgOPSZ were determined in atmospheric conditions at 25 C using a sessile drop measuring machine (FTA 125; First Ten Angstroms, Inc.). In order to estimate the inuence of contaminant layers on the measurement results, the specimens of the untreated MgOPSZ were cleaned with acetone in an ultrasonic bath for 2 hours, rinsed with distilled water several times and dried in a vacuum oven at 90 C for 12 hours. All of the ve control test liquids were used to measure y on the cleaned samples. It was observed that the value of y on the cleaned samples was lower than on the as-received (not cleaned) samples by 1.5, 1.2, 1.0, 0.9 and 0.8 for glycerol, formamide, etheneglycol, polyglycol E-200 and polyglycol 15-200, respectively. It appears that any contaminants on the surface of the MgOPSZ have only a slight inuence on y. Because the contaminant is a minor factor active in the wettability characterisation, it is reasonable to preclude any cleaning pre-treatment for the practical application of CO2 laser treatment. Therefore, to explore the potential of CO2 laser treatment as an industrial and economical processing technique for altering the wettability characteristics of MgOPSZ, the work was conducted in a normal atmospheric environment without pre-cleaning.
Table 4.1 Total surface energy (glv ), dispersive (gd ) and polar lv (gp ) components for the selected test liquids [143] lv Liquid Glycerol Formamide Etheneglycol Polyglycol E-200 Polyglycol 15-200 glv (mJ/m2) 64.0 58.3 48.3 43.5 36.6 gd (mJ/m2) lv 34 32.3 29.3 28.2 26.0 glv (mJ/m2) 30 26.0 19.0 15.3 10.6
p
The Effects of CO2 Laser Radiation
41
Each measurement of y lasted for 3 minutes with prole photographs of the sessile drop being obtained every minute and a mean value being subsequently determined. After the test liquid drops for each liquid attached and rested on the MgOPSZ surface, the drops consistently reached an equilibrium state in around 6 seconds. Thereafter they remained motionless and the magnitude of the y changed little with time. On average only 0.5 deviation of the y for each test liquid was observed during the 3 minutes of measuring when photographs were taken every minutes, indicating that the shape of the drop was stable in its equilibrated state. The difference between the y value on the left-hand side and the right-hand side of the sessile drop is very small, which generates a 0.36 deviation. In turn, the average total deviation for the y measurement was 0.86 .
4.3 The Effects of CO2 Laser Radiation on Wettability Characteristics 4.3.1 Contact Angle When a drop of liquid is brought into contact with a at solid surface, the nal shape taken by the drop, and thus whether or not it will wet the surface, depends upon the relative magnitudes of the molecular forces that exist within the liquid (cohesive) and between the liquid and the solid (adhesive) [95]. The index of this effect is y, the angle at which the liquid subtends with the solid. In practice, for wetting to occur y should be less than 90 . If y is greater than 90 then the liquid does not wet the solid surface and no adhesion takes place [95]. Optical micrographs of sessile drops of glycerol placed on an MgOPSZ sample before and after CO2 laser irradiation are shown in Figure 4.2. The y values and other characteristics of the sessile drops are also provided.
Figure 4.2 Contact angels for glycerol on (a) the untreated MgOPSZ and (b) CO2 laser treated MgOPSZ (power density of 1.6 kW/cm2 and traverse speed of 2000 mm/min)
42
Table 4.2 Mean values of contact angles formed between the untreated and CO2 laser treated MgOPSZ for various power densities (traverse speed of 2000 mm/min) and the selected wetting control test liquids at 25 C Contact angle, y (deg) CO2 laser treated (kW/cm2) Untreated 0.5 0.9 1.6 1.9 2.5 79 73 61 53 35 76 71 60 51 33 62 57 48 40 28 40 36 29 26 19 50 44 38 33 22 54 50 41 36 27
Test liquid Glycerol Formamide Etheneglycol Polyglycol E-200 Polyglycolycol 15-200
All the values of y obtained with the ve control test liquids and the MgO PSZ when untreated and CO2 laser treated at various power densities are given in Table 4.2. As Table 4.2 shows, with all the wetting control test liquids used the MgOPSZ consistently experienced a signicant reduction in y as a result of interaction with the CO2 laser beam. Since the effects of any contamination on the surface of the MgOPSZ has been shown to have a negligible effect on the value of y, then the sharp reduction in y is clearly the product of the CO2 laser treatment instead of pollution elimination. 4.3.2 The Effect of Surface Oxygen Content The observed increase in the wetting performance of the MgOPSZ would have certainly been inuenced by the increase in the surface oxygen content of the MgOPSZ (as a result of CO2 laser treatment), as this is known to increase the likelihood of wetting [13, 144147]. Wetting is governed by the rst atomic layers of the surface of a material. Therefore, in order to determine accurately the element content of oxygen on the surface of the MgOPSZ, it was necessary to examine the surface using XPS. As can be seen from Figure 4.3, augmentation of the surface oxygen content of the MgOPSZ after interaction with the CO2 laser beam was observed. The values obtained show that the surface oxygen content increased from an initial value of 41.6 to 64.3 at %, while the y for glycerol decreased from an initial value of 79 to 40 . It can be seen from Figure 4.3 that an overall increase of 22.7 at % in the amount of surface oxygen on the CO2 laser treated MgOPSZ occurred due to the oxidisation of the MgOPSZ surface during melting and re-solidication. When the surface oxygen content increased to a value of 64.3 at %, the y decreased to its minimum
43
Figure 4.3 Relationship between the y (glycerol) for the untreated and CO2 laser treated MgOPSZ and surface oxygen content
value. This indicates that oxygen enrichment of the CO2 laser treated MgO PSZ surface was active in promoting wetting and adhesion. Such a nding is similar to that of Song and Netravali [146, 147], who observed that surface oxygen content increased after laser treatment and, in turn, effected a reduction in the y. However, when the surface oxygen content increases beyond 64.3 at %, y increased despite the further increase in surface oxygen content. This suggests that other mechanisms are active and more dominant, thereby causing y to increase. 4.3.3 The Effect of Surface Roughness By varying the CO2 laser operating parameters it was possible to obtain a narrow range of surface roughness values. The values of y for glycerol in contact with the CO2 laser treated MgOPSZ samples were obtained at various points across this narrow range of surface roughness values. After CO2 laser treatment, the roughness of the MgOPSZ surface increased with power density. This is likely to be the result of turbulent convection in the melt pool caused by the TEM01 mode of the CO2 laser beam. Indeed, the power density distribution of the CO2 laser appears to be multimode. It is evident that the CO2 laser beam does not display a maximum peak in the middle of the beam; rather the peak is around the midpoint of the beam (see Figure 4.4). Based on the convective currents caused by a Gaussian beam prole [148, 149] and the intensity distribution of a TEM01 beam prole, a schematic diagram of the convection currents within the CO2
44
Figure 4.4 Schematic diagram of the convection currents generated within the CO2 laser meltpool
laser treated track on the MgOPSZ was generated and is given in Figure 4.4. When a laser beam has a sufcient power density, convective currents will be generated by the surface tension gradients resulting from the temperature gradient at the free surface. The strong turbulent convection in the centre of the TEM01 laser beam will be very complex and will change with the power density of the CO2 laser beam and the diameter of the beam. A model similar to that for heterogeneous solid surfaces can be developed in order to account for surface irregularities, being given by Wenzels equation [150] ra gsv gsl glv cos yW 4:1
where ra is the roughness factor dened as the ratio of the true to apparent surface areas and yW is the contact angle for the wetting of a rough surface. If ra is large, i.e. the surface is rough, then cos yW is large and yW will decrease if y was originally less than 90 . Therefore, when ra increases yW decreases if y was originally less than 90 . In the instance when y is originally greater than 90 , the situation is vice versa. As one can see from Figure 4.5, the surface of the untreated MgOPSZ sample was very smooth, with an average surface roughness value (Ra ) of around 0.295 mm. At this Ra value, y was measured as 79 . As shown in Figure 4.5, the CO2 laser treatment consistently generated a surface that was rougher, to varying degrees, and gave rise to values of y that were always much lower than on the untreated MgOPSZ surface. Having said that, Figure 4.5 presents some very interesting ndings that give tremendous insight into the mechanisms and effects of the CO2 laser surface treatment of the MgOPSZ. First, with only a relatively small increase in the surface
45
Figure 4.5 Relationship between y (glycerol) for the untreated and CO2 laser treated MgOPSZ and surface roughness
roughness of less than 0.5 mm, the minimum y value of 40 occurred at a surface roughness of 0.717 mm. Although this observation is in line with Equation (4.1), which states that an increase in surface roughness ought to effect a decrease in y when it is below 90 , this increase in surface roughness of the MgOPSZ occasioned after CO2 laser treatment is extremely small and is unquestionably not proportional to the considerable reduction in y. An explanation for this observation lies in the fact that the CO2 laser treatment simultaneously effects changes in many other surface properties of the MgOPSZ besides surface roughness. Second, it can be seen from Figure 4.5 that even with signicant increases in Ra , beyond 0.717 mm the value of y, rather than decreasing, actually experienced a slight increase from 40 to 54 . This observation not only patently contradicts Equation (4.1) but a previous nding [151] in which higher surface roughness corresponded to a lower value of y on ZrO2. Again, this is further evidence that aspects of the surface properties of the MgOPSZ other than surface roughness are active. 4.3.4 The Effects of Solidied Microstructures and Surface Melting on Wettability Characteristics Exposure of the MgOPSZ to CO2 laser irradiation resulted in rapid heating of the surface, for most materials typically 103105 K/s [152], which subsequently led to the melting and re-solidication of the MgOPSZ surface.
46
Figure 4.6 Typical SEM surface images of the MgOPSZ (a) before and (b) after CO2 laser treatment at power density of 1.6 kW/cm2
Figure 4.7 Relationship between y (glycerol) on the untreated and CO2 laser treated MgOPSZ and power density and solidied microstructure
Indeed, after the CO2 laser treatment a distinctive microstructure was generated on the MgOPSZ surface, as can be seen in Figure 4.6. Such microstructures are indicative of the incidence of rapid solidication. The various degrees of rapid solidication generated by the different laser power densities employed resulted in the diverse range of microstructures generated on the MgOPSZ surface, as shown in Figure 4.7. In order to
Surface Energy and Its Component Parts
47
simplify the analysis, the structures were dened according to the main structures on the MgOPSZ after CO2 laser treatment. It can be seen from Figure 4.7 that the surface structures of the MgOPSZ play a signicant role in determining y. The mere re-ordering of the crystals that occurred at low power densities appears to have only a slight effect on y, reducing it from 79 to 76 . At around 0.9 kW/cm2, y decreases markedly from 77 to 62 , with a hexagonal structure being generated on the MgOPSZ. With further increases in power density to around 1.6 kW/cm2, y decreases from 62 to 40 , with some cells beginning to form in the region due to the high temperature peak caused by the intensity distribution of the CO2 laser beam. One reason for this sharp reduction in y may be related to the onset of melting at this power density, which therefore implies that melting is an essential prerequisite for a signicant reduction in y. Further increases in power density resulted initially in an increase in y from 40 to 50 , which corresponds to the formation of uniform cell structures, and then a further increase to 54 , at which point dendrites and coral structures were observed. Indeed, work conducted by Zhang, Yue and Man [153] found that considerable improvement in the bond strength of an Si3N4 ceramic could be realised only when excimer laser treatment of a structural alloy steel (SAE 4340) resulted in surface melting. Similarly, Lawrence [154] observed a sharp reduction in y at the point of melting for an Al2O3/SiO2-based oxide compound after high-power diode laser (HPDL) treatment.
4.4 Surface Energy and Its Component Parts It is possible to adequately estimate the dispersive component of the MgOPSZ surface energy, gd , by plotting the graph of cos y against sv 1 (gd )2 =glv according to Equation (3.8). Thus, according to Fowkes [100], the lv 1 value of gd is estimated by the gradient ( 2(gd )2 ) of the line that connects sv sv the origin (cos y 1) with the intercept point of the straight line (cos y 1 against (gd )2 =glv ) correlating the data point with the abscissa at cos y 1. The lv values of gd for the untreated and CO2 laser treated MgOPSZ could be sv calculated from Figure 4.8. Comparing the ordinate intercept points of the untreated and CO2 laser treated MgOPSZliquid systems in Figure 4.8, it can be seen clearly that for the untreated and CO2 laser treated MgOPSZ at lower power density of 0.5 kW/cm2, the best-t straight line intercepts the ordinate closer to the origin. This is noteworthy since the intercept of the ordinate close to the origin is characteristic of the dominance of dispersion forces acting on the MgOPSZ materialliquid interfaces of the untreated and low CO2 laser power density treated sample, resulting in poor adhesion [100, 101]. On the other hand, the best-t straight line of the samples treated at higher
48
0.9 0.6 0.3 0.0 0.3 0.6 0.9 0.00
cos
Untreated 0.5 kW/cm2 0.9 kW/cm2 1.6 kW/cm2 1.9 kW/cm2 2.5 kW/cm2 0.03 0.06 0.09 0.12 0.15 0.18
( lv)
1 2
d 1/ 2
/ l
Figure 4.8 Plot of cos y against (gd ) =glv for the MgOPSZ in contact with the wetting lv control test liquids, before and after CO2 laser treatment with various power densities
CO2 laser power densities (>0.5 kW/cm2) intercept the ordinate considerably high above the origin. The highest intercept point is found for the sample CO2 laser treated with 1.6 kW/cm2 power density. An interception of the ordinate above the origin is indicative of the action of polar forces across the interface, in addition to dispersion forces, and hence improved wettability and adhesion is promoted [100, 101]. Furthermore, because none of the best-t straight lines intercept below the origin, it can be said that the development of an equilibrium lm pressure of adsorbed vapour on the MgOPSZ surface (untreated and CO2 laser treated) did not occur [101]. p It is not possible to determine the gsv value of the MgOPSZ directly from Figure 4.8. This is because the intercept of the straight line (cos y against 1 1 p (gd )2 =glv ) is at 2(gsv )(gd )2 =glv , and so only refers to individual control liquids lv lv and not the control liquid system as a whole. Still, it has been established that the entire amount of the surface energies due to dispersion forces either of the solids or the liquids are active in the wettability performance [100, 103]. As such, it is possible to calculate the dispersive component of the work d of adhesion, Wad , by Equation (3.9). Table 4.3 shows the values of Wad calculated using Equation (3.4) and the d values of Wad calculated using Equation (3.9) for both the untreated and CO2 laser treated MgOPSZ with various power densities. Figures 4.9 and 4.10 d show the best-t straight line plots of Wad against Wad for the MgOPSZ when it is both untreated and CO2 laser treated. From the plots of Wad d against Wad one can see that the experimental results reveal that for each
49
d Table 4.3 Values of Wad and Wad for the control test liquids and the determined d constant, a, from the plots of Wad against Wad for untreated (UT) and CO2 laser treated MgOPSZ with various power densities d Power Work of adhesion Wad Dispersive work of adhesion Wad density a Glyc Form Ethel P1 P2 Glyc Form Ethel P1 P2 (kW/cm2)
UT 0.5 0.9 1.6 1.9 2.5
2.41 2.37 3.03 4.25 3.42 3.17
76.2 79.4 92.8 113.3 105.0 101.8
75.2 77.5 89.8 105.5 98.5 95.6
71.5 72.5 80.7 90.3 86.5 84.5
69.6 70.9 77.0 82.7 80.0 78.7
66.6 67.3 68.8 71.4 70.6 70.3
76.1 77.2 77.7 80.9 80.5 77.7
74.2 75.2 75.7 78.8 78.4 75.2
70.7 71.6 72.1 75.1 74.7 71.6
69.3 70.3 70.7 73.7 73.3 70.3
66.6 67.5 67.9 70.8 70.4 67.5
Note: Glyc, glycerol; Form, formamide; Ethel, Etheneglycol; P1, polyglycol e-200; P2, polyglycol 15-200.
d Figure 4.9 Plot of Wad against Wad for the untreated MgOPSZ
particular control liquid in contact with both the untreated and CO2 laser treated MgOPSZ surfaces, Wad , determined from Equation (3.4), can be d correlated with Wad , determined from Equation (3.9), by the straight line relationship presented by Equation (3.10). Consequently, the values of a for various CO2 laser parameters shown in Table 4.3 were determined by the d best-t straight line of Wad against Wad . p As Figure 4.11 shows, a linear relationship between gd and glv of the lv control test liquids surface energies was observed which satised Equation (4.2) and thereby allowed the constant, c, to be calculated. Since gd has already been determined for the untreated and CO2 laser treated sv
50
d Figure 4.10 Plot of Wad against Wad for the CO2 laser treated MgOPSZ (1.6 kW/cm2 and 2000 mm/min)
8 7 6 5
( )
d lv
1/2
4 3 2 1 0 0 1 2 3 4 5
1/2
( )
d lv
1
Figure 4.11 Plot of (gp )2 against (gd )2 for the control test liquids lv lv
1
MgOPSZ from Figure 4.8, then it is possible to calculate gsv for untreated and CO2 laser treated MgOPSZ using Equation (3.14). The values for gsv , gd sv p and gsv of the untreated and CO2 laser treated MgOPSZ are given in Figure 4.12. As one can see from Figure 4.12, CO2 laser treatment of the
51
Figure 4.12 Relationship between surface energy (gd , gp and gsv ) for the CO2 laser sv sv treated MgOPSZ and power density
surface of the MgOPSZ leads to an overall increase in the gsv , while, more p importantly, also signicantly increasing gsv . The increase in gsv of the MgO p PSZ was primarily attributed to the increased gsv value, since gd was almost sv p similar for all the samples. The increase, in particular the increase in gsv , had a positive effect upon the action of wetting and adhesion [103] as primarily both dispersion and polar forces were active to a greater extent [100, 155]. The changes in the surface energy are thought to be due to the fact that the CO2 laser treatment of the MgOPSZ results in the melting of the surface, a p transition that is known to cause an increase in gsv [141] and hence an improvement in the wettability characteristics. p Moreover, as can be seen from Figure 4.12, gsv and gsv changed depending on the microstructures obtained at different CO2 laser parameters. When a hexagonal microstructure was obtained on the surface of the MgOPSZ, a p marked increase in gsv and gsv occurred, as is evident from Figure 4.12. When the MgOPSZ was treated with a relatively medium CO2 laser power density, cell formation on the MgOPSZ surface was induced and the p maximum value of gsv and gsv was achieved. Figure 4.12 suggests that the onset of melting initiated the cell formation. With relatively high CO2 laser power densities, coral and dendritic microstructures were apparent on the
52
surface of the MgOPSZ. The formation of these microstructures was p accompanied by a reduction in gsv and gsv from the maximum value. 4.5 Identication of the Predominant Mechanisms Active in Determining Wettability Characteristics Regardless of the CO2 laser power density employed, noticeable differences in surface roughness, microstructure, surface oxygen content and surface energy of the MgOPSZ were occasioned simultaneously. All these surface properties will have been a factor in determining the wettability characteristics of the MgOPSZ. First, CO2 laser treatment of the surface of the MgO PSZ generated a rougher surface and thereby reduced y. Second, the increase in the surface oxygen content of the MgOPSZ resulting from CO2 laser treatment will be inuential in the promotion of wetting, because an increase in surface oxygen content inherently effects a decrease in y and vice versa. p Lastly, an increase in gsv resulting from the melting and re-solidication of the surface of the MgOPSZ occurred. This naturally created a different microstructure that quite possibly improved the action of wetting and adhesion. Still, the foregoing sections have revealed that each of these factors did not play an equal role in governing the wettability characteristics of the MgOPSZ. It is, therefore, essential to identify the effect of each factor and nd the predominant mechanism active in governing the wettability characteristics of the MgOPSZ. Figure 4.13 shows the relationship between the wettability characteristics (denoted by cos y for glycerol) p and the inuential factors of surface roughness, gsv and surface oxygen content. As is evident from Figure 4.13, the rougher surface of the modied sample has a higher value of cos y than the smooth, untreated sample. Nevertheless, the change in cos y is not proportional to the alteration in the surface roughness, with cos y increasing sharply up to a surface roughness of 0.717 mm, then decreasing despite a considerable increase in surface roughness. This signies that other mechanisms, namely the surface oxygen p content and gsv , may play a more predominant role in inuencing the wettability characteristics of the CO2 laser treated MgOPSZ than surface roughness. It is apparent from Figure 4.13 that cos y increased with increasing surface oxygen content below 63.4 at %. This is in accord with the established theory for the relationship between the surface oxygen content of a material and their propensity for wetting. However, when the value of the surface oxygen content exceeded 63.4 at %, cos y decreased, indicating that the surface oxygen content is not a major factor active in changing the wettability of the MgOPSZ.
Identication of the Predominant Mechanisms Active
53
Figure 4.13 Relationship between cos y for glycerol and the surface roughness, the surface oxygen content, gp and microstructures of the untreated and CO2 laser sv treated MgOPSZ
A clear relationship between the value of cos y and gsv is observed in p Figures 4.13. Figure 4.14 reveals that an increase in gsv will in turn cause a p rise in cos y. From this it is evident that gsv inuences the wettability characteristics of the MgOPSZ. Indeed, it was found by Lawrence [154] that surface energy was the most predominant factor governing the wetting characteristics of a SiO2/Al2O3-based ceramic following irradiation with an HPDL. From the above discussion it is not clear whether the surface roughness, the surface energy (by way of microstructural changes) or the surface oxygen content alone, or a combination thereof, are the principal factors inuencing the observed changes in the wettability characteristics of the MgOPSZ after CO2 laser surface treatment. Therefore, several stages of ne grinding were used to isolate the various inuential factors detailed above and thus analyse and establish qualitatively the effect each one had on the wettability characteristics of the MgOPSZ. In the rst stage, the surfaces of the untreated MgOPSZ and CO2 laser treated MgOPSZ (1.6 kW/cm2) with the largest change in y were ground with grinding paper (180 grit SiC)
54
Figure 4.14 Relationship between cos y for glycerol and gp on the untreated and sv CO2 laser treated MgOPSZ
for 3 minutes, while still retaining the CO2 laser treated microstructure. In this way it was possible to investigate the effects of the surface oxygen content, which exists within the rst atomic layers of the material. In order to evaluate the inuence of the CO2 laser induced microstructure, an intermediate grinding stage using grinding paper (400 grit SiC) for 3 minutes was used to remove the microstructure. In the nal stage, both the untreated and the CO2 laser treated samples were ground down further with grinding paper (800 grit SiC) for 3 minutes to study the effect of surface roughness. The observed changes to surface roughness, O2 content and y (glycerol) effected by these steps are given in Table 4.4. After the rst grinding stage, a large difference in y for glycerol was observed between the untreated and the CO2 laser treated samples, with y
Table 4.4 The contact angle (for glycerol), surface roughness and surface oxygen content of the untreated and CO2 laser treated MgOPSZ following the ne grinding stages Untreated Ra (mm) O2 (at %) y (deg) 0.30 0.22 0.08 0.06 41.6 41.5 41.7 41.8 79.1 81.9 82.3 82.3 CO2 Laser Treated Ra (mm) O2 (at %) y (deg) 0.72 1.90 1.46 1.23 64.3 42.0 41.8 41.7 39.4 43.7 77.3 77.9
Polishing steps Unpolished 180 grit SiC 400 grit SiC 800 grit SiC
Identication of the Predominant Mechanisms Active
55
increasing slightly from 39.4 to 43.7 for the CO2 laser treated sample, while y for the untreated sample increased from 79 to 82 (Table 4.4). From this observation it is reasonable to suggest that the retained CO2 laser induced microstructure in the CO2 laser treated MgOPSZ could be the mechanism responsible for the large different y from the untreated sample. Furthermore, the surface oxygen content of the CO2 laser treated sample was found to have reduced from 64.3 to 42.0 at %, a level similar to that of the untreated sample, 42.2 at %. It is, therefore, quite possible that the decreased surface oxygen content could be the factor inuencing the general increase in y. It is interesting to note that although the surface roughness of the CO2 laser treated sample increased from 0.72 to 1.90 mm, the value of y increased, thus implying that surface roughness does not have as great an inuence on the wetting characteristics of the MgOPSZ as that of the surface oxygen content. This proposition was borne out somewhat when the samples were ground further. In this subsequent stage, the CO2 laser induced microstructure and heat affected zone (HAZ) were removed from the CO2 laser treated sample. The surface oxygen content on the untreated and CO2 laser treated samples were practically the same as the original untreated value of 41.6 at %. Signicantly, y for the CO2 treated samples was 77.3 , a value close to the original untreated value of 79.1 . Basically, the removal of the CO2 laser induced microstructure alone appears to have brought about an increase in y to around the original level, since the surface oxygen content was almost the same value in both ground stages. Such ndings reveal unequivocally that the microstructure is by far the predominant mechanism governing the wettability characteristics of the MgOPSZ. The effect of the surface roughness was studied through a further step. This step generated a smoother surface by reducing the Ra from 1.72 to 1.40 mm. For the CO2 laser treated sample, this step caused y to change slightly from 77.3 to 77.9 . For the untreated sample, the nal ground stage caused Ra to change considerably, reducing from 0.30 to 0.08 mm. However, as seen from Table 4.4, y only increased very marginally from 79.1 to 82.3 . Despite the magnitude change in surface roughness, y had varied only slightly in this range of surface roughness. Such a nding indicates that changes in surface roughness are insignicant, as reected by the corresponding change in y. It is therefore reasonable to assume that the surface energy difference brought about by microstructural changes is the primary inuential factor governing changes in y and, in turn, the wettability characteristics of the MgOPSZ. What is more, the surface oxygen content was also found to inuence changes in the wettability characteristics of the MgO PSZ but to a much lesser extent, while surface roughness was shown to play a very minor role in inducing changes in the wettability characteristics of the MgOPSZ.
56
4.6
The Role Played by Microstructures in Terms of Crystal Size and Phase in Effecting Surface Energy Changes
The surface energy has been identied as the main mechanism governing the modication of wettability characteristics of the MgOPSZ and is varied with the surface microstructure. It is believed that the changes in surface energy of the MgOPSZ were attributed to the change in microstructures in the form of crystal sizes and phase changes. 4.6.1 The Role of Crystal Size on Surface Energy After CO2 laser radiation, the modied surface of the MgOPSZ exhibits a typical microstructure of rapid solidication. As the XRD patterns of the untreated and CO2 laser treated MgOPSZ given in Figure 4.15 show, after
Figure 4.15 XRD analysis of the MgOPSZ surface (a) before and (b) after CO2 laser treatment (1.6 kW/cm2) (c cubic, t tetragonal, m monoclinic)
The Role Played by Microstructures in Terms of Crystal Size and Phase
57
CO2 laser treatment a distinct phase change took place. The diffraction patterns of 2-theta between 28 and 32 are of particular relevance (shown in Figure 4.15) and should be emphasised [156]. The peak at 29 belongs to the overlap diffraction of the (111) cubic phase and (101) tetragonal phase (denoted as c(111) and t(101)). The peaks of the cubic and tetragonal phases are all overlapped with others for the untreated and CO2 laser treated samples (see Figure 4.15). The peaks at 28.2 and 31.5 belong to the (111) monoclinic phase (m (111)). An increase in the peak at 29 signies the increase in the tetragonal phase. After CO2 laser treatment, the peak at 29 , which shifts to 30.5 , was seen to rise while the peaks at 28.2 and 31.5 disappeared (see Figure 4.15), indicating that the tetragonal phase was increasing while the monoclinic phase was decreasing in the MgOPSZ surface. As shown in Figure 4.16, the relative intensity of the tetragonal phase on the MgOPSZ treated with a CO2 laser power density below 1.6 kW/cm2 increased with the power density and then decreased slightly as the power density increased beyond 1.6 kW/cm2. In contrast, the monoclinic phase decreased as the tetragonal phase increased.
Figure 4.16 The XRD pattern of the MgOPSZ with various power densities between 27 and 32 2-theta angle
58
According to the equilibrium phase diagram for MgOZrO2 [157], the monoclinic phase is stable below 1240 C and the tetragonal phase is stable between 1240 and 1400 C. Above 1400 C, tetragonal and cubic phases coexist, with increasing temperature, tetragonal transforms to the cubic phase. From 2370 C to the melting temperature (2600 C) the stable phase is a cubic structure. With the increase in CO2 laser power density, the surface temperature of the MgOPSZ will increase and create the phase transformation on the surface. As is evident from Figure 4.16, the decrease in the monoclinic phase is obvious while the tetragonal phase increases greatly when the power density is above 0.9 kW/cm2. Indeed, pyrometer readings in the range 2502000 C showed that the surface temperature of the MgOPSZ was above the 2000 C when the CO2 laser power density was 1.6 kW/cm2. Since the monoclinictetragonal transformation temperature is 1240 C, then under these conditions the monoclinic phase transformed to the tetragonal phase and the tetragonal phase reached the highest density. It has been reported that the CO2 laser cladding of ZrO2 composite coating showed a higher tetragonal phase in the laser-clad ZrO2 ceramic layer than that in the original ZrO2 powder [158]. Moreover, the plasma sprayed 8 wt % Y2O3PSZ coating generated the metastable tetragonal phase (peak at 29 ) in the as-sprayed condition after Nd:YAG laser treatment [159]. Moreover, the intensity of the peak at 30.5 assigned as the (111) plane of the tetragonal lattice overwhelms the others, which indicated the tendency of crystal orientation. The crystal size in the direction perpendicular to the hkl plane, Dhkl , is expressed by the Scherrer equation [160] Dhkl Kl b cos a

4:2
where l is the wavelength of the X-ray (1.54056 A for the Ka line of Cu in this experiment), a is the Bragg angle, b is the expansion of the XRD peak caused by the crystal size and K is the Scherrer constant. We take the full-width halfmaximum (FWHM) of the peak at (111) in the XRD analysis as b, the crystallite size as Dhkl and K as 0.91. The crystallite sizes in the untreated and CO2 laser treated MgOPSZ at various power densities are listed in Table 4.5. As shown in Table 4.5, the crystal sizes in the MgOPSZ following CO2 laser radiation are consistently larger than the untreated sample, implying that the crystal grew after CO2 laser radiation. A high heat input from a laser beam facilitates surface localized melting at a very high efciency. Its ability to maintain a cold substrate while melting a thin surface layer of material results in rapid quenching of the molten layer once the beam is removed. Thermal gradients at the liquidsolid interface layer are very steep and cause crystal growth taking place along the thermal gradient. The power
The Role Played by Microstructures in Terms of Crystal Size and Phase Table 4.5 Crystallite size calculated from the XRD of the untreated and CO2 laser treated MgOPSZ on the basis of the Scherrer equation Power Density (kW/cm2 ) Untreated 0.5 0.9 1.6 1.9 2.5 FWHM 4.187 3.489 2.318 1.396 2.415 2.268 a (deg) 15.26 15.25 15.22 15.20 15.24 15.18 D (nm) 34.7 41.5 65.4 103.1 60.1 64.0
59
density of the CO2 laser treatment has a signicant effect on the crystal size. Generally, the crystal sizes in the MgOPSZ increase with the increasing power density, with the largest crystal size of around 103.1 nm occurring at a power density of 1.6 kW/cm2. Furthermore, it was found that the crystal size varies similarly as surface energy with power density, as shown in Figure 4.17, signifying that the crystal size is correlated with the surface energy of the MgOPSZ. According to the classical theory of nucleation and growth in solids [161], a crystallite nucleate in the form of critical embryos grows by accreting atoms from the surroundings. Assuming the embryo has a spherical shape
Figure 4.17 Relationship between surface energy and crystal size of the CO2 laser treated MgOPSZ and power densities
60
of radius, r, the free surface energy of an unstrained spherical particle, G, is expressed as Gr 4pr3 GV 4pr2 gt 3 4:3
where G is the free energy of an spherical particle, GV is the free energy per unit volume of a crystal and gt is the surface energy of the tetragonal crystal as Figure 4.16 showed, the crystals of the CO2 laser treated MgOPSZ are tetragonal. Therefore, by assuming the number of crystals in the CO2 laser treated surface to be n in unit area, the total surface energy of MgOPSZ, gsv (considering that half of the surface of the crystal is covered by neighbour crystals), may be estimated by the expression gsv 1npr2 gt 1npD2 gt 2 8 4:4
where D is crystal size. Since the main crystals remain as tetragonal structures in the MgOPSZ, it is reasonable to assume that gt does not change with the crystal size. During CO2 laser processing, the n could remain the same as the crystal grows. If this is so, then gsv is proportional 1 to D2 , which is in agreement with the linear relationship between g2 and sv D as shown in Figure 4.18. Therefore, the larger crystal size can be attributed to the increase in the surface energy of the MgOPSZ. Indeed, Man et al. [153, 162] found that excimer laser treatment induced a conical structure, a microscale of the peak and valley structure, providing an extra adherent surface area for a strong adhesion joint on Si3N4 and LT35 surfaces. The joint
Figure 4.18 Relationship between g2 and crystal size D of the MgOPSZ SV
Investigation of Wettability and Work Adhesion
61
strength increased with the height of the cones and the laser energy density. Kappel [128] showed that the improved adhesion strength occasioned by excimer laser texturing of ceramics is due to the formation of raised microscopic protrusions over the surface. 4.6.2 The Role of Phase Change on Surface Energy The previous XRD analysis revealed that the (101) tetragonal phase increased and the (111) monoclinic phase decreased in the CO2 laser interaction layer on the MgOPSZ. Furthermore, the relative intensity of the tetragonal phase increased when power density was below 1.6 kW/cm2 and then decreased slightly as the power density increased further. The MgOPSZ CO2 laser treated at 1.6 kW/cm2 power density has the highest surface energy (108.9 mJ/m2), as discussed previously, and corresponds to the highest intensity of the tetragonal phase. When the power density is at 1.9 and 2.5 kW/cm2, the surface temperature of the MgOPSZ increased to well above the melting temperature, thereby generating more cubic phase and reducing the tetragonal phase. Associated with this phenomenon, the surface energy begins to decrease from 108.9 to 80.7 mJ/m2 and then nally to 74.9 mJ/m2. These phase changes could be represented by the different microstructures on the CO2 laser treated MgOPSZ with various power densities. It is noticeable that tetragonal intensity varies, in a similar manner to the surface energy, with the CO2 laser power density. This indicates that the tetragonal intensity is closely correlated with the surface energy of the MgOPSZ. In fact, it has been found that at T 0 K, the surface energy of the (101) tetragonal phase is 45 % higher than that of the (111) monoclinic phase and 95 % higher than that of the (111) cubic phase [163]. Based on this it is believed that the phase change (an increase in the tetragonal phase and a decrease in the monoclinic phase) resulted in the higher surface energy of the MgOPSZ following the CO2 laser irradiation. 4.7 Investigation of Wettability and Work Adhesion Using Physiological Liquids In order to simulate the biological environment, the physiological uids and simulated physiological liquids used for the wetting experiments were human blood, human blood plasma, simulated body uid (SBF) and SBF BSA (bovine serum albumin). The SBF was prepared by dissolving reagentgrade chemicals, NaCl, NaHCO3, KCl, K2HPO4 3H2O, MgCl2 6H2O, CaCl2 and Na2SO4 in ion-exchanged and distilled water, buffered at pH 7.25 at 37 C with tris(hydroxymethyl) aminomethane ([CH2OH]3CNH2) and 1 m hydrochloric acid (HCl). The SBF has an inorganic ion concentration close to that found in human blood plasma, as shown later in Table 5.1.
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Table 4.6 Mean values of contact angles formed between the selected and simulated physiological test liquids and the untreated and CO2 laser treated MgOPSZ Contact angle, y (deg) Human blood Human blood plasma SBF SBFBSA 54.7 35.8 57.9 39.2 78.8 56.2 67.9 48.5
MgOPSZ Untreated CO2 laser (1.6 Kw/cm2 )
SBF BSA was prepared using BSA (Sigma A-9306, lot 106H0300) dissolved in SBF at pH 7.25 with a concentration of 4 mg/ml, to be used for protein studies to simulate human albumin because it is very similar to the sequence of amino acid units. The values of y formed between the selected and simulated physiological test liquids and untreated and CO2 laser treated MgOPSZ at power density of 1.6 kW/cm2 alloy are shown in Table 4.6. It clearly reveals that the y values of all body uids on the CO2 laser treated MgOPSZ are lower than the untreated specimens, indicating the wettability characteristics of the material with the body uids improved obviously after CO2 laser treatment. Further, according to Equation (3.4), the decrease in y resulted in an increase in the Wad of the MgOPSZ towards the physiological and simulated physiological liquids. Using the referenced glv value of human blood (47.5 mJ/m2), human blood plasma (50.5 mJ/m2) [141], SBF (72.5 mJ/m2) and SBFBSA (54.0 mJ/m2) [164], the work adhesion, Wad , of the Ti6Al4V alloy towards these body uids were determined through Equation (3.4), as shown in Figure 4.19. A discernable increase in Wad of body uids can be
Figure 4.19 Work adhesion of body uids for untreated, mechanically roughened and CO2 laser treated MgO-PSZ
Summary
63
seen on the MgOPSZ following CO2 laser treatment. Moreover, Wad increased as the CO2 laser power density increased. Since biomaterials rst contact a proteinaceous liquid phase, almost aqueous in nature, leading to surface reorganization of proteins followed by cell attachment on biomaterials, wettability characteristics, by controlling the interaction with physiological uids, would primarily inuence cell behaviour on biomaterials. Wetting of the solid surface is a predictive index of cytocompatibility [165]. Moreover, the improvements of Wad towards these uids would imply better suitability of titanium in a biological environment.
4.8 Summary The results presented in this chapter are a clear indication that CO2 laser surface treatment of the MgOPSZ brought about a reduction in the y formed between the MgOPSZ and the control test liquids, indicating that the wettability characteristics of the material were modied. The extent of this wettability characteristics modication was varied by manipulation of the CO2 laser parameters. Changes in the wettability characteristics of the MgOPSZ were attributed to the following factors: (a) an increase in surface roughness; (b) incorporation of oxygen at the MgOPSZ surface resulting from CO2 laser treatment; p and (c) the increase in the polar component, gsv , of the surface energy resulting from the melting and re-solidication of the MgOPSZ surface. The p changes in gsv resulted from the melting and solidication of the MgOPSZ surface, with the value varying with the solidied microstructure. The cellular microstructure obtained by the CO2 laser induced rapid solidicap tion corresponded to the maximum value of gsv . Indeed, it was found that the surface energy of the MgOPSZ increased as the crystal size and tetragonal phase present increased after the CO2 laser treatment. Further analysis revealed that surface energy, by way of microstructure, was the primary inuential factor governing changes in y and hence the wettability characteristics of the MgOPSZ. Incorporation of oxygen at the surface was also shown to inuence, to a lesser extent, changes in the wettability characteristics, while surface roughness was found to play a varying minor role in inducing changes in the wettability characteristics of the MgOPSZ.
5
In vitro Biocompatibility Evaluation of CO2 Laser Treated Magnesia Partially Stabilised Zirconia
This chapter is concerned with comparatively evaluating the biocompatibility of the CO2 laser treated MgOPSZ. An investigation of the bioactivity of the CO2 laser modied MgOPSZ in simulated body uids (SBF) was conducted. Thereafter the effect of the CO2 laser treatment on the OH groups, the correlation between OH groups and polar surface energy and the effect of the OH groups on the apatite formation were studied. In addition, ellipsometry was used to investigate the albumin and bronectin adsorption on the untreated and CO2 laser treated MgOPSZ bioceramic. The relationship between the protein adsorption and surface properties of the MgOPSZ was discussed. Finally, the in vitro behaviour of human fetal osteoblastic (hFoB) cells on the untreated and CO2 laser treated MgOPSZ was studied. An evaluation of osteoblast cell adhesion and proliferation was also conducted to determine the effect of surface properties on the osteoblast cell adhesion and growth, elucidating the mechanisms active in the osteoblast cell response and thus deducing that the main factors are active.
5.1 Introduction The biological activity of most orthopaedic and dental biomaterials is related to their ability to promote the formation of a neoformed layer of carbonate apatite crystals analogous to bone mineral. This layer also associates specic bone proteins and is the starting point in bone reconstruction [42]. The
66
In vitro Biocompatibility Evaluation
nucleation of apatite on the surface of a material is induced by the functional groups on its surface [166]. The adsorption of proteins on to a biomaterial surface from the surrounding uid phase is rapid, with the surface properties of the biomaterial determining the type, amount and conformation of the adsorbed proteins [167]. The structure and composition of the adsorbed protein layer determine the type and extent of the subsequent biological reactions, such as osseointegration [168]. The adsorption of the protein layer may also be critical in terms of providing attachment sites for bone cells such as osteoblasts and their progenitors [169]. The cellular behaviour on a biomaterial is an important factor determining the biocompatibility. Osteoblasts are anchorage-dependent cells that must adhere to substrate surfaces prior to undergoing subsequent cell functions such as proliferation, synthesis of collagen and other extracellular matrix proteins, etc. Cell adhesion is one of the initial events essential to subsequent proliferation and differentiation of cells before tissue formation. The whole process of adhesion and spreading of the cell after contact with biomaterials consists of cell attachment, growth of lopodia, cytoplasmic webbing and attening of the cell mass, and the rufing of peripheral cytoplasm, which progress in a sequential fashion [170]. With the aim being to improve the biocompatibility (bioactivity and biointegration) of a magnesiapartially stabilised zirconia (MgOPSZ), CO2 laser radiation was used to generate surface properties that would promote a better biological response. For an articial material to bond to living bone, it is essential that the material has the ability to form a biologically active, bone-like, apatite layer on its surface in the human body. On account of this the bioactivities of the untreated and CO2 laser treated MgOPSZ at different laser power densities were evaluated by observing the bone-like apatite formation on their surface after soaking in simulated body uids (SBF). Protein adsorption and osteoblast cell response were performed on the untreated and CO2 laser treated MgOPSZ in order to assess their propensity for biointegration, because protein adsorption is an almost immediate event occurring upon implantation of metals and mediates, prior even to cell response and tissueimplant interactions [9]. In addition, it is widely acknowledged that a major determinant of the bonebiomaterial interfacial response is the initial attachment, spreading and growth of osteoblasts on the implant surface and that improvements in these processes may lead to faster and more extensive implant integration and higher long-term stability [171]. Indeed, Vitale Brovarone et al. [172] investigated the in vitro behaviour of samples coated with a glass-matrix/zirconia particle composite by means of soaking in SBF followed by scanning electron microscopy (SEM) observation and X-ray diffraction (XRD) analysis. Rosengren et al. [173] studied in vitro the adsorption of proteins from diluted human plasma on hydroxyapatite,
Bone-Like Apatite Formation
67
alumina and zirconia with regard to total protein binding capacity, relative binding capacity for specic proteins and ow-through and desorption patterns. Josset et al. [174] evaluated the biocompatibility of two implantable materials, zirconia and alumina ceramics, in vitro using human osteoblast cell cultures. Furthermore, Bosetti et al. [61] used methods of soaking in SBF, protein adsorption and cell culture for an in vitro characterisation of a biomedical device.
5.2 Sample Preparation The MgOPSZ, with properties as described in Section 4.2.1, was subjected to various in vitro evaluations to determine the effects of CO2 laser surface treatment on bioactivity. In order to perform all of the in vitro tests, the MgOPSZ sheet was cut into 30 blocks each of 50 12 12:15 mm3 with a cutting machine (Miniton; Struers, GmbH) using a diamond-rimmed cutting blade, used as received prior to CO2 laser treatment. The 30 blocks were then divided into two groups of 15 samples, with the groups being untreated and CO2 laser treated. The CO2 laser processing of the materials was conducted in the same manner as described in Section 4.2.2. For the in vitro apatite formation test, the samples used were CO2 laser treated power densities ranging from 0.6 to 2.5 kW/cm2. For the protein adsorption test, only samples that were CO2 laser treated with laser power densities of 0.9 and 1.6 kW/cm2 were used. Samples CO2 laser treated with power densities ranging from 0.6 to 2.5 kW/cm2 were used in the in vitro osteoblast cell adhesion and proliferation test, while samples CO2 laser treated with power densities of 0.9 and 1.6 kW/cm2 were used in the evaluation of cell functions. Untreated samples were used as control in all of the in vitro tests.
5.3 Bone-Like Apatite Formation Histological examinations in vivo show that an apatite layer is formed on the ceramic [41] surface early in the implantation period and thereafter the bone matrix integrates into the apatite. This apatite layer consists of nanocrystals of carbonate-ion-containing apatite that has a defective structure and low crystallinity. These features are, in fact, very similar to those of the mineral phase in bone and hence bone-producing cells (osteoblasts) can preferentially proliferate on the apatite and differentiate to form an extracellular matrix composed of biological apatite and collagen. As a result, the surrounding bone comes into direct contact with the surface apatite layer. When this process occurs a chemical bond is formed between the bone mineral and the surface apatite to decrease the interfacial energy between them. It can be
68
concluded from these ndings that an essential requirement for an articial material to bond to living bone is the formation of a layer of biologically active bone-like apatite on its surface in the body [41]. There have been considerable efforts for improving the bioactivity of zirconia inert bioceramics. It has been revealed that a zirconia gel forms an apatite on its surface in SBF, indicating that the ZrOH group is able to induce apatite nucleation [166]. The investigation of the apatite-forming ability of zirconia gels with different structures shows that specic structures of the ZrOH group in tetragonal or monoclinic zirconia are effective for inducing apatite nucleation [175177]. For the purpose of the apatite formation, the chemical treatment has been used to produce the ZrOH group on a zirconia/alumina composite subjecting the composite to H3PO4, H2SO4, HCl or NaOH aqueous solution treatments [178] and on zirconium metal treated with aqueous NaOH [179]. 5.3.1 Experimental Procedures FTIR Analysis The optical adsorption spectra were measured at room temperature by means of a Fourier transfer infrared (FTIR) (FTS135; Bio-Rad, Inc.) spectrometer over the 5005000 cm1 range at a resolution of 1 cm1. Soaking in Simulated Body Fluid The samples were soaked in an acellular SBF [41], having an ion concentration nearly equal to that of human blood plasma. This solution, whose composition is reported in Table 5.1, was prepared by dissolution of highpurity reagents in distilled water, and was buffered at 7.25 with 50 mM trishydroxymethyl amino ethane and 45 mM hydrochloric acid. The untreated and CO2 laser treated samples (treated with various power densities) were immersed in 30 mL SBF in a polyethylene bottle at 37 C, without stirring. After 14 days they were removed from the solution, gently washed in distilled water and dried at room temperature. The soaked samples were then characterised by SEM and EDX, the details of which
Table 5.1 Ionic concentration and pH of SBF in comparison with those in human blood plasma [41] Concentration Ion SBF Blood plasma Na 142.0 142.0 K 5.0 5.0 Mg2 1.5 1.5 Ca2 2.5 2.5 Cl 148.8 103.8 HCO 3 4.2 27.0 HPO2 4 1.0 1.0 SO2 4 0.5 0.5 pH 7.40 7.40
69
0.6 arb. units Absorbance (arb. units)
2.5 kW/cm2 1.9 kW/cm2 1.6 kW/cm2 0.9 kW/cm2 0.6 kW/cm2 Untreated 1000 1500 2000 2500 3000 3500 4000
Wavenumber (cm1)
Figure 5.1 Infrared spectra of the untreated and CO2 laser treated MgOPSZ (treated with different power densities)
are given in Section 4.2.3. The samples for SEM observations were simply dried and covered by a thin gold layer to guarantee the conductivity. 5.3.2 Spectral Analysis and Hydroxyl Group The main regions in the 750950 cm1 range are ascribed to ZrO2 stretching modes, as shown in Figure 5.1, owing to the fact that it is similar to infrared (IR) peak of the 20 mol % Al2O3-doped ZrO2 nanoparticles reported previously [180] and the IR peak of the Al2O3ZrO2 nanopowders after laser ablation [181]. The vibration around 33003500 cm1 in the FTIR spectra (see Figure 5.2) could be attributed to the OH groups. Indeed, OH stretching vibrations around this region have been observed by other workers on Al2O3ZrO2 nanopowders after Nd:YAG laser ablation [181] and on the Fe-doped crystals after laser irradiations [182]. As one can see from Figure 5.2, the absorption coefcient of the OH group on the MgOPSZ in this region increased after CO2 laser irradiation and varied with the power density employed. For the untreated sample and the CO2 laser treated sample (power density of 0.6 kW/cm2), the absorption peaks from 3200 to 3600 cm1 are not obvious, indicating that no OH groups bonded on these samples. In contrast, the absorption peaks at this region can be clearly observed on the samples following the CO2 laser irradiation with power densities of 0.9, 1.6 and 1.9 kW/cm2, denoting that OH groups existed on these samples. The highest absorption coefcient of OH groups was obtained on the sample that had been treated at 1.9 kW/cm2. This nding shows that the OH groups increased with the CO2 laser power density used. This relationship was also seen by Zeng, Yung and Xie [183] on the OH
70
2.5 kW/cm2 Absorbance (arb. units) 0.05 arb.units
1.9 kW/cm2 1.6 kW/cm2 0.9 kW/cm2 0.6 kW/cm2 Untreated 3000 3500 Wavenumber (cm1) 4000
Figure 5.2 Infrared spectra of the hydroxyl groups presents on the surface of the untreated and CO2 laser treated MgOPSZ (treated with different power densities)
groups bonded on to copper after CO2 laser treatment; nevertheless, the absorption peaks in this region on the sample treated with a CO2 laser power density of 2.5 kW/cm2 was not obvious, suggesting that the OH bond does not increase linearly with the CO2 laser power density. The explosive evaporation due to the super-high temperature on the MgOPSZ surface treated at this power density caused water vaporisation and disappearance of the OH band. The phenomena of losing OH groups was also found on the human dentine after Er:YAG (erbium-doped YAG) laser irradiation [184]. CO2 is a weak acid and is known to absorb on ZrO2 in the form of both carbonate and bicarbonate species [185]. Carbonate structures are formed via the interaction of CO2 with zirconium cations in the lattice, as well as with a surface oxygen atom, whereas bicarbonate structures are formed via the interaction of CO2 with a hydroxyl group. The peak from 2800 to 3000 cm1 testied to the existence of carbonate structures on the MgOPSZ surface. The change of carbonate structures has the same trend as the OH groups discussed above. The formation of the hydroxyl groups on the MgOPSZ is due to the reactions of the zirconia with water vapour in air during CO2 laser processing. The hydroxyl ion is a common impurity in insulating crystals and by interacting with other impurities it gives rise to new complexes. The OHstretching frequency is a very sensitive probe of the hydroxyl environment. Proper thermal treatments and isotopic substitutions allowed the stretching mode absorption lines to be assigned to the defects in which OH is embedded and to supply possible models for them [185]. Crystal growth
71
from the melt is commonly carried out in air atmosphere as air always contains a certain degree of humidity from which OH ions are incorporated into the lattice [186]. CO2 laser irradiation is a thermal process. When the laser uence exceeds the ablation threshold, the irradiated surface experiences melting, followed by evaporation, whereupon the particles emit from the surface. At a higher uence, the amount of particles increases and they break out quickly from the superheated surface to produce a high-density vapour plume wherein a portion of the particles are ionised due to the thermal ionisation. The main reactions to occur in the melt ceramic and vapoured ume are ZrO2 , Zr4 2O 2 5:1
and the following oxidoreduction reactions occur at the meltatmosphere interface: O 2 ! 1 O2 2e 2 H2 O e ! 1 H2 OH 2 the whole reaction being O 2 2H2 O ! 2OH H2 1 O2 2 5:4 5:2 5:3
Finally, the OH ions produced according to Reaction (5.4) would be incorporated with one, two and three and four surface Zr4 respectively. According to the classication proposed by Tsyganenko and Filimonov [187], the OH groups bonded in the spectral ranges at %3770 and %3680 cm1 are typical one and three surface Zr4 cations, respectively, in tetragonal zirconia while the OH groups at %3775 and 3675 cm1 bonds are one and more than one (possibly three) surface Zr4 ions, respectively. It has been speculated that surface melting was occasioned on the MgO PSZ treated by the CO2 laser treatment with 1.6 kW/cm2 power density. In turn the Zr4 ion and OH were produced and reaction between these ions brought about the ZrOH group on the MgOPSZ. The relatively high amounts of the hydroxyl groups bonded on to the modied samples with 1.6 and 1.9 kW/cm2 were associated with the melting and chemical reaction on the MgOPSZ surface. 5.3.3 The Correlation between OH Groups and Wettability Characteristics The values of the surface energy have been calculated for the MgOPSZ treated by the various power densities in detail (see Chapter 4) and are
72
Table 5.2 Determined surface energy values for the MgOPSZ before and after CO2 laser treatment (treated with various power densities and traverse speed of 2000 mm/min) Surface Energy (mJ/m2) gd sv p gsv gsv CO2 laser treated (kW/cm2) 0.5 0.9 1.6 1.9 2.5 43.8 10.4 54.2 44.4 21.9 66.3 48.2 60.7 108.9 47.5 33.2 80.7 48.2 26.7 74.9
Untreated 42.7 10.1 52.8
shown in Table 5.2. It is found that the absorption coefcient of the p OH group (see Figure 5.2) and gsv on the MgOPSZ increased after CO2 laser irradiation (see Table 5.2) and varied with the power density employed. The untreated sample and the samples treated with the lower power densities had relatively low absorption peaks of the OH groups and lower p gsv . On the other hand, the relatively high absorption peaks of the OH groups and higher surface energy existed on the samples following the CO2 laser irradiation with power densities of 1.6 and 1.9 kW/cm2. Moreover, when the OH groups decreased on the sample treated at the power density p of 2.5 kW/cm2, there was a corresponding decrease in gsv . It is also interesting to notice that the melting of the MgOPSZ was the fundamental reason for the induction of the OH groups and improvement of the surface energy. This nding implied that there was a correlation between the OH p groups and gsv . Indeed, Takeda et al. found that the surface OH groups governed the wettability of commercial glasses [188] and adsorption properties of metal oxide lms [189]. A previous study [190] indicated that in the case of cassiterite its wettability strongly depends on the acidbase interactions (polar component) resulting from the presence of OH groups and physically adsorbed water on it. For the surface of the dry cassiterite its surface free energy practically results only from Lifshitzvan der Walls (dispersive component) intermolecular interactions. Most metal oxides are hydroxylated under normal conditions, i.e. at room temperature and when water or its vapour has had access to the surface. It was stated that the acid base component of surface energy of the zirconia probably depends on the density of OH groups on the surface of the solids studied [191]. Indeed, the acidbase component of surface energy presented the majority of the forces as the functions of the particular chemical nature of a certain material p corresponding to gsv [192]. As such, it is believed that the CO2 laser induced p hydroxyl groups could be a major factor inuencing gsv and, in turn, the wettability characteristics of the MgOPSZ.
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5.3.4 The Effects of CO2 Laser Treatment on the MgOPSZ in Simulated Body Fluids As one can see from Figure 5.3, very small amounts of sediment are apparent on the surface of the untreated MgOPSZ (Figure 5.3(a)) and the 0.6 kW/ cm2 CO2 laser treated MgOPSZ (Figure 5.3(b)). In contrast, considerable amounts of sediment were clearly discernible on the samples that were CO2 laser treated with power densities of 0.9 (Figure 5.3(c)), 1.6
Figure 5.3 SEM images of the MgOPSZ soaked in the SBF: (a) untreated, (b) 0.6 kW/ cm2, (c) 0.9 kW/cm2, (d) 1.6 kW/cm2, (e) 1.9 kW/cm2 and (f) 2.5 kW/cm2
74
Figure 5.4 SEM image and EDX analysis of the apatite formed on the surface of the MgOPSZ when treated with power densities of (a) 1.6 kW/cm2 and (b) 1.9 kW/cm2
(Figure 5.3(d)), 1.9 (Figure 5.3(e)) and 2.5 kW/cm2 (Figure 5.3(f)), with the highest amount of sediment being observed on the sample that was CO2 laser treated at 1.9 kW/cm2. The EDX analysis shows that most particles on the soaked surface are NaCl sediments as the elements of Na and Cl, as shown in the Figure 5.4(a). Apatites were only found on the samples treated at power densities of the 1.6 and 1.9 kW/cm2. As shown in Figure 5.4(a), only a few apatites formed on the sample treated at a power density of 1.6 kW/cm2, with only a small amount of Ca element shown in the EDX analysis given in Figure 5.4(a). However, on the sample treated with a CO2 laser power density of 1.9 kW/ cm2, some apatites were observed. One of them is shown in Figure 5.4(b) with the Ca:P ratio about 1.65. This Ca:P ratio exhibits the calcium phosphate transforms into apatite [41]. The Effect of OH Groups There was no occurrence of apatite formation on the untreated and certain CO2 laser treated samples (0.6, 0.9 and 2.5 kW/cm2) that displayed few hydroxyl groups. On the other hand, some apatites formed on other CO2
Protein Adsorption
75
laser treated samples (1.6 and 1.9 kW/cm2) that did display any hydroxyl groups. This nding suggests that the hydroxyl group on the MgOPSZ could be the predominant factor governing the formation of the apatites. The hydroxyl groups on the MgOPSZ surface certainly generate ZrOH groups, which have been shown to be functional groups for the formation of the apatite [166]. It is suggested that ZrOH functional groups formed on the samples in the CO2 laser processing at certain parameters and that such functional groups naturally brought about the nucleation of the apatite on these samples in the simulated body uid environment. The nucleation of the apatite could yield the apatite formation and bone-bonding ability to the MgOPSZ modied to have ZrOH groups on the surface. The Effect of Wettability It was found that there were more sediments and apatites on the surface with the higher surface energy than on the surface with the lower surface energy. In the process of CaP precipitation, the variations of the Gibbs function G of the MgOPSZ with the higher surface energy should be greater, compared to that of the MgOPSZ surface with lower surface energy. This nding, agreeing with the study by Feng et al. [193], suggested that the adsorption and reaction would more easily have occurred on the surface with the higher surface energy, especially the polar component of surface energy, which would be benecial to the chemical force and bonding. 5.4 Protein Adsorption The molecules involved in cell adhesion and spreading include extracellular matrix molecules, transmembrane receptors and intracellular cytoskeletal components. Among the extracellular matrix proteins shown to mediate cell attachment to substrates, bronectin is protein found in many extracellular matrices and in blood plasma and serves as an attachment molecule between the substrate and cell membrane of anchorage-dependent cells. It is known that the ligand bronectin connects to the cell membrane via integrin receptors. The activation of integrins triggers cytoplasmic reactions, and thereby stimulates the intracellular signalling pathway and subsequently cellular functions such as proliferation and differentiation [169]. On the other hand, human albumin is a nonadhesive protein for osteoblasts [194]. Albumin is the major protein component of serum and dominates the adsorption of phenomena on medical implants in the rst stage of contact with body uids. Human serum albumin or bovine serum albumin (BSA) coatings are often used as a passivating agent to prevent the adhesion of cells and thrombus formation [195].
76
An important fact to highlight is that different surfaces provide very different opportunities for protein binding. The investigation of competitive protein adsorption showed that BSA in a single-component solution adsorbed on to a hydrophobic surface two times more than that on to a hydrophilic surface [196]. The surface wettability of biomaterials affects the ability of cells to reorganise pre-adsorbed bronectin and to form their own matrix by secreted bronectin. Moderate wettable and hydrophilic surfaces are ideal for better interactions with cells while hydrophobic substrata inhibit early and late matrix formations. It is possible that there is a critical value in the strength of bronectin adsorption that regulates the ability of cells to construct a bronectin matrix [197]. 5.4.1 Experimental Procedures Protein Adsorption The proteins used for this study were human serum albumin and human plasma bronectin (Calbiochem, Inc.). Prior to the adsorption of 1 mg/ml albumin in phosphate buffered salines (PBS), MgOPSZ samples were rinsed with deionised water. The individual samples were transferred into a 24-well tissue culture plate. Thereafter, 2.5 ml of prepared albumin solution was added into each well. Adsorption proceeded for 1 hour in an incubator at 37 C. After adsorption was complete, the samples were dried with N2 and immediately transferred to an ellipsometer for measurement of the adsorbed protein layer. The above procedure was repeated with a 0.2 mg/ml concentration of bronectin in PBS. Ellipsometric Measurement Human plasma bronectin was measured using an automatic ellipsometer equipped with a 633 nm heliumneon laser (L117F; Gaertner, Inc). The thickness and refractive indices of protein lms were determined using an ellipsometer computer program with an accuracy of 3 A. Four ellipsometer measurements at different locations on each sample were taken and the average value was calculated. Statistics Statistical analysis was performed with an SPSS v.12 software package (SPSS/PC, Inc.). Data are reported as mean SD (standard deviation) at a signicance level of p < 0:05. After having veried normal distribution and homogeneity of variances, one-way ANOVA and Scheffes post hoc multiple comparison tests were done.
Protein Adsorption
77
5.4.2 Albumin and Fibronectin Adsorption on CO2 Laser Treated MgOPSZ The thickness of the absorbed bronectin layer was less on the untreated sample than that on the CO2 laser treated sample, as shown in Figure 5.5. The statistical analysis reveals that the thickness of absorbed bronectin on the untreated sample was similar to that on the sample CO2 laser treated at the power density of 0.9 kW/cm2, but signicantly less than that on the sample CO2 laser treated at the power density of 1.6 kW/cm2 p < 0:01. On the other hand, the thickness of the absorbed albumin layer on the untreated MgOPSZ was higher than that on the CO2 laser modied sample, as shown in Figure 5.5. The statistical analysis reveals that the thickness of the absorbed albumin layer on the untreated sample was signicantly higher than on the CO2 laser treated samples p < 0:01. From Figure 5.5 it is apparent that the CO2 laser power density applied in the experiments was negatively correlated to the amounts of albumin, but positively correlated with the bronectin. The results showed that the CO2 laser treatment promoted the adsorption of the bronectin on the MgOPSZ and the amount of the adsorbed bronectin was positively correlated with the CO2 laser power density applied in the experiments, as shown in
700 Fibronectin Thickness of Adsorbed Protein Layer () 600 * * 500 * * Albumin
400
300
Untreated CO2 laser CO2 laser 0.9 kW/cm2 1.6 kW/cm2 Untreated CO2 laser CO2 laser 0.9 kW/cm2 1.6 kW/cm2
Figure 5.5 Thickness of the adsorbed bronectin and albumin layer on the untreated and CO2 laser treated MgOPSZ (treated with different power densities). For the bronectin adsorption, there was a signicant statistical difference in thickness between the untreated MgOPSZ and the sample CO2 laser treated at 1.6 kW/cm2, and no statistical difference between the untreated MgOPSZ and the sample CO2 laser treated at 0.9 kW/cm2. For the albumin adsorption, there was a signicant statistical difference in thickness between the untreated MgOPSZ and the samples that were CO2 laser treated at 0.9 and 1.6 kW/cm2, and no statistical difference between the CO2 laser treated samples p < 0:05
78
Figure 5.5. However, the thickness of the adsorbed human serum albumin layer on the untreated MgOPSZ is higher than that on the CO2 laser modied MgOPSZ and was negatively correlated with the CO2 laser power densities. This nding is perhaps not so surprising as the various CO2 laser power densities used brought about changes in wettability characteristics and surface roughness of the MgOPSZ: it is known that protein adsorption is inuenced by the surface topography (roughness) [198] and the surface chemistry (wettability characteristics) [199]. The Effects of Surface Roughness By altering the CO2 laser power density, it was possible to obtain the narrow range of surface roughness values detailed in Figure 5.6 (see also Section 4.3.3). The experimental results given in Figure 5.6 reveal that the amount of bronectin adsorption increased, while the amount of albumin adsorption decreased with the surface roughness of the MgOPSZ. These trends in absorption for the bronectin and the albumin were veried to a large extent by the results of the statistical analysis.
700 Fibronectin Albumin
Thickness of Adsorbed Protein Layer ()
600 * 500 * * *
400
300
0.2
0.4
0.6
0.8
0.2
0.4
0.6
0.8
Surface Roughness, Ra (m)
Figure 5.6 The relationship between the thickness of adsorbed bronectin and albumin layer and Ra of the MgOPSZ. For bronectin adsorption, there was a signicant statistical difference in thickness between the sample with Ra of 0.295 mm and the sample with Ra of 0.717 mm, and no statistical difference between the sample with Ra of 0.295 mm and the sample with Ra of 0.313 mm. For albumin adsorption, there was a signicant statistical difference in thickness between the sample with Ra of 0.295 mm and the samples with Ra of 0.313 and 0.717 mm, and no statistical difference between the sample with Ra of 0.313 mm and the sample with Ra of 0.717 mm p < 0:05
Protein Adsorption
79
The relationship between the albumin adsorption and surface roughness given in Figure 5.6 is consistent with the ndings of other researchers, insofar as bovine serum was adsorbed preferentially on to the smooth substratum [200, 201], thus implying that the surface roughness of the MgOPSZ is one of the factors active in albumin adsorption. It was explained, however, by MacDonald et al. [202] that by roughing the surface of Ti one would obtain a more hydrophilic surface. This increase in surface hydrophilicity of the Ti, according to Serro et al. [201], will consequently result in lower albumin adsorption. The relationship between the bronectin adsorption and surface roughness given in Figure 5.6 is in agreement with previous reports. Deligianni et al. [200] found that a roughened Ti alloy sample adsorbed much more bronectin than a smooth sample. The much higher afnity of rough substrata to bronectin could be the driving force for preferential adsorption of bronectin; however, other researchers have reported that the amounts of immobilised bronectin on the rough titanium were 50 % lower than those adsorbed on the smooth one [203]. This decrease was noticed when the roughness was produced by polishing or sandblasting, followed by acid attack, which is an indication that the chemical or mechanical manufacturing process, used to achieve the surface texture, might inuence the protein adsorption behaviour of a surface [203]. Hence, a simple conclusion would be difcult to execute for the relationship between the amplitude of surface roughness and protein adsorption. It must be noted that in this work the CO2 laser treatment effects change in other surface properties besides roughness. It is most likely that the surface roughness plays a role in the protein adsorption, but its effects correlate to and are less than the wettability characteristics of the MgOPSZ. The Effects of Wettability Characteristics The previous results are a clear indication that interaction of the CO2 laser beam with the MgOPSZ brought about a decrease in y, which in some instances was considerable. This in turn naturally gave rise to improved wettability characteristics. As one can see from Figure 5.7, as the wettability characteristics of the MgOPSZ increased, the adsorbed amounts of bronectin increased, while the adsorbed amounts of albumin decreased. Indeed, this observation was supported somewhat by the results of the statistical analysis. The results of the albumin adsorption are consistent with the previous nding that the increase in surface hydrophilicity of Ti results in lower albumin adsorption [201], showing that the wettability characteristics of the MgOPSZ could be the main factor active in the albumin adsorption. The results of the adsorption of bronectin show that it increased on the hydrophilic surface. The previous investigation [204] on the extent of bronectin adsorption as
80
700 Fibronectin 600 *
Albumin
* 500
400
300 0.0 0.2 0.4 0.6 0.8 0.0 0.2 0.4 0.6 0.8 Wettability, cos (glycerol)
Figure 5.7 The relationship between the thickness of adsorbed bronectin and albumin layer and wettability characteristics cos y of the MgOPSZ. For the bronectin adsorption, there was a signicant statistical difference in thickness between the sample with cos y 0:19 and the sample with cos y 0:77, and no statistical difference between the sample with cos y 0:19 and the sample with cos y 0:47. For the albumin adsorption, there was a signicant statistical difference in thickness between the sample with cos y 0:19 and the sample with cos y 0:77, and no statistical difference between the sample with cos y 0:47 and the sample with cos y 0:77 p < 0:05
compared to its biological activity on hydrophobic and hydrophilic surfaces suggested the possibility that bronectin was adsorbed in two different conformations when incubated with the surfaces at low concentrations, with the more active conformation on the hydrophilic surfaces. The results showed that the antiplasma bronectin antibody appeared to bind to the conformation of bronectin adsorbed on hydrophilic surfaces much better than the conformation of bronectin adsorbed on hydrophobic surfaces [204]; therefore, the wettability characteristics of the MgOPSZ could be the predominant mechanism governing the bronectin adsorption. It is p noticeable that considerable change in the gsv , instead of the minor difference in gd , was the main mechanism governing the wettability characteristics of sv the MgOPSZ after CO2 laser irradiation, indicating that the albumin and bronectin adsorption on the MgOPSZ surfaces was probably due to the polar and chemical interactions [205].
5.5 Osteoblast Cell Response The development of boneimplant interfaces depends on the direct interactions of bone matrix and osteoblasts with the biomaterial. There is a
Osteoblast Cell Response
81
substantial body of literature based on the premise that improved initial attachment of osteoblasts or osteoblast precursor cells to orthopaedic implant surfaces may lead to improved bone integration of the implant and longer-term stability [206]. Osteoblast adhesion is a prerequisite for bonebiomaterial interaction and depends on the surface aspect of materials. A cell in contact with the surface of a material will rstly attach, adhere and then nally spread. The quality of this adhesion will inuence their morphology and their future capacity for proliferation and differentiation. The attachment of anchorage-dependent cells such as osteoblasts to biomaterial surfaces is a complex process involving cell attachment and spreading [207], focal adhesion formation, and extracellular matrix formation and reorganisation [208]. 5.5.1 Experimental Procedures Osteoblast Cell and Cell Culture The human osteoblastic cell line hFOB 1.19 was obtained from American Type Culture Collection (ATCC, Inc.). The hFOB cell line was established by transfection of limb tissue obtained from a spontaneous miscarriage. The cells have the ability to differentiate into mature osteoblasts expressing the normal osteoblast phenotype and provide a homogeneous, rapidly proliferating model system for study of normal human osteoblast cells. Moreover, it overcomes the disadvantages of earlier in vitro model systems, namely the unknown species-specic phenotype characteristics of animal osteoblast cultures and the very slow rates of proliferation, as well as the short lifetime of primary cultures derived from normal human bone [209]. The cells were cultured in a medium containing a 1:1 mixture of Dulbeccos modied Eagles medium without phenol red and Hams F-12 medium with 2.5 mM L-glutamine (D-MEM/F-12 medium), supplemented with 10 % fetal bovine serum (ATCC, Inc.) and 0.3 mg/ml G418 (Calbiochem, Inc.) at 37 C in a humidied 5 % CO2 incubator. Osteoblasts at passage numbers 24 were used in this experiment. Cell Cytotoxicity Cytotoxicity tests consisted of the quantication of the activity of lactate dehydrogenase (LDH) in culture medium of cells in contact with the samples. The activity of the LDH enzyme rises when cells are damaged. Thus the LDH activity induced by the untreated and selected CO2 laser treated specimens (0.9 and 1.6 kW/cm2) in triplicate were compared to that induced by a toxic agent (Triton X100 0.05 % in PBS) and to that induced by a culture polystyrene plate (NUNC, Inc.). The cell culture plate was used as a negative control and a Triton toxic agent as a positive control.
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Cell Adhesion and Morphology The MgOPSZ samples were placed in a 24-well tissue culture polystyrene plate (NUNC, Inc.), sterilised in 70 % alcohol and rinsed in PBS solution. To analyse the osteoblast cell attachment and morphology, one untreated sample and one CO2 laser modied sample (power density of 1.6 kW/cm2) were used in the assessment of cell morphology. The specimens were seeded with the 0.5 ml cell suspension of 1 105 cell/mL and analysed by SEM after 24 hours of cell culture. For a 7 day osteoblast cell adhesion analysis, the samples were rinsed in PBS, whereupon they were seeded with 0.5 mL cell suspension (4 105 cell/ml). After culturing, the cells were xed in 2.5 % glutaraldehyde solution for 1 hour, washed with PBS and then dehydrated in increasing concentrations of alcohol (70, 85 and 100 %). Thereafter, the osteoblast cells were dried in a critical point dryer (CPD030; BAL-TEC, GmbH). Then the samples were examined with by SEM after sputter gold coating. For the cell adhesion analysis, three images were taken for the each sample at different areas and a typical one was chosen for the analysis. Cell Proliferation Each group of specimens used for cell proliferation tests in triplicate was measured after cell culturing for 14 days. Osteoblast cells were cultured on specimen surfaces with the 0.5 ml cell suspension of 1 105 cell/ml in 6-well culture plates. The cell culture medium was changed every 3 days. At every harvest time point, cells were detached from specimen surfaces by incubation with trypsin/EDTA (ethylene diamine tetraacetic acid) (0.5 g/l trypsin and 0.2 g/l EDTA) (GIBCO, Ltd) for 5 minutes at 37 C and each specimen was washed with PBS. Released cells were counted with a hemocytometer, and on every specimen counting was repeated three times. Alkaline Phosphatase Assay For the staining of human osteoblast cells an alkaline phosphatase (ALP) assay kit (Sigma Diagnostics, Inc.) was used. After rinses with PBS, the samples with cells were xed by immersing in a citrate buffered acetone for 30 seconds and rinsed gently with deionised water for 45 seconds. Alkaline dye mixture was added and the samples were incubated at 24 C for 30 minutes protected from direct light. They were then rinsed thoroughly in deionised water for 2 minutes and placed in Mayers hematoxylin solution for 10 minutes. Positive staining for alkaline phosphatase (redviolet) was identied by light microscopy and evaluated by scoring cell rating and count according to the characterisation method provided.
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Statistics The statistical analysis of the results was performed with an SPSS v.12 software package in the same manner as discussed in Section 5.4.1. 5.5.2 Osteoblast Cell Response on CO2 Laser Treated MgOPSZ Cell Cytotoxicity LDH is a kind of enzyme in the cell. When cells are damaged or broken, the LDH will leak into the culture medium. As the concentration of LDH in the medium is proportional to the numbers of dead/damaged cells, the concentration of LDH in the medium can reect the cytotoxicity. The LDH activity in the culture media obtained from cells cultured on all the tested materials was found to be not signicantly different from the negative control, as shown in Figure 5.8, indicating that untreated and CO2 laser treated MgOPSZ were not cytotoxic. Cell Attachment Figure 5.9(a) shows that no osteoblast cells were observed on the untreated MgOPSZ after 24 hours of cell incubation, whereas a few cells attached on
60
50
40 LDH Activity * * 30 * *
20
10
Positive Control
Negative Control
Untreated MgO-PSZ
CO2 Laser CO2 Laser MgO-PSZ MgO-PSZ 0.9 kW/cm2 1.6 kW/cm2
Figure 5.8 Results provided by assessment of cell membrane damage are expressed as LDH activity (U/L) SD at 340 nm. There was a signicant statistical difference between the positive control and MgOPSZ samples, and no statistical difference between the negative control and the untreated and CO2 laser treated MgOPSZ p < 0:05
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Figure 5.9 SEM image of hFOB human osteoblast cells after 24 hours on (a) the untreated MgOPSZ and (b) the CO2 laser treated MgOPSZ at power densities of 1.6 kW/cm2
to the CO2 laser treated MgOPSZ (Figure 5.9(b)). It is quite clear that the osteoblast cell attachment on the MgOPSZ was inuenced by the CO2 laser treatment, indicating that the surface properties generated by the CO2 laser treatment were more favourable for the osteoblast cell attachment. Furthermore, the cells on the CO2 laser treated samples showed lopodia and spread well (Figure 5.9(b)), denoting good cell attachment. Moreover, it is evident from Figure 5.10 that the osteoblast cells had different morphologies in different regions of the CO2 laser treated track. Figure 5.10(a) shows that the osteoblast cells at the edge underwent initial spreading and the individual cell was found to cover an area of about 30 40 mm, as shown in Figure 5.10(b). Short lopodia protruded and elongated about 510 mm from the osteoblast cell (see Figure 5.10(b)). The elongation direction of the short lopodia implies the direction of the migration process. Conversely, the osteoblast cells at the centre reached a stage where they grew and spread to cover a region of 60150 mm (Figure 5.10(a)). One typical osteoblast cell (see Figure 5.10(c)) had spread completely and attened, with the cytoplasmic spread to cover an area of about 3050 mm as well as forming four lopodias, two of them elongated to a length of 5060 mm. Likewise, another two osteoblast cells (Figure 5.10(d)) had a at cytoplasm with two lopodias elongated to 5060 mm. The morphologies of these osteoblast cells display the nal stage of cell attachment. In general, osteoblast cells in the centre spread better and reached a higher stage of cell attachment than those at the edge of the CO2 laser treated track. Since the CO2 laser treatment exerted a higher photochemical effect at the centre than on the edge due to intensity distribution of the CO2 laser TEM01 beam mode (see Section 4.3.3), it could be concluded that the osteoblast cell spreading and attachment is inuenced by the level of the CO2 laser treatment.
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Figure 5.10 SEM images of hFOB human osteoblast cells after 24 hours (a) at the interface region, (b) at the circled area at the edge and (c) and (d) at the circled areas at the centre of the CO2 laser treated MgOPSZ with 1.6 kW/cm2 power density
Cell Growth After a 7 day incubation period, the hFOB cells grew well and formed a layer on all of the samples (see Figure 5.11). The degree of osteoblast cell adhesion and growth in terms of cell coverage area varied with the CO2 laser power density. The cover density is dened by the ratio of the osteoblast cell adhesion area to the whole surface area and is used as an indication of the osteoblast cell adhesion and growth. It has been found that the CO2 laser power density used in the treatment had a signicant inuence on the cover density of the osteoblast cells (see Figure 5.12). For instance, compared with the untreated sample, a power density of 0.9 kW /cm2 generated double cover density, while the higher power densities of 1.6, 1.9 and 2.5 kW/cm2 brought about triple cover density on the CO2 laser treated MgOPSZ. Generally, the osteoblast cell coverage area was found to increase as the power density increased when the power density is less than 1.9 kW/cm2.
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Figure 5.11 SEM images of hFOB cells on (a) the untreated MgOPSZ and CO2 laser treated MgOPSZ at power densities of (b) 0.5 kW/cm2, (c) 0.9 kW/cm2, (d) 1.6 kW/ cm2, (e) 1.9 kW/cm2 and (f) 2.5 kW/cm2
Figure 5.13 indicates that the number of osteoblast cells after 14 days on the untreated MgOPSZ is less than the CO2 laser treated MgOPSZ. Especially, compared with the untreated sample, the osteoblast cell grows signicantly faster on the samples that were CO2 laser treated at relatively high power densities of 1.6, 1.9 and 2.6 kW/cm2. Indeed, both cell adhesion and growth in 7 days investigated by SEM and cell proliferation after
87
100
80 Cell Cover Density (%)
60
40
20
0.0
0.5
1.0
1.5
2.0
2.5
CO2 Laser Power Density (kW/cm2)
Figure 5.12 The relationship between the cover density of the hFOB cells and CO2 laser power density. There was a signicant statistical difference between the untreated and CO2 laser treated MgOPSZ samples, and no statistical difference among the samples CO2 laser treated at 1.6, 1.9 and 2.6 kW/cm2 p < 0:05
14 12 Total No of Cells (105) 10 8 6 4 2 0
0.0
0.5 1.0 1.5 2.0 CO2 Laser Power Density (kW/cm2)
2.5
Figure 5.13 Total number of osteoblast cells on the untreated and CO2 laser treated MgOPSZ after 14 days. There was a signicant statistical difference between the untreated sample and the samples that were CO2 laser treated at 1.6, 1.9 and 2.6 kW/cm2, and no statistical difference among the untreated sample and the samples CO2 laser treated at 0.6 and 0.9 kW/cm2 p < 0:05
88
Figure 5.14 Optical image of the positive staining for alkaline phosphatase on (a) the untreated and (b) the CO2 laser treated samples (c) at 0.9 kW/cm2 and (d) at 1.6 kW/cm2
14 days counted by the hematocytometer show a similar trend in the cell growth on the untreated and CO2 laser treated MgOPSZ. Alkaline Phosphatase (ALP) Activity Optical images of the positive staining for ALP on the untreated and selected samples that were CO2 laser treated at 0.9 and 1.6 kW/cm2 are shown in Figure 5.14. The cell rating was determined on the basis of quantity and intensity of precipitated dye within the cytoplasm of these cells. As can be seen from Figure 5.14, the granule on the untreated sample is small in size and shows faint to moderate intensity of staining. On the other hand, the granule is medium to large in size and shows strong intensity of staining on the sample CO2 laser treated at 0.9 kW/cm2 and brilliant intensity of staining on the sample CO2 laser treated at 1.6 kW/cm2. One granule evidenced spreading of the cell on the sample CO2 laser treated at 0.9 kW/cm2, as shown in Figure 5.14(c). The leukocyte alkaline phosphatase activity (LAPA) scores, determined by the number of cells counted and multiplying by the

120 100 80 LAPA Score 60 40 20 0 Untreated CO2 laser 0.9 kW/cm2 CO2 laser 1.6 kW/cm2
89
Figure 5.15 LAPA scores of the untreated and CO2 laser treated MgOPSZ. There was a signicant statistical difference between the untreated sample and samples CO2 laser treated at 0.9 and 1.6 kW/cm2 at p < 0:05
value of the cell rating, are shown in Figure 5.15. The LAPA score of the CO2 laser treated sample is clearly much higher than that of the untreated sample. Furthermore, the CO2 laser treated MgOPSZ samples had LAPA scores that were statistically signicantly higher than that of untreated samples, as shown in Figure 5.15. 5.5.3 The Effect of CO2 Laser Treatment on the Osteoblast Cell Response The results show that the CO2 laser treatment brought about the considerable change in the response of osteoblast cells. Moreover, the osteoblast cell response varied on the MgOPSZ with the power density of CO2 laser treatment applied. As discussed in Chapter 4, the variation of the power density of CO2 laser treatment resulted in the different changes in surface properties. The surface topography and wettability characteristics chemistry have been shown to be the factors inuencing the osteoblast cell response in previous studies [210, 211] and are believed to effect the osteoblast cell response on the CO2 laser treated MgOPSZ. The Effect of Topography on the Osteoblast Cell Response The topography has been shown to be one of the factors in inuencing the osteoblast cell response [212]. As demonstrated in Chapter 4, CO2 laser treatment generated a consistently rougher surface on the MgOPSZ when compared with the untreated sample and Ra increased with the power
90
100 80 60 40 20 0 0.0
Cell Cover Density (%)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Figure 5.16 The relationship between the cover density of the hFOB cells and Ra
density of CO2 laser treatment. In this way it was possible to obtain a narrow range of surface roughness values (see Section 4.3.3). As shown in Figure 5.16, the CO2 laser treated MgOPSZ with rougher surfaces has a higher osteoblast cell cover density compared with the smooth untreated sample. This is in agreement with some reports that the rougher surface of titanium promoted more osteoblast-like cell attachment [213]. Even so, there is no linear relationship between the osteoblast cell cover density and Ra , as shown in Figure 5.16. Furthermore, the different microstructures and increase in the crystal sizes were postulated to be the factors inuencing the osteoblast cell cover density, as shown in Figure 5.17. The degrees of the cell adhesion improved markedly when an obvious microstructure change happened on the MgO PSZ. Surface microtopography has been cited as an important factor inuencing proteinsurface and cellsurface interactions [80]. A number of reasons have been suggested for an increased differentiation of osteoblasts on microstructured surfaces, such as the inuence of surface structure on cell shape or the fact that the surface topography creates a specic biochemical microenvironment around each cell [214]. Figure 5.17 shows that the crystal sizes in all CO2 laser treated MgOPSZ are larger than the untreated sample. The osteoblast cell cover density generally increased with the increased crystal size when the power density was lower than 1.9 kW /cm2, indicating that crystal size could possibly inuence the osteoblast cell adhesion. It is most likely that the greater nanosurface area created by the larger crystal size may promote interactions (such as adsorption, conguration, bioactivity, etc.), of select serum proteins(s), which, subsequently, enhance osteoblast adhesion. The study of osteoblast adhesion on nanophase ceramics has elucidated the fact that a critical grain size (between 49 and 67 nm for
91
Figure 5.17 The relationship between the cover density of hFOB cells, microstructure and crystal size of the MgOPSZ (treated with various CO2 laser power densities)
alumina and between 32 and 56 nm for titania) played a crucial role in mediating osteoblast adhesion to nanophase ceramics by creating a greater surface area and promoting the interaction of protein [140]. However, a linear relationship does not exist between the cell cover density and crystal size (see Figure 5.17). Owing to this lack of a linear relationship, a simple conclusion would be difcult to elucidate the relation between the topography and cell behaviours, because the surface roughness is only one of the factors affecting cell behaviours. Indeed, Hallab et al. [215] demonstrated that surface free energy was a more important surface characteristic than surface roughness for cellular adhesion strength and proliferation. Thus it is reasonable to postulate that the surface roughness does inuence the human osteoblast cell response; however, its effect is less than that of the surface energy. The Effect of Wettability Characteristics on the Osteoblast Cell Response As demonstrated in Chapter 4, the wettability characteristics of the MgO PSZ improved after CO2 laser treatment. It is believed that the changes in wettability characteristics resulted in modication of the osteoblast cell response. The result of the one-day cell culture on the MgOPSZ showed
92
that there were no osteoblast cells attached on the untreated MgOPSZ with low wettability characteristics, whereas some cells had already attached and /cm2) spread on the CO2 laser treated MgOPSZ (power density of 1.6 kW with high wettability characteristics. The difference in wettability characteristics and surface energy must be the mechanism determining the difference in the osteoblast cell attachment. The CO2 laser used in the experiment is a TEM01 multimode. The level of CO2 laser beam interaction is higher in the centre than at the edge of the CO2 laser beam; consequently the modication level of surface energy would be higher at the centre than at the edge of the CO2 laser treated track. The different wettability characteristics generated by CO2 laser treatment across the track brought about the different levels of osteoblast spreading between cells at the edge and cells at the centre (see Figure 5.10). As can be seen from Figure 5.15, the enhanced cell functions represented by the LAPA increase with the wettability characteristics. It is believed that the wettability characteristics of the MgOPSZ was the primary factor governing the cell response. The effects of wettability characteristics on cell functions could result from their inuence on the protein adsorption and cell adhesion. The adsorption of the proteins is the net result of various types of interactions that depend on the nature of the protein aqueous solution. The difference in cellular response of different materials suggests that there are differences in the organization of the adsorbed protein layer. Protein adsorption mediated cell behaviours are regarded as fundamental reactions at the biomaterialtissue interface [216]. The value of cos y (glycerol) was used to express the wettability characteristics of the MgOPSZ. The higher the value of cos y, the higher is the wettability characteristics. It is evident from Figure 5.18 that the cell growth
100 Cell Cover Density (%) 80 60 40 20 0 0.0
0.2
0.4
0.6
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1.0
Wettability, cos (glycerol)
Figure 5.18 The relationship between the cover density of hFOB cells and the wettability characteristics of the MgOPSZ
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increased when the wettability characteristics changed from a lower value to a moderate value (cos y from 0.2 to 0.65), indicating that generation of the wettability characteristics is the main mechanism accounting for the different amounts of osteoblast cell adhesion and growth. The nding agrees with previous studies showing the inuence of wettability on the attachment and spreading of various cells [213, 217219]. These studies showed good cell attachment and spreading on high-energy substrata and poor cell attachment and spreading on low-energy substrata, which accounts for the minimal energetic state of a system in equilibrium. It was noticed that the value of cos y ranged between 0.6 and 0.8 and did not present a great disparity in cell cover density. This implied that after a certain value, further increases in the wettability characteristics would not improve the cell response. This is similar to the nding that the highest levels of cell attachment were found on a moderately hydrophilic surface using a model surface [220]. As demonstrated in Chapter 4, the surface roughness, surface oxygen content and surface energy are the mechanisms governing the wettability characteristics of the MgOPSZ. The correlation between the wettability characteristics and the osteoblast cell response implies that the surface p roughness, surface oxygen content and gsv have some bearing on the cell response owing to the fact that these surface properties inuence the wettability characteristics of the MgOPSZ. In the consideration of surface roughness in Section 5.5.3, it was found that surface roughness does inuence the response of osteoblast cells, but its effect is slight. The surface oxide, among the surface characteristics, is shown to be a main factor inuencing the cell response besides surface roughness [52, 221]. In vivo studies show that a high degree of bone contact and bone formation are achieved with titanium implants which are modied with respect to oxide thickness and surface topography [222]. As shown in Chapter 4, the CO2 laser processing of the MgOPSZ with the oxygen shield gas resulted in the incorporation of oxygen atoms on the materials surface layer. It is postulated that the surface oxygen content is one of the factors inuencing cell growth. It can be seen from Figure 5.19 that the cell cover density on the material is higher when there is a higher surface oxygen content, indicating that the increase in surface oxygen content is attributed to better cell growth. However, there is no linear relationship between the surface oxygen content and cell cover density, implying that some other mechanism is more dominant in governing the cell response. In Chapter 4 it was found that changes in the wettability characteristics of p the MgOPSZ were primarily inuenced by gsv of the MgOPSZ. As one can p see from Figure 5.20, the cell cover density statistically increases as gsv increases from 10 to 25 mJ/m2. Hence, the higher surface energy induced by the CO2 laser treatment resulted in better proliferation of human osteoblast
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100 80 60 40 20 0
40
45 50 55 60 65 Surface Oxygen Content (at%)
70
75
Figure 5.19 The relationship between the cover density of the hFOB cells and the surface oxygen content of the MgOPSZ
100 80 60 40 20 0
10
20
30
40
50
60
p sv (mJ/m2)
Figure 5.20 The relationship between the cover density of the hFOB cells and gp for sv the MgOPSZ
cells on the MgOPSZ surface than on the untreated MgOPSZ. A further p increase in gsv from 25 to 60 mJ/m2 did not bring about an increase in cell p proliferation that was statistically signicant. As the gsv did not change p markedly after CO2 laser treatment, the results indicated that gsv inuenced the behaviour of the osteoblasts on MgOPSZ surfaces more strongly compared to gd following CO2 laser treatment, which was probably attribsv uted to the fact that the composition and the culture medium all are polar, and thus cells and the MgOPSZ should interact mainly by polar force.
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The behaviour of osteoblastic cells at the surface of hydroxyapatite [218] p and at the surface of titanium [213] demonstrated that gsv plays a critical role.
5.6 Predictions for Implantation in an in vivo Clinical Situation A relationship between the bioactivity/biocompatibility of a material in vivo and the materials ability to form an apatite-like layer in vitro when soaked in aqueous solutions that imitate the inorganic components of human plasma was propounded recently by Vallet-Regi [223]. This proposition extends to ceramics, for once implanted into the body, bioactive ceramics are able to bond to bone through the formation of a hydroxyapatite surface layer. These hydroxyapatite layers that are formed in vivo can be closely mimicked through in vitro testing, usually by using SBF, which contains ionic concentrations similar to that of human blood plasma. This allows for an evaluation of the potential for biocompatibility/bioactivity of a new ceramic, or a novel processing technique for a ceramic can be conducted before performing any animal tests. As the preceding sections of this chapter show, CO2 laser treatment could generate functional groups and subsequently facilitate the formation of bone-like apatites on the surface of the MgOPSZ. Building on this nding by considering the view of Vallet-Regi [223], it is reasonable to assume that, if implanted, the in vivo performance of the CO2 laser treated MgOPSZ would be acceptable. There is certainly a considerable body of evidence that supports this supposition. In comprehensive studies by Aldini et al. [224, 225] and Torricelli et al. [226], yttriastabilised tetragonal zirconia (Y-STZ), either coated with a bioactive glass termed RKKP or uncoated, was evaluated in vitro using normal and osteopenic bone-derived osteoblasts and in vivo using healthy bone in female Sprague Dawley rats. The in vitro results suggested that the RKKP-coated YSTZ was biocompatible and enhanced proliferation, activation and differentiation of normal bone-derived osteoblasts and stimulation of osseopenic bone-derived osteoblasts when compared with the uncoated YSTZ. To assess the in vivo performance of the RKKP-coated and uncoated YSTZ, samples of each were implanted into the distal femurs of the rats and then removed after 30 and 60 days from surgery, whereupon they were subjected to a histomorphometrical analysis to assess osseointegration and bone quality around the implants. The histomorphometrical analysis showed that the RKKP coating ensured a better osseointegration rate with higher afnity index values than the uncoated YSTZ, even when the osseopenic rate were used. No differences were observed at the bone biomaterial interfaces for either material. In this instance there was a direct
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correlation between the ndings of the in vitro and the in vivo investigations in terms of biocompatibility and osseointegration, but not for the case of bone-biomaterial interfacial characteristics. In a study conducted to evaluate the biocompatibility of MgO-doped HA (hydroxyapatite)/b-TCP (tricalcium phosphate) biphasic ceramics, Ryu et al. [227] performed in vitro and in vivo tests. The in vitro tests were carried out with a murine broblast L929 cell culture of extract from a 1 wt % MgOdoped HA/b-TCP ceramic with the aim of establishing whether the ceramics were biocompatible or cytotoxic. The presence or absence of any cytotoxic effects was determined qualitatively using SEM analysis. The SEM analysis following the cell culture from the 1 wt % MgO-doped HA/b-TCP ceramic revealed no morphological change, no vacuolisation or cell lysis and hence the absence of any cytotoxicity. In the rst of two in vivo tests small samples of the 1 wt % MgO-doped HA/b-TCP ceramic were inserted into the back muscles of mature rabbits and then removed after 8 weeks for XRD analysis. The XRD analysis showed that the 1 wt % MgO-doped HA/ b-TCP ceramic was indeed biocompatible as the b-TCP phase had completely dissolved and an apatite layer had formed on the surface by means of cellular activity and a resultant dissolution/precipitation process. The second in vivo test involved implanting the same small 1 wt % MgOdoped HA/b-TCP ceramic samples that were inserted into the back muscles of the rabbits for 8 weeks across the proximal tibial metaphysis of mature rabbits for a further 8 weeks. From a histological examination of the samples no inammation or foreign body reaction such as the formation of an intervening brous tissue was observed; rather the new bone formed and bonded directly to the implanted 1 wt % MgO-doped HA/b-TCP ceramic. Clearly, there was a direct link between the in vitro results and the observations made from both of the in vivo tests, with the in vitro tests indicating that the 1 wt % MgO-doped HA/b-TCP ceramic was biocompatible, the rst in vivo tests demonstrating that the 1 wt % MgO-doped HA/ b-TCP ceramic could form an apatite layer and the nal in vivo tests showing that the 1 wt % MgO-doped HA/b-TCP ceramic could support the bonding of new bone to its surface. Literature exists that shows that the in vitro bioactivity of a material has been observed to be dependent upon the type of aqueous solution used and does not always correlate to the observed in vivo behaviour. For example, apatitewollastonite (AW) glassceramics, which form a hydroxyapatite surface layer when immersed in SBF, are unable to form such layers in tris buffer [228]. In addition, SBF has been reported to affect the rate at which crystalline hydroxyapatite layers develop [229]. Indeed, the work of Gil-Albarova et al. [230] studied the in vivo behaviour of an SiO2P2O5 CaO sol-gel glass and an SiO2P2O5CaOMgO glassceramic, both of which are bioactive when soaked in SBF but display different rates of apatite layer
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formation. The ceramics were implanted into mature and immature New Zealand rabbits and histological results after 6 and 12 weeks revealed that both ceramics allowed bone growth over their surface in similar quantities and at similar rates by means of mesenchymal cell recruitment from the surrounding bone. Similarly, the in vitro and in vivo behaviour of certain ceramics has been seen to differ on account of the ceramic material itself. For instance, sintered hydroxyapatite exhibits in vivo bioactivity although the formation kinetics of an apatite-like layer on its surface is very slow under in vitro conditions [228]. Additionally, Li et al. [8] reported that that Al2O3 gel did not induce apatite formation when immersed in SBF for 21 days, whereas both pure SiO2 gel and gel-derived TiO2 were hydroxyapatite inducers. Kobayashi et al. [231] developed a composite (designated ABC) consisting of Al2O3 bead powder as an inorganic ller and bisphenol-a-glycidyl methacrylate (bis-GMA) based resin as an organic matrix, which allows direct bone formation on its surface in vivo. Although bioactive materials such as bioglass or apatite and wollastonite-containing glassceramic have previously been reported to form bone-like apatite on their surfaces in vitro under acellular conditions via simple chemical reactions, ABC did not present such characteristics. Indeed, no apatite formation was detected on the surfaces of the ABC composite after soaking in SBF for 28 days in vitro. Histological examination of rat tibiae after 8 weeks revealed that the ABC composite bonded to bone directly via a layer of calcium, phosphorus and alumina with no interposed soft-tissue layer. Moreover, the amount of bone directly apposed to the ABC composite surface was seen to increase with time. These results imply that the ABC composite has the ability to bond directly with bone through a calciumphosphorus-rich layer. It was concluded that this layer was induced by some property of the ABC composite that encouraged calcication or apatite formation due to the actions of proteins and cells in vivo. Although the aqueous solutions employed to replicate inorganic body uids are able to reproduce the process of bone-like apatite formation on the surfaces of bioactive ceramics in vitro, they are only capable of evaluating the bone-bonding capacities of bioactive ceramics by means of simple chemical reactions. However, once a ceramic is implanted into the body, they elicit several responses from living tissue that cannot be simulated in vitro. These responses include protein adsorption, cell attachment and adhesion, as well as ionic exchange. Therefore, depending upon the actual ceramic itself and the aqueous solution used, a ceramic could well be biocompatible in vivo despite appearing to be cytotoxic in vitro. However, importantly for this work, it is clear that if a ceramic presents itself to be biocompatible after in vitro testing, then it is highly likely that it will perform satifactorily in vivo.
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5.7 Summary A CO2 laser was used to modify the surface of the MgOPSZ for the purpose of acquiring surface properties favouring interaction at the implantbone tissue interface. The bioactivity of the CO2 laser modied MgOPSZ was investigated in SBF. Protein adsorption and hFOB cells were used to examine the in vitro biological response on the MgOPSZ following CO2 laser treatment. It was demonstrated that CO2 laser treatment could improve the bioactivity of the MgOPSZ surface by generating functional groups to facilitate the formation of bone-like apatites. The apatite formed readily on the MgOPSZ with relatively high amounts of hydroxyl groups, which were generated by CO2 laser treatment with power densities of 1.6 and 1.9 kW/cm2. No apatite was observed on the untreated and CO2 laser modied samples (0.6, 0.9 and 2.5 kW/cm2), which exhibited few hydroxyl groups. These observations indicate that ZrOH groups on the MgOPSZ surface are the functional groups required to facilitate apatite formation. The melting and re-solidication on the surface of the MgOPSZ induced by CO2 laser processing provides the Zr4 ion and OH ion and therefore creates the ZrOH group on the surface. In comparison with the untreated MgOPSZ, CO2 laser treatment brought about a thinner adsorbed albumin layer and a thicker adsorbed bronectin layer on the MgOPSZ. As the wettability characteristics of the MgOPSZ increased, the albumin adsorption decreased while the bronectin adsorption increased, indicating that wettability is a major factor in governing p protein adsorption. Further, the correlative effect of gsv observed on the protein adsorption suggested that protein adsorption on the MgOPSZ was probably due to the polar and chemical interactions. Better osteoblast cell responses were found on the CO2 laser treated MgO PSZ when compared with the untreated sample. The change in topography induced by the CO2 laser treatment was identied as being one of the factors inuencing the hFOB cell response, but in a minor capacity only. The improved wettability characteristics of the MgOPSZ due to enhanced surface energy, especially the polar component, brought by the CO2 laser treatment, played a signicant role in the number of initial cells that attached and spread, thereby enhancing the long-term cell adhesion and growth potential. There is a reasonable body of literature to support the concept that if a ceramic presents itself to be biocompatible after in vitro testing then it is highly likely that it will perform satisfactorily in vivo. This being the case, then it is reasonable to assume that the in vivo performance of the CO2 laser treated MgOPSZ would be acceptable due to its excellent in vitro performance demonstrated herein.
6
The Effects of CO2 Laser Radiation on the Wettability Characteristics of a Titanium Alloy
This chapter describes the modication of the wettability characteristics of a titanium alloy (Ti6Al4V ELI) following CO2 laser irradiation. The Ti6Al4V alloy is often used for the fabrication of dental and orthopaedic implants. To study the change in the wettability characteristics of the Ti6Al4V alloy, contact angles between selected control test liquids and the surfaces of the untreated and CO2 laser treated Ti6Al4V alloy were measured. The surface properties of the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy were characterised and the effect of surface roughness, surface oxygen content and surface energy on the wettability characteristics of the Ti6Al4V alloy were analysed. It was apparent that CO2 laser treatment brought about signicant changes in the wettability characteristics of the Ti6Al4V alloy. Furthermore, the predominant mechanisms active in determining the wettability characteristics were analysed and the primary mechanism was identied.
6.1 Introduction During the last decade, numerous materials have been used for the fabrication of dental and orthopaedic implants. The materials of choice have predominantly been metals. Stainless steel, cobalt chromium molybdenum alloy, titanium and a multitude of titanium alloys were the materials of
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choice. The following criteria dene an ideal bone contacting material for orthopaedic surgery: a biocompatible chemical composition to avoid adverse tissue reactions, acceptable strength, a high wear resistance to minimise wear debris, excellent corrosion resistance in the physiological milieu and a modulus of elasticity similar to that of bone to minimise bone resorption around the device. Titanium and its alloys, including the Ti6Al4V alloy, are now being used as a common material for bone implants under biomechanical loading conditions. None of these bioinert titanium based materials, however, bonds to bone and subsequently their stable xations to the surrounding bone have long been considered as a fundamental problem in clinical uses [232]. It is generally accepted that early surface events that occur rapidly upon implantation of a biomaterial into biological uids determine a subsequent response. These involve wetting by physiological liquids, followed by adsorption of proteins and cells to the biomaterials surface [233]. Numerous research groups have studied the interactions of different types of cultured cells with biomaterials with different wettability characteristics to correlate the relationship between surface wettability and blood, cell or tissue compatibility for polymeric materials [59, 115, 234]. Furthermore, the surface wettability has a signicant inuence on the friction behaviour of a tribological system and gives an indication of its biotolerance: in a rst approximation, the more wettable the material, the better the human body tolerates it [235]. Clearly, techniques to control the wettability characteristics of a biomaterials surface and thereby improve the materials biocompatibility are of great interest. Due to the rapid and specic modication of organic and inorganic materials, laser surface processing has aroused growing interest and been proven to be a controllable and exible technique for modifying the surface properties of materials. It is recognised within the currently published work that laser irradiation of material surfaces can affect their wettability characteristics. Previously Heitz et al. [133], Henari and Blau [134] and Olfert et al. [135] had found that excimer laser treatment of metals results in improved coating adhesion, attributed to the fact that the excimer laser treatment resulted in a smoother surface and as such enhanced the action of wetting. It was demonstrated that ve pulses per area of CO2 laser treatment were sufcient to produce a fully wettable mild steel surface. The wettability was inuenced by the surface exposing time (SET) after laser treatment [136]. Self-uxing FeCrNiBSi alloy powders with various Ni contents were laser clad on medium carbon steel substrates [137]. Lawrence and Li [138] revealed that the interaction of CO2, Nd:YAG, HPDL and excimer laser radiation with the surface of a selected mild steel gave rise to changes in the wettability characteristics of the material. It was observed that interaction of the mild steel with Nd:YAG and HPDL
Experimental Procedures
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radiation brought about an improvement in the wettability characteristics of the steel. In contrast, interaction of the mild steel with CO2 and excimer laser radiation resulted in a depreciation of the wettability characteristics of the steel [236]. However, despite a growing amount of work conducted with metal, no work has been conducted so far on the feasibility of the laser surface treatment process for the modication of the wettability characteristics of biograde metals.
6.2 Experimental Procedures 6.2.1 Material Specications and Preparation Medical grade titanium alloys have a signicantly higher strength-to-weight ratio than competing stainless steels. The range of available titanium alloys enables medical specialist designers to select materials and forms closely tailored to the needs of the application. The natural selection of titanium for implantation is determined by a combination of most favourable characteristics, including immunity to corrosion, biocompatibility, strength, low modulus and density and the capacity for joining with bone and other tissue (osseointegration). The mechanical and physical properties of titanium alloys combine to provide implants that are highly damage tolerant. Forms and material specications of titanium and its alloy for medical application are detailed in a number of international specications. In this study a Ti6Al4V ELI alloy (F136) was used. The as-received Ti 6Al4V alloy (ground annealed) was in the form of a round bar with a diameter of 28.5 mm (Carpenter, Inc.). For experimental purposes, the round bar was divided into 15 sections, each of 3 mm thickness, by a cutting machine (Miniton; Struers, GmbH) using a diamond-rimmed blade. The 15 divided sections were then separated into three groups of ve samples, with the groups being: untreated, mechanically roughened and CO2 laser treated. For the mechanically roughened group, the samples were roughened by evenly abrading the entire surface of the sample with grinding paper (180 grit SiC). This was achieved by applying the grinding paper to the surface of the sample with moderate pressure and drawing it across the surface in different directions eight times. The composition of the Ti6Al4V alloy was: 88.390.8 wt % Ti, 5.56.5 wt % Al, 3.54.5 wt % V, <0.08 wt % C, 0.0125 wt% H, <0.25 wt % Fe, <0.05 wt % N and <0.13 wt % O. The main physical properties of the Ti6Al4V alloy are: density of 4.42 g/cm2, melting point of 1649 15 C, a specic heat at 25 C of 560 J/kg C and a thermal conductivity at 25 C of 7.2 W/mK. The mechanical properties are: tensile strength of 1000 MPa, an elastic modulus of 114 GPa and a Rockwell hardness of 36 C.
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6.2.2 CO2 Laser Surface Treatment The surface treatment of the Ti6Al4V alloy was completed with the 3 kW CO2 laser described in Section 4.2.2. As before, the laser was run in the CW mode. The CO2 laser beam was defocused to a 7 mm spot diameter and operated with laser powers that yielded laser power densities of 1.3 and 1.6 kW/cm2. The CO2 laser beam was traversed across the surface of the Ti 6Al4V alloy at a speed of 4800 mm/min. The fumes produced were removed with an extraction system while an O2 assist gas was supplied at 2 bar pressure to shield the laser optics. The set-up of the CO2 laser experiment was as described in Chapter 4 and is shown in Figure 4.1. 6.2.3 Morphological, Chemical and Phase Analysis Procedures The morphological characteristics of the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy samples were examined by means of surface roughness measurements of the samples and through SEM analysis. The chemical composition of the Ti6Al4V alloy before and after CO2 laser treatment was determined using SEM, EDX and XPS analysis, while the phase was observed with XRD across a range of 30 to 80 2-theta. The equipment details and manner in which they were operated are described in Section 4.2.3. 6.2.4 Wettability Characteristics Analysis Procedure To examine the wetting and surface energy characteristics of the Ti6Al4V alloy when in the untreated, mechanically roughened and CO2 laser treated conditions, wetting experiments were conducted. A set of sessile drop control experiments was carried out using glycerol, formamide, etheneglycol, polyglycol E-200 and polyglycol 15-200. The characteristics of the control test liquids are given in Table 4.1. The y values for the control test liquids on the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy were determined in atmospheric conditions at 25 C using a sessile drop measuring machine in the same manner as described in Section 4.2.4. In order to estimate the inuence of contaminant layers on the measured y results, the untreated Ti6Al4V alloy samples were cleaned in the same manner as described in Section 4.2.4. The values of y on the cleaned samples were lower than on the as-received (not cleaned) samples by 0.8, 0.7, 0.5, 0.3 and 0.2 for glycerol, formamide, etheneglycol, polyglycol E-200 and polyglycol 15-200, respectively. It is therefore reasonable to conclude that the presence of any contaminants on the surface of the Ti6Al4V alloy do not have a great inuence on y. This being the case, the work was conducted in a normal atmospheric environment without pre-cleaning with the same justication as that given in Section 4.2.4.
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6.3 The Effects of CO2 Laser Radiation on Wettability Characteristics 6.3.1 Contact Angle The mean values of y for the ve control test liquids measured on the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy are given in Table 6.1. As one would expect, in accord with Equation (4.1), reductions in y for all of the control test liquids were occasioned by the mechanical roughening of the surface of the Ti6Al4V alloy. It is clear, however, that these reductions were only moderate. In contrast, CO2 laser treatment of the surface of the Ti6Al4V alloy at both 1.3 and 1.6 kW/cm2 resulted in marked decreases in y with all of the control test liquids, with the greatest reductions in y occurring when the power density was 1.6 kW/cm2. 6.3.2 Morphological Analysis and Its Effect on Wettability Characteristics The typical surface view of the untreated Ti6Al4V alloy, as shown in Figure 6.1(a), exhibited regularly ordered parallel and longitudinal grooves. From Figure 6.1(b) it can be seen that the mechanically roughened Ti6Al 4V alloy samples also presented a grooved surface. However, in contrast with the untreated Ti6Al4V alloy surface, the mechanically roughened samples typically displayed no surface ordering, with the grooves being orientated in various directions. The surfaces of both of the CO2 laser treated Ti6Al4V alloy samples retained a similar morphology to that of the untreated sample insofar as parallel and longitudinal grooves that are regular in size and periodicity are present (see Figures 6.1(c) and (d)). However, as one can see from Figures 6.1(c) and (d), it appears that CO2 laser radiation, when incident with the Ti6Al4V alloy surface, resulted in
Table 6.1 Measured contact angle values for the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy Contact angle, y (deg) CO2 laser treated (kW/cm2) Mechanically Untreated roughened 1.3 1.6 85.8 83.6 77.3 71.1 67.1 78.7 74.5 70.9 64.5 55.3 74.4 72.6 68.4 63.2 53.5 70.1 67.6 62.9 57.3 48.1
Control test liquid Glycerol Formamide Etheneglycol Polyglycol E-200 Polyglycolycol 15-200
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Figure 6.1 Typical SEM surface images of the Ti6Al4V alloy (a) untreated, (b) mechanically roughened and CO2 laser treated at power densities of (c) 1.3 kW/cm2 and (d) 1.6 kW/cm2
partial melting and re-solidication. By comparing Figures 6.1(c) and (d) it is evident that the melting and re-solidication of the surfaces of the Ti6Al 4V alloy occurred in varying degrees. The extent to which melting and re-solidication differs on the surface of the CO2 laser treated Ti6Al4V alloy samples is due entirely to the difference in the CO2 laser power density used to treat the surfaces. In the case of the sample treated at the CO2 laser power density of 1.6 kW/cm2, the higher power used would result in more energy being obsorbed, thereby causing more surface melting to occur. In contrast, the sample treated at the CO2 laser power density of 1.3 kW/cm2 was treated with less power and so less energy was absorbed. It is obvious that the specimen treated at the CO2 laser power density of 1.6 kW/cm2 experienced more melting and resolidication and so the depth of the grooves was much greater (see Figure 6.1(d)), whereas the specimen treated at the CO2 laser power density of 1.3 kW/cm2 underwent melting and re-solidication to a lesser extent and
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0.5 Surface Roughness, Ra (m)
0.4
0.3
0.2
0.1
0.0
Untreated
Mechanically Roughened
CO2 laser 1.3 kW/cm2
CO2 laser 1.6 kW/cm2
Figure 6.2 Mean values of surface roughness for the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy
thus the depth of the grooves was shallower (see Figure 6.1(c)). It is worth remarking that since the surface morphology of the two CO2 laser treated samples, while being different to the untreated sample, are not dramatically different from each other, it is therefore possible to assert that the energy absorbed was neither insufcient nor excessive. From Figure 6.2 one can see that the surface of the Ti6Al4V alloy in the untreated condition was reasonably smooth, having an average surface roughness (given in terms of Ra ) value of 0.35 mm. Figure 6.2 reveals that the average surface roughness value of the Ti6Al4V alloy did not alter a great deal as a result of CO2 laser treatment, increasing to 0.39 mm when treated with a laser power density of 1.3 kW/cm2 and to 0.42 mm when treated with a laser power density of 1.6 kW/cm2. Conversely, Figure 6.2 shows that mechanical roughening of the Ti6Al4V alloy surface had more of a marked effect on surface roughness. In this instance, CO2 laser treatment brought about an increase in the average surface roughness to 0.47 mm. In both instances these reductions in y with corresponding increases in surface roughness are in accord with Equation (4.1). These values of average surface roughness are reected by the images shown in Figure 6.1. 6.3.3 Phase and Chemical Analysis and Its Effects on Wettability Characteristics The results of an XRD analysis on the Ti6Al4V alloy surface before and after CO2 laser treatment are shown in Figure 6.3. The mechanically roughened Ti6Al4V alloy samples were not subjected to an XRD analysis
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160 140 120 100 80 60 40 20 0 30

Ti (110) Ti (101) Al (110) Ti (201) Ti (102) Al (220) Al V (211) (311)
Intensity
V Al (110) (200)
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50 2 theta (a)
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350 300 Intensity 250 200 150 100 50 0 30 40 50 2 theta (b) 450 400 350 Intensity 300 250 200 150 100 50 0 30 40 50 2 theta (c) 60 70 80
V (110) Ti Al (200) (102) Al (220) Rutile Ti (110) Ti (101) Ti (110) Rutile Al (110) V (110) Ti Al (102) (200) Al (220) Ti (101)
Ti (201) V Al (211) (311)
60
70
80
Al (110) Ti V (201) (211) Al (311)
Figure 6.3 XRD analysis of the Ti6Al4V alloy (a) untreated and CO2 laser treated at power densities of (b) 1.3 kW/cm2 and (c) 1.6 kW/cm2
as the roughening process would not have induced any phase change. On the whole, CO2 laser treatment appears not to have altered the phases present in the Ti6Al4V alloy (see Figures 6.3(b) and (c)). The results of an EDX analysis on the surface of the Ti6Al4V alloy are shown in Figure 6.4. Again, it appears that CO2 laser treatment did not alter the chemical
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Figure 6.4 EDX analysis of the Ti6Al4V alloy (a) untreated and CO2 laser treated at power densities of (b) 1.3 kW/cm2 and (c) 1.6 kW/cm2
composition of the surface of the Ti6Al4V alloy, since Ti, Al and V, the basic elements of Ti6Al4V alloy, were still present in similar proportions on the surface of the alloy before and after CO2 laser treatment. Having said that, the XRD analysis did reveal that an oxide diffraction peak at 39.5 was generated after CO2 laser treatment (see Figures 6.3(b) and (c)). This nding is evidence that the surface of the Ti6Al4V alloy was oxidised after the CO2 laser process, with the oxide layer consisting of rutile. This oxidation of the Ti6Al4V alloy samples came about because the surface was supercially melted by the CO2 laser beam during treatments. This in turn led to oxygen diffusion through the molten material and subsequently to the oxidation of the Ti6Al4V alloy surface. Of particular noteworthiness is the observation that can be made by comparing
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60
Surface Oxygen Content (at%)
50
40
30
20
10
0 Untreated Mechanically Roughened CO2 laser 1.3 kW/cm2 CO2 laser 1.6 kW/cm2
Figure 6.5 Mean values of the surface oxygen content for the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy
Figures 6.3(b) and (c), namely that the level of oxidation, as indicated by the diffraction peak at 39.5 , increases with increasing CO2 laser power density. Such an observation supports the proposition given earlier that the oxidation of the Ti6Al4V alloy surface following CO2 laser treatment is brought about by oxygen diffusion through the molten material. Hence the more molten material there is and the longer the material remains in the molten state, conditions that result from an increase in CO2 laser power density, the greater the degree of oxidation. XPS was used to examine the surface oxygen content on the surface of the Ti6Al4V alloy before and after CO2 laser treatment (see Figure 6.5). As is evident from Figure 6.5, the surface oxygen content on the Ti6Al4V alloy increased after CO2 laser treatment. This increase in the oxygen content on the surface of the Ti6Al4V alloy was naturally due to oxidisation of the CO2 laser treated surfaces, which was shown to occur from the XRD analysis (see Figures 6.3(b) and (c)). A similar observation was found in the work of Lawrence and Li [138], where the surface oxygen content of a carbon steel increased after CO2 laser treatment. The specimen CO2 laser treated at a power density of 1.6 kW/cm2 had the highest surface oxygen content. This nding further supports the proposition that with an increase in laser power comes a greater degree of melting, thereby inducing a larger amount of oxygen to be absorbed into the Ti6Al4V alloy surface. From the results of y measurements between the control test liquids and the Ti6Al4V alloy in various conditions of treatment (see Table 6.1), it is
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clear that the CO2 laser treatment increased the wettability characteristics of the Ti6Al4V alloy. This observed increase in the wetting performance of the Ti6Al4V alloy would have certainly been inuenced by the increase in the oxygen content of the Ti6Al4V alloy surface as a result of the CO2 laser treatment (see Figure 6.5), since this is known to increase the likelihood of wetting [13, 146].
6.4 Surface Energy and Its Component Analysis Measurements of y between the Ti6Al4V alloy and the control test liquids (see Table 6.1) shows that CO2 laser surface treatment improved the wettability characteristics of the material. As has already been demonstrated in Chapter 4, it is possible to estimate reasonably accurately the gd of the Ti sv 1 6Al4V alloy by plotting the graph of cos y against (gd )2 =glv ) in accordance lv with Equation (3.8). Figure 6.6 shows the best-t plot of cos y against 1 (gd )2 =glv ) for the untreated, mechanically roughened and CO2 laser treated lv Ti6Al4V alloyexperimental control liquids system. Comparing the ordinate intercept points of the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloyliquid systems, as shown in Figure 6.6, it can be seen clearly that for the untreated and mechanically roughened Ti6Al4V alloy, the best-t straight line intercepts the ordinate
1.0 0.8 0.6 0.4 0.2 cos 0.0 0.2 0.4 0.6 0.8 1.0 0.00 0.03 0.06 0.09 0.12 Untreated Mechanically Roughened CO2 laser (1.3 kW/cm2) CO2 laser (1.6 kW/cm2)
( )
0.15
0.18
0.21
0.24
d 1/ 2 lv
/ lv
Figure 6.6 Plot of cos y against (gd )2 =glv for the untreated, mechanically roughened lv and CO2 laser treated Ti6Al4V alloy in contact with the control test liquids
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closer to the origin. This is noteworthy as the intercept of the ordinate close to the origin is characteristic of the dominance of dispersion forces acting on the Ti6Al4V alloy materialliquid interfaces of the untreated and mechanically roughened sample, resulting in poor adhesion [100, 101]. On the other hand, the best-t straight line of samples treated by the CO2 laser intercepts the ordinate considerably higher above the origin. An interception of the ordinate above the origin is indicative of the action of polar forces across the interface, in addition to dispersion forces, and hence improved wettability and adhesion are promoted [100, 101]. Furthermore, because none of the best-t straight lines intercepts below the origin, it can be said that the development of an equilibrium lm pressure of adsorbed vapour on the Ti 6Al4V alloy surface (untreated, mechanically roughened and CO2 laser treated) did not occur [101]. p As was shown previously in Chapter 4, in order to determine the gsv of the Ti6Al4V alloy, it is necessary to calculate values of Wad calculated d using Equation (3.4) and Wad calculated using Equation (3.9). Both Wad d and Wad are related by the straight line relationship represented by Equation (3.10). On account of this it is possible from the best-t straight line plots of d Wad against Wad to determine the constant, a, for each separate condition of the Ti6Al4V alloy: 2.38 in the untreated condition, 2.66 when mechanically roughened, 2.80 when CO2 laser treated at 1.3 kW/cm2 and 2.84 when CO2 laser treated at 1.6 kW/cm2. Since a linear relationship exists between the dispersive and polar components of the control liquids surface energies that satises Equation (3.11), the constant, c, is determined to be 2.9 p (see Section 4.4). It is therefore possible to calculate gsv directly for the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy using Equation (3.14) with the determined values of the constants, a and c. As can be seen from Figure 6.7, CO2 laser treatment of the surface of the Ti6Al4V alloy led to an increase in the total surface energy. In particular, the CO2 laser treatment brought about a considerable increase in the p value of gsv , which is known to have a positive effect upon the action of wetting and adhesion [237]. The changes in the surface energy values of the Ti6Al4V alloy are thought to be due to the fact that CO2 laser treatment results in the melting and re-solidication of the Ti6Al4V alloy surface, a p transition that is known to effect an increase in gsv [103, 130, 131]. This explains why, after CO2 laser treatment, the Ti6Al4V alloy specimens have a higher propensity for wetting. As before, owing to the higher degree of melting on the Ti6Al4V alloy surface occasioned by CO2 laser treatment at p a power density of 1.6 kW/cm2, the increase in the value of gsv was more than for the Ti6Al4V alloy sample CO2 laser treated at a power density of 1.3 kW/cm2.
Identication of the Predominant Mechanisms
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40
Dispersive Component Polar Component Total
Surface Energy (mJ/m2)
30
20
10
Figure 6.7 Measured total surface energy gsv , dispersive (gd ) and polar (gp ) sv sv components for the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy
6.5 Identication of the Predominant Mechanisms Active in Determining Wettability Characteristics Based on the ndings of the preceding sections it is apparent that modications to the wettability characteristics of the Ti6Al4V alloy following CO2 laser treatment are attributable to changes in the surface roughness, surface O2 content and surface energy. The dependency of y to surface roughness is well known and, moreover, surface roughness has been identied as the predominant factor governing changes in wettability characteristics of steel after surface treatment with various lasers [138, 236]. Additionally, the surface oxygen content has been found to be a factor contributing to the enhancement of the wettability characteristics of a number of materials: MgOPSZ after CO2 laser treatment (see Chapter 4) and steel following p irradiation with a HPDL [236]. Further, increases in gsv due to laser-induced surface melting and re-solidication of steel [138, 236] and various ceramic materials have been seen to be inuential in effecting improvements in the p wettability characteristics of the materials. What is more, the increase in gsv of MgOPSZ after CO2 laser surface treatment was identied as the major factor in determining the wettability characteristics of MgOPSZ (see Chapter 4). Naturally, it would be advantageous to determine whether the changes to the surface roughness, the surface oxygen content or the surface
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p
energy, namely the increases in gsv (which is determined by the microstructure), inuenced the observed increase in the wettability of the Ti6Al 4V alloy after CO2 laser surface treatment, either independently or in combination with one another. To do this, several stages of ne grinding were used to isolate the various inuential factors detailed above and thus analyse and qualitatively establish the effect each one had on the wettability characteristics of the Ti6Al4V alloy. The fact that the value of y was seen to decrease by simply roughening the surface mechanically (see Table 6.1) suggests that the surface roughness may play a major part in determining the wettability characteristics of the Ti 6Al4V alloy. Having said that, Table 6.1 shows that the value of y was consistently lower on the CO2 laser treated samples than on the mechanically roughened sample, despite the fact that the surface roughness value was higher on the mechanically roughened sample. This implies that not only are other factors besides surface roughness active in promoting a reduction in y, they are signicant in their inuence. In the rst ne grinding stage the surfaces of the untreated and CO2 laser treated Ti6Al4V alloy (1.6 kW/cm2) were ground down to an Ra value of around 0.30 mm with grinding paper (800 grit SiC), while still retaining the CO2 laser treated microstructure. In this way it was possible to isolate and assess the effect of surface roughness. In addition the rst ne grindingdown stage would allow one to investigate the effects of the surface oxygen content as it is present only within the rst atomic layers of the alloy. In order to evaluate the inuence of the CO2 laser induced microstructure, and in turn the surface energy, a second ne grinding-down stage was undertaken using grinding paper (800 grit SiC) to remove the microstructure but retain the surface roughness value obtained after the rst ne grinding stage. Following on from this, previously un-ground samples of both the untreated and the CO2 laser treated Ti6Al4V alloy were ground up with grinding paper (400 grit SiC) to an Ra value of around 0.45 mm in order to conrm the effect of surface roughness and investigate the role played by surface energy. In this rst ne grinding-up stage the CO2 laser induced microstructure was retained. A second ne grinding-up stage was then carried out to remove the CO2 laser induced microstructure and hence conrm the effect of surface roughness. The observed changes to surface roughness, surface oxygen content and y for glycerol brought about after each of these ne grinding stages are given in Table 6.2. Conrmation of the predominant role played by surface roughness is clearly evident by the results obtained after the rst ne grinding-down stage. From Table 6.2 it can be seen that for both the untreated and CO2 laser treated Ti6Al4V alloy samples, y increases and in the case of the CO2 laser treated sample, the increase is considerable. However, despite the fact that the surface roughness values for the untreated and CO2 laser treated
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Table 6.2 The contact angle, surface roughness and surface oxygen content of the untreated and CO2 laser treated (1.6 kW/cm2) Ti6Al4V alloy following the ne grinding stages Untreated O2 (at %) 23.32 23.33 23.31 23.32 23.32 y (glycerol) 85.8 87.1 87.1 76.5 76.4 CO2 laser treated O2 (at %) 41.80 23.34 23.33 23.33 23.33 y (glycerol) 74.4 86.2 86.9 74.6 76.4
Fine polishing stages Unpolished Grinding-down stage 1 Grinding-down stage 2 Grinding-up stage 1 Grinding-up stage 2
Ra (mm) 0.35 0.30 0.30 0.45 0.46
Ra (mm) 0.39 0.31 0.30 0.46 0.46
Ti6Al4V alloy samples are similar after the rst ne grinding-down stage, there is a discernible difference in the value of y, being 0.9 lower for the CO2 laser treated sample. Whereas the surface oxygen content of the untreated Ti6Al4V alloy sample remained around the original value of 23.32 at %, the surface oxygen content of the CO2 laser treated Ti6Al4V alloy sample reduced to a level similar to that of the untreated sample. Because the rst ne grinding-down stage does not remove the CO2 laser induced microp structure, this difference in y can, therefore, be taken as an indication that gsv is active in determining the wettability characteristics of the Ti6Al4V alloy. The situation after the rst ne grinding-up stage presented revealing results. As one would expect, the increase in surface roughness occasioned by the rst ne grinding-up stage caused the y measured for the untreated Ti6Al4V alloy sample to decrease. For the CO2 laser treated Ti6Al4V alloy sample the rst ne grinding-up stage actually caused the value of y to increase by 0.2 , despite the increase in the surface roughness to virtually the same value as the untreated Ti6Al4V alloy sample. Since the surface oxygen content of the untreated Ti6Al4V alloy sample remained around the original value, while the surface oxygen content of the CO2 laser treated Ti6Al4V alloy sample reduced to a level similar to that of the untreated sample, then this observation implies that the surface oxygen content is indeed active in determining y. Moreover, because the CO2 laser induced microstructure was retained after the rst ne grinding-up stage, then it is p reasonable to assert that gsv is the factor responsible for the fact that y is 1.9 degrees lower on the CO2 laser treated Ti6Al4V alloy sample than that of p the untreated Ti6Al4V alloy sample. Thus the claim that gsv plays a role in inuencing changes in the wettability characteristics of the Ti6Al4V alloy is substantiated. p Further conrmation of the action of gsv with regard to the wettability characteristics of the Ti6Al4V alloy can be found from an examination of
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the results of the second ne grinding-down stage. The values obtained for y given in Table 6.2 show that after the second ne grinding stage, which removed the CO2 laser induced microstructure, y increased slightly from 86.2 to 86.9 . No change in the value of y for the untreated Ti6Al4V alloy sample was observed after the second ne grinding-down stage, nor was any change in the surface oxygen seen. This suggests that the increase in y was due to the removal of the CO2 laser induced microstructure and, in turn, p the reduction in gsv , which was presumably reduced to a value around that of the untreated Ti6Al4V alloy sample. When the CO2 laser induced microstructure was removed by the second grinding-up stage, the value of y increased again to 76.4 , a value almost equal to that of the untreated Ti 6Al4V alloy sample. Although the rst and second ne grinding stages of the grinding-down p and grinding-up processes revealed that gsv inuenced the wettability characteristics of the Ti6Al4V alloy, a comparative examination of the surface roughness, surface oxygen content and y values for the untreated and CO2 laser treated Ti6Al4V alloy samples shows that its inuence is relatively small. If one considers the rst ne grinding-down stage, for the untreated Ti6Al4V alloy sample the surface roughness was decreased from 0.35 to 0.30 mm, which gave rise to an increase in y of 1.3 , with the surface oxygen content remaining around the original value of 23.32 at %. In contrast, the surface roughness of the CO2 laser treated Ti6Al4V alloy sample after the rst ne grinding-down stage was reduced from 0.39 to 0.31 mm, which in turn caused an increase in y of some 11.8 . This increase in y for the CO2 laser treated Ti6Al4V alloy sample is clearly disproportionate to the reduction in the surface roughness. However, unlike the case with the untreated Ti6Al4V alloy, the surface oxygen content of the CO2 laser treated sample was found to have reduced from 41.8 to 23.34 at %, a level similar to that of the untreated samples. This suggests that the relatively large increase in y experienced by the CO2 laser treated Ti6Al 4V alloy sample is correlated with the change in the surface oxygen content. As such, it is reasonable to conclude that the wettability characteristics of the Ti6Al4V alloy are, after surface roughness, inuenced predominantly by the surface oxygen content and, to some extent, by the microstructure.
6.6 Investigation of Wettability and Work Adhesion Using Physiological Liquids In order to simulate the biological environment, the physiological uids and simulated physiological liquids used for the wetting experiments were human blood, human blood plasma, simulated body uid (SBF) and SBF BSA (bovine serum albumin), as described in Section 4.7.
Investigation of Wettability and Work Adhesion
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Table 6.3 Mean values of contact angles formed between the simulated physiological test liquids and the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy Contact angle, y (deg) Human blood Human blood plasma SBF SBFBSA 58.3 56.9 50.6 48.3 63.2 61.7 55.4 53.6 82.8 80.2 71.0 68.2 60.9 59.1 53.5 51.2
Ti6Al4V alloy Untreated Mechanically roughened CO2 laser (1.3 kW/cm2) CO2 laser (1.6 kW/cm2)
The values of y formed between the selected and simulated physiological test liquids and untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy are shown in Table 6.3. It clearly reveals that the y values of all body uids on the CO2 laser treated Ti6Al4V alloy are lower than the untreated and mechanically roughened specimens, indicating that the wettability characteristics of the material with the body uids obviously improved after CO2 laser treatment. The results detailed previously show clearly that interaction of the CO2 laser beam with the Ti6Al4V alloy had resulted in the lower y formed between the physiological liquids. Since biomaterials rst contact a proteinaceous liquid phase, almost aqueous in nature, leading to surface reorganization of proteins followed by cell attachment on biomaterials, wettability characteristics, by controlling the interaction with physiological uids, would primarily inuence cell behaviour on biomaterials. Wetting of the solid surface is a predictive index of cytocompatibility [165]. Further, according to Equation (3.4), the decrease in the y resulted in the increase of the work adhesion of Ti6Al4V towards the physiological and simulated physiological liquids. Using the referenced glv value of human blood (47.5 mJ/m2), human blood plasma (50.5 mJ/m2) [141], SBF (72.5 mJ/ m2) and SBFBSA (54.0 mJ/m2) [164], the work adhesion, Wad , of the Ti 6Al4V alloy towards these body uids was determined through Equation (3.4), as shown in Figure 6.8. A discernable increase in Wad of body uids can be seen on the Ti6Al4V alloy following CO2 laser treatment. Moreover, Wad increased as the CO2 laser power density increased. Owing to the fact that biomaterials rst contact a proteinaceous liquid phase, almost aqueous in nature, leading to surface reorganization of proteins followed by cell attachment on biomaterials, wettability characteristics, by controlling the interaction with physiological uids, would primarily inuence cell behaviour on biomaterials. Wetting of the solid surface is a predictive index of cytocompatibility [165]. Moreover, the improvements of Wad towards these uids would imply better suitability of titanium in the biological environment.
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120 Human blood Human blood plasma SBF SBF+BSA
100 Work Adhesion, Wad(mJ/m2)
80
60
40
20
Figure 6.8 Work of adhesion of body uids for the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy
6.7 Summary The results presented in this chapter are a clear indication that CO2 laser surface treatment of the Ti6Al4V alloy brought about a reduction in the y formed between the Ti6Al4V alloy and simulated physiological liquids, indicating that the wettability characteristics of the material were modied. Such results reveal that the CO2 laser could be a suitable tool to modify the biograde metals for improved biocompatibility. It was found that the modication of surface roughness, surface oxygen content and surface energy of the Ti6Al4V alloy following laser treatment were the factors inuencing the wettability characteristics. The predominant mechanisms active in determining the wettability characteristics of the Ti6Al4V alloy following CO2 laser irradiation were identied. It was found that the wettability characteristics of the Ti6Al4V alloy were, after surface roughness, inuenced predominantly by the surface oxygen content and, to some extent, by the microstructure. A reduction in y contributes to enhancement in work adhesion of physiological liquids on the Ti6Al4V alloy following CO2 laser treatment. The improvements in work adhesion of the Ti6Al4V alloy surface towards these uids would mean better suitability of the Ti 6Al4V alloy to be used as a biomaterial after laser treatment.
7
In vitro Biocompatibility Evaluation of CO2 Laser Treated Titanium Alloy
This chapter is concerned with comparatively evaluating the biocompatibility of a CO2 laser treated titanium alloy (Ti6Al4V). An investigation of the bioactivity of the CO2 laser modied Ti6Al4V alloy in simulated body uid (SBF) was conducted and the formation of bone-like apatite was found on samples that were CO2 laser treated at certain power densities. In addition, ellipsometry was used to investigate the albumin and bronectin (protein) adsorption on the untreated and CO2 laser treated Ti6Al4V alloy and signicant changes in protein adsorption were observed on the samples following CO2 laser treatment, being the result of the CO2 laser modied surface properties. Finally, the in vitro behaviour of the hFOB cell response was conducted to determine the effect of surface properties on the osteoblast cell adhesion and growth, thereby elucidating the mechanisms active in the osteoblast cell response and correlating them with the enhanced wettability characteristics of the Ti6Al4V alloy after CO2 laser treatment.
7.1 Introduction Titanium and some of its alloys are now dominant biomaterials because of their good biocompatibility. Commercially pure titanium (cp Ti) implants are alloplastic materials used as the foundation for replacing teeth in dentistry and are also used for orthopaedics; however, this interaction does not involve a chemical bond with bone. The lack of ability to bond
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chemically may lead to slow xation of cp Ti dental implants and to their gradual loosening over a long period [238]. Different approaches are being used in an effort to obtain the desired boneimplant interface. The implant should present a surface conductive to or that will induce osseointegration [9]. It was recently demonstrated by Kokubo et al. [239] that an in vitro chemical deposited bone-like apatite on cp Ti could be induced by an alkali and heat treatment process followed by a simulated body uid (SBF) soaking. This apatite layer is an essential requirement for articial materials to bond to living bone [41]. Furthermore, wettability, which controls the way biological uids interact with materials, is among the physicochemical characteristics that have been altered with the aim of improving the boneimplant interface. The cells attached to carboxylicylic-acid-terminated hydrophilic monolayers were about two times more than those attached to methyl-terminated hydrophobic monolayer over 90 minutes [217]. Radiofrequency glow discharge has been used to increase surface energy and to enhance cell binding [240, 241]. Alterations in surface morphology and roughness have been used to inuence cell and tissue response. Classically, to improve bone tissue integration on implant surfaces, various techniques have been used to increase the roughness of the implant surfaces [242244]. Many in vivo studies have compared the efciency of various surface treatments in mechanically and morphologically improving bone tissue integration of implants. Various results have been obtained, depending on the roughness amplitude but also on the method used to produce the surface roughness [242246]. Due to the rapid and specic modication of organic and inorganic materials, laser surface processing has aroused growing interest and been proven to be a controllable and exible technique for modifying the surface properties of materials. Yet little work has been carried out to investigate employing lasers to modify the surface properties of biomaterials in order to improve their biocompatibility. Having said that, it is recognised within the currently published work that laser irradiation of material surfaces can affect changes in cell adhesion on biomaterials. Lately, several publications have investigated the modication of biocompatibility of biomaterials surfaces following laser irradiation. A CO2 pulsed laser were used to graft a polymer [17] and a rubber [18]. The results showed a marked reduction of the platelet adhesion and aggregation for the modied polymer surface and cell attachment with a greater degree of spreading and attening on the unmodied rubber surface. Dadsetan et al. [19] found that L929 broblast cells attached and proliferated extensively on CO2 and KrF laser treated PET lms in comparison with unmodied PET, with surface morphology and wettability being found to affect cell adhesion and spreading. More recently, Hao and Lawrence found that the laser generated surface properties on magnesia partially stabilised zirconia (MgOPSZ) resulted in bone apatite formation in
Sample Preparation
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stimulated body uid [23], favourable albumin [24] and bronectin adsorption [25] and better human broblast response [247] and human osteoblast cell adhesion [248] and functions [26]. However, no work has so far investigated the use of lasers to alter the biocompatibility of the biograde metals. With the aim of improving the biocompatibility (bioactivity and biointegration) of a titanium alloy (Ti6Al4V), a CO2 laser was used to generate the favourable surface properties of the material for better biological response. The bioactivities of untreated and CO2 laser treated Ti6Al4V alloy were evaluated by observing the bone-like apatite formation on their surface after soaking in SBF, because for an articial material to bond to living bone, it is essential that the material has the ability to form a biologically active, bone-like, apatite layer on its surface in the human body. The biointegrations of the untreated and CO2 laser treated Ti6Al 4V alloy were assessed by protein adsorption and osteoblast cell response. Protein adsorption is the almost immediate event that occurs upon implantation of metals and mediates subsequent cell response and tissueimplant interactions [9]. In addition, it is widely acknowledged that a major determinant of the bonebiomaterial interfacial response is the initial attachment, spreading and growth of osteoblasts on the implant surface and that improvements in these processes may lead to faster and more extensive implant integration and higher long-term stability [171]. Indeed, the investigation of apatite formation in SBF was applied by Gil et al. [238] and Wang et al. [249] to evaluate the bioactivity of titanium alloys. In vitro tests of protein adsorption and osteoblast cell interactions are now well established to assess the biocompatibility of biomaterials and are widely used to evaluate the osseointegration of titanium alloys [171, 221]. In addition, the better performance of apatite formation on titanium alloy in SBF in vitro was conrmed by the benecial effect on the osteoconduction in vivo experiment conducted by Lu et al. [250]. Of great signicance is the fact that in vitro cell culture models have the potential to help elucidate events at the bone implant interface (reviewed by Davies [251]), by providing morphological, biochemical and molecular information regarding osteoblastic development and synthesis of the matrix at the interface with various biomaterials.
7.2 Sample Preparation Full details of the properties of the Ti6Al4V alloy used for in vitro evaluation of the potential of CO2 laser surface treatment to enhance bioactivity are to be found in Section 6.2.1. The Ti6Al4V alloy was supplied as a round bar with a diameter of 28.5 mm. To prepare the Ti6Al4V alloy for the in vitro tests, it was sectioned to produce 30 samples
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(discs) each of 3 mm thickness with a cutting machine (Miniton; Struers, GmbH) using a diamond-rimmed cutting blade. As in Chapter 6, the Ti6Al4V alloy samples were used as received prior to CO2 laser treatment. The 30 discs were then divided into two groups of 15 samples, with the groups being: untreated and CO2 laser treated. The procedure adopted for the CO2 laser processing of the Ti6Al4V alloy is described in Section 6.2.2. In this instance, however, only CO2 laser power densities of 1.3 and 1.6 kW/ cm2 were used, since the ndings of Chapter 6 showed these two CO2 laser power densities were the most effective. Untreated samples were used as the control for the in vitro apatite formation and protein adsorption tests. Mechanically roughened samples were used as a control for the osteoblast cell culture as the aim in this test was to compare the effect of CO2 laser treatment with the traditional method employed to improve cell adhesion (surface roughening by mechanical means). Details of the mechanical roughening procedure are given in Section 6.2.1.
7.3 Bone-Like Apatite Formation on Titanium Alloys In a different model, the mechanism of apatite formation on an amorphous sodium titanate formed on titanium metal was also examined by XPS. In the SBF, the sodium titanate releases Na ions via exchange with the H3O ions in the uid to form TiOH groups on its surface. The TiOH groups formed immediately combine with Ca2 ions in the uid to form amorphous calcium titanate. This calcium titanate later combines with phosphate ions in the uid to form amorphous calcium phosphate with a low Ca:P ratio. The calcium phosphate transforms into apatite, which exhibits a Ca:P ratio of 1.65, and contains a small concentration of Mg and Na, similar to bone mineral [41]. To reveal the reasons why this complex process is required for apatite formation, the zeta potential of the surface of sodium titanate was measured by laser electrophoresis at various SBF soaking times. It was found that the surface of the sodium titanate was highly negatively charged immediately after it was soaked in the SBF. The surface potential increased with increasing soaking time up to a maximum positive value. Thereafter, it decreased with increasing soaking time, reached a negative value again and nally converged to a constant negative value. On the basis of this nding, the complex process of apatite formation described above is well interpreted in terms of the electrostatic interaction of the functional groups with the ions in the uid. The TiOH groups formed on the surface of sodium titanate after soaking in SBF are negatively charged and, hence, combine selectively with the positively charged Ca2 ions in the uid to form calcium titanate. As the calcium ions accumulate on the surface, the surface gradually gains an overall positive charge. As a result, the positively charged surface
Bone-Like Apatite Formation on Titanium Alloys
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combines with negatively charged phosphate ions to form amorphous calcium phosphate. This calcium phosphate spontaneously transforms into the apatite, because the apatite is the stable phase in the body environment [41]. 7.3.1 Experimental Procedures Soaking in Simulated Body Fluid The untreated and CO2 laser treated Ti6Al4V alloy samples were soaked in an acellular SBF [41] having an ion concentration nearly equal to that of human blood plasma. This solution was prepared by dissolution of highpurity reagents in distilled water and was buffered at 7.25 with 50 mM trishydroxymethyl amino ethane and 45 mM hydrochloric acid. The untreated and CO2 laser treated Ti6Al4V alloy samples with various power densities were immersed in 30 ml of SBF in a polyethylene bottle at 37 C, without stirring. After 14 days they were removed from the solution, gently washed in distilled water and dried at room temperature. The soaked samples were then characterised by SEM and EDX. The samples for SEM observations were simply dried and covered by a thin gold layer to guarantee the conductivity. 7.3.2 The Effects of CO2 Laser Treatment on the Ti6Al4V in Simulated Body Fluid Figure 7.1 shows the morphologies of the untreated and CO2 laser treated Ti6Al4V alloys after 7 days of immersion in SBF. On the untreated sample, very small precipitants could be observed and no apatite was found (see Figure 7.1(a)). While on the CO2 laser treated Ti6Al4V alloys, some apatite nuclei were observed in Figures 7.1(b) and (c), indicating that laser processing brought about the bioactivity to the nonbioactive Ti6Al4V alloy. The EDX analysis revealed an average Ca:P ratio of 1.57 in these apatite nuclei. Hydroxide ions (OH) attach to metal cations. It has been reported that the hydrated surface of titanium contains more hydroxyl groups, thus improving the bioactivity of titanium [249]. The titanium alloy oxide surface in contact with water is believed to be highly hydroxylated [211]. It is demonstrated that the laser surface processing brought about the oxidation of the Ti6Al4V alloy as the surface oxygen content of the laser modied Ti6Al 4V alloy is much higher than the untreated sample. Feng et al. [193] found that during heat treatment, oxygen and water molecules in atmosphere reacted with the surfaces, resulting in an increase in the amount of OH groups as well as thickening of the oxide lms. The OH groups originated from chemisorption. Since chemisorption belongs to the molecular layer,
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Figure 7.1 SEM images of the Ti6Al4V alloy soaked in SBF for 7 days: (a) untreated, (b) CO2 laser treated at a power density of 1.3 kW/cm2 and (c) CO2 laser treated at a power density of 1.6 kW/cm2
when active sites on titanium surfaces that could induce chemisorption of water were saturated, the amount of hydroxyl groups would tend to be constant. Oxide-covered titanium is simple in terms of composition, and the possible species that may be released are H or OH ions and the ions related to titanium. Healy and Ducheyne [252] have suggested that when in SBF titanium was subjected to passive dissolution and, within a soaking period of up to 400 hours, the passive dissolution was governed by the hydrolysis of the titanium oxide and the equilibration of the surface with the 4n . The OH SBF, which resulted in the formation of OH and Ti(OH)n ions were adsorbed on the oxide surface, while the titanium hydroxide ions entered into the SBF. In the present specimen set-up, these ions could be accumulated inside the conned space between the two contact surfaces, where the SBF was much more stagnant. Obviously, the accumulation of OH ions on the surface would lead to a more negatively charged surface that was believed necessary for apatite nucleation [253].
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7.4 Protein Adsorption The molecules involved in cell adhesion and spreading include extracellular matrix molecules, transmembrane receptors and intracellular cytoskeletal components. Among the extracellular matrix proteins shown to mediate cell attachment to substrates, bronectin is protein found in many extracellular matrices and in blood plasma and serves as an attachment molecule between the substrate and cell membrane of anchorage-dependent cells. It is known that the ligand bronectin connects to the cell membrane via integrin receptors. The activation of integrins triggers cytoplasmic reactions, and thereby stimulates the intracellular signalling pathway and subsequently the cellular functions such as proliferation and differentiation [169]. On the other hand, human albumin is a nonadhesive protein for osteoblasts [194]. Albumin is the major protein component of serum and dominates the adsorption of phenomena on medical implants in the rst stage of contact with body uids. Human serum albumin or bovine serum albumin (BSA) coatings are often used as a passivating agent to prevent the adhesion of cells and thrombus formation [195]. 7.4.1 Experimental Procedures Protein Adsorption The proteins used for this study were human serum albumin and human plasma bronectin (Calbiochem, Inc.). Prior to the adsorption of 1 mg/ml of albumin in phosphate buffered salines (PBS), Ti6Al4V samples were rinsed with deionised water. The individual samples were transferred into a 24-well tissue culture plate. Thereafter, 2.5 ml of prepared albumin solution was added into each well. Adsorption proceeded for 1 hour in an incubator at 37 C. After adsorption was complete, the samples were dried with N2 and immediately transferred to an ellipsometer for measurement of the adsorbed protein layer. The above procedure was repeated with a 0.2 mg/ml concentration of bronectin in PBS. Ellipsometric Measurement The thickness of the adsorbed human serum albumin and human plasma bronectin were measured using an automatic ellipsometer equipped with a 632.8 nm heliumneon laser (L2W16SF.544; Gaertner, Inc.). The thickness and refractive indices of protein lms were determined using an ellip someter computer program with an accuracy of 3 A. Four ellipsometer measurements at different locations on each sample were taken and the average value was calculated.
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Statistics Statistical analysis was performed with an SPSS v.12 software package (SPSS/PC, Inc.) in the same manner as described in Section 5.4.1. 7.4.2 Albumin and Fibronectin Adsorption on CO2 Laser Treated Titanium Alloy The thickness of the adsorbed human serum albumin and human plasma bronectin layer, which indicate the amount of the adsorbed protein on the untreated and CO2 laser treated Ti6Al4V alloy, are shown in the Figure 7.2. It was found that the thickness of the albumin layer on the untreated Ti6Al4V is higher than on the CO2 laser modied sample, whereas the thickness of the bronectin layer is less on the untreated one than on the modied sample, as shown in Figure 7.2. The CO2 laser power density applied in the experiment is negatively correlated with amounts of albumin, while positively correlated with the bronectin (see Figure 7.2). On the one hand, the CO2 surface treatment promoted the adsorption of the bronectin and the amount of the adsorbed bronectin on the Ti6Al4V was positive
800 Fibronectin 700 Thickness of Adsorbed Protein Layer () * 600 * 500 * * Albumin
400
300
Figure 7.2 Thickness of the adsorbed bronectin and albumin protein layer on the untreated and CO2 laser treated Ti6Al4V alloys (treated with different power densities). For bronectin adsorption, there was a signicant statistical difference in thickness between the untreated Ti6Al4V alloy and the sample CO2 laser treated at 1.6 kW/cm2, and no statistical difference between the untreated Ti6Al4V alloy and the CO2 laser treated sample at 1.3 kW/cm2. For albumin adsorption, there was a signicant statistical difference in thickness between the untreated Ti6Al4V alloy and samples CO2 laser treated at 1.3 and 1.6 kW/cm2 and no statistical difference among the samples CO2 laser treated at 1.3 and 1.6 kW/cm2 ( p < 0:05)
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with the CO2 laser power density; on the other hand, it inhibited albumin adsorption and the amount of the adsorbed albumin was negative with CO2 laser power density. In addition, the statistical analysis reveals that the thickness of absorbed bronectin on the untreated sample was signicantly less than on the sample CO2 laser treated at the power density of 1.6 kW/ cm2, but not signicantly less than on the sample CO2 laser treated at the power density of 1.3 kW/cm2 (p < 0:01); on the other hand, the thickness of the absorbed albumin layer on the untreated Ti6Al4V alloy was signicantly higher than that on the CO2 laser modied samples, as shown in Figure 7.2. As stated in Section 5.4.2, the previous study shows that protein adsorption is inuenced by the surface topography [198] and the surface chemistry (wettability characteristics) [199]. The Effects of Surface Roughness As discussed previously in Section 6.3.2, the change in surface roughness of the Ti6Al4V following CO2 laser irradiation is very slight, increasing by only 3 mm. Further, there is no obvious relationship between the surface roughness and protein adsorption due to the fact that the CO2 laser treated samples have more or less surface roughness than the untreated samples, but both treated samples have a similar trend for an increase in bronectin adsorption or a decrease in albumin adsorption. It is therefore reasonable to postulate that the surface roughness is not a factor of importance when it comes to inuencing the adsorption of these proteins. The Effects of Wettability Characteristics As one can see from Figure 7.3, as the wettability characteristics of the Ti6Al4V increased, the adsorbed amounts of albumin decreased. The results of the albumin adsorption are consistent with the previous nding that the increase in surface hydrophilicity of Ti results in lower albumin adsorption [201], showing that the wettability characteristics of the Ti6Al 4V could be an active factor in albumin adsorption, much better than the conformation of bronectin adsorbed on hydrophobic surfaces [204]. The results of the adsorption of bronectin given in Figure 7.3 show that it increased with the increasingly wettable characteristics (hydrophilic) of the Ti6Al4V alloy surface, implying that wettability characteristics of the Ti 6Al4V alloy provide the predominant mechanism governing bronectin adsorption. In addition, the statistical analysis revealed that the bronectin adsorption on the sample with a cos y value of 0.07 was signicantly less than on the sample with a cos y value of 0.34 and was not signicantly less on the sample with cos y of 0.27, the albumin adsorption on the sample with a cos y value of 0.07 was signicantly higher than on the samples with cos y values of 0.27 and 0.34 and no signicant difference in albumin adsorption
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700
Fibronectin Albumin
600
500
400
300 0.0 0.1 0.2 0.3 0.4 Wettability, Cos (Glycerol)
Figure 7.3 The relationship between the thickness of the adsorbed albumin and bronectin layer and wettability characteristics (cos y) of the Ti6Al4V alloy. For bronectin adsorption, there was a signicant statistical difference in thickness between the sample with cos y of 0.07 and the sample with cos y of 0.34, and no statistical difference between the sample with cos y of 0.27 and the sample with cos y of 0.34. For albumin adsorption, there was a signicant statistical difference in thickness between the sample with cos y of 0.07 and the samples with cos y of 0.27 and 0.34, and no statistical difference between the sample with cos y of 0.27 and the sample with cos y of 0.34 ( p < 0:05)
was observed between the sample with a cos y value of 0.27 and the sample with a cos y value of 0.34. Indeed, a previous study by Hao and Lawrence [25] on albumin adsorption also revealed that albumin absorption decreased on the MgOPSZ following CO2 laser treatment due to the increased wettability characteristics. Moreover, a previous investigation [204] on the extent of bronectin adsorption as compared to its biological activity on hydrophobic and hydrophilic surfaces suggested the possibility that bronectin was adsorbed in two different conformations when incubated with the surfaces at low concentrations, with the more active conformation on the hydrophilic surfaces. The results showed that the antiplasma bronectin antibody appeared to bind to the conformation of bronectin adsorbed on hydrophilic surfaces. p As discussed previously in Section 6.5, the considerable change in gsv as opposed to the minor difference in gd was a driver for the improvement in sv the wettability characteristics of the Ti6Al4V alloy after CO2 laser irradiation. The observed signicant correlation between wettability characteristics
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and protein adsorption implies that albumin and bronectin adsorption on the Ti6Al4V alloy surfaces was probably due to the polar and chemical interactions [205]. This nding is similar to a previous investigation that p albumin and bronectin adsorption is mainly inuenced by the change in gsv of the MgOPSZ following CO2 laser irradiation [25]. It is therefore possible p to maintain that the change in gsv of the Ti6Al4V alloy generated by the CO2 laser irradiation contributes to the inhabitation of albumin adsorption and enhancement of bronectin adsorption, and in turn its potential for a favourable bone cell response on the Ti6Al4V alloy surface.
7.5 Osteoblast Cell Adhesion The development of boneimplant interfaces depends on the direct interactions of bone matrix and osteoblasts with the biomaterial. There is a substantial body of literature based on the premise that improved initial attachment of osteoblasts or osteoblast precursor cells to orthopaedic implant surfaces may lead to improved bone integration of the implant and longer-term stability [206]. Osteoblast adhesion is a prerequisite for bonebiomaterial interaction and depends on the surface aspect of materials. A cell in contact with a material surface will rstly attach, adhere and then spread. The quality of this adhesion will inuence their morphology and their future capacity for proliferation and differentiation. The attachment of anchorage-dependent cells such as osteoblasts to biomaterial surfaces is a complex process involving cell attachment and spreading [207], focal adhesion formation, and extracellular matrix formation and reorganisation [208]. 7.5.1 Experimental Procedure Cell Culture The human osteoblastic cell line hFOB 1.19 was obtained from the American Type Culture Collection (ATCC) (Manassas, Inc.). These cells were cultured in a medium containing a 1:1 mixture of Dulbeccos Modied Eagles medium without phenol red and Hams F12 medium with 2.5 mM L-glutamine (D-MEM/F-12 Medium), supplemented with 10 % fetal bovine serum (ATCC) and 0.3 mg/ml G418 (Calbiochem, Inc.) in a 37 C, humidied, 5 % CO2/95 % air incubator. Cell Cytotoxicity Cytotoxicity tests consisted of the quantication of the activity of lactate dehydrogenase (LDH) in a culture medium of cells in contact with the samples. The activity of the LDH enzyme rises when cells are damaged: the
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LDH activity induced by the untreated and selected CO2 laser treated specimens (0.9 and 1.6 kW/cm2) in triplicate were compared to that induced by a toxic agent (Triton X100 0.05 % in PBS) and to that induced by a culture polystyrene plate (NUNC, Inc.). The cell culture plate was used as a negative control and a Triton toxic agent as a positive control. Cell Adhesion and Morphology For cell adhesion analysis, osteoblasts were enzymatically lifted from polystyrene tissue culture asks until cell conuence using 1 ml Trypsin-EDTA (0.25 % Trypsin/0.53 mM EDTA solution) before suspension in the culture medium. The Ti6Al4V alloy samples were placed in a 24-well tissue culture polystyrene plate (Falcon, BP) under a sterile environment and sterilized in 70 % ethanol for 24 hours. The samples were rinsed by PBS and then were seeded with cell suspension. To analyse the cell attachment and morphology, the untreated and laser treated specimens were seeded with a 0.5 ml cell suspension of 1 105 cell/ml for 24 hours of cell culture, then dehydrated in a graded ethanol series, critical point dried with CO2 and gold coated for SEM analysis. Cell Proliferation Cell proliferation on each specimen was measured by MTT assay. The osteoblast cells cultured for 7 days on each specimen were gently washed with PBS and were measured by MTT assay using 3-(4,5-dimethyl-thiazole2-yl)-2, 5-diphenyl tetrazolium bromide (MTT; Sigma, Inc.). The MTT solution was added to each specimen and the cells were incubated for 4 hours at 37 C, before replacing the medium with dimethylsulfoxide. Absorbance of the solution was measured by an instrument plate reader (EL312; Bio-Tek, Inc.) at 490 nm. Statistics Statistical analysis was performed with an SPSS v.12 software package (SPSS/PC, Inc.) in the same manner as described in Section 5.4.1. 7.5.2 Osteoblast Cell Response on CO2 Laser Treated Titanium Alloy Cell Cytotoxicity The cell culture plate was used as a negative control and a Triton toxic agent as a positive control for the assessment of cell membrane damage expressed as LDH activity. LDH activity in the culture media obtained from cells cultured on all the tested materials was found to be not signicantly

60
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50 LDH Activity (U/L)
40 *
* * *
30
20
10
0
+ve
ve
Untreated
Mech. Rough
CO2 laser CO2 laser 1.3 kW/cm2 1.6 kW/cm2
Figure 7.4 LDH activity on the positive and negative controls, untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloys. There was a signicant statistical difference between the positive control and the untreated, mechanically roughened and CO2 laser treated samples, and no statistical difference between the negative control and the untreated and CO2 laser treated Ti6Al4V alloy samples ( p < 0:05)
different from the negative control, as shown in Figure 7.4, indicating that untreated and CO2 laser treated Ti6Al4V were not cytotoxic. Cell Attachment Generally, cells in contact with a materials surface will rstly attach, then adhere and nally spread. From Figure 7.5, it is quite clear that the adhesion and spreading of osteoblast cells was inuenced by the CO2 laser treatment. The cells on the untreated and mechanical roughened surface present the initial stage of the adhesion, with individual cells covering a small surface area and not spreading; on the other hand, the cells on the CO2 laser treatment show a good state of adhesion and attening to cover more surface area. This means that the cells showed a better adhesion on the CO2 laser treated sample than the untreated and mechanical roughed samples, suggesting that the surface properties generated by the CO2 laser treatment were more favourable for the osteoblast cell adhesion. In addition, the number of cells on the untreated Ti6Al4V alloy is nearly the same as on the mechanical roughened material, but is much lower than on the CO2 laser treated Ti6Al4V alloy (for both power densities). Hence, the CO2 laser
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Figure 7.5 SEM images of osteoblast cells on the Ti6Al4V alloy when (a) untreated, (b) mechanically roughened and CO2 laser treated (c) at 1.3 kW/cm2 and (d) at 1.6 kW/cm2
treatment has a signicant effect on the cell adhesion and growth, and plays a more important role than when the surface is roughened mechanically. Cell Proliferation The MTT results in Figure 7.6 reveal the cell proliferation on the surface of untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy specimens for 7 days. It can be seen that the CO2 laser treated samples had a signicantly higher cell proliferation compared to the untreated sample and mechanically roughened sample. A slight improvement in cell proliferation was observed on the mechanically roughened sample compared to the untreated sample. It is observed that the cell proliferation generally increases as power density of the CO2 laser treatment increases; however, the

0.18
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0.15 MTT Optical Density
0.12 * *
0.09
0.06
0.03
0.00 Untreated Mechanically Roughened CO2 laser 1.3 kW/cm2 CO2 laser 1.6 kW/cm2
Figure 7.6 MTT optical density of osteoblast cells grown on untreated and CO2 laser treated Ti6Al4V alloys after 7 days of cell culture. There was a signicant statistical difference between the untreated sample and the samples CO2 laser treated at power densities of 1.3 and 1.6 kW/cm2, and no statistical difference among the untreated and the mechanically roughened samples ( p < 0:05)
statistical analysis shows that there are no signicant differences in cell proliferation between the samples CO2 laser treated at the power densities of 1.3 and 1.6 kW/cm2. In addition, the statistical analysis reveals that cell proliferation signicantly improved for the CO2 laser treated samples compared with that for the untreated sample, but was not signicantly increased for the mechanically roughened sample. The results apparently revealed that the surface generated by CO2 laser treatment is more favourable for cell proliferation than the untreated and mechanical roughened surfaces. 7.5.3 The Effect of CO2 Laser Treatment on the Osteoblast Cell Response As discussed in Chapter 6, the CO2 laser surface treatment of the Ti6Al4V alloy was shown to result in alteration of the surface topography, oxide chemistry, wettability characteristics as well as albumin and bronectin protein adsorption. The changes in these surface properties certainly inuenced the osteoblast cell response. It is demonstrated that CO2 laser surface processing brought about not only better cell adhesion but also higher cell proliferation to the treated Ti6Al4V alloy compared with the untreated and mechanically roughened samples, indicating that this surface processing
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technique is effective and better than the traditional mechanical roughing method. In this study, the osteoblast cell response increases with the power density of CO2 laser treatment. Such ndings may further understanding of the particular surface properties that are most closely related to osteoblast cell attachment and function. For this reason it is suggested that either the surface modications investigated here or knowledge gained from their study may be used to help improve the biocompatibility and osteoinductive properties of titanium based implants. The Effect of Topography on the Osteoblast Cell Response In this study there was no distinguishable difference in the cell adhesion between the untreated and mechanically roughened samples. In addition, statistical analysis shows that the cell proliferation on the mechanically roughened sample did not signicantly increase compared to the untreated samples. On the other hand, the sample CO2 laser treated at the power density of 1.6 kW/cm2, which had the lowest surface roughness as demonstrated in Chapter 6, exhibits better cell adhesion and a signicant increase in cell proliferation compared to the mechanically roughened sample, which had the highest surface roughness. This in turn suggests that the stimulatory effect of surface properties followed by CO2 laser irradiation on osteoblast cell attachment is most likely to be attributable to changes in surface properties other than roughness alone. Furthermore, since the CO2 laser induced change in surface roughness is of such small magnitude compared to the accompanying changes in oxygen chemical and wettability characteristics, the change in surface roughness is also unlikely to explain the observed effects of CO2 laser treatment on osteoblast cell adhesion and proliferation. Indeed, the work by Macdonald et al. [254] examining a thermally modied Ti6Al4V alloy revealed that the surface topography was less important for determining protein and cell attachment than surface chemistry. The Effect of Wettability Characteristics on the Osteoblast Cell Response From the foregoing results it is clear that the CO2 laser treatment of the Ti6Al4V alloy enhanced the wettability characteristics of the material and that following CO2 laser treatment the Ti6Al4V alloy presented improved bioactivity in terms of osteoblast cell adhesion and proliferation. It is not possible, however, to conclude from these ndings that the increased osteoblast cell bioactivity of the Ti6Al4V alloy is correlated to the improvement of the wettability characteristics of the material. The reason that it is not possible to claim the existence of a correlation between wettability and osteoblast cell bioactivity for the CO2 laser treated Ti6Al4V
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Table 7.1 Wettability characteristics and MTT optical density of osteoblast cells grown on the untreated, mechanically roughened and CO2 laser treated Ti6Al4V alloy after 7 days of cell culture Wettability characteristics y (glycerol) Roughness Oxygen p (deg) (mm) gsv (mJ/m2) (at %) MTT 85.8 78.7 74.4 70.1 0.35 0.46 0.39 0.41 4.85 5.10 9.43 10.11 23.32 23.35 41.80 48.78 0.073 0.080 0.115 0.130
Ti6Al4V alloy Untreated Mechanically roughened CO2 laser treated (1.3 kW/cm2) CO2 laser treated (1.6 kW/cm2)
alloy is that the CO2 laser treatment resulted in an increase in the surface roughness of the alloy. Because surface roughness is known to inuence osteoblast cell adhesion and proliferation, it is possible that the surface roughness increase alone could be responsible for the increased osteoblast cell bioactivity. Therefore, for any correlation between wettability characteristics and osteoblast cell bioactivity for the CO2 laser treated Ti6Al4V alloy to be conrmed or refuted, attention needs to be paid to the effects of increasing only the surface roughness of the alloy. This was done by including the mechanically roughened Ti6Al4V alloy sample in the analysis. A comparison of the MTT values for the untreated and mechanically roughened Ti6Al4V alloy samples given in Table 7.1 conrms that surface roughness does indeed affect osteoblast cell adhesion. As one can see from Table 7.1, the MTT value on the Ti6Al4V alloy increased markedly from 0.073 to 0.080 when the surface roughness was increased by mechanical roughening from 0.35 to 0.46 mm. In addition, y was observed to decrease from 85.8 to 78.7 , which is perhaps not surprising as surface roughness is known to be a factor inuential in determining y, as demonstrated by Equation (4.1). Signicantly, Table 7.1 shows that the surface oxygen content p and gsv of the sample subjected to mechanical roughening remained virtually the same as the untreated sample, thus indicating that the decrease in y and the increase in surface roughness alone caused the MTT value to increase. The MTT value for the sample CO2 laser treated at 1.3 kW/cm2 was considerably larger than that of the mechanically roughened sample, 0.130 compared to 0.080, despite the similar values of surface roughness for the p two samples; however, the surface oxygen content and gsv of the sample CO2 2 laser treated at 1.3 kW/cm were much higher. The value of y was also found p to be smaller by 4.3 . Since the surface oxygen content and gsv , along with surface roughness, have been shown to play a role in governing the wettability characteristics of the Ti6Al4V alloy (see Chapter 6), it is
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to be expected that the CO2 laser treated sample displays the lowest value of y. Owing to the extensive body of literature attesting to the efcacy of surface roughness in enhancing biocompatibility, it is clear that the increase in surface roughness of the Ti6Al4V alloy following CO2 laser treatment was responsible, in a large part, for the observed improvement in osteoblast cell bioactivity. The wetting of a solid surface can, nevertheless, be a predictive index of the biocompatibility of materials involved. As such, improvements in the wettability characteristics of the Ti6Al4V alloy would most possibly result in better biocompatibility. Indeed, a reduction in y contributes to the enhancement in Wad of SBF and SBFBSA on the Ti6Al4V alloy following CO2 laser treatment. As both SBF and SBFBSA have close chemical compositions to human body uids, the improvements to Wad of the Ti6Al4V alloy surface towards these uids would mean better suitability of the Ti6Al4V alloy as a biomaterial after CO2 laser treatment. Also, Hallab et al. [215] demonstrated that surface free energy was a more important surface characteristic than surface roughness for cellular adhesion strength and proliferation. Schakenraad et al. [115] found that, despite the great number of parameters interfering with cellular adhesion and spreading, the solid surface energy is apparently a dominated factor in cellular attachment to a polymer surface and remains so, even if the solid surface has been covered by a protein layer. Moreover, as discussed previously, improvement of the wettability characteristics resulted in a decrease of albumin adsorption and an increase of bronectin. The phenomena would be benecial to cell adhesion since albumin is a nonadhesive protein and bronectin is an adhesion protein. A previous study by Hao and Lawrence [247, 248] also shows that enhancement of the wettability characteristics of MgOPSZ after CO2 laser treatment resulted in a better response of the human broblast cell and human osteoblast cell. The surface oxygen content of the Ti6Al4V alloy following CO2 laser irradiation is much higher than the untreated sample. The improved performance of cell adhesion and proliferation is very likely to be due to the augmentation of the surface oxygen content. It has been found that the biocompatibility of titanium implants is associated with the oxide on its surface [58]. The surface oxide layer on titanium is biocompatible and capable of interacting with surrounding biological uids and cells when implanted in situ [58, 255]. This layer, composed primarily of TiO2, is found supercially on both cp Ti and Ti6Al 4V alloy [58, 256]. Based on the results of this analysis it appears that the aspects of p wettability characteristics, surface oxygen content and gsv , play an important role in promoting cell proliferation, particularly when surface roughness is simultaneously increased. Moreover, it would be reasonable to postulate that a correlation exists between the CO2 laser induced wettability
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characteristics of the Ti6Al4V and osteoblast cell bioactivity. Further support for this postulation comes when one considers the results given in Table 7.1 for the Ti6Al4V alloy sample that was CO2 laser treated at 1.6 kW/cm2. In this instance the CO2 laser treatment gave rise to the highest MTT value, 0.130. At the same time, in comparison with the sample CO2 p laser treated at 1.3 kW/cm2, the surface oxygen content and gsv increased appreciably, while the surface roughness increased slightly. These increases in the factors known to affect wettability led in turn to a decrease in y. Since the surface roughness of the sample CO2 laser treated at 1.6 kW/cm2 was 0.04 mm less than that of the mechanically roughened sample, it is clear that the better wettability characteristics of the CO2 laser treated sample were responsible for the improved MTT value. It is also evident that CO2 laser treatment could be a more effective way to improve osteoblast cell adhesion than the traditional methods currently available, especially mechanical roughening.
7.6 Predictions for Implantation in an in vivo Clinical Situation The orthopaedic and dental implant industry is based entirely on favourable interaction at the bonetitanium interface (osseointegration). What is more, it is clear that osseointegration is a property of titanium implant surfaces. The surface modication of titanium implants is an active area of research for two reasons. The rst reason is to increase the rate of successful implantation from satisfactory, as it is today, to excellent. The second is to induce acceleration of normal bone healing phenomena as this would allow early immediate loading of the implant, which would have signicant implications in terms of decreased patient morbidity, patient physicology and health care costs. According to Puleo and Nanci [9], there are basically three different approaches to the surface modication of bone-contacting titanium implants: physicochemical methods, morphological methods and biochemical methods. Of the three, morphological methods are the most widely applied. Whichever approach is adopted, cellular behaviour, namely adhesion, morphologic change, functional alteration, proliferation and differentiation, is greatly affected by surface properties, such as roughness, chemical composition, wettability and morphology of the oxide on the titanium. A rapid osseointegration is associated with improved secondary stability and, in turn, with a favourable prognosis for long-term success of the implant. To this end, initial stability has to be achieved through the reduction of micromotion so as to allow early brin adhesion, blood vessel growth and eventually new bone formatin [257259]. If micromotion cannot be reduced then, as Branemark [1] and Brunski [260] found, a brous tissue
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instead of a bony interface will result at the implant surface. In order to reduce micromotion initially, and improve osseointegration thereafter, many variations of surface geometry have been developed [261263], for it is well known that surface geometry governs the interactions of proteins and cells with the implant surface [66, 264]. Moreover, many workers [259, 265268] have demonstrated that increased surface roughness is associated with better cell adherence, higher boneimplant contact (BIC) and improved biomechanical interaction. Using implants fabricated from the Ti6Al4V alloy, Gotz et al. [269] conducted a detailed study of the effects of various laser induced surface nishes on osseointegration. The Ti6Al4V alloy implants were lasertextured with an Nd:YAG laser treated to generate pores of various sizes, while corundum-blasted (CB) Ti6Al4V alloy implants were used as controls. An in vivo evaluation of the control and laser-textured implants was conducted using a rabbit transcortical implantation model. Initially, the surface between the pores was polished after laser-texturing, which was found to result in a BIC lower than that of the CB control implants. However, when the laser-textured surfaces were blasted with 500 to 710 mm of Al2O3 grit to generate a rougher surface between the pores, the osseointegration was markedly improved, with the BIC being higher than that of the CB control implants. This observation suggests that the biological mechanisms involved in the bone ingrowth on the laser-textured and surface-blasted Ti 6Al4V alloy implants preferentially used the pores to initially anchor and implant within the newly formed bone. The roughened surface due to the Al2O3 grit blasting came into play in the latter stages of osseointegration when bone growth spread on to the implant surface. The effect of surface topography, in particular surface roughness, on osseointegration with cp Ti was investigated by Li et al. [270]. The study took the form of a biomechanical comparison of cp Ti cylindrical solid-screw dental implants with surfaces roughened by two procedures. The two procedures formed two testing groups, with the rst group being subjected to acid etching (sulfuric acidhydrochloric acid) and termed MA, while the second group was sandblasted (large grit of 250500 mm) and then acid etched (sulfuric acidhydrochloric acid) and was termed SLA. Samples in each of the two groups exhibited similar topographical features. In each case the acid etching generated micropits of around 2 mm in diameter. For the SLA samples, however, the micropits were accommodated within the macrorough texture produced by the sandblasting, whereas for the MA samples the micropits were superimposed on a at machined surface. These differing surface topographies naturally resulted in different values of surface roughness: an Ra of 1.57 mm for the MA samples and an Ra of 2.18 mm for the SLA samples. The efcacy of each surface in terms of interfacial shear strength was assessed using an established animal model for
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implant removal torque testing by means of a split-mouth experimental design. The ndings revealed that the SLA surface displayed enhanced interfacial shear strength in comparison with the MA surface, with the removal torque value being 30 % higher. Also, the SLA surfaces achieved a better bone anchorage than the MA surfaces owing to the fact that the sandblasting carried out before the acid etching generated a rougher surface. The biocompatibility of titanium and its alloys as an implant material is attributed to surface oxides spontaneously formed in air and/or physiological liquids. The natural oxide that exists on the surface of titanium and titanium alloys is thin, approximately 38 nm in thickness, and is amorphous, as well as being stoichiometrically defective. It is known that the protective and stable oxides on titanium surfaces are able to provide favourable osseointegration. Studies, both in vitro and in vivo, by Sul et al. [271] have revealed that alterations in the surface oxide on cp Ti implants strongly inuence the tissue response. The nature of the surface oxides can be manipulated by thermal oxidation and/or anodic oxidation. In terms of osteoblast cell responses, Zhu et al. [272] studied the effects of the topography and composition of the surface oxides of cp Ti in vitro. The surface oxides of the cp Ti were modied in terms of composition and topography using anodic oxidation in two kinds of electrolytes: the rst being 0.2 M H3PO4 and the second being 0.03 M calcium glycerophosphate (Ca-GP) and 0.15 M calcium acetate (CA). Depending upon the type of anodic oxidation, phosphorus (ca.10 at %) or calcium (16 at %) and phosphorus (36 at %) were incorporated into the surface of the cp Ti in the form of phosphate and calcium phosphate. Using the SaOS-2 human osteoblastlike cell line, the study revealed that cell attachment and cell proliferation were enhanced by the anodic oxidation. Moreover, the cell attachment was seen to increase as the thickness of the oxide layers increased. In contrast, ALP activity was not found to be inuenced by the presence of the oxides. Yang, Ong and Tian [273] evaluated in vivo the biological response of a cp Ti surface modied by sandblasting and ion implantation of amino (NH2) using an established dog model. The study found that the porous nature of the cp Ti surface after sandblasting encouraged osseous tissues to grow into the pores and thereby allow the formation of a gradual calciumphosphate (CaP) interface layer. In addition, it was concluded that ion implantation of the cp Ti surface with the amino groups induced high concentrations of Ca and P precipitation and more mineralization as compared with the non-ionimplanted surfaces. A comprehensive in vitro and in vivo investigation of the bioactivity of uncoated and collagen-coated cp Ti was conducted by Morra et al. [274]. In this study collagen was covalently linked to the surface of cp Ti by the deposition of a thin lm from hydrocarbon plasma followed by acrylic acid
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grafting. Cell growth experiments conducted in vitro using osteoblast-like SaOS-2 cells revealed that the growth rate was lower on the collagen-coated cp Ti than on the uncoated cp Ti. No signicant difference in ALP production was detected between the collagen-coated and the uncoated cp Ti. Importantly, the in vivo study using a rabbit model in which samples were implanted into rabbit femurs revealed a signicant increase in bone growth and bone-to-implant contact in the case of the collagen-coated cp Ti implants. In addition, an in vivo study of metal implants coated with small, synthetic peptides revealed the stimulation of bone formation and supported in vitro studies showing that adhesion and spreading of osteoblasts on RGD-modied surfaces was quite substantial [275]. In a study conducted by Wu et al. [276], the bioactivity of a new kind of anodic oxidized titanium metal was investigated in vitro and in vivo. After being immersed in SBF solution for 7 days in vitro, apatite formed and covered almost all the surfaces of the anodic oxidized samples. Furthermore, after an in vivo animal experiment the apatite also precipitated on the interface of the tissue and materials after 12 weeks post-operation. Of great signicance was the observation that there were no brous capsules formed around the materials. The materials bonded with the bone very tightly and attached to the skin very closely, which would result in the achievement of the biological sealing for the bone-anchored percutaneous implants. These positive results might be attributed to the precipitated apatite layer formed on the surface of the bioactive oxidized titanium. For implants fabricated from titanium or titanium alloys, the existing literature reveals that should a surface treatment process applied to improve the bioactivity prove not to be cytotoxic in an in vitro analysis, then it is more than likely that the in vivo functioning of the implant will be improved. The treatments in current usage change only one surface characteristic, such as surface topography, surface chemistry, wettability, etc. Even so, it is evident from this section how effective these treatments are at enhancing the in vivo performance. The results presented in this chapter show that the biocompatibility of the Ti6Al4V alloy was enhanced considerably as a result of CO2 laser treatment, which changes surface topography, surface chemistry and wettability simultaneously. Thus, since the CO2 laser treatment has been shown in vitro to be an actual means of improving the bioactivity of the Ti6Al4V alloy, it would appear that the CO2 laser treatment would be effective in vivo.
7.7 Summary This study investigated the apatite formation on the untreated and CO2 laser treated Ti6Al4V alloy after soaking in simulated body uid. The protein
Summary
139
adsorption and hFOB cells were used to examine the in vitro biological response on the Ti6Al4V alloy following CO2 laser treatment. It has been demonstrated that the CO2 laser treatment could improve the bioactivity of the Ti6Al4V alloy, as some apatite nuclei were only observed on the CO2 laser modied Ti6Al4V alloy, while no apatite nuclei appeared on the untreated sample. It is believed that the CO2 laser oxided surface layer generated the hydroxide ions in the water and resulted in the nucleation of apatite. The CO2 laser treatment brought about a lower amount of the adsorbed albumin layer and a higher amount of the adsorbed bronectin layer on the modied Ti6Al4V alloy compared with the untreated sample. The albumin adsorption decreased, while the bronectin increased with the increased wettability characteristics of the material, indicating that the wettability characteristic is a predominant factor governing protein adsorpp tion. Further, the effect of gsv on protein adsorption implied that the protein adsorption on the Ti6Al4V alloy was probably due to the polar and chemical interactions. The one-day cell adhesion test showed that cells not only adhered and spread better, but also grew faster on the CO2 laser treated sample than the untreated and mechanically roughed sample. Further, the MTT cell proliferation analysis revealed that the roughed surface resulted in a slight enhancement, while CO2 laser treatment brought about the considerable increase in cell proliferation compared with the untreated sample. Despite the fact that surface roughness is certainly one of the factors inuencing cell adhesion and proliferation, the results presented herein suggest that the aspects of wettability characteristics, surface oxygen content p and gsv , play an important role in promoting cell proliferation. Indeed, it was evident that the better wettability characteristics of the CO2 laser treated Ti6Al4V alloy were responsible for the improved MTT value. Thus it would be reasonable to maintain that a correlation exists between the CO2 laser induced wettability characteristics of the Ti6Al4V and osteoblast cell bioactivity. It is also evident that the CO2 laser treatment could be a more effective way of improving osteoblast cell adhesion than the traditional methods currently available, especially mechanical roughening.
8
Enquiry into Possible Generic Effects of the CO2 Laser Treatment on Bone Implant Biomaterials
If the previously observed improvements to the surface characteristics and the biocompatibility of the MgOPSZ and the Ti6Al4V alloy after CO2 laser treatment are common across a whole range of bioinert ceramics and biometals, then the viability of CO2 laser surface treatment of biomaterials will be reinforced. This chapter aims to establish generic relationships in terms of enhanced surface properties and biocompatibility of biomaterials using CO2 laser surface treatment. Studies of the generic effects of CO2 laser treatment on the modication of wettability characteristics and biocompatibility of a yttria partially stabilised zirconia (YPSZ) bioinert ceramic and a biograde stainless steel were conducted. The ndings of these studies were correlated with one another and extended not only to other bioinert ceramics and biometals but also to ceramics and metals in general. In this way it was possible to establish the generic nature of the advantageous effects of the CO2 laser surface treatment of ceramics and metals with regard to improving wettability and biocompatibility.
8.1 Introduction Although laser surface processing has aroused growing interest and been proven to be a controllable and exible technique for modifying the surface properties of materials, the applications of lasers in surface processing of
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Enquiry into Possible Generic Effects
biomaterials are still limited. Having said that, it is recognised within the currently published work that laser irradiation of material surfaces can effect changes in the biocompatibility of biomaterials. Lately, several publications have investigated the modication of biocompatibility of biomaterial surfaces following laser irradiation. A CO2 pulsed laser was used to graft a polymer [17] and a rubber [18]. The results showed a marked reduction in platelet adhesion and aggregation for the modied polymer surface and cell attachment with a greater degree of spreading and attening on the unmodied rubber surface. Furthermore, L929 broblast cells attached and proliferated extensively on the CO2 and KrF laser treated lms [19] in comparison with the unmodied PET, with surface morphology and wettability being found to affect cell adhesion and spreading. More recently, Hao and Lawrence found that the laser generated surface properties on magnesiapartially stabilised zirconia (MgOPSZ) resulted in bone apatite formation in stimulated body uid [23], favourable albumin [24] and bronectin adsorption [25] as well as a better human broblast response [247] and human osteoblast cell adhesion [248] and functions [26]. In addition, Hao and Lawrence demonstrated that surface treatment using an HPDL brought about higher wettability and an improved stimulated physiological liquid response on a biograde 316 LS stainless steel [277] and better osteoblast cell adhesion and proliferation on a Ti6Al4V alloy [278]. With the aim being to establish the laser as a generic processing technique for improving the biocompatibility of biomaterials, CO2 laser surface processing was applied to a yttriapartially stabilised zirconia (YPSZ) bioceramic and a biograde stainless steel.
8.2 Ascertaining the Generic Effects of CO2 Laser Treatment on Bioinert Ceramics The YPSZ was introduced in the biomaterials world several years ago [279] and is frequently used in high-load-bearing sites such as articial knee and bone screws in an orthopaedic application and dental post-crown in a dental application due to their attractive mechanical properties [139]. Y-PSZ ceramics, whose minimal requirements as implants for surgery are now described by the standard ISO 13356 [3], are the materials selected by almost all manufacturers that are introducing zirconia ball heads into the market (see Figure 2.1). Fini et al. [280] tested various materials in healthy and osteopenic bone, and YPSZ performed better when implanted in the healthy bone of rats. More than 300 000 YPSZ ball heads have been implanted [4], and only two failures were reported [5] up to now. The reason for the large and rapid development of the zirconia hip joint head is a result of reduced polyethylene wear rate of the mating component and
Generic Effects of CO2 Laser Treatment on Bioinert Ceramics
143
improved mechanical properties when compared to alumina ceramics. The outstanding fracture strength and toughness of YPSZ ceramics, which are double that of surgical grade alumina, allow the manufacture of a large variety of femoral heads with a combination of various head diameters, down to 22 mm. Also, several taper designs with a high reliability are achieved. Such a high reliability is a consequence not only of the improved mechanical properties of YPSZ ceramics but also of the optimization of the manufacturing process from designing to nal quality controls. The properties of laser treated ceramic surfaces are very important in determining the effectiveness of the treatment of such materials for various applications. For the ceramics used as linings in high-temperature waste incinerators, the surface roughness and wettability inuence the interaction characteristics of the treated ceramic surfaces with the corrosive species. In general, the effects of laser treatment on the wetting characteristics of ceramics have not been fully explored yet, with only limited research work being conducted [129131]. The main factors that affect the wetting characteristics of a surface are its composition, the content of surface oxygen, the surface morphology, surface energy and the temperature [1, 23]. As for the Al2O3 based oxide ceramics, the change in surface morphology mainly affects wettability characteristics following laser surface treatment as no signicant changes in the composition and oxygen content caused laser induced melting and re-solidication [281]. Interactions of the bioceramics with biouid and cells are inuenced by their surface properties, such as surface roughness and wettability characteristics. The laser can be a highly controllable tool to modify the surface roughness and wettability characteristics of the ceramics and can be used to improve the performance of the MgOPSZ bioinert ceramics. It is reasonable to expect that the laser can be an effective surface processing technique for bioinert ceramics and can alter the interaction between the materials and the biological response. 8.2.1 Experimental Procedures The material under investigation was a 5 % yttria partially stabilised zirconia (YPSZ) sheet with dimensions of 10 50 5 mm3 (Dynamic Ceramic, Ltd). The main mechanical and thermal properties of the YPSZ used in this study are: a density of 6.05 g/cm3, a compressive strength of 2000 MPa, a Vickers hardness of 1400 kgf/mm2, a tensile modulus of 205 GPa, a specic heat at /m 25 C of 400 J/K kg and a thermal conductivity at 20 C of 2 W K. The YPSZ sheet was CO2 laser treated as received in the same manner as described in Section 4.2.2. To investigate the effects of the CO2 laser irradiation on the wetting and surface energy characteristics of the YPSZ, a set of sessile drop control experiments was carried out under the same conditions as described in Section 4.2.4.
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The human osteoblastic cell line hFOB 1.19 was obtained from the American Type Culture Collection (Manassas, Inc.). These cells were cultured using the same procedure given in Section 5.4.1. The samples and osteoblast cells were prepared and examined using SEM in the same manner as described in Section 5.4.1. The specimens were seeded with a 0.5 ml cell suspension of 4 105 cell/ml and then cultured with cell culture medium and maintained in the incubator for one week. The cell culture medium was changed every 3 days. 8.2.2 Modication of the Surfaces Properties and Wettability Characteristics of a YPSZ Bioinert Ceramic As one can see from Table 8.1, the YPSZ experienced marked reductions in y with all of the control test liquids as a result of interaction with the CO2 laser beam. The CO2 laser treatment brought about a consistently rougher surface on the YPSZ sample compared with the untreated sample. Evidence of this rougher surface can be seen in Figure 8.1, as well as the fact that the Ra value increased in a linear fashion as the power density of the laser treatment increased. The higher the power density applied, the lower the y obtained. Wenzels equation (4.1) [150] states that when the solid surface is rough, cos yw is large and yw decreases when yw < 90 . In this study, the rougher surface generated by the CO2 laser treatment at 1.8 kW/cm2 brought about a reduction in y in accordance with Equation (4.1) and previous work conducted by Uelzen and Muller [282]. An additional reduction in y was observed when the surface of the YPSZ was roughened further by CO2 laser treatment at 2.3 kW/cm2. In a similar way, Figure 8.2 reveals that the surface oxygen content of the YPSZ increased following CO2 laser treatment and increased in a linear fashion as the laser power density increased. This observed increase in the surface oxygen content of the YPSZ would have effected better wettability characteristics, since oxidation is known to increase the likelihood of wetting [13, 144147].
Table 8.1 Mean values of the contact angle formed between the untreated and CO2 laser treated YPSZ for various power densities and selected control test liquids at 25 C Contact angle, y (deg) Polyglycol Polyglycol Glycerol Formamide Etheneglycol E-200 15-200 82.4 74.2 70.5 76.2 70.9 67.7 64.4 59.2 57.0 56.8 53.2 49.8 40.2 38.0 36.9
YPSZ Untreated CO2 laser (1.8 kW/cm2) CO2 laser (2.3 kW/cm2)

0.6
145
0.5 Surface Roughness, Ra (m)
0.4
0.3
0.2
0.1
0.0 Untreated CO2 Laser 1.8 kW/cm2 CO2 Laser 2.3 kW/cm2
Figure 8.1 Relationship between the surface roughness and the CO2 laser power density of the YPSZ
80 Surface Oxygen Content (at%)
60
40
20
0 Untreated CO2 Laser 1.8 kW/cm2 CO2 Laser 2.3 kW/cm2
Figure 8.2 Relationship between the surface oxygen content and the CO2 laser power density of the YPSZ
The untreated YPSZ presented only a grooved surface, as evidenced in Figure 8.3(a), which may have been generated in the manufacturing processing. In contrast, a hexagonal microstructure appeared on the surface of the sample CO2 laser treated at 1.8 kW/cm2(see Figure 8.3(b)), while a cellular microstructure appeared on the surface of the sample CO2 laser treated at
146
Figure 8.3 Optical images of the morphology of (a) the untreated YPSZ and the CO2 laser treated YPSZ with laser power densities of (b) 1.8 kW/cm2 and (c) 2.3 kW/cm2
2.3 kW/cm2 (see Figure 8.3(c)). In both instances, the surfaces of the modied YPSZ displayed a microstructure typical of rapid solidication after CO2 laser irradiation. With a CO2 laser power density of 1.8 kW/cm2, the cappedhexagonal formation becomes the interface (see Figure 8.3(b)). When marked supercooling is generated with a CO2 laser power density of 2.3 kW/cm2, the cellular structure developed as shown in Figure 8.3(c). Indeed, different re-solidied microstructures generated by laser irradiation have been reported by a number of workers conducting research into the laser treatment of various ceramics. Pei, Omyang and Lei [158] noted that both equiaxed and dendritic microstructures were obtained in different regions of the same laser clad ZrO2 layer, concluding that the differences were related to different cooling rates in the various regions of the laser clad ZrO2 layer. Liu [283] obtained similar results in the laser sealing Y2O3ZrO2 and MgO ZrO2 ceramic coatings. Hao and Lawrence found the different microstructures on the MgOPSZ generated at the different CO2 laser power densities [21]. Such differences in microstructure type and size were ascribed to

1.0 0.8 0.6 0.4 0.2 Cos 0.0 0.2 0.4 0.6 0.8 1.0 0.00 0.02 0.04 0.06 0.08 0.10 Untreated CO2 Laser (1.8 kW/cm2) CO2 Laser (2.3 kW/cm2) 0.12 0.14 0.16 0.18
147
(
1
d lv 1/2
/ lv
Figure 8.4 Plot of cos y against (gd )2 =glv for the untreated and CO2 laser treated YPSZ lv in contact with the wetting control test liquids
the varying degrees of constitutional supercooling, which, according to McCallum, Kramer and Weir [284], are inherent in laser processes. As has already been demonstrated in Section 4.4, it is possible to estimate 1 gd for the YPSZ, by plotting the graph of cos y against (gd )2 =glv according to sv lv Equation (3.8), as shown in Figure 8.4. Comparing the ordinate intercept points of the untreated and CO2 laser treated YPSZliquid systems in Figure 8.4, it can be seen clearly that for the untreated YPSZ, the best-t straight line intercepts the ordinate closer to the origin while the best-t straight line of samples treated by the laser intercepts the ordinate considerably higher above the origin. As with the MgOPSZ studies described in Chapter 4, this shows that polar forces are active across the YPSZ interface after CO2 laser treatment, and hence improved wettability and adhesion is p promoted. As was shown previously in Section 4.4, in order to determine gsv d for the YPSZ, it is necessary to calculate Wad by using Equation (3.9). Both d Wad and Wad are related by the straight-line relationship represented by d Equation (3.10). Thus, from the best-t straight line plots of Wad against Wad for the YPSZ when it is both untreated and CO2 laser treated, it is possible to determine the constant a for each separate condition of the YPSZ. For the YPSZ the value of a is 2.28 (untreated), 2.85 (1.8 kW/cm2) and 3.10 (2.3 kW/cm2). Since it is already known that from Equation (3.11) c is 2.9 for the set of p control test liquids, it is possible to calculate gsv directly for the untreated
148
75 Surface Energy (mJ/cm2) 60 45 30 15 0
svd svp sv
Untreated
CO2 Laser 1.8 kW/cm2
Figure 8.5 Relationship between the surface energy and CO2 laser power density for the YPSZ
and CO2 laser treated YPSZ using Equation (3.14). The determined results of surface energies of the untreated and CO2 laser treated YPSZ (at the selected CO2 laser power densities) are given in Figure 8.5. As is evident from Figure 8.5, the CO2 laser treatment increased gsv of the YPSZ by p primarily increasing gsv , as gd was similar for all the samples. It is important sv to note that because of the long-range ionic interactions between the YPSZ and the control test liquids, it is highly likely that the thermodynamically dened total solid surface energy will be higher than the sum of the gd and sv p gsv components of the surface energy. A clear relationship between the value of cos y and the surface energy can be observed in Figure 8.6, which reveals that an increase in the surface energy will cause a rise in the value of cos y. It is therefore deemed that the surface energy primarily inuences the wettability of the YPSZ. Indeed, it was found by Lawrence [154] that the surface energy was the most predominant factor governing the wetting characteristics of the SiO2/ Al2O3 based ceramic following irradiation by the high power diode laser. What is more, Hao and Lawrence recently found that the change in surface energy, represented by the change in microstructure features [285], was identied as the main mechanism governing the wettability characteristics of the MgOPSZ following CO2 laser irradiation [22]. Since gd only changed sv slightly after laser treatment, as shown in Figure 8.5, an appreciable increase p in the total surface energy were governed by the marked enhancement in gsv . p The increase in gsv , in particular the increase in gsv , has a positive effect upon the action of wetting and adhesion [286], since primarily both dispersion and polar forces are active to a greater extent [100, 101]. The changes in gsv are thought to be due to the fact that CO2 laser treatment of the YPSZ results in

0.5
149
0.4
0.3 cos
0.2
0.1
0.0 30
40
50 60 sv (mJ/mm2)
70
80
Figure 8.6 Relationship between wettability characteristics (cos y, glycerol) and gsv for the YPSZ
the surface melting, a transition that is known to effect an increase in gsv [155], and thus an improvement in the wettability and an increase in the adhesion at the interface in contact with the control liquids. In fact, the re-solidied microstructure generated by laser surface melting might be attributed to the changes in the surface energy and thereof in wettability characteristics. The total surface energy values of samples with the hexagonal structure and cellular structure were 60 and 67.3 mJ/cm2, respectively, being higher than the untreated sample, which had a total surface energy value of 46.7 mJ/cm2. Indeed, work conducted by Zhang, Yue and Man [153] found that considerable improvement in the bond strength of an Si3N4 ceramic could be realised only when excimer laser treatment of a structural alloy steel (SAE 4340) resulted in surface melting. Similarly, Lawrence [154] observed a sharp reduction in y at the point of melting for an Al2O3/SiO2 based oxide compound after HPDL treatment. 8.2.3 Identication of the Predominant Mechanism Active in the Wettability Characteristics Modication of a YPSZ Bioinert Ceramic It is apparent from the above discussion that the surface roughness, the surface energy (by way of microstructural changes) and the surface oxygen content govern the observed changes in the wettability characteristics of the YPSZ after CO2 laser surface treatment. To determine the extent to which each of these factors affect the wettability characteristics of the YPSZ, several stages of grinding were used to isolate the various inuential factors and thus analyse and qualitatively establish their role. The grinding
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Table 8.2 The contact angle (for glycerol), surface roughness and surface oxygen content of the untreated and CO2 laser treated YPSZ following the ne grinding stages Untreated Ra (mm) O2 (at %) y (deg) 0.35 0.24 0.10 0.09 44.1 44.7 45.2 45.2 82.4 83.1 83.3 83.3 CO2 laser treated (2.3 kW/cm2) Ra (mm) O2 (at %) y (deg) 0.49 0.47 0.44 0.43 59.3 44.4 44.2 44.1 70.5 73.7 80.7 81.9
Polishing steps Unpolished 180 grit SiC 400 grit SiC 800 grit SiC
procedures were similar to those used for MgOPSZ which are described in Section 4.5. The observed changes to surface roughness, surface oxygen content and y (glycerol) caused by these grinding steps are given in Table 8.2. After the rst grinding stage, a large difference in y for glycerol was observed between the untreated and the CO2 laser treated samples. However, the decrease in the surface oxygen content of the CO2 laser treated sample from 59.3 to 44.1 at % resulted in only a small increase in the y, increasing from 70.5 to 73.7 , implying that the decreased surface oxygen content could be the factor inuencing the general increase in y. In this subsequent stage, the CO2 laser induced microstructure was removed from the CO2 laser treated sample and y for the CO2 treated samples was 80.7 and close to the original untreated value of 79.1 . Basically, the removal of the CO2 laser induced microstructure alone appears to have brought about an increase in y to around the original level, since the surface oxygen content was almost the same value in both ground stages and the surface roughness only changed slightly. In addition, the change in the surface roughness of the untreated sample brought about only a very slight increase in y, revealing that surface roughness played a very minor role in inducing changes in the wettability characteristics of the YPSZ. 8.2.4 Generic Effects of CO2 Laser Treatment on the Wettability Characteristics of Bioinert Ceramics As one can see from Figure 8.7, CO2 laser surface treatment of both the MgOPSZ and YPSZ bioinert ceramics caused a general reduction in y, which was decreased further as the CO2 laser power density was increased. Such decreases in y following laser surface treatment and the attendant increase in wettability were observed by Triantafyllidis, Li and Stott [281], found for the alumina based ceramics. These results suggest that laser surface treatment of ceramic materials generally brings about a change in the wettability characteristics.

100 Contact Angle, (deg) 80 60 40 20 0
MgO-PSZ YPSZ
151
Untreated
0.9 1.8 CO2 Laser (kW/cm2)
Figure 8.7 Contact angle (for glycerol) for the MgOPSZ and the YPSZ following CO2 laser treatment
As was discussed previously in Sections 4.3.3 and 8.2.2, the further reduction in y observed for the MgOPSZ and YPSZ when surface treating with the CO2 laser is due in part to the increase in surface roughness. This is in accord with Equation (4.1) and is therefore to be expected. As shown in Table 8.3, the surface roughness of both the MgOPSZ and YPSZ increased with increasing CO2 laser power density and as a consequence y decreased accordingly. The reasons for such an observation have been discussed in Sections 4.3.2 and 8.2.2. In fact, it was found by Triantafyllidis, Li and Stott [281] that an almost linear increase in surface roughness with laser power density was seen for laser treated Al2O3 based ceramics and y decreased with increasing laser power density. It is therefore speculated that the laser irradiation brought about the rougher surface and thereby generically contributed to the better wettability characteristics of zirconia and alumina. Similarly, the improvement in the wettability characteristics of the YPSZ after CO2 laser treatment (see Section 8.2.2) will have been inuenced by the increase in surface oxygen content. This is likewise the case for the MgO PSZ (see Section 4.3.2). Indeed, it was reported by Lawrence [287] that the
Table 8.3 The surface roughness, surface oxygen content and polar surface energy of the untreated and CO2 laser treated MgOPSZ and YPSZ Bioinert ceramic CO2 laser power density (kW/cm2) Surface roughness, Ra (mm) Surface oxygen content (at %) p gsv (mJ/m2) Untreated 0.29 41.5 10.1 MgOPSZ 0.9 0.33 44.0 21.0 1.6 0.72 64.3 60.7 YPSZ Untreated 0.35 44.1 46.7 1.8 0.40 52.2 60.0 2.3 0.49 59.3 67.3
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increase in surface oxygen content was one of the factors inuencing the enhanced wettability of an alumina bioceramic following HPDL surface treatment. As with the studies of MgOPSZ described in Section 4.4, Figure 8.5 reveals that polar forces are active across the YPSZ interface p after CO2 laser treatment. The observed increases in gsv resulted from the melting and solidication of the CO2 laser treated MgOPSZ and YPSZ surfaces, with the value varying with the re-solidied microstructure. The cellular microstructure on the MgOPSZ and YPSZ resulting from the CO2 laser induced rapid solidication corresponded to the maximum value of p p gsv . This increase in gsv has been found to play an important role in the wettability characteristics of the bioinert ceramics examined in this work, as discussed in Sections 4.4 and 8.2.2. Indeed, Bradley, Li and Stott [288] found that CO2 laser surface treatment generated a denser, more uniform surface layer on alumina based materials, with the re-solidied microstructure and ensuing increase in surface energy resulting in improved wettability and bonding characteristics. Similar ndings were made by Lawrence [287, 289] when using CO2, Nd:YAG and HPDLs to treat the surface of an alumina bioceramic. From the above discussion it would seem reasonable to postulate that the effects of laser surface treatment on the surface roughness, surface oxygen p content and gsv of the bioinert ceramics studied in this work, as well as the ceramics studied by other workers, are generic. Furthermore, based on this postulation it is perhaps possible to assume that modication of the wettability characteristics of the ceramics caused by the changes to these factors is also generic. This assertion regarding the generic nature of the changes to the wettability characteristics of ceramics as a result of laser surface treatment can be substantiated somewhat by considering the information given in Table 8.3. Here one can see that the increases in the surface roughness, p surface oxygen content and gsv of both the MgOPSZ and YPSZ following CO2 laser treatment appear to have altered in relation to one another, an occurrence that could only take place if the changes were generic. Further support for the proposition that CO2 laser surface treatment brings about generic modication of the wettability characteristics of the MgOPSZ and YPSZ can be obtained from consideration of the ndings arising from the discussions in Sections 4.5 and 8.2.3. These ndings established that the predominant factor governing modication of the wettability characteristics p of both the MgOPSZ and YPSZ was the increase in gsv , which was yielded by the CO2 laser induced re-solidied microstructure. In addition, Lawrence p [132, 154, 289] has identied that increases in gsv as the result of laser induced re-solidied microstructures as being the predominant mechanism governing the wettability characteristics modication of a number of cerap mic materials. Again, the incidence of gsv being the primary mechanism in determining the wettability characteristics of such a wide range of ceramics
153
following surface treatment with a number of different lasers could only be if the changes were actually generic. 8.2.5 CO2 Laser Induced Effects on the Cell Response on a YPSZ Bioinert Ceramic The hFOB cell adhesions improved considerably on the YPSZ after CO2 laser treatment. As presented in Figure 8.8, few osteoblast cells were found on the untreated YPSZ and covered less than 10 % of the surface area of the sample. On the other hand, highly dense osteoblast cells appeared and covered about 70 and 90 % of the surface area of the CO2 laser treated YPSZ at 1.8 and 2.3 kW/cm2, respectively. In addition, the morphology of osteoblast cells on the CO2 laser treatment showed better spreading on the CO2 laser treated sample compared with the cells on the untreated sample. Typical osteoblast cells on the CO2 laser treated sample at 1.8 kW/cm2, as observed in Figure 8.8(c), had spread completely and attened, with the
Figure 8.8 SEM image of hFOB cells on (a) the untreated YPSZ and the CO2 laser treated YPSZ at laser power densities of (b) 1.8 kW/cm2, (c) 1.8 kW/cm2 at high magnication and (d) 2.3 kW/cm2
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Figure 8.9 SEM image of two hFOB cells adhering and spreading within one of the ever-present grooves on the untreated YPSZ
cytoplasmic material spreading to cover a larger area and elongated lopodias. On the untreated YPSZ sample the situation was different. It is discernible from Figure 8.8(a) that the osteoblast cells did not exhibit good spreading and lopodias. It is very interesting to note that two osteoblast cells adhered in one of the grooves which were generated by the polishing methods employed by the manufacturers of the YPSZ. The groove in question had a width of about 10 mm and the two osteoblast cells spread along the direction of the groove and connected with each other, as shown in Figure 8.9. Such an observation implies that the surface topography could inuence the direction of osteoblast cell spreading and growth. Cell coverage density, represented by the ratio of the osteoblast cell covered area and the whole surface area, is used to express the degree of osteoblast cell adhesion. There are higher cell cover densities on the samples following the CO2 laser treatment than on the untreated sample, as shown in Figure 8.10. In the power density range employed and in the specied experimental conditions, the increase in power density caused an increase in cell cover density. It is certain that the levels of power densities of the CO2 laser treatment are a signicant factor in promoting the hFOB cell adhesion, implying that this technique is able to improve the response of the cells to the YPSZ. Indeed, the CO2 laser treatment creates the changes in the topography and surface energy synchronously. These changes, which primarily determined the wettability characteristics, could be the factors inuencing the response of the hFOB cells. As discussed previously, the CO2 laser treatment generated a consistently rougher surface on the YPSZ compared with the untreated sample, with the value of Ra increasing with increases in power density of the CO2 laser treatment. As shown in Figure 8.10, the CO2 laser treated YPSZ with a rougher surface had a higher cell cover density compared with the smooth untreated sample. This agrees with some reports demonstrating that the

80 Cell Cover Density (%) 60 40 20 0 0.45 Ra (m) 0.30 0.15 0.00 100 sv (mJ/m2) 75 50 25 0 Untreated CO2 Laser 1.8 kW/cm2 CO2 Laser 2.3 kW/cm2
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Figure 8.10 Relationship between the osteoblast cell coverage density, surface roughness (Ra ), surface energy and CO2 laser power density for the YPSZ
rougher surface promoted more osteoblast-like cell attachment on titanium [213], apatitewollastonite glassceramic [290] and hydroxyapatite [291]. Hence, one could assume that surface roughness plays a role in inuencing human osteoblast cell adhesion on the laser treatment of the YPSZ. In addition, the microtopography could affect cell spreading, as shown in Figure 8.9. The spreading of the cells could be dened in the groove and along the direction of the groove for about 10 mm. A previous nding revealed that the surface topography also caused the alignment of cells parallel to the 10 mm grooves present on the metal surfaces [292]. Surface microtopography has been cited as an important factor inuencing protein surface and cellsurface interactions [80]. Curved surfaces, pits, protrusions, cavities, etc., that have sizes and radii comparable with those of the biological entities (proteins $110 nm, cells 1100 mm) induce biological interactions different from those on a at surface [80]. It is noted that the degrees of the cell adhesion improved markedly when obvious microstructural change occurred on the YPSZ. The 70 and 90 % cell cover densities occurred on the laser modied surface with hexagonal and cellular microstructures, respectively. A number of reasons have been suggested for an increased differentiation of osteoblasts on the microstructured surface, such as the inuence of surface structure on cell shape or the fact that the surface topography creates a specic biochemical microenvironment around each cell [214]. Microstructures of about 1020 mm induced by the
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CO2 laser treatment on the polyethylene terephthalate (PET) were regarded as one of the factors for broblast cell attachment and spreading [293]. As can be seen from Figure 8.10, cell adhesion increases as the surface energy of the YPSZ increases. There were only sparsely adhered cells on the untreated sample with a surface energy of 46.7 mJ/cm2, and an obvious improvement of cell adhesion was found on the CO2 laser treated YPSZ with a surface energy of 60 mJ/cm2, while maximum cell adhesion occurred on the YPSZ with a surface energy of 67.3 mJ/cm2. The work on polymers and glass revealed that cell spreading and substratum surface free energy showed a characteristic sigmoid relationship both in the presence and in the absence of serum proteins; good spreading only occurred when the surface energy was higher than approximately 57 mJ/cm2 [115]. It was found that the critical parameter for osteoconduction was the initial number of wellattached osteoblastic cells to the bone substitute [218]. The difference in cell adhesion was therefore attributed to the difference in wettability characterp istics, which is determined by the surface energy, particularly gsv between the untreated and CO2 laser treated YPSZ, since the change in wettability characteristics was primarily inuenced by the surface energy of the YPSZ, p especially gsv . The nding agrees with previous studies showing the inuence of wettability on the attachment and spreading of various cells [213, 217219]. These studies showed good cell attachment and spreading on high-energy substrata and poor cell attachment and spreading on lowenergy substrata, which accounts for the minimal energetic state of a system in equilibrium. The behaviour of osteoblastic cells at the surface of HA [218] and at the surface of titanium [213] demonstrated that gp plays a critical role. As shown in Figure 8.5, the dispersion components (gd) of the surface energy p were similar, whereas the gsv values were signicantly different for the untreated and CO2 laser treated YPSZ at various power densities. Therefore it is possible to say that the change in osteoblast cell adhesion was mainly related to gp. These results indicate that gp inuenced the behaviour of osteoblasts on YPSZ surfaces more strongly when compared to gd, which can probably be attributed to the fact that the composition of the culture medium is all polar; thus osteoblast cells and the YPSZ should interact mainly by polar force. Moreover, the osteoblast cells showed better spreading and attening at the CO2 laser treated sample. It is likely that the more attened osteoblast cells produced more collagen than less attened osteoblast cells [218] on the untreated sample. One important aspect to be considered in this study is the kinetics of osteoblast cell events. The difference in morphology of osteoblast cell attachment might lead to the difference in terms of osteoblast cell growth. It was suggested that enhancing p gsv would promote the initial osteoblast cell attachment and spreading, and thereby could bring about a large bone-like matrix synthesis.
Ascertaining the Generic Effects of CO2 Laser Treatment on Metal Implants

100
MgO-PSZ YPSZ
157
Cell cover density (%)
80
60 40
20
Untreated
Figure 8.11 Cell cover density (hFOB osteoblast cell) on the MgOPSZ and YPSZ following CO2 laser treatment at various laser power densities
8.2.6 Generic Effects of CO2 Laser Treatment on the Cell Response on Bioinert Ceramics As one can see from Figure 8.11, the CO2 laser surface treatment resulted in both the MgOPSZ and the YPSZ displaying an increase in the cell cover density. As discussed in Section 8.2.4, the CO2 laser surface treatment yields generic enhancement of the surface roughness and wettability characteristics of these bioinert ceramics. Further, Chapter 5 and Section 8.2.2 revealed that improvements in the cell response of the MgOPSZ and the YPSZ after CO2 laser surface treatment were correlated directly to the CO2 laser induced enhancement of the wettability characteristics of these ceramics. On account of the generic effects of the laser surface treatment on the surface roughness and wettability for other ceramics such as alumina, by extension it would appear that improvements in the cell response of the bioinert ceramics after CO2 laser surface treatment are generic. 8.3 Ascertaining the Generic Effects of CO2 Laser Treatment on Metal Implants Currently, 316 LS stainless steel is still the most used metal for internal xation devices thanks to a favourable combination of mechanical properties, acceptable biocompatibility and cost effectiveness when compared to other metallic implant materials [294]. Still, a disadvantage seen for stainless steel is its tendency towards corrosion under physiological conditions,
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causing a release of metal ions such as those of nickel (Ni) and chromium (Cr). Stainless steels are actually not immune to crevice corrosion in the human body, which can increase ion release in the surrounding tissues by several orders of magnitude. To date, very little published work exists pertaining to the use of lasers for altering the surface properties of stainless steel in order to improve its wettability characteristics. Notwithstanding this, Lawrence and Li [138] compared the interaction of CO2, Nd:YAG, HPDL and excimer laser radiation with the surface of the mild steel and found that changes took place to the wettability characteristics. HPDL surface treatment of a common engineering carbon steel (EN8) was found to effect appreciable changes to the wettability characteristics of the metal [295]. These modications have been investigated in terms of the changes in the surface roughness of the steel, the presence of any surface melting, the polar component of the steel surface energy and the relative surface oxygen content of the steel. Surface properties of biometals, such as surface roughness and wettability characteristics, inuence their interactions with biouids and cells. As a laser was used to modify the surface properties of a mild steel in order to improve the wettability characteristics, it is reasonable to expect that the CO2 laser treatment can be used to modify the surface of 316 LS stainless steel and thereby improve its biological response. It is possible that successful application of the laser surface treatment to titanium alloys and stainless steels would provide evidence of the lasers potential for application to other biometals such as pure titanium and cobalt alloys. 8.3.1 Experimental Procedures The as-received biodur consumet type 316 LS stainless steel (of implant quality, ground annealed and cold worked) was in the form of a round bar with a diameter of 19 mm (Carpenter, Inc.). The composition and main properties of the steel are detailed in Table 8.4. It was cut into disks of approximately 3 mm thickness with a diamond-rimmed blade cutter.
Table 8.4 Composition and selected physical and mechanical properties of 316 LS stainless steel Composition 0.030 C, 2.00 Mn, 0.75 Si, 0.025 P, 0.010 S, 17.00/19.00 Cr , 13.00/15.00 Ni, 2.25/3.50 Mo , 0.50 Cu, 0.10 N, balance Fe ( 3.3 Mo Cr 26.00) Physical properties Density 7.95 g/cm3 Melting range 1649 15 C Specic heat 502 J/kg C Thermal conductivity 16.3 W/m K
Mechanical Tensile strength properties 883 MPa
Elastic modulus Hardness Rockwell 190 GPa 36 C
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The 316 LS stainless steel was used as received prior to CO2 laser treatment. The surface treatment was carried out with a 3 kW CO2 laser in the same manner as described in Section 4.2.2. A series of experiments was conducted with a wide range of CO2 laser power densities with a 5.5 mm beam spot diameter, while the traverse speed was set at 800 mm/min. To protect the CO2 laser optics and assist the surface treatment of the 316 LS stainless steel, 2 bar pressure O2 process gas was supplied off-axis. The surface properties of untreated and CO2 laser modied stainless steel were examined in the same manner as in Section 6.2.3. To investigate the effects of laser irradiation on the wetting and surface energy characteristics of the 316 LS stainless steel, a set of sessile drop control experiments were carried out in the same way as in Section 4.2.4. The proteins used for this study were human serum albumin and human plasma bronectin (Calbiochem, Inc.). The adsorption of proteins was conducted in the same manner as described in Section 5.4.1 and was measured by the ellipsometer described in Section 5.4.1. The human osteoblastic cell line hFOB 1.19 was obtained from the American Type Culture Collection (Manassas, Inc.). These cells were cultured following the same procedure described in Section 5.5.1. The samples and osteoblast cells were prepared in the same way as explained in Section 5.5.1. The specimens were seeded with a 0.5 ml cell suspension of 1 105 cell/ml and then cultured with cell culture medium and maintained in the incubator for one week. The cell culture medium was changed every 3 days. After incubation and gently rinsing with PBS, the samples were processed and investigated using SEM in the same manner as in Section 5.5.1. Cell proliferation on each specimen was measured by MTT assay in the same manner as Section 5.5.1. The statistical analysis of the results was performed with a SPSS v.12 software package (SPSS/PC, Inc.) in the same manner as discussed in Section 5.5.1. 8.3.2 Modication of Surfaces Properties and Wettability Characteristics of a 316 LS Stainless Steel As one can see from Table 8.5, the CO2 laser treated 316 LS stainless steel experienced a consistent reduction in y with all the control test liquids used, showing that the wettability characteristics improved. The CO2 laser treatment at a power density of 1.8 kW/cm2 yielded the maximum decrease in y. In addition, y on the mechanically roughened 316 LS stainless steel was seen to be only slightly lower than on the untreated sample. As evident in Figure 8.12, both mechanical roughening and CO2 laser treatment had a marked effect on the surface roughness of the 316 LS stainless steel. For the CO2 laser treated samples, the Ra value increased with increasing laser power density. The CO2 laser treatment at 1.8 kW/cm2 generated a surface with an increase in the Ra value of 65 %, compared with
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Table 8.5 Mean values of contact angle formed between the untreated and CO2 laser treated 316 LS stainless steel and the control test liquids at 25 C Contact angle, y (deg) Polyglycol Polyglycol Glycerol Formamide Etheneglycol E-200 15-200 73.8 70.2 69.0 67.9 68.1 64.9 63.4 61.7 57.4 55.6 53.2 51.3 52.3 50.2 48.6 45.5 36.4 34.7 33.2 31.5
Stainless steel Untreated Mechanically roughened CO2 laser (1.6 kW/cm2) CO2 laser (1.8 kW/cm2)
0.4
0.3
0.2
0.1
0.0
Untreated Mechanically Roughened CO2 Laser 1.6 kW/mm2 CO2 Laser 1.6 kW/mm2
Figure 8.12 Surface roughness values for the untreated, mechanically roughened and CO2 laser treated 316 LS stainless steel
the untreated specimen. Its effect on surface roughness is approximate to the mechanical roughening, which resulted in the roughest surface and increased the surface roughness by 80 %. It is worth remarking that even a cursory cross-referencing of the results given in Table 8.5 and Figure 8.12 shows that despite the higher surface roughness occasioned on the 316 LS stainless steel by mechanical roughening, the CO2 laser treatment is more effective in enhancing the wettability characteristics of the 316 LS stainless steel. As one can see from Figure 8.13(a), the untreated 316 LS stainless steel sample presented a surface that appeared to be smooth and without depressions or holes. Some irregular marks can be seen, however, which are scratch marks produced on the surface by the rolling process involved during manufacture of the sheets. It is clear from Figures 8.13(c) and (d) that the surface morphology of the 316 LS stainless steel was altered markedly
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Figure 8.13 Typical SEM images of (a) the untreated, (b) the mechanically roughened and the CO2 laser treated at power densities of (c) 1.6 kW/cm2 and (d) 1.8 kW/ cm2 316 LS stainless steel
from the untreated state after the CO2 laser treatment, regardless of the laser power density used. Between the two CO2 laser treated samples, the sample treated with a laser power density of 1.6 kW/cm2 exhibited a more uneven surface, which seemed to have a rougher and groove-like texture, than the sample treated at 1.8 kW/cm2. The surface of the mechanically roughened sample was also altered noticeably from the untreated state, displaying a generally rougher surface with deep, wide grooves (see Figure 8.13(b)). Figure 8.12, together with Figures 8.13(c) and (d) clearly show that interaction of the CO2 laser beam with the surface of the 316 LS stainless steel effected an increase in the surface roughness. This occurrence is due to the melting and re-solidication of the 316 LS stainless steel surface. Because the melt ow within the meltpool is under the inuence of Marangoni forces, a more random surface morphology will result. It is well known that, on a wetting surface, the rougher the surface, the higher is the wettability of the surface [150]. However, a review of the results given in Table 8.5 and
162
Surface Oxygen Content (mass%)
30
20
10
0
Untreated Mechanically Roughened CO2 Laser 1.6 kW/mm2 CO2 Laser 1.8 kW/mm2
Figure 8.14 Surface oxygen content of the untreated, mechanically roughened and CO2 laser treated 316 LS stainless steel
Figure 8.12 shows that the CO2 laser treatment is more effective in enhancing the wettability characteristics of the 316 LS stainless steel, in spite of the fact that mechanical roughening generated a rougher surface. This would suggest that the changes in the other surface properties associated with the CO2 laser treatment (surface oxygen content and surface energy, which are modied simultaneously) play a signicant role in modifying the wettability characteristics of the 316 LS stainless steel. An XPS analysis was conducted to ascertain the oxygen content on the surface of the untreated and CO2 laser treated 316 LS stainless steel. As one can see from Figure 8.14, mechanical roughening occasioned no discernible change in the surface oxygen content of the 316 LS stainless steel. In contrast, the CO2 laser treatment brought about, to varying degrees, an increase in the surface oxygen content. In particular, the sample CO2 laser treated at a power density of 1.8 kW/cm2 experienced a signicant increase in the surface oxygen content, from 15.9 to 27.6 mass %. Such a relatively large increase in the surface oxygen content implies that sufcient melting on the surface of the 316 LS stainless steel took place, which in turn gave rise to more oxygen being adsorbed into the material surface. The surface oxygen content of the sample CO2 laser treated at a power density of 1.6 kW/cm2 increased marginally from 15.9 to 17.7 mass %, an indication of an insufcient degree of melting of the surface of the 316 LS stainless steel to effect substantial oxygen absorption. Based on previous work by Lawrence and Li [20, 295], which revealed that the surface oxygen content of a carbon steel increased after the CO2 laser treatment and contributed to higher wettability characteristics, it is reasonable to suppose that changes in the surface oxygen content for the CO2 laser treated samples are responsible in part

1.0 0.8 0.6 0.4 0.2 Cos 0.0 0.2 0.4 0.6 0.8 1.0 0.00 0.03 0.06 0.09 Untreated Macanically rounded CO2 laser (1.6 kW / cm2) CO2 laser (1.8 kW / cm2) 0.12
/ lv
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0.15
0.18
0.21
( )
1
d 1/2 lv
Figure 8.15 Plot of cos y against (gd )2 =glv for the untreated, mechanically roughened lv and CO2 laser treated stainless steel in contact with the wetting test control liquids
for the overall increase in the wettability characteristics of the 316 LS stainless steel. The gd value for the 316 LS stainless steel are estimated by plotting the sv 1 graph of cos y against (gd )2 =glv according to Equation (3.8), as shown in lv Figure 8.15. Thus, according to Fowkes [100], the value of gd is estimated by sv 1 the gradient ( 2(gd )2 ) of the line that connects the origin (cos y 1) with sv 1 the intercept point of the straight line (cos y against (gd )2 =glv ) correlating the lv data point with the abscissa at cos y 1. Comparing the ordinate intercept points of the untreated and CO2 laser treated 316 LS stainless steel-liquid systems in Figure 8.15, it can be seen clearly that for the untreated 316 LS stainless steel, the best-t straight line intercepts the ordinate closer to the origin. This is noteworthy for the intercept of the ordinate close to the origin is characteristic of the dominance of dispersion forces acting on the 316 LS stainless steelliquid interfaces of the untreated and low-power density treated sample, resulting in poor adhesion [100, 101]. On the other hand, the best-t straight line of samples treated by the CO2 laser intercept the ordinate considerably higher above the origin. An interception of the ordinate above the origin is indicative of the action of polar forces across the interface, in addition to dispersion forces, and hence improved wettability and adhesion is promoted [100, 101]. Furthermore, because none of the best-t straight lines intercept below the origin, it can be said that the development of an equilibrium lm pressure of adsorbed vapour on
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the 316 LS stainless steel surface (untreated and CO2 laser treated) did not occur [101]. d From the best-t straight line plots of Wad against Wad for the 316 LS stainless steel it is possible to determine the constant, a, for each separate condition of the untreated, mechanically roughened and CO2 laser treated 316 LS stainless steel. For the 316 LS stainless steel the value of a is 2.10 (untreated), 2.29 (mechanically roughened), 2.48 (1.6 kW/cm2) and 2.5 (1.8 kW/cm2). As a linear relationship satisfying Equation (3.11) exists between the dispersive and polar components of the surface energy of the test control liquids, then c is 2.9 for the set of test control liquids. As was p shown previously, it is possible to calculate gsv directly for the untreated and CO2 laser treated 316 LS stainless steel using Equation (3.14). The calculated results of surface energies of the untreated and CO2 laser treated stainless steel (at various laser power densities) are given in Figure 8.16. This overall change in the surface energy of the 316 LS stainless steel is perhaps one of the most important outcomes of the CO2 laser treatment. As amply demonstrated by Lawrence and Li [13] and Hao and Lawrence [2022], the surface energy of ceramic and metals, which results from a variety of intermolecular forces existing within the materials elements, can be increased by laser treatment. This fact is borne out by Figure 8.16, where the surface energy of the 316 LS stainless steel changed by varying degrees after mechanical roughing and CO2 laser irradiation. It is interesting to note that the mechanically roughened sample experienced a slight increase in surface energy, suggesting that surface roughness inuences the surface p energy. However, the marked changes in gsv were brought about by CO2 laser treatment. From Figure 8.16 it can be seen that gd of the sample CO2 sv
svd svp sv
40 Surface Energy (mJ/m2)
30
20
10
Untreated
CO2 Laser CO2 Laser 1.6 kW/mm2 1.8 kW/mm 2
Figure 8.16 Surface energy values of the untreated, mechanically roughened and CO2 laser treated 316 LS stainless steel
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laser treated with a power density of 1.6 kW/cm2 is lower than the untreated sample. This sample, however, has higher wettability characteristics than the p untreated sample, denoting that enhancement in gsv played a major role in governing the improvement in the wettability characteristics of the 316 LS p stainless steel. This is in accord with the fact that an increase in gsv has a positive effect upon the action of wetting and adhesion [237]. Indeed, the /cm2 has the highest sample CO2 laser treated with a power density of 1.8 kW p d value of gsv and gsv and its wettability characteristics are, unsurprisingly, the highest of the sample group. Based on the work of Lawrence, Li and Spencer [129], it is believed that the melting and re-solidication of the 316 LS stainless steel surface caused by CO2 laser irradiation of the surface effected p the observed increases in gsv . Furthermore, it seems highly likely that the p increase in gsv was inuential in precipitating enhancement of the wettability characteristics of the 316 LS stainless steel. In order to simulate the biological environment, physiological uids and simulated physiological liquids were used for wetting experiments. The selected physiological and simulated physiological liquids were: human blood, human blood plasma, simulated body uid (SBF) and SBFBSA (bovine serum albumin). The values of y formed between the selected physiological and simulated physiological liquids, and the untreated, mechanically roughened and CO2 laser treated 316 LS stainless steel are given in Table 8.6. Here it is evident that the values of y for all the selected physiological and simulated physiological liquids on the surface of the CO2 laser treated 316 LS stainless steel samples were lower than on either the untreated or mechanically roughened specimens. This is a clear indication that the wettability characteristics of the 316 LS stainless steel improved with regard to the selected physiological and simulated physiological liquids. Because the wetting effect of a solid surface can be a predictive index of the biocompatibility of the material involved, improvements in the wettability characteristics of the 316 LS stainless steel would no doubt result in better biocompatibility.
Table 8.6 Mean values of contact angles formed between the selected and simulated physiological test liquids and the untreated, mechanically roughened and CO2 laser treated 316 LS stainless steel Contact Angle, y (deg) Human Human blood blood plasma SBF SBFBSA 46.3 45.2 43.6 39.2 72.1 68.3 66.9 63.5 82.9 80.6 78.2 73.2 61.4 60.0 60.4 57.1
Stainless steel Untreated Mechanically roughened CO2 laser (1.6 kW/cm2) CO2 laser (1.8 kW/cm2)
166
100 Human blood Human blood plasma SBF SBF+BSA
Work Adhesion,Wad (mJ/m2)
90
80
70
60
50
Untreated
Figure 8.17 Work of adhesion of body uids for the untreated, mechanically roughened and CO2 laser treated 316 LS stainless steel
Any reduction in y would contribute to an enhancement in Wad of the SBF and SBFBSA on the 316 LS stainless steel following the CO2 laser treatment. As both SBF and SBFBSA have close chemical compositions to human body uids, the augmentation of Wad towards these uids would mean better suitability of the 316 LS stainless steel for use as a biomaterial after the CO2 laser treatment. Using the referenced glv value for human blood (47.5 mJ/m2), human blood plasma (50.5 mJ/m2) [141], SBF (72.5 mJ/m2) and SBFBSA (54.0 mJ/m2) [164], the value of Wad for the 316 LS stainless steel towards the selected physiological and simulated physiological liquids was determined by means of Equation (3.4). As Figure 8.17 shows, the decrease in y following the CO2 laser treatment did indeed yield an increase in the value of Wad for the 316 LS stainless steel with respect to the physiological and simulated physiological uids. Moreover, Wad can be seen to increase as the CO2 laser power density increases. Perhaps as one would expect, Figure 8.17 reveals that the mechanically roughened sample experienced only a very minor increase in Wad in relation to the selected physiological and simulated physiological liquids. 8.3.3 Identication of the Predominant Mechanism Active in the Wettability Characteristics Modication of a 316 LS Stainless Steel It is evident from the preceding discussion that the increases in the surface roughness, surface oxygen content and surface energy of the 316 LS stainless
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steel, which take place concurrently during the CO2 laser treatment, are responsible for the increased potential of the steel to wet. Indeed, although the surface roughness of the CO2 laser treated 316 LS stainless steel samples was less than that of the mechanically roughened sample, the CO2 laser treated samples presented a surface with a greater propensity to wet. This reinforces the assertion that the changes in other surface properties associated with the CO2 laser treatment (surface oxygen content and surface energy) play a signicant role in modifying wettability characteristics of the 316 LS stainless steel. Even so, it is highly likely that surface roughness plays a major part in determining the wettability characteristics of the 316 LS stainless steel as y was seen to decrease by simply roughening the surface mechanically (see Table 8.5). To ascertain the extent to which each of these factors affect the wettability characteristics of the 316 LS stainless steel, several stages of grinding were used to isolate the various inuential factors and thus analyse and qualitatively establish their role. The grinding procedures were similar to those used for the Ti6Al4V alloy described in Section 6.5. The observed changes to the surface roughness, surface oxygen content and y caused by these grinding steps are given in Table 8.7. The principal role played by surface roughness is certainly afrmed by the results obtained after the rst ne grinding-down stage. From Table 4.7 it can be seen that for both the untreated and CO2 laser treated stainless steel samples, y increases, and in the case of the CO2 laser treated sample, the increase is considerable. The combination of a decrease in the surface oxygen content of the CO2 laser treated sample from 27.66 to 15.98 at % to a similar original level to the untreated one and surface roughness from 0.31 to 0.19 mm resulted in an increase in y, increasing from 67.9 to 72.8 . On the other hand, the change in Ra for the untreated sample ground up from 0.19 to 0.38 mm generated a similar change in y from 73.8 to 68.4 . The fact that the
Table 8.7 The surface roughness, surface oxygen content and contact angle (for glycerol) of the untreated and CO2 laser treated 316 LS stainless steel (1.8 kW/cm2) following the ne grinding stages Untreated Ra (mm) 0.19 0.17 0.15 0.35 0.38 O2 (at %) 15.96 15.92 15.91 15.90 15.92 y (glycerol) (deg) 73.8 74.2 74.4 70.5 68.4 Ra (mm) 0.31 0.19 0.17 0.34 0.36 CO2 laser treated O2 (at %) 27.66 15.98 15.96 15.92 15.92 y (glycerol) (deg) 67.9 72.8 74.0 70.7 69.7
Fine polishing stages Unpolished Grinding-down stage 1 Grinding-down stage 2 Grinding-up stage 1 Grinding-up stage 2
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combined effect of the surface oxygen content and a small change in Ra nearly equals the effect of a large change in Ra on the modication of y reveals that the oxygen surface content does inuence y and its effect is less than the considerable change in surface roughness. In addition, despite the fact that surface roughness values for the untreated and CO2 laser treated stainless steel samples are similar after the rst ne grinding-down stage, there is a discernible difference in the value of y, being 1.4 lower for the CO2 laser treated sample. Whereas the surface oxygen content of the untreated stainless steel sample remained around the original value, the surface oxygen content of the CO2 laser treated stainless sample reduced to a level similar to that of the untreated sample. As the rst ne grinding-down stage does not remove the CO2 laser induced microstructure, this difference in y p can, therefore, be taken as an indication that gsv is active in determining the wettability characteristics of the stainless steel. Thus it is reasonable to conclude that the wettability characteristics of the stainless steel alloy are, after surface roughness, inuenced predominantly by the surface oxygen content and, to some extent, by the microstructure. The dependency of y on surface roughness is well known and, moreover, surface roughness has been identied as the predominant factor governing changes in wettability characteristics of steel after surface treatment with various lasers [138, 236]. 8.3.4 Generic Effects of CO2 Laser Treatment on the Wettability Characteristics of Biometals As one can see from Figure 8.18, CO2 laser surface treatment of both the Ti 6Al4V and 316 LS stainless steel caused a general reduction in y which was decreased further as the laser power density was increased. A similar
100 Ti6Al4V alloy Stainless steel 80
Contact Angle, (deg)
60
40
20
Untreated
1.3 1.6 1.6 1.8 CO2 Laser (kW/cm 2) CO2 Laser (kW/cm2)
Figure 8.18 Contact angle (for glycerol) for the Ti6Al4V alloy and the 316 LS stainless steel following CO2 laser treatment
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observation was made by Verdier et al. [296], who conducted a study on topographic and wettability modications induced by laser treatment on an aluminium alloy, a Ti6Al4V alloy and a 2C22 ferritic steel. In that work the laser treatment led to better wetting for the aluminium alloy and the Ti6Al 4V alloy at certain laser beam energy densities, but did not produce better wetting for the 2C22 ferritic steel. The reason cited for the lack of improvement in the wettability characteristics of the 2C22 ferritic steel after laser treatment was that the laser power used was limited to 40 W and no oxygen processing gas was employed. Additionally, Lawrence and Li [295] successfully improved the wettability characteristics of carbon steel using an HPDL. It would seem reasonable to infer from such results that laser surface treatment of metallic materials is an effective and controllable means for the modication of wettability characteristics. As was discussed previously in Sections 6.3.2 and 8.3.2, the reduction in y observed for the Ti6Al4V alloy and 316 LS stainless steel when surface treated with the CO2 laser is inuenced a great deal by the enhancement of surface roughness, which is in accord with Equation (4.1). As shown in Table 8.8, the surface roughness of both the Ti6Al4V alloy and 316 LS stainless steel increased with increasing CO2 laser power density. This in turn led to a natural reduction in y. The reasons for such an observation have been discussed in Sections 6.3.2 and 8.3.2. Such modications to the surface roughness for metallic materials have been observed in previous studies [138, 236, 295, 296]. The improvement in the wettability characteristics of the Ti6Al4V alloy and the 316 LS stainless steel after CO2 laser treatment (see Figure 8.18) would have been inuenced by the increase in the surface oxygen content (see Table 4.8). Indeed, it was reported by Lawrence and Li [138, 236, 295] that an increase in the surface oxygen content was one of the factors inuencing the enhanced wettability of a carbon steel following laser surface treatment. The discussions in Sections 6.4 and 6.5, along with those of Sections 8.3.2 and 8.3.3, revealed that the increase in wettability characteristics of the Ti6Al4V alloy and the 316 LS stainless steel following the CO2 laser treatment were effected partially by p the increase in gsv .
Table 8.8 The surface roughness, surface oxygen content and polar surface energy of the untreated and CO2 laser treated Ti6Al4V alloy and 316 LS stainless steel Ti6Al4V alloy CO2 laser power density (kW/cm2) Surface roughness, Ra (mm) Surface oxygen content (at %) p gsv (mJ/m2) Untreated 0.35 23.0 4.9 1.3 0.39 41.6 9.3 1.6 0.42 49.1 10.4 Stainless steel Untreated 0.19 15.9 3.3 1.6 0.27 15.7 6.4 1.8 0.31 27.7 6.7
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Following on from this, it would seem reasonable to postulate that the effects of the CO2 laser surface treatment on the surface roughness, surface oxygen p content and gsv of the bioinert metals studied in this work are generic. Furthermore, by extension of this postulation it is possible to maintain that modication of the wettability characteristics of the bioinert metals caused by the changes to these factors is also generic. This assertion regarding the generic nature of the changes to the wettability characteristics of the biometals as a result of the CO2 laser surface treatment can be substantiated somewhat by considering the information given in Table 4.8. Here, one can see that the increases in the surface roughness, surface oxygen content and p gsv of both the Ti6Al4V alloy and the 316 LS stainless steel following the CO2 laser treatment appear to have altered in relation to one another. This similar alteration could only have occurred if the changes were generic. More verication of the assertion that the CO2 laser surface treatment brings about generic modication of the wettability characteristics of both the Ti6Al4V alloy and the 316 LS stainless steel can be obtained from consideration of the ndings developed from the discussions in Sections 6.5 and 8.3.3. These ndings established that the leading factor governing the modication of the wettability characteristics of both Ti6Al4V alloy and the 316 LS stainless steel was the increase in surface roughness. The surface roughness was also identied as the predominant mechanism active in inuencing the wettability characteristics of other laser treated metallic materials [236]. It seems inevitable then to conclude that because surface roughness is the primary mechanism in determining the wettability characteristics of such a range of metals following surface treatment with a number of different lasers, the laser induced changes to the wettability characteristics of these metals were actually generic. 8.3.5 CO2 Laser Induced Effects on Protein Adsorption and the Cell Response on a 316 LS Stainless Steel The thickness of the human serum albumin layer on the untreated 316 LS stainless steel sample was found to be higher than on either of the CO2 laser modied samples, as shown in Figure 8.19. Conversely, Figure 8.19 shows that the thickness of the human plasma bronectin layer was less on the untreated 316 LS stainless steel sample than on both of the CO2 laser modied samples. What is more, whereas the thickness of the adsorbed human serum albumin layer was seen to decrease as the CO2 laser power density applied increased, the thickness of the adsorbed human plasma bronectin layer increased with increasing laser power density (see Figure 8.19). In addition, the statistical analysis revealed that the thickness of the absorbed bronectin on the untreated 316 LS stainless steel sample was signicantly less than on the sample CO2 laser treated with a power

1800 1500 1200 900 600 300 0
171
Fibronectin
Albumin
Figure 8.19 Thicknesses of the adsorbed bronectin and albumin layers on the untreated and CO2 laser treated 316 LS stainless steel with different laser power densities. For the bronectin adsorption, there was a signicant statistical difference in thickness between the untreated stainless steel and CO2 laser treated sample at a power density of 1.8 kW/cm2, and no statistical difference between the untreated stainless steel and CO2 laser treated sample at a power density of 1.6 kW/cm2. For the albumin adsorption, there was a signicant statistical difference in thickness between the untreated stainless steel and CO2 laser treated samples at power densities of 1.6 and 1.8 kW/cm2 p < 0:05
density of 1.8 kW/cm2, but not signicantly less than on the sample CO2 laser treated with a power density of 1.6 kW/cm2 p < 0:01. For the untreated 316 LS stainless steel sample a disparate situation existed, with the thickness of the absorbed albumin layer being signicantly higher than on either of the CO2 laser modied samples, as shown in Figure 8.19. As stated in Section 5.4.2, protein adsorption is inuenced by the surface topography [198] and the surface chemistry (wettability characteristics) [199]. It was found by Deligianni et al. [200] that human serum albumin was adsorbed preferentially on to a smooth substratum, while the rough substratum bounded a higher amount of total protein (from a culture medium supplied with 10 % serum) and bronectin (10-fold) over a smooth substratum. These ndings are reected by those of the 316 LS stainless steel in this study so it is possible to declare that the increasing surface roughness of the 316 LS stainless steel associated with increases in the CO2 laser power densities used brought about directly the observed reduction in the albumin adsorption and enhancement of the bronectin adsorption. As one can see from Figure 8.20, as the wettability characteristics of the 316 LS stainless steel increased, the adsorbed amounts of albumin decreased. The results of the albumin adsorption tests for the 316 LS stainless steel are
172
1800 Thickness of Absorbed Protein Layer () 1500 1200 900 600 300 0 0.05 0.00 0.05 0.10
Fibronectin Albumin
0.15
0.20
0.25
Wettability, cos (glycerol)
Figure 8.20 The relationship between the thickness of the adsorbed bronectin layer and wettability characteristics (cos y) of the 316 LS stainless steel
consistent with a previous nding by Serro et al. [201], in which the increase in surface hydrophilicity of Ti was seen to result in lower albumin adsorption, implying that the wettability characteristics of the 316 LS stainless steel might be a factor active in promoting albumin adsorption. Also, Janocha et al. [297] found that the amount of adsorbed BSA protein decreased with increasing surface energy of the substrate. The interfacial energy of the solidliquid interface increases for substrates of high surface energy, which indicates that the decrease in interfacial energy is not the only driving force of this adsorption, for expulsion of the proteins from the solution also takes place (hydrophobic interaction). This would therefore suggest that the enhancement of the surface energy of the 316 LS stainless steel after CO2 laser treatment inhibited the adsorption of the protein. Hence the wettability characteristics of the material chiey governed the adsorption of human serum albumin. The results of the adsorption of bronectin show that it increased with the increasingly wettable characteristics (hydrophilic) of the 316 LS stainless steel surface. A previous investigation by Grinnell and Feld [204] on the extent of bronectin adsorption as compared to its biological activity on hydrophobic and hydrophilic surfaces suggested the possibility that bronectin was more actively adsorbed on the hydrophilic surfaces. The results showed that the antiplasma bronectin antibody appeared to bind to the conformation of bronectin adsorbed on hydrophilic surfaces much better than the conformation of bronectin adsorbed on hydrophobic surfaces [204]. It is therefore possible to maintain that for the 316 LS stainless steel, the modication of human plasma bronectin was inuenced predominantly by the wettability characteristics of the steel. It is noticeable that a p considerable change in gsv affected the wettability characteristics of the
173
Figure 8.21 Typical SEM images of one-day osteoblast cell adhesion on (a) the untreated, (b) the mechanically roughed and the CO2 laser treated 316 LS stainless steel at a laser power density of (c) 1.6 kW/cm2 and (d) 1.8 kW/cm2
316 LS stainless steel after CO2 laser irradiation, signifying that the albumin and bronectin adsorption on the 316 LS stainless steel surfaces was probably due to the polar and chemical interactions [205]. Generally, cells in contact with the surface of a material will rstly attach, adhere and spread. From Figure 8.21 it is quite clear that the adhesion and spreading of osteoblast cells was inuenced by the CO2 laser treatment. The cells on the untreated and mechanically roughed surfaces shown in Figures 8.21(a) and (b) present the initial stage of the adhesion, with individual cells covering a small surface area and not spreading. For the CO2 laser treated surfaces the situation was different: the cells showed a good state of adhesion and attening, leading to coverage of more surface area (see Figures 8.21(c) and (d)). This means that the cells showed better adhesion on the CO2 laser treated samples than on the untreated and mechanically roughened samples, suggesting that the surface properties generated by the CO2 laser treatment were more favourable for osteoblast
174
0.16
MTT Optical Density
0.12
0.08
0.04
0.00
Untreated Mechanically Roughened CO2 Laser 1.6 kW/cm2 CO2 Laser 1.8 kW/cm2
Figure 8.22 MTT optical density of osteoblast cells grown on untreated and CO2 laser treated 316 LS stainless steel after 7 days of cell culture. There was a signicant statistical difference between the untreated sample and samples CO2 laser treated at laser power densities of 1.6 and 1.8 kW/cm2, and no statistical difference among the untreated sample and mechanically roughened samples ( p < 0.05)
cell adhesion. Moreover, the number of cells on the untreated 316 LS stainless steel sample is nearly the same as on the mechanically roughened sample, but much lower than on both of the CO2 laser treated samples (see Figure 8.21). It can therefore be propounded that the CO2 laser treatment of the 316 LS stainless steel surface has more of an effect on cell adhesion and growth than mechanical roughening of the surface and thus plays a more important role than the surface roughness alone. From the MTT results given in Figure 8.22, it can be seen that the mechanically roughened 316 LS stainless steel sample and both of the CO2 laser treated 316 LS stainless steel samples experienced better cell proliferation than the untreated sample. Further, Figure 8.22 shows that the cell proliferation increased as the CO2 laser power density increased. In addition, the statistical analysis revealed that cell proliferation improved signicantly on the CO2 laser treated samples compared with the untreated sample. A comparison of the cell proliferation on the untreated sample with the mechanically roughened sample revealed that, statistically, mechanical roughening of the surface produced no signicant increase. It therefore seems apparent from these results that the surface generated by the CO2 laser treatment was more favourable for cell proliferation than either the untreated or the mechanically roughened surfaces. In fact, the cells not only adhered better on the surface of the CO2 laser treated 316 LS stainless steel samples than on either the untreated or the
175
mechanically roughened samples (see Figure 8.21), but also grew better (see Figure 8.22). Additionally, the cell response increased with the power density of the CO2 laser treatment. The observation that the growth of osteoblast cells was marginally better on the mechanically roughened surface compared with the smoother, untreated 316 LS stainless steel surface is similar to that reported by Feng et al. [213], who noted that a rougher surface promotes more osteoblast-like attachment. This demonstrates that surface topography does have an effect on osteoblast cell proliferation. However, the surface generated by the CO2 laser treatment at a power density of 1.8 kW/ cm2 exhibited the best cell adhesion and proliferation, yet its surface was smoother than that of the mechanically roughened sample. The superior performance of the CO2 laser treatment 316 LS stainless steel sample in terms of cell adhesion and proliferation is most certainly due to the higher wettability characteristics of this sample. Furthermore, the results reveal that the cell proliferation increased as the wettability characteristics of the samples increased. Indeed, Hallab et al. [215] demonstrated that the surface free energy was a more important surface characteristic than surface roughness for cellular adhesion strength and proliferation. Schakenraad et al. [115] found that, despite the great number of parameters interfering with cellular adhesion and spreading, the solid surface energy is apparently a dominated factor in cellular attachment to a polymer surface and remains so, even if the solid surface has been covered by a protein layer. Previous work by Hao and Lawrence [247, 248] also showed that enhancement of the wettability characteristics of the MgOPSZ after CO2 laser treatment resulted in a better response of human broblast and human osteoblast cells. Owing to this, it is reasonable to postulate that the wettability characteristics of the 316 LS stainless steel play a vital role in initiating cell proliferation, and is the key factor in producing the observed improved cell adhesion and proliferation over mechanical roughening alone. 8.3.6 Generic Effects of CO2 Laser Treatment on Protein Adsorption and the Cell Response on Biometals As one can see from Figure 8.23, the CO2 laser surface treatment resulted in both the Ti6Al4V alloy and the 316 LS stainless steel displaying an increase in cell proliferation (higher MTT optical density). From the discussion in Section 8.3.4 it was established that CO2 laser surface treatment caused generic enhancement of the surface roughness and, perhaps more importantly, the wettability characteristics of these biometals. Furthermore, Chapter 7 and Section 8.3.2 revealed that favourable protein adsorption and improvements in the cell response of the Ti6Al4V alloy and the 316 LS stainless steel after the CO2 laser surface treatment were correlated directly to the CO2 laser induced enhancement of the surface roughness and
176
0.16
Ti6Al4V alloy Stainless steel
MTT Optical Density
0.12
0.08
0.04
0.00
Untreated
1.3 1.6 1.6 1.8 CO2 Laser (kW/cm2 ) CO2 Laser (kW/cm2 )
Figure 8.23 Cell cover density (hFOB osteoblast cell) on the Ti6Al4V alloy and the 316 LS stainless steel following CO2 laser treatment
wettability characteristics of these biometals. Because of the generic effects of the laser surface treatment on the surface roughness and wettability characteristics of other metals, it seems valid to say that improvements in the cell response of these biometals after the CO2 laser surface treatment are generic. 8.4 Summary To ascertain the presence of generic features of the CO2 laser surface processing technique for improving the biocompatibility of ceramic and metal biomaterials, surface modications of the YPSZ and the 316 LS stainless steel were conducted. Changes in the surface properties of the YPSZ and the 316 LS stainless steel following CO2 laser irradiation were analysed and the in vitro biological responses of the untreated and the laser modied materials were evaluated. It was found that improvement in the wettability characteristics of the YPSZ as a result of the CO2 laser treatment was primarily due to enhancep p ment of the surface energy, particularly gsv . This increase in gsv was attributed to microstructural changes induced on the surface of the YPSZ by the melting and re-solidication. An in vitro test using hFOB cells revealed that cell adhesion was better on CO2 laser treated samples than on the untreated YPSZ. Although microtopography, especially surface grooves, inuenced the osteoblast cell spreading, the results suggest that CO2 laser induced changes in the wettability characteristics of the YPSZ could be the main mechanism governing osteoblast cell adhesion.
Summary
177
For the MgOPSZ and YPSZ bioinert ceramics examined, the CO2 laser surface treatment of both caused a general reduction in y. This observation, coupled with the ndings of others, suggests that not only the CO2 laser surface treatment but also the surface treatment of a wide range of ceramic materials with a variety of lasers is inherently capable of effecting changes in wettability characteristics. Moreover, the ndings allow one to postulate that modication of the wettability characteristics of the ceramics are generic. It is possible to claim this because increases in the surface roughness, surface p oxygen content and gsv of both the MgOPSZ and YPSZ following the CO2 laser treatment altered in relation to one another, an occurrence that could only take place if the changes were generic. Further support for this proposition comes from consideration of the predominant factors governing modication of the wettability characteristics of both the MgOPSZ and YPSZ. In each case the predominant factor was identied as the increase in p p gsv . An increase in gsv for other ceramic materials has been found by others to be the predominant factor responsible for wettability characteristics modp ication. Again, the incidence of gsv being the primary mechanism in determining the wettability characteristics of such a wide range of ceramics following surface treatment with a number of different lasers could only be if the changes were actually generic. Improvements in the cell response of the MgOPSZ and YPSZ after the CO2 laser surface treatment were correlated directly to CO2 laser induced enhancement of the wettability characteristics. On account of the generic effects of the laser surface treatment on the surface roughness and wettability for other ceramics, the improvements in the cell response of the bioinert ceramics after the CO2 laser surface treatment appear to be generic. The predominant mechanism active in determining changes in the wettability characteristics of the 316 LS stainless steel following CO2 laser treatment was identied as being the increase in surface roughness, while p increases in the surface oxygen content and gsv were shown to be active to a lesser extent. An in vitro analysis using osteoblast cells showed better adhesion on the CO2 laser treated samples than on the untreated and mechanically roughened samples, suggesting that the surface properties generated by the CO2 laser treatment were more favourable for osteoblast cell adhesion. The proliferation of the osteoblast cells was found to be better on the mechanically roughened sample and both of the CO2 laser treated 316 LS stainless steel samples. Furthermore, cell proliferation increased as the wettability characteristics of the samples increased. Therefore, the increased wettability of the 316 LS stainless steel played a vital role in initiating cell proliferation and was the key factor in producing the observed improved cell adhesion and proliferation over mechanical roughening alone. The observed changes in the wettability characteristics of the Ti6Al4V alloy and the 316 LS stainless steel when surface treated with the CO2 laser
178
were deemed to be generic as the increases in the surface roughness, surface p oxygen content and gsv of both biometals appear to have altered in relation to one another. Additional verication of the generic nature of the wettability characteristics modication of both biometals comes from the fact that the leading aspect governing the modication of the wettability characteristics of both biometals was the increase in surface roughness. Since surface roughness is the predominant mechanism active in inuencing the wettability characteristics of other laser treated metallic materials, then it seems highly likely that the laser induced changes to the wettability characteristics of most metals will actually be generic. Favourable protein adsorption and improvements in the cell response of the Ti6Al4V alloy and the 316 LS stainless steel after the CO2 laser surface treatment were correlated directly to CO2 laser induced enhancement of the surface roughness and wettability characteristics of these biometals. Because of the generic effects of the laser surface treatment on the surface roughness and wettability characteristics of other metals, it seems valid to say that improvements in the cell response of these biometals after the CO2 laser surface treatment are generic.
Conclusions
To investigate the efcacy of the CO2 laser as a means for improving the bioactivity and biointegration of bone implants materials, work was conducted to alter the surface properties of two widely used bioinert ceramics, magnesiapartially stabilised zirconia (MgOPSZ) and yttriapartially stabilised zirconia (YPSZ), and two established biograde metals, Ti6Al4V alloy and 316 LS stainless steel. More specically, the ability of the CO2 laser to modify the wettability characteristics of the materials and induce functional groups, thereby allowing bone-like apatite formation, protein adsorption and cells to be manipulated, was studied. Valuable inroads have been made as a result of this work for establishing the CO2 laser as a novel and viable technique for improving the biocompatibility of implant materials. The CO2 laser surface treatment of the MgOPSZ brought about a reduction in the contact angle, y, formed between the MgOPSZ and the control test liquids, providing a clear indication that the wettability characteristics of the material were modied. Moreover, the extent of this wettability characteristics modication was demonstrated to be variable and controllable by means of manipulation of the CO2 laser operating parameters. Changes in the wettability characteristics of the MgOPSZ were attributed to the following factors: (a) an increase in surface roughness, (b) incorporation of oxygen at the MgOPSZ surface resulting from CO2 p laser treatment and (c) the increase in the polar component, gsv , of the surface energy resulting from the melting and re-solidication of the MgO p PSZ surface. In addition, gsv for the MgOPSZ was seen to increase as the crystal size and the presence of the tetragonal phase increased after the CO2 p laser treatment. Further analysis revealed that gsv , by way of the re-solidied microstructure, was the primary inuential factor governing changes in y and hence the wettability characteristics of the MgOPSZ. Incorporation of
180
Conclusions
oxygen at the surface was also shown to inuence, to a lesser extent, changes in the wettability characteristics, while surface roughness was found to play a very minor role in inducing changes in the wettability characteristics of the MgOPSZ. The bioactivity of the untreated and CO2 laser modied MgOPSZ was investigated in SBF, while protein adsorption and hFOB cells were used to examine the in vitro biological response. It was demonstrated that the CO2 laser treatment could improve the bioactivity of the MgOPSZ surface by generating functional groups to facilitate the formation of bone-like apatites. The apatite formed readily on the CO2 laser treated MgOPSZ samples, with relatively high amounts of hydroxyl groups being generated. In contrast, no apatite formation was observed on the untreated MgOPSZ samples and consequently few hydroxyl groups were generated. Further analysis revealed that the ZrOH groups on the surface of the CO2 laser treated MgOPSZ samples were the functional groups facilitating the apatite formation; the surface melting on the MgOPSZ induced by the CO2 laser processing provided the Zr4 ion and OH ion and in turn created the Zr OH group. Compared with the untreated MgOPSZ, the CO2 laser treatment brought about a thinner adsorbed albumin layer and a thicker adsorbed bronectin layer on the MgOPSZ surface. Whereas the albumin adsorption decreased, the bronectin increased with increased wettability, indicating that the wettability of the MgOPSZ was the predominant factor governing p protein adsorption. Further, the observed effect of gsv on protein adsorption implied that protein adsorption on the MgOPSZ surface was probably due to polar and chemical interactions. Better hFOB osteoblast cell responses were witnessed on the CO2 laser treated MgOPSZ samples in comparison with untreated samples. Generally, the cell cover density increased with increasing CO2 laser power density. The change in topography induced by the CO2 laser treatment is certain to be one of the factors inuencing the hFOB osteoblast, but its role will be minor. The improved wettability characteristics of the MgOPSZ due to enhanced surface energy brought p by the CO2 laser treatment, especially gsv , played a signicant role in precipitating initial cell attachment and spreading in high numbers, consequently enhancing long-term cell adhesion and growth. The CO2 laser surface treatment of the Ti6Al4V alloy brought about a reduction in the y formed between the Ti6Al4V alloy and the simulated physiological liquids, signifying that the wettability characteristics of the material were modied. It was found that modication of the surface roughness, surface oxygen content and surface energy of the Ti6Al4V alloy following the CO2 laser treatment were the factors inuencing the wettability characteristics. It was found that the wettability characteristics of the Ti6Al4V alloy were, after the surface roughness, inuenced by the surface oxygen content and, to some extent, by the microstructure. The
Conclusions
181
reductions in y occasioned by the CO2 laser treatment were found to contribute to an augmentation in the work adhesion of selected physiological liquids (SBF and SBF BSA) on the Ti6Al4V alloy. As both SBF and SBF BSA have close chemical compositions to human body uids, the increase in the work adhesion of the Ti6Al4V alloy surface towards these uids would mean better suitability of the Ti6Al4V alloy for use as a biomaterial after the CO2 laser treatment. Apatite formation on the untreated and CO2 laser treated Ti6Al4V alloy after soaking in SBF was used to investigate bioactivity. In addition, protein adsorption and the hFOB cell response were used to examine the in vitro biological response on the untreated and CO2 laser treated Ti6Al4V alloy. The fact that apatite nuclei were observed only on the CO2 laser modied Ti6Al4V alloy samples demonstrated that the CO2 laser treatment was capable of improving the bioactivity of the Ti6Al4V alloy; no apatite nuclei appeared on the untreated samples. It is believed that the CO laser-induced oxidised surface layer on the Ti6Al4V alloy generated the hydroxide ions in the water and resulted in the nucleation of the apatite. The CO2 laser treatment brought about a thinner adsorbed albumin layer and a thicker adsorbed bronectin layer on the Ti6Al4V alloy compared with the untreated samples. Moreover, the albumin adsorption was seen to decrease, while the bronectin increased, with increasing wettability of the Ti6Al4V alloy. This would suggest that the wettability characteristics of the Ti6Al 4V alloy are the chief driver for protein adsorption. Further, the observed p effect of gsv on protein adsorption implied that the protein adsorption on the Ti6Al4V alloy surface was most likely due to the polar and chemical interactions. One-day cell adhesion tests showed that cells not only adhered and spread better but also grew faster on the CO2 laser treated Ti6Al4V alloy sample than on either the untreated or mechanically roughened (the traditional method of improving cell adhesion) samples. Additionally, compared with the untreated sample, MTT cell proliferation analysis revealed that mechanical roughening of the surface of the Ti6Al4V alloy resulted in only a slight enhancement, while the CO2 laser treatment brought about a considerable increase. Although surface roughness is surely one of the factors inuencing cell adhesion and proliferation, certain other aspects of wettability characteristics surface oxygen content and were found to play an important role in promoting cell proliferation. Indeed, it was evident that the better wettability characteristics of the CO2 laser treated Ti6Al4V alloy were responsible for the improved MTT value. Thus it would be reasonable to maintain that a correlation exists between the CO2 laser induced wettability characteristics of the Ti6Al4V and the hFOB osteoblast cell bioactivity. Moreover, it is apparent from the results that the CO2 laser treatment could be a more effective way to improve osteoblast cell adhesion than the traditional methods currently available.
182
Conclusions
To determine the presence of generic features of the CO2 laser surface processing technique for improving the biocompatibility of ceramic and metal biomaterials, surface modications of the YPSZ and the 316 LS stainless steel were conducted. Changes in the surface properties of the YPSZ and the 316 LS stainless steel following the CO2 laser irradiation were analysed and the in vitro biological responses of the untreated and the laser modied materials were evaluated. The CO2 laser surface treatment of both the MgOPSZ and the YPSZ bioinert ceramics caused a general reduction in y, suggesting that CO2 laser induced changes to the wettability characteristics of these bioinert ceramic materials are generic. It is possible to claim this as increases in the surface p roughness, surface oxygen content and gsv of both the MgOPSZ and the Y PSZ following the CO2 laser treatment altered in relation to one another, an occurrence that could only take place if the changes were generic. The predominant factor governing modication of the wettability characteristics p of both the MgOPSZ and the YPSZ was identied as the increase in gsv , further reinforcing the proposition that the changes are generic. Improvements in the cell response of the MgOPSZ and the YPSZ after the CO2 laser surface treatment were correlated directly to CO2 laser induced enhancement of the wettability characteristics. On account of the generic effects of the CO2 laser surface treatment on the surface roughness and wettability, improvements in the cell response of the bioinert ceramics after the CO2 laser surface treatment appear to be generic. Observed changes in the wettability characteristics of the Ti6Al4V alloy and the 316 LS stainless steel when surface treated with the CO2 laser were deemed to be generic as increases in the surface roughness, surface oxygen p content and gsv of both biometals appear to have altered in relation to one another. Additional verication of the generic nature of the wettability characteristics modication of both biometals comes from the fact that the leading aspect governing the modication of the wettability characteristics of both biometals was the increase in surface roughness. Because surface roughness is the predominant mechanism active in inuencing the wettability characteristics of other laser treated metallic materials, it seems highly likely that the laser induced changes to the wettability characteristics of most metals will actually be generic. Favourable protein adsorption and improvements in the cell response of the Ti6Al4V alloy and the 316 LS stainless steel after the CO2 laser surface treatment were correlated directly to CO2 laser induced enhancement of the surface roughness and wettability characteristics of these biometals. Because of the generic effects of the laser surface treatment on the surface roughness and wettability characteristics of other metals, it seems valid to say that improvements in the cell response of these biometals after the CO2 laser surface treatment are generic.
Conclusions
183
The major focus of this work was the employment of a CO2 laser for the surface processing of bioinert ceramics and biograde metals that are widely used as load-bearing bone implants. This contemporary research, in conjunction with the ndings of others, rmly establishes the potential of the CO2 laser for improving the biocompatibility of a variety of other biomaterials.
References
1. P. I. Branemark, Osseointegration and its experimental background, Journal of Prosthetic Dentistry, 50, 399410 (1983). 2. R. E. Smallman, Modern Physical Metallurgy and Materials Engineering: Science, Process and Applications, Boston: Butterworth-Heinemann (1999), pp. 394402. 3. M. J. Yaszemski, R. G. Payne, W. C. Hayes, R. Langer and A. G. Mikos, Evolution of bone transplantation: molecular, cellular and tissue strategies to engineer human bone, Biomaterials, 17, 17585 (1996). 4. R. J. Friedman, J. Black, J. O. Galante, J. J. Jacobs and H. B. Skinner, Current concepts in orthopaedic biomaterials and implant xation, Journal of Bone and Joint Surgery, Series A, 75, 1086109 (1993). 5. G. W. Hastings and F. A. Mahmud, Intelligent orthopaedic materials, Journal of Intelligent Material Systems and Structures, 4, 4526 (1993). 6. P. Li, X. Ye, I. Kangasniemi, J. M. de Blieck-Hogervorst, C. P. Klein and K. de Groot, In vivo calcium phosphate formation induced by sol-gelprepared silica, Journal Of Biomedical Materials Research, 29, 3258 (1995). 7. M. Tanahashi, T. Yao, T. Kokubo, M. Minoda, T. Miyamoto, T. Nakamura and T. Yamamuro, Apatite coated on organic polymers by biomimetic process: improvement in its adhesion to substrate by glowdischarge treatment, Journal of Biomedical Materials Research, 29, 34957 (1995). 8. P. Li, C. Ohtsuki, T. Kokubo, K. Nakanishi, N. Soga, T. Nakamura and T. Yamamuro, Process of formation of bone-like apatite layer on silica gel, Journal of Materials Science: Materials in Medicine, 4, 12731 (1993). 9. D. A. Puleo and A. Nanci, Understanding and controlling the boneimplant interface, Biomaterials, 20, 231121 (1999).
186
References
10. C. D. McFarland, S. Mayer, C. Scotchford, B. A. Dalton, J. G. Steele and S. Downes, Attachment of cultured human bone cells to novel polymers, Journal of Biomedical Materials Research, 44, 111 (1999). 11. R. N. S. Sodhi, Application of surface analytical and modication techniques to biomaterial research, Journal of Electron Spectroscopy and Related Phenomena, 81, 26984 (1996). 12. E. A. Vogler, Structure and reactivity of water at biomaterial surfaces, Advances in Colloid and Interface Science, 74, 69117 (1998). 13. J. Lawrence and L. Li, Laser Modication of the Wettability Characteristics of Engineering Materials, London: Professional Engineering (2001). 14. R. G. Nuzzo, L. H. Dubois and D. L. Allara, Fundamental studies of microscopic wetting on organic surfaces. I. Formation and structural characterization of a self-consistent series of polyfunctional organic monolayers, Journal of the American Chemical Society, 112, 55869 (1990). 15. P. E. Laibinis, G. M. Whitesides, D. L. Allara, Y.-T. Tao, A. N. Parikh and R. G. Nuzzo, Comparison of the structures and wetting properties of self-assembled monolayers of n-alkanethiols on the coinage metal surfaces, Cu, Ag, Au, Journal of the American Chemical Society, 113(11), 715267 (1992). 16. W. M. Steen, Laser Material Processing, London: Springer-Verlag (1991). 17. H. Mirzadeh, A. A. Katbab and R. P. Burford, CO2-laser graft copolymerization of HEMA and NVP onto ethylenepropylene rubber (EPR) as biomaterial-(III), Radiation Physics and Chemistry, 46, 85962 (1995). 18. H. Mirzadeh, A. A. Katbab, M. T. Khorasani, R. P. Burford, E. Gorgin and A. Golestani, Cell attachment to laser-induced AAm- and HEMAgrafted ethylenepropylene rubber as biomaterial: in vivo study, Biomaterials, 16, 6418 (1995). 19. M. Dadsetan, H. Mirzadeh, N. Shari-Sanjani and M. Daliri, Cell behavior on laser surface-modied polyethylene terephthalate in vitro, Journal of Biomedical Materials Research, 57, 1839 (2001). 20. L. Hao and J. Lawrence, CO2 laser modication of the wettability characteristics of a magnesia partially stabilised zirconia (MgOPSZ) bioceramic, Journal of Physics D: Applied Physics, 36, 12929 (2003). 21. L. Hao and J. Lawrence, On the role of laser induced microstructures in inuencing the wettability characteristics of magnesia partially stabilised zirconia (MgOPSZ) bioceramic, Proceedings of the Institution of Mechanical Engineers, Part B: Journal of Engineering Manufacture, 218(B1), 5976 (2004). 22. L. Hao and J. Lawrence, Identication of the mechanisms governing modications of the wettability characteristics of a magnesia partially stabilised zirconia bioceramic following CO2 laser treatment, Journal of Laser Applications, 16, 2527 (2004).
References
187
23. L. Hao, J. Lawrence, D. K. Y. Low, K. S. Chian, G. C. Lim and H. Y. Zheng, The formation of a hydroxyl bond and the effects thereof on bone-like apatite formation on a magnesia partially stabilised zirconia (MgOPSZ) bioceramic following CO2 laser irradiation, Journal of Materials Science: Materials in Medicine, 15, 96775 (2004). 24. L. Hao and J. Lawrence, The adsorption of human serum albumin (HSA) on CO2 laser modied magnesia partially stabilised zirconia (MgO PSZ), Colloids and Surfaces B: Biointerfaces, 34, 8794 (2004). 25. L. Hao and J. Lawrence, On the role of CO2 laser treatment in the human serum albumin and human plasma bronectin adsorption on zirconia (MgOPSZ) bioceramic surface, Journal of Biomedical Materials Research, 69A, 74856 (2004). 26. L. Hao, D. R. Ma, J. Lawrence and X. Zhu, Enhancing osteoblast functions on a magnesia partially stabilised zirconia bioceramic by means of laser irradiation, Materials Science and Engineering C: Biomimetic and Supramolecular Systems, 25, 496502 (2005). 27. B. A. Dalton, C. D. McFarland, T. R. Gengenbach, H. J. Greisser and J. G. Steele, Polymer surface chemistry and bone cell migration, Journal of Biomaterials Science, Polymer Edition, 9, 78199 (1998). 28. K. E. Healy, C. H. Thomas, A. Rezania, J. E. Kim, P. J. McKeown, B. Lom and P. E. Hockberger, Kinetics of bone cell organization and mineralization on materials with patterned surface chemistry, Biomaterials, 17, 195208 (1996). 29. A. F. von Recum and T. G. van Kooten, The inuence of microtopography on cellular response and the implications for silicone implants, Journal of Biomaterials Science, Polymer Edition, 7, 18198 (1995). 30. D. F. Williams, Denitions in Biomaterials, Amsterdam: Elsevier (1987). 31. P. Thomsen and L. E. Ericson, Inammatory cell response to bone implant surfaces, in The BoneBiomaterial Interface (Ed. J. E. Davies), Toronto, Ontario, Canada: University of Toronto Press (1991), pp. 53 164. 32. R. R. Tarr and D. A. Wiss, The mechanics and biology of intramedullary fracture xation, Clinical Orthopaedics, 212, 107 (1986). 33. J. E. Davies, Early extracellular matrix synthesis by bone cells, in The BoneBiomaterial Interface (Ed. J. E. Davies), Toronto, Ontario, Canada: University of Toronto Press (1991), pp. 21425. 34. W. C. Vrouwenvelder, C. G. Groot and G. K. de Groot, Histological and biochemical evaluation of osteoblasts cultured on bioactive glass, hydroxylapatite, titanium alloy, and stainless steel, Journal of Biomedical Material Research, 27, 46575 (1993). 35. D. M. Brunette, In vitro models of biological responses to implants, Advanced Dental Research, 13, 357 (1999).
188
References
36. S. J. Northup, Mammmalian cell culture models, in Handbook of Biomaterials Evaluation: Scientic, Technical and Clinical Testing of Implant Materials (Ed. A. F. von Recum), New York: Macmillan (1986), pp. 209 25. 37. L. F. Cooper, T. Masuda, P. K. Yliheikkila and D. A. Felton, Generalizations regarding process and phenomenon of osseointegration. Part II. In vitro studies, International Journal of Oral and Maxillofacial Implants, 13, 16374 (1998). 38. K. G. Nichols and D. A. Puleo, Effect of metal ions on the formation and function of osteoclastic cells in vitro, Journal of Biomedical Materials Research, 35, 26571 (1997). 39. K. Gomi, B. Lowenberg, G. Shapiro and J. E. Davies, Resorption of sintered synthetic hydroxyapatite by osteoclasts in vitro, Biomaterials, 14, 916 (1993). 40. J. E. Davies, In vitro modeling of the bone/implant interface, The Anatomical Record, 245, 42645 (1996). 41. T. Kokubo, H. M. Kim and M. Kawashita, Novel bioactive materials with different mechanical properties, Biomaterials, 24, 216175 (2003). 42. C. Rey, Orthopedic biomaterials, bioactivity, biodegradation; a physicalchemical approach, Journal of Biomechanics, 31, 182 (1998). 43. T. Carlsson, B. Rostlund, T. Albrektson, P. Albrektson and I. Branemark, Osseointegration of titanium implants, Acta Orthopedic Scandinavian, 57, 285 (1986). 44. K. Anselme, Osteoblast adhesion on biomaterials, Biomaterials, 21, 667 81 (2000). 45. L. L. Hench, Biomaterials: a forecast for the future, Biomaterials, 19, 1419 23 (1998). 46. B. D. Boyan, T. W. Hummert, D. D. Dean and Z. Schwartz, Role of material surfaces in regulating bone and cartilage cell response, Biomaterials, 17, 13746 (1996). 47. K. D. Chesmel, C. C. Clark, C. T. Brighton and J. Black, Cellular responses to chemical and morphologic aspects of biomaterial surfaces. Part II. The biosynthetic and migratory response of bone cell populations, Journal of Biomedical Materials Research, 29, 110110 (1995). 48. W. J. Grzesik and P. G. Robey, Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro, Journal of Bone and Mineral Research, 9, 487 96 (1994). 49. A. Naji and M. F. Harmand, Study of the effect of the surface state on the cytocompatibility of a CoCr alloy using human osteoblasts and broblasts, Journal of Biomedical Materials Research, 24, 86171 (1990). 50. K. Anselme, M. Bigerelle, B. Noel, E. Dufresne, D. Judas, A. Iost and P. Hardouin, Qualitative and quantitative study of human osteoblast
References
189
51.
52.
53.
54.
55.
56.
57.
58. 59. 60.
61.
62.
adhesion on materials with various surface roughnesses, Journal of Biomedical Materials Research, 49, 15566 (2000). K. T. Bowers, J. C. Keller, B. A. Randolph, D. G. Wick and C. M. Michaels, Optimization of surface micromorphology for enhanced osteoblast responses in vitro, International Journal of Oral and Maxillofacial Implants, 7, 30210 (1992). J. Y. Martin, Z. Schwartz, T. W. Hummert, D. M. Schraub, J. Simpson, J. J. Lankford, D. D. Dean, D. L. Cochran and B. D. Boyan, Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63), Journal of Biomedical Materials Research, 29, 389401 (1995). C. M. Michaels, J. C. Keller, C. M. Stanford and M. Solursh, In vitro cell attachment of osteoblast-like cells to titanium, Journal of Dental Research, 68, 27886 (1989). J. Lincks, B. D. Boyan, C. R. Blanchard, C. H. Lohmann, Y. Liu, D. L. Cochran, D. D. Dean and Z. Schwartz, Response of MG63 osteoblast-like cells to titanium and titanium alloy is dependent on surface roughness and composition, Biomaterials, 19, 221932 (1998). J. L. Ong, D. L. Carnes, H. L. Cardenas and R. Cavin, Surface roughness of titanium on bone morphogenetic protein-2 treated osteoblast cells in vitro, Implant Dentistry, 6, 1924 (1997). U. Meyer, D. H. Szulczewski, K. Moeller, H. Heide and D. B. Jones, Attachment kinetics and differentiation of osteoblasts on different biomaterials, Cells and Materials, 3, 12940 (1993). K. Webb, V. Hlady and P. A. Tresco, Relationships among cell attachment, spreading, cytoskeletal organization and migration rate for anchorage-dependent cells on model surfaces, Journal of Biomedical Material Research, 49, 3628 (2000). B. Kasemo and J. Lausmaa, Surface science aspects on inorganic biomaterials, Critical Review of Biocomptability, 2, 33580 (1986). Y. Tamada and Y. Ikada, Cell adhesion to plasma-treated polymer surfaces, Polymer, 34, 220812 (1993). S. Fujimori, Surface characterization of titanium plates with different surface treatments and cellular proliferation and expression of osteoblast-like cells in vitro on their surface, Journal of Dental Materials, 14, 15568 (1995). M. Bosetti, E. Verne, M. Ferraris, A. Ravaglioli and M. Cannas, In vitro characterisation of zirconia coated by bioactive glass, Biomaterials, 22, 98794 (2001). D. R. Sumner, T. M. Turner, A. F. Purchio, W. R. Gombotz, R. M. Urban and J. O. Galante, Enhancement of bone ingrowth by transforming growth factor-beta, The Journal of Bone and Joint Surgery, American Volume, 77, 113547 (1995).
190
References
63. A. Piattelli, A. Scarano, M. Corigliano and M. Piattelli, Effects of alkaline phosphatase on bone healing around plasma-sprayed titanium implants: a pilot study in rabbits, Biomaterials, 17, 14439 (1996). 64. S. Q. Liu, Y. Ito and Y. Imanishi, Cell growth on immobilized cell growth factor. Part 5: interaction of immobilized transferrin with broblast cells, International Journal of Biological Macromolecules, 15, 2216 (1993). 65. B. D. Ratner, Biomaterials Science: An Introduction to Materials in Medicine, San Diego, California: Academic Press (1996), p. 484. 66. B. Kasemo and J. Gold, Implant surfaces and interface processes, Advances in Dental Research, 13, 820 (1999). 67. B. D. Ratner, The engineering of biomaterials exhibiting recognition and specicity, Journal of Molecular Recognition, 9, 61725 (1996). 68. A. S. Blawas and W. M. Reichert, Protein patterning, Biomaterials, 19, 595609 (1998). 69. S. F. Hulbert, The use of alumina and zirconia in surgical implants, in An Introduction to Bioceramics (Ed. J. Wilson), Singapore: World Scientic (1993), pp. 2540. 70. C. Piconi and G. Maccauro, Zirconia as a ceramic biomaterial, Biomaterials, 20, 125 (1999). 71. L. L. Hench, Bioceramic: from concept to clinic, Journal of the American Ceramic Society, 74, 1487510 (1991). 72. V. Saikko, Wear of polyethylene acetabular cups against zirconia femoral heads studies with a hip joint simulator, Wear, 176, 20712 (1994). 73. M. Oka, P. Kumar, K. Ikeuchi, T. Yamamuro and T. Nakamura, Low wear rate of UHMWPE against zirconia, in Bioceramics (Ed. T. Nakamura), Kyoto, Japan: Kyoto University Press (1992), pp. 3739. 74. M. Long and H. J. Rack, Titanium alloys in total joint replacement: a materials science perspective, Biomaterials, 19, 162139 (1998). 75. P. Christel, A. Meunier and A. J. C. Lee, Biological and Biomechanical Performance of Biomaterials, Amsterdam: Elsevier (1997), pp. 8186. 76. R. S. Benson, Use of radiation in biomaterials science, Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms, 191, 7527 (2002). 77. S. Mandl and B. Rauschenbach, Plasma immersion ion implantation. A new method for homogeneous surface modication of complex forms of medical implants. Biomedizinische Technik, 45, 1938 (2000). 78. P. K. Chu, J. Y. Chen, L. P. Wang and N. Huang, Plasma-surface modication of biomaterials, Materials Science and Engineering R: Reports, 36, 143206 (2002). 79. F. Z. Cui and Z. S. Luo, Biomaterials modication by ion-beam processing, Surface and Coatings Technology, 112, 27885 (1999).
References
191
80. B. Kasemo and J. Gold, Implant surfaces and interface process, Advance Dental Research, 13, 820 (1999). 81. A. C. Duncan, F. Weisbuch, F. Rouais, S. Lazare and C. Baquey, Laser microfabricated model surfaces for controlled cell growth, Biosensors and Bioelectronics, 17, 41326 (2002). 82. B. R. Ringeisen, D. B. Chrisey, A. Pique, H. D. Young, R. Modi, M. Bucaro, J. Jones-Meehan and B. J. Spargo, Generation of mesoscopic patterns of viable Escherichia coli by ambient laser transfer, Biomaterials, 23, 1616 (2002). 83. A. Prina Mello, M. A. Bari and P. J. Prendergast, A comparison of excimer laser etching and dry etching process for surface fabrication of biomaterials, Journal of Materials Processing Technology, 124, 28492 (2002). 84. C. M. Cotell, Pulsed laser deposition and processing of biocompatible hydroxylapatite thin lms, Applied Surface Science, 69, 1408 (1993). 85. M. D. Ball, S. Downes, C. A. Scotchford, E. N. Antonov, V. N. Bagratashvili, V. K. Popov, W.-J. Lo, D. M. Grant and S. M. Howdle, Osteoblast growth on titanium foils coated with hydroxyapatite by pulsed laser ablation, Biomaterials, 22, 33747 (2001). 86. L. DAlessio, D. Ferro, V. Marotta, A. Santagata, R. Teghil and M. Zaccagnino, Laser ablation and deposition of Bioglass 45S5 thin lms, Applied Surface Science, 183, 107 (2001). 87. L. DAlessio, R. Teghil, M. Zaccagnino, I. Zaccardo, D. Ferro and V. Marotta, Pulsed laser ablation and deposition of bioactive glass as coating material for biomedical applications, Applied Surface Science, 138139, 52732 (1999). 88. P. K. Wu, B. R. Ringeisen, J. Callahan, M. Brooks, D. M. Bubb, H. D. Wu, A. Pique, B. Spargo, R. A. McGill and D. B. Chrisey, The deposition, structure, pattern deposition, and activity of biomaterial thin-lms by matrix-assisted pulsed-laser evaporation (MAPLE) and MAPLE direct write, Thin Solid Films, 398399, 60714 (2001). 89. D. B. Chrisey, A. Pique, R. Modi, H. D. Wu, R. C. Y. Auyeung and H. D. Young, Direct writing of conformal mesoscopic electronic devices by MAPLE DW, Applied Surface Science, 168, 34552 (2000). 90. F. Villermaux, M. Tabrizian, L. H. Yahia, M. Meunier and D. L. Piron, Excimer laser treatment of NiTi shape memory alloy biomaterials, Applied Surface Science, 109110, 626 (1997). 91. T. M. Yue, J. K. Yu and H. C. Man, The effect of excimer laser surface treatment on pitting corrosion resistance of 316LS stainless steel, Surface and Coatings Technology, 137, 6571 (2001). 92. T. M. Yue, J. K. Yu, Z. Mei and H. C. Man, Excimer laser surface treatment of Ti6Al4V alloy for corrosion resistance enhancement, Materials Letters, 52, 20612 (2002).
192
References
93. P. Cheang, K. A. Khor, L. L. Teoh and S. C. Tam, Pulsed laser treatment of plasma-sprayed hydroxyapatite coatings, Biomaterials, 17, 19014 (1996). 94. J. T. Davies and E. K. Rideal, Interfacial Phenomena, New York: Academic Press (1963). 95. M. J. Jaycock and G. D. Partt, Chemistry of Interfaces, Chichester, Great Britain: John Wiley & Sons, Ltd (1981), pp. 23447. 96. W. A. Zisman, Inuence of constitution on adhesion, Industrial and Engineering Chemistry, 55, 1938 (1963). 97. W. A. Zisman, Relationship of equilibrium contact angle to liquid and solid constitution, in Contact Angle, Wettability and Adhesion. Advances in Chemistry Series 43 (Ed. R. F. Gould), Washington DC: American Chemical Society (1964), pp. 151. 98. P. R. Chidambaram, G. R. Edwards and D. I. Olson, Thermodynamic criterion to predict wettability at metalalumina interfaces, Metallurgical Transactions B: Process Metallurgy, 23, 21522 (1992). 99. N. K. Adam and G. E. P. Elliott, The effects of high temperature variations on contact angle measurements of vitreous enamels, Journal of the Chemical Society, 18, 220615 (1958). 100. F. M. Fowkes, Attractive forces at interface, Industrial Engineering Chemistry, 56, 4052 (1964). 101. J. R. Dann, Forces involved in the adhesive process. Part I. Critical surface tensions of polymeric solids as determined with polar liquids, Journal of Colloid and Interface Science, 32, 30220 (1970). 102. L. A. Girifalco and D. J. Good, A theory for the estimation of surface and interfacial energies. Part I. Derivation and application to interfacial tension, Journal of Physical Chemistry, 61, 9049 (1957). 103. R. J. Good and L. A. Girifalco, A theory of the estimation of surface and interfacial energies. Part III. Estimation of surface energies of solids from contact angle data, Journal of Physical Chemistry, 64, 5615 (1960). 104. R. E. Baier, Blood vessels: problems arising at the borders of natural and articial blood vessels, in Proceedings of the 8th Scientic Conference of Gesellschaft Deutscher Naturforscher, 1216 May 1976, Hamburg, Germany, pp. 455461, Berlin, Germany: Springer-Verlag (1977). 105. R. E. Baier and R. C. Dutton, Initial events in interactions of blood with a foreign surface, Journal of Biomedical Material Research, 3, 191 (1969). 106. R. E. Baier, The role of surface energy in thrombosis, Academic Medical, 257, 25772 (1972). 107. R. E. Baier, Surface properties inuencing biological adhesion, in Adhesion in Biological Systems (Ed. R. S. Manly), New York: Academic Press (1970), pp. 1548. 108. C. J. van Oss, C. F. Gillman and A. W. Neumann, Phagocytic Engulfment and Cell Adhesiveness, New York: Marcel Dekker (1975), pp. 7152.
References
193
109. C. J. van Oss, W. Zingg, O. S. Hum and A. W. Neumann, Platelet activation on agar/agarose gel surfaces: variation correlated with casting technique and hydrophobic/hydrophilic balance as reected in contact angle measurements, Thrombosis Research, 11, 18391 (1977). 110. E. A. Vogler, Interfacial chemistry in biomaterials science, in Wettability (Surfactant Science Series 49) (Ed. J. Berg), New York: Marcel Dekker (1993), pp. 184250. 111. B. V. Derjaguim and L. D. Landau, Theory of the stability of strongly charged lyophobic sols and of the adhesion of strongly charged particles in solution of electrolytes, Acta Physic Chemica, 14, 63362 (1941). 112. E. J. W. Verway and J. H. G. Overbeek, Theory of Stability of Lyophobic Colloids, Amsterdam: Elsevier (1948). 113. C. J. van Oss, R. J. Good and M. K. Chaudhury, The role of van der Waals forces and hydrogen bonds in hydrophobic interactions between biopolymers and low energy surfaces, Interface Science, 111, 3789 (1986). 114. M. G. Stewart, E. Moy, G. Chang, W. Zingg and A. W. Neumann, Thermodynamic model for cell spreading, Colloids Surfaces, 42, 21532 (1989). 115. J. M. Schakenraad, H. J. Busscher, C. R. H. Wildevuur and J. Arends, Inuence of substratum surface free energy on growth and spreading of human broblasts in the presence and absence of serum proteins, Journal of Biomedical Materials Research, 20, 77384 (1986). 116. J. S. Jeffrey and S. F. James, Chemically modied polymeric resins for high-performance liquid chromatography, Journal of Chromatography A, 522, 95105 (1990). 117. R. Bischoff and G. Bischoff, Chemical surface modications of silicone biomaterials, Journal of Biomechanics, 31, 58 (1998). 118. M. Iwaki, Ion surface treatments on organic materials, Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms, 175177, 36874 (2001). 119. J. Kurdi, H. Ardelean, P. Marcus, P. Jonnard and F. Are-Khonsari, Adhesion properties of aluminium-metallized/ammonia plasmatreated polypropylene: spectroscopic analysis (XPS, EXES) of the aluminium/polypropylene interface, Applied Surface Science, 189, 11928 (2002). 120. L. Carrino, G. Moroni and W. Polini, Cold plasma treatment of polypropylene surface: a study on wettability and adhesion, Journal of Materials Processing Technology, 121, 37382 (2002). 121. J. S. Cho, Y.-W. Beag, S. Han, K.-H. Kim, J. Cho and S.-K. Koh, Hydrophilic surface formation on materials and its applications, Surface and Coatings Technology, 128129, 6670 (2000). 122. K.-H. Kim, J.-S. Cho, D.-J. Choi and S.-K. Koh, Hydrophilic group formation and cell culturing on polystyrene Petri-dish modied by
194
References
123.
124.
125.
126.
127.
128. 129.
130.
131.
132.
133.
134.
ion-assisted reaction, Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms, 175177, 5427 (2001). V. Haddadi-Asl and R. P. Burford, Radiation graft modication of ethylenepropylene rubber. Part III. Effect on water uptake, wettability and biocompatibility, Radiation Physics and Chemistry, 47, 907 12 (1996). I. Mathieson and R. H. Bradley, Improved adhesion to polymers by UV/ozone surface oxidation, International Journal of Adhesion and Adhesives, 16, 2931 (1996). H.-Y. Nie, M. J. Walzak, B. Berno and N. S. McIntyre, Atomic force microscopy study of polypropylene surfaces treated by UV and ozone exposure: modication of morphology and adhesion force, Applied Surface Science, 144145, 62732 (1999). J. H. Lee, G. Khang, J. W. Lee and H. B. Lee, Interaction of different types of cells on polymer surfaces with wettability gradient, Journal of Colloid and Interface Science, 205, 32330 (1998). M. Vallet, B. Berge and L. Vovelle, Electrowetting of water and aqueous solutions on poly(ethylene terephthalate) insulating lms, Polymer, 37, 246570 (1996). J. Kappel, Excimers help ceramic stick together, Opto and Laser Europe, 34 (1998). J. Lawrence, L. Li and J. T. Spencer, The effects of high-power diode laser radiation on the wettability, adhesion and bonding characteristics of an alumina/silica-based oxide and vitreous enamel, Surface and Coatings Technology, 115, 27381 (1999). J. Lawrence and L. Li, Wettability characteristics of an Al2O3/SiO2based ceramic modied with CO2, Nd:YAG, excimer and highpower diode lasers, Journal of Physics D: Applied Physics, 32, 107582 (1999). J. Lawrence, L. Li and J. T. Spencer, Diode laser modication of ceramic material surface properties for improved wettability and adhesion, Applied Surface Science, 138139, 38893 (1999). J. Lawrence, Identication of the principal elements governing the wettability characteristics of ordinary Portland cement following high power diode laser surface treatment, Materials Science and Engineering A, Structural Materials: Properties, Microstructure and Processing, 356, 16272 (2003). J. Heitz, E. Arenholz, T. Kefer, D. Bauerle, H. Hibst and A. Hagemeyer, Enhanced adhesion of metal lms on PET after UV-laser treatment, Applied Physics A (Solids and Surfaces), A55, 3912 (1992). F. Henari and W. Blau, Excimer-laser surface treatment of metals for improved adhesion, Applied Optics, 34, 5814 (1995).
References
195
135. M. Olfert, R. E. Mueller, W. Duley, T. North, J. Hood and D. Sakai, Enhancement of adhesion in coated steels through excimer laser surfacing, Journal of Laser Applications, 8, 7987 (1996). 136. G. W. Critchlow, C. A. Cottam, D. M. Brewis and D. C. Emmony, Further studies into the effectiveness of CO2 laser treatment of metals for adhesive bonding, International Journal of Adhesion and Adhesives, 17, 14350 (1997). 137. W. Song, P. Zhu and K. Cui, Effect of Ni content on cracking susceptibility and microstructure of laser-clad FeCrNiBSi alloy, Surface and Coatings Technology, 80, 27982 (1996). 138. J. Lawrence and L. Li, Wettability characteristics of carbon steel modied with CO2, Nd:YAG, excimer and high power diode lasers, Applied Surface Science, 154155, 6649 (2000). 139. J. Li and G. W. Hastings, Oxide bioceramics: inert ceramic materials, in Medicine and Dentistry Handbook of Biomaterial Properties (Ed. G. W. Hastings), New York: Chapman & Hall (1998), pp. 34053. 140. T. J. Webster, R. W. Siegel and R. Bizios, Osteoblast adhesion on nanophase ceramics, Biomaterials, 20, 12217 (1999). 141. S. Agathopoulos and P. Nikolopoulos, Wettability and interfacial interactions in bioceramic-bodyliquid systems, Journal of Biomedical Materials Research, 29, 4219 (1995). 142. J. Lawrence, L. Li and J. T. Spencer, A two-stage ceramic tile grout sealing process using a high power diode laser. Part I. Grout development and materials characteristics, Optics and Laser Technology, 30, 205 14 (1998). 143. A. J. Kinloch, Adhesion and Adhesives: Science and Technology, New York: Chapman & Hall (1998), p. 30. 144. M. Ueki, M. Naka and I. Okamoto, Wettability of some metals against zirconia ceramics, Journal of Materials Science Letters, 5, 12612 (1986). 145. J. Li, Microscopic approach of adhesion and wetting of liquid metal on solid ionocovalent oxide surface, Rare Metals, 12, 8496 (1993). 146. Q. Song and A. N. Netravali, Excimer laser surface modication of ultrahigh-strength polyethylene bers for enhanced adhesion with epoxy resins. Part 1. Effect of laser operating parameters, Journal of Adhesion Science and Technology, 12, 95782 (1998). 147. Q. Song and A. N. Netravali, Excimer laser surface modication of ultrahigh-strength polyethylene bres for enhanced adhesion with epoxy resins. Part 2. Effect of treatment environment, Journal of Adhesion Science and Technology, 12, 9837 (1998). 148. K. Rivindram, J. Srinivasan and A. G. Marathe, Finite element study on the role of convection in laser surface melting, Numerical Heat Transfer, 26, 60118 (1994).
196
References
149. D. Morvan, F. D. Cipriani and P. Bournot, Thermocapillary convection during laser surface melting, International Journal of Heat and Mass Transfer, 37, 197383 (1994). 150. R. N. Wenzel, Resistance of solid surfaces to wetting by water, Industrial Engineering Chemistry, 28, 98894 (1936). 151. A. Feng, B. J. McCoy, Z. A. Munir and D. Cagliostro, Wettability of transition metal oxide surfaces, Materials Science and Engineering A, Structural Materials: Properties, Microstructure and Processing, 242, 506 (1998). 152. I. Spalding, Modern laser applications, Proceedings of the Institution of Mechanical Engineers, Part B: Management and Engineering Manufacture, 201, 16574 (1987). 153. X. M. Zhang, T. M. Yue and H. C. Man, Enhancement of ceramic-tometal adhesive bonding by excimer laser surface treatment, Materials Letters, 30, 32732 (1997). 154. J. Lawrence, On the predominant mechanisms active during the highpower diode-laser modication of the wettability characteristics of an SiO2/Al2O3 based ceramic material, Proceedings of the Royal Society of London, Series A: Mathematical, Physical and Engineering Sciences, 458, 244563 (2002). 155. D. K. Chattoraj and K. S. Birdi, Adsorption and the Gibbs Surface Excess, New York: Plenum Press (1984), p. 95. 156. Q. Liu, S. An and W. Qiu, Study on thermal expansion and thermal shock resistance of MgOPSZ, Solid State Ionics, 121, 615 (1999). 157. C. F. Grain, Phase relations in the ZrO2MgO system, Journal of the American Ceramic Society, 50, 288 (1967). 158. Y. T. Pei, J. H. Ouyang and T. C. Lei, Laser cladding of ZrO2(Ni alloy) composite coating, Surface and Coatings Technology, 81, 1315 (1996). 159. S. O. Chwa and A. Ohmori, The inuence of surface roughness of sprayed zirconia coatings on laser treatment, Surface and Coatings Technology, 148, 8794 (2001). 160. P. Scherrer, Estimation of size and internal structure of colloidal particles by means of Rontgen rays, Gottinger Nachrichten, 2, 98100 (1918). 161. K. Lu, Interfacial structural characteristics and grain-size limits in nanocrystalline materials crystallized from amorphous solids, Physical Review B, 51, 1827 (1995). 162. H. C. Man, X. M. Zhang, T. M. Yue and W. S. Lau, Excimer laser surface modication of engineering ceramics for adhesive bonding, Journal of Materials Processing Technology, 66, 1239 (1997). 163. A. Christensen and E. A. Carter, First-principles study of the surfaces of zirconia, Physical Review B (Condensed Matter), 58, 805064 (1998).
References
197
164. M. Amaral, M. A. Lopes, J. D. Santos and R. F. Silva, Wettability and surface charge of Si3N4bioglass composites in contact with simulated physiological liquids, Biomaterials, 23, 41239 (2002). 165. M. Lampin, R. Warocquier-Clerout, C. Legris, M. Degrange and M. F. Sigot-Luizard, Correlation between substratum roughness and wettability, cell adhesion, and cell migration, Journal of Biomedical Materials Research, 36, 99108 (1997). 166. M. Uchida, H.-M. Kim, T. Kokubo, F. Miyaji and T. Nakamura, Bonelike apatite formation induced on zirconia gel in a simulated body uid and its modied solutions. Journal of the American Ceramic Society, 84, 20414 (2001). 167. T. A. Horbett and L. A. Klumb, Cell culturing: surface aspects and considerations, in Interfacial Phenomena and Bioproducts (Eds J. L. Brash and P. W. Wojciechowski), New York: Marcel Dekker (1996), pp. 351 445. 168. J. Hong, J. Andersson, K. N. Ekdahl, G. Elgue, N. Axen, R. Larsson and B. Nilsson, Titanium is a highly thrombogenic biomaterial: possible implications for osteogenesis, Thrombosis and Haemostasis, 82, 5864 (1999). 169. P. Ducheyne and Q. Qiu, Bioactive ceramics: the effect of surface reactivity on bone formation and bone cell function, Biomaterials, 20, 2287303 (1999). 170. J. H. Lee, H. W. Jung, I.-K. Kang and H. B. Lee, Cell behaviour on polymer surfaces with different functional groups, Biomaterials, 15, 705 11 (1994). 171. C. A. Scotchford, M. Ball, M. Winkelmann, J. Voros, C. Csucs, D. M. Brunette, G. Danuser and M. Textor, Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein- and cell-interactions. Part II. Protein adsorptionand early cell interactions, Biomaterials, 24, 114758 (2003). 172. C. Vitale Brovarone, E. Verne, A. Krajewski and A. Ravaglioli, Graded coatings on ceramic substrates for biomedical applications, Journal of the European Ceramic Society, 21, 285562 (2001). 173. A. Rosengren, E. Pavlovic, S. Oscarsson, A. Krajewski, A. Ravaglioli and A. Piancastelli, Plasma protein adsorption pattern on characterized ceramic biomaterials, Biomaterials, 23, 123747 (2002). 174. Y. Josset, Z. OumHamed, A. Zarrinpour, M. Lorenzato, J. J. Adnet and D. Laurent-Maquin, In vitro reactions of human osteoblasts in culture with zirconia and alumina ceramics, Journal of Biomedical Materials Research, 47, 48193 (1999). 175. M. Uchida, H.-M. Kim, T. Kokubo, K. Tanaka and T. Nakamura, Structural dependence of apatite formation on zirconia gels in a simulated body uid, Journal of the Ceramic Society of Japan, 110, 7105 (2002).
198
References
176. M. Uchida, H.-M. Kim, T. Kokubo, M. Nawa, K. Tanaka and T. Nakamura, Apatite-forming ability of zirconia gels with different structures, in The 14th International Symposium on Ceramics in Medicine, Annual Meeting of the International Society for Ceramics in Medicine, 1417 November 2001, Palm Springs, California, pp. 126135, Zurich, Switzerland: Trans Tech Publications (2002). 177. M. Uchida, H.-M. Kim, T. Kokubo, S. Fujibayashi and T. Nakamura, Structural dependence of apatite formation on titania gels in a simulated body uid, Journal of Biomedical Materials Research, 64, 16470 (2003). 178. M. Uchida, H.-M. Kim, T. Kokubo, M. Nawa, T. Asano, K. Tanaka and T. Nakamura, Apatite-forming ability of a zirconia/alumina nanocomposite induced by chemical treatment, Journal of Biomedical Materials Research, 60, 27782 (2002). 179. M. Uchida, H.-M. Kim, F. Miyaji, T. Kokubo and T. Nakamura, Apatite formation on zirconium metal treated with aqueous NaOH, Biomaterials, 23, 3137 (2002). 180. Z. Qian and J. L. Shi, Characterization of pure and doped zirconia nanoparticles with infrared transmission spectroscopy, Nanostructured Materials, 10, 23544 (1998). 181. X. C. Yang, W. Riehemann, M. Dubiel and H. Hofmeister, Nanoscaled ceramic powders produced by laser ablation, Materials Science and Engineering B: Solid-State Materials for Advanced Technology, 95, 299 307 (2002). 182. M. Cochez, M. Ferriol, P. Bourson and M. Aillerie, Inuence of the dopant concentration on the OH absorption band in Fe- doped LiNbO3 single-crystal bers, Optical Materials, 21, 77581 (2002). 183. D. W. Zeng, K. C. Yung and C. S. Xie, UV Nd:YAG laser ablation of copper: chemical states in both crater and halo studied by XPS, Applied Surface Science, 217, 17080 (2003). 184. B.-S. Lee, Y.-L. Hung and W.-H. Lan, Compositional and morphological changes of human dentin after Er:YAG laser irradiation, International Congress Series, 1248, 14352 (2003). 185. K. T. Jung and A. T. Bell, The effects of synthesis and pretreatment conditions on the bulk structure and surface properties of zirconia, Journal of Molecular Catalysis A: Chemical, 163, 2742 (2000). 186. L. Malicsko and A. Watterich, Changes of OH bands induced by laser irradiation in LiNbO3:Fe single crystals, Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms, 191, 1069 (2002). 187. A. A. Tsyganenko and V. N. Filimonov, Infrared spectra of surface hydroxyl groups and crystalline structure of oxides, Spectroscopy Letters, 5, 47787 (1972).
References
199
188. S. Takeda, K. Yamamoto, Y. Hayasaka and K. Matsumoto, Surface OH group governing wettability of commercial glasses, Journal of Noncrystalline Solids, 249, 416 (1999). 189. S. Takeda, M. Fukawa, Y. Hayashi and K. Matsumoto, Surface OH group governing adsorption properties of metal oxide lms, Thin Solid Films, 339, 2204 (1999). 190. B. Janczuk, J. M. Bruque, M. L. Gonzalez-Martin and J. M. del Pozo, Determination of components of cassiterite surface free energy from contact angle measurements, Journal of Colloid and Interface Science, 161, 20922 (1993). 191. M. L. Gonzalez-Martin, L. Labajos-Broncano, B. Janczuk and J. M. Bruque, Wettability and surface free energy of zirconia ceramics and their constituents, Journal of Materials Science, 34, 59236 (1999). 192. B. Janczuk, M. L. Gonzalez-Martin and J. M. Bruque, Inuence of mixture anionic and non-ionic surfactants on the surface free energy of cassiterite, Journal of Materials Science, 29, 317784 (1994). 193. B. Feng, J. Y. Chen, S. K. Qi, L. He, J. Z. Zhao and X. D. Zhang, Characterization of surface oxide lms on titanium and bioactivity, Journal of Materials Science: Materials in Medicine, 13, 45764 (2002). 194. D. A. Puleo and R. Bizios, Mechanisms of bronectin-mediated attachment of osteoblasts to substrates in vitro, Bone and Mineral, 18, 21526 (1992). 195. M. Taborelli, L. Eng, P. Descouts, J. P. Ranieri, R. Bellamkonda and P. Aebischer, Bovine serum albumin conformation on methyl and amine functionalized surfaces compared by scanning force microscopy, Journal of Biomedical Materials Research, 29, 70714 (1995). 196. P. Ying, Y. Yu, G. Jin and Z. Tao, Competitive protein adsorption studied with atomic force microscopy and imaging ellipsometry, Colloids and Surfaces B: Biointerfaces, 32, 110 (2003). 197. G. Altankov, F. Grinnell and T. Groth, Fibronectin matrix formation and signaling via integrins on biomaterials, in Proceedings of the 1996 5th World Biomaterials Congress. Part 1 (of 2), 29 May2 June 1996, Toronto, Ontario, Canada, pp. 25769, St Louis Park, Minnesota: Society for Biomaterials (1996). 198. C. Galli, M. Collaud Coen, R. Hauert, V. L. Katanaev, P. Groning and L. Schlapbach, Creation of nanostructures to study the topographical dependency of protein adsorption, Colloids and Surfaces B: Biointerfaces, 26, 25567 (2002). 199. J. Olsson, A. Carlen, N. L. Burns and K. Holmberg, Modied pellicle formation and reduced in vitro bacterial adherence after surface treatment with different siloxane polymers, Colloids and Surfaces B: Biointerfaces, 5, 1619 (1995).
200
References
200. D. D. Deligianni, N. Katsala, S. Ladas, D. Sotiropoulou, J. Amedee and Y. F. Missirlis, Effect of surface roughness of the titanium alloy Ti6Al4V on human bone marrow cell response and on protein adsorption, Biomaterials, 22, 124151 (2001). 201. A. P. Serro, A. C. Fernandes, B. Saramago and W. Norde, Bovine serum albumin adsorption on titania surfaces and its relation to wettability aspects, Journal of Biomedical Materials Research, 46, 37681 (1999). 202. D. E. MacDonald, B. Marcovic, M. Allen, P. Somasundaran and A. L. Boskey, Surface analysis of human plasma bronectin adsorbed to commercially pure titanium materials, Journal of Biomedical Materials Research, 41, 12030 (1998). 203. P. Francois, P. Vaudaux, M. Taborelli, M. Tonetti, D. P. Lew and P. Descouts, Inuence of surface treatments developed for oral implants on the physical and biological properties of titanium. Part 2. Adsorption isotherms and biological activity of immobilized bronectin, Clinical Oral Implant Research, 8, 21725 (1997). 204. F. Grinnell and M. K. Feld, Fibronectin adsorption on hydrophilic and hydrophobic surfaces detected by antibody binding and analyzed during cell adhesion in serum-containing medium, The Journal of Biological Chemistry, 257, 488893 (1982). 205. B. Feng, J. Weng, B. C. Yang, J. Y. Chen, J. Z. Zhao, L. He, S. K. Qi and X. D. Zhang, Surface characterization of titanium and adsorption of bovine serum albumin, Materials Characterization, 49, 12937 (2002). 206. D. A. Puleo and R. Bizios, Formation of focal contacts by osteoblasts cultured on orthopedic biomaterials, Journal of Biomedical Materials Research, 26, 291301 (1996). 207. F. Grinnell and D. G. Hays, Induction of cell spreading by substratumadsorbed ligands directed against the cell surface, Experimental Cell Research, 116, 27584 (1978). 208. K. Burridge, K. Fath, T. Kelly, G. Nuckolls and C. Turner, Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton, Annual Review of Cell Biology, 4, 487525 (1988). 209. G. Heinrich, T. Grogler, R. F. Singer and S. M. Rosiwal, CVD diamond coated titanium alloys for biomedical and aerospace applications, Surface and Coatings Technology, 9495, 51420 (1997). 210. H. Liao, A.-S. Andersson, D. Sutherland, S. Petronis, B. Kasemo and P. Thomsen, Response of rat osteoblast-like cells to microstructured model surfaces in vitro, Biomaterials, 24, 64954 (2003). 211. D. E. MacDonald, B. E. Rapuano, N. Deo, M. Stranick, P. Somasundaran and A. L. Boskey, Thermal and chemical modication of titanium aluminumvanadium implant materials: effects on surface properties, glycoprotein adsorption and MG63 cell attachment, Biomaterials, 25, 313546 (2004).
References
201
212. K. Anselme, M. Bigerelle, B. Noel, E. Dufresne, D. Judas, A. Lost and P. Hardouin, Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses, Journal of Biomedical Materials Research, 49, 15566 (2000). 213. B. Feng, J. Weng, B. C. Yang, S. X. Qu and X. D. Zhang, Characterization of surface oxide lms on titanium and adhesion of osteoblast, Biomaterials, 24, 466370 (2003). 214. B. Chehroudi, J.-L. Qu and D. M. Brunette, Effects of implant surface topography on osteogenesis, in Proceedings of the 1996 5th World Biomaterials Congress. Part 1 (of 2), 29 May2 June 1996, Toronto, Ontario, Canada, pp. 44452, St Louis Park, Minnesota: Society for Biomaterials (1996). 215. N. J. Hallab, K. J. Bundy, K. OConnor, R. L. Moses and J. J. Jacobs, Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion, Tissue Engineering, 7, 5571 (2001). 216. J. E. Davies, The importance and measurement of surface charge species in cell behavior at the biomaterial interface, in Surface Characterization of Biomaterials (Ed. B. D. Ratner), Amsterdam: Elsevier (1988), pp. 21922. 217. C. A. Scotchford, E. Cooper, G. J. Leggett and S. Downes, Growth of human osteoblast-like cells on alkanethiol on gold self-assembled monolayers: the effect of surface chemistry, Journal of Biomedical Materials Research, 41, 43142 (1998). 218. S. A. Redey, M. Nardin, D. Bernache-Assolant, C. Rey, P. Delannoy, L. Sedel and P. J. Marie, Behavior of human osteoblastic cells on stoichiometric hydroxyapatite and type A carbonate apatite: role of surface energy, Journal of Biomedical Materials Research, 50, 35364 (2000). 219. R. L. Price, M. C. Waid, K. M. Haberstroh and T. J. Webster, Selective bone cell adhesion on formulations containing carbon nanobers, Biomaterials, 24, 187787 (2003). 220. K. Webb, V. Hlady and P. A. Tresco, Relative importance of surface wettability and charged functional groups on NIH 3T3 broblast attachment, spreading, and cytoskeletal organization, Journal of Biomedical Materials Research, 41, 42230 (1998). 221. K. Anselme, P. Linez, M. Bigerelle, D. Le Maguer, A. Le Maguer, P. Hardouin, H. F. Hildebrand, A. Iost and J. M. Leroy, The relative inuence of the topography and chemistry of TiAl6V4 surfaces on osteoblastic cell behaviour, Biomaterials, 21, 156777 (2000). 222. C. Larsson, P. Thomsen, B.-O. Aronsson, M. Rodahl, J. Lausmaa, B. Kasemo and L. E. Ericson, Bone response to surface-modied titanium implants: studies on the early tissue response to machined and electropolished implants with different oxide thicknesses, Biomaterials, 17, 60516 (1996).
202
References
223. M. Vallet-Regi, Ceramics for medical applications, Journal of the Chemical Society Delton Transactions, 2, 97108 (2001). 224. N. N. Aldini, M. Fini, G. Giavaresi, L. Martini, B. Dubini, M. G. PonziBossi, F. Rustichelli, A. Krajewski, A. Ravaglioli, M. Mazzocchi and R. Giardino, Osteointegration of bioactive glass-coated and uncoated zirconia in osteopenic bone: an in vivo experimental study, Journal of Biomedical Materials Research, 68, 26472 (2004). 225. N. N. Aldini, M. Fini, G. Giavaresi, P. Torricelli, L. Martini, R. Giardino, A. Ravaglioli, A. Krajewski, M. Mazzocchi, B. Dubini, M. G. Ponzi-Bossi, F. Rustichelli and V. Stanic, Improvement in zirconia osseointegration by means of a biological glass coating: an in vitro and in vivo investigation, Journal of Biomedical Materials Research, 61, 2829 (2002). 226. P. Torricelli, E. Verne, C. V. Brovarone, P. Appendino, F. Rustichelli, A. Krajewski, A. Ravaglioli, G. Pierini, M. Fini, G. Giavaresi and R. Giardino, Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone, Biomaterials, 22, 253543 (2001). 227. H.-S. Ryu, K. S. Hong, J.-K. Lee, D. J. Kim, J. H. Lee, B.-S. Chang, D.-H. Lee, C.-K. Lee and S.-S. Chung, Magnesia-doped HA/[beta]-TCP ceramics and evaluation of their biocompatibility, Biomaterials, 25, 393401 (2004). 228. T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi and T. Yamamuro, Solutions able to reproduce in vivo surface-structure changes in bioactive glassceramic A-W3, Journal of Biomedical Materials Research, 24, 72134 (1990). 229. M. R. Filgueiras, G. La Torre and L. L. Hench, Solution effects on the surface reactions of a bioactive glass, Journal of Biomedical Materials Research, 27, 44553 (1993). 230. J. Gil-Albarova, R. Garrido-Lahiguera, A. J. Salinas, J. Roman, A. L. Bueno-Lozano, R. Albarova and M. M. Vallet-Regi, The in vivo performance of a solgel glass and a glassceramic in the treatment of limited bone defects, Biomaterials, 25, 463945 (2004). 231. M. Kobayashi, T. Kikutani, T. Kokubo and T. Nakamura, Direct bone formation on alumina bead composite, Journal of Biomedical Materials Research, 37, 55465 (1997). 232. S. Nishiguchi, T. Nakamura, M. Kobayashi, H.-M. Kim, F. Miyaji and T. Kokubo, The effect of heat treatment on bone-bonding ability of alkali-treated titanium, Biomaterials, 20, 491500 (1999). 233. P. E. Baldwin, The determination of cell behavior at the biomaterial interface, in Surface Characterization of Biomaterials, Progress in Biomedical Engineering (Ed. B. D. Ratner), Amsterdam: Elsevier (1998), pp. 21934. 234. P. B. van Wachem, T. Beugeling, J. Feijen, A. Bantjes, J. P. Detmers and W. G. van Aken, Interaction of cultured human endothelial cells
References
203
235.
236.
237.
238.
239.
240.
241.
242.
243.
244.
245.
246.
with polymeric surfaces of different wettabilities, Biomaterials, 6, 4038 (1985). C. C. Annarelli, J. Fornazero, R. Cohen, J. Bertand and J. L. Besse, Colloidal protein solutions as a new standard sensor for adhesive wettability measurements, Journal of Colloid and Interface Science, 213, 38694 (1999). J. Lawrence and L. Li, On the mechanisms of wetting characteristics modication for selected metallic materials by means of high power diode laser radiation, Journal of Laser Applications, 14, 10713 (2002). A. W. Neumann and R. J. Good, Thermodynamics of contact angles. Part I. Heterogeneous solid surfaces, Journal of Colloid Interface Science, 38, 34158 (1972). F. J. Gil, A. Padros, J. M. Manero, C. Aparicio, M. Nilsson and J. A. Planell, Growth of bioactive surfaces on titanium and its alloys for orthopaedic and dental implants, Materials Science and Engineering C: Biometric and Supramolecular Systems, 22, 5360 (2002). T. Kokubo, F. Miyaji, K. Hyun-Min and T. Nakamura, Spontaneous formation of bonelike apatite layer on chemically treated titanium metals, Journal of the American Ceramic Society, 79, 11279 (1996). C. M. Michaels, J. C. Keller and C. M. Stanford, In vitro periodontal ligament broblast attachment to plasma-cleaned titanium surfaces, The Journal of Oral Implantology, 17, 1329 (1991). D. Kawahara, J. L. Ong, G. N. Raikar, L. C. Lucas, J. E. Lemons and M. Nakamura, Surface characterization of radio-frequency glow discharged and autoclaved titanium surfaces, The International Journal of Oral and Maxillofacial Implants, 11, 43542 (1996). M. Bigerelle, K. Anselme, B. Noel, I. Ruderman, P. Hardouin and A. Iost, Improvement in the morphology of Ti-based surfaces: a new process to increase in vitro human osteoblast response, Biomaterials, 23, 156377 (2002). K. Suzuki, K. Aoki and K. Ohya, Effects of surface roughness of titanium implants on bone remodelling activity of femur in rabbits, Bone, 21, 507 14 (1997). P. R. Klokkevold, R. D. Nishimura, M. Adachi and A. Caputo, Osseointegration enhanced by chemical etching of the titanium surface. A torque removal study in the rabbit, Clinical Oral Implants Research, 8, 4427 (1997). V. M. Goldberg, S. Stevenson, J. Feighan and D. Davy, Biology of gritblasted titanium alloy implants, Clinical Orthopaedics and Related Research, 1229 (1995). S. Vercaigne, J. G. C. Wolke, I. Naert and J. A. Jansen, Histomorphometrical and mechanical evaluation of titanium plasma-spray-coated
204
References
247.
248.
249.
250.
251. 252.
253.
254.
255.
256.
257. 258.
implants placed in the cortical bone of goats, Journal of Biomedical Materials Research, 41, 418 (1998). L. Hao and J. Lawrence, Effects of CO2 laser irradiation on the wettability and human skin broblast cell response of magnesia partially stabilised zirconia, Materials Science and Engineering C: Biometric and Supramolecular Systems, 23, 62739 (2003). L. Hao, J. Lawrence and K. S. Chian, On the effects of CO2 laser irradiation on the surface properties of a magnesia partially stabilised zirconia (MgOPSZ) bioceramic and the subsequent improvements in human osteoblast cell adhesion, Journal of Biomaterials Application, 19, 81105 (2004). X.-X. Wang, W. Yan, S. Hayakawa, K. Tsuru and A. Osaka, Apatite deposition on thermally and anodically oxidized titanium surfaces in a simulated body uid, Biomaterials, 24, 46317 (2003). X. Lu, Y. Leng, X. D. Zhang, J. R. Xu, L. Qin and C. W. Chan, Comparative study of osteoconduction on micromachined and alkalitreated titanium alloy surfaces in vitro and in vivo, Biomaterials, 26, 1793 801 (2005). J. E. Davies, In vitro modelling of the bone/implant interface, The Anatomical Record, 245, 42645 (1996). K. E. Healy and P. Ducheyne, Mechanisms of passive dissolution of titanium in a model physiological environment, Journal of Biomedical Materials Research, 26, 31938 (1992). P. Li, C. Ohtsuki, T. Kokubo, K. Nakanishi, N. Soga and K. de Groot, The role of hydrated silica, titania and alumina in inducing apatite on implants, Journal of Biomedical Materials Research, 28, 715 (1994). D. E. MacDonald, B. E. Rapuano, N. Deo, M. Stranick, P. Somasundaran and A. L. Boskey, Thermal and chemical modication of titanium aluminumvanadium implant materials: effects on surface properties, glycoprotein adsorption and MG63 cell attachment, Biomaterials, 25, 313546 (2004). B. D. Ratner, D. G. Castner, T. A. Horbett, T. J. Lenk, K. B. Lewis and R. J. Rapoza, Biomolecules and surfaces, Journal of Vacuum Science and Technology A: Vacuum, Surfaces, and Films, 8, 230617 (1990). I. Milosev, M. Metikos-Hukovic and H.-H. Strehblow, Passive lm on orthopaedic TiAlV alloy formed in physiological solution investigated by X-ray photoelectron spectroscopy, Biomaterials, 21, 210313 (2000). R. K. Schenk and D. Buser, Osseointegration: a reality, Periodontol 2000, 17, 2235 (1998). K. Donath, Vergleichende histologische Untersuchung verschiedener enossaler Implantattypen, Zeitschrift Zahnaerztl Implantol, 4, 1068 (1998).
References
205
259. J. E. Davies, Mechanisms of endosseous integration, International Journal of Prosthodontics, 11, 391401 (1998). 260. J. B. Brunski, In vivo bone response to biomechanical loading at the bone/dental-implant interface, Advance Dental Research, 13, 99119 (1999). 261. Y.-S. Chang, H.-O. Gu, M. Kobayashi and M. Oka, Inuence of various structure treatments on histological xation of titanium implants, The Journal of Arthroplasty, 13, 81625 (1998). 262. D. D. Dlima, S. M. Lemperle, P. C. Chen, R. E. Holmes and C. W. Colwell, Bone response to implant surface morphology, The Journal of Arthroplasty, 13, 92834 (1998). 263. J. Li, H. Liao, B. Fartash, L. Hermansson and T. Johnsson, Surfacedimpled commercially pure titanium implant and bone ingrowth, Biomaterials, 18, 6916 (1997). 264. Z. Schwartz and B. D. Boyan, Underlying mechanisms at the bone biomaterial interface, Journal of Cellular Biochemistry, 56, 3407 (1994). 265. K. A. Thomas, J. F. Kay, S. D. Cook and M. Jarcho, The effect of surface macrotexture and hydroxylapatite coating on the mechanical strengths and histologic proles of titanium implant materials, Journal of Biomedical Materials Research, 21, 1395414 (1987). 266. D. Buser, R. K. Schenk, S. Steinemann, J. P. Fiorellini, C. H. Fox and H. Stich, Inuence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs, Journal of Biomedical Materials Research, 25, 889902 (1991). 267. K. A. Thomas and S. D. Cook, Relationship between surface characteristics and the degree of boneimplant integration, Journal of Biomedical Materials Research, 26, 8313 (1992). 268. J. E. Feighan, V. M. Goldberg, D. Davy, J. A. Parr and S. Stevenson, The inuence of surface-blasting on the incorporation of titanium-alloy implants in a rabbit intramedullary model, Journal of Bone and Joint Surgery, Series A, 77, 138095 (1995). 269. H. E. Gotz, M. Muller, A. Emmel, U. Holzwarth, R. G. Erben and R. Stangl, Effect of surface nish on the osseointegration of laser-treated titanium alloy implants, Biomaterials, 25, 405764 (2004). 270. D. Li, S. J. Ferguson, T. Beutler, D. L. Cochran, C. Sittig, H. P. Hirt and D. Buser, Biomechanical comparison of the sandblasted and acid-etched and the machined and acid-etched titanium surface for dental implants, Journal of Biomedical Materials Research, 60, 32532 (2002). 271. Y.-T. Sul, C. B. Johansson, Y. S. Jeong, A. Wennerberg and T. Albrektsson, Resonance frequency and removal torque analysis of implants with turned and anodized surface oxides, Clinical Oral Implants Research, 13, 2529 (2002).
206
References
272. X. Zhu, J. Chen, L. Scheideler, R. Reichl and J. Geis-Gerstorfer, Effects of topography and composition of titanium surface oxides on osteoblast responses, Biomaterials, 25, 4087103 (2004). 273. Y. Yang, J. L. Ong and J. Tian, In vivo evaluation of modied titanium implant surfaces produced using a hybrid plasma spraying processing, Materials Science and Engineering C: Biometric and Supramolecular Systems, 20, 11724 (2002). 274. M. Morra, C. Cassinelli, G. Cascardo, P. Cahalan, L. Cahalan, M. Fini and R. Giardino, Surface engineering of titanium by collagen immobilization. Surface characterization and in vitro and in vivo studies, Biomaterials, 24, 463954 (2003). 275. J. Schwartz, M. J. Avaltroni, M. P. Danahy, B. M. Silverman, E. L. Hanson, J. E. Schwarzbauer, K. S. Midwood and E. S. Gawalt, Cell attachment and spreading on metal implant materials, Materials Science and Engineering C: Biometric and Supramolecular Systems, 23, 395400 (2003). 276. Y. Wu, B. Yang, J. Chen and X. Zhang, In vivo and in vitro studies on the bioactivity of anodic oxidized titanium metal, Key Engineering Materials, 2846 (2004). 277. L. Hao, Y. F. Phua, M. W. Koo, J. Lawrence and L. Li, The wettability modication of bio-grade stainless steel in contact with simulated physiological liquids by the means of laser irradiation, in International Conference on European Materials Research Society Spring Meeting (E-MRS): Symposium N, Laser Interactions in Materials: Nanoscale to Mesoscale, 2426 May 2004, Strasbourg, France. 278. L. Hao, T. H. Wang, J. Lawrence and L. Li, The manipulation of the osteoblast cell response to the Ti6Al4V titanium alloy using high power diode laser (HPDL), in International Conference on European Materials Research Society Spring Meeting (E-MRS): Symposium N, Laser Interactions in Materials: Nanoscale to Mesoscale, 2426 May 2004, Strasbourg, France. 279. P. S. Christel, Zirconia. The second generation of ceramics for total hip replacement, Bulletin of the Hospital for Joint Diseases Orthopaedic Institute, 49, 1707 (1989). 280. M. Fini, N. Aldini, M. G. Gandol, M. Mattioli, M. Belmonte, G. Giavaresi, C. Zucchini, A. De Benedittis, S. Amati, A. Ravaglioli and E. A. Krayewski, Biomaterials for orthopedic surgery in osteoporotic bone: a comparative study in osteopenic rats, The International Journal of Articial Organs, 20, 2917 (1997). 281. D. Triantafyllidis, L. Li and F. H. Stott, The effects of laser-induced modication of surface roughness of Al2O3-based ceramics on uid contact angle, Materials Science and Engineering A, Structural Materials: Properties, Microstructure and Processing, 390, 2717 (2005).
References
207
282. T. Uelzen and J. Muller, Wettability enhancement by rough surfaces generated by thin lm technology, Thin Solid Films, 434, 3115 (2003). 283. Z. Liu, Surface modication of materials using high power lasers and an arc image intensier, PhD Thesis, University of Liverpool (1991). 284. R. W. McCallum, M. J. Kramer and S. T. Weir, Phase diagram effects in rapid thermal processing of REBa2Cu3O7-d, IEEE Transactions on Applied Superconductivity, 3, 11479 (1993). 285. L. Hao and J. Lawrence, CO2 laser induced microstructural features in a magnesia partially stabilised zirconia bioceramic and the effects thereof on wettability characteristics, Materials Science and Engineering A, Structural Materials: Properties, Microstructure and Processing, 364, 17181 (2003). 286. A. W. Neumann, Contact angles and their temperature dependence: thermodynamic status, measurement, interpretation and application, Advances in Colloid and Interface Science, 4, 10691 (1974). 287. J. Lawrence, Wetting and bonding characteristics of selected liquidmetals with a high power diode laser treated alumina bioceramic, Proceedings of the Royal Society, Series A: Mathematical, Physical and Engineering Sciences, 460, 172335 (2004). 288. L. Bradley, L. Li and F. H. Stott, Characteristics of the microstructures of alumina-based refractory materials treated with CO2 and diode lasers, Applied Surface Science, 138139, 2339 (1999). 289. J. Lawrence, An analysis of the beam interaction characteristics of selected lasers with an alpha-alumina bioceramic, Optics and Lasers in Engineering, 41, 50514 (2004). 290. H. Ohgushi, Y. Dohi, T. Yoshikawa, S. Tamai, S. Tabata, K. Okunaga and T. Shibuya, Osteogenic differentiation of cultured marrow stromal stem cells on the surface of bioactive glass ceramics, Journal of Biomedical Materials Research, 32, 3418 (1996). 291. D. D. Deligianni, N. D. Katsala, P. G. Koutsoukos and Y. F. Missirlis, Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength, Biomaterials, 22, 8796 (2001). 292. M. Ahmad, D. Gawronski, J. Blum, J. Goldberg and G. Gronowicz, Differential response of human osteoblast-like cells to commercially pure (cp) titanium grades 1 and 4, Journal of Biomedical Materials Research, 46, 12131 (1999). 293. H. Mirzadeh and M. Dadsetan, Inuence of laser surface modifying of polyethylene terephthalate on broblast cell adhesion, Radiation Physics and Chemistry, 67, 3815 (2003). 294. C. Schmidt, A. A. Ignatius and L. E. Claes, Proliferation and differentiation parameters of human osteoblasts on titanium and steel surfaces, Journal of Biomedical Materials Research, 54, 20915 (2001).
208
References
295. J. Lawrence and L. Li, Carbon steel wettability characteristics enhancement for improved enamelling using a 1.2 kW high power diode laser, Optics and Lasers in Engineering, 32, 35365 (1999). 296. M. Verdier, S. Costil, C. Coddet, R. Oltra and O. Perret, On the topographic and energetic surface modications induced by laser treatment of metallic substrates before plasma spraying, Applied Surface Science, 205, 321 (2003). 297. B. Janocha, D. Hegemann, C. Oehr, H. Brunner, F. Rupp and J. GeisGerstorfer, Adsorption of protein on plasmapolysiloxane layers of different surface energies, Surface and Coatings Technology, 142144, 10515 (2001).
Index
Absorption coefcient, 69, 72 Adhesion, 5, 23, 24, 61, 82, 114, 127, 128 Air incubator, 127 Alkaline phosphatase assay, 82 Alumina, 14 Attractive van der waals force, 30, 31 Bioactivity, 1, 3, 66, 119 Biochemical methods, 7, 135 Biocompatibility, 2, 65, 117 Bioinert ceramic, 13, 142, 144, 149, 150, 153, 157 Biointegration, 1, 5, 66, 119 Biological Response, 2 Biomaterial interface, 29 Biomaterials, 2, 11, 12, 17, 18, 19, 23, 29, 31, 141 Bio-metals, 141, 158, 168, 175 Biomolecule, 8, 9, 12 Bonding, 23, 28 Bone cell adhesion, 5 Bone-implant interface, 3, 6 Bone Implants, 3, 141 Bone like apatite, 67, 120 Bovine serum albumin, 61, 75, 114, 123, 165 Cell Cell Cell Cell adhesion, 82, 128 attachment, 83 cover density, 90, 91, 93, 154 culture, 81, 127 Cell cytotoxicity, 81, 83, 127, 128 Cell formation, 51 Cell growth, 85 Cell morphology, 82 Cell proliferation, 82, 128, 130 Ceramic materials, 35 Chemical bonding, 28 Chemical reactions, 33 CO2 laser, 37, 39, 41, 65, 77, 89, 99, 102, 103, 117, 121, 124, 131, 141, 142, 150, 153, 157, 168, 170, 175 Compressive strength, 38, 143 Contact angle, 24, 41, 103 Continuous wave (CW), 20, 39 Conversion coating, 19 Corona discharge, 35 Corrosion, 16, 21 Cross-section, 39 Crystal size, 56 Culture polystyrene plate, 81, 82, 128 Dendrite structure, 47 Density, 7, 19, 38, 43, 44, 47, 48, 51, 52, 5759, 6163, 6972, 74, 77, 78, 82, 85, 8993, 101, 103105, 108, 110, 115, 124, 125, 130, 132, 143, 144, 146, 150, 151, 154, 157, 159, 161 163, 165, 166, 168171, 174, 175 Dental implant, 1, 5, 11 Diamond rimmed cutting blade, 38, 39, 67, 120 Dispersive component of surface energy, 26, 47
210 Electrowetting, 35 Ellipsometer, 76, 123, 159 Energy dispersive X-ray analysis, 39 Etheneglycol, 40, 102 Excimer laser, 2022, 35, 36, 38, 47, 60, 61, 100, 101, 149, 158 Fetal bovine serum, 81, 127 Filopodia, 66, 84, 154 Formamide, 40, 102 Fourier Transform Infrared Spectrometer (FTIR), 68, 69 Functional group, 4 Generic effect, 141, 142, 150, 157, 168, 175 Glycerol, 4043, 52, 54, 92, 102, 112, 150 Hemocytometer, 82 Hexagonal structure, 47, 149 High power diode laser, 36, 47, 148 Host response, 2, 11 Human blood, 61, 62, 114, 115, 165, 166 Human blood plasma, 61, 62, 68, 95, 114, 115, 121, 165, 166 Human plasma bronectin, 76, 123, 124, 159, 170, 172 Human serum albumin, 75, 76, 78, 123, 124, 159, 170, 171, 172 Hydrophilic, 2, 33, 172 Hydrophobic, 2, 33 Hydroxide ions, 121, 122, 139 Hydroxyl group (OH group), 69, 70 In vitro, 2, 65, 117 In vivo, 95, 135 Infrared (IR), 69 Instrument plate reader, 128 Interfacial biophysics, 30 Ion-beam assisted deposition, 18, 34 Ion beam processing, 18, 34 Ion implantation, 18, 34, 137 LS stainless steel, 142, 157159, 166, 170 Lactate dehydrogenase, 81, 127 Langmuir-blodgett deposition, 19 Laser, 19, 20, 21, 22, 35, 36, 37, 39, 41, 67, 89, 99, 103, 117, 121, 124, 131, 141, 142, 150, 153, 157, 168, 170, 175
Index Laser grafting, 22 Laser patterning, 20 Lasers, 19, 35, 38, 111, 118, 119, 141, 153, 158, 168, 170, 177 Magnesia partially stabilised zirconia, 37, 65, 118 MAPLE direct write, 21 Matrix-assisted pulsed laser evaporation, 21 Mechanical bonding, 28 Mechanical properties, 1, 16 Metallic materials, 36 Microstructure, 45, 56 Morphology, 82, 128 MTT assay, 128, 159 Nd:YAG laser, 58, 69, 136 Ordinary Portland cement, 36 Orthopaedic implant, 1, 5, 11 Osseointegration, 5, 12, 38, 101, 135 Osteoblast cell, 6, 6567, 8086, 8991, 93, 98, 117, 119, 120, 127, 128, 131, 132 Oxidation, 34, 107, 108, 121, 137, 144 Parylene coating, 19 Phase, 39, 56, 61, 102, 105 Phosphate buffered salines, 76, 123 Photografting, 17 Physical bonding, 28 Physicochemical methods, 7 Physiological liquids, 61, 114 Plasma surface modication, 18, 33 Polar component of surface energy, 63, 75, 164 Polyglycol 15-200, 40, 102 Polyglycol E-200, 40, 102 Predominant mechanisms, 52, 111, 149, 166 Prolometer, 39 Protein adsorption, 75, 76, 123, 170, 175 Pulsed laser deposition, 20 Radiation grafting, 17, 34 Radiation, 17, 34, 41, 99, 103 Rapidly solidied microstructure, 45 Repulsive electrostatic force, 24, 31 Rutile, 107 Scanning electron microscopy, 12, 66 Self-assembled monolayers, 4, 19 Sessile drop, 40, 41, 102, 143, 159
Index Silanization, 19 Simulated body uid, 119, 142 Specic heat, 38, 101, 143 Stainless steel, 159, 166, 170 Statistics, 76, 83, 124, 128 Stress shielding, 16 Surface analysis, 12, 39 Surface energy, 25, 47, 56, 61, 109 Surface melting, 45 Surface modication, 7, 8, 11, 17, 19, 33, 35, 36 Surface-modifying additive, 19 Surface oxygen content, 42 Surface properties, 12, 65, 99, 117, 141 Surface roughness, 43, 78, 125 Surface roughness, 43, 78, 125 Tensile modulus, 38, 143 Tensiometry, 29 Test control liquids, 164 Thermal conductivity, 38, 101, 143 Thermodynamic, 31 Titanium alloys (Ti6Al4V), 99, 109, 117, 119, 120, 124, 128 Topography, 6, 89, 132
211 Total interaction energy, 31 Transverse electromagnetic multimode, 39 Tribological, 17, 100 Trypsin-EDTA, 82, 128 Ultraviolet (UV), 17, 18, 34 Uniform cell structure, 47 Vickers hardness, 38, 143 Wavelength, 39, 58 Wettability characteristics, 35, 37, 40, 41, 45, 52, 71, 79, 91, 99, 102, 103, 105, 111, 125, 132, 144, 149, 150, 159, 166, 168 Work of adhesion, 24 X-ray diffraction (XRD), 40, 66 X-ray photoemission spectroscopy (XPS), 12, 40 Yttria partially stabilised zirconia, 141, 142, 143 Zirconia, 14, 37, 65

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To Yan-jun and My Parents For the world they bring to me.

To Louise and Ethan, Always there; beyond compare.

3.4.3 3.4.4 3.4.5 3.4.6 3.4.7 3.5 Laser 3.5.1

141 141 142 143 144

149 150 153 157 157 158

166 168 170 175 176 179 185 209

Lasers and Their Application for Modication of the Biomaterials

Lasers and Their Application for Modication of the Biomaterials

Bioactivity and Biointegration

Bioactivity of Bone Implants

Bioactivity and Biointegration

Biointegration of Orthopaedic and Dental Implants

Bioactivity and Biointegration

Controlling the BoneImplant Interface

Bioactivity and Biointegration

Controlling the BoneImplant Interface

Surface Modication of Biomaterials

Ceramic Implants [65]

Surface Modication of Biomaterials

Surface Modication of Biomaterials

Surface Modication of Biomaterials

Laser Surface Modication of Biomaterials

Surface Modication of Biomaterials

Laser Surface Modication of Biomaterials

Surface Modication of Biomaterials

Wettability in Biomaterials Science

Wettability, Adhesion and Bonding

Wettability in Biomaterials Science

Figure 3.2 substrate

Wettability, Adhesion and Bonding

or, alternatively, p 1 p 1 1 1 b gsv 2 glv 2 a 1 gd 2 gp 2 sv sv 2 3:13

Wettability in Biomaterials Science

Wettability in Biomaterial Science

Wettability in Biomaterials Science

Wettability in Biomaterial Science

Wettability in Biomaterials Science

Relatively Cell Spreading

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Current Methods of Wettability Modication

Wettability in Biomaterials Science

Laser Wettability Characteristics Modication

Wettability in Biomaterials Science

CO2 Laser Modication of the Wettability Characteristics

CO2 Laser Modication of the Wettability Characteristics

The Effects of CO2 Laser Radiation

CO2 Laser Modication of the Wettability Characteristics

Test liquid Glycerol Formamide Etheneglycol Polyglycol E-200 Polyglycolycol 15-200

The Effects of CO2 Laser Radiation

CO2 Laser Modication of the Wettability Characteristics

The Effects of CO2 Laser Radiation

CO2 Laser Modication of the Wettability Characteristics

Surface Energy and Its Component Parts

CO2 Laser Modication of the Wettability Characteristics

Surface Energy and Its Component Parts

UT 0.5 0.9 1.6 1.9 2.5

2.41 2.37 3.03 4.25 3.42 3.17

76.2 79.4 92.8 113.3 105.0 101.8

75.2 77.5 89.8 105.5 98.5 95.6

71.5 72.5 80.7 90.3 86.5 84.5

69.6 70.9 77.0 82.7 80.0 78.7