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Surface Engineering of
Polymeric Biomaterials
Todorka G. Vladkova
Surface Engineering of
Polymeric Biomaterials
Todorka G. Vladkova
A Smithers Group Company
Shawbury, Shrewsbury, Shropshire, SY4 4NR, United Kingdom

Telephone: +44 (0)1939 250383 Fax: +44 (0)1939 251118
http://www.polymer-books.com
First Published in 2013 by
Smithers Rapra Technology Ltd

Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK
©2013, Smithers Rapra Technology Ltd
All rights reserved. Except as permitted under current legislation no part

of this publication may be photocopied, reproduced or distributed in any
form or by any means or stored in a database or retrieval system, without
the prior permission from the copyright holder.
A catalogue record for this book is available from the British Library.
Every effort has been made to contact copyright holders of any material reproduced
within the text and the authors and publishers apologise if
any have been overlooked.
ISBN: 978-1-84735-658-1 (hardback)

978-1-84735-659-8 (softback)
978-1-84735-660-4 (ebook)
Typeset by Argil Services

P
reface
Biomaterials work in contact with living matter and this raises a number of specific
requirements for their surface properties, such as bio-inertness or bioactivity, anti-
biofouling, and so on. These properties are usually lacking in the materials used for
fabrication of biomedical devices, such as implants, scaffolds for tissue engineering,
diagnostic sensors, targeted drug delivery carriers and other. Surface engineering based
on physical, chemical, physical-chemical, biochemical or biological principles, is an
important tool for the preparation of biomaterials with desired biocontact properties.
Tissue engineering, based on human cells seeded onto polymeric scaffolds, is a new
technique, which is developing very quickly with the potential to create tissues and
organs de novo. The surface property tailoring in this case, is aimed at the creation
of a biologically relevant microenvironment for the corresponding cells, because
they are sensitive to the environment in which they exist and respond to chemical
cues and the morphologic aspects of the surfaces with which they are in contact.
The common synthetic polymeric scaffolds have great advantages in terms of their
manufacturing process and the reproducibility of their degradability and other
properties but they suffer from a lack of those finer cues. One approach to providing
them is surface modification by physical adsorption of suitable compounds or by
chemical modification. Another common type of surface modification is micro- or
nano-patterning for influencing cell behaviour or for creating structured cellular arrays.
Over the last few decades, we have witnessed an extraordinary development of surface
engineering techniques, such as plasma and chemical treatments, polymer/organic thin
film and coating deposition, biomolecule immobilisation, micro- and nano-pattern
surface modification, especially tuned to respond to the requirements of the specific
biomaterial surface design. The need for better characterisation of surface engineered
surfaces and especially of micro- or nano-structured and modified nanosize surface
layers motivates the development of new instruments and apparatus for investigating
structural and chemical nanosize features.
The large number of contemporary scientific papers and reviews dedicated to the
surface engineering of polymeric biomaterials needs to be collected and summarised.
Detailed presentation and discussion of all known and advanced surface engineering
iii
Surface Engineering of Polymeric Biomaterials
and surface characterisation methods used so far for biocontact properties, and
improvement of existing and advanced polymeric biomaterials in one book is difficult
because of their huge amount and large variety. The aim of this book is to provide
an introduction to help the reader quickly step into such an impetuously developing
area with references given as a starting point. It provides a brief survey of strategies
for tailoring the biological response through the creation of biomaterial surfaces
resistant to fouling by proteins, cells, or microorganisms, as well as of ones capable
of eliciting specific biomolecular interactions that can furthermore be combined with
micro- and nano-patterning techniques to engineer adhesive areas in a non-interactive
background. The theoretical base of surface engineering for improvement of biocontact
properties of polymeric biomaterials as well as the current state-of-the-art of the
surface engineering of polymeric biomaterials for all main applications: including
artificial implants and organs, blood contacting devices, biosensors, controlled drug
delivery, microfluidics, tissue engineering, and others outlining the surface problems
of the corresponding biomaterial and the possible solutions by surface engineering
are briefly presented. This book also includes the most often used conventional and
advanced surface engineering methods including micro- and nano-structuring as well
as biomaterial surface characterisation techniques.
The book is targeted at a broad range of experts, researchers, post-doctorates, PhD

and MSc students, entering or working in the field of biomaterials with special interest
in the creation of polymeric materials with improved biocontact properties via surface
engineering. The author hopes that this book can strongly prompt those who are
already involved in this type of research to extend their activities and to contribute
to the advances in the field of surface engineering of biomaterials.
T. Vladkova
2013
iv
C
ontents
1 Introduction................................................................................................ 1
1.1 Specific Objectives of Biomaterial Surface Engineering..................... 2
1.2 Theoretical Basis of Biomaterial Surface Engineering ...................... 5
1.2.1 Protein Adsorption ............................................................ 5
1.2.1.1 Specific Protein Adsorption................................ 5
1.2.1.2 Non-specific Protein Adsorption ....................... 6
1.2.2 Initial Cell/Biomaterial Surface Interactions ....................... 8
1.3 Biomaterial Surface Engineering Approaches................................. 12
2 Surface Engineering Methods.................................................................... 23

2.1 Introduction................................................................................... 23
2.2 Physicochemical Methods.............................................................. 24
2.2.1 Blending............................................................................ 24
2.2.2 Acid Etching..................................................................... 24
2.2.3 Surface Grafting................................................................ 25
2.2.3.1 Graft Polymerisation . ..................................... 26
2.2.3.2 Polymer Brushes............................................... 27
2.2.4 Plasma Techniques............................................................ 31
2.2.5 Photon Irradiation............................................................ 35
2.2.6 Ion-beam Modification..................................................... 37
2.2.7 Adsorption from Solution (Thin Film/Coating
Preparation Methods)....................................................... 37
2.2.7.1 Dip Coating..................................................... 39
2.2.7.2 Spin Coating.................................................... 39
v
2.2.7.3 Langmuir–Blodgett Films................................. 39

2.2.7.4 Self-assembled Monolayers.............................. 41
2.2.7.5 Self-assembled Monolayers with Molecular
Gradients......................................................... 41
2.2.7.6 Layer-by-Layer Assembly................................. 41
2.3 Biological Methods........................................................................ 42
2.3.1 Biomolecule Immobilisation by Physical Adsorption......... 43
2.3.2 Biomolecule Immobilisation by Blending.......................... 43
2.3.3 Electrostatic Attachment of Biomolecules......................... 44
2.3.3.1 LbL Technique using Polyelectrolytes............... 44
2.3.3.2 Electrochemical Polymerisation Using
Conducting Polymers....................................... 45
2.3.4 Covalent Bonding of Biomolecules.................................... 45
2.3.4.1 Thiol-mediated Bonding................................... 47
2.3.4.2 Hydroxyl Group-Mediated Bonding................ 49
2.3.4.3 Carboxylate Group-Mediated Bonding ........... 50
2.3.4.4 Photoinitiated Coupling of Biomolecules......... 51
2.3.4.5 Enzymic Coagulation of Biomolecules............. 52
2.3.4.6 iomolecules Bonding with ‘Click’ Reactions..... 52
2.4 Surface Micro- and Nano-structuring............................................ 53
2.4.1 Photolithography.............................................................. 54
2.4.2 Ion Lithography and Focused Ion Beam Lithography....... 56
2.4.3 Electron Lithography........................................................ 56
2.4.4 Soft Lithography............................................................... 56
2.4.5 Dip Pen Nanolithography................................................. 60
2.4.6 Near-field Scanning Methods............................................ 63
2.4.7 General Methods of Nano- and Micro-bioarray
Patterning......................................................................... 63
3 Surface Engineering of Biomaterials Reducing Protein Adsorption............ 87

3.1 Introduction................................................................................... 87
vi
Contents
3.2 Surface Engineering of Biomaterials to Reduce

Undesirable/Uncontrolled Responses to Implants and
Extracorporeal Devices.................................................................. 91
3.2.1 Polyethylene Glycol-coated Surfaces................................. 91
3.2.1.1 Photopolymerised or Photocrosslinked
Coatings........................................................... 93
3.2.1.2 Chemical Coupling of PEG.............................. 96
3.2.1.2.1 Chemical Coupling based on the
Reactivity of the Terminal
Hydroxyl Groups............................ 96
3.2.1.2.2 Covalent Attachment by
Employment of Functionalised
PEG (Derivative Terminal OH
Groups)........................................... 97
3.2.1.3 Non-covalent Immobilisation......................... 102
3.2.2 Non PEGylated Hydrophilic Surfaces............................. 104
3.2.3 Zwitterionic Polymer Thin Layers.................................. 108
3.2.4 Hydrophilic Surfaces of Hyperbranched Polymers.......... 111
3.2.5 Multi-layer Thin Films . ................................................. 112
3.2.6 Hydrogels and Hydrogel Coatings.................................. 114
3.2.6.1 PEG-based Hydrogel Coatings....................... 115
3.2.6.2 Hydrogel Coatings of Other Polymers........... 120
3.2.6.3 Hydrogels of Zwitterionic Polymers............... 123
3.2.7 Patterned Surfaces........................................................... 126
3.2.7.1 Backfill Non-fouling Polymers and
Procedures .................................................... 127
3.2.7.2 Micro- and Nano-patterning Techniques........ 129
3.3 Surface Engineering of Biomaterial Surfaces
Reducing/Eliminating Non-specific Adsorption on
Biosensors and Bioassays............................................................. 142
3.4 Surface Engineering of Microfluidic Devices................................ 158
3.4.1 Dynamic Coating . ......................................................... 159
3.4.2 Permanent Coatings........................................................ 162
vii
3.4.2.1 Plasma Treatments......................................... 163

3.4.2.2 Laser Treatments............................................ 164
3.4.2.3 Surface Graft Polymerisation......................... 165
3.4.2.4 Patterning of Microfluidics............................. 169
3.4.2.5 Covalent Modification................................... 169
3.4.2.6 Self-assembled Monolayers............................ 175
3.4.2.7 Polyelectrolyte Multi-layer Coatings.............. 176
4 Surface Engineering of Blood Contacting Polymeric Biomaterials........... 231

4.1 Introduction . .............................................................................. 231
4.2 Strongly Hydrophilic and Strongly Hydrophobic Surfaces........... 234
4.2.1 Strongly Hydrophilic Surfaces......................................... 234
4.2.2 Strongly Hydrophobic Surfaces....................................... 237
4.3 Biomaterials with Micro- and Nano-domain Surfaces . ............... 237
4.4 The Immobilisation of Heparin and Other Bioactive Molecules... 241
4.4.1 Heparinised Surfaces....................................................... 241
4.4.2 Immobilisation of Other Bioactive Molecules................. 248
4.5 Albumin Coating......................................................................... 251
4.6 Endothelial Cells Attachment . .................................................... 257
4.7 Natural Biomembrane Mimetic Surfaces...................................... 261
4.8 Polyelectrolyte Multi-layers ........................................................ 266
4.9 Micro- and Nanostructured Blood Contacting Surfaces............... 268
5 Surface Engineering of Bio-interactive Biomaterials................................. 295

5.1 Introduction................................................................................. 295
5.2 Surface Engineering of Biomaterials Promoting Cell
Attachment/Adhesion ................................................................. 298
5.2.1 Cell/Biomaterial Surface Interaction................................ 299
5.2.2 Surface Engineering of Cell Adhesive Biomaterials
via Physicochemical Modification................................... 301
5.2.2.1 Control over the Surface Energy
(Hydrophilic/Hydrophobic Balance) ............. 302
viii
Contents
5.2.2.2 Creation of Positively Charged Surfaces......... 311

5.2.2.3 Surface Micro-architecture Manipulation...... 311
5.2.2.4 Creation of Polyelectrolyte Multi-layers......... 317
5.2.2.5 Temperature-responsive Polymer Coatings . .. 321
5.2.2.6 Other Functional Polymer Coatings............... 323
5.2.2.7 Multi-layer Thin Films for Cell
Encapsulation................................................ 328
5.2.3 Surface Engineering of Cell Adhesive Biomaterials
via Biomolecule Immobilisation...................................... 329
5.2.3.1 Cell Adhesion Ligands .................................. 331
5.2.3.2 Non-covalent Immobilisation of
Biomolecules.................................................. 336
5.2.3.3 Covalent Bonding of Biomolecules ................ 347
5.2.3.4 Patterning of Biomolecules on Biomaterial
Surfaces.......................................................... 366
5.3 Surface Engineering of Drug Delivery Systems ............................ 371
5.3.1 Drug Delivery Systems.................................................... 373
5.3.1.1 Hydrogel Controlled Release Formulations.... 373
5.3.1.2 Functionalised Electrospun Nanofibres
Drug Delivery Carriers................................... 376
5.3.1.3 Drug Loaded Micro- and Nano-particles....... 377
5.3.1.4 Drug Loaded Magnetic Nanoparticles .......... 379
5.3.1.5 Electrostimulated Drug Release Systems........ 379
5.3.2 Polymeric Thin Films and Coatings for Drug and
Gene Delivery................................................................. 381
5.3.3 Protein Delivery in Tissue Engineering............................ 389
5.3.3.1 Matrices and Scaffolds for Protein Delivery
in Tissue Engineering..................................... 390
5.3.3.2 Bioactive Proteins and Peptides...................... 392
5.3.3.3 Strategies for Bioactive Factors Controlled
Delivery ........................................................ 394
ix
5.4 Surface Engineering of Biomaterials Reducing Bacterial

Adhesion...................................................................................... 405
5.4.1 Biomaterials Resistant to Bacterial Adhesion ................. 407
5.4.2 Nanocomposite Polymer Coatings Containing
Inorganic Biocides........................................................... 411
5.4.3 Antibiotic Conjugated Polymer Coatings........................ 412
5.4.4 Biomimetic Antibacterial Coatings.................................. 413
5.4.5 Antibacterial Coatings Based on Cationic Polymers ....... 414
6 Biomaterial Surface Characterisation...................................................... 477

6.1 Introduction................................................................................. 477
6.2 Surface Morphology Observation ............................................... 477
6.3 Contact Angle Measurements...................................................... 478
6.3.1 Surface Tension and Determination of its Components . 479
6.3.2 Methods of Contact Angle Measurement........................ 480
6.3.2.1 Drop and Bubble Methods for Contact
Angle Measurement....................................... 481
6.3.2.2 Wilhelmy Plate Method.................................. 482
6.4 Surface Forces Measurement........................................................ 483
6.5 Ellipsometry Measurements......................................................... 484
6.6 Surface Chemical Composition Characterisation ........................ 488
6.6.1 Spectroscopy Methods (ATR-FTIR, TOF-SIMS
and XPS)......................................................................... 488
6.6.2 Colorimetric Determination of Surface Functional
Groups Density............................................................... 491
6.6.3 Radiotracer Method....................................................... 492
6.6.4 Estimating the Thickness of Grafted Polymer Layers...... 494
6.7 Characterisation of Protein Layers on Biomaterial Surfaces......... 494
6.7.1 Estimating the Density and Thickness of Protein
Layers on Biomaterial Surfaces....................................... 494
6.7.2 Characterisation of Biomolecules Attachment
to/Detachment from Biomaterial Surfaces ...................... 497
x
Contents
6.7.3 Bioactivity Evaluation of Proteins Immobilised on

Biomaterial Surface . ...................................................... 498
6.7.4 Spatial Distribution of Proteins and Adhering Cell
Characterisation............................................................. 500
6.8 Evaluation of Cell Behaviour on Biomaterial Surfaces ................ 500
6.8.1 Cell Proliferation ........................................................... 501
6.8.2 Cell Imaging................................................................... 502
6.8.3 Cell Migration................................................................ 503
6.8.4 Cell Function Analysis.................................................... 503
6.9 Tests for Biocompatibility ........................................................... 504
7 Summary and Outlook............................................................................ 517
Abbreviations..................................................................................................... 531
Index ............................................................................................................... 553
xi
xii
1
Introduction
Introduction
In many ways, for a lot of parameters, polymeric materials most fully satisfy the
requirements of biomedical applications. The unique combination of hierarchical
structuring, with many levels of different complexities, the ability to control
mechanical properties and chemical composition, combined with low cost and ease
processing, make polymers the most widely applied biomaterial.
In such applications, materials interact directly with biological systems, human tissues,
organs, cells and blood and they are categorised as ‘biomaterials’ [1]. A variety of
devices made by synthetic and natural polymeric biomaterials are currently used in
medical applications such as vascular grafts, coronary stents, heart valves, blood bags,
catheters, blood oxygenators, renal dialysers, hip and knee prostheses, intraocular
and contact lenses, cochlear and dental implants, and so on. Advances in protein-
based drugs, gene therapy, targeted drug delivery and tissue engineering are expected
to revolutionise contemporary medicine, and polymeric materials will play a major
role in the construction and assembly of a new generation of medical devices and in
tissue engineering.
However, biomedical applications are limited in most cases by the poor biocontact
properties of the polymers. In general, whenever an object comes into contact with a
biological system such as tissues or cells, or with fluids derived from them (e.g., blood
or serum), the processes that occur at the newly formed interface determine whether
that object will fulfil its function or will fail. Biomaterials interact with biological
systems through their surfaces. Therefore, the biomaterial surface properties are of
vital importance for the control of these interactions. Surface engineering leading to
creation of micro- or nano-size layers with controlled parameters such as chemical
composition, surface tension and related parameters, surface roughness, topography
and patterns emerged as a simple, useful and versatile approach to solving the problem.
Surface engineering can be considered in terms of surface treatments (modification of

a surface by changing its composition or microstructure, or both) or surface coating
(addition of a distinct layer of material to the original surface). The surface engineered
1
system can, therefore, be considered as a composite system, consisting of a near-surface

modified region, which is optimised for the working environment, and the substrate
or bulk material, which is usually selected according to the mechanical and physical
characteristics and manufacturability. The objective of creating such a composite
system is for it to exhibit a performance superior to the surface or substrate alone.
The newly formed interfaces, where the initial contact between material and the
biological system occurs, are frequently referred to as biointerfaces [2–4]. Although
the role of biointerfaces was first recognised many years ago, biointerface science
emerged quite recently.
From the mid-1900s to the end of the last century, biomaterials were petroleum-derived
synthetic polymers designed to be inert in vivo, that is, to perform their function
without interacting with the organism [5]. These biomaterials are characterised by
exclusively low protein adsorption and weak interactions with blood, living tissues
and cells. Over recent decades, many new synthetic and biologically derived polymers
have been studied and applied, altering this paradigm [6, 7]. Now material scientists
are focused on the design of bio-interactive materials that integrate with biological
molecules or cells and regenerate tissues [8–10]. In response to this, the meaning of
the term ‘biocompatibility’ has changed. In early studies, biocompatibility is simply
equated to inertness. In 1986, Williams gave a more specific definition of ‘the ability of
a material to perform with an appropriate host response in a specific application’ [11].
In 1993, Ratner defined biocompatibility as ‘the body’s acceptance of the material, i.e.,
the ability of an implant surface to interact with the cells and fluids of the biological
system and to cause exactly the reactions that the analogous body tissue would bring
about’ [12]. Biomaterials for regenerative medicine and tissue engineering are of
especial interest. Tissue engineering is based on cell seeding of a substrate followed
by culturing in a bioreactor or directly in the human body. The substrate is often a
polymeric biomaterial that stimulates not only the cell attachment and differentiation
but also the extracellular matrix (ECM) formation and tissue regeneration. Advanced
biospecific and biomimetic materials consisting of a bio-inert environment enriched
with ligands for adhesive receptors, usually short amino acid sequences such as arg-
gly-asp (RGD), or carbohydrates and/or functional parts of hormones, enzymes or
growth factors, are currently under intense investigation [13, 14].
1.1 Specific Objectives of Biomaterial Surface Engineering
In most cases, the interface interaction between biomaterial and living matter is
accompanied by undesirable response reactions, called foreign body reactions, leading
to inflammatory processes and necroses, blood coagulation and thrombi formation,
and to the rejection of the implant in some cases. The degree of this reaction depends
2
Introduction
on the properties of the device, such as shape, size, surface chemistry and roughness,
morphology and porosity, composition, contact duration and degradation [15]. In
other cases, the specificity of the biosensor interaction or cell adhesion to the tissue
engineering scaffolds, biomaterial surface wettability, friction, wear resistance, and
so on, are insufficient and have to be optimised by biomaterial surface modification.
Therefore, the design and manufacture of biomedical surfaces is of increasing interest
[16].
Control over the interface interaction between biomaterial surface and living matter
(tissue, blood, cells) is the main purpose of biomaterial surface engineering, aimed
at enhancing their performance in the biological environment while retaining the
bulk properties of the biomedical device. The main purpose of biomaterial surface
engineering has a number of specific objectives, such as:
• Reduction/elimination of protein adsorption, to reduce both undesirable responses

to implants and extracorporeal devices and non-specific adsorption on biosensors
and bioassays.
• Reduction/elimination of cell adhesion at blood contacting surfaces.
• Promotion of cell attachment/adhesion for ingrowing implants and tissue

engineering.
• Reduction of bacterial adhesion.
• Control of transport properties, to optimise the passage of therapeutic agents,

water, and so on.
• Increase in lubricity, hardness and resistance to corrosion/wear.
Many surface treatments and coating processes are available to enhance the
performance of biomedical materials. The specific objectives of biomaterial
surface engineering can be achieved by producing special functional groups on the
biomaterial surface; control over surface energy and related parameters; surface
crosslinking; modification of surface morphology and roughness; surface micro- and
nano-patterning. Polymer films and coatings are among the most popular and most
successful tools for modulating the surface properties of biomaterials, and especially
of tissue responses and fouling behaviour. Over the past decade, surface coatings in
surface-mediated drug delivery have been widely investigated. In these applications,
deposited polymer films act as both a coating to modulate surface properties and a
reservoir for the active therapeutic cargo [17].
Recently, new bio-inspired and biomimetic approaches have been investigated for the
creation of biomaterial surfaces with improved biocontact properties. These consist of
3
attempts to mimic the nanostructuring and function of natural surfaces, for example
non-fouling lotus leaf or dolphin skin surface, and others [18]. New biomaterial
generations include surface modification of the material to overcome non-specific
protein adsorption in vivo, precise immobilisation of signalling groups on the surface,
synthetic materials with controlled properties for drug and cell carriers, biologically
inspired materials that mimic natural processes, and sophisticated three-dimensional
architectures to produce well-defined patterns for diagnostics, e.g., biological
microelectromechanical systems, and tissue engineering. However, recently a variety
of new approaches have been introduced, so that the level of control achieved and the
level of complexity that can be addressed in the fabrication of advanced biointerfaces
extends from the molecular to macroscopic length scales. Linear and crosslinked
polymers, which are tunable over a broad range mechanical properties, such as the
elastic modulus, are used. Depending on the choice of backbone, co-monomers, side
chains and crosslink density, the entire spectrum of required moduli can be achieved.
A number of books are available that highlight different aspects of biomaterial surface
engineering [19–27]. Due to the broad scope and rapid development of biomaterial
surface science, there are dozens of review articles on various aspects of this topic; some
of the more general include: surface modification of polymers for medical applications
[28], surface treatments of polymers for biocompatibility [29], influence of surface
hydrophilicity/hydrophobicity on biocompatibility [30], interactions of biomaterial
surfaces with water and protein molecules, cells and tissues [2], current approaches to
engineering nanoscale material’s surfaces to mimic the nanoscale patterns of chemical
and topographical cues of natural ECM [31], biomaterial surface characterisation
and modification approaches to bone-contacting devices [32], RGD-modified polymer
surfaces [33], laser surface engineering for bioimplants [34], vapour-based polymer
coatings [35], plasma-induced micropatterning [36], plasma-based ion implantation
[37], surface modification and property analysis of biomedical polymers used for
tissue engineering [38], biocompatibility of implantable devices: current challenges
to overcoming foreign body response [16], surface engineering to control non-specific
protein adsorption of biomaterials for biosensors and biomedical applications [39],
specific techniques for biosurface analysis such as time-of-flight - secondary ion
mass spectroscopy, ellipsometry, surface matrix-assisted laser desorption/ionisation
mass spectroscopy, x-ray photoelectron spectroscopy [40–44], polymer films and
coatings for drug delivery [17], from bio-inspired to synthetic design and fabrication
of bio-interfacial topography at the micro/nanoscale [18]. It is worth noting that
biomaterial surface science is a very broad area, with an explosion of techniques and
approaches. Thus, it is impossible to give a complete review of it in a single book.
The references and the techniques quoted here are just a fraction of those actually
available in this area.
4
Introduction
1.2 Theoretical Basis of Biomaterial Surface Engineering
It has been known for a long time that the chemical and physical nature of surfaces
drastically affects various macroscopic properties. However, the earliest publications
about surface properties and surface modification deal with biomaterial surfaces
with insufficiently well-characterised chemical or physical features to permit valid
conclusions about the relationship between the molecular constitution and the
macroscopic behaviour of the surface. As a technology, surface engineering has been
used for a long time, but recent decades have seen its application extended hugely
because of the development of high-speed, precise and relatively easily implemented
chemical, physicochemical, physical and plasma methods for surface engineering and
characterisation of biomaterial surfaces.
Current knowledge about surface forces: dispersion (van der Waals), solvation,
hydrophobic, electrochemical, double electrical layer, and interface interactions, as
well as limited knowledge about interface phenomena on the border of living and
non-living matter, such as protein adsorption, initial cellular interactions, bioadhesion
and biofouling are the theoretical basis for the development of surface engineered
biomaterials with the desired biocontact properties, both bio-inert and bio-interactive.
The mechanisms of protein adsorption, cell/biomaterial surface interaction and
bioadhesion are a key question in many studies but, despite enormous effort, they
are still not fully understood.
1.2.1 Protein Adsorption
Protein-biomaterial surface interactions play a key role in the performance of

biomedical devices in areas such as drug delivery, solid face diagnostics, extracorporeal
therapy, orthopaedic implants, dental applications, biosensors, and biotechnical
separation methods. The biological cascade of all non-desirable foreign body reactions
against biomaterials begins with the deposition of proteins. Protein adsorption is
the primary event in biofouling. Secreted by cells, adhesive proteins mediate their
interaction with the biomaterial surface. Therefore, many investigations are devoted
to studying the adsorption mechanism (both structural and compositional aspects)
of single, well-defined proteins or of concurrent adsorption from double and multi-
component systems on different surfaces [45–48].
1.2.1.1 Specific Protein Adsorption
Specific molecular interactions are the fundamental processes governing control of

biological form and function, and these biological phenomena are mediated mainly by
5
proteins [39, 49]. These specific interactions are generally non-covalent and between both
biomolecules, and the molecules presenting at the cell surface. Strong (although non-
covalent) bonding occurs between such macromolecules due to a unique combination
of steric and ionic interactions, and directional bonds. Such specific interactions are
referred to as ‘complementary’, ‘ligand-receptor’ or ‘recognition’ interactions, and
they form the foundation of a molecular imprinting technology. The binding energy
of specific interactions depends strongly on the local geometry and chemistry [50].
The affinity and uniqueness of the binding sites are the two major characteristics for
specific interactions. Specific protein adsorption on biomaterial surfaces is utilised
in the development of biosensors for protein detection, chromatographic protein
separation, and in the development of protein microarrays. The specific interactions
between biomaterial surfaces and proteins are a very complicated process. Many
different forces may act at different locations and/or at different times, which can affect
the specific molecular interaction event. Rearrangement of the molecules can happen
both normally and laterally, i.e., different events can occur at different locations on
the surface or away from the surface, either simultaneously or sequentially [39]. In
most cases, the molecular interactions taking place in a biological environment are
not the only interaction of energy or force happening between two entities, but the
overall effect on the whole system [51].
1.2.1.2 Non-specific Protein Adsorption
Non-specific protein adsorption is the key, complex event taking place when the
biomaterial surface contacts the biological environment, often leading to a failure or
loss of function of the biomaterial [52]. Because of their versatile nature (Figure 1.1),
many proteins can be adsorbed via many mechanisms when they are in front of
complementary surfaces [53], making it difficult to control protein adsorption.
Non-specific protein adsorption is governed on the one hand by the protein properties
(structure, size, distribution of charge and polarity), and on the other hand by the
biomaterial surface properties (chemical nature, charge, roughness and surface energy),
as well as by environmental conditions (pH, ionic strength and temperature) [54].
Based on widespread research, some general correlations between protein properties

and their adsorption on different surfaces are known [54–58]. Protein adsorption
is greatly influenced by the charge and distribution of charges on the surface of the
protein molecule. Adsorption depends also on the stability of the protein structure.
An unfolding protein structure leads to an increased conformational freedom of the
peptide chain, which in turn makes more sites available for protein-surface contact
[39].
6
Introduction
POLAR DONOR - ACCEPTOR INTERACTIONS
+
+++
+
+
HYDROPHOBIC DOMAIN
++
+++
++
++
++
IONIC INTERACTIONS +
HYDROPHOBIC DOMAIN
+ ++ +
SOLID SURFACE
Figure 1.1 The versatile nature of proteins. Adapted from reference [53]
The protein adsorption process is generally divided into three stages: initial,
intermediate (of reversible bonding) and final, yielding an irreversibly adsorbed
protein layer [59, 60]. Different molecular forces participate in the protein adsorption
process (hydrophobic interactions, electrostatic forces and hydrogen bonding, as
shown in Figure 1.1), depending on the nature of the biomaterial surface and the
protein, the orientation of the protein approaching the biomaterial surface, and the
overall binding energetics [55, 56, 58].
The initial adsorption is determined by the tertiary or quaternary structure of the

protein, followed by an energetically driven unfolding process [55, 56, 61, 62]. The
hydrophobic interactions driving protein adsorption could be generalised as the
interfacial free energy driven adsorption [54, 57, 63, 64]. Electrostatic attraction
theory has also been verified, but only with limited success due to two reasons: (i)
the force is too small under physiological conditions and too short range to exert any
major effect on the binding event, and (ii) the heterogeneous charge distribution on
the protein surface makes the analysis too complex, if it is possible at all [54, 58].
Other factors that affect the adsorption process include the solution conditions and the
adsorption kinetics. The pH value of the liquid affects the charge on the biomaterial
surface as well as the overall protein charge, and thus affects the adsorption process
7
[65]. The well-known ‘Vroman effect’ describes the competitive adsorption of plasma
proteins, which is dependent on the concentration and the contact time [66]. This
phenomenon was later confirmed by the surface plasmon resonance (SPR) technique,
using protein-specific antibodies [67, 68]. Additionally, in situ measurement of SPR
and its high sensitivity indicates that the protein adsorption tendency corresponds with
its properties such as molecular weight, surface affinity and bulk concentration; the
latter believed to affect the rate of mass transportation. Thus, the adsorption kinetics
also affects the final composition of an adsorbed protein layer [29].
As a result of many investigations [45, 54, 57, 63, 64, 69–72], it is well known today
that the protein adsorption on polymer surfaces depends significantly on the surface
physicochemical properties: surface chemical composition and topography, surface
hydrophilic/hydrophobic balance and charge, surface functional groups and their
mobility, modifying layer thickness, density and adhesion to the substrate, and so on.
Thus, by knowing the protein properties and the surrounding environment parameters,
one can control the protein adsorption by changing some of the physicochemical
characteristics of the substrate biomaterial surface.
When a biomaterial surface is exposed to a complex biological environment such as

blood or body fluid, the surface is instantaneously contaminated by adsorption of
small molecules (water and ions), resulting in the formation of a water layer and an
electrical double layer, followed by adsorption of larger molecules such as proteins
[55, 73–75]. Often the adsorbed protein layer interacts with other biomolecules and
cells, leading also to their adsorption on the surface. An example is the formation
of a biofilm to yield a colony of immobilised bacteria on the surface of a biomedical
implant, which then shows an enhanced resistance to antibiotics due to its robust
structure [56].
It is expected that new analytical techniques and direct measurement of interfacial

forces between proteins and surfaces will help the deeper understanding of the protein/
surface interaction and thus, will open up new prospects for improved, guided design
of surfaces intended to resist protein adsorption, and cell bioadhesion.
1.2.2 Initial Cell/Biomaterial Surface Interactions
Knowledge of the mechanism of cell/surface interaction is very important for the

design of biomaterial surfaces with desirable biocontact properties. The interaction
of adherent cells with their surroundings can ultimately determine the cell’s fate.
The cells require a minimal contact area with the substrate to survive [76], and the
nature of this contact area can control the formation of connections with the outside
environment [77]. Normally cells exist within a complex and changing environment
8
Introduction
that includes soluble molecules such as growth factors, an ECM containing adhesive
proteins and carbohydrates, and other neighbouring cells. They actively sense and
respond to changes in this environment, existing in a state of physiological equilibrium
with it. The information content in the adhesive environment is encoded both in
its composition and its organisation on the nanometer to micrometre length scales.
When taken out of this physiological context and cultured in plastic tissue culture
dishes, the cells lose the cues that maintain their in vivo identity or phenotype, and
dedifferentiate. For example, hepatocytes – the principal cell type in the liver –
perform several critical liver-specific functions such as production of bile, metabolism
of urea, and the synthesis of important serum proteins such as albumin, fibrinogen
and transferrin. When hepatocytes are cultured in vitro and isolated from the liver
microenvironment, they rapidly down-regulate the liver-specific phenotype. Similarly
chondrocytes, which are required for the secretion and maintenance of cartilage, lose
their differentiated function when cultured in vitro, down-regulating the synthesis and
secretion of cartilage-specific collagens and proteoglycans. Obviously, tissue-specific
cell function is closely related to the microstructural organisation of the tissue itself
[78].
There is to date no general theory of bioadhesion, although the fundamental

understanding of its molecular mechanisms can lead to the creation of material surfaces
that can reduce or support the cell/biomaterial interface interaction [48, 79]. On an
artificial substrate, such as a biomaterial, the interaction with cells proceeds via the
following steps [80]: (i) initial cell attachment, (ii) cell spreading, (iii) organisation
of actin cytoskeleton; and (iv) formation of specific focal contacts. The initial
attachment of cells onto artificial substrates results in an immobilisation, preventing
detachment in response to mild shear forces. Once attached to the surface, the cell
membrane begins to spread along the available surface area. This is followed by the
creation of a filamentous actin cytoskeleton. Finally, in response to force applied by
the cytoskeleton, integrins form clusters known as focal adhesion sites that trigger
signalling pathways, which can influence cell function, viability and proliferation [81].
It is well-known that different cell types use different attachment mechanisms to

different surfaces, and as a rule, cells do not interact with the surface directly but via
adhesive proteins secreted by them. The initial interface interaction when cells contact
biomaterial surfaces resembles to some extent the natural adhesive interaction of
the cells with their ECM. According to a ‘classical scheme’, adhesive factors such as
fibronectin and vitronectin present in the serum adsorbed on the substrate, and the
adhesion is in fact an interaction between them. This interaction is ligand-receptor
because the cells have specialised receptors (integrins) through which they identify
the adsorbed adhesive protein ligands [54, 82]. Guided by the substrate-surface
properties, conformational alterations of the adsorbed proteins possibly change their
biological behaviour [82]. In this context, the initial cellular interaction depends on the
9
surface physicochemical properties such as surface wettability, charges, heterogeneity,

topography, roughness and presence of functional groups [79]. It is not clear why,
but it is well known that some materials with a hydrophobic surface adsorb proteins
in a way that decreases their native bioactivity [82]. An adequate adhesive protein
adsorption is essential for initial cell adhesion. However, the cells not only interact
with the adsorbed soluble matrix proteins, they also tend to reorganise them in fibrils.
This cellular activity depends significantly on different biomaterial surface parameters,
such as hydrophilicity, chemical composition and charges [79].
Although the mechanism of cell/biomaterial surface interaction is not fully understood,

surface physicochemical parameters that influence it are known and could be
summarised as follows [46, 48, 79, 83]: surface free energy and related parameters,
hydrophilic/hydrophobic balance, polarity, water contact angle (WCA) and its
hysteresis; surface charge and related electrostatic interactions; type and mobility
of the surface functional groups; micro- and nano-topography features and surface
roughness [83, 84]; thickness, density and adhesion of the modifying layer; surface
crystallinity [85]. The shape and size of biomaterial surface structure can also control
the cell proliferation and orientation [86]. The shape and size of the biomaterial
particles influence the cell recognition ability and interaction [87].
The effect of surface topography and chemistry on cellular response is of fundamental

importance, especially where living systems encounter device surfaces in medical
implants, tissue engineering and cell-based sensors. There is widespread interest in the
tailoring of surface-active materials produced by suitable surface chemistry coupled
with advanced patterning processes to understand the biological processes on the
surfaces [88]. Most biomolecules have immense recognition power (specific binding)
and at the same time demonstrate a tendency to physically adsorb onto solid substrate
without specific receptor recognition (non-specific adsorption). Therefore, interfaces
are required that have both enhanced specific binding and reduced non-specific binding
to create useful materials for many biomedical and biotechnological applications. In
applications such as sensors, the tailoring of surface chemistry and the use of micro-
and nano-fabrication techniques became an important direction for the production
of surfaces with specific binding properties and minimal background interference.
Both self-assembled monolayers and polymer brushes attract considerable attention
as surface-active materials [89].
It is known that the cellular activity depends greatly on the biomaterial surface
hydrophilicity [45, 79], together with steric hindrance, the existence of a ‘conditioning
layer’, surface chemical composition and charge, surface topography and roughness
[90]. Studying a number of model surfaces, Altankov [79] concludes that the
hydrophilic surfaces support cell adhesion and proliferation, cell growth and the
organisation of the focal adhesion complex delivering the signal via integrin receptors.
10
Introduction
An optimum interaction with cells usually appears at moderate hydrophilicity (WCA of

~50–65°). The synthesis and organisation of the fibronectin matrix by cells is better on
surfaces weakly bonding fibronectin and other matrix proteins. The conformation of
the adsorbed adhesive proteins also plays an important role in the adhesive interaction
on strong hydrophilic non-charged surfaces [76, 91].
Many chemical functional groups, such as hydroxyl, carbonyl, carboxyl and amine,
are important for the fate modulation of the attached cells [92]. For example, the
macrophage’s ability to form giant multi-nuclear cells (granular reaction) on some
hydrogel surfaces correlates with the presence of some functional groups. The
probability of macrophages joining, decreases in the following order [93]:
–N(CH3)2> –OH> –CONH– > –SO3H> –COOH (–COONa)
A similar interaction hierarchy is observed at cell incubation onto functionalised

surfaces, in which the cell attachment and growth decrease in the following sequence
[94]:
–CH2NH2> –CH2OH> –CONH2> –COOH
The cell responses are controlled by intracellular signalling pathways that are originally
triggered by transmembrane proteins interacting with the engineered surface[95]. The
surface chemistry characterised by the type of cell binding ligands (peptides, proteins
and so on), their surface density [96–98] and spatial distribution [95, 99, 100], as
well as their conformation [101], are important surface cues.
In order to be viable, anchorage-dependent cells require an adhesive surface to stick

to and consequently spread. The ability to constrain the spreading to a specific cell-
surface contact area has been shown to dramatically affect cellular development [77,
102, 103]. Mechanical compliance of cell-adhering substrates can also substantially
affect the cellular response and development [104]. The ability to spatially and
temporally control the chemistry, the pattern geometry and the local substrate stiffness
will continue to provide new insights into the fundamental aspects of cell-surface
interactions [105, 106].
11
It is well known that cells interact with their external environment via transmembrane
proteins, many of which are receptors functioning not only as transmitters of
information but also as transporters of molecules from the outside to the inside of
the cell. As so-called ‘membrane spanning proteins’, these receptors are characterised
by an extracellular ligand binding domain and an intracellular signalling domain.
The binding of a ligand with the receptor causes an intracellular cascade of enzyme-
mediated reactions that in turn leads to the signal being amplified. This process
is triggered either by a change in the receptor’s conformation, or by a change in
its affinity for other molecules. As this so-called ‘signal transduction’ affects gene
regulation, it is not surprising that a variety of cell functions, including survival,
proliferation, migration and differentiation, are governed by integrated signals
from many types of molecules. Interestingly, these signalling cascades and cellular
processes can also be controlled or triggered by more or less specific interactions
with tailored surfaces (interfaces). In their early investigations, Whitesides and
co-workers patterned monolayers by microcontact printing [107–109] with cell
adhesive and cell repellent functionalities, and further investigated the effect of the
feature dimensions of these patterns on cell behaviour [77]. In addition to surface
chemistry [110], the topographical structures [111] and control of the substrate
modulus are important in determining the cell behaviour on the surface [106,
112–120].
1.3 Biomaterial Surface Engineering Approaches
The contact of biomaterial surface with any living matter – tissue, cells, blood and
other biological fluids inside or outside the human body – is usually accompanied
by a foreign body response that could be overcome by suitable surface engineering
of the biomaterial. In particular, the use of a biocompatible outer modified surface
layer or coating can minimise these negative responses while maintaining the device
functionality. Different surface engineering approaches to create biomaterials with
improved biocontact properties are based on the relationship between the tissues,
blood and other living matter contacting surface properties and the interactions
on the interface. A variety of surface engineering methods known so far are briefly
presented in this book, divided into three main groups: (i) physicochemical methods,
including acid etching/oxidation, grafting of surface functional groups [121], surface
coating by deposition and ionising irradiation treatments (‘dry methods’): various cold
plasmas, ion or electron beams, and laser; (ii) biological methods based on biomolecule
immobilisation such as matrix proteins, peptides or short peptide sequences (RGD
and gly-arg-gly-asp as well as different growth factors, and (iii) patterned surface
modification, i.e., creation of surface micro- or nano-structure by lithography and
near field scanning methods.
12
Introduction
Plasma treatment is usually accompanied by so-called ‘surface reconstruction’

(basically due to relaxation processes in the polymer), tending to return the surface to
its initial state. Therefore, the plasma treatment is usually employed as a preliminary
procedure, followed by chemical grafting and/or biomolecule immobilisation [89,
122–126]. Matrix proteins and different growth factors are immobilised, chemical
functional groups are grafted and topographic features are created to design a support
mimicking the natural ECM-specific features or functions [127–132], including
simple physical pre-adsorption of proteins, peptides and/or growth factors, enzyme
immobilisation, and cell pre-seeding.
This book covers the general techniques of surface engineering, then examines
strategies and gives selected examples concerning surface modifications of a variety of
polymeric biomaterials with different specific applications. Finally, biomaterial surface
characterisation methods are presented. Emphasis is placed on the combination of
both material and surface engineering aspects of the biomaterial, the physicochemical
properties of the surface, and its protein adsorption and cell adhesion characteristics.
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16
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heirlooms, and these were pucka (good old Indian word!) and not
those of other people.
If he could only hold on to the end, and put in his time fairly and
squarely, he might yet see Aurea at Wynyard—though at present his
prospects were blank; all he had to his name was his weekly wages,
and these wages, figuratively, bore him into the presence of Miss
Parrett. What an old bully she was! how she brow-beat and hectored
her unfortunate sister, and what a jabbering impostor! talking
incessantly of all she did, and was going to do, but leaving
everything in the way of work to Miss Susan and her niece—whilst
she trotted round spying and scolding.
As Wynyard reclined against the bank smoking, absorbed in his
reflections—and Joss was equally engrossed in an adjacent ditch—a
far-away sound broke faintly on their ears. In a few seconds this had
resolved itself into the regular “thud, thud, thud,” of a galloping horse,
and here he came into sight—a chestnut in a lather, with streaming
reins, and exultant tail, carrying an empty side saddle.
Wynyard instantly recognised Aurea’s weedy thoroughbred, and,
flinging away his cigar, ran forward, but the animal, bound for his
stable, was not thus to be captured and detained; with a snort of
defiance, he made a violent swerve, and tore on, hotly pursued by
Joss.
CHAPTER XXIV
ON YAMPTON HILL
It was not the horse, but the horse’s rider that was of consequence.
Where was she? What had happened? Spurred by an agony of
apprehension, Wynyard ran in the direction from which the runaway
had appeared. In five minutes’ time a speck, and then a figure came
into sight, and this presently resolved itself into Miss Morven—
apparently unhurt. She, too, had been running; her habit was
splashed, she carried her hat in her hand, her beautiful hair was
becomingly loosened, and she had a brilliant colour.
As Wynyard slowed down to a walk, she called to him—
“Have you seen my horse?”
“Yes; he must be in Ottinge by this time,” was the comforting
rejoinder.
“Why didn’t you stop him?”
“It would have wasted a lot of time, and I wanted to see what had
happened to you.—I was afraid you’d had a spill.”
This was not the ever silent and respectful chauffeur to whom Miss
Morven had been hitherto accustomed; but no less a person than
Lieutenant Wynyard, late of the Red Hussars, who, in a cheery
voice, addressed her as an equal—as no doubt he was. So be it.
She instantly decided to abandon herself to the situation. Possibly he
would now confide something about himself, and how and why he
came to be in her aunts’ service. So, after a momentary hesitation,
she replied—
“Oh no, I only got off to open a gate, and Rufus broke away. I
suppose I shall have to walk home!”
To this Wynyard secretly and joyfully agreed, but merely said—
“I see you are alone.”
“Yes; father and I rode over to Shrapton-le-Steeple; he wanted to
see Mr. Harnett, a literary friend, and Mr. Harnett had so much to
show and to say that he persuaded father to stay and dine, as there
is a moon, and I came home by the short-cut. I must be three miles
from Ottinge?” and she halted and deliberately looked about her.
“Yes,” he replied; “a good three miles.”
(Oh, a very good three miles, during which he would have Aurea’s
undivided company—what a piece of luck!)
For some little time the couple proceeded in silence—a sensitive
silence. During the interval since their last meeting, they had
accomplished a vast amount of very special thinking—many
disturbing, dominating, and dangerous thoughts had entered the
young lady’s brain, and she said to herself—
“I must keep perfectly composed, and if ever he intends to speak
freely, now—now is the time! To think of us two alone on Yampton
Hills, three miles away from home!”
Somehow those three miles held a thrilling prospect. Wynyard, for
his part, was longing to utter what was in his mind; here was his one
grand opportunity; and yet for several hundred yards a strange
silence hung between them, though the man was burning to speak
and the girl was longing to listen; meanwhile moments, precious as
life itself, were ebbing fast! At last the conversation began to trickle;
the topics were the choir, the boy scouts, old Thunder’s pig, and Mrs.
Hogben’s face-ache—a spent cartridge in the path introduced sport
and shooting.
“I wonder why men are so keen on killing things?” said Aurea.
“I believe we inherit it from our ancestors, who had to kill wild
creatures or starve. I must say I like shooting.”
“Oh, do you!”—a blank pause—“the only sport I can imagine any
pleasure in, is hunting.”
“Do you hunt?”
“No; I only wish I did; but Aunt Bella thinks it so improper for a
woman to follow the hounds, and father could not escort me.”
“But parsons do hunt.”
“They did; a vicar of Ottinge actually kept hounds. Father says he
only left a dozen dusty books in the library, but a hundred dozen of
sound wine in the cellar.”
“Yes, those were the good old days!”
“I’m not so sure that they were superior to our own times. What do
you say?”
“That I hope you will always have a good time, Miss Morven.”
Miss Morven coloured and bit her lip, but resumed—
“If I only might hunt, I would be bound to have a good time.”
“Is your horse a clever jumper?”
“No; he either blunders on his head, or sits down.”
“Doesn’t sound very promising!” and they both laughed. “Anyway, it’s
a rotten, bad country,” said Wynyard, with a contemptuous wave of
his hand; “the uplands are full of rabbit holes, and as for the lowlands
—you’d want a boat! You should see Leicestershire—big fields and
sound turf.”
“Yes; but I’m afraid I can’t hunt in Leicestershire from Ottinge,” she
answered, with a smile; “and I have some hopes of sport this winter.
Mrs. Waring, who is tremendously keen, wants me to go out with
her.”
“On a pillion?”
“No; her brother has a capital horse, not up to his weight, that would
just carry me. He is so anxious that I should try it; it jumps
beautifully.”
“And what does Miss Parrett say?”
“I think Mrs. Waring may talk her over, and Mr. Woolcock promises to
look after me.”
This information roused Wynyard’s ire, his face hardened, and his
tone was dry as he said—
“Woolcock is too heavy to hunt, except pounding along the road. He
must weigh seventeen stone!”
“Very likely; but he is going to do a cure before the season opens.”
“Why not a couple of hours with the garden roller, and save the
donkey?”
Miss Morven took no notice of this impudent suggestion—merely
flicked her habit with her hunting crop, and he continued—
“Westmere is a fine old place.”
“Yes, isn’t it? The hall and galleries are real Tudor, and the park is
lovely.”
“How would you like to live there?”
“I?” and as she turned to him her air was lofty. “What a—a—an
extraordinary question!”
“Yes,” he replied, with hasty penitence; “please forgive me, it was
more than extraordinary, it was impertinent.”
“By the way (it was, after all, the girl who broke the ice), I must ask
you to excuse me for my inquisitive question the other day in the
garden.”
“You wanted to know why I hung on at Ottinge, with little or nothing
to do?” and he paused. “I think you do know, Miss Morven, in fact,
I’m sure you know. I’d be only too glad to speak out, but my hands
and tongue are tied. I’ve given a promise I’m bound to keep, and
between you and my absolute confidence, there stands at present
an enormous obstacle.”
“Oh!”
“I ought to tell you that I’m not what I seem.”
“Of course,” with a touch of impatience, “you are a gentleman by
birth; I’ve always known that.”
“Nor am I here in my own name—only my christian name; but I’ve
never done anything to disgrace it, I give you my word of honour.” As
he came to a halt and faced her, and the setting sun shone into his
truthful eyes and touched his crisp brown locks, the glow of the
evening air seemed to give added force to his personality. “I’ve
played the fool—the silly ass—and I’ve got to pay. How I wish I might
talk to you openly, and tell you all about myself!”
“I wish you might,” repeated the girl, and her voice shook; an
emotional tension had crept into the situation—her pulses beat
wildly, and her mind was in a tumult.
“You cannot imagine what it is to be in my fix,” he continued,
speaking with low, passionate intensity; “for months and months to
love some one with all my soul, and never be able to open my lips.”
“It must be trying,” she answered, now moving on, with her eyes on
the ground.
“And when I’m free, I may be too late!” he said gloomily.
“You may,” she assented; “for how could some one guess?”
“That’s it! That is what is the awful part of the whole thing; but, look
here, Miss Morven, let me state a case. Supposing you knew a
fellow in such a hole, and felt that you cared for him, and could trust
him and stick to him, as it were, blindly for a time; supposing he were
your social equal, and had a clean record, and that you knew he
worshipped your very shadow—would”—and here he looked straight
into her face—“you wait?” To this question, impetuously delivered,
there followed a silence.
“This is a sort of problem, isn’t it?” she faltered at last, “like the Hard
Cases in Vanity Fair?”
“No, by Jove, part of it is God’s truth! but I’m only talking like an idiot.
Of course no girl that ever was born could do it.”
“I’m not so sure,” she murmured, with her eyes on the ground, her
heart beating in hurried thumps.
“Miss Morven—Aurea,” he went on, now moved out of all discretion,
and casting self-control to the winds, “you are the only girl I’ve ever
cared for in all my life. I fell in love with you the first moment I ever
saw you, when you danced with Mackenzie in the Manor drawing-
room. This meeting to-day has been the one good turn luck has
done me in three years—and I seize upon it perhaps unlawfully;
perhaps it’s not just cricket, my talking to you in this way, but it’s my
only chance, so I snatch it, for I may never see you alone again—
and all is fair in love and war.”
At this moment he caught sight of a stout figure, far below, labouring
up the winding lane; it was Miss Morven’s maid, Norris. He
recognised her bright blue gown. Oh, the precious moments were
numbered, and it was now—or never!
“What do you say?” he demanded, coming to a standstill.
“But what can I say?” she rejoined, lifting her startled eyes to his. “I
don’t know anything about you. You cannot even tell me your name.”
(Naturally she did not mention that it was already known to her.) “It’s
all rather bewildering, isn’t it?”
“Yes, it’s simply crazy,” he admitted; “here am I, your aunts’
chauffeur, receiving weekly wages, living like a working man, telling
you, with the most audacious and astounding impertinence, that I’ve
been in love with you for months. You know that I’m of your own
class, but who I am, or where I come from, I refuse to disclose. No
wonder you feel dazed.” They were now within sight of the village
and of Norris. “Look here, Miss Morven,” he continued, and his voice
was a little hoarse, “I see your maid coming, and my priceless,
precious time is running out. Let me ask you one question;
supposing I were not Owen, the chauffeur, but had fair prospects,
good friends, and say a thousand a year to start with—would you—
marry me?”
Aurea knew perfectly well that she would marry him if he had only a
few hundreds a year, no prospects, and no friends; but she took
refuge in that nice, useful, and evasive word “Perhaps.”
“Perhaps”—he stooped and kissed her gloved hand—“perhaps—will
keep me going! Even if I don’t see you, I shall live on that one little
word for the next eighteen months. I don’t suppose I shall have
another opportunity of speaking to you.”
Here he was interrupted by Norris, who suddenly appeared round a
bend in the lane, puffing like a grampus, her hat on the back of her
head, and her face crimson from exertion.
“Oh, Miss Aurea!” she screamed, as she halted and held her fat
sides, “such a cruel fright as you give me—and the three men gone
by the road looking to find your corpse! and I ran this way, after your
horse come home all loose and wild. Are you hurt?”
“No; he only broke away when I was opening a gate, and I had to
walk,” replied the young lady with wonderful composure.
Norris threw a sharp glance at the chauffeur.
“And couldn’t he have helped you? What was he about?”
“I’m afraid, like yourself, I was looking for Miss Morven,” he replied.
Norris turned from him with a sniff of disparagement, and,
addressing herself to her mistress, said—
“And where’s the Rector?”
“He is dining with Mr. Harnett.”
“Tut, tut, tut! And he will stay half the night talking books, and there
are a brace of grouse for dinner—kept to the hour—and all he will
get at Harnett’s will be green vegetables, like a goat—he’s a
sexagenarian!”
At this Aurea laughed and the chauffeur smiled; he was now walking
alongside of Miss Aurea, as much at his ease as if he were a
gentleman! Norris turned on him abruptly, and said—
“Look here, young man, you’d better be getting on—it’s your supper-
time, and Mother Hogben won’t keep it hot for you.”
“It’s very good of you to think of my supper,” he replied, with a touch
of hauteur; “but I’m not in any hurry.”
He spoke to her as her superior; his was the voice and air of the
ruling and upper class, and Norris’ dislike to the insolent young
ne’er-do-well suddenly flared into active hatred. Meanwhile, he
walked with them to the very end of the lane, and opened the side
gate for Miss Morven and herself; and as he held it, he took off his
cap to Miss Aurea and said—
“Good-night—miss.”
As mistress and maid crossed the lawn, the latter burst out—
“I can’t abide that young fellow, with his fine manners and his taking
off his cap like a lord! Miss Aurea dear, I’m thinking the Rector would
not be too well pleased to see you in the lanes a-walking out like any
village girl along of your aunts’ chauffeur.”
“Norris, how dare you speak to me in such a way!” cried Aurea
passionately. And yet, why be furious? She had been “a-walking out”
precisely like any other country girl.
“Well, well, well, dearie, don’t be angry. I’m only giving you a hint for
your good, and I know you are a real lady, as proud as proud, and as
high-minded as a queen or an angel. Still and all, I’m mighty glad
that none of our talkers happened to come across you!”
CHAPTER XXV
LADY KESTERS AT THE DRUM
Jane Norris, who had been Aurea’s nurse, was now her maid and
housekeeper, a most efficient individual in both capacities. Jane was
a woman of fifty, with a round, fat face, a complacent double chin, a
comfortable figure, and a quantity of ginger-coloured hair—of which
she was unreasonably vain. Jane had also a pair of prominent brown
eyes (which gave the impression of watchfulness), a sharp tongue, a
very sincere affection for her child, and an insatiable appetite for
gossip. She was left in sole charge of the Rector and Rectory when
Aurea was absent, and considered herself a person of paramount
importance in the community, not only on account of her position at
the Rectory, but also for being the happy possessor of a real fur
coat, a gold watch, and, last, but by no means least, considerable
savings. Her circle was naturally contracted and select; her
intimates, the village dressmaker, Miss Poult—who had many clients
in the neighbourhood—Mrs. Frickett, of the Drum; and Mrs. Gill, the
schoolmistress. (Mrs. Hogben, who took in washing, needless to say,
was not in her set.) Miss Norris had a flair for uncloaking scandals,
and was a veritable Captain Cook in the way of making marvellous
and unsuspected discoveries. She had always been particularly
anxious to explore the chauffeur’s past and to learn what she called
the “geography” of this young man. Hitherto the young man had
defeated her efforts, and baffled her most insidious inquiries. He did
not drink or talk or give himself away; he did not carry on with girls,
or encourage them. Oh, it was an old head on young shoulders, and
there was something about him that was not fair and square—and
she was bound to know it!
Miss Norris had been occasionally disturbed by a vague
apprehension (resembling some persistent and irritating insect) that
her mistress was interested in this good-looking stranger, but she
thrust the idea angrily aside. Miss Aurea was not like those bold,
chattering minxes who were always throwing themselves in his way!
She was really ashamed of herself, and her wicked mind. Of course,
Miss Aurea would make a grand match, and marry young Woolcock
—who was just crazy about her, as all the world knew—and she
would go with her as maid to Westmere Park. But the vision of her
young lady and the chauffeur talking to her so earnestly in the hill
lane had excited her fears, and she resolved to give Miss Aurea
something to think of, and put her from speaking to the upsetting,
impudent fellow—who got more notice and made more talk in
Ottinge than the Rector himself!
Aurea, who had been accustomed to Norris ever since the days of
socks and strapped shoes, regarded her as a friend, and even
suffered her to gossip (mildly) as she dressed her hair, for she said
to herself—
“The poor thing has no one else to talk to all day long”—Simple
Aurea!—“being set in authority over the other servants, and must
have some safety-valve.”
The night after her walk with Wynyard, Aurea slept but little; she was
thinking, and wondering, and happy. As she dressed, she was
unusually abstracted, and when Norris began her coiffure, she did
not as usual read the Psalms for the day, but sat with crossed hands
in a trance of meditation, whilst her maid brushed her soft and
lustrous locks. After twice clearing her throat with energetic
significance, Norris began—
“So Mrs. Ramsay is letting the house for six months, I hear?”
“Yes,” was the languid reply.
“To a sort of county inspector; the chauffeur fellow showed him in—
he has a finger in every one’s pie.”
“I don’t know what you mean, Norris.”
“Well, anyway, he did a lot for Mrs. Ramsay,” she answered, with
significance. “He was in and out at all hours—some think he is good-
looking—and ladies like him.”
“What ladies?”
“Well, now, Miss Aurea, you know I don’t intend any harm, but the
talk is that your aunt, Miss Susan, makes too great a pet of him.
Why, half his day he’s helping her in the garden or potting plants in
the greenhouse; and she lends him books, and talks and makes a
fuss of him, just as if he were in her own station.”
Norris’ speech was so rapid, such a cataract of words, that her
young mistress had not been able to interrupt; at last she broke in—
“How wicked of people!” endeavouring to wrench her hair away.
“Poor Aunt Susan—so good, unselfish, and kind—not even spared!
Oh, it’s too abominable! I’m ashamed of you, Norrie; how can you
listen to such things?”
“Indeed, Miss Aurea, I said just what you said, and that Miss Susan
was too old; but they say there is no fool like an old one—and some
folk will gossip. And there was Mrs. Lambert, who married a boy that
was at school with her own son. You know there’s not much to talk of
here—now the Ramsays are gone. As for the young man, as I told
you to-night, I never held a good opinion of him; he’s too secret and
too off-hand to please me. He goes out of a night for exercise, so he
says, walking the country till daybreak; but that’s just a blind. Who is
he with?—tell me that?”
Aurea remembered, with a sudden stinging pang, how she and her
father had overtaken him one evening escorting Dilly Topham. Dilly
had been crying, and she was holding his hand!
“Why, I saw him myself in the theatre at Brodfield,” resumed Norris,
“and he had a young woman with him—so he had.”
“And why not?” bravely demanded Aurea, but her lips were white.
“The two were in a box, and he sat back—but I knew him—and
afterwards they walked together to the Coach and Horses Hotel, the
best in Brodfield. She was tall and slim, and wore a long coat and
black lace scarf over her head—I call it very bold in the public street.”
“One of his friends,” explained Aurea, with a stoical indifference her
heart belied; and to cut short any further disclosures, she released
herself from her handmaiden’s clutches and knelt down to say her
prayers.
By a disagreeable and curious coincidence, Miss Morven received
that same evening ample confirmation of Norris’ arraignment!
Lady Kesters had decided to pay her brother another visit, and wrote
to announce that, as she and Martin were within fifty miles, she
would fly down to see him for a few hours.
“I’ll come to Brodfield by train and motor over. Don’t
breathe a word to the Parretts. I can put up at the Drum
and meet you there. I’ve ever so much to say and hear;
your letters are miserable, and I’ve not seen you for more
than two months. Martin is off to America in October—he
has to look after some business—and I am going with him,
as I want to see the country, but I shudder to think of the
crossing. Uncle Dick is at Carlsbad. If you come over to
the churchyard about six to-morrow, I shall be there. I’ll
hire a car for the day and get back to Brodfield for the
night, and rush to Rothes next morning with the milk; if
you will make an appointment, I can meet you, and go for
a stroll and a talk.”
A smart Napier and a motor-veiled lady were not now a startling
novelty in Ottinge—it was the highway to many places; but the 40
h.p. motor and lady who put up at the Drum was a refreshing novelty
—and a novelty invested in mystery.
The Drum jutted out obtrusively; the front faced down the road
towards the Manor, and one side was parallel to the street, and
whoever entered or left was well in evidence. Lady Kesters asked for
dinner and a sitting-room, as if such were a matter of course! The
sole sitting-room was just across the passage from the bar and
overlooked the street. It was oak-panelled, very low, the walls were
decorated with cheap prints and faded photographs of cricket
groups, there was a round table, three or four chairs, and an
overpowering atmosphere of stale beer.
“Oh, let me see—I’ll have some tea and roast chicken,” announced
the traveller.
“Chicken, ma’am?” repeated Mrs. Frickett, and her tone was
dubious. “I don’t know as I can run to that. The hens is roosting now.”
“Oh, well,”—impatiently—“bacon and eggs. I’ll go and take a turn
about the village.”
With her veil drawn over her face, Lady Kesters walked out, went
slowly up to the church, and critically inspected the Parsonage.
Then, just inside the churchyard, she discovered her brother sitting
on a tombstone. As he sprang to meet her, she exclaimed—
“Are you smiling at Grief?”
“Hullo, Sis, this is most awfully good of you! How are you? Very fit?”
“Yes. Do come out of this horribly dismal rendezvous, and let us go
down one of the lanes, and talk.”
“Aren’t you tired?”
“No, only hungry. I’ve ordered a meal at the Drum. I’m tired of sitting
in a train or motor, and glad of a walk. Well, Owen, so far so good—
six months are gone—hurrah!”
“Yes, thank goodness, but it’s been a pretty stiff job.”
“An uphill business, and terribly dull! Again I repeat, would you like to
move? You could so easily better yourself.”
“No, I stop on till the car breaks up.”
Lady Kesters raised her eyebrows.
“Well, I can only hope that blest epoch will be soon! I met Miss
Susan, you know, and the crafty old thing was fishing to find out who
you are? She has her suspicions, but I gave her no assistance. The
niece was with her—Miss Aurea——” She paused expressively, then
went on, “Owen—she’s a remarkably pretty girl.”
He nodded.
“Yes, I understand your reason for remaining in Ottinge; it is beautiful
—simplicity itself.” She looked at her brother attentively. “Are you
making love to her?”
“I—her aunts’ chauffeur?”
“Nonsense! Are you in love with her?” she persisted. “Come, tell the
truth, my dear boy. Why should you not take me into your
confidence? Are you?”
“Well—I am.”
“And she?”
“Don’t I tell you that I’m only her aunts’ chauffeur, and my tongue is
tied? All the same, Sis—it’s beastly hard lines.”
“Then, Owen, you really ought to go away; you’ll soon forget her and
Ottinge. I’ll find you another opening at once.”
“No, I won’t stir yet,” he answered doggedly.
“You are wrong, and on your head be it! I wish you could come out to
America with us; but foreign countries are barred.”
“Why are you and Martin off there?” artfully changing the subject.
“Partly business—chiefly, indeed. He has not been well, and I can’t
allow him to go alone; but, anyway, I’m looking forward to the trip.
Tell me, how are you off for money?”
“All right; I fare sumptuously on a pound a week and washing extra.”
“I suppose you live on bacon? That’s to be my dinner.”
“Bacon—eggs—fowl—steak. Mrs. Hogben is a mother to me, and a
real good sort.”
“I must say I think you look rather thin, Owen.”
“I’m glad of it; I’m as fit as a fiddle, and made sixty runs last week for
Ottinge. They little dream that I was in the Eton Eleven! Hullo! here
are some people coming. I say—what a bore!”
No less than two couples now approached arm in arm; as they
passed, they stared hard, and even halted to look back.
“What will they think, Owen?” and she laughed gaily.
“I don’t care a blow what they think!” he answered recklessly; “but all
the same you’d better return to the Drum alone.”
“Well, mind you come in this evening—I start at nine; you can
pretend my chauffeur is your pal—pretend anything!”
“Oh, I’m good enough at pretending; it’s now my second nature!
Joking apart, you ought to be going back to the inn, and getting
something to eat.”
CHAPTER XXVI
THE OBSTACLE
At seven o’clock Wynyard went boldly to the Drum and inquired for
the lady who was stopping there.
Mrs. Frickett stared at him with a stony expression in her dull grey
eyes. She had heard of his airs and his impudence from Norris.
“Will she see you?” she asked, and her tone was aggressively
insolent.
“Oh yes,” was the ready answer; “it’s business.”
“Oh, if it’s business——” and she gave an incredulous sniff and,
flinging open the parlour door, ushered him into the presence of his
sister.
Lady Kesters had removed her cap and motor coat, and was seated
at the table in a careless attitude, leaning her head on her hand and
smoking a cigarette. The door was exactly opposite to the taproom,
and the assembled crowd enjoyed a rare and unexpected spectacle.
A woman smoking—ay, and looking as if she were well used to it
and enjoying herself—a lady, too—there was a string of pearls round
her throat, and the hand that supported her dark head was ablaze
with diamonds. Ottinge had heard and read that females were taking
to tobacco, and here was the actual demonstration before their
gloating eyes. A fine, handsome young madam, too, with a car in the
yard—ay, and a friend to visit her! They craned over to catch a
glimpse of the figure ushered in by Mrs. Frickett. The man’s back
and shoulders had a familiar look. Why, if it wasn’t Owen, Miss
Parrett’s chauffeur! The immediate result of this astounding
discovery was a deadly and expressive silence.
Since Wynyard had parted with his sister he had made up his mind
to tell her all about Aurea. He longed to share his secret with some
one, and who could be better than Leila? She would give him her
sympathy and—what was more—a helping hand; if any one could
unravel a hopeless tangle, it was she. After a little commonplace
talk, in a few abrupt sentences he commenced to state his case.
“Ah!” she exclaimed as he paused, and she dabbed the end of her
cigarette on the old oak table, “so it’s all coming out now! You show
your good sense, Owen, in confiding in me—two heads are better
than one. I’ve seen the young lady; she is distractingly pretty—and I
think I approve.”
“Think!” The words were a text upon which her brother delivered to
his astounded listener an address of such emotional eloquence, that
she sat and stared in bewildered silence.
As he spoke, he strode about the room, carried away by his adorable
subject—Aurea’s beauty, her cleverness, her unselfishness, her
simple and single-hearted disposition, her good influence in Ottinge,
her delicious voice, and her entrancing smile. Oh, it was a wonderful
relief to share with another the raptures so long bottled up in his own
breast!
In the middle of his discourse, the door, which was flung open to
admit “two lemonades,”—Owen had warned his sister against the
deadly Drum coffee,—revealed to a profoundly interested tap, young
Owen, the shover, “a-walkin’ and a-talkin’ and a-carryin’ on like old
Billy, and in such a takin’ as never was seen.”
“She’s his sweetheart, ’tis sure!” suggested one sightseer.
“Nay, more likely his missus,” argued another; “she was a-laughin’ at
him!”
As the door closed Leila threw her cigarette into the grate with a
quick, decided gesture, and, leaning both elbows on the table, said,
as she looked up at her brother—
“It’s an extraordinary entanglement, my dear boy. You are in love—
for the first and only time in your life. Of course I can believe as
much of that as I like!”
“You can!” His voice was sharp and combative.
“In love with an angel. I may tell you that she really is a fellow-
creature! You think she likes you, but for one solid year and a half
you may not impart to her who you are, or where you come from, or
even your name—I mean your surname. You are at liberty to inform
her that you are ‘Owen St. John Willoughby FitzGibbon’—a nice long
string!—but must never breathe the magic word ‘Wynyard.’”
“No, you know I can’t,” he answered irritably.
“You are her aunts’ servant now, though you will be, if you live, Sir
Owen Wynyard of Wynyard; but you may not give her the faintest
hint, as you must stick to your bargain with Uncle Dick and he to his
with you. Now, let me consider,” and she held up a finger: “if you
speak, and reveal your identity, and become engaged, you lose a
fortune.”
“Yes,” he agreed, a trifle dryly.
“If you don’t speak, you run a great chance of losing the young lady!
Mr. Woolcock is on the spot, and as willing as Barkis. Westmere is
close by—an ever-enticing temptation—and he has the goodwill of
the girl’s relations.”
“Yes, that’s a true bill; it’s wonderful how you grasp things.”
“What grounds have you for supposing the girl would wait for
eighteen months in absolute ignorance of who you were? Have you
ever spoken to her, as her equal?”
“Yes, once,” and he described their walk two days previously. “I
stated a similar case; I made the most of my time, and asked her
what she would do under such circumstances.”
“My dear Owen,”—and she looked at him with an expression of
wonder in her eyes—“I am simply staggered at your presumption!”
“Yes, so was I; but, you see, it was my only chance, and I snatched
it.”
“And what did she answer. That it was evident you were an
uncertified lunatic!”
“No; she said ‘Perhaps.’”
“‘Je m’en vais, chercher un grand peut-être,’ as some one said on
his death-bed.”
“Don’t talk French—or of death-beds, Sis.”
“No, I won’t. I see that your divinity is a clever, modern young
woman, who refuses to commit herself. Look here, Owen, I won’t
tease you any more; this situation is such that it even baffles the
activity of my clever and contriving mind! I’m afraid I can do nothing
at present; but when we return from America, I shall make a point of
cultivating General and Mrs. Morven, on account of the girl. I’ll
cultivate the girl for your sake, and ask her to stay in Mount Street.
Possibly she may open her heart to me, and tell me everything! I
have a wonderful knack of extracting similar confidences even from
my housemaids! I shall listen sympathetically, advise sagaciously,
and urge her to stick to you!”
“Yes, I know that once you take a thing in hand, Sis, it goes like an
express train; but you will be away for six months—six months is a
long time.”
“Time!”—springing to her feet—“and talking of time, I must be off.
Ring the bell, my dear boy, and order the car at once.”
Miss Morven had been dining at the Manor. She had endured a long,
leaden evening playing draughts with her Aunt Bella; she played so
carelessly that Bella had repeatedly huffed her, and eventually won
with six kings to the good! After their niece’s departure, the sisters
were for once unanimous in their opinion: they had never seen
Aurea looking so well, as that night.
“What a rose-blush complexion, what clear, glowing eyes!” said
Susan, with enthusiasm.
“Yes,” agreed Miss Parrett, who was putting away the draught-board,
“she’s got my skin, and her mother’s eyes. I’ve often been asked if I
were painted!” she announced, with serene complacency.

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2 Surface Engineering Methods.................................................................... 23

2.2.7.3 Langmuir–Blodgett Films................................. 39

3 Surface Engineering of Biomaterials Reducing Protein Adsorption............ 87

3.2 Surface Engineering of Biomaterials to Reduce

3.4.2.1 Plasma Treatments......................................... 163

4 Surface Engineering of Blood Contacting Polymeric Biomaterials........... 231

5 Surface Engineering of Bio-interactive Biomaterials................................. 295

5.2.2.2 Creation of Positively Charged Surfaces......... 311

5.4 Surface Engineering of Biomaterials Reducing Bacterial

6 Biomaterial Surface Characterisation...................................................... 477

6.7.3 Bioactivity Evaluation of Proteins Immobilised on

7 Summary and Outlook............................................................................ 517

Index ............................................................................................................... 553

Surface engineering can be considered in terms of surface treatments (modification of

system can, therefore, be considered as a composite system, consisting of a near-surface

1.1 Specific Objectives of Biomaterial Surface Engineering

• Reduction/elimination of protein adsorption, to reduce both undesirable responses

• Reduction/elimination of cell adhesion at blood contacting surfaces.

• Promotion of cell attachment/adhesion for ingrowing implants and tissue

• Reduction of bacterial adhesion.

• Control of transport properties, to optimise the passage of therapeutic agents,

• Increase in lubricity, hardness and resistance to corrosion/wear.

1.2 Theoretical Basis of Biomaterial Surface Engineering

1.2.1 Protein Adsorption

Protein-biomaterial surface interactions play a key role in the performance of

1.2.1.1 Specific Protein Adsorption

Specific molecular interactions are the fundamental processes governing control of

1.2.1.2 Non-specific Protein Adsorption

Based on widespread research, some general correlations between protein properties

POLAR DONOR - ACCEPTOR INTERACTIONS

The initial adsorption is determined by the tertiary or quaternary structure of the

When a biomaterial surface is exposed to a complex biological environment such as

It is expected that new analytical techniques and direct measurement of interfacial

1.2.2 Initial Cell/Biomaterial Surface Interactions

Knowledge of the mechanism of cell/surface interaction is very important for the

There is to date no general theory of bioadhesion, although the fundamental

It is well-known that different cell types use different attachment mechanisms to

surface physicochemical properties such as surface wettability, charges, heterogeneity,

Although the mechanism of cell/biomaterial surface interaction is not fully understood,

The effect of surface topography and chemistry on cellular response is of fundamental

An optimum interaction with cells usually appears at moderate hydrophilicity (WCA of

–N(CH3)2> –OH> –CONH– > –SO3H> –COOH (–COONa)

A similar interaction hierarchy is observed at cell incubation onto functionalised

–CH2NH2> –CH2OH> –CONH2> –COOH

In order to be viable, anchorage-dependent cells require an adhesive surface to stick