02 Whole01

Chapter 2
Literature review
Chapter 2
Literature review
2.1 Introduction
This chapter contains the background information of surface
modification of biomedical materials based on reviews of literature. The
content includes an introduction of PIII and general properties of
biomedical materials such as metals, alloys, polymers and ceramics, and
details of NiTi and UHMWPE are also presented.
Furthermore,
commonly used methods for surface modification of biomedical materials

and some other applications of PIII to biomedical materials are discussed in
this chapter.
2.2 Plasma immersion ion implantation (PIII)

Plasma immersion ion implantation (PIII) was first introduced in the
late 1980s by Conrad et al.1 and Tendys et al.2.
PIII was first called
plasma source ion implantation (PSII) and later also referred to as

plasma-based ion implantation (PBII) or plasma immersion ion
implantation (PIII).
The PIII technique was developed from as an
alternative to conventional line-of-sight ion beam implantation.
The
plasma generated in the treatment chamber is utilized directly to conduct
Chapter 2
Literature review
ion implantation instead of using an ion extracted beam.
The plasma can
be generated by a number of methods such as hot filament (HF) discharge3,

inductively coupled plasma (ICP) 4 , radio frequency (RF) 5 , electron
cyclotron resonance (ECR)6 system or high voltage bias self- ignition7 for
example.
During PIII8, the specimen (substrate) is placed in the chamber and

immersed in the plasma, and a high negative pulsed voltage9 is applied to
the sample stage. Based on the potential difference, positive ions in the
plasma are accelerated towards the substrate surface resulting in ion
implantation.
Because the plasma sheath through which the ions implant
confirms to the shape of the substrate PIII is a non-line of sight technique

suitable for treating complex shaped samples.
It is also capable of
providing a high ion flux die to the absence of mass selection and losses
associated with ion transport optics. An additional advantage is that a PIII
instrument is simpler and cheaper than a traditional beam-line ion
implanter.
Ion implantation is a method which can be used to alloy the near

surface region of substrate irrespective of the solubility and diffusivity10 of
the implanting species in the substrate material.
In addition, the
possibility of low-temperature processing has prompted more widespread

applications.
Limitations such as dimensional changes and delamination
associated with conventional coatings such as thin films can be avoided in

by using PIII to create surface alloy. PIII is regularly performed to modify
the surface of metals to improve mechanical properties such as hardness,
Chapter 2
Literature review
friction coefficients, wear resistance, and corrosion resistance 11,12 .

addition, PIII is used in semiconductor processing13.
In
In the semiconductor
industry, high implantation efficiency and small equipment footprint are

two main advantages for manufacturing14.
The technique has been used to
fabricate shallow junctions in sub-micrometer integrated circuits 15 ,

silicon-on-insulator (SOI) 16 , SPIMOX 17 and silicone 18 .
Recently,
modification of polymers by PIII has received more attention and is used to

improve the friction 19 , wear 20 , 21 and wettability19, 22 , 23 in optical and
biomedical engineering.
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Literature review
2.3 Biomedical materials

The term biomedical materials is interpreted as materials used in
biological and medical applications.
This class of materials is quite broad,
including for example, implant biomaterials such as artificial heart valves

and single-use medical implement such as syringes.
Biomaterials can be
made of metals, metal alloys, polymers, ceramics, and composites.

Examples of applications of some of the materials together with their
advantages and disadvantages are listed in Table 2.1 (reference24).
Applications of biomedical materials are generally divided into two

main streams: internal and external uses.
The internally used materials are
usually referred to as biomaterials such as replacements of damaged organs,

blood vessels or tissues to improve the quality and length of life24.
External applications are those outside the human body.
Some of them
are single-use items such as syringes, blood pouches, catheters, sterile

packaging, drug delivery kit and some long-life items, for example, wound
clips and stethoscopes25.
There is a lot of room for improvement in biomedical materials and

engineering due to the short history of this field and the ever increasing
needs and demands of patients. In order to meet the requirements for
advanced surgical applications, biomedical materials that interface well
living tissue and possess good biocompatibility are required.
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Table 2.1. Examples of different types of biomedical use materials
Materials
Advantages
Disadvantages
Applications
Polymers:
Polyethylene,
Polyesters,
Polystyrene,
Polyurethane,
Silicone rubber
Low density and

easy to fabricate.
Flexible and tissue
equivalent density.
Good
biocompatibility
due to organic
constituents.
Low mechanical
strength and wear
resistance. Wetting
characteristics are
often not optimal.
Cardiovascular,
ELISA dish
surface, soft
skeletal tissue,
dental implants,
bone cement,
intraocular lens,
catheters and
tissue adhesive
Metals and Alloys:

Titanium, Stainless
steel, Nickel
titanium,
Cobalt-chromium
High impact
strength and wear
resistance
Corrosion in
human body
environment, low
biocompatibility
Orthopedic,
fixation devices
and dental
implants
Ceramics:
Alumina, Zirconia,
Calcium Phosphates
Good
biocompatibility,
corrosion
resistance
Undesirable
surface properties
and special
techniques
required for
fabrication
Improve
biocompatibility
as an interface
Composite
materials:
Fibres
Designer physical
or chemical
properties.
Possible by
varying
components
Complicated
synthesis
Assist
regeneration
natural tissue
of
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2.3.1 Metal Nickel Titanium
Nickel titanium was first developed in early 1960s by the Naval

Ordnance Laboratory with the commercial trade name Nitinol (an acronym
for Nickel Titanium Naval Ordnance Laboratories).
The concentrations of
Ni and Ti are about the same in these materials. NiTi is now a well known
shape memory alloy which possess super-elasticity and shape memory
effect.
These two outstanding properies make NiTi ideal in many medical
applications.
The notable properties were discovered by accident26 when the alloy
was subjected to strength tests by being pounded with hammers to see how
much force was necessary to deform it.
After several dents had been
created, the researchers left the samples on a windowsill and went to lunch.
Upon their return, they discovered that the dents had "repaired" themselves.
This phenomenon was explained by Dr. Frederick E. Wang that Nitinol
undergoes phase changes while remaining a solid.
Normally these phase
changes occur in an alloy when heated to its melting point.

phase changes occur at different temperatures.
Different
These phase changes,
known as between martensite and austenite, involve the rearrangement of

the position of particles within the crystal structure of the solid.
In shape
memory alloys, these phase transformations occur below its melting point.
Thus, the alloys can retain their shape without melting.
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2.3.1.1 Shape memory and Super elasticity effects

Shape memory alloy is a metal that exhibits one shape when cold and
another when heated27.
Therefore, the geometry is "remembered".
The
changed shape at low temperature will return to the original or restored

geometry when the temperature is elevated.
This is called the shape
memory effect which arises from a soft and easy to deform martensite
phase at low temperature and transformed from a low-symmetry to a rigid
and highly symmetrical crystallographic remembered structure called the
austenitic phase.
The relationship between that martensite and austenite
phases manifested by the shape memory effect in NiTi is illustrated in Fig.

2.1 (reference28).
Fig. 2.1. Schematic diagram for explanation of transformation from

martensitic to austenitic phase by shape memory effect of NiTi. (from [23])
The martensite phase is a crystalline structure that can be found when

NiTi is cooled from the austenite structure.
The martensite phase emerges
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when the temperature cools to a temperature, Ms. The phase transition is

complete when the temperature reaches the martensite finish temperature,
Mf .
On the contrary, when the temperature increases above the austenite
starting temperature (As), the martensite structure starts to convert to

austenite and the transition finishes at an austenite finish temperature (Af).
The relationship among the four temperatures is displayed in Fig. 2.2
(reference
29
).
It should be mentioned that the phase transition
temperatures depend on composition, heat treatment, cold work and ternary

alloying27.
Md
Fig. 2.2. Transition of martensite and austenite by temperature change. Ms:

martensite starting temperature; Mf: martensite finish temperature; As:
austenite starting temperature; Af: austenite finish temperature. (from [29])
Superelasticity is a rubber like function behaviour that returns the

deformed shape to original shape under the condition of stress-induced
martensite. The effect occurs when the temperature wis above Ms but a
pseudo martensite structure exists locally by loading as a result deformation.
This phenomenon is also called pseudo-elasticity.
Since it is a reversible
Chapter 2
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effect, it returns to the original shape as austenite during unloading.

Normally, the range of deformation (tensile strains) can be as high as 8%.
This effect facilitates advanced medical applications such less invasive
surgical procedures. It is possible to perform operation related to trocars
and vascular through small, leak-tight portals in the body.
2.3.1.2 Medical applications of NiTi
Early commercial applications of NiTi are in non-medical fields such as

couplings for industrial oil line pipes. Water pipes and similar types of
piping have also been adopted.
In the last two decades, NiTi has been
used for medical purposes such as dentistry, orthopedics, and

cardiovascular operation, for instance, invasive endovascular medical
applications. The shape memory properties of NiTi allow phases change
at the human body temperature.
Therefore it provides an attractive
alternative in orthopedic and cardiovascular applications. Although NiTi

is more costly than stainless steel, on average half of all peripheral vascular
stents are currently manufactured with NiTi26.
NiTi has been used in dentistry due to its resilience and shape memory
effect.
Dental products include wire clasps, prestretched wire, double
wedge materials for rapid wedging or separation of teeth30.
Furthermore, the shape memory effect is useful in other clinical

applications. One example is the Vena Cava filter to trap blood clots.
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10
The filter is collapsed before inserting into the Vena Cave and expands to
an umbrella shape at the human body temperature.
Another well know
application of NiTi that makes use of the shape memory effect is the suture
anchor used in orthopedic surgery in order to attach temdons or soft tissue
to bones. The NiTi anchor is inserted through a small hole drilled in the
bone and it expands at body temperature.
2.3.1.3 Biocompatibility of NiTi
Generally, the body reacts to foreign materials and sometimes rejects

them. The type of reaction varies widely depending on the implanted
materials.
here.
Three similar descriptions of biocompatibility are discussed
Biocompatible materials are those that do not induce acute or
chronic inflmmatory response and do not prevent a proper differentition of

implant surrounding tissues31.
Another definition is that biocompatibility
describes good or harmonious behavior of implants in contact with tissue24.

The definition of biocompatibility from the European Society for
Biomaterials is the ability of a biomaterial to induce the appropreiate
answer in a specific application25.
The controversial issue related to biocompatibility of NiTi is related to

the high content of Ni.
The high risk triggered by Ni sensitive reactions is
a problem when equiatomic NiTi is implanted into human beings.
NiTi is
an internetallic compound with a well defined regular crystal lattice order

that exhibits high atomic bonding forces with mixed covalent and metallic
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11
character meaning that it is not easy to remove atoms from the bulk
structure 32 .
Therefore, the most critical point with regard to the
biocompatibility of NiTi as a implant is the interface between NiTi and

body fluids.
It is well know that the main reason for cytotoxicity is
related to metal ion release33 due to the relatively poor corrosion resistance
of the materials in the an aggressive medium found in the human body.
Jia34 found a high cell death rate in cultures on NiTi. Similar results were
obtained by Shih35 who found smooth muscle cell growth was impaired
significantly when the Ni ion concentration was above 9 ppm.
Furthermore, the processes of osteogenesis and steonectin synthesis activity
were found to be unfavorable on NiTi compared to alternative materials
like stainless steel and titanium.
Although oxides that are usually formed
on the metal surface can act as an anti-corrosion barrier, it is not strong

enough to act as a protective layer to prevent extensive corrosion.
On the
contrary, good biocompatibility36,37,38,39,40 on surface modified NiTi has

been reported.
For example, enhancement of fibroblast human cell
proliferation and decrease of the fibrous capsule surrounding the treated

NiTi have been observed41. From the point of view of safety, there is no
doubt that the enhancement of anti-corrosion and anti wear properties are
required before the materials can be widely applied clinically.
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12
2.3.2 Polymer Ultra high molecular weight polyethylene

2.3.2.1 Composition and structure
Understanding the basic chemical structure and morphology is

important to the investigation of the key properties of UHMWPE. First of
all, UHMWPE is a member of the family of polyethylene in which the
polymer is formed from ethylene (C2H4) which is a gas with a molecular
weight of 28.
The molecular chain can consist of as many as 200,000
ethylene repetitive units and up to 400,000 carbon atoms.
The basic
chemical formula of polyethylene is (C2H4)n, where n is the degree of

polymerization.
A schematic of the chemical structures for ethylene and
polyethylene is illustrated in Fig. 2.3 (reference42).
Fig. 2.3. Chemical structures for ethylene and polyethylene. (from [42])
The four common members of the family of polyethylene are LDPE,

LLDPE, HDPE, UHMWPE. They have different molecular weights and
chain structures. LDPE and LLDPE stand for Low Density Polyethylene
and Linear Low Density Polyethylene, respectively.
Generally, LDPE
and LLDPE have branches and a linear structure with typical molecular
Chapter 2
Literature review
weights less than 50,000 g/mol whereas high density polyethylene (HDPE)
has a linear structure with a molecular weight up to 200,000 g/mol. The
average molecular weight of UHMWPE is 6,000,000 g/mol. In medical
applications, high density polyethylene is useful and the physical and
mechanical properties of HDPE and UHMWPE are summarized Table 2.2
(reference42).
13
Chapter 2
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14
Table 2.2. Typical physical and mechanical properties of HDPE and

UHMWPE. (from [42})
(*Testing conducted at 23C )
Property
HDPE
UHMWPE
Molecular Weight (106
0.05-0.25
2-6
Melting Temperature (C)
130-137
125-138
Poisson's Ratio
0.40
0.46
Specific Gravity
0.952-0.965
0.932-0.945
Tensile Modulus of
0.4-4.0
0.8-1.6
26-33
21-28
Tensile Ultimate Strength* 22-31
39-48
g/mole)
Elasticity* (GPa)
Tensile Yield Strength*
(MPa)
(MPa)
Tensile Ultimate
10-1200
350-525
60-80
39-75
Elongation* (%)
Degree of Crystallinity
(%)
2.3.2.2 Medical applications of UHMWPE

Generally speaking, polymers are promising biomedical materials since
they can be readily combined physically or chemically with biomolecules
or cells to yield biologically functional systems 43 .
Immobilization of
Chapter 2
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15
biomolecules such as enzymes and antibodies or further extension to drugs

and cells within the polymeric surface are the basic applications in
therapeutics, diagnostics, and bioprocesses.
Examples of biomolecules
immobilized on polymeric biomedical materials and the corresponding

applications are summarized in Table 2.343.
UHMWPE has been used in orthopedics as a bearing material in

artificial joints for 40 years42.
As UHMWPE possesses outstanding
properties such as high impact resistance, chemical resistance and

biocompatibility, it is an excellent articulating surface in artificial joint
replacements.
It has been used in the acetabular cup of the artificial hip as
well as a replacement for cartilage in artificial knee prostheses.
Since
UHMWPE shows no harmful effects in contact with body fluids and cells
as an implant, it is a potential candidate for further application as the
surface interacting with biomolecules.
Chapter 2
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16
Table 2.3. Examples of biomolecules immobilized on polymeric biomedical

materials and their applications. (from [43])
Biomolecules immobilized on
Application of immobilized
polymers
biomolecules surfaces
Proteins/Peptides:
Bioreactors (industrial, biomedical)
Enzymes; Antibodies; Antigens etc.
Bioseparations; Biosensors;
Diagnostic assays; biocompatible
surfaces; Target drug delivery; Cell
culture
Thrombo-resistant surfaces;
Lipids:
Fatty
acids;
Phospholipids; Albuminated surfaces
Glycolipids
Thrombo-resistant surfaces; Drug
Drugs
delivery systems
Nucleic acids and nucleotides
DNA probes; Gene therapy
2.4 Surface modification of biomedical materials
Most of the applications of biomedical materials require particular

interactions with the biological environment or biomolecules.
Besides
suitable physical properties, biocompatibility is a crucial criteria for

materials selection.
Since the reaction at the interface is related to direct
contact between the biological medium and biomedical materials, surface

modification is a viable technique to improve the surface properties while
Chapter 2
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17
the favourable bulk properties of the materials can be retained.
Therefore,
different surface modification techniques have been investigated.
Various
methods of surface treatment are available for the modification of polymers

and metals and they include physical or chemical means such as chemical
treatment, ultraviolet (UV) irradiation, plasma spraying, laser irradiation,
and plasma treatment including PIII.
Chemical treatment is one of the most widely accepted and

commercialized surface modification methods.
Chemical treatments
include surface cleaning by organic solvents, acidic or alkaline etching of

metal surfaces, and chemical coating.
The advantages are simple
processes, low costs, and suitable for continuous manufacturing.

However, the drawbacks are that they are wet processes and by-products
such as volatile organic compounds may lead to pollution, environmental
hazards, and waste disposal issues.
UV irradiation is an environmental friendly surface treatment44. The

attractive characteristic of the process is that it is simple and can be
conducted at atmospheric pressure.
Usually UV treatment is used to
activate the surface and is followed by grafting of functional groups on the

excited sites.
Nahar et al.45,46,47 reported the enhancement of protein
immobilization on inert polymer by photochemical reaction catalysed by

sunlight or UV irradiation on a chemically coated polymer surface.
The
main limitation of this technique is the difficulty of treating

three-dimensional targets due to the line-of-sight nature of the UV source.
Chapter 2
Literature review
18
Plasma spraying is a physical method in which a surface modified layer

or coating is produced.
Plasma spraying is a subset of thermal spraying
which utilizes thermal energy to melt the coating materials into droplets
that bombard the surface with high thermal energy.
The principal
difference between plasma and thermal spraying is the maximum

temperature since thermal spraying is restricted by the combustion
temperature of the fuel.
or atmospheric pressure.
Plasma spraying can be performed under vacuum

Various kinds of coating materials have been
found to foster the growth of bones, such as hydroxyapatite on titanium,

calcium silicate on glass or ceramics and titanium sprayed as a porous
structure48. However, plasma spraying is not suitable for materials with a
low melting point because of the high temperature operation during the
treatment.
Laser surface treatment has a unique advantage that the applied energy
can be controlled by precise placement of the treated depth without
overheating the rest of the bulk materials.
Furthermore, it is a dry process
and chemically clean. Hao and Lawrence49 observed the improvement of

biocompatibility of magnesia-partially stabilised zirconia (MgO-PSZ) and
titanium by carbon dioxide (CO2) laser treatment.
Bone-like apatite could
be formed on the treated samples but not on the untreated surfaces by

soaking in simulated body fluid (SBF).
Although surface heating is not
severe, it is still a shortcoming especially when materials with a low

melting point are processed.
In addition, the line-of-sight nature of the
laser makes it difficult to treat orthopedic implants with a complex

geometry.
Chapter 2
Literature review
19
Plasma treatments typically utilize energetic ions and molecules created

in a vacuum chamber to alter the surface properties of the substrate
immersed in the plasma.
Nitrogen, oxygen, argon and other elements
such as calcium and phosphate are commonly introduced to enhance the

performance of biomedical materials. The plasma contains not only ions
but also electrons, UV radiation and photons that can cause morphological
and chemical changes in the substrate. Plasma treatments have unique
advantages such as low temperature during treatment as well as
non-line-of-sight and dry process. They are thus suitable for polymers
which usually require a low processing temperature.
Furthermore, the
non-line-of-sight nature of the process makes it possible to treat complex

geometry biomedical components.
Furthermore, a dry process is always
preferred since a wet process gives rise to contamination on the treated

surface and pollution problems.
It should be mentioned that plasma
treatment is able to sterilize surfaces on implants.
A number of examples
of biomedical applications of PIII are described in the next section.
In summary, different techniques have their own advantages and the

choice of the surface modification method depends on particular needs.
Plasma treatment is generally suitable for biomedical materials due to the
flexibility and other desirable attributes.
Chapter 2
Literature review
2.5 Applications of PIII to biomedical materials

PIII surface modification has drawn a lot attention from researchers.
Fig. 2.4 (reference50) displays a number of the possible surface properties
that can be modified by PIII.
Some applications of PIII to surface
modification on biomedical materials including diamond like carbon (DLC)

deposition on artificial heart valve, hydrogenation of silicon, deposition of
titanium nitride and oxide, and modification of polymeric components in
total joint replacements are described.
Fig. 2.4. Surface properties of biomedical materials which can modified by

PIII.(from [50])
20
Chapter 2
Literature review
21
DLC
DLC layers can be synthesized by plasma deposition on orthopedic
components used in total hip and knee joints and prostheses.
DLC is
commonly applied as a surface coating materials for artificial joints to

enhance the wear and tribological properties. It is an ideal materials in
orthopedics due to not only the mechanical advantages but also good
biomedical compatibility.
Besides orthopedic applications, DLC can be
used in other biomedical areas such as coatings on artificial heart valve51.
LTIC (low temperature isotropic carbon) is currently the most

acceptable materials for use in artificial heart valves.
Unfortunately, LTIC
is brittle and the blood compatibility is still not good enough for prolonged
clinical use. As a result, patients must continuously take anti-coagulation
medicine to prevent the occurrence of thrombus.
DLC coating is an
alternative to improve blood compatibility since some reports52,53 have

mentioned that a DLC layer with the proper structure exhibits good blood
compatibility.
Furthermore, biological friendly elements such as
phosphorus54,55 and nitrogen56,57 can be introduced into DLC by PIII to

improve the surface bioactivity.
Consequently, PIII is an effective
technique to enhance the surface bioactivity.
Hydrogenated silicon
Hydrogen in silicon has been studied in microelectronics applications
for a number of years and the properties and structure of hydrogenated
silicon have been reported.
Besides applications to microelectronics,
hydrogenated silicon is a potential bioactive material.
Silicon is the major
Chapter 2
Literature review
22
component in microelectronic component devices and hence it is frequently

used in biosensors.
Therefore, the biocompatibility of a silicon surface
may be the key to successful biomedical applications. There have not been
many publications about hydrogenated silicon from the perspective of
biological research. Hydrogen PIII treated silicon was found to improve
the bioactivity or bone conductivity58,59.
Liu showed that hydroxyapatite
(HA) formed on a PIII hydrogen treated silicon surface soaked in a

simulated body fluid (SBF) for 14 and 28 days while there was no HA
formation on the untreated silicon surface.
The formation of HA is
believed to be due to the hydrogen PIII since no HA was observed on the

Ar PIII treated silicon. Therefore, hydrogen PIII is an important step.
Titanium nitride and oxide

Titanium is one of the most biocompatible materials and are commonly
used for orthopedic and dental purposes.
However, titanium has relatively
poor wear resistance and a protective coating or surface hardening is

necessary to improve prosthesis longevity. Titanium nitride (TiN) and
oxide (TiOx) are suitable protective coatings since TiN possesses excellent
mechanical properties like high hardness and TiOx has excellent
biocompatibility and chemical stability 60 .
Nitrogen PIII of titanium
improves the wear rate by 20%50. Besides the wear resistance, TiN has
good corrosion resistance and intrinsic biocompatibility. Therefore, TiN
coated surfaces are useful to cutting tools as well as hip prostheses. TiOx is
a promising biomedical coating in blood-contacting applications since it
has better blood compatibility than LTIC.
A number of methods can be
used to form nitride or oxide on a Ti surface, and among these techniques,
Chapter 2
Literature review
23
PIII is particularly useful because it can generate a gradual and mixed

interface that minimizes the risk of film delamination.
Polymeric components in joint replacements

The total joint replacement is a common remedy to restore joints
functions after injury or severe arthritis.
Usually, the artificial hip or knee
joints consist of a metallic cup coupled with a polymeric cup to provide

elasticity and joint comfort.
However, serious wear of the polymer has
been found due to its soft nature compared to metals.
The debris cause
biological reactions that lead to bone resorption and eventually joint

loosening or failure. Nitrogen PIII can improve the surface properties61
by reducing the wear rate of the polymeric cup. In fact, a better solution to
minimize wear is to modify the contacting metallic parts of the prothesis in
order to reduce the friction.
Chapter 2
Literature review
2.6 References
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10
P. K. Chu, B. Y. Tang, Y. C. Cheng and P. K. Ko, Principles and

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11
X. B. Tian, S. C. H. Kwok, L. P. Wang, and P. K. Chu, Corrosion resistance

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24
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12
X. B. Tian, B. Y. Tang, and P. K. Chu, Tribological behavior of the modified

layers of AISI304 stainless steel implanted with low-voltage PSII, Tribology
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13
P. K. Chu and C. Chan, Applications of PIII in Microelectronics - A Brief

Review, Surface and Coating Technology 136 (1-3) (2001), 151-156.
14
P. K. Chu, Recent developments and applications of plasma immersion ion

implantation, Journal of Vacuum Science and Technology B 22 (2004),
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15
P.K. Chu, X. Lu, S.S. K. Iyer, et al., A new way to make SOI wafer, Solid
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16
P. K. Chu, S. Qin, C. Chan, et al., Instrumental and process considerations

for the fabrication of silicon-on-insulator (SOI) Structures by PIII, IEEE
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17
X. B. Tian, D. T. K. Kwok, P. K. Chu, and A. Anders, Two-dimensional

sample temperature modeling in separation by plasma implantation of
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(2002), 423-427.
18
I. F. Husein, C. Chan, and P. K. Chu, Chemical structure modification of

silicone surfaces by plasma immersion ion implantation, Journal of Materials
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D. Hegemann, H. Brunner and C. Oehr, Plasma treatment of polymers for

surface and adhesion improvement, Nuclear Instruments and Methods in
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20
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21
P. Firkins, J.L. Hailey, J. Fisher, A.H. Lettington and R. Butter. Wear of

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22
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23
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25
Chapter 2
Literature review
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S. V. Bhat, Biomaterials, Boston , Kluwer Academic Publishers, New Delhi,

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L. Hao and J. Lawrence, Laser surface treatment of bio-implant materials,

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C. C. Shih, S. J. Lin, Y. L. Chen, Y. Y. Su, S. T. Lai, G. J. Wu, C. F. Kwok, K.

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36
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37
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26
Chapter 2
Literature review
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shape memory alloy on bone formation, Biomaterials (2001) 22, 2475-2480.
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G. S. Firstov, R. G. Vitchev, H. Kumar, B. Blanpain, J. Van Humbeeck,

Surface oxidation of NiTi shape memory alloy, Biomaterials (2002) 23,
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42
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46
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polymer surface for covalent immobilization of a protein, Analytical
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47
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photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent
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X. Y. Liu, P. K. Chu and C. X. Ding, Surface modification of titanium,

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49
L. Hao and J. Lawrence, Laser Surface Treatment of Bio-implant Materials,

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50
P. K. Chu, J. Y. Chen, L. P. Wang and N. Huang, Plasma-surface

modification of biomaterials, Materials Science and Engineering Reports 36
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27
Chapter 2
Literature review
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S. C. H. Kwok, Haemocompatibility of doped diamond-like carbon (DLC)

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28
Chapter 3
Equipment and Characterization methods
29
Chapter 3
3.1 Introduction
This chapter describes the equipment used to modify NiTi and

UHMWPE in my research activities.
The plasma immersion ion
implantation (PIII) systems in the Plasma laboratory of City University of

Hong Kong and School of Physics of Sydney University of Australia are
introduced. The first half of my study is surface modification of NiTi by
PIII which was performed in City University of Hong Kong.
During the
studying period in the University of Sydney, my research focus was plasma

modification of UHMWPE.
In order to characterize the samples, a number of techniques are

employed.
XPS and FTIR-ATR are utilized to analyze the chemical
properties of the sample surface such as depth profiles and elemental

concentrations.
ICPMS is the tool used to determine elemental
concentration in solution. AFM, Form Talysurf PGI, and SEM are used to
study the morphology of the sample surface.
Surface hardness, elasticity
and
by
corrosion
resistance
are
examined
nanoindentation
and
electrochemical tests. For wettability, surface hydrophilicity, and surface

energy, contact angle tests are conducted.
Chapter 3
In order to investigate the biocompatibility and functionality of the

sample surfaces, in vitro cell adhesion tests, horseradish peroxidase
attachment, and functionality assay are used. The cell adhesion test on
NiTi is conducted in collaboration with the Department of Orthopedic &
Traumatology (Division of Spine Surgery), University of Hong Kong.
Furthermore, the horseradish peroxidase attachment and functionality assay
are conducted under guidance from the Muscle Research Unit of
Department of Anatomy and Histology, The University of Sydney.
3.2 Plasma immersion ion implantation system
The PIII instrument in the plasma laboratory of City University of

Hong Kong consists of a stainless steel cylindrical chamber with a large
rectangular front door. This system is used for the modification of NiTi.
The typical base vacuum is about 1x10-6 Torr.
The plasma described in
this report is generated by radio frequency (RF) discharge of 13.56 MHz

with power 1 kW.
The setup includes an RF generator, a matching
network, and an antenna. Fig. 3.1 shows the schematic diagram of the PIII
system.
Samples for PIII treatment (substrate) are placed on a sample stage

which is connected to the negative pulse bias source.
The sample stage is
oil cooled and connected to a high voltage (-10 kV to -40 kV). Nitrogen
30
Chapter 3
or oxygen gas can be bled into the chamber through two individual flow
controls and inlets on top of the chamber.
The specimen is pulse-biased to
a high negative potential relative to the chamber wall and ions generated in
the plasma are accelerated across the sheath formed around the specimen
and implanted into the surface of the specimen.
Because of the absence of
ion transport optics and mass selection, PIII can provide a high ion flux.
It is also a non-line-of-sight process and has advantages over conventional
beam-line ion implantation.
In this technique, ions bombard the specimen
from all sides with approximately normal incident angles. The detailed
experimental parameters are discussed in Chapter 5.
31
Chapter 3
32
Gas Inlets
RF Antenna
system
Plasma
Cluster
Vacuum
Chamber
Substrate
Sample Stage
Negative Pulse Bias

To Vacuum
pump
Fig. 3.1. Schematic diagram of PIII system for surface modification of NiTi in
City University of Hong Kong.
Chapter 3
33
The PIII system in the laboratory of Applied and Plasma Physics of the
School of Physics, The University of Sydney is used for the plasma surface
modification of UHMWPE. Fig. 3.2 shows the schematic diagram of the
plasma treatment chamber.
The system consists of an inductively coupled
plasma source and a treatment chamber.
The plasma is generated in the
plasma source by supplying RF power (13.56 MHz) to a single loop

antenna wound around a borosilicate glass tube.
The RF power is fed to
the antenna via a CPM-2000 matching network.
A magnetic field of
approximately 50 G generated by 2 pairs of copper coils guides the plasma

into the aluminium treatment chamber.
The sample holder is mounted on
the axis of the treatment chamber in a face down orientation. The sample
holder consists of a stainless steel plate mounted on a vacuum feed-through
which is electrically isolated from the chamber and can therefore be biased
or left floating.
The holder is electrically connected to a mesh which is
located 30 mm in front of the sample position. This prevents the samples

from arching during plasma immersion ion implantation treatment.
The
typical vacuum pressure of the chamber is in 5x10-6 Torr. The experimental

parameters are discussed in details in Chapter 6.
Chapter 3
34
feed-through to high voltage

pulsed power supply
mesh sample holder
3.2 A
gas flow
controller
magnetic field coils

treatment chamber
6.0 A
matching network
glass tube
RF power supply
single loop antenna
to turbo pump
Fig. 3.2. Schematic diagram showing the plasma treatment system used to
modify the polymer surfaces.
Chapter 3
35
3.3 Characterization techniques
3.3.1 X-ray Photoelectron spectroscopy (XPS)
XPS is a surface specific1 technique which is used to investigate the

surface composition and elemental depth profiles in NiTi and UHMWPE.
Compositional and structural information can be obtained and generally the
technique probes only a few molecular layers about 10 nm from the surface.
On the contrary, reflection infra-red techniques typically probe 103 nm into
the sample. Therefore, XPS is a complementary technique for the analysis
of the modified layer.
Ultra-high vacuum system (pressure 10-5 Pa) is employed during sample

analysis.
The X-ray impinges the surface of the sample and the irradiation
gives rise to photoelectrons as well as Auger electrons 2 .
Figure 3.3
(reference3) illustrates the photoelectron emission process.

2p3/2 (L3)
2p1/2 (L2)
2s (L1)
h
Photoelectron
1s (K)
Fig. 3.3. Schematic diagram of the emission process of photoelectrons excited

by X-rays.
Chapter 3
36
The kinetic energy of the emitted photoelectrons is given by:

EK = h Eb Ws
where h is the energy of the photon, Eb the binding energy of the atomic
orbital from which the electron originates, and Ws is the spectrometer work
function.
As each element has a unique value of binding energies,
identification of the element can be achieved by measuring the value of the

kinetic energy of the emitted photoelectrons within the escape depth (near
surface, less than 10 nm) of photoelectrons.
Therefore, XPS can identify
all elements except hydrogen. In addition, variations in the elemental

binding energies (chemical shifts) arise from the differences in the
chemical environment and polarizability of the atoms.
These chemical
shifts can provide information on the chemical states of the elements.
The
output of spectra are plotted as binding energy against number of counts of

signal received.
A typical survey spectrum from as-received UHMWPE is
shown in Fig. 3.4, where some trace oxygen is detected on the surface.
The peak intensity is proportional to the number of atoms on the sample
and can be quantified using the appropriate sensitivity factor.
Besides the
survey spectrum, elemental depth profiling is another useful output from

XPS as it displays the chemical composition across the depth of the
modified layer into the bulk substrate. Depth profiles are obtained by
progressively sputtering away the top surface and then collecting XPS
spectra from the new surfaces they are exposed.
In this report, the
elemental depth profile shows the interface between the modified layer of
NiTi and the substrate. During irradiating X-ray on polymers, the surface
tends to charge up, and so an external source of charge-compensating
Chapter 3
37
C1s
electrons is commonly used for neutralizing those charges.
120000
100000
60000
O1s
c/s
80000
40000
20000
0
1000
1200
1400
1600
1800
2000
Binding Energy (eV)
Fig. 3.4. XPS survey spectrum of as-received UHMWPE.
A PHI Model 5802 of XPS system is utilized in this study to analyze

the composition and bond structure of Ni and UHMWPE.
The survey
spectra and elemental depth profiling are obtained using a monochromatic

Al K radiation source operated at 14 kV and 350 W.
3.3.2 Fourier Transform Infrared Attenuated Total

Reflectance Spectroscopy (FTIR-ATR)
FTIR-ATR is a widely used technique in polymer research for

investigation of the chemical composition and structure 4 near surface.
Surface sensitivity is achieed by sampling with the evanescent wave, the
near surface of a polymer in contact with a crystal, set at a total internal
Chapter 3
reflection condition with respect to the IR beam.
38
The detection region is
within 103 nm underneath the surface depending on the refractive index

different between the crystal and the polymer under investigation.
This
technique is based on the vibration of atoms or molecules. Therefore, the

type of bonding present can be revealed5. The information is obtained by
passing the evanescent wave of the infrared radiation through a sample and
determining which fraction of the incident radiation is absorbed at a
particular each energy6 in the IR spectrum.
FTIR ATR spectra in this report are recorded using a Digilab FTS7000
FTIR spectrometer fitted with an ATR accessory with trapezium
germanium crystal and incident angle of 45. The required signal/noise
ratio and resolution are obtained by using 500 scans and a resolution of 1
cm-1.
The output is in the form of a spectrum which plots the inverse
wavelength (cm-1) against percentage of absorbance.
A background
spectrum that is collected without the sample is subtracted from the sample
spectrum.
The peak occurring in the absorption spectrum corresponds to
the frequency of molecule vibration of a particular part of the sample.
Fig.
3.5 shows the spectrum of the untreated UHMWPE showing peaks found in
the ranges of 600-900 cm-1, and those at 1460, 2850 and 2925 cm-1 which
are the signature vibrational peaks of UHMWPE7.
Chapter 3
500
1000
1500
2000
2500
3000
3500
4000
39
4500
-1
Wavelength (cm )
Fig. 3.5. FTIR-ATR spectrum of an as-received UHMWPE sample.
As mentioned before, infrared spectroscopy is able to analyze collect

data from a volume up to a few micro below the surface depending on the
penetration of
the evanescent field. Usually, the plasma modified layer
is less than the detection volume.
Therefore, some of the bulk structure is
recorded and then subtracted using the control (untreated) sample.
This
technique can acquire information from the whole modified volume and
does not require a vacuum environment. The application of FTIR-ATR in
this report is to study the surface chemistry of modified UHMWPE.
3.3.3 Inductively Coupled Plasma Mass Spectroscopy

(ICPMS)
ICPMS is a highly sensitive analytical technique for detecting metals.

ICPMS is able to identify the various isotopes of an element in which more
Chapter 3
40
than one stable isotope exist8. The advantage of using ICPMS is that it is
suitable for using for all concentrations from ultratrace levels to major
components and the detection limits are generally low for most elements.
ICPMS instrument include two pieces of hardware, inductively coupled

plasma (ICP) and mass spectroscopy (MS). ICP is designed to generate
plasma from a gas in which atoms are present in an ionized state.
Since
argon gas is used to generate plasma, ICPMS is able to identify and

quantify all elements with the exception of argon. Singly charged positive
ions are typically produced in ICP, and a series of ion lenses maintained at
appropriate voltages are used to direct the ions into the quadrupole mass
analyzer8.
The ICMPS instrument employed in this research is Perkin Elmer, PE

SCIEX ELAN 6100, USA. The application of ICPMS in this research is
to measure the amount of Ni ion out diffused from NiTi immersed in
simulated body fluid (SBF) for certain periods.
In this way, the Ni
concentration in SBF can be determined.
3.3.4 Atomic Force Microscopy (AFM)
AFM is one of the members of scanning probe microscopy (SPM).

AFM is a powerful tool to study the surface topography of insulators,
conductors, and semiconductors.
dimensions.
AFM measures surfaces in three
The roughness over a scanned surface area (referring to the
Chapter 3
41
total distance the probe scanned) can be obtained.
An AFM consists of a sharp tip with a cantilever, a laser, a photodiode

and controller electronics such as a piezoelectric scanner.
In AFM, a
sharp tip at the end of cantilever that scans the surface. The distance
between the tip and the surface is controlled by maintaining a constant
intermolecular force between the tip and atoms on the surface.
The tip
thus moves up or down in order to maintain the constant force. The

vertical motion of the tip is detected by the signal from the laser source and
reflection from the back of cantilever to the receiver which is the
photodiode, followed by processing by the controller electronics.
Fig. 3.6
(reference9) shows the schematic diagram of the working principle of AFM.
Fig. 3.6. Schematic diagram of the working principle of AFM. (from [9])
Chapter 3
42
The AFM used in the present research is the Autoprobe CP produced by

Park Scientific Instrument (1171 Borregas Avenu, Sunnyavale, CA 94089).
The measurement performance depends on the scanner type.
As a
multitask probe head is employed, the maximum lateral scan range is 100
m while the vertical one is 7.5 m.
The technique can be operated under
ambient air, liquid or vacuum environments.
Contact mode AFM is used in this report to measure the roughness of

NiTi and UHMWPE before and after PIII treatment.
Fig. 3.7 shows a
typical three dimensional image output from AFM.
AFM is used to
examine the microscopic scale from 10 nm to 0.1 nm while the FTPGI

(introduced in the next section) is used for examining roughness over
macroscopic length scales on the order of mm.
Fig. 3.7. Three dimensions AFM image of untreated UHMWPE of size 1m

1m.
Chapter 3
43
3.3.5 Form Talysurf PGI (FTPGI)
Form Talysurf PGI is a surface profilometer which maps the surface

morphology by putting a stylus in mechanical contact with the sample.
FTPGI is used to determine the surface roughness and a three dimensional
topographic map of a surface.
In the present study, FTPGI from Taylor Hobson, Leicester, England is

used.
It consists of a stylus, a PGI gauge (transducer), a precision x-y
stage and a computer system.
The surface is monitored by the upward and
downward movement of the stylus. Signals from the stylus movement are
sent to the computer and converted to data and visual profiles.
Fig. 3.8
(Talyor Hobson Ltd. 200310) shows the schematic stylus movement of

FTPGI.
Fig. 3.8. Schematic diagram illustrating the stylus movement of the FTPGI.
(from [10])
Chapter 3
44
PGI stands for Phase Grating Inteferometric transducer.
This
transducer is housed within a gauge and converts the moments of the stylus
into electrical signals. The resolution of the scan is limited by the size of
the tip and the diameter of stylus tip is 2m.
In contrast to AFM, FTPGI
has more flexibility with regards to the scanned region size.
It is used to
examine larger areas than AFM such as 1 mm2 and a typical output from
FTPGI is displayed in Fig. 3.9.
Fig. 3.9. A three dimensional FTPGI image of nitrogen plasma treated

UHMWPE of scan size 1mm 1mm.
3.3.6 Scanning electron microscopy (SEM)
SEM is one of the most commonly used surface analysis techniques in

which a wide range of scales and features can be observed. The basic
concept of SEM is that the injected electron interacts with the substrate
surface and secondary electrons get away within from an escape volume of
dimension ~10nm.
The incident electrons are produced by using the
filaments (W or LaB6) and the emitted electrons are collimated and focused
Chapter 3
by the electron optics in the condenser and objective lenses.
45
Secondary
electrons are emitted when the incident electrons interact with the sample
surface.
Those secondary electrons are the signals carrying information of
the top 10 nm of surface topography. The final image of the sample is

assembled by processing and amplifying signals collected by the detector
according to the corresponding X-Y coordinates of the scanning electron
beam.
3.3.7 Nanoindentation
The nanoindentation test is used to determine the hardness (H) and

elasticity (E) of the thin film, coating or surface layer.
The indenter is
pushed into the top 1-2 m of substrate which causes elastic or plastic
deformation.
The elastic recovery, depth of contact, and residual depth
after unloading are analyzed.
MTS Nano Indenter XP is employed to
study nanoindentation in this report. T he test is conducted on five areas to

determine the average hardness and Youngs modulus of the treated and
untreated NiTi.
Chapter 3
46
3.3.8 Electrochemical test
The electrochemical tests11 based on ASTM G5-94 (1999) and G61-86

(1998) are performed by a potentiostat (VersaStat II EG&G) using a
standard simulated body fluid (SBF) at a pH of 7.42 and temperature of
37+0.5oC.
The ion concentrations in the SBF and blood plasma are
shown in Table 3.112.
A cyclic potential spanning between -400 mV and
+1600 mV is applied at a scanning rate of 600 mV per hour.
Before the
electrochemical tests, the medium is purged with nitrogen for 1 hour to

remove dissolved oxygen and nitrogen purging continued throughout the
measurements.
Table 3.1. Ion concentration in SBF in comparison with human blood plasma.
Concentration (mMole)
Na+
K+
Ca2+
Mg2+
HCO3-
Cl-
HPO42-
SO42-
142.0
5.0
2.5
1.5
4.2
148.5
1.0
0.5
Blood plasma 142.0
5.0
2.5
1.5
27.0
103.0
1.0
0.5
SBF
3.3.9 Contact angle measurement
The sessile drop contact angle measurement method is utilized to study

the wettability of surfaces.
The contact angle is measured by the sessile
drop method with a goniometer.
When the water drops on the surface, it
spreads upon contact. The angle made by the edge of water drop with the
solid surface is termed the contact angle, and the side view schematic is
Chapter 3
shown in Fig. 3.10.

in this research.
A Kruss contact angle equipment DS10 is employed

In each measurement, 50 l of de-ionised water is
dropped onto the sample and the angle is measured.
Water drop
Fig. 3.10. Schematic side view diagram of water drop with contact angle ()
3.4 Biomedical tests
3.4.1 Simulated body fluid (SBF) immersion test
In order to estimate the amount of Ni ion out diffusion after

implantation into the human body, immersion test is designed for the NiTi
samples immersed in simulated body fluid in incubator at 370.1C for 5
weeks.
The SBF is prepared under an environment with pH value of 7.42
and temperature of 37+0.5oC.
The ion contents of SBF are the same as
those in human body fluids as listed in Table 4.1. The NiTi samples
(untreated or treated) are immersed in 25 ml of simulated body fluids
(SBF)12 in polypropylene (pp) bottles. After five weeks, the NiTi samples
are removed and the SBF in the bottles is analyzed by inductively-coupled
plasma mass spectrometry (ICPMS) to determine the amount of Ni leached
47
Chapter 3
48
from the untreated or treated samples.
3.4.2 In vitro cyto-compatibility test

Besides Ni leaching from the substrate of NiTi, there is a concern on
the cyto-compatibility of the untreated and treated NiTi surfaces. The
biocompatibility of orthopedic implant such as NiTi in this research
depends on the effect from it has on behaviour of the bone-forming cell,
osteoblasts13. Therefore, Osteoblasts are cultured on the surface of NiTi
in this study.
A description of cell culturing on the substrate is presented
here and the detailed procedures and experimental setup are discussed in
section 5.2.6.
Osteoblasts in contact with treated and untreated NiTi surface interact,

attach, and proliferate on the surface.
the surface is cyto-compatible.
If they do not denature, that means
Therefore, the number of cells adhered
after a fixed culturing period is used to compare the cyto-compatibility

among different surfaces.
In the experiment, approximately the same
number of osteoblast cells is initially cultured on the untreated and treated

samples. After a fixed period, the NITi samples are taken out and cells on
top of the surface are released and counted.
The cell morphology is then
observed by fluorescent microscopy to observe cell proliferation.
The
cyto-compatability test is conducted in collaboration with the Department

of Orthopedic & Traumatology (Division of Spine Surgery), the University
of Hong Kong.
Chapter 3
49
3.4.3 Horseradish peroxidase (HRP) attachment and

functionality assay
Horseradish peroxidase is used to examine the protein attachment

ability of the untreated and PIII treated UHMWPE.
it is a kind of commonly used enzymes.
HRP is chosen since
For immobilization of protein for
sensor assay and applications, high yield of functional protein attachment is

one of the goals and of interest to industry.
Therefore, the assay is
designed to determine the quantity of functional active protein immobilized

on the surface.
The detailed procedures and experimental setup are described in section

6.3.2. The concept of the HRP attachment and functionality assay is
presented here.
First of all, the treated and untreated surface are soaked in
the 10 mM phosphate buffer solution containing HRP, so that HRP can

attach on those surfaces and some of the loosen HRP can be washed by
rinsing with fresh buffer solution without HRP repeatedly.
Then, the
samples are taken out from the fresh buffer solution and soaked in a
colourless TMB solution (substrate of HRP). The TMB solution gradually
changed to yellow as a result of a diimeine oxidation product14. The
degree of color change depends on the number of functional HRP that
reacts with TMB.
Finally, UV/VIS spectrophotometry is used to read the
optical density (O.D.) passing through the color solution in accordance with
the concentration of the functional HRP.
Chapter 3
3.5 References
1
Characterization of solid polymers: new techniques and developments edited

by S.J. Spells, Chapman & Hall, London (1994).
J. Chastain (ed.), J.F. Moulder, W.F. Stichle, P.E. Sobol, K.D. Bomben,
Handbook of X-ray Photoelectron Spectroscopy, Perkin-Elmer Corporation,
Physical Electronics Division (1995), p. 10.
S. C. H. Kwok, Haemocompatibility of doped diamond-like carbon (DLC)

thin films synthesized by plasma treated method, PhD thesis, City University
J. Meichsner, M. Nitschke, R. Rochotzki and M. Zeuner, Fundamental

investigations in plasma modification of polymers, Surface Coating &
Technology 74-75 (1995), 227-231.
D. Klee, Z. Ademovic, A. Bosserhoff, H. Hoecker, G. Maziolis and H. J. Erli,

Surface modification of poly(vinylidenefluoride) to improve the osteoblast
adhesion, Biomaterials 24 (2003), 3663-3670.
6
B. Stuart, Polymer Analysis. John Wiley & Sons Ltd., New York (2002).
M. Veres, M. Fule, S. Toth, I. Pocsik, M. Koos, A. Toth, M. Mohai and I.

Bertoti, Raman scattering of ultra-high molecular weight polyethylene treated
by plasma-based ion implantation, Thin Solid Films 482 (2005), 211-215.
http://ewr.cee.vt.edu/environmental/teach/smprimer/icp/icp.html. received at
22 Dec., 2006.
9
10
http://www.molec.com/what_is_afm.html. received at 22 Dec., 2006.

http://www.talyor-hobson.com/talysurfpgi.htm.
11
L. Tan, R. A. Dodd, and W. C. Crone, Corrosion and wear-corrosion

behavior of NiTi modified by plasma source ion implantation, Biomaterials 24
(2003), 3931-3939.
12
T. Kokubo, F. Miyaji, H. M. Kim, T. Nakamura, Spontaneous formation of

bonelike apatite layer on chemically treated titanium metals, Journal of the
American Ceramic Society 79 (4) (1996), 1127-1129.
50
Chapter 3
13
A. Kapanen, A. Kinnunen, J. Ryhanen and J. Tuukkanen, TGF-1 secretion

of ROS-17/2.8 cultures on NiTi implant material, Biomaterials 23 (2002),
3341-3346.
14
P. D. Josephy, T. Eling and R. P. Mason, The horseradish

peroxidase-catalyzed oxidation of 3,5,3,5- tetramethylbenzidine, Journal of
Biological Chemistry 257 (7) (1982), 3669-3675.
51
Chapter 4
Theory
52
Chapter 4
Theory - Interactions between energetic ions
and materials in surface modification
processes
4.1 Introduction
In this chapter, the theoretical study of the interactions between
energetic ions and target materials is presented.
First of all, the
fundamental concepts related to atomic collisions in a matter stated,

followed by computational calculation of interaction paths using SRIM
2003 (TRIM included in the same software) to estimate the penetration
depth of the ions in NiTi and UHMWPE.
Lastly, the expected structural
changes in the target materials are deduced theoretically.
4.2 Theory PIII ion and matter interaction
As energetic ions penetrate a solid and slow down by losing energy in

nuclear and electronic collisions during the collision cascades.
A major
description of an ion-solid interaction includes estimation of the the depth

of penetration, distribution of energy deposited, sputtering, new chemical
phase formation, and damage caused by collision between incoming ions
Chapter 4
Theory
53
and substrate atoms. Surface modification is a consequence of the collision

cascade and the results are generally arise from both physical and chemical
interactions.
The schematic diagram of the process of ion implantation is depicted in

Fig. 4.1 (reference1).
Since collisions are random processes, the incoming
ions may be deflected when interacting with atoms in the substrate.

Therefore, ions having the same energy and incident angle may not end up
at the same place.
The depth of penetration of ion can be described as a
probability distribution.
Incoming ion
+
Sputtered
particle
Vaccum
Original surface
Solid
After sputtering
surface
Ion penetration
depth, R
Implanted ion
Fig. 4.1. Schematic diagram of ion-solid interaction. (from [1])
Chapter 4
Theory
4.2.1 Sputtering and implanted ion distribution
While the incoming ion is injected into the near surface region, the
subsequent near surface collisions remove surface atoms from the substrate
and this process is called sputtering. One of the applications of sputtering
is surface cleaning.
The sputtering yield is the ratio of number of
sputtered atom per incident ion which typically lies between 0.5 to 20
depending on the ion species, energy, target composition, and incident
angle.
The sputtering yield of most materials is less than two2. High
sputtering yields lead to thinner modified layers and smaller retained

doses.3
Sputtering limits the maximum concentration of implanted particles that

can be retained in the substrate4.
When sputtering is minimal and thermal
diffusion is not significant, the in-depth distribution of the implanted

species is likely to be Gaussian.
However, sputtering and diffusion due to
higher substrate temperature may become substantial particularly during

high-dose rate implantation.
When the sputtering rate is less than that of
implantation, a high fluence can be implanted given sufficient time5. The

schematic view of the maximum concentration achieved is sketched in Fig
4.2 (reference5).
54
Chapter 4
Theory
55
Beginning of
implantation
Depth
Sputter direction
Transition stage
Number of
implanted ion
Origin surface
Depth
Maximum
concentration
Depth
Etched thickness
Fig. 4.2. Schematic diagram of the development of concentration profile of ions
implanted. (from [5])
4.2.2 Ion stopping and Ion range
When the incident ions penetrate the target, they interact with neighbor
particles and lose energy.
The average energy loss per unit traversed
distance is defined as stopping power dE/dx. There are two types of

interactions between the ions and substrate atoms.
The first one is nuclear
collision that causes different ranges of energy loss and significant angular
Chapter 4
Theory
deflection of the ion trajectory.
56
The other one is electronic collision
which does not play a significantly role in atom displacement or change in

the ion trajectory.
However, the loss can result in heat formation and
electronic collisions are more dominant at higher implantation energy.

The average energy loss resulting from elastic collision with target atoms is
called the nuclear stopping power.
On the other hand, the average energy
loss resulting from inelastic interactions with electrons of the substrate

atom is the electronic stopping power. The total stopping power is the
sum of the nuclear and electronic components.
Nuclear stopping
predominates when the ion energy is under 10 keV and electronic stopping
power becomes more dominant when the ion energy exceeds 100 keV.
Hence, these two types of stopping are energy dependent. The electronic
stopping power is approximately proportional to the ion velocity if the ion
is below the Bohr velocity (the orbital speed of an electron in the ground
state of a hydrogen atom) of the atomic electrons4 (2.188106m/s).
In this
study, both nuclear and electronic stopping are important since the ion
energies are between several to 40 keV.
The projection depth of the ion trajectory in the solid is called the ion
range (R). The ion range can be plotted as a distribution of number of
ions against depth and Fig. 4.3 is a typical ion range distribution graph
simulated by TRIM for 20 keV nitrogen ion implanted into UHMWPE.
Chapter 4
Theory
57
Fig. 4.3. Ion range distribution graph of 20keV nitrogen ion on UHMWPE
4.2.3 Damage by implantation
An incident ion with an initial kinetic energy of 100 keV will come to
rest in about 10-13s via electronic and nuclear stopping.
Besides sputtering,
collisions may cause displacement of atoms and re-orientation of lattice in

the case of metals or chain scission or polymerization in polymers resulting
in structural change in the substrate. In these collisions, the target atoms
displaced by the incident ions are called primary knock-on atoms and
those that are knocked out may in turn displace other atoms creating a
cascade of atomic collision4.
Such displacement from the original atomic
position are viewed as damage or defects resulting from collisions. The

degree of damage is determined by the mutual interaction between the ions
and target materials, which is in turn related to the ionizing power, stopping
Chapter 4
Theory
58
power, and initial energy of the incoming ions.
For instance, Fig. 4.4 shows the distribution curve of the nuclear
stopping power of nitrogen ions with initial energy 100 keV impinging into
UHMWPE simulated by TRIM. The nuclear stopping power governs the
structural damage by describing the energy deposited along the trajectory
of the ions (the ions travel from right to left in Fig. 4.4).
A higher
stopping power is found for lower energy ions leading to a higher rate of
energy deposition.
Hence, the most serious damage is caused by ions with
low energy, that is, at the end of the collision cascade or ion trajectory.
Fig. 4.5 displays the damage in terms of collision events in UHMWPE

caused by a nitrogen ion implanted based on as calculated by TRIM.
higher collisions frequency occurs in the subsurface than the surface.
A
The
highest number of collision happens close to the ion range (R) and the
damage is concentrated in this region as well.
Chapter 4
Theory
N in UHMWPE (Nuclear stopping)
1.6
Nuclear stopping power
59
1.4
1.2
1.0
0.8
0.6
0.4
0
20
40
60
80
100
Ion Energy (keV)
Fig. 4.4. Nuclear stopping power of nitrogen ion in UHMWPE with energy
range from 0 to 100keV
Fig. 4.5. Collision eventsleading to target atom displacement distribution

graph for 20keV of nitrogen ion in UHMWPE.
Chapter 4
Theory
60
4.3 Computational calculation by TRIM
The program called transport of ions in matter (TRIM) is one the most
commonly use programs to calculate ranges or damage distributions. In
this report, SRIM-2003 is used to study the interactions between the
implanted ions like argon, nitrogen and oxygen and target substrates such
as NiTi and UHMWPE.
The aim of this study is to predict the profiles
and compare them to experimental results to be discussed in the following

chapters.
4.3.1 Nickel Titanium (NiTi)
Fig. 4.6 shows the distribution of ion ranges for nitrogen ions incident
into NiTi with ion energies of: (a) 40 keV, (b) 30 keV and (c) 20 keV.
These energies were used in our experiments as well.
The average ion
range in each case is also stated in the graphs. For instance, the average
ion range of 40 keV in (a) is 53.4 nm and the straggle distance is 23.6 nm.
From the graph (a), some of the ions penetrate over 100 nm.
Furthermore,
Figs. 4.6 (d) (f) show similar distribution curves for oxygen instead of
nitrogen.
Chapter 4
Theory
(a)
(d)
(b)
(e)
(c)
(f)
Fig. 4.6. Depth profile of nitrogen ion with incident energy of (a) 40keV, (b)
30keV and (c) 20keV. Oxygen ion with incident energy of (d) 40keV, (e) 30keV
and (f) 20keV.
61
Chapter 4
Theory
62
By comparing different energies and types (nitrogen and oxygen) of

incident ions, the more energetic ions have larger ion ranges.
The ion
ranges of oxygen and nitrogen ions with different energies up to 100 keV
are plotted versus ion energy in Fig. 4.7.
The region bounded by the dash
lines is the experimental window (20 keV 40 keV) used in the

experiments.
The ion ranges of nitrogen are larger than those of oxygen at
the same energy.
It can be explained by the difference of stopping power
of the two different ions as shown in Fig. 4.8.
Based on the figure,
nitrogen has a higher electronic stopping power and oxygen ion has a
higher nuclear stopping power.
higher total stopping power.
Nevertheless, oxygen has an overall
The higher stopping power means the ions
deposit more energy per unit length and so they stop sooner.
Since
nitrogen has a lower total stopping power than oxygen, nitrogen ions travel
longer distances thus resulting in a larger ion range.
200
Experiment window
Ion range (nm)
160
120
O
80
40
0
20
40
60
80
100
Ion Energy (keV)
Fig. 4.7. Ion range of nitrogen and oxygen ions with energy from 0 to 100keV
NiT substreate.
Chapter 4
Theory
63
-2
Stopping power (keV/gcm )
1.0
Total O
0.8
Elec N
0.6
Total N
Elec O
0.4
Nucl O
0.2
Nucl N
0.0
0
20
40
60
80
100
Ion Energy (keV)
Fig. 4.8. Stopping power of nitrogen and oxygen ion with different energies.
Elec N, Elec O, Nucl N and Nucl O are electronic or nuclear stopping power of
nitrogen and oxygen ion respectively. Total N and Total O are sum of nuclear
and electronic stopping power.
The number of sputtered atoms from NiTi per incident nitrogen or

oxygen ion with energy from 20 keV to 40 keV is simulated by TRIM and
the results are shown in Table 4.1. The sputtering yield decreases when
the incident energy increases.
Furthermore, oxygen has a higher
sputtering ability than nitrogen. The probabilities of Ni or Ti being

sputtered are dissimilar indicate the presence of preferential sputtering6,7.
According to literature, the sputtering yield of Ni is higher than Ti for wide
range of ion incident energies. However, in other research8 reverse results
obtained Ti became the preferential sputtered element in NiTi. It should be
noted that sputtering of metal alloys may lead to changes in the surface
elemental
concentrations
if
preferential
sputtering
is
present
Nonetheless, the mechanism of reduction of Ni concentration from the NiTi
Chapter 4
Theory
64
surface due ion implantation process is further investigated.
Table 4.1. The number of sputtered atoms of NiTi per incident nitrogen or
oxygen ion with energy from 20k-40keV.
Energy (keV)
20
30
40
Type of implanted ion

Nitrogen
Oxygen
1.149
0.874
0.770
1.358
1.186
1.006
4.3.2 Ultra high molecular weight polyethylene

(UHMWPE)
Fig. 4.9 shows the ion range of nitrogen and argon ions with energies
up to 100 keV in UHMWPE. The nitrogen ion (82.6 nm) has twice the
ion range than argon (42.5 nm) at 20 keV which is the energy used in this
report.
The discrepancy becomes larger as the ion energy increases. The
phenomena can be explained by the difference of stopping power of the

two kinds of ions.
Fig. 4.10 shows the stopping power in UHMWPE for
the two incident ions. The stopping power of argon ion at 20 keV is at
least double that of nitrogen at the same energy.
Therefore, argon ions
have a shorter ion range as they deposit more energy per unit distance
traversed.
Chapter 4
Theory
65
Ion range (nm)
400
300
200
Ar
100
0
0
20
40
60
80
100
Ion Energy (keV)
Fig. 4.9. Ion range of nitrogen and argon ion with energy from 0 to 100keV
NiTi substreates.
Stopping power (MeV/(mg/cm ))
Total Ar
6
5
Nucl Ar
4
3
Total N
Elec Ar
Nucl N
1
Nucl N
0
0
20
40
60
80
100
Ion Energy (keV)
Fig. 4.10. Stopping power of nitrogen and argon ions with various energies.
Elec N, Elec Ar, Nucl N and Nucl Ar are electronic or nuclear stopping power
of nitrogen and argon ion respectively. Total N and Total Ar are sum of nuclear
and electronic stopping power.
Chapter 4
Theory
66
Fig. 4.11 shows the ion ranges and damage caused by collisions along
the incident ion trajectory in UHMWPE simulated by TRIM. Nitrogen
and argon ions show different behaviors in the polymer.
The results
shown in Fig. 4.11 are simulated based on the same number of incident ions
of nitrogen and argon in Fig. 4.11 (c) and (d). Nitrogen has a broader
region of damage, as nitrogen may cause a higher number of collisions due
to the longer ion range than argon. However, the argon ion causes intense
surface damage as collisions are more concentrated close to the surface
region.
(a)
(b)
(c)
(d)
Fig. 4.11. Depth profiles with 20keV incident ion energy of (a) nitrogen and (b)
argon in UHMWPE are depicted. The damages caused by these ions are also
shown in (c) nitrogen and (d) argon.
Chapter 4
Theory
67
The different sputtering yields of nitrogen and argon at 20 keV and 90

incident angle are simulated by TRIM and the results are summarized in
Table 4.2. The argon sputtering yield of argon is about 3 times higher
than that of nitrogen.
In both nitrogen and argon PIII, hydrogen is
preferential sputtered at 5 times the rate compared to carbon.
As a result,
dehydrogenation and carbonization is expected after PIII treatment of

UHMWPE.
Table 4.2. The number of sputtered atoms of UHMEPE per incident nitrogen or
oxygen ion with energy 20keV.
Type of implanted ions
Nitrogen
Argon
Sputtering yield of C
0.056
0.163
Sputtering yield of H
0.379
1.10
Total sputtering yield
0.435
1.268
4.4 Structural change in NiTi and UHMWPE

Tthe
morphological
and
functional
properties
as
well
biocompatibility of NiTi and UHMWPE can be changed by PIII.
as
In this
section, the expected and theoretical chemical and physical reactions

occurring in the two biomaterials are described.
Chapter 4
Theory
68
4.4.1 Nickel titanium (NiTi)
The aims of modifying medical NiTi are to mitigate Ni out diffusion

and enhance the wear resistance to achieve good biocompatibility.
Before
the discussion of experimental results of modification of NiTi in chapter 5,

the expected structural changes such as surface compositional change and
mechanical strength are first discussed in this section.
There are two types of gases used to generate the plasma in this
research, nitrogen and oxygen.
Some bonds between Ni and Ti break
when energetic ions bombard the materials and atoms are re-distributed in
reducing the surface energy.
The heat of formation of titanium nitride
(TiN) and titanium oxides (TiOx) are -305.6 kJ is -913 kJ mole-1 whereas
that of nickel oxide (NiO) is -244 kJ mole-1 and nickel nitrides such as Ni3N
are unstable10,11,12.
As Ti forms stronger bonds with N and O due to the
high affinities of Ti with N or O compared to Ni, (TiN) and (TiOx)

preferentially form on the surface. Consequently, the concentration of Ni
in this new layer is reduced.
Furthermore, TiN has outstanding mechanical properties13 such as high

corrosion and wear resistance, chemical inertness, high hardness and some
reports have indicated good biocompatibility as well 14 .
known for its good biocompatibility15.
TiOx is well
Combined with excellent adhesion
between the newly formed layer and substrate, promising surface properties
of N-PIII and O-PIII NiTi can be achieved.
Chapter 4
Theory
69
4.4.2 UHMWPE
Functionality of polymer can be inserted or activated by plasma

modification. Chemical and morphological changes are expected on
polymer surfaces as energetic ions from the plasma can react with the outer
atomic layers of the substrate up to few nanometers.
The effects caused
by ion bombardment are chain scission and introduction of elements to

form new chemical groups, radical formation, cross linking16 and oxidation
when exposed to air17.
First of all, ion bombardment during PIII results in preferential

sputtering of hydrogen and consequently dehydrogenation 18 , 19 .
The
density of carbon-carbon bonding increases which in turn carbonizes by

recombination of radicals or cross linking among carbon atoms.
Some
reports have mentioned that diamond-like carbon (DLC) or a graphite layer

can be obtained by PIII of UHMWPE 20,21 . Enhancement of the wear
resistance and surface hardening can be realized here.
Plasma modification is one of the common methods to enhance or

reduce the wetting properties of polymers 22 .
Most biocompatible
materials have a hydrophilic nature but as-received UHMWPE is

hydrophobic with a water contact angle of about 110. The hydrophilicity
of UHMWPE can be improved by introduction of polar groups23 such
carboxyl, carbonyl and hydroxy groups19 from oxidation.
Most of the
oxygen containing groups can are introduced by post treatment when the
sample surface reacts with air or residual air in the treatment chamber.
Chapter 4
Theory
4.5 References
1
K. Y. Fu, Plasma implantation and deposition for advanced materials surface

modification, City University of Hong Kong, PhD Thesis (2004) p.10.
O. Auciello and R. Kelly, Ion bombardment modification of surfaces:

fundamentals and applications, Elsevier Science Publishing Company INC.,
New York, 1984. p.325.
3
R. K. Y. Fu and P. K. Chu, Plasma modification of materials, Encyclopedia of

Biomaterials and Biomedical Engineering (2005), 1-12.
Andre Anders, Handbook of plasma immersion ion implantation and

deposition, John Wiley & Sons, Inc., New York, 2000.
5
W. Moller and S. Mukherjee, Plasma-based ion implantation, Current

Science 83 (2002), 237-253.
6
L. Tan, R. A. Dodd, and W.C. Crone, Corrosion and wear-corrosion behavior

of NiTi modified by plasma immersion ion implantation, Biomaterials 24
(2003), 3931-3939.
7
N. Matsunami, Y, Yamamura, Y. Itikawa, N. Itoh, Y. Kazumata, S. Miyagawa,

K. Morita, R. Shimizu and H. Tawara, Energy dependence of the ion-induced
sputtering yields of monatomic, Atomic Data and Nuclear Data Tables 31 (1)
(1984), 1-80.
8
N. Shevchenko, M. T. Pham and M. F. Maitz, Studies of surface modified

NiTi alloy, Applied Surface Science 235 (2004), 126-131.
9
O. Auciello and R. Kelly, Ion bombardment modification of surfaces:

fundamentals and applications, Elsevier Science Publishing Company INC.,
New York, 1984. p.399.
10
X. Tian, R. K. Y. Fu, L. Wang and P. K. Chu, Oxygen-induced nickel

segregation in nitrogen plasma implanted AISI 304 stainless steel, Materials
Science and Engineering A 316 (2001), 200-204.
11
R. Mientus and K. Ellmer, Reactive DC magnetron sputtering of elemental

targets in Ar/ N2 mixtures: relation between the discharge characteristics and
the heat of formation of the corresponding nitrides, Surface Coating and
Technology 116-119 (1999), 1093-1101.
12
K. Ylkota, K. Nakamur, T. Kasuya, S. Tamura, T. Sugimoto, K. Akamatsu,

K Nakao and F. Miyashita, Compositional structure of dual TiNO layers
deposited on SUS 304 by an IBAD technique, Surface Coating and Technology
158-159 (2002), 568-572.
70
Chapter 4
Theory
13
Y. Cheng and Y. F. Zheng, Formation of TiN films on biomedical NiTi shape

memory alloy by PIIID, Materials Science and Engineering A 434 (2006),
99-104.
14
S. Piscanec, L. C. Ciacchi, E. Vesselli, G. Comelli, O. Sbaizero, S. Merizani

and A. D. Vita, Acta Materialia 52 (2004), 1237-1245.
15
G. S. Firstov, R. G. Vitchev, H. Kumar, B. Blanpain and J. Van Humbeeck,

Surface oxidation of NiTi shape memory alloy, Biomaterials 23 (2002),
4873-4871.
16
B. K. Gan, M. M. M. Bilek, A. Kondyurin, K. Mizuno and D. R. McKenzie,

Etching and structural changes in nitrogen plasma immersion ion implanted
polystyrene films, Nuclear Instruments and Methods in Physics Research B
247 (2006), 254-260.
17
A. Kondyurin, V. Karmanov and R. Guenzel, Plasma immersion ion

implantation of polyethylene, Vacuum 64 (2002), 105-111.
18
W. Shi, X. Y. Li and H. Dong, Improved wear resistance of ultra-high

molecular weight polyethylene by plasma immersion ion implantation, Wear
250 (2001), 544-552.
19
I. Bertoti, M. Mohai, A. Toth and T. Ujvari, Nitrogen-PBII modification of

ultra-high molecular weight polyethylene: composition, structure and
nanomechanical properties, Surface Coating and Technology, in press.
20
K. G. Kostov, M. Ueda, I. H. Tan, N. F. Leite, A. F. Beloto and G. F. Gomes,

Structural effect of nitrogen plasma-based ion implantation on ultra-high
molecular weight polyethylene, Surface Coating and Technology 186 (2004),
287-290.
21
J. S. Chen, S. P. Lau, Z. Sun, B. K. Tay, G. Q. Yu, F. Y. Zhu, D. Z. Zhu and

H. J. Xu, Structural and mechanical properties of nitrogen ion implanted ultra
high molecular weight polyethylene, Surface Coating and Technology 138
(2001), 33-38.
22
S. H. Han, Y. H. Lee, Yo H. D. Kim, G. H. Kim, J. H. Lee, J. H. Yoon and G.

W. Kim, Polymer surface modification by plasma source ion implantation,
Surface Coating and Technology 93 (1997), 261-264.
23
V. Svorcik, I. Micek, V. Rybka and V. Hnatowicz, Surface properties of

polyethylene implanted with N+, F+, Ar+ ions, Materials Letters 23 (1995),
167-171.
71
Chapter 5
Nickel Titanium shape memory alloy
72
Chapter 5
Surface modification of Nickel Titanium
shape memory alloy application in
orthopedics
5.1 Introduction
Nickel-titanium shape memory alloys (NiTi) are promising orthopedic

materials in human implants because of their super-elastic properties and
shape memory effects which outperform other common orthopedic
materials such as titanium and stainless steels. Moreover, the mechanical
properties of NiTi are closer to those of cortical bone and a number of
reports have indicated that the materials possess good biocompatibility1,2,3
and better wear resistance than CoCrMo shape memory alloy4. However,
deleterious effects caused by out-diffusion of harmful Ni 5 , 6 from the
substrate have been found.
cell death rate 7 .
Toxic substance from NiTi may lead to higher
Shih et al. have reported cell death when the Ni
concentration is more than 9 ppm 8 .
Furthermore, osteogenesis and
osteonectin synthesis is prohibited9 on the surface of NiTi. The noxious

effects can cause allergic reactions in hyper-sensitive patients.
The main
reason for these undesirable effects and cytotoxicity are believed to be the
consequence of the poor corrosion resistance of the materials in an
Chapter 5
aggressive medium found in the human body.
Leaching of nickel from
NiTi thus raises a safety concern during prolonged use of the materials
inside a human body and the problem must be solved before the materials
can be more widely used in orthopedics.
Es-souni et al.10 have reviewed
the biocompatibility of NiTi and suggested that the amount of released Ni

depends on the surface conditions such as roughness and structure. Ni
ion leaching from a smooth surface with a protective outermost firmly
attached is negligible. Plasma immersion ion implantation (PIII) is one of
the means to fabricate a surface barrier layer with good adhesion to the
substrate. Since PIII is a conformal process and the treatment temperature
is low, it is particularly useful for complex-shaped NiTi orthopedic implants
materials that must retain the shape memory and superelastic effects to be
useful.
In this study, PIII surface modification was conducted on two kinds of

NiTi structures, dense NiTi and porous NiTi.
This chapter describes the
experimental details and the results obtained from surface analysis and
biological tests.
73
Chapter 5
74
5.2 Dense Nickel Titanium
Incorporation of nitrogen or oxygen is a good way to improve the

mechanical and anti-corrosion properties of metals such as NiTi.
For
example, titanium nitride possesses excellent mechanical properties like

high hardness and titanium oxide has excellent biocompatibility and
chemical stability11.
Both titanium nitride and oxide have been produced
on the surface of NiTi substrates by various means12,13,14,15,16 to improve

their corrosion resistance.
In this work, TiN and TiOx barrier layers
synthesized by PIII were studied. The modified surface properties, ion

leaching from the materials, anti-corrosion ability, cytocompatibility, and
surface morphology of the materials are presented here.
Chapter 5
75
5.2.1 Sample preparation
In this section, the sample preparation of dense NiTi is described (Porous

NiTi preparation details in the section 5.3.1).
A cylindrical dense NiTi rod
with a Ni atomic percent of 50.8% (model number: SE508) purchased from

Nitinol Device Company, Fremont, USA. Discs 1 mm thick and 5 mm in
diameter (cross section area is 2.5 x 10-5 m2) were cut, grinded, polished to a
shiny surface finish and then ultrasonically cleaned with acetone and ethanol
and dried in ambient air before being inserted into the plasma immersion ion
implanter17,18,19.
All the implanted samples were ultrasonically cleaned after
the treatment.
The optimal PIII conditions were first determined by doing experiments

using several sets of implantation and annealing conditions.
parameters are shown in Tables 5.1 and 5.2.
These
Different bias voltages from -40
to -20 kV were applied to the substrates during nitrogen or oxygen PIII.

Table 5.3 lists the conditions for the -40 kV samples with and without
annealing at a higher temperature (600oC). Table 5.4 lists the conditions of
the samples prepared by different frequencies (50 to 200 Hz).
Chapter 5
76
Table 5.1. Nitrogen PIII treatment parameters for samples #1 -4.

Sample group
#1 Control
#2 (40 N)
Gas of Plasma
#3 (30 N)
#4 (20 N)
Nitrogen
Working Voltage
40kV
30kV
20kV
Base Pressure
7.010-6
7.010-6
7.010-6
Torr
Torr
Torr
Pulse Width
50s
100s
100s
Treatment duration
4hrs
2hrs
2hrs
Frequency
200Hz
200Hz
200Hz
Working Pressure
6.410-4
6.410-4
6.410-4
Torr
Torr
Torr
8.010-4
8.010-4
8.010-4
Torr
Torr
Torr
450 oC
450 oC
450 oC
9.61016
5.91016
4.01016
cm-2
cm-2
cm-2
Annealing Pressure
Annealing
Temperature
Annealing duration
Dose
Chapter 5
77
Table 5.2. Oxygen PIII treatment parameters for samples #5 -7.

Sample group
#5 (40 O)
Gas of plasma
Working Voltage
#6 (30 O)
#7 (20 O)
Oxygen
40kV
30kV
20kV
7.010-6 Torr
7.010-6 Torr
7.010-6 Torr
Pulse Width
50s
100s
100s
Treatment duration
4hrs
2hrs
2hrs
200Hz
200Hz
200Hz
Working Pressure
6.410-4 Torr
6.410-4 Torr
6.410-4 Torr
Annealing Pressure
8.010-4 Torr
8.010-4 Torr
8.010-4 Torr
450oC
450 oC
450 oC
1.01017 cm-2
6.31016 cm-2
3.81016 cm-2
Base Pressure
Frequency
Annealing
Temperature
Annealing duration
Dose
Chapter 5
78
Table 5.3. Nitrogen and Oxygen PIII conditions with different annealing
temperatures for samples #8 -11.
Sample group
#8
#9
#10
#11
Nitrogen
Nitrogen
Oxygen
Oxygen
40kV
40kV
40kV
40kV
7.010-6 Torr
7.010-6 Torr
7.010-6 Torr
7.010-6 Torr
Pulse Width
50s
50s
50s
50s
Treatment duration
4hrs
4hrs
4hrs
4hrs
200Hz
200Hz
200Hz
200Hz
Working Pressure
6.410-4 Torr
6.410-4 Torr
6.410-4 Torr
6.410-4 Torr
Anneal Pressure
8.010-4 Torr
8.010-4 Torr
Anneal Temperature
600oC
600oC
Annealing duration
9.61016 cm-2
9.61016 cm-2
1.01017 cm-2
1.01017 cm-2
Plasma type
Working Voltage
Base Pressure
Frequency
Dose
Chapter 5
79
Table 5.4.Nitrogen PIII treatment conditions for different pulsing frequencies

for samples #12-14.
Sample group
#12
Plasma
Working Voltage
#13
#14
Nitrogen
40kV
40kV
40kV
7.010-6 Torr
7.010-6 Torr
7.010-6 Torr
Pulse Width
30s
30s
30s
Treatment duration
4hrs
4hrs
4hrs
Working Pressure
4.510-4 Torr
4.510-4 Torr
4.510-4 Torr
Dose
1.61016 cm-2
3.21016 cm-2
6.41016 cm-2
50Hz
100Hz
200Hz
Base Pressure
Frequency
Chapter 5
5.2.2 Depth profile and chemical state analysis
X-ray photoelectron spectroscopy (XPS)

In order to reveal the chemical composition of the new phases formed
after the PIII treatment, XPS was used.
The sputtered depth was estimated
by the sputtering rate calibrated using SiO2 sputtered under similar conditions.
Since it is well known that the sputtering rate changes in the surface region
with changing elemental compositions and is different from that of SiO2, the
thicknesses of the implanted zone are approximate, but comparison among
different samples is more valid.
5.2.3 Dissolution Test
The release of Ni ion

In order to determine the amount of Ni leached from the substrate (NiTi)
into the simulated body fluid (SBF) in a mimetic human body environment,
the dissolution test was used to measure the ions in the SBF from the
samples had been immersed for 5 weeks.
The total surface area of the
sample contact with SBF was about 55 mm2 which is less than the area of
real implant materials, but it is adequate for the purpose to compare the Ni
suppression effect among different treatments (Table 5.1 and 5.2 and 5.3) on
samples with the same dimensions.
80
Chapter 5
81
Two samples of each type of treated and control samples were immersed
in 25 ml of simulated body fluids (SBF)20 in polypropylene (pp) bottles. The
pp bottles were closed tightly and incubated in a thermostatic chamber at
370.1oC for 5 weeks. All the bottles were shaken gently for a few seconds
every 3 days.
After five weeks, the SBF in the bottles were analyzed by
inductively-coupled plasma mass spectrometry (ICPMS) to determine the

amount of Ni leached from each specimen.
5.2.4 Electrochemical Corrosion Test
To evaluate the corrosion resistance of the modified layer,

electrochemical corrosion tests were performed.
The electrochemical
tests 21 , 22 , 23 based on ASTM G5-94 (1999) and G61-86 (1998) were

conducted on a potentiostat (VersaStat II EG&G) in a standard simulated
body fluid (SBF) at a pH of 7.42 and temperature of 37+0.5oC. The
working electrode (the modified layer facing to the medium) was immersed
in the SBF. A cyclic potential spanning between -400 mV and +1600 mV
was applied at a scanning rate of 600 mV per hour.
Before the
electrochemical tests, the medium was purged with nitrogen for 1 hour to
remove dissolved oxygen and nitrogen purging continued throughout the
measurements. The resulting potential values of corrosion potential (Ecorr)
and breakdown potential (Eb) were recorded. The two values of each
samples compared and display anti-corrosion ability under more aggressive
medium than human body environment.
Chapter 5
5.2.5 Hardness Measurement

Mechanical properties in terms of hardness and elastic modulus of the
modified layer were measured by a nano-indenter (MTS Nano Indenter XP).
The nano-indentation tests were conducted on five areas to determine the
average hardness and elastic modulus of the treated and control samples.
5.2.6 Biomedical test
To investigate the cyto-compatibility of the plasma-treated and

untreated samples, osteoblasts isolated from calvarial bones of 2-day-old
mice that ubiquitously expressed an enhanced green fluorescent protein
(EGFP) were used to culture in a Dulbeccos Modified Eagle Medium
(DMEM) (Invitrogen) supplemented with 10% (v/v) fetal bovine serum
(Biowest, France), antibiotics (100 U/ml of penicillin and 100 g/ml of
streptomycin), and 2 mM L-glutamine at 37C in an atmosphere of 5% CO2
and 95% air.
The specimens (1 mm thick and 5 mm in diameter) were
fixed onto the bottom of a 24-well tissue culture plate (Falcon) using 1%
(w/v) agarose. A cell suspension consisting of 5,000 cells was seeded
onto the surface of the control (#1), N treated (#2), and O treated (#5) PIII
treated NiTi and the wells without any metal discs serving as a control for
normal culturing condition.
changed every three days.
Cells were grown in 1 ml of medium and

Cell attachment was examined after the second
day of culture, and cell proliferation examined after 4, 6 and 8 days of

culture. Each sample at each time point would have four identical samples
82
Chapter 5
83
as for statistical significance. In this study, cells were allowed to reach

confluence during the examination period.
To determine the cell number,
the attached cells were released by digestion with trypsin-EDTA

(Invitrogen) and counted using a haematocytometer (Tiefe Depth
Profondeur, Marienfeld, Germany).
Cell viability was assessed by
staining with 0.2% Trypan blue (Sigma). The number of cells was
expressed as mean value standard deviation (SD).
The data were
analyzed by using unpaired two-sample t-test and the statistical analysis

was performed using the SPSS program (SPSS for Windows, Release
11.0.0). The cell morphology was assessed using a fluorescent microscope
(Axioplan 2, Carl Zeiss, Germany). The attached living EGFP-expressing
osteoblasts were visualized using a 450-490 nm incident filter and the
fluorescence images emitted at 510 nm captured using a Sony DKS-ST5
digital camera.
5.2.7 Surface roughness
The surface roughness affects the biocompatibility and corrosion

resistance of orthopaedic materials. The surface topography of the control
and PIII treated NiTi samples were examined by atomic force microscopy
(AFM) (Autoprobe, CP).
Chapter 5
5.2.8 Results and Discussion
5.2.8.1 Depth profiles and chemical states analysis
Figure 5.1 shows the typical elemental depth profiles of (a) control NiTi
(#1), (b) 40 kV nitrogen and implanted and 450oC samples (#2), (c) 40 kV
oxygen implanted and 450oC annealed (#5), and (d) 40 kV oxygen
implanted without annealing (#10) samples.
The Ni depleted region in #2
can be observed from the top surface (0 nm) to a depth of 70 nm, in

comparison to a thickness of 5 nm determined from the control sample with
only a native oxide surface layer. Oxygen was also found in the top 30
nm of the N treated sample as a result of the non-UHV (ultra high vacuum)
conditions in the implanter and also that surface oxidation is accelerated at
high temperature.
The surface barrier layer in the oxygen treated sample
#5 (~130 nm) (Fig. 5.1c) is about twice that in the nitrogen treated sample
#2 (~70 nm).
Furthermore, the oxide layer on the sample without
annealing (#10) is thinner (90 nm) than that on the annealed samples.
Furthermore, the depth profiles of N or O treated samples do not show
abrupt interfaces.
The broad region which is about 40 nm reduces the
possibility of film delamination.
This gradual transitional zone also bodes
well for adhesion between the modified layer and substrate.
84
Chapter 5
(a)
Atomic Concentration %
70
60
Ni
50
Ti
40
30
20
10
0
0
20
40
60
Depth (nm)
(b)
60
Ni
50
40
Ti
30
20
N
10
O
0
0
20
40
60
80 100 120 140 160 180 200

Depth (nm)
Fig. 5.1. (a) XPS depth profile of control sample (sample #1), (b) XPS depth
profile of N treated NiTi by 40 kV bias voltage and 450 oC (sample #2), (c) XPS
depth profile of O treated NiTi by 40 kV bias voltage and 450 oC (sample #5),
(d) XPS depth profile of O treated NiTi by 40 kV bias voltage without annealing
(sample #10).
85
Chapter 5
(c)
80
70
O
60
50
Ti
40
30
20
10
Ni
0
0
20
40
60
80 100 120 140 160 180 200

Depth (nm)
(d)
70
60
O
50
40
Ti
30
20
Ni
10
0
0
20
40
60
80 100 120 140 160 180 200

Depth (nm)
Fig. 5.1(c) XPS depth profile of O treated NiTi by 40 kV bias voltage and 450
o
C (sample #5) (d) XPS depth profile of O treated NiTi by 40 kV bias voltage
without annealing (sample #10)
86
Chapter 5
87
It should be noted that all the PIII treated NiTi shows a Ni depletion
region overlapping with the implanted (either N or O rich) region. In Fig.
5.2, the combined depth profiles of different implantation voltages of
individual elements (Ni, N or O) show the existence of a 60 nm thick Ni
depletion region in all the plasma treated samples.
The quality and
thickness of newly formed layers depend on the PIII treatment parameters

such as bias voltage and annealing temperature. In general, the thickness
of the modified layer increases with higher bias voltage at the same
annealing temperature.
Figures 5.2 (a) and (b) compare the Ni depth
profiles among the 40, 30 and 20 kV samples treated by nitrogen or oxygen

PIII, respectively and Figures 5.2 (c) and (d) show the N and O depth
profiles likewise.
Fig. 5.2(a) clearly demonstrates that the Ni
concentration is below 10% in #2 (40 kV) in the top 60 nm from the

surface but more than 20% Ni in #3 and #4.
In addition, Fig. 5.2(c) shows
a higher concentration of N within the newly formed layer as the

implantation voltage increases.
oxygen treatment.
Another phenomenon is observed for
That is, there is a thicker Ni depletion region but
similar distribution of Ni for high voltage implantation as shown in Fig.

5.2(b).
Combining the results discussed above and Fig. 5.2 (d), it can be
deduced that the N treatment can better suppress the Ni concentration

whereas the O treatment gives rise to a thicker Ni depletion layer.
Chapter 5
(a)
60
50
40
30
#4
#2 - 40kV N
#3 - 30kV N
#4 - 20kV N
20
#3
10
#2
0
0
20
40
60
80
100
120
140
160
Depth (nm)
Fig 5.2
(b)
#5 - 40kV O
#6 - 30kV O
#7 - 20kV O
60
50
40
30
#7
#6
#5
20
10
0
0
20
40
60
80
100
120
140
160
Depth (nm)
Fig. 5.2. (a) XPS depth profiles of Ni in samples #2 40kV N, #3 30kV N and
#4- 20kV N (b) XPS depth profiles of Ni in samples #5 40kV O, #6 30kV O
and #4- 20kV O (c) XPS depth profiles of N in samples #2 40kV N, #3 30kV
N and #4- 20kV N (d) XPS depth profiles of O in samples #5 40kV O, #6
30kV O and #4- 20kV O.
88
Chapter 5
(c)
#2 - 40kV N
#3 - 30kV N
#4 - 20kV N
50
#2
#3
40
30
#4
20
10
0
0
20
40
60
80
100
120
140
160
Depth (nm)
(d)
#5 - 40kV O
#6 - 30kV O
#7 - 20kV O
80
70
60
#6
50
40
#5
30
#7
20
10
0
0
20
40
60
80
100
120
140
160
Depth (nm)
Fig. 5.2. (c) XPS depth profiles of N in samples #2 40kV N, #3 30kV N and
#4- 20kV N (d) XPS depth profiles of O in samples #5 40kV O, #6 30kV O
and #4- 20kV O.
89
Chapter 5
90
Furthermore, the small Ni content in the implanted region (N or O rich

region) can be explained by the higher affinity of Ti towards N or O to
form chemical bonds than Ni.
As more favorable chemical bond
formation is preferred in the implantation region, Ni is segregated away

from the surface.
In view of the heat of formation, that of the lowest
titanium oxide is 913 kJmole-1 whereas that of NiO is 244 kJmole-1.

The heat of formation of TiN is 305.6 kJmole-1 while nickel nitrides such
as Ni3N are unstable compared to TiN24,25,26.
Therefore, the formation of
titanium nitride and oxide is energetically favorable over that of the nickel
counterparts.
Titanium nitride and oxide are good surface barrier layers.
Our
previous study27,28 shows the implanted layer is mainly TiN with some
surface oxide due to natural oxidation.
However in the oxygen implanted
samples, Ti2+, Ti3+, and Ti4+ are present at different depths.
The major
oxidation states of Ti change gradually from 4+ to 3+ and then 2+ with

depths. It is probably due to oxygen in-diffusion towards the bulk of the
sample resulting in more Ti3+ and Ti2+ at the expense of surface Ti4+.
The
Ti chemical states are consistent with the variation of the oxygen profiles
along the depth of the implanted layers.
As shown in Fig. 5.3(a), both the N and O treated samples have

broadened Ni depletion regions at higher annealing temperature.
The N
and O treated samples have similar Ni suppression regions when annealed

at 600oC.
In the case of nitrogen, annealing at 600oC increases the
Chapter 5
thickness of this low level Ni region by a factor of two. The higher Ni

concentration in the topmost 10-30 nm is shown in Fig. 5.2(b) and the Ni
peak disappears when the annealing temperature reaches 600oC (#11) as
shown in Fig. 5.3(a). The oxygen concentration in the implanted region is
even distributed as shown in Fig. 5.3(b).
A thicker Ni depleted region can
better reduce the toxicity. Annealing broadens the implant profile via a
diffusion mechanism further suppressing the surface Ni content and
perhaps mending the implantation-induced damaged zone resulting in a
surface layer that is more impervious to Ni.
Based on the XPS depth profiles, a Ni barrier layer is formed by either

N or O PIII treatment to impede Ni out-diffusion.
The thickness of this Ni
suppressed region and the Ni content depend on the bias voltage applied
during the PIII treatment and annealing temperature.
A higher voltage and
annealing temperature create a thicker barrier layer and lower Ni

concentration near the surface.
However, a high temperature such as
600oC can degrade the mechanical properties of the materials and Wong29
have recommended that the heat treatment temperature should be between
250 to 350oC in order that the desirable mechanical and shape recovery
properties can be retained.
Therefore, although a higher annealing
temperature increases the Ni depletion region, it is not appropriate for the

application in orthopedic materials.
A proper annealing temperature must
be chosen in order to balance the mechanical properties with Ni

suppression.
91
Chapter 5
92
(a)
60
#11
#9
#5
50
#2
40
#10
30
#8
#2 - N 450 C
#8 - N without anneal
o
#9 - N 600 C
o
#5 - O 450 C
#10 - O without anneal
o
#11 - O 600 C
20
10
0
0
50
100
150
200
250
300
Depth (nm)
(b)
o
#5 - 450 C
#10 - without anneal
o
#11 - 600 C
80
70
60
50
40
#11
#5
#10
30
20
10
0
0
50
100
150
200
250
300
Depth (nm)
Fig. 5.3. (a) XPS depth profile of Ni at different annealing temperature or

without anneal NiTi treated by N or O (b) XPS depth profile of O at different
annealing temperature or without anneal NiTi treated by O.
Chapter 5
93
5.2.8.2 Dissolution test
Dissolution is one of two techniques used in this work to evaluate the

corrosion resistance of the surfaces. Orthopedic samples must tolerate in
vivo conditions that are well known to be corrosive. Figure 5.4 shows
the Ni concentration in the SBF measured by ICPMS with (#1) and
without PIII treatment (#2-#11) after immersion in SBF for 5 weeks.
The treated surfaces exhibit improved resistance to out-diffusion of Ni.
The amount of Ni leached from the control sample is much more than that
of the treated samples. According to the results, PIII treatment is effective
in suppressing Ni release from the substrate to body fluids.
It should be
noted that Ni leaching from the PIII treated samples is consistently lower
than that from the control (#1).
The amount of Ni released from the
control is about 30 times higher than that from the treated samples.
The
degree of Ni suppression depends on the implantation parameters as Tian

et al.24 have reported Ni seggregation in stainless steel. In our study, the
higher implantation voltage and milder annealing temperature such as 40
kV and 450 oC yield better performance.
It is believed that the thick and
strong barrier layer (TiN or TiOx) and Ni depletion region up to 60 nm in

thickness can sufficiently impede out-diffusion of Ni in simulated in vitro
conditions.
The significant mitigation of Ni leaching from the treated NiTi in the

dissolution test attests to the enhancement of the anti-corrosion ability in
aggregative medium such as in SBF at body temperature.
Concentration of Ni ion in SBF
Chapter 5
800
600
400
200
0
#1
#2
#3
#4
#5
#6
#7
#8
#9 #10 #11
Sample group number
Fig. 5.4. Ni ion concentration in SBF leached from samples (#1- #11) after 5
week immersion in SBF as detected by ICPMS.
94
Chapter 5
95
5.2.8.3 Electrochemical corrosion analysis
The electrochemical test is another technique used to assess the

corrosion properties in this study.
This method accelerates surface
corrosion by applying a high voltage.
It should be noted that the
electrochemical test is an accelerated corrosion method which is more

extreme than situations under in vivo conditions.
However, it gauges the
long term anti-corrosion capability of the materials.
Figure 5.5 shows the essential readings from the corrosion test.
In this
figure, Ecorr is a symbol of the corrosion potential indicating spontaneous

corrosion of the materials.
Eb represents the breakdown potential or the
largest potential within the passive region.
These two terms are important
to assess the corrosion resistance and higher values suggest better corrosion
resistance. Among the samples, the control sample (#1) shows the lowest
Ecorr and Eb.
By comparing the treated samples, the N treated and 450oC
annealed samples (#2, 3, 4, 8 and 9) have higher Eb than the O treated

samples (#5, 6, 7, 10 and 11). However, there is no obvious trend based
on the scattered data points collected for Ecorr.
Nonetheless, both the N
and O treated samples show significant improvement over the control.

The enhancement of the anti corrosion properties is due to the strong and
inert barrier such as nitride and oxide formed.
Based on the result of electrochemical corrosion test and dissolution

described in the previous section, N and O PIII treatment can synthesis
high quality barrier layers with good corrosion resistance that suppress out
Chapter 5
diffusion of Ni ions from substrate.
96
Furthermore, annealing at 450C
results in a consolidated barrier layer which has a higher density.
Our N
treatment results indicate that a proper annealing procedure gives rise to a

denser layer for enhanced corrosion resistance and diffusion barrier
characteristics.
0
1200
1000
-100
800
-150
600
Ecorr
-200
Eb
400
200
-250
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11
Sample group number
Fig. 5.5. Summary of the results from electrochemical corrosion test.
EbPotential (mV)
Ecorr Potential (mV)
-50
Chapter 5
97
5.2.8.4 Nano-indentation
The surface hardness (H) and elastic modulus (E) of some of the treated
and untreated NiTi are plotted in Fig. 5.6.
The control sample has
constant H and E values throughout the indented depths. All the treated
samples have higher H and E values than the control samples but they
approach those of the control at large depths. It shows the transition from
the modified layer to the bulk substrate.
Comparing the treated samples,
sample #2 (N treated with 450 oC annealed) shows the maximum H and E

values whereas the O treated (annealed or unannealed) samples have
relatively small H and E.
The outstanding performance can be attributed
to TiN which has high hardness. The annealing process also repairs the
damages caused by ion bombardment.
We have also conducted the
measurement on the 600 oC annealed sample (not shown in the figure), and
both H and E diminish and with large error bars, implying that the surface
is unstable. The reason may be that the high annealing temperature leads
to crystallization and grains size growth in the TiN phase.
As a result, the
hardness and strength values decrease based on the Hall-Petch equation30.

The N treatment coupled with 450oC annealing is recommended as the
optimal treatment conditions from the perspective of the best mechanical
properties.
Chapter 5
(a)
#2 - 40kV N 450 C
#8 - 40kV N without anneal
o
#5 - 40kV O 450 C
#10 - 40kV O without anneal
#1 - control
13
12
11
Hardness (GPa)
10
9
8
7
6
5
4
3
2
0
20
40
60
80
100
Depth (nm)
(b)
o
#2 - 40kV N 450 C
#8 - 40kV N without anneal
o
#5 - 40kV O 450 C
#10 - 40kV O without anneal
#1 -Control
160
Elastic modulus (GPa)
140
120
100
80
60
40
0
20
40
60
80
100
Depth (nm)
Fig. 5.6. (a) Hardness depth profile of treated and untreated NiTi from surface
to 100nm acquired by nano-indenter (b) Elastic modulus depth profile of
treated and untreated NiTi from surface to 100nm acquired by nano-indenter.
98
Chapter 5
99
5.2.8.5 Cell culture
The average numbers of cells proliferating in different days in the

culture medium are shown in Fig. 5.7.
The results show that all the
treated and untreated samples are well tolerated by osteoblasts.
After
culturing for 2 days (D2), the cells start to attach and proliferate on all
sample surfaces.
After days 4 and 6, the number of viable cells on the
untreated (#1) samples is larger than the 2 treated samples (#2 and 5).
However, the N treated samples exhibit the highest proliferation after 8
days.
Furthermore, the rate of cell proliferation on the untreated sample
shows signs of levelling off starting from day 6 but the cells continue to
multiply in all three other wells.
By comparing the well with NiTi
(untreated or treated) and the empty well (without NiTi), the cell
proliferation is suppressed to some degrees on NiTi.
In addition, among
the NiTi with the culturing wells, cells on the N or O treated samples seem
a little less than that on the untreated sample, but the results demonstrate no
immediate cytotoxic effect caused by the treated surface.
The in vitro study indicates good biocompatibility of the untreated and

treated surfaces, particularly the N treated sample showing better cell
proliferation than the O treated sample.
It has been explained by
Piscanec31 that TiN benefits the growth of calcium phosphate. This layer
is favorable to the formation of bone like hydroxyapatites. Therefore, the
N treated sample shows the best proliferation of osteoblasts.
On the other hand, there are other factors affecting the cell attachment
Chapter 5
and growth on surfaces such as surface stress created by ion implantation.

Es-Souni et al.32 have reported that the surface morphology plays a role as
lower cyto-compatibility is observed on rougher surfaces . Furthermore, a
rough surface is more prone to corrosion than a smooth surface.
Consequently, toxic ion leaching due to corrosion leads to increased cell
death rates. It should be noted that only short term cell tests are described
here and more work needs to be done to assess the longer term
biocompatibility.
30
Number of cells (x 10 )
25
#1 - control
o
#2 - 40kV N 450 C
o
#5 - 40kV O 450 C
without NiTi well
20
15
10
5
0
D2 D4 D6 D8 -- D2 D4 D6 D8 -- D2 D4 D6 D8 -- D2 D4 D6 D8
Day
Fig. 5.7. Viable cell number versus days. (D2 - Day 2, D4 Day 4, D6 Day 6
and D8 Day 8).
100
Chapter 5
Figure 5.8 captured the cells on sample #2 (N treated) on Day 2 and 8.

The cell morphology of the untreated and treated surfaces was observed
and all of the samples show the cells attached and proliferated on the
samples.
(a)
50m
(b)
50m
Fig. 5.8. (a) Osteoblasts microscopic views (200) of (N treated) sample #2

after 2 days cell culturing. (b) after 8 days cell culturing.
101
Chapter 5
102
5.2.8.6 AFM
Since surface roughness is one of the important factors leading to
corrosion and cyto-compatibility, the topography and roughness of the
untreated and treated samples were measured by AFM.
The root mean
square values of the roughness are listed in Table 5.5 and the AFM 3D
images are displayed in Fig 5.9(a) - (d). It should be mentioned that the
roughness estimations exclude the artefact circled at (a) and (c).
By comparing the root mean square values among the samples, the
rougher surface is found for higher frequency or dose implantation while
low frequency shows a smoother surface compared to the control sample.
A rough surface is created by ion bombardment with relatively high
implant fluence or ion current for the same treatment time.
On the other
hand, ion bombardment with a low fluence gives a smoother surface

perhaps due to less sputtering. Further investigation of roughening effect
by PIII can be achieved by examining effects of implantation frequencies
for the same fluence.
Nevertheless, the difference in the absolute
roughness is small from the perspective of biomedical materials. Based

on the electrochemical corrosion and dissolution tests, the rougher surface
(treated) shows better corrosion resistance over the smoother surface
(control). However, it is believed that the roughness increase arising from
the treatment process is not sufficient to degrade the anti-corrosion
capability of NiTi.
Chapter 5
Table 5.5. Roughness of 40kV N treated by different pulse frequency of

implantation (#12-#14) and control sample.
Samples
RMS roughness ()
#1 Control
44.3
#12 50 Hz
28.0
#13 100 Hz
58.8
#14 200 Hz
71.2
103
Chapter 5
(a)
(b)
Fig. 5.9. AFM 3D topographies acquired (5m5m): (a) #1 control; (b)

#12 50Hz; (c) #13 100Hz; (d) #14 200Hz; (c) #13 100Hz; (d) #14
200Hz.
104
Chapter 5
(c)
(d)
Fig. 5.9 AFM 3D topographies acquired (5m5m): (c) #13 100Hz; (d)
#14 200Hz.
105
Chapter 5
106
5.2.9 Summary Dense NiTi

The successful surface modification of NiTi by PIII for biomedical uses
has been investigated using elemental depth profiling, in vitro dissolution
test,
electrochemical
corrosion
analysis,
mechanical
properties
determination, in vitro cyto-compatibility and surface morphology

measurement.
The superior properties observed from the modified
surfaces are confirmed.
Eight important results are listed here.
1. TiN and TiOx phases are formed in the modified layers by PIII.
The
graded interface promotes strong adhesion between the newly form layers
to substrate.
2. The thickness of the TiN or TiOx layer depends on the bias voltage applied
and annealing temperature.
3. The PIII treatment effectively suppresses Ni ion leaching from the NiTi
substrate into SBF by at least 10 times compared to the untreated samples.
4. All the N or O PIII treated samples possess improved corrosion resistance
compared to the untreated samples indicating that the barrier layers are
strong and robust in our simulated in vitro dissolution test and accelerated
electrochemical corrosion test.
5. The mechanical properties such as hardness and elastic modulus are
enhanced by PIII, particularly in conjunction with annealing at 450oC for
the N-PIII sample.
6. The untreated and PIII treated surfaces have good biocompatibility and cell
proliferation.
Cells attached and grow well especially on the N treated
and untreated sample.
Chapter 5
7. The surface roughness increases after high frequency (200 Hz) PIII
treatment. However, it is not sufficient to degrade corrosion resistance.
8. Comparing the PIII treated samples, the optimal modification conditions
are N-PIII at 40 kV and post-annealing at 450oC.
107
Chapter 5
108
5.3 Porous Nickel Titanium
Recently, porous structure NiTi has attracted attention for the potential use
in bone implants.
Similar to the dense counterpart, the materials exhibit the
shape memory effect, superelasticity, biocompatibility

mechanical properties35.
33 , 34
and good
The exceptional feature of porous structure is that it
allows ingrowth of bone tissues36 and favors bone osseointegration37,38 as well

as transport of body fluids through open and interconnected pores.
Similar to
other metals, unsatisfactory surgical outcome has been found from materials
with higher elastic modulus mismatch with human bones.
Uneven
distribution of stresses of high elastic modulus material carries most of the load
giving the risk of loosening the implant. In this connection, the appropriate
elastic modulus can be obtained from porous NiTi through adjustment of the
synthesis process.
Furthermore, the porosity and pore size can be adjusted to
fulfil the suitable mechanical strength (1-2 GPa for cancellous bone, 17.0-18.9
GPa for compact bone39) and pore sizes in the range from 100-500 m benefit
ingrowth of bone tissues40. On the other hand, corrosion of orthopedic metals
in body fluid is common.
Despite good corrosion resistance of dense NiTi
mentioned in the literature, Li41 has shown crevice and localized corrosion on
porous NiTi due to the complicated porous network and electrolyte trapped in
the pores.
Rhanlmi37 has demonstrated in the in vitro biocompatibility
evaluation that there is no adverse effect regardless of both implantation and

post-surgery recovery time.
The carcinogenic and other potential deleterious
effects stemming from Ni ion release that have restricted more widespread
applications of NiTi in orthopedic use are more serious for porous NiTi due to
Chapter 5
the larger surface area of the porous structure. Also, it is difficult to apply
conventional surface modification technologies such as laser or ion
implantation directly to the porous materials. As PIII is non-line of sight
technique, it is a more effective technique. We have successfully applied PIII
to dense NiTi to produce a surface barrier layer to reduce Ni out-diffusion and
improve corrosion resistance.
In this part of the work, we do similar things on
porous NiTi by means of oxygen PIII.
Nickel out-diffusion from the porous
NiTi SMA and the surface characteristics of oxygen plasma-implanted samples

before and after PIII treatment are investigated.
109
Chapter 5
5.3.1 Sample preparation

The porous NiTi SMAs were synthesized by equiatomic nickel and
titanium powders (NI006021 Nickel, Goodfellow Corporation; Titanium,
Shanghai Reagent Corporation) by capsule-free hot isostatic pressing
methods42 in a HIP unit (ABB Company). The sample with average 40%
porosity was cut and machined into plates with diameter of 4.8 mm and
thickness of 1.5 mm.
They were grinded and polished until a shiny
surface was achieved and then ultrasonically cleaned with acetone and
ethanol.
Implantation was conducted in the plasma immersion ion
implanter in City University of Hong Kong.
The parameters of the
oxygen PIII experiments are listed In Table 5.6. Before the oxygen PIII
treatment, the surface of the porous NiTi samples was cleaned using argon
(Ar) plasma ion sputtering for 5 minutes.
Both sides of the samples were
implanted
Table 5.6. Oxygen plasma implantation parameters
Plasma Gas
Oxygen
Working Voltage
40 kV
Pulse Width
50 s
Frequency
Treatment Duration
Base Pressure
Working Pressure
200 Hz
2 hrs
110-5 Torr
6.010-4 Torr
110
Chapter 5
111
5.3.2 Surface Characterisation
SEM
The typical surface morphology of porous NiTi used in this report was
investigated by scanning electron microscopy (SEM).
XPS
The surface elemental structure of the treated and untreated samples
was characterized by X-ray photoelectron spectroscopy (XPS).
The
elemental depth profiles of the untreated and O implanted porous NiTi

samples before and after simulated body fluid (SBF) immersion tests
(described in 5.3.3) were obtained.
5.3.3 Dissolution Test
The release of Ni ion

The degree of nickel release was assessed by simulated body fluid (SBF)
immersion tests.
Two samples of each type of O implanted and control
samples were immersed in 25 ml of simulated body fluid (SBF) in

polypropylene (pp) bottles.
The pp bottles were closed tightly and
incubated in a thermostatic chamber at 370.1oC for 7, 14, 21 and 28 days.

The
nickel
concentrations
in
the
SBF
were
inductively-coupled plasma mass spectrometry (ICPMS).
measured
using
Chapter 5
112
5.3.4 Mechanical properties test
Compression test
In order to reveal the superelasticity of porous NiTi before and after
implantation, mechanical compression tests were conducted.
The
specimens were cut to 12 mm in length and 6 mm in diameter. The

uniaxial compression test was carried out at a constant rate of 0.06 mm/min
at room temperature (22 oC) using an Instron 4206.
5.3.5 Biomedical test
To investigate the cyto-compatibility of the O implanted and untreated

porous NiTi, osteoblasts attachment was examined.
described in section 5.2.6.
The procedures were
A cell suspension consisting of 5000 cells was
seeded onto the untreated, O treated porous NiTi, and empty wells (without
metal discs) acting as the control for normal culturing conditions.
identical samples were used for each group.
Five
Cell proliferation was
examined after 8 days of culture. The attached living osteoblasts were

visualized using fluorescent microscopy and the images were captured
using a digital camera.
Chapter 5
5.3.6 Results and Discussion
5.3.6.1 SEM
Figure 5.10 shows the typical microstructure of the porous NiTi before
implantation.
The size of most of the interconnecting pores varies from
about 50 to 500 m which falls in the conventional range for bone tissue
ingrowth43.
Some of the big pores are interconnected with each other via
small pores which enhance bone tissue ingrowth.
Ayers38 reported that
there was no significant difference in bone ingrowth for different porosities

(40 55 %) and pore sizes (180 360 m). Therefore, the morphology of
the porous NiTi made by CF-HIP in this work is appropriate for bone
implants.
Fig. 5.10. Surface morphology of typical porous NiTi before implantation.
113
Chapter 5
114
5.3.6.2 XPS
Nickel suppression was investigated by studying the depth profile of the

samples before and after oxygen plasma treatment.
Fig. 5.11 shows the
XPS depth profiles obtained from: (a) oxygen plasma treated porous NiTi
and immersed in SBF for 28 days, (b) porous NiTi without oxygen PIII
treatment and immersed in SBF for 28 days, (c) oxygen plasma treated
porous NiTi and not immersed in SBF and (d) porous NiTi without oxygen
PIII treatment and not immersed in SBF. The depth profiles are plotted as
atomic percentage against sputtering time.
The sputtering rate is
approximately 22.74 nm/ min based on a silicon dioxide standard.
Due to
the depletion of Ni from the surface, the sputtering rate changes in this
region and so the depth scale may be over-interpreted.
Nonetheless,
comparison among different samples is valid and the thickness of the

Ni-depleted layer is found to be about 100 nm.
Comparing the treated
(Figs. 5.11a and c) and untreated samples (Figs. 5.11b and d), the Ni
content is greatly reduced in the implanted region.
In addition, there is
very little difference in the depth profiles between the as implanted and
implanted and SBF immersed samples.
The results confirm that the
barrier layer remains unchanged in a biomimetic environment and there is

no obvious chemical reaction between the implanted layer and SBF which
is similar to blood plasma.
It should be emphasized that PIII differs from
conventional coating techniques in that there is no abrupt interface and so

problems associated with film delamination are greatly mitigated.
surface barrier layer composed of titanium oxide, titanium nitride, or

titanium carbide has been shown to improve the wear resistance, corrosion
Chapter 5
resistance and biocompatibility of dense NiTi44,45,46. The PIII technique

has been successfully used to modify the surface properties of dense
NiTi47,48,49. Here, we show evidence that the technology is successfully
extended to porous NiTi.
115
Chapter 5
(a)
O1s
Ti2p
Ni2p
Atomic concentration (%)
80
60
40
20
0
0
50
100
150
200
Depth (nm)
(b)
O1s
Ti2p
Ni2p
Atomic concentratrion (%)
80
60
40
20
0
0
50
100
150
200
Depth (nm)
Fig. 5.11. XPS depth profiles acquired from the different porous NiTi samples:
(a) oxygen plasma implanted and immersed in SBF for 28 days, (b) without
plasma treatment and immersed in SBF for 28 day, (c) oxygen plasma
implanted and not immersed in SBF and (d) without plasma treatment and not
immersed in SBF.
116
Chapter 5
(c)
O1s
Ti2p
Ni2p
80
60
40
20
0
0
50
100
150
200
Depth (nm)
(d)
O1s
Ti2p
Ni2p
80
60
40
20
0
0
20
40
60
80
100
120
Depth (nm)
Fig. 5.11. XPS depth profiles acquired from the different porous NiTi samples:
(c) oxygen plasma implanted and not immersed in SBF, and (d) without plasma
treatment and not immersed in SBF.
117
Chapter 5
5.3.6.3 Dissolution test
The effectiveness of the barrier layer against Ni out-diffusion was

evaluated by determining the Ni concentrations in the SBFs after
immersion tests.
Fig. 5.12 summarizes the Ni concentrations in the SBFs
of the untreated, oxygen implanted, dense NiTi and porous NiTi. The
higher Ni concentration from the untreated porous NiTi than the dense NiTi
is probably caused by the larger exposed surface area on the porous
samples.
The amount of Ni leached from the untreated porous sample is
at least 3 times higher than that from the treated porous samples.
Therefore, the barrier layer is effective in mitigating out-diffusion of Ni
from the substrate.
It should also be mentioned that the oxygen plasma
induced layer has strong anti-corrosion properties50.
This is evidenced by
that the amount of leached Ni from the untreated dense NiTi is similar to
that from the porous oxygen PIII treated sample in the beginning but is
dramatically higher after immersion in SBF for twenty one days. Thus,
our results clearly show that PIII is useful in mitigating harmful Ni ion out
diffusion from both dense and porous NiTi.
This is in part due to the
unique non-line-of-sight capability of the technique which has been

demonstrated on much smaller features such as sub-micrometer trenches in
microelectronic devices51. Hence, the pores with size from 50 to 500 m
are sufficiently big for plasma to penetrate into and the sidewalls can be
adequately implanted.
118
Chapter 5
119
Concentration of Ni ion in SBF (ppb)
250
200
Dense
(O PIII)
150
Dense
(untreated)
100
Porous
(O PIII)
50
Porous
(untreated)
0
7D
14D
21D
28D
Number of Day immersed in SBF
Fig. 5.12. Ni ion concentration in SBFs for dense and porous NiTi samples
after immersion for 7 to 28 days determined by ICPMS.
Chapter 5
120
5.3.6.4 Compression test
Fig. 5.13 shows the stress-strain result of the compression test of the
untreated and O PIII treated porous NiTi samples strained up to 4.7%.
The compressive strength values of the untreated and the O PIII treated
NiTi are about 330 and 300M Pa, respectively. In addition, the width of
the hysteretic loop of the two curves is quite small thus demonstrating the
superelastic capability. The remaining strain in the untreated and O PIII
treated samples is only 0.4 and 0.5%, respectively after unloading.
Unlike
the porous NiTi prepared by other methods52,53,54, both the untreated and
O-PIII treated porous samples in our experiments exhibit an obvious stress
plateau similar to that of dense NiTi. It means that after reaching a critical
stress of about 50 MPa, the porous NiTi samples start to transform into
martensite. Upon further straining, the stress is almost constant until the
porous samples are fully transformed. The reverse transformation occurs
during unloading, indicating a stress-induced transformation in the porous
NiTi SMAs produced by CF-HIP during loading or unloading.
Our data
reveal that both the untreated and O-PIII porous NiTi SMAs have good
mechanical properties and excellent superelasticity, and O-PIII has minor
degradation on the mechanical properties of porous NiTi alloys.
Chapter 5
121
Untreated
350
Stress (MPa)
300
250
O-PIII
treated
loading
200
150
100
unloading
50
0
0
Strain (%)
Fig. 5.13. Compression stress against strain of untreated and O PIII treated
porous NiTi.
Chapter 5
5.3.6.5 Cytocompatibility
Figures 5.14 and 5.15 display the fluorescent and SEM images showing
the results of cell proliferation on the untreated and O-PIII porous samples
after 8 days of culture. Both the O-PIII and untreated samples are well
tolerated by the EGFP-expressing osteoblasts. The cells clearly attach
well and proliferate on both samples. The data indicate no immediate
cyto-toxic effects.
(a)
(b)
Fig. 5.14. Images of proliferation clusters (4magnification) of osteoblasts on

porous NiTi after culturing for 8 days. (a) Untreated and (b) O PIII treated.
122
Chapter 5
Fig. 5.15. Cell morphology image captured by using SEM (after culturing for 8
days).
123
Chapter 5
5.3.7 Summary Porous NiTi
We have successfully extended the PIII treatment process to porous

NiTi.
PIII is demonstrated to be an effective treatment to create a barrier
layer on porous NiTi to mitigate Ni out-diffusion.
The superelasticity,
mechanical properties and cytocompatibility of the porous NiTi are retained

after PIII surface modification.
Five important results are listed below:
1. The thickness of TiOx barrier layer remains the same after 28 days
immersion in SBF.
2. The PIII treatment is effective on porous NiTi and Ni ion leaching is
reduced in simulated in vitro conditions.
3. The PIII treated porous NiTi exhibits good corrosion resistance.
4. Both the untreated and O-PIII treated samples have good mechanical
properties and superelasticity.
5. Good cytocompatibility is observed from both the treated and untreated
samples.
124
Chapter 5
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nickel titanium shape memory alloys, Biomaterials 26 (2005), 2265-2272.
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C. Trepanier, M. Tabrizian, L. H. Yahia, L. Bilodeau and D. L. Piron, Effect

of modification of oxide layer on NiTi stent corrosion resistance, Journal of
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128
Chapter 5
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C. T. Kao, S. J. Ding, Y. C. Chen and T. H. Huang, The anticorrosion ability

of titanium nitride (TiN) plating on an orthodontic metal bracket and its
biocompatibility, Journal of Biomedical Materials Research 63 (2002),
786-792.
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N. Shevchenko, M. T. Pham and M. F. Maitz, Studies of surface modified

NiTi alloy, Applied Surface Science 235 (2004), 126-131.
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S. M. Green, D. M. Grant and J. V. Wood, XPS characterization of surface

modified Ni-Ti shape memory alloy, Materials Science & Engineering A 224
(1997) 21-26.
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L. Tan and Crone W. C., Surface characterization of NiTi modified by

plasma source ion implantation, Acta Materialia 50 (2002), 4449-4460.
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R. W. Y. Poon, J. P. Y. Ho, X. Y. Liu, C. Y. Chung, P. K. Chu, K.W. K. Yeung,

W. W. Lu and K. M. C. Cheung, Anti-corrosion performance of oxidized and
oxygen plasma- implanted NiTi alloys, Materials Science & Engineering A 390
(2005), 444-451.
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P. K. Chu and C. Chan, Applications of plasma immersion ion implantation

in microelectronics - a brief review, Surface Coating and Technology 136(1-3)
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B. Y. Li, L. J. Rong and Y. Y. Li, Stress-strain behavior of porous Ni-Ti

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129
Chapter 6
Ultra High Molecular Weight Polyethylene
Chapter 6
Surface modification of Ultra High Molecular
Weight Polyethylene for protein binding
6.1 Introduction
Immobilization of proteins on surfaces whilst preserving their
biological functionality is important in many areas of bioengineering and
medical biotechnology 1,2 , 3 .
For instance, enzymes bound to polymer
membranes are used to catalyse chemical processes in the beverage, food,

textile and chemical industries. New applications of immobilised proteins
are emerging including fuel cells, diagnostic arrays and biosensors.
Biosensors and diagnostic arrays require active antibodies or enzymes
attached to a surface to detect molecules of interest by means of their
specific interaction. Durable surfaces that can retain immobilized protein in
a functional state for long periods are therefore of considerable value.
Most of the commercially available protein binding surfaces are based

on polymeric materials. Polymers are inexpensive, chemically inert and
have good mechanical properties (light weight, rigidity, flexible shape and
low surface roughness etc.). However, some surface properties relevant to
protein attachment such as wettability are often not optimal. Surface
modification is one of the ways to enhance the surface functionality.
Various methods, including chemical 4 , physical 5 , radiation 6 (e.g. UV
130
Chapter 6
irradiation) and plasma deposition and etching7, have been reported to

enhance protein attachment on polymeric surfaces. Combinations of
methods, such as plasma grafting with polyacrylic acid8,9 and UV radiation
with solvent coating 10 have been suggested. Some reviews 11,12,13 have
highlighted plasma surface modification (PSM) as an outstanding surface
treatment technique owing to its non-line of sight, low temperature and dry
processing. PSM on polymers has been intensively studied14,15,16,17,18 in
terms of physical and chemical properties for many years. However, there
are only a few studies19,20 which investigate protein attachment to plasma
treated polymers.
Ultra high molecular weight polyethylene (UHMWPE) is employed in

this work as the polymer surface for study as it has a relatively good
biocompatibility and simple chemical structure and it is widespread in
biomedical applications 21 , 22 , 23 . The protein chosen for this study was
horseradish peroxidise (HRP), a widely researched24,25 enzyme, used in
many applications such as labelling in immunoassays and waste water
treatment.
In this chapter, we investigate the use of PSM to prepare UHMWPE

surfaces for binding active proteins. The experimental procedures and
results of protein binding after plasma treatment in two gases, nitrogen and
argon, with and without energetic ion bombardment are presented.
Furthermore, a comparison of the performance of the PIII treated
UHMWPE with that of a popular commercial protein binding surface is
presented. A special focus of this work is to assess the shelf life of the
131
Chapter 6
treated surfaces in terms of the retention of protein binding performance.
6.2 Materials and sample preparation
6.2.1 Materials
Ultra high molecular weight polyethylene (UHMWPE) with 200m

thickness was purchased from Goodfellow Cambridge Limited (cat no
ET301200/1). The polymer sheets were cut to 1.3 cm x 1.5 cm size and the
surfaces were used as supplied.
NUNC Maxisorp MicroArray Slides, used as benchmark binding
surfaces, were purchased from Nalge NUNC International US (cat no
230302).
Horseradish peroxidase (HRP) was purchased from Sigma (product
number: P6782) and it was dissolved in a buffered solution of 10 mM PO4
at pH 7.
6.2.2 Sample preparation Plasma surface modification

(PSM) methods
The plasma surface modifications of UHMWPE were carried out at the

University of Sydney.
Two types of plasma treatments were used. The first, to which refer to
132
Chapter 6
as plasma treatment, involves exposure of the sample to the RF plasma

with no electrical connections made to the sample holder. This means that
the holder assumes a floating potential determined by the potential at which
the current due to the more mobile electrons is balanced by ion current
drawn from the plasma. The floating potential was in the range of -20 to
-40 volts. This potential determines the energy of ion bombardment during
the treatment process.
The second type of plasma treatment, to which we refer to as PIII

treatment, involves the application of high voltage pulses periodically to
the conducting sample holder. This results in a fraction of the ions incident
having energies of the order of tens of keV. This process is known as
Plasma Immersion Ion Implantation (PIII)26,27. The high voltage pulses are
supplied by a pulsed power supply connected to the sample holder
feedthrough. In our PIII treatment process, we used a 20 kV bias voltage
applied for pulse lengths of 20 s at a repetition rate of 50 Hz.
For both types of plasma treatment, process gases were delivered to the
chamber using MKS mass flow rate controllers. The radiofrequency power
used was 100 W. The gases used were high purity (99.99% pure) argon and
nitrogen. In order to obtain the optimal treatment condition, different
pressure and treatment duration were attempted and the parameters used are
listed in Table 6.1. Some of the treated surfaces were stored in a desiccator
at atmospheric pressure and temperature for 1, 2 and 4 weeks, 6 months
and 1 year.
133
Chapter 6
134
Table 6.1. Plasma and PIII treatment parameters. For the PIII processes the
high voltage bias applied was 20 kV for a pulse length of 20 and with a
frequency of 50 Hz.
Gas type (Ar or
Working
Treatment time
N) in chamber
pressure (mTorr)
(second)
Ar
2.0
800
Ar
2.0
1200
Ar
2.0
20
Ar
0.3
20
2.0
800
2.0
100
2.0
20
0.3
20
Name
Ar PIII 2mTorr 800s
Ar Plasma 2mTorr 800s
Ar PIII 2mTorr 1200s
Ar Plasma 2mTorr
1200s
Ar PIII 2mTorr 20s
Ar Plasma 2mTorr 20s
Ar PIII 0.3 mTorr 20s
Ar Plasma 0.3 mTorr
20s
N PIII 2mTorr 800s

N Plasma 2mTorr 800s
N PIII 2mTorr 100s
N Plasma 2mTorr 100s
N PIII 2mTorr 20s
N Plasma 2mTorr 20s
N PIII 0.3mTorr 20s
N Plasma 0.3mTorr 20s
Chapter 6
6.3 Experimental methods
6.3.1 Surface characterizations
Surface morphology
Atomic force microscopy (AFM, Autoprobe CP) and a profilometer
called Form Talysurf PGI (FTPGI, Taylor Hobson Leicester, England)
were employed to measure the topography of plasma, PIII-treated and
as-received (referred to as untreated) UHMWPE surfaces. AFM was used
to examine the microscopic scale from 10nm to 0.1nm while the FTPGI
was used for macroscopic length scales of order mm. Surface roughness
and 3 dimensional images were examined using atomic force microscopy
(AFM) in contact mode. Scans were taken over squares of various side
lengths and root mean square roughness was calculated over each scan.
Wettability
The wettability was studied by measuring of contact angle using the
sessile drop method. Kruss contact angle equipment DS10 was employed to
measure the water contact angle after treatment starting at 5 min after the
sample was removed from the treatment chamber and repeated at intervals
for times up to 11days. In each measurement, 50l of de-ionised water was
dropped onto the sample and the angle between the edge of the water drop
and the surface was measured.
135
Chapter 6
FTIR
Fourier transform infrared (FTIR) spectroscopy was used to determine
the chemical bonding on the surface of the untreated and treated UHMWPE.
It is a conventional accepted technique for the chemical characterization of
polymers. Used with total internal reflection in a Ge crystal in the
attenuated total reflection (ATR) mode it is able to investigate the entire
thickness of modified layer since the measurement depth into the surface is
in the order of 2 m.
XPS
X-ray photoelectron spectroscopy (XPS) is another powerful tool to
provide information on chemical content and bonding at the surface. XPS
has been used to study the surface (5-10nm) chemical changes induced by
argon or nitrogen plasma surface modification on UHMWPE. The survey
scan acquires a wide spectrum from 0 to 1000eV. It provides information
on chemical species present on the surface. Furthermore, high resolution
spectra from XPS with 0.1eV resolution can reveal the chemical bonding at
the surface. This data can corroborate the analysis of FTIR.
The chemical structure of modified UHMWPE was characterized by

FTIR and XPS spectroscopies. The combination of these two techniques
gives the whole picture of the surface and interface (modified layer) of the
treated samples for comparison with the untreated sample.
136
Chapter 6
6.3.2 Protein attachment process and functionality assay

for HRP
The treated and untreated surfaces were incubated overnight in a buffer

solution containing 50 g/ml HRP protein and then rinsed in fresh 10 mM
phosphate buffer of pH 7.0, six times for 20 minutes each time. After
washing, each sample was clamped between two stainless steel plates
separated by an O ring (inner diameter 8 mm, outer diameter 11 mm)
which sealed to the sample surface. The top plate contained a 5 mm
diameter hole, enabling the addition of 75 l TMB (3,3',5,5'
tetramethylbenzidine, Sigma T0440), an HRP substrate, to an area of
polymer surface defined by the diameter of the O ring. After 30 seconds, 25
l of reacted liquid was taken and added to 50 l of 2 M hydrochloric acid
to stop the reaction. 25 l of unreacted TMB was also added to make up the
volume for a 100 l cuvette. Concurrently, the liquid changes to yellow and
that colour remains stable for an hour. The optical density (O.D.) in
transmission at 450 nm was then measured using a Beckman DU530 Life
Science UV/VIS spectrophotometer. To improve the statistical significance,
each measurement was repeated on three samples and the data points
shown are the means and error bars corresponding to one standard
deviation. To ensure that the optical density (O.D.) measured in this HRP
functionality assay is not affected by chemical processes occurring on the
buffer soaked polymer surfaces, the assay was carried out in PO4 buffer in
the absence of HRP. The optical density measured was zero.
HRP from two bottles from the same manufacturer and lot number was
137
Chapter 6
used for all of the studies presented in this paper. The protein was dissolved
in fresh buffer prior to each incubation with surfaces. We confirmed that
the activity level of the protein was the same each time a fresh solution was
prepared from the same bottle. The experiments, shown in Figures 6.9-6.10,
6.12-6.15, 6.17-6.19 used HRP from one shipment while the experiments
shown in Figure 6.11 and 6.16 used HRP from another with the same lot
number. The activity measured in solution was approximately 20% higher
for protein from the second shipment (bottle two) as compared to the first
shipment (bottle one) for the same concentration of HRP dissolved.
6.3.3 Stability of attached protein activity

To assess the stability of the attached protein over time, samples were
kept in buffer solution which was replaced with fresh buffer each day. The
assay was carried out on samples removed from the solution on the day
following incubation (day 0), the day after that (day 1) and then every other
day (days 3 and 5). In some of the experiments the activity was assessed
daily.
6.3.4 Shelf life of treated UHMWPE
To assess the shelf life of the plasma and PIII treated surfaces, the
above procedure was repeated with surfaces that had been stored in a
desiccator in dry air at room temperature and atmospheric pressure for 2
138
Chapter 6
and 4 week, 6 month and 1 year periods prior to incubation in the protein
solution.
6.3.5 Plasma treatment on commercial protein binding

surface
The commercial protein binding surface, NUNC MaxisorpTM, was

treated using PIII in argon and nitrogen gas for 800 seconds at 2mTorr
working pressure. The nitrogen treated surfaces were stored in a
desiccator at atmospheric pressure and temperature for 6 months which is
the same storage condition as used for the treated UHMWPE mentioned
in previous section (6.2.2 Sample preparation).
139
Chapter 6
6.4 Results
6.4.1 Surface characterizations
This section contains a comparison between the physical and chemical

properties of the untreated and treated surfaces. In particular, the surface
roughness over micro and macro scales, the wettability and the chemical
structure of surface are compared and interpreted in terms of the protein
binding characteristics.
6.4.1.1 Surface morphology

Two magnitudes of scan were performed to determine the microscopic
and macroscopic changes in surface roughness caused by the plasma
surface treatments. Small magnitude scans over areas with 10, 1 and 0.1m
side lengths were obtained by AFM. Figure 6.1 shows surface morphology
images from a series of 10 and 1 m side length scans of untreated and
argon/nitrogen treated surfaces. The 3-D images show mountain and valley
topographies for all scans including untreated surfaces. The root mean
square (RMS) roughness and surface area of the corresponding scans are
listed in Table 6.2 and the uncertainties for the roughness and surface area
measurements are estimated to be 20% and 1% respectively. The roughness
values of the 10m side-length scans of the Ar Plasma 2mTorr 1200s and
the N PIII 2mTorr 800s samples are greater than that of the Ar PIII 2mTorr
1200s sample. This outcome is attributed to the obvious furrow in Fig. 6.1
140
Chapter 6
(e) and (g) but not in Fig. 6.1 (a) and (c). The furrow is a feature which
appears on the untreated samples and is believe to originate in the
processing of the polymer sheet during production. The furrows are parallel
and oriented along one dimension of the surface, can be observed more
clearly in the macro scale scans. The micro scale roughness values are not
consistent in the different sized scans - the roughest surface on the scale of
10m scan is the Ar Plasma-treated sample while for the 1m scan it is the
Ar PIII-treated sample. The differences are between roughening are small
and apparently due to statistical variations between regions on any given
sample rather to differences in the surface roughness of the various
treatment methods. The untreated surfaces are over all slightly smoother
than the treated surfaces, although the difference on the microscale is less
than a factor of 2. This observation shows that the ion bombardment and
plasma etching associated with the plasma surface modification results in a
slight increase in roughness on the micro scale.
The microscopic view of the surface is representative of the local

surface for HRP attachment. Especially the smallest size of scan (0.1m
scan length) is well-match to the dimension of the HRP molecule a factor
of 10 or so larger than the molecules approximately 7nm length. The
different in roughness of all samples is only 1.2 in this smallest scan.
Thus, the plasma surface modification induced roughness increase is
insignificant compared to the size of HRP. The change in surface area after
treatment is less than 4%, 5% and 2% in 10, 1 and 0.1m scan lengths
respectively. Hence there is no significant increase of effective surface area
which should be taken into account when comparing the results of the HRP
141
Chapter 6
attachment assay between sample surfaces. As will be demonstrated in

section 6.4.2 the difference in attachment of active HRP between the
treated and untreated samples is much greater than could be explained by
variation in effective surface areas. Hence, roughening by the plasma
treatments can not explain the increase of HRP attachment observed on
treated surfaces.
142
Chapter 6
A
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Fig. 6.1. Surface morphology of UHMWPE scans in side length size 10m
(column A) and 1m (column B). (a) and (b) Untreated; (c) and (d) Ar PIII
1200s; (e) and (f) Ar Plasma 1200s; (g) and (h) N PIII 800s.
143
Chapter 6
144
Table 6.2. A summary of the roughness and surface area of untreated and
treated UHMWPE AFM scans of different side length size.
Sample/Scan
10m
size
RMS surface
area
()
(m2)
1m
RMS
0.1m
RMS
()
surface
area
(m2)
()
surface
area
(m2)
Untreated
528
101.9
84.1
1.019
2.40
0.01063
Ar PIII 1200s
606
103.7
179
1.074
3.04
0.01063
Ar Plasma
1150
106.3
136
1.034
3.61
0.01079
1130
105.6
130
1.019
3.13
0.01086
1200s
N PIII 800s
To obtain a macroscopic view of surface roughness, a Form Talysurf

PGI was used to scan an area of 1mm1mm. Fig 6.2 shows the three
dimensional (3D) surface profiles and cross sections (along the dotted lines
on fig. 6.2a and 6.2c) of untreated and Ar PIII 1200s UHMWPE. The RMS
roughness measured by the FTPGI is presented in Table 6.3. The variance
of three measurements of roughness is about 20%. The major contribution
to roughness of the surfaces on both the treated and untreated samples are
the stripes which are features of the as-received surfaces. Hence on this
scale similar RMS roughness is found on the untreated and the Ar Plasma
1200s surfaces. Although some surface roughening was observed in the
microscopic view by AFM, the plasma surface modification does not
significantly change the roughness values in macroscopic view as measured
Chapter 6
by FTPGI. This is due to the rough surface of the as-received UHMWPE.

Similar results were obtained by other researchers28, who also reported that
the surface roughness remains unchanged after ion implantation.
The surface (Ar 2mTorr 1200s) treated with the highest ion fluence,
which is the roughest sample, was chosen to compare with the untreated
surface. The mean value roughness of the Ar PIII 2mTorr 1200s treated
surface is slightly higher than that of the untreated surface. However, the
difference is only 8%, which is within the error bars and therefore
statistically insignificant. Furthermore, roughness of plasma-treated
samples overall is not significantly different from that of the untreated
surface. The Ar Plasma 2mTorr 1200s surface for example has the same
roughness as the untreated surface. The N PIII 2mTorr 800s surface, on the
other hand, has a roughness that is lower than that of both the sample
treated with the highest fluence and untreated surfaces. This implies that
the roughness created by plasma surface modification is relatively minor
compared to that of the original bumpy surface. No significant change of in
surface morphology and roughness can be observed on the macroscopic
scale.
145
Chapter 6
(a)
(b)
(c)
(d)
Fig. 6.2. Surface morphology of UHMWPE captured by Form Talysurf PGI

with a scan side length of 1mm, (a) is a 3D profile of the untreated surface and
(b) is the cross section taken along the dotted line in (a); (c)is a 3D profile of
the Ar PIII 1200s treated UHMWPE surface and (d) shows the cross section
along the dotted line in (c).
146
Chapter 6
Table 6.3. Macroscopic surface roughness of untreated and treated surfaces

measured by FTPGI.
Sample
RMS roughness (m)
Untreated UHMWPE
0.794
Ar PIII 1200s
0.861
Ar Plasma 1200s
0.795
N PIII 800s
0.634
147
Chapter 6
6.4.1.2 Wettability
Figure 6.3 shows the water contact angles measured for the argon and
nitrogen, plasma and PIII treated samples (all samples shown were treated
with 2mTorr working pressure for 800 seconds) as a function of time since
treatment while being stored in ambient laboratory atmosphere. The contact
angle of the as received untreated UHMWPE is about 110. All of the
treated samples have lower water contact angles than the untreated surfaces.
All of the treated surfaces also show a hydrophobic recovery, in which
the contact angle increases gradually over time. The nitrogen treated (for
both plasma and PIII treatments) surfaces have lower contact angles than
the argon treated samples. The samples treated using the PIII process in
nitrogen consistently have the lowest contact angles.
110
Contact angle (degree)
100
90
80
70
60
50
40
30
0
50 100 150
6000
9000
12000
15000
Time (minutes)
Fig. 6.3. Water contact angles of plasma treated UHMWPE surfaces as a

function of time since removal from the treatment chamber. Measurements are
from surfaces treated using the Ar plasma (8), the Ar PIII process (7), the N
plasma () and the N PIII process (!).
148
Chapter 6
One factor which may influence the accuracy of contact angle

measurements is the surface roughness. According to Wenzels equation
(Eq. 6.1), contact angles below 90 are expected to be underestimated if
measured on rough surfaces compared to the angle on a smooth surface29.
cos r = r cos 0 (Eq. 6.1)

r - Contact angle as measured on a rough surface
0 Real thermodynamic contact angle on smooth surface
r - Ratio of the actual roughened area of interface to the geometric
surface area
It should be noted that in order to apply the Wenzel equation, the size of
the water droplet used in the measurement must be much larger than the
surface roughness30. The correction can be ignored if the roughness on the
surface is less than 100nm. In the case of this study, the water droplet has a
diameter of about 1mm which is much larger than the roughness of both the
treated and untreated surfaces (about 800nm). The results in Fig. 6.3 are
presented without correction by the Wenzel equation, whereas the corrected
contact angle of N and Ar PIII-treated listed in Fig. 6.4. The lower contact
angle is more sensitive to the correct than the higher. Nonetheless, the
correction shows no different for the rate of hydrophobic recovery. The N
PIII treated samples still have the lowest contact angle among different
treatments. Therefore, the low contact angles obtained from the treated
surfaces are thought to associate with chemical changes on the surface
rather than surface roughening.
149
Chapter 6
(a)
N PIII treated
50
40
30
Measured data
20
Corrected data
10
0
0
50
100
150
200
Time (mintues)
(b)
Ar PIII treated
100
80
60
Measured data
40
Corrected data
20
0
0
50
100
150
200
Time (minutes)
Fig. 6.4. Correction of water contact angle of (a) N PIII-treated and (b) Ar
PIII-treated samples by the Wenzel equation.
150
Chapter 6
6.4.1.3 ATR-FTIR
The chemistry of the nitrogen-treated UHMWPE surface was studied

by ATR-FTIR and the results are shown in Fig 6.5. The characteristic peaks
of UHMWPE are observed at 719, 1463, 1473, 2848 and 2917cm-1 and are
clearly seen in the spectra taken from the untreated surface. The peak at
719cm-1 is associated with the C C skeletal vibration and the reduction of
intensity seen in the treated surface is associated with the destruction of the
polymer backbone of the UHMWPE. The peaks at 1463, 1473, 2848 and
2917 cm-1 are associated with C H bending and stretching vibrations31.
The intensity of these peaks also decreases after treatment and the
reduction indicates the removal of hydrogen from the structure during
treatment. The arrows in the figure show features in two spectral regions
associated with oxidized structures 32 , 33 introduced after post-treatment
exposure to atmosphere. These included the characteristic spectral band in
the region 1710 1745 associated with carbonyl and carboxyl groups, and
a broad band in the region 3200 3600 cm-1 due to the hydroxyl (OH)
group. These peaks are all absent in the untreated spectra as is the 1650
cm-1 signature associated with carbon double bonds (C=C)).
151
Chapter 6
152
(a)
(b)
(c)
(d)
(e)
(f)
2000
1800
1600
1400
1200
1000
-1
Wave number (cm )
(a)
(b)
(c)
(d)
(e)
(f)
4000
3500
3000
2500
2000
1500
1000
-1
Wave number (cm )
Fig. 6.5. FTIR spectra of nitrogen-treated and untreated UHMWPE. (a) N PIII
2mTorr 800s; (b) N PIII 2mTorr 100s; (c) N Plasma 2mTorr 800s; (d) N PIII
2mTorr 20s; (e) N PIII 0.3mTorr 20s; (f) Untreated UHMWPE.
Chapter 6
Fig. 6.5 shows that the nitrogen PIII-treated samples with 100 and 800s
treatment (highest fluence - line (a) and (b) respectively in Fig. 6.5) contain
significant oxidized structures and hydroxyl functional groups. On the other
hand the nitrogen plasma treated surface (line (c)) and the lower fluence
PIII-treated surfaces with 20s treatment times (line (d) and (e)) show only a
very slight signal above background for the oxidised structures and
hydroxyls. These results indicate that modification by plasma treatment and
short PIII treatments are either superficial (relative to the ATR-FTIR
sampling thickness) or that they impact insufficient dose to exhibit strong
chemical structure changes in ATR-FTIR. We note here however that the
oxidized structures are seen on plasma-treated and 20 seconds PIII-treated
surfaces using XPS (results in section 6.4.1.4), which is sensitive only to
the top several monolayers as opposed to sampling hundreds of microns as
in ATR-IR. The majority of oxygen containing groups are believed to arise
from oxidation during atmospheric exposure after the samples have been
removed from the vacuum chamber34,35. Some oxidation may also be due to
residual oxygen in treatment chamber.
Because the result from ATR-FTIR shows the content of the entire
modified layer (thickness less than 100nm from TRIM result obtained from
Chapter 4) together usually with some of the bulk material under the
modified layer, it is not unusual to overlook changes occurring only on the
surface or with insufficient volume density for detection such as for the
plasma-treated sample.
153
Chapter 6
Some of the argon-treated samples were examined and the same

oxidized structures (depicted in Fig 6.6) were found. The argon PIII-treated
samples demonstrate have similar ATR-IR spectra as the nitrogen
PIII-treated samples showing the same clear peaks corresponding to
oxidized structures and OH groups. Unlike the case for nitrogen treatment,
the argon plasma-treated (1200s and 800s) sample spectra (Fig. 6.6 line (a)
and (c)) also contain the same oxidized and OH structures as the
PIII-treated samples. This indicates that argon treatment has a higher
damage creation rate with than a nitrogen treatment with the same
parameters. This is most likely due to the fact that the plasma density is
higher in 2 mTorr of argon than it is in nitrogen at the same working
pressure as well as the fact that argon is a heavier ion which produces
higher levels of damage16. Argon would therefore produce a higher
concentration of active species and free radicals in the polymer than
nitrogen.
154
Chapter 6
155
(a)
(b)
(c)
(d)
(e)
(f)
2000
1800
1600
1400
1200
1000
-1
Wave number (cm )
Fig. 6.6. FTIR spectra of argon-treated and untreated UHMWPE. (a) Ar

Plasma 2mTorr 1200s; (b) Ar PIII 2mTorr 800s; (c) Ar Plasma 2mTorr 800s;
(d) Ar PIII 2mTorr 20s (e) Ar PIII 0.3mTorr 20s; (f) Untreated UHMWPE.
Chapter 6
156
6.4.1.4 XPS
XPS complements the information on chemical structure obtained from

ATR-FTIR because it samples only the top-most 5-10nm from the surface
while ATR-IR samples microns into the surface. XPS was therefore also
used to obtain information about the chemical structure of treated and
untreated UHMWPE. A survey scan spectrum to identify the chemical
species present was taken from each sample, followed by high resolution
spectra of the peaks associated with each species to determine their
bonding states. Fig. 6.7 shows a survey scan identifying carbon, oxygen
and nitrogen as the three main components of the surface of the N PIII
C1s
2mTorr 800s sample.
O1s
40000
Counts
N1s
30000
20000
10000
0
1000
800
600
400
200
Binding Energy (eV)
Fig. 6.7. XPS survey scan of the nitrogen treated UHMWPE (N PIII 2mTorr
800s).
Chapter 6
157
The high resolution spectrum of the C1s signal was collected to identify
the chemical bond present at the surface. A high resolution spectrum of the
C1s line for an untreated UHMWPE sample is shown in Fig 6.8 (a). It
shows a symmetric peak centred at 284.0 eV, indicating that C C and C
H are predominant bonding states of carbon. Similar scans taken from the
nitrogen and argon treated samples show that the C1s peak is shifted
slightly towards lower energies and a shoulder develops on the higher
energy side (peak location listed in Table 6.4).
8000
(a)
4000
(b)
6000
3000
4000
2000
2000
0
292
288
284
280
276
1000
292
Binding Energy (eV)
3000
288
284
280
Binding Energy (eV)
(c)
(d)
3000
2000
2000
1000
1000
296
292
288
284
Binding Energy (eV)
280
296
292
288
284
280
Binding Energy (eV)
Fig. 6.8. XPS C1s spectra of untreated, argon or nitrogen treated UHMWPE.
(a) Untreated; (b) Ar PIII 2mTorr 800s; (c) Ar Plasma 2mTorr 1200s; (d) Ar
PIII 2mTorr 1200s; (e) N Plasma 2mTorr 800s; (f) N PIII 2mTorr 800s; (g) N
Plasma 2mTorr 20s; (h) N PIII 2mTorr 20s.
Chapter 6
2500
(e)
158
(f)
3000
2000
1500
2000
1000
1000
500
296
292
288
284
296
280
288
284
280
Binding Energy (eV)
Binding Energy (eV)
4000
292
3000
(g)
(h)
3000
2000
2000
1000
1000
296
292
288
284
Binding Energy (eV)
280
296
292
288
284
280
Binding Energy (eV)
Fig. 6.8. XPS C1s spectra of untreated, argon or nitrogen treated UHMWPE.
(e) N Plasma 2mTorr 800s; (f) N PIII 2mTorr 800s; (g) N Plasma 2mTorr 20s;
(h) N PIII 2mTorr 20s.
The asymmetric peaks from spectra of the nitrogen and argon-treated

samples (Fig. 6.8 (b)-(h)) show new chemical components present on the
surface. In Fig. 6.8 (b)-(h), several new peaks overlapping with the main
C1s have appeared. They can be attributed to the functional groups C O,
C = O and O C O 36 , 37 and additionally a C N peak in the
nitrogen-treated samples38. This shows once again that oxygen functional
groups are incorporated in the surface after exposure to the plasma
treatment and subsequently to an oxygen containing atmosphere. Such a
result is supported by many published reports39,40,41.
Chapter 6
159
Table 6.4. Surface chemical composition atomic percentage, oxygen to carbon

ratio (obtained by fitting the survey scan data) and peak location of the C1s
peak for untreated, argon and nitrogen-treated UHMWPE
Sample
C%
O%
Untreated 94.9
5.1
Ar
PIII 79.7
2mTorr
800s
20.3
Ar
Plasma
2mTorr
1200s
81.9
Ar PIII
2mTorr
1200s
N%
Peak (eV) O/C ratio

284.0
0.054
283.6
0.254
18.1
283.5
0.222
79.8
20.2
283.3
0.254
N Plasma 71.5
2mTorr
800s
17.8
10.7
283.6
0.249
N PIII
2mTorr
800s
71.1
15.5
13.4
283.5
0.219
N Plasma 73.6
2mTorr
20s
16.8
9.7
283.5
0.228
N PIII
2mTorr
20s
20.6
8.5
283.5
0.289
71.0
The surface composition to a depth of order 10nm as evaluated from the

survey scans is presented in Table 6.4. The percentage of oxygen in the
treated samples is over 15% but only 5% on the untreated UHMWPE. For
the nitrogen-treated samples, there is some nitrogen incorporated but the
oxygen percentage is higher than that of nitrogen. Moreover, the oxygen to
Chapter 6
carbon (O/C) ratio is similar for both argon and nitrogen treated surfaces
(between 0.22 and 0.29). These results are in agreement with data stated
and discussed in literature42,43.
The spectra obtained from XPS provide strong evidence to corroborate

the results of ATR-FTIR indicating that oxygen containing groups appear
after both plasma and PIII treatments. The oxidation is however least
intense for the 20s nitrogen-treated (both plasma and PIII) and 800s
nitrogen plasma-treated samples. These samples did not show oxidized
structures in their ATR-FTIR spectra but nevertheless had quite high
percentages of oxygen (17, 21 and 18% respectively) detected by XPS on
topmost surface. Overall, the oxygen percentage in argon-treated samples is
higher than in nitrogen-treated in agreement with the analysis from
ATR-FTIR and indicating that argon has produces more damage.
In conclusion for the XPS results, in the treatment of polymers with

inert gases such as argon, the plasma process predominantly leads to the
formation of free radicals and crosslinking which is known as
CASTING44,45 (Crosslinking by activated species of inert gas) but not the
addition of new chemical functionalities from the treatment process itself.
Incorporation of oxygen occurs when the treated samples are exposed to
atmosphere46. Treatment of polymers with active gas, such as nitrogen,
however leads to new nitrogen containing functionalities formed during
treatment. At the same time, free radicals are created by the treatment and
oxygen containing functional groups are introduced by post-treatment
reaction with atmospheric oxygen or water.
160
Chapter 6
6.4.2 Protein attachment
Figure 6.9 shows the results of the colorimetric assay used to assess the
activity of HRP attached to argon treated (Ar 2mTorr 800s, PIII and Plasma
treatment as indicated) UHMWPE as a function of time over a five day
period after attachment. Throughout the experiment the samples and
attached enzymes were stored in fresh buffer and the buffer was changed
daily. Both the argon plasma and PIII treated surfaces have more active
protein attached immediately after incubation than the untreated surface,
but there are no differences outside the measurement uncertainty between
the data points for the plasma and PIII treated surfaces. All of the surfaces
show a reduction in the quantity of active protein over time but both types
(PIII and Plasma) of argon treatment on surfaces maintain significantly
higher protein activity than the untreated surfaces over the 5 day duration
of the experiment.
161
Chapter 6
O.D. in transmission at 450nm
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
Fig. 6.9. Optical density measurements from the HRP activity assay on
UHMWPE surfaces treated using Ar Plasma 2mTorr 800s () and Ar PIII
2mTorr 800s () as well as on untreated control surfaces (). The day 0 data
points correspond to activity measured on samples which were incubated in
HRP solution overnight and then washed in fresh buffer before testing by a
colorimetric activity assay. For data taken on subsequent days, the buffer
solution was changed each day and further activity assays were conducted
from day 1 to 5. This data was measured using HRP from bottle 1.
To investigate the effect of treatment time in the argon plasma and PIII
treatments, we repeated the above enzyme activity time course study for
plasma and PIII surfaces treated for 20 and 1200 seconds (s). The results
for the 20s treatments are shown in Fig. 6.10 while Fig. 6.11 shows the
results for the 1200s treatments. Comparing Fig. 6.9 (800s treatment) and
Fig. 6.10 (20s treatment), the initial (Day 0) HRP attachment on the surface
is decreased for the lower treatment time. A difference in activity remains
up until 5 days of immersion in buffer with daily washing showing that the
20 second treatments are not as effective as the 800 second treatments. The
162
Chapter 6
results depicted in Fig. 6.11 shows that there is no significant difference

between the performances of surfaces treated for 800 and 1200 seconds.
Note that because a different shipment of HRP was used for the 1200
seconds study, the 800 seconds study on PIII treated surfaces was repeated
as a calibration between the two HRP bottles. The repeated 800 second
treatment optical density measurements are also shown in Fig. 6.11.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
Day
Fig. 6.10. Optical density measurements from the HRP activity assays on
surfaces treated for 20 seconds, Ar Plasma 2mTorr 20s () and Ar PIII 2mTorr
20s (). The activities on untreated () surfaces are also shown. This data
was measured using HRP from bottle 1.
163
Chapter 6
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Day 0
Day 1
Day 3
Day 5
surfaces treated for 1200 seconds Ar Plasma 2mTorr 1200s () and Ar PIII
2mTorr 1200s (U). Since bottle 2 of HRP was used for these measurements, an
800 seconds PIII treatment (Ar PIII 2mTorr 800s, ) was repeated and is
shown for comparison. The results from untreated samples () are also shown.
164
Chapter 6
Besides comparison between treatment duration, different working

pressures are investigated. Fig. 6.12 shows the results of the HRP
colorimetric activity assay on samples treated with argon plasma and PIII
for 20s at 0.3mTorr and at 2mTorr. For both the plasma and PII treatments a
pressure of 2 mTorr is significantly more effective. The 0.3 mTorr
treatments produce only a slight improvement in attachment of active
protein over the untreated surfaces.
O. D. transmission at 450nm
0.6
Untreated
Ar 2mT PIII
Ar 0.3mT PIII
Untreated
Ar 2mT P
Ar 0.3mT P
0.5
0.4
0.3
0.2
0.1
0.0
Day 0Day 1Day 3Day 5 -- Day 0Day 1Day 3Day 5
surfaces treated in Argon at two different working pressures over the same
treatment time (20s). Circles show the results for the 2mTorr treatments and
triangles represent those done using 0.3mTorr. This data was measured using
HRP from bottle 1.
165
Chapter 6
A similar comparative study of 20, 100 and 800 seconds treatment

times was repeated using a nitrogen gas instead of argon for the treatment
process. The HRP used in this study was from the bottle one. The results in
Figure 6.13 indicate the retention of bound protein activity is improved
gradually from 20s, 100s and then 800s. The initial day 0 activity is slightly
lower but the activity retention observed at day 5 for the 800 seconds
nitrogen treatment is slightly higher than that achieved by argon treatment
for the same treatment time (see Figure 6.9).
0.7
0.6
0.5
800U
100U
20U
0.4
0.3
0.2
0.1
800P
100P
20P
800PIII
100PIII
20PIII
0.0
D 0 D 1 D 3 D 5 -- D 0 D 1 D 3 D 5 -- D 0 D 1 D 3 D 5
PIII
Plasma
untreated
surfaces treated in N PIII 2mTorr 800s (800PIII), N PIII 2mTorr 100s (100PIII)
and N PIII 2mTorr 20s (20PIII) and in N Plasma 2mTorr 800s, N Plasma
2mTorr 100s and N Plasma 2mTorr 20s (800P, 100P and 20P). Results from
three sets of untreated samples used as controls (800U, 100U and 20U) are
shown for comparison. This data was obtained using protein sourced from
bottle 1.
166
Chapter 6
Fig. 6.14 shows that, unlike the case for argon, (Figure 6.12), there is
no apparent effect of changing working pressure from 0.3 mTorr to 2 mTorr
for the nitrogen treatment. The lack of dependence on pressure was also
apparent for a nitrogen treatment of duration 800s.
0.6
Untreated
N 2mT PIII
N 0.3mT PIII
Untreated
N 2mT P
N 0.3mT P
0.5
0.4
0.3
0.2
0.1
0.0
Day 0Day 1Day 3Day 5
--
Day 0Day 1Day 3Day 5
surfaces treated in Nitrogen at two different working pressures over the same
treatment time (20s). Circles show the results for the 2mTorr treatments and
triangles represent those done using 0.3mTorr. This data was measured using
HRP from bottle 1.
167
Chapter 6
6.4.3 Aging of Plasma treated surfaces
Figure 6.15 shows the results from the HRP activity assay for samples
stored in a desiccator for 2 and 4 weeks compared with results from freshly
treated samples. Both the freshly treated surfaces show slightly higher
levels of functional attachment than the aged samples in terms of the mean
values plotted, but not all of the differences are significant given the
standard deviation error bars. The differences between the 2 and 4 week
aged surfaces are not significant. These results show that any aging effect
in treated samples is small and has stabilized after 2 weeks. The results of
protein attachment to treated surfaces stored for 1 year are shown in Fig.
6.16. Although neither the plasma nor the PIII surfaces bind significantly
more active protein than the untreated surface at day 0, there retention of
activity over many days seems to be better than that of the untreated
surface. The PIII has the highest retention if activity at day 5 while the
plasma treated sample has decayed almost to the level of the untreated
sample at this point.
168
Chapter 6
(a)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0
Day
(b)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0
Day
surfaces incubated within 5 hours of the treatment process (), 2 weeks after
treatment (), and 4 weeks after treatment (). (a) shows results by the Ar
Plasma 2mTorr 800s treatment and (b) shows results by the Ar PIII 2mTorr
800s treatment. HRP was taken from bottle 1.
169
Chapter 6
0.7
0.6
0.5
0.4
0.3
0.2
0.1
D0
D1
D3
D5
Day
surfaces aged in desiccators for 1 year after undergoing plasma ( ) and PIII
( ) treatments are compare with untreated controls (U, ).and a freshly
treated (Ar PIII 2mTorr 800s) which was incubated in protein solution within 5
hours (T) of the treatment. All treatments were conducted in 2 mTorr of Argon
for 800s. HRP was taken from bottle 2.
170
Chapter 6
Aging of nitrogen-treated samples was also studied. Fig. 6.17 shows the
results from the HRP activity assay for samples stored in a desiccator for 4
weeks and 6 months compared with results from freshly treated samples.
The activity of the HRP attached to the plasma treated surfaces which had
been stored for 4 weeks and 6 months prior to incubation in protein
solution decayed faster than that of HRP attached to the freshly treated
samples. However, the PIII treated surfaces show significantly better
activity retention over time than the surfaces treated with the plasma alone.
171
Chapter 6
4wP
Plasma
6mP
O. D. in transmission at 450nm
0.7
0.6
0.5
4wPIII
PIII
6mPIII
4wU
U
6mU
0.4
0.3
0.2
0.1
0.0
D 0 D 1 D 3 D 5 -- D 0 D 1 D 3 D 5 -- D 0 D 1 D 3 D 5
Day
Fig. 6.17. Optical density measurements from HRP activity assays on surfaces
incubated in HRP solution within 5 hours of the treatment process (), 4 weeks
() and 6 months after treatment (). Since the assays were done at different
times, one set of control samples was analysed together with each, hence three
sets of data for untreated surfaces are shown in the figure. The controls are
shown on the left; the surfaces treated with the nitrogen PIII process on the
right and the nitrogen plasma treated surfaces in the centre. HRP was taken
from bottle 1.
172
Chapter 6
6.4.4 Commercial protein binding surface
The HRP binding properties of a commercial protein binding surface

(NUNC Maxisorp) were compared with our treated UHMWPE surfaces.
Some of the NUNC surfaces were modified by argon PIII treatment under
the same conditions as employed for the UHMWPE. Untreated NUNC,
argon PIII-treated NUNC, untreated PE, and argon PIII-treated PE surfaces
are compared in Fig. 6.18, showing HRP activity at days 0 and 3. The
results indicate that untreated PE has the lowest (O.D.~0.30) HRP
attachment and the argon PIII-treated PE (O.D.~0.70) has more functional
HRP attachment than the NUNC (O.D.~0.60 for both treated and untreated)
surfaces. After immersion in buffer for 3 days with daily washing, argon
PIII-treated PE (O.D.~0.50) retained more activity than the untreated
NUNC (O.D.~0.40) surface. The PIII-treated NUNC surface (O.D.~0.55)
was marginally better than the PIII treated PE (O.D.~0.50) at day 3. The
PIII treatment process significantly enhances the activity retention
characteristics for the NUNC surfaces.
173
Chapter 6
NUNC
NUNC + PIII
PE
PE + PIII
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Day 0
Day 3
Fig. 6.18. Optical density measurements from the HRP activity assay on argon
PIII treated and untreated NUNC and UHMWPE surfaces. Activity assays
were conducted on the day after incubation in protein solution day 0 and on
day 3. The buffer was replaced each day, even on days when the assay was not
carried out. HRP was taken from bottle 1.
174
Chapter 6
Subsequently, an aging study of nitrogen PIII-treated NUNC samples

was conducted. Fig. 6.19 shows the result of the untreated and PIII-treated
NUNC stored for 6 months before being incubated in HRP buffer solution.
For easy comparison, the data is plotted together with a freshly treated
NUNC sample and its untreated control extracted from Fig. 6.18. The 6
month old PIII-treated UHMWPE sample data from Fig. 6.17 is also shown
for comparison. The treated NUNC samples stored for 6 months show
decrease in the activity from HRP attachment at both day 0 and 3. There is
little difference outside the error bars between all of the aged samples and it
is particularly noteworthy that the treated polyethylene sample which has
been stored for 6 months prior to protein attachment performs at least as
well as the NUNC commercial surface.
175
Chapter 6
6m NUNC
6m NUNC+PIII
NUNC
NUNC+PIII
6m UHMWPE+PIII
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
D0
D3
Day
untreated and PIII treated NUNC and PIII treated UHMWPE surfaces stored
in ambient for 6 months (6m NUNC, 6m NUNC+PIII and 6m UHMWPE+PIII)
and for easy comparison the NUNC and NUNC+PIII from Fig. 6.18 are also
shown. The HRP used came from bottle 1.
176
Chapter 6
6.5 Discussion
In this section, an integrated and general discussion of the results and

the analysis are presented. First of all, the main observations are listed in
point form. The text that follows explores the relationships between various
surface treatments and the observed protein binding properties including
the shelf life. Lastly, a discussion of the optimal treatment conditions found
is presented.
The data support the following observations:

1. The PIII and plasma treatments significantly enhance active protein
retention compared to untreated surfaces. They also significantly lower
the water contact angle.
2. The small increase in surface area induced by surface roughing after
plasma surface modification is not sufficient to explain the increase in
protein attachment observed. Therefore chemical changes to the surface
must be responsible.
3. Oxygen containing functional groups are introduced by the argon and
nitrogen treatments. Some nitrogen containing groups also appear in the
case of the nitrogen treated surfaces.
4. A conversion to hydrophilic surface property is observed after both the
PIII and plasma treatments. This can be attributed to the polar
functional groups introduced when the surface is oxidised. The minor
surface roughening observed will not have a significant effect on the
water contact angle.
177
Chapter 6
5. Surfaces PIII treated for 800 seconds showed improved attachment of

active protein compared to those treated with equivalent 20 second
treatments. No further benefit was gained when the argon treatments
were extended to 1200 seconds, indicating that a saturated level of
polymer surface modification was attained within 800 seconds.
6. The 800 seconds nitrogen PIII-treated surface maintained the lowest
contact angle after treatment and showed the best retention of bound
protein activity.
7. The nitrogen PIII-treated surfaces can be stored in laboratory ambient
without any loss of protein attachment density and activity for at least
four weeks. They show a very small reduction active protein binding
performance after 6 months. Although both of the argon treatment
processes and the nitrogen plasma treatment showed some loss of
protein binding and activity retention after storage, they had
significantly improved performance over the untreated polyethylene.
8. A PIII-treated UHMWPE surface outperforms the untreated NUNC
Maxisorp MicroArray Slide in both initial binding capability and in its
ability to retain the HRP activity over 3 days immersion in buffer with
daily buffer changes.
9. The activity retention of the NUNC MicroArray Slides can be
significantly enhanced by treating the surfaces with the PIII treatment
process.
10. The shelf life under ambient conditions of the PIII treated surfaces is at
least six months.
178
Chapter 6
Our experiments indicate that plasma surface modifications can

improve the protein binding characteristics of a polymer surface
considerably. In particular the retention of bioactivity of the bound protein
when exposed to repeated washing is greatly improved over untreated
surfaces. We attribute the improved retention of activity to the increased
hydrophilic character of the surface as demonstrated by the decrease in
measured water contact angles. The surface treated with the nitrogen PIII
process had the lowest water contact angle and performed the best in terms
of its ability to retain protein activity. Analogous results of increased
wettability of surface led to higher cell adhesion have been reported47,48.
It is well known that the effects on surface energy of plasma surface

modification of polymers often decay with time and in many cases almost
completely lost after 20 days49,50,51. The nitrogen PIII treated surfaces
which showed the highest levels of activity retention also had the lowest
rate of hydrophobic recovery (i.e. the rate of increase in water contact angle
over time stored under ambient conditions). Reports in the literature52
indicate that the application of the PIII method (also known as plasma
source ion implantation or PSII) reduces the rate of hydrophobic recovery
in plasma-treated polymers. Behnisch et al. 53 suggested that surface
crosslinking induced by repeated PIII treatment may retard the hydrophobic
recovery. The crosslinking effect by PIII treatment is more severe than that
induced by plasma treatment. The progressive removal of hydrogen54 and
formation of amorphous carbon regions in the polymer is correlated with
the observed colour change from white to light grey of the PIII treated
samples. The plasma-treated samples where PIII was not applied remained
179
Chapter 6
white after treatment.
The XPS and ATR-FTIR characterization of the plasma or PIII treated

surfaces shows that oxygen containing groups are introduced on the treated
surfaces. A prior FTIR spectroscopy study of PIII treated polystyrene also
showed that this process produced surface damage that oxidised in air to
create new C=O groups
55
. These groups, (e.g. aldehydes and carboxyls)
can react 56 with groups such as primary amines commonly found on

proteins. The C=O groups are highly polar and would result in the
significant reduction in water contact angle reported here. The enhanced
retention of protein activity observed on the nitrogen PIII-treated surfaces
may be attributed to the low contact angle. The reduced hydrophobic
recovery imparted by the nitrogen PIII treatment is likely to be due to the
ion damage induced carbonisation (conversion to amorphous carbon) that
occurs at the surface to a depth of a few tens of nm for the ion energies
used in this study57,58. The fact that the nitrogen PIII-treated surface showed
no loss of protein attachment and activity retention capability after 4 weeks
and 6 months can be attributed to the stabilisation of the modified surface
by subsurface carbonisation. If the subsurface is strongly crosslinked, the
gradual removal of polar groups from the surface by molecular
interdiffusion or reptation is reduced or prevented59. It should be noted that
crosslinking process also happens during argon treatment45. However,
damage density by argon ion is heavier than nitrogen and different gas
plasmas have different physical and chemical energies and reaction with
substrate. Therefore, the inward diffusion of surface polar component may
not block by the crosslinked layer in argon-treated case. The crosslinked
180
Chapter 6
layers in both nitrogen and argon treatments on UHMWPE were found in

literature that either graphite like layer 31 or DLC layer36,37,38,60.
The benefits of the plasma surface modification processes increased

with fluence for short treatment times, but saturated at higher fluences.
Treatments over 800 seconds yielded no further improvement in protein
binding properties. This is consistent with the development of a saturated
surface modified layer in which the structure is no longer modified by
additional ion impacts. The depth and damage level of such a layer would
be determined by equilibrium between etching and ion modification.
Furthermore, the protein binding effectiveness of the surfaces was
influenced also by the working pressure. Two different working pressures
were tried in this report, but the optimal condition can not be decided at the
present stage. Nonetheless, optimization of treatment parameters could be
achieved by a further systematic study employing a wider range of different
conditions.
181
Chapter 6
182
6.6 Conclusion
We applied a plasma immersion ion implantation treatment process to

polymer surfaces and found dramatically improved protein-binding
characteristics. Both the amount of active protein bound and the activity of
the bound protein over time were greatly increased by the treatment process.
Significant improvements were obtained both on UHMWPE and the
NUNC
Maxisorp
commercial
surface.
However,
the
PIII-treated
UHMWPE outperformed the as received NUNC Maxisorp surface on both

these metrics.
These data suggest that the strong binding to the treated surfaces may
be associated with interactions of the protein with long-lived free radicals
in the damaged polymer layer or with C=O groups introduced on the
polymer surface by oxidation in atmosphere of the plasma activated surface.
We attribute the improved protein activity on the treated surfaces to their
increased hydrophilic character.
Although improvements were observed for short treatment times no

further benefit was observed for treatment time beyond 800 sec. We
attribute this to the development of an equilibrium surface modified layer
which cannot be further carbonised or oxidised by the impinging ions due
to establishment of equilibrium between the etching rate and the
modification rate of buried layers.
Chapter 6
Our PIII-treated surfaces retained their properties for 2 to 4 weeks with

only minimal loss of the protein binding and activity. The best performing
treatment showed no reduction in performance after 4 weeks and continued
to show excellent binding and activity retention after six months of storage.
The treatment that achieved the best performance was the plasma
immersion ion implantation (PIII) using nitrogen plasma. It had the highest
level of retained HRP activity and no degradation of performance after
months of storage. This surface also had the lowest water contact angle and
the lowest level of hydrophobic recovery.
183
Chapter 6
6.7 References
1
. P. Angenendt, Progress in Protein and Antibody Microarray Technology,

Drug Discovery Today 10 (2005), 503-511.
. J. R. Sydor and S. Nock, Protein expression profiling arrays: tools for the
multiplexed high-throughput analysis of proteins, Proteome Sciences 1 (2003),
3.
3
. S. P. Lal, R. I. Christopherson and C. G. dos Remedios, Antibody arrays: an

embryonic but growing technology, Drug Discovery Today 7 (2002),
S143-S149.
4
S. Bousalem, S. Benabderrahmane, Y. Y. C. Sang, C. Mangeney and M. M.

Chehimi, Covalent immobilization of human serum albumin onto reactive
polypyrrole-coated polystyrene latex particles, Journal of Materials Chemistry
15 (2005) 3109-3116.
5
S. H. Lee and E. Ruckenstein, Adsorption of protein onto polymeric surfaces

of different hydrophiilicities a case study with bovine serum albumin,
Journal of Colloid and Interface Science 125(2) (1988), 365-379.
6
V. N. Vasilets, A. V. Kuznetsov and V. I. Sevastianov, Vacuum ultraviolet

treatment of polyethylene to change surface properties and characteristics of
protein adsorption, Journal of Biomedical Materials Research 69A (2004),
428-435.
7
F. Bretagnol, A. Valsesia, G. Ceccone, P. Colpo, D. Gilliland, L. Ceriotti, M.

Hasiwa and F. Rossi, Surface functionalization and patterning techniques to
design interfaces for biomedical and biosensor applications, Plasma Processes
and Polymers 3 (2006), 443-455.
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189
Chapter 7
Conclusion and Future work
190
Chapter 7 Conclusion and Future work
7.1 Conclusion
In this study, the feasibility of utilizing plasma surface modification to

enhance the surface properties of NiTi and UHMWPE biomaterials is
demonstrated.
The modified surface has excellent properties in terms of
mechanical, cyto-compatibility and functionality.
In order to simplify the
presentation, the conclusion of the work from this research is presented

according to the numbered objectives in Chapter 1.4.
7.1.1 NiTi
Modified layer impeding out-diffusion of Ni ions
(1) For NiTi used as orthopaedic materials, out-diffusion of Ni ions is

the primary concern.
Therefore, creating a durable barrier layer in order
to impede the out-diffusion of Ni ions is the objective. Using surface

modification by nitrogen or oxygen PIII treatment, TiN and TiOx phases are
formed in the modified layers. The thickness of the TiN or TiOx layer
depends on the bias voltage applied and annealing temperature.
The film
formation is clearly revealed by chemical analysis and depth profiling by

XPS.
Based on the results of dissolution test, the PIII treatment can
Chapter 7
191
effectively suppress Ni ion leaching from the NiTi substrate by at least 10

times compared to the untreated samples.
Anti-corrosion properties
(2) The anti-corrosion ability of the modified NiTi is greatly improved

as disclosed by the electrochemical corrosion analysis.
Furthermore, the
graded interface promotes strong adhesion between the newly form layer
with the substrate.
All the N-PIII and O-PIII samples possess improved
corrosion resistance compared to the untreated samples indicating that the

barrier layers are strong and robust in our simulated in vitro dissolution test
and accelerated electrochemical corrosion test.
Optimization of PIII treatment
(3) As the results after surface modification by PIII are promising,

systematic optimization of the instrumental parameters is necessary.
The
parameters studied include the applied bias voltage from -40 to -20 kV,
treatment duration from 2 to 4 hours, and a range of post-annealing
temperature. The corrosion resistance and dissolution test show that all
the treated samples outperform the untreated ones.
However, there is no
significant difference among the treated samples using these parameters

ranges, although some minor differences are observed. For instance, all
the treatments enhance the mechanical properties such as hardness and
Chapter 7
192
elastic modulus, but the best properties are achieved on the 450oC annealed
N-PIII sample.
Furthermore, the N-PIII samples show the best
proliferation of osteoblasts.
The results indicate that the optimal
modification conditions are N-PIII at -40 kV and post-annealing at 450oC.
Cyto-compatibility of NiTi
(4)
The
untreated
and
PIII
treated
biocompatibility and cell proliferation.
surfaces
exhibit
good
Cells attached and grow well
especially on the N treated and untreated sample.
It should be noted that
only short term cell tests are described here and more work needs to be
done to assess the longer term biocompatibility. Nevertheless, the results
demonstrate no immediate cytotoxic effect caused by the treatment.
Further applications on porous NiTi
(5) This study successfully extends PIII to porous NiTi.
The
capability of PIII on porous material is verified by XPS depth profiling.

From the results of the dissolution test, PIII is useful in mitigating harmful
Ni ion out diffusion from both dense and porous NiTi. In addition, the
PIII treated porous NiTi soaked in SBF for 28 days exhibits better
corrosion resistance than the untreated dense NiTi. Only minor degradation
in the superelasticity is observed from the PIII samples.
Similar to dense
NiTi, good cyto-compatibilty is observed from both the untreated and PIII
Chapter 7
193
porous NiTi in terms of cell proliferation and cell morphology.
7.1.2 UHMWPE
Immobilization of protein
(6) Plasma surface modification can dramatically improve the

protein-binding characteristics on UHMWPE surfaces.
The amount of
active protein bound is greatly increased after the treatments.
PIII-treated UHMWPE surface outperforms the untreated NUNC Maxisorp

MicroArray Slide in both initial binding capability and the ability to retain
the HRP activity after 3 days of immersion.
Furthermore, significant
improvements are obtained both on the treated UHMWPE and the NUNC
Maxisorp. The activity retention of the NUNC MicroArray Slides can
also be significantly enhanced by PIII.
protein
binding
ability
on
Superior results pertaining to the
PIII-treated
UHMWPE
are
obtained
substantiating the application as a protein binding surface.
Shelf life and binding retention of treated UHMWPE
(7) The study reveals that the nitrogen PIII-treated UHMWPE can be
stored for at least 4 weeks in ambient conditions without loss of protein
attachment density and activity and only a very small reduction active
protein binding performance is observed after six months.
Therefore, the
Chapter 7
194
shelf life under ambient conditions of the nitrogen PIII-treated is at least six
months.
The number of active protein binding on UHMWPE after 6
months is no less than that on NUNC Maxisorp.
Although both the argon treatment and nitrogen plasma treatment show
some loss of protein binding and activity retention after storage, they are
significantly improved compared to the untreated polyethylene. All the
surfaces including the untreated UHMWPE show a reduction in the
quantity of active protein over time when exposed to repeated washing.
The treated UHMWPE is greatly improved from the perspective of
retention of bound active proteins.
Optimization of PIII conditions
(8) Several treatment durations are attempted in the experiments.

Argon PIII for 800 seconds shows improved attachment of active proteins
compared to those treated for 20 s.
No further benefit is gained when the
argon treatment is extended to 1200 s, indicating a saturated level of

surface modification within 800 s. In the case of nitrogen treatments,
nitrogen-PIII for 800 s shows the best overall properties.
There is slightly
higher protein binding compared to the argon PIII sample treated for 800 s.
In general, the benefits of plasma surface modification increases with ion
fluence for short treatment times but saturate with larger fluences.
Chapter 7
195
The effectiveness of protein binding on the treated surfaces is studied at

two different pressures, but the optimal conditions cannot yet be
definitively determined at this stage.
Further optimization of the
treatment parameters requires further systematic studies employing a wider

range of conditions.
Physicochemical change on polymer surface
(9) XPS and ATR-FTIR characterization of the plasma or PIII treated

surfaces shows that oxygen containing groups are introduced onto the
treated surfaces.
According to the wettability test, the 800 s nitrogen PIII
surface possesses the lowest contact angle and best retention of bound
protein activity.
We attribute the improved protein activity on the treated
surfaces to their increased hydrophilic characteristic.
Benchmark PIII treated protein binding surface
(10) The outstanding protein binding performance of PIII treated

UHMWPE is consolidated by comparison with the popular commercial
product NUNC MicroArray as mentioned in (6).
In summary, the studies show good consistency.
PIII is one of the
convenient and effective methods to obtain a high quality protein binding

surface on UHMWPE.
Chapter 7
196
7.2 Future work
The experiments have shown promising results and further works are
required to further refine the treatment process. For NiTi as orthopedic
materials,
1.
Event though the preliminary test on mechanical properties and

biocompatibility
has
been
studied,
tests
involved
with
international standards must be performed before clinical

applications.
2.
Short-term in vitro cytocompatability test has been demonstrated.

Long-term in vitro cytocompatability must be studied since
possible cyto-toxic Ni ion may leak out during prolonged
soaking in SBF.
3.
Evaluation of short and long-term in vivo experiments of the

NiTi obtained from the refined treatment is necessary after the in
vitro test.
For UHMWPE as protein binding surfaces,
4.
The optimal parameters still need refinement.
In addition ways
to shorten the PIII process should be researched in order to

improve the process efficacy and degradation of polymers.
5.
In order to claim the PIII-treated UHMWPE as a practical

protein binding surfaces, the protein binding ability of other
types of proteins must be determined.
Chapter 7
6.

Selective binding of protein is useful in disease diagnostic
technology. Attempts to be made include grafting of different
functional groups by combination of surface treatment processes
and different kinds of polymers as the substrate.
197

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Chapter 2

commonly used methods for surface modification of biomedical materials

2.2 Plasma immersion ion implantation (PIII)

PIII was first called

plasma source ion implantation (PSII) and later also referred to as

The PIII technique was developed from as an

alternative to conventional line-of-sight ion beam implantation.

plasma generated in the treatment chamber is utilized directly to conduct

ion implantation instead of using an ion extracted beam.

The plasma can

be generated by a number of methods such as hot filament (HF) discharge3,

During PIII8, the specimen (substrate) is placed in the chamber and

Because the plasma sheath through which the ions implant

confirms to the shape of the substrate PIII is a non-line of sight technique

Ion implantation is a method which can be used to alloy the near

possibility of low-temperature processing has prompted more widespread

Limitations such as dimensional changes and delamination

associated with conventional coatings such as thin films can be avoided in

friction coefficients, wear resistance, and corrosion resistance 11,12 .

industry, high implantation efficiency and small equipment footprint are

The technique has been used to

fabricate shallow junctions in sub-micrometer integrated circuits 15 ,

modification of polymers by PIII has received more attention and is used to

2.3 Biomedical materials

This class of materials is quite broad,

including for example, implant biomaterials such as artificial heart valves

made of metals, metal alloys, polymers, ceramics, and composites.

Applications of biomedical materials are generally divided into two

The internally used materials are

usually referred to as biomaterials such as replacements of damaged organs,

are single-use items such as syringes, blood pouches, catheters, sterile

There is a lot of room for improvement in biomedical materials and

Table 2.1. Examples of different types of biomedical use materials

Low density and

Metals and Alloys:

2.3.1 Metal Nickel Titanium

Nickel titanium was first developed in early 1960s by the Naval

These two outstanding properies make NiTi ideal in many medical

After several dents had been

Normally these phase

changes occur in an alloy when heated to its melting point.

These phase changes,

known as between martensite and austenite, involve the rearrangement of

2.3.1.1 Shape memory and Super elasticity effects

Therefore, the geometry is "remembered".

changed shape at low temperature will return to the original or restored

This is called the shape

The relationship between that martensite and austenite

phases manifested by the shape memory effect in NiTi is illustrated in Fig.

Fig. 2.1. Schematic diagram for explanation of transformation from

The martensite phase is a crystalline structure that can be found when

The martensite phase emerges

when the temperature cools to a temperature, Ms. The phase transition is

On the contrary, when the temperature increases above the austenite

starting temperature (As), the martensite structure starts to convert to

It should be mentioned that the phase transition

temperatures depend on composition, heat treatment, cold work and ternary

Fig. 2.2. Transition of martensite and austenite by temperature change. Ms:

Superelasticity is a rubber like function behaviour that returns the

effect, it returns to the original shape as austenite during unloading.

2.3.1.2 Medical applications of NiTi

Early commercial applications of NiTi are in non-medical fields such as

In the last two decades, NiTi has been

used for medical purposes such as dentistry, orthopedics, and