physics muhammad ahsan shafiqueprr.hec.gov.pk/jspui/bitstream/123456789/10379/1... · stainless...
TRANSCRIPT
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Fabrication of Biomaterials for orthopedic applications
Doctor of Philosophy
in
PHYSICS
by
Muhammad Ahsan Shafique
2012-PhD-Phy-33
DEPARTMENT OF PHYSICS
GC UNIVERSITY LAHORE
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Fabrication of Biomaterials for orthopedic applications
Submitted to GC University Lahore
In partial fulfillment the requirements
For the award of
DOCTOR OF PHILISOPHY
In
PHYSICS
by
Muhammad Ahsan Shafique
2012-PhD-Phy-33
DEPARTMENT OF PHYSICS
GC UNIVERSITY LAHORE
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Dedication
Dedicated to one of my favorite verse "My Lord!
Enrich me with knowledge...” (Quran, 20:114)
and
To the tolerance and consistent support of all my
teachers, family members and supervisor.
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Acknowledgement
“Sufficient for us is Allah, and he is the best disposer of affairs” Quran 3:173
All the praise and glory to God, for whom nothing is hard.
“There is no beauty better than the intellect”
Prophet Muhammad (PBUH)
I would like to express my deep gratitude to the kind and humble Prof. Dr. Riaz
Ahmad, as a supervisor, he gave me the opportunity to think out of the box for my
projects and different experiments, I am particularly thankful for his guidance regarding
my academic writing skill.
I am thankful to all my lab fellows Dr. G Murtaza, MrAtharNaeem, MrShahzadSaadat,
Mr Muhammad Shahnawaz and Mr. Muhammad Khalil for their support and
coordination regarding the experimentation in accelerator lab, they were always ready to
help. I must acknowledge the tolerance and patience of my family because they had
difficulties due to my busy schedule during PhD studies.
Last but not least I would like to acknowledge the financial support of HEC Pakistan and
the office of research of innovation (ORIC) GC University Lahore, for the financial
support.
Muhammad Ahsan Shafique
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Abstract
The surface properties of functional materials are often more important than the bulk
properties because of its varied applications. In case of biomaterials, the surface
properties become even more important because the surfaces of biomaterials are either in
close contact with the body or the surfaces are exposed to an in-vivo biological system.
Therefore it is highly desirable to tailor the surface properties of currently used and
potential candidate biomaterials. Surface properties of metallic biomaterials are improved
using two techniques; ion implantation by 2MV pelletron accelerator and by plasma
focus system. The experiments based upon ion implantation are presented in this
thesis. Currently Stainless steel and nearly equiatomic nickel-titanium alloy are selected
to study different mechanical and biomedical properties after ion implantation. Stainless
steel samples are selected to investigate for corrosion properties, hardness,
hydroxyapatite growth and cell viability likewise nickel-titanium alloy samples are tested
for toxic ion releases in the simulated body fluid, corrosion potential and hardness.
Stainless steel 306 is implanted with various doses of nitrogen ions using a 2MV
pelletron accelerator for the improvement of its biomedical surface properties biomedical.
Raman spectroscopy reveals incubation of hydroxyapatite (HA) on all the samples and it
is found that the growth of incubated HA is greater in higher ion dose samples. SEM
profiles depict uniform growth and greater spread of HA with higher ion implantation.
Human oral fibroblast response is also found consistent with Raman spectroscopy and
SEM results; the cell viability is found the maximum in the samples treated with the
highest (more than 300%) dose. XRD profiles signified greater peak intensity of HA with
ion implantation; a contact angle study revealed the hydrophilic behavior of all the
samples but the treated samples were found to be lesser hydrophilic compared to the
control samples. Nitrogen implantation yields greater bioactivity, improved surface
affinity for HA incubation and improved hardness of the surface.
The effect of hydrogen ion implantation on surface wettability and biocompatibility of
stainless steel is investigated. Hydrogen ions are implanted in the near-surface of
stainless steel to facilitate hydrogen bonding at different doses with constant energy of
500 KeV, which consequently improve the surface wettability. Treated and untreated
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sample are characterized for surface wettability, incubation of hydroxyapatite and cell
viability. Contact angle (CA) study reveals that surface wettability increases with
increasing H-ion dose. Raman spectroscopy shows that precipitation of hydroxyapatite
over the surface increase with increasing dose of H-ions. Cell viability study using MTT
assay describes improved cell viability in treated samples as compared to the untreated
sample. It is found that low dose of H-ions is more effective for cell proliferation and the
cell count decreases with increasing ion dose. Our study demonstrates that H ion
implantation improves the surface wettability and biocompatibility of stainless steel.
Carbon ions are implanted on nickel titanium alloy (nitinol) and nickel ion release is
investigated along with affinity of calcium phosphate precipitation on nickel titanium
alloy. Four annealed samples are chosen for the present study; three samples with
oxidation layer and the fourth without oxidation layer. X-ray diffraction (XRD) spectra
reveal amorphization with ion implantation. Proton-induced X-ray emission (PIXE) result
shows an insignificant increase in Ni release in simulated body fluid (SBF) and calcium
phosphate precipitation up to 8 × 1013 ions/cm2. Then Nickel contents show a sharp
increase for greater ion doses. Corrosion potential decreases by increasing the dose but all
the samples passivate after the same interval of time and at the same level of VSCE in
Ringer lactate solution. The hardness of samples initially increases at a greater rate (up to
8 × 1013 ions/cm2 and then increases with the lesser rate. It is found that 8 ×
1013 ions/cm2 (≈1014) is a safer limit of implantation on nickel titanium alloy; this limit
gives us lesser ion release, better hardness and reasonable hydroxyapatite incubation
affinity.
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Table of Contents
Chapter 1 ............................................................................................................................. 2
Introduction ......................................................................................................................... 2
1.1 Synthetic Biomaterials .................................................................................................. 3
1.2 Metallic biomaterial ...................................................................................................... 4
1.2.1 Biodegradable metals ................................................................................................. 5
1.3 Biomedical properties of materials ............................................................................... 6
Mechanical Properties ......................................................................................................... 7
1.4 Biomaterial market........................................................................................................ 8
1.5 Three generations of biomaterials ................................................................................. 9
1.6 ASTM standard for materials...................................................................................... 10
1.7 Introduction to particle Accelerators .......................................................................... 10
1.71 Electrostatic Accelerators ......................................................................................... 11
1.8 Ion implantation using accelerator .............................................................................. 13
1.9 The motivation of this thesis ....................................................................................... 14
Thesis layout ..................................................................................................................... 15
References ......................................................................................................................... 17
2 Literature Review....................................................................................................................... 20
2.1 Surface modification by ion implantation using particle accelerator ..................................... 22
2.2 Surface modification by Plasma ................................................................................. 22
2.3 Stainless steel surface treatment for biomedical application. ..................................... 23
2.5 Processing of magnesium and magnesium alloys for biomedical applications. ......... 28
References ......................................................................................................................... 31
Chapter 3 ........................................................................................................................... 35
Experimental details and characterization techniques ...................................................... 35
3. Introduction to pelletron Accelerator ............................................................................ 35
3.1 Working principle of Pelletron accelerator ................................................................. 35
3.2. Working and different parts of Pelletron accelerator ................................................. 35
3.3Characterization techniques ......................................................................................... 43
3.3.1 X-ray diffractometer (XRD) .................................................................................... 44
3.3.2Scanning electron microscope (SEM) ...................................................................... 46
3.3.3Raman spectroscopy ................................................................................................. 49
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3.3.4Biocompatibility study .............................................................................................. 51
3.3.5 Bioactivity study ...................................................................................................... 52
3.4 Stopping range of ions in matter (SRIM) ................................................................... 53
Sample preparation ........................................................................................................... 56
References ......................................................................................................................... 57
Chapter 4 ....................................................................................................................................... 58
Results and Discussions .................................................................................................... 58
Ion implantation in stainless steel and nitinol ................................................................... 58
4.1 Effect of nitrogen ion implantation in stainless steel .................................................. 59
4.1.2 Earlier work ............................................................................................................. 60
4.1.3 Nitrogen Ion implantation ........................................................................................ 61
4.1.4 Immersion in simulated body fluid .......................................................................... 63
4.1.5 Results and Discussions ....................................................................................................... 63
4.1.5.1 Raman Spectroscopy profiles ........................................................................................... 63
4.1.5.2 XRD studies ...................................................................................................................... 65
4.1.5.2.1 Estimation of range of ions in material lattice (SRIM study) ............................ 66
4.1.5.3 SEM Results.......................................................................................................... 67
4.1.5.4 in- vitro Cell Viability Studies .............................................................................. 69
4.1.5.5 Contact Angle studies ........................................................................................... 71
4.1.5.6 Hardness Results ............................................................................................................... 72
4.2 Effect of Hydrogen ion implantation in stainless steel ............................................... 73
4.2.1 Introduction .............................................................................................................. 73
4.2.2 Ion implantation. ...................................................................................................... 74
4.2.3 Immersion in simulated body fluid .......................................................................... 74
4.2.4 Results and Discussions ........................................................................................... 75
4.2.4.1 Contact Angle Studies........................................................................................... 75
4.2.4.2 Raman Spectroscopy ............................................................................................. 76
4.2.4.3 Mass of incubated species ..................................................................................... 77
4.2.4.4 in- vitro Cell Viability Studies .............................................................................. 79
4.4 Effect of carbon ion implantation in nitinol lattice ................................................................. 80
4.4.2 Ion implantation and heat treatment of samples. ................................................................. 81
4.4.2.1 Immersion of samples in SBF and Sample Preparation for PIXE Analysis ..................... 82
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4.4.3 Results and Discussion ........................................................................................................ 82
4.4.3.1 XRD Analysis ................................................................................................................... 82
4.4.3.2 FTIR Analysis ................................................................................................................... 83
4.4. 3.3 PIXE Analysis .................................................................................................................. 83
4.4. 3.4 Corrosion potential and passivation time ............................................................. 85
4.4.3.5 Hardness Test ........................................................................................................ 87
Refrences........................................................................................................................... 88
Chapter 5 ........................................................................................................................... 93
5. Conclusion and future work .......................................................................................... 93
5.1 Conclusions ............................................................................................................................. 93
5.2 Future work ................................................................................................................. 94
List of Publications ........................................................................................................... 95
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Chapter 1
Introduction
Material science is a versatile field of research which actively caters the needs of almost
all fields of applied sciences such as spintronics, optoelectronics, mining, petroleum,
water purification, renewable energy automobile etc. The biomaterial research is an
important field of science. 100 years before Lane (1895) laid the foundation of this
discipline when he introduced metal plate inside the body for bone fracture fixation. The
biomaterials are such materials which come in close contact with body i.e. contact lenses
and dental implants etc. or the materials which are implanted inside the body to assist
and/or to repair a body organ i.e. heart valve, cardiovascular stents, pacemakers, hip and
knee prostheses etc. are formally known as biomaterials.
American National Institute of Health presented the most acceptable definition of
biomaterials which defines biomaterial as “any substance or combination of substances,
other than drugs, synthetic or natural in origin, which can be used for any period of time,
which augments or replaces partially or totally any tissue, organ or function of the body,
in order to maintain or improve the quality of life of the individual’’.
There are some essential requirements for biomaterials
Ideal biomaterial must possess appropriate stability
Ideal biomaterial must be compatible with tissue or organ of body
Ideal biomaterial should not trigger any unwanted body response
Ideal biomaterial must have set of suitable mechanical properties (strength,
tensile)
Ideal biomaterial must have high wear resistance
Biomaterials are broadly classified into two main types:
Natural biomaterials
Synthetic biomaterials
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Naturally, derived biomaterials include cellulose, chitin/chitosan, glucose
(polysaccharides based) and collagen, gelatin, silk (Protein based). The synthetic
biomaterials include metals, alloys, ceramics and polymers. There are some advantages
and disadvantages associated with each class of biomaterial.
1.1 Synthetic Biomaterials
Natural biomaterials are biologically recognized by the body and the introduction of these
materials inside the body does not trigger any unwanted biological response, while
synthetic biomaterial has some drawbacks: their structure, composition and mechanical
properties are not similar to the biological system. The synthetic biomaterials when
applied in-vivo are stranger to body therefore body immune system responses to these
materials which create complications. Metallic biomaterials corrode in-vivo and release
toxic ion e.g. Ni, Cr, etc.
Synthetics biomaterials do have benefits such as strength and durability. The synthetic
biomaterials are easy to manufacture and these materials can be given a desired shape.
Each type of synthetic biomaterial possesses its specific advantages and disadvantages.
Natural biomaterial
cellulose, chitin/chitosan,
glucose,collagen, gelatin, silk , etc.
biocompatible, does not trigger immune
responce, not hostile to body
synthetic biomaterials
metals, alloys, ceramics and
polymers
strong, resistant to fatigue, easy to
manufacture,can be stirlize easily
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1.2 Metallic biomaterial
Several medical purposes are being served with metallic materials e.g. titanium and
stainless steel has been used in cardiovascular stent fabrication and orthopedic implant
applications. Nickel-titanium shape memory alloy for orthodontics and stent applications.
Cobalt chromium and platinum chromium are also being utilized for stent applications
because of their strength. Some specific properties and applications of different are
described below [1].
Stainless steel
Stainless steel has been used during last few decades in orthopedics as bone implant, in
orthodontics as braces and as cardiovascular stent. Stainless steel is useful biomaterial
because of its supporting properties like biocompatibility, cost effectiveness, corrosion
resistance and suitable mechanical properties. But the mismatch of mechanical properties
with bone give rise stress shielding effect, release of carcinogenic ion i.e. Ni and Cr from
the surface of stainless steel into blood plasma and high density (greater mass to volume
ratio) hinders the use of stainless steel as perfect material [2, 3].
Nickel titanium Alloy
Nitinol is an alloy having a nearly equal atomic ratio of Ni and Ti elements
(approximately 50 each). This alloy contains wonder properties like shape memory
effect and super elasticity also the elastic modulus of nitinol is nearly equal to the elastic
modulus of human bone. The mentioned thee properties makes nitinol an exceptional
material to make novel surgical implants [4, 5]. Currently, nitinol is being used in
orthopedics for bone fixation, in orthodontics for teeth alignment and in cardiology for
stent application. Along with mentioned advantages, the higher Ni content makes this
material somehow less biocompatible.
Titanium
Titanium metal is very important biomedical material, some of its relevant supporting
properties are given below:
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Titanium metal is low density, biocompatible, good corrosion resistance, and suitable
mechanical properties and has a high strength to weight ratio. The fact that the
mechanical properties of titanium are similar to bone makes titanium a natural choice for
orthopedic implant application. Titanium and its alloys used in hard tissue replacement,
in dental implants, in artificial bone joints [6, 7].
Cobalt Chromium Alloy
Cobalt chromium is an important biomedical material. High corrosion resistance,
biocompatibility and formation of a passive film over the surface make this material a
promising candidate for biomedical application. Cobalt chromium alloy has mechanical
properties similar to that of stainless steel. This material is being widely used in dental
and orthopedic implant applications. Cobalt chromium alloy is also being employed for
cardiovascular stent application [8, 9].
1.2.1 Biodegradable metals
The biomedical implants are often needed inside the human body for a specific period
time, from six months to 12 months, after the healing the implant materials are no more
needed by the body, therefore a second surgery is needed to remove these materials from
the body. Biodegradable stents and orthopedic implants are being studied which could
leave the body after the healing of affected body part so that the second surgery may be
prevented. Iron and manganese are the biodegradable metallic materials, they are
biocompatible and possess excellent mechanical properties. Both the metals are tested in
the lab for degradation and other properties however magnesium showed rapid
degradation rate, rapid enough to degrade before sufficient healing while biodegradability
of iron is too slow. Researcher are making alloys of these metals with other elements to
tailor degradation rate of these metals [10-12].
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1.3 Biomedical properties of materials
The candidate material for biomedical applications should have an appropriate set of
mechanical and biomedical properties [13-15], some key properties of a candidate
biomaterial must possess are explained below.
Biocompatibility
Property of a biomaterial being compatible with the biological system. A biocompatible
material does not produce any toxicity and does not trigger an immunological response of
the biological system. According to ASTM (American Society for Testing and
Materials), definition biocompatibility is “Comparison of the tissue response produced
through the close association of the implanted candidate material to its implant site within
the host animal to that tissue response recognized and established as suitable with control
materials”
Corrosion resistance
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The resistance of a material to withstand unwanted chemical reaction with surrounding
elements, the reaction cause leakage of atoms from the material surface. Corrosion is the
loss of atom from the metal surface due to chemical reaction with an environment.
There are various types of corrosion
The electrochemical corrosion is the most common form of corrosion, this type of
corrosion occurs when electron transfers from surface atom to an electron acceptor
species through an electrolyte. Corrosion from localized depassivated small pits or
cavities is called, this is one of the most damaging form of corrosion.
When two metal are brought into electrical contact then one metal corrode faster than the
second one preferentially this type of corrosion is called galvanic corrosion.
Mechanical Properties
Hardness is an important mechanical property of a material, hardness can be defined as
the ability of a solid material to retain its shape against permanent shape changing or
deforming forces. There are various methods to measure the hardness e.g. Rockwell
Hardness Test, Brinell hardness Test and Vickers Hardness Test. Ductility is the ability
of a material to undergo permanent change i.e. elongation or bending without breaking.
The maximum amount of stress produced in a material just before plastic deformation or
this is the stress at which some plastic deformation is produced in a material. The
maximum pulling or stretching force which a material can bear before failure, in other
words, the ability of a material to resist failure under tensile stress. The ratio of applied
force to a material to resulting strain within elastic limit is called modulus of elasticity
Surface wettability
The surface wettability an important biomedical property, according to some reports cell
viability of a material is closely interconnected to surface wettability
(hydrophilicity). The tendency of a material surface to adhere a liquid is called surface
wettability. The drop of a liquid spread over the surface of wetting material while liquid
drop tends to have a spherical shape on the surface of a non-wetting material. Wettability
of a surface is usually measured by the contact angle of a liquid drop with the surface.
8
Lesser the contact angle greater is the wettability or in case of water drop word,
hydrophobicity is used. The contact angle is usually measured by different methods.
The static sessile drop method
The dynamic sessile drop method
The pendant drop method
The Wilhelmy balance method
The degree of Irregularity in the topography of a material is called surface roughness, or
surface roughness may be defined as “deviation in the direction of the normal vector of a
real surface from its normal form”. The surface roughness is also an important parameter
that sometimes determines the surface wettability and cell viability[16].
1.4 Biomaterial market
The global market for the biomaterials is growing very fast, due to the fact that almost all
the sections of biomedical sciences are looking for artificial material and/or devices for
the treatment of different disorders. It is estimated that the global market of biomaterial
will reach the value of $139 billion by 2022. It is predicted by some reports that the
metallic material contributes a momentous volume in future biomaterial market. Today a
large number of different biomaterials are being used, the number of different
biomaterials used per year is listed below:
The people of America are the big user of biomaterials, more than 50% of total produced
biomaterials are being used in the USA, then Germany, Japan and other parts of the
world. From given data it is clear that rich countries are proportionally big users of
biomaterial while economically developing countries share a relatively smaller portion of
biomaterial, therefore low cost and medically safer biomaterial are desired so low-income
group people can have equal advantages of biomaterials.
Table 1. Biomaterial being use per year.
Sr.No Type of Biomaterial Used per year
1. Catheters 300,000,000
2. Contact lenses 75,000,000
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3. Cardiovascular stents 2,000,000
4. Renal dialyzer 25,000,000
5. Hip and knee prosthesis 1.000,000
6. Dental implants 500,000
7. Intra ocular lenses 7,000,000
8. Heart valve 200,000
1.5 Three generations of biomaterials
First generation biomaterials
Biomaterial researchers were rather much contained initially as they were after simpler
requirements for biomedical applications. The goal of first generation biomaterial
research was to develop biomaterial having suitable physical and chemical properties
with the minimal toxic response of host body, it was desired as well that biomaterials
should not interact with a biological system. Therefore biologically inert materials were
chosen for the fabrication of first generation biomaterials in order to prevent the immune
response to foreign implanted material and leaked toxic ions from implant surface. By the
end of 1980, the researchers were able to develop more than 50 materials for implant
prosthesis from 40 different materials
Titanium and its alloys, stainless steel, cobalt-chromium are the examples of first
generation metallic biomaterials while the polymers and ceramic materials were Alumina
Al2O3, Zirconia ZrO2, silicone and acrylic resins etc.
Second generation biomaterials
First generation biomaterials were prohibited to interact with the biological system while
in second generation biomaterials the term bioactivity was introduced, bioactivity refers
to the ability of a biomaterial to interact with the biological system to improve body
response without triggering the immune system. Metallic materials are not biomaterial
but researchers opted two approaches to fabricate bioactive biomaterials, both the
methodologies depend upon surface modification of materials, the first approach relies
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upon coating of substrate material with bioactive ceramic material e.g. hydroxyapatite,
bio-glass while the second approach is to chemically modify the material surface to make
the material bioactive.
Examples of second generation biomaterials include ceramic (calcium phosphate,
bioglass, glass ceramics), polymers (polyglycolide, polylactide).
Third generation biomaterials
Currently, third generation biomedical materials are being considered: these materials are
being designed to stimulate a specific cellular response, the concept of bioactivity and
resorbability is converged. Researchers are trying to have both the properties in a
material, bioactive materials are being made resorbable and resorbable materials are
being made bioactive.
Some porous metallic and nonmetallic structures, artificial skin, some resorbable bone
repair cements etc. are the examples of third generation biomaterials [17, 18].
1.6 ASTM standard for materials
ASTM stands for American society for testing and materials. ASTM International is an
organization that develops and publish technical standards for a variety of materials,
systems and products. Moreover, ASTM provides protocols for different procedures, the
purpose is to provide a compendium for a particular method. ASTM does not enforce the
compliance of standards however the standards become mandatory when referenced by
some agency or government. ASTM standards have been adopted in many states of USA,
other governments also refer the ASTM standards. ASTM international was founded
in1898. The headquarter is situated in Pennsylvania, Philadelphia, USA. More than
12000 standards of ASTM operate worldwide.
1.7 Introduction to particle Accelerators
A particle accelerator is a device which accelerates charged particle to high energy, the
charged particles may be elementary particle electron, proton or ions of different
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elements e.g. Cu, Fe, Au etc. the energy that a particle accelerator imparts to charged
particles ranges from few KeV to 100s of GeV or TeV.
Accelerators accelerates the charged particles between two electrodes which are at
different potential
Particle accelerators are divided into two basic types:
Electrostatic Accelerator
Electromagnetic Accelerator/ electrodynamic Accelerators (oscillating field
accelerators)
1.71 Electrostatic Accelerators
Electrostatic accelerators are simpler in design as compare to oscillating field
accelerators, the first electrostatic accelerator was realized by Ernest Walton and John
Cockcroft. Electrostatic accelerators use the principle of electrostatic attraction and
repulsion to accelerate charged particles. The charged particles are accelerated in an
evacuated tube or high gas pressure tank between two oppositely charged plates. These
are the initial accelerator manufactured however these accelerators are still very popular
for low energy acceleration requirements because of their simple working principle and
design.
The main disadvantages of an electrostatic accelerator are the energy constraints: the
limited number of eVs can be provided to the charged particle with the help of these
accelerators because it is difficult to generate and maintain high electrostatic potential.
There are two types of electrostatic accelerator
I. Cockcroft Walton Accelerator
II. Van de Graaff Accelerator
Cockcroft Walton accelerator is a type of electrostatic linear accelerator, the first nuclear
disintegration was done by Cockcroft Walton accelerator when lithium atom was
bombarded with fast-moving proton:
P + Li 2 He
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Cockcroft Walton Accelerator uses an electrical circuit called voltage doubler circuit
which produces high DC voltage from low AC input voltage.
Van de Graaff accelerator is a type of electrostatic linear particle accelerator. It uses Van
de Graaff generator to develop high accelerating potential difference. The charges are
transported mechanically with the help of a moving belt from one point to other to
develop a high voltage, often two generators are used an in pair to double the applied
voltage, this type of accelerators are known as tandem accelerators.
Fig 1: schematic diagram of Van de graaf generator (source: http://helios.augustana.edu/~dr/203/van-de-
graaff.html)
Electrodynamic accelerators use periodically changing electric field, the changing electric
field imparts extremely high energy to charged particles. Electrodynamic acceleration can
arise from either of two mechanisms: non-resonant magnetic induction or resonant
circuits or cavities excited by oscillating RF fields. Some popular particle accelerators are
mentioned below.
Linac (Linear Accelerator)
The cyclotron
Betatron
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1.8 Ion implantation using accelerator
Ion implantation is a process by which energetic ions of different elements are created,
accelerated and then bombarded on the surface of a target material. The bombarded ions
produce different phenomenon in target lattice [19, 20]. Depending upon the energy of
and nature ions (atomic radius, charge state etc.) of bombarded ions, moreover, the
impact depends upon the nature of target material [21, 22]. The bombarded ions damage
the surface lattice by producing surface roughness, craters and other irregularities [23].
Incident ions cover some distance in target lattice, lose their energy by multiple
phenomena and get implanted. The incident ions sometimes they cross the target depth,
which is called ion irradiation. The incident energetic charged particle undergoes multiple
interactions in target lattice, it transfers energy to target atom and knock it down, the
knocked out atom is called primary knock down atom (PKA). The primary knock down
atom has ample of energy it further knocks down the lattice atoms while passing through
the lattice which are called secondary knock down atoms. In this way the energy of
incident ion gets distributed within a certain region of target lattice called displacement
cascade [24, 25]. The displaced lattice atoms travel through crystal system, collide with
the atoms in their way and displace these atoms from their original sites. The displaced
atoms contain energy they collide further and this is the way a collision cascade is
created. Finally, an equal number of displaced atoms and vacancies are created in the
crystal system. Thus defects are created as a result of ion implantation or irradiation
results if crystal defects, therefore, annealing is needed to restore the crystal
symmetry[26].
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Fig 2: Schematic of ion implantation process (source: Ion implantation Physical encyclopedia by A.M.
Prokhorov. 1990)
Ion implantation has various technological applications e.g. CMOS fabrication,
modification of compounds (TiO2), doping in a semiconducting material, surface
modification of materials and many more. The surface modification by ion implantation
is a useful phenomenon in material science. Surface amorphization, point annealing,
surface hardening surface functionalization by incorporation of implanted ion in the near-
surface region of a materials etc. are the examples of surface modification. The implanted
ion changes the chemical, electrical and mechanical properties of target material[27].
1.9 The motivation of this thesis
The aim of this thesis is to improve the biomedical properties of various currently used
biomaterials by accelerated ion implantation in near surface region. There are some
specific important issues with currently used metallic biomaterials which compromise the
safety of that material to be used in-vivo: Nickel titanium alloy (nitinol) is an important
biomedical material, it contains some wonderful mechanical and biomedical properties
18, therefore it is being widely in dentistry as orthodontic arch wires, in orthopedic
surgery as bone implant and in cardiovascular treatments as stent, but nitinol contains
approximately 50% nickel content which is a toxic material. The nickel titanium alloy
15
when implanted inside the body it experiences some chemical interactions with human
blood plasma, the human blood plasma is a corrosive fluid, therefore the toxic nickel ions
are released into blood plasma which consequently causes allergy 19, activates immune
response and some other disorders similar is the case with stainless steel, the stainless
steel also contains a considerable fraction nickel and other toxic ions these issues hinder
the safe use of these materials for in-vivo biomedical applications 20. Therefore the
biomaterial should be stable, the safer use of nitinol and stainless steel requires high
corrosion resistance and some other properties.
Various studies revealed that ion implantation in material lattice enhances the corrosion
resistance [28-30] produce amorphization and some other properties. It was hypothesized
that the chemical interaction of specific implanted ion in the material surface can improve
the bonding between material surface and bone (hydroxyapatite) which will consequently
lead to rapid healing of bone tissue. In first experiment nickel titanium alloy was
bombarded with C+ ions, the ion implanted samples were studied for nickel ion release,
surface amorphization, corrosion potential of prepared samples and incubation of
hydroxyapatite over the surface.
In the second experiment, the stainless steel was implanted with different doses of
nitrogen ions, the prepared samples were characterized for incubation of hydroxyapatite,
surface wettability, hardness and cytocompatibility.
In third experiment, the stainless steel surface was bombarded with different doses of
protons H+, the prepared samples were studied for surface wettability, incubation of
hydroxyapatite, and cytocompatibility.
Thesis layout
The thesis is divided into four chapters. Chapter 1 contains an introduction to
biomaterials, some major types and generations of biomaterials are also introduced.
Moreover, the essential properties of a candidate material are explained. Next sections of
chapter 1 describe the magnitude of the worldwide market of biomaterial and description
of the technique used for material modification.
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Chapter2 (literature review) describes the current focus of researchers working in the
field biomaterials and related field. The section 2.1 and 2.2 describe different
experiments, their objectives, the techniques used and results of surface modification by
ion implantation and plasma treatment. Section 2.3 and 2.4 reveals the attempts made by
researchers to tailor the properties of stainless steel and nickel titanium alloy using
different techniques. Finally, the last section contains the literature review of
biodegradable magnesium and magnesium based alloys.
The experimental details and characterization techniques are explained in chapter 3. This
chapter describes pelletron accelerator and explains the functions of different parts of a
pelletron accelerator. The second part of chapter 2 describes the principles and working
of different techniques used for characterization.
The first part of chapter 4 explains the associated problems of potential biomaterials and
possible harmful effects of these materials .e. nickel titanium alloy and stainless steel,
when applied in physiological conditions. Section 4.1 reveals the detail of the first
experiment. Effect of nitrogen ion implantation in stainless steel, the experimental
parameters, results of characterization techniques and findings are mentions similarly the
4.2 and 4.3 contain the details of next two experiments.
17
References
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interface between human tissue and implants of titanium and stainless steel. Journal of
Colloid and Interface Science 1986;110:9-20.
[3] Eschbachz JADaL. Stainless steel in bone surgery Injury, Int I Care Injured
2000;31:S-D2-4
[4] Ryhanen J, Niemi E, Serlo W, Niemela E, Sandvik P, Pernu H, et al. Biocompatibility
of nickel-titanium shape memory metal and its corrosion behavior in human cell cultures.
Journal of biomedical materials research 1997;35:451-7.
[5] D.J. Wever AGV, M.M. Sanders, J.M. Schakenraad, Horn aJRv. Cytotoxic, allergic
and genotoxic activity of a nickel-titanium alloy Biomoteri0ls
1997;18: 1115-20.
[6] Wen CE, Yamada Y, Shimojima K, Chino Y, Asahina T, Mabuchi M. Processing and
mechanical properties of autogenous titanium implant materials. Journal of Materials
Science: Materials in Medicine 2002;13:397-401.
[7] Long M, Rack HJ. Titanium alloys in total joint replacement—a materials science
perspective. Biomaterials 1998;19:1621-39.
[8] Jacobs JJ, Skipor AK, Doorn PF, Campbell P, Schmalzried TP, Black J, et al. Cobalt
and chromium concentrations in patients with metal on metal total hip replacements.
Clinical orthopaedics and related research 1996:S256-63.
[9] Lucchetti MC, Fratto G, Valeriani F, De Vittori E, Giampaoli S, Papetti P, et al.
Cobalt-chromium alloys in dentistry: An evaluation of metal ion release. The Journal of
Prosthetic Dentistry 2015;114:602-8.
[10] Moravej M, Mantovani D. Biodegradable Metals for Cardiovascular Stent
Application: Interests and New Opportunities. International Journal of Molecular
Sciences 2011;12:4250-70.
[11] Yun Y, Dong Z, Lee N, Liu Y, Xue D, Guo X, et al. Revolutionizing biodegradable
metals. Materials Today 2009;12:22-32.
18
[12] Hermawan H, Dubé D, Mantovani D. Developments in metallic biodegradable
stents. Acta biomaterialia 2010;6:1693-7.
[13] Katti KS. Biomaterials in total joint replacement. Colloids and Surfaces B:
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[15] Kokubo T, Kim HM, Kawashita M. Novel bioactive materials with different
mechanical properties. Biomaterials 2003;24:2161-75.
[16] Amjed J, Manish K, Seokyoung Y, Jung Heon L, Satomi T, Masaru H, et al. Role of
surface-electrical properties on the cell-viability of carbon thin films grown in
nanodomain morphology. Journal of Physics D: Applied Physics 2016;49:264001.
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[18] Mousa HM, Tiwari AP, Kim J, Adhikari SP, Park CH, Kim CS. A novel in situ
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magnesium alloy surface towards third generation biomaterials. Materials Letters
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[19] Wei R, Wilbur PJ, Sampath WS, Williamson DL, Wang L. Effects of Ion
Implantation Conditions on the Tribology of Ferrous Surfaces. Journal of Tribology
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[20] Wahl KJ, Dunn DN, Singer IL. Effects of ion implantation on microstructure,
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[21] Pope LE, Picraux ST, Follstaedt DM, Knapp JA, Yost FG. Effect of ion implantation
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Energy Systems 1985;7:27-37.
[22] Jin-Liang Z, Zhao-Min L, Zhen-Wen Y, Ye-Ping G, Zue-Teh M, Rui-Zhong B.
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Instruments and Methods in Physics Research Section B: Beam Interactions with
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19
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[25] Porte L, Villeneuve CHd, Phaner M. Scanning tunneling microscopy observation of
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1988;38:8444-50
20
2 Literature Review
Researchers from various disciplines are struggling to modify the surface of materials
using different methods, the most frequently used method for surface modification is thin
film fabrication, thin film is applied over the surface of a material to serve some
purposes: primarily to protect underlying material from deteriorating hostile environment
or some times to separate one type of material from another type, this type of coating is
called protective coating, other types of coating includes optical coatings: the optical
coatings are used to tailor the interference, reflection of light and some other purposes.
Thin films are also deposited for the photovoltaic cell, for batteries, to induce some
magnetic and electrical properties in a material etc.
Along with film fabrication, ion implantation is also a very important technique for
surface modification of materials. The basic difference between ion implantation and thin
is the number density and energy of implanted ions, generally the number density of
implanted material is much more in case of thin film: therefore thin film covers the whole
surface of substrate while in case of ion implantation the number density is lesser and the
ions are more energetic, in case of ion implantation, the implanted ions penetrate in
substrate lattice, they don’t cover the whole surface, the ions are sprinkled on the surface
of a material.
2.1 Surface modification by ion implantation using particle accelerator
Surface modification of materials using particle accelerator is relatively less famous
technique as compare to some other techniques, although this technique is being used
from many decades for surface engineering[1-3], researchers have been using focused ion
beam for various purposes: Kant et al modified TiN thin film using nitrogen ions, they
found reduced oxygen contamination in N ion implanted coatings as compare to un-
implanted coatings, they also found significantly reduced hardness and ductile behavior
of TiN ion implanted coatings[3, 4]. Ensinger et al demonstrated that ion beam assisted
deposition is appropriate for adherent coating, they also found that these coatings are
suitable for long-term corrosion protection[5]. Natishan et al prepared Mo-Al surface
alloy using accelerated ion implantation and vapor deposition on aluminum substrate,
they studied pitting corrosion resistance in prepared samples, the studies reveal
approximately six time higher pitting potential of ion beam mixed Mo-Al surface alloy
than pure aluminum and ion implanted Mo-Al alloy[6].
Iwaki et al modified the surface of various organic materials by ion implantation: they
used substrates poly-tetra-fluoroethylene (PTFE), Silicone rubber, poly styrene (PS), poly
21
imide (PI), poly acetylene and different kind of protein for ion bombardment or ion
implantation, the substrates were bombarded with inert ions, chemically active ions and
metallic ions. They investigated surface wettability, cell adhesion, and electrical
conductivity of prepared samples, finally, they concluded that surface properties of
organic materials can be tailored by using certain ion implantation or bombardment[7].
Colli et. al. [8] implanted high dose (1015ions/cm2) of Phosphorous and Boron ions in
silicone nano wires (SiNW), they found only limited amount of amorphization and they
recovered fully crystalline structure after annealing as prepared samples at 800 0C, they
concluded the results by Raman spectroscopy and electrical transport properties
measurements.
Chu et.al. [9] studied cobalt implanted ZnO nano wires and heat treated cobalt doped
ZnO nanowires, they found a high degree of structural disorder in implanted samples as
compared to annealed samples using hard X-ray nano probe. They estimated the average
content of cobalt in nanowires using XRF analysis, they concluded structural distortion in
nano wires by ion implantation may affect the performance optoelectronics and
spintronics devices.
Since ion implantation is a versatile technique for a material scientist, it has been used for
diverse purposes, Ghicov et al fabricated TiO2 nano tubes by electrochemical self-
organized oxidation of titanium, and they implanted two doses of nitrogen ions in
prepared samples i.e. 1×1015 ions/cm2 and 1×1016 ions/cm2 . The ions were implanted in
both crystalline and amorphous phases, successful doping was confirmed using XRD,
SEM and photo-electrochemical measurements, they concluded that the crystalline tubes
were amorphized by ion implantation while the amorphous tube lost their morphological
integrity moreover they found decreased photo response in UV range in amorphized
tubes, and N-doping yield strong sub band-gap response[10].
22
2.2 Surface modification by Plasma
Plasma ion implantation is being used frequently to improve surface properties of
metallic, polymers and other materials, plasma film deposition is an effective and
economical method for surface modification. This technique allows depositing a thin film
of various compositions and thickness over different substrates. The deposited thin films
are used for various applications:
Han et al modified the surface of polymers by oxygen implantation, they evaluated the
wetting properties of untreated samples and oxygen implanted surface they found
contrasting results: the surface modified samples showed high degree of hydrophilicity as
compare to untreated samples, they also modified the surface by CF4 implantation: they
found this group of sample highly hydrophobic the contact angles exceeds 100 degrees,
they concluded that plasma ion implantation is highly effective technique for surface
modification [11].
Larisch et al nitrided four grades of stainless steel at different temperatures between
2500C to 5000C, they prepared nitrogen enriched surface layers. The nitrided samples
were found harder as compared to pristine samples, they also concluded that there are no
precipitations of CrN if the samples are treated at low temperature i.e. 4000 C[12].
Suh et al also performed a similar experiment on stainless steel, they carburized the
surface of AISI- 316 stainless steel using CH4/H2 to increase surface hardness. They
analyzed the carburized samples by scanning electron microscopy (SEM), micro hardness
tester, optical microscopy and Auger electronscopy, the hardness in prepared samples
found linearly increasing with carbon content. They divide the carburized surface into
three regions: the near surface named as white zone, they observed elongated carbon
structures and found carbon concentration about 4% by weight, the dark zone had carbon
concentration 1.5 to 4 % by weight and they found fine carbides in this zone, finally the
third one is the core in this zone the carbides were observed only at the grain
boundaries[13].
23
Jamesh et al modified the surface of magnesium alloy ZK-60 by plasma ion implantation
for biomedical application, the purpose of experiment was to tailor the corrosion behavior
of samples, they implanted Zr, O and both Zr and O ions in the surface of magnesium
alloy, the prepared samples were studied by electrochemical impedance spectroscopy
(EIS) after 30 hrs. of immersion. They observed 37 times decrease in corrosion current
(Icorr) and 62 times lesser Rp. they finally concluded that plasma ion implantation is an
effective method for modification of initial corrosion behavior[14].
Hosseni et al studied the corrosion behavior of plasma spray aluminum coated nickel-
titanium alloy (nitinol) modified. They investigated the prepared samples by X-ray
diffraction studies (XRD) and scanning electron microscopy (SEM) to investigate
morphology and microstructure of samples and electrochemical impedance spectroscopy
was used to study corrosion behavior of plasma coated and untreated samples. They
found slightly decreased corrosion resistance but the nickel release in simulated
biological fluid was significantly hindered, they concluded that the stability of coated
samples in simulated biological fluid was enhanced as compare to untreated samples [15]
2.3 Stainless steel surface treatment for biomedical application.
Stainless steel is an important material for various biomedical applications, the
conventional stainless steel alloy has been modified largely for biomedical application
during last few decades: initially, stainless steel contains vanadium but it was replaced
with nickel and chromium then molybdenum was added to reduce carbon content and to
achieve high corrosion resistance[16]. Stainless steel is a low cost, biocompatible and
corrosion resistant material. Typically stents, artificial valves, bone plates, artificial
joints, pins, screws orthodontic wires and any other applications are being catered with
stainless steel, therefore, researchers are working actively to tailor some properties of
stainless steel.
Qin et al fabricated silver nano particles of different sizes and distributions on the surface
of stainless steel by plasma ion immersion implantation (PIII), they treated the samples
for 0.5h and 1.5h respectively. The prepared samples were then evaluated by in-vivo and
24
in-vitro tests, they found enhanced antibacterial activity of stainless steel moreover they
observed improved osteogenic differentiation of human bone marrow stromal cells[17].
Braceras et al selected 316LVM austenitic stainless steel for their study, stainless steel
316 MVM is a potential choice for the temporal musculoskeletal implant. They aimed the
study to improve surface resistance of stainless steel to bacterial colonization, they
implanted 50KeV silicon ions (flounce 2.5-5 ×1016, at angle 45 to 900), and they found
decreased adhesion of bacterial species to the surface of medical grade stainless steel
without compromising the biocompatibility, they also concluded that bacterial adhesion
is dependent on implantation conditions[18].
Corrosion is an important biomedical property, appropriate corrosion resistance and
stability of biomedical implant are crucial for in-vivo application of the material. There
are several studies to improve the corrosion behavior.
Galvan et al also selected surgical stainless steel 316LVM and they implanted same ions
as Braceras et al did, but the aim of the study was different. They aimed the study to
investigate the effect if silicon ion implantation on short-term corrosion resistance and
ion release. They varied three parameters: ion doses, accelerating voltage and angle of
incidence. They carried out corrosion test using electrochemical impedance spectroscopy
(EIS), They demonstrated that at certain value to dose and voltage the corrosion
resistance is enhanced while they found worst contrasting results of corrosion resistance
in higher ion dose samples: they observed corrosion protection at ion dose equal to
1×1016 ions/ cm2 and accelerating voltage 50KV while at 1×1017 ions/ cm2 and
accelerating voltage 80KV they found worst corrosion resistance, they also concluded
that enhanced corrosion resistance yields reduced ion leakage from the surface of surgical
stainless steel [19].
Muthukumaran et al modified the surface of surgical AISI 316L stainless steel by ion
implantation: in their study they implanted 1×10 17 nitrogen and helium ions/ cm2 at
100KeV energy, they studied surface morphology and crystallographic orientations by
SEM and XRD respectively moreover they evaluated the corrosion behavior of fabricated
samples in 0.9% of NaCl solution using electrochemical test, the Tafel scan revealed that
25
the ion implanted samples are more corrosion resistant as compare to virgin samples, they
also observed improved micro hardness by Vickers method by varying load [20].
Zou et al investigated the mechanism of corrosion and wear improvement of stainless
steel 316 L by low energy and high current pulsed electron beam. They used
potentiodynamic polarization analysis and electrochemical impedance spectroscopy to
model the corrosion behavior of prepared samples. They concluded that corrosion
resistance was improved by 3 order of magnitude after sufficient pulses and the wear
resistance was also improved by sub surface work hardening (over 100 micrometer)[21].
Kheirkhah et al coated Nanostructured forsterite (Mg2SiO4) over the surface of AISI
316L stainless steel by the sol gel dip coating method, they utilized X-ray diffraction
(XRD, scanning electron microscope (SEM) and energy dispersive spectroscopy for
structural morphological and elemental composition analysis. Electrochemical corrosion
was studied in simulated body fluid (SBF) finally the in-vitro bioactivity was evaluated
by soaking the prepared samples in SBF. They observed lesser corrosion current density,
which shows improved corrosion resistance, the deposition of Calcium phosphate
products confirmed bioactivity of prepared samples, they concluded that deposition of
nano structured forsterite may be beneficial for dental and orthopedic implant
applications [22].
Shih et al passivated the surface of stainless steel 316L using different techniques for
improved corrosion resistance for, both in-vivo and in-vitro application. They used
different characterization techniques i.e. tunneling electron microscope (TEM), auger
electron spectroscopy (AES), X- ray photoelectron spectroscopy (XPS) and anodic
polarization test, their results showed that only amorphous oxidation improves the results,
all other techniques do not improve corrosion resistance, the author attributed the
improved results to the removal of plastically deformed oxide layer and development of
new layer, the author concluded that the properties of oxide layer determine the in-vitro
stability of prepared sample surfaces rather than the thickness of oxide films [23].
2.4 Surface treatment of nickel titanium alloy (nitinol)
26
Nitinol is nearly equi-atomic, super elastic shape memory alloy, this super elastic alloy of
nickel and titanium is being extensively studied for various biomedical applications,
along with some favorable mechanical properties there are also some potential drawbacks
e.g. toxic nickel ion release from the surface of nitinol is one of the major problems,
researchers are employing different techniques to tailor corrosion and some other related
biomedical properties of nitinol [24].
Gill et al applied magneto-electropolishing technique to alter surface characteristics of
nitinol, i.e. biocompatibility, surface wettability, roughness and corrosion resistance of
nickel titanium alloy, they motivated by the fact that magneto- electropolishing alter the
composition and morphology of surface films, which improves corrosion resistance on
nitinol. They observed improved mechanical properties by addition of alloying element.
Improved corrosion resistance and cell viability was observed by potentiodynamic
polarization test and endothelial cell response in magneto-electropolished samples [25].
Tape et al compared different surface modification approaches. They evaluated heparine,
aluminum and polyurethane coating, they found improved results in polyurethane coated
samples: coagulation and inflammation was improved compared to other designs [26].
Ghaley et al bombarded the surface of nitinol with three different doses of nitrogen ion,
they aimed the study to improve biological properties of alloy while preserving
mechanical properties. The prepared samples were investigated by the small angle x- ray
diffraction study to evaluate newly generated phases on the surface. Electrochemical
impedance spectroscopy and potentiodynamic polarization test in simulated body fluid
were applied to investigate the corrosion behavior of prepared samples, atomic absorption
spectroscopy was applied to estimate nickel ion release in simulated body fluid within the
period of two months. The ion implanted surfaces were also evaluated for cellular
response, the results showed improved cytocompatibility and corrosion protection in
modified samples, they found highest corrosion resistance and lowest nickel ion release
in simulated body fluid by the sample bombarded with 1.4×1018 ions/ cm2[27].
Sun et al utilized cathodic electrophoretic deposition (EPD) to deposit composite films on
the surface of NiTi shape memory alloy, they deposited chitosan-heparin films from the
solution of non-stoichiometric Chitosan–heparin, and they found that addition of anionic
27
heparin to the of chitosan yields a significant increase in film thickness. Their results
showed that ability of chitosan- heparin films to bind antithrombin was enhanced [28].
Poon et al proposed enhanced corrosion properties of Nickel titanium alloy by carbon
plasma immersion ion implantation (PIII), but the release of toxic nickel ions from the
surface of NiTi alloy into human body was a great concern. They deposited amorphous
hydrogenated carbon thin film and implanted carbon ion into NiTi target using plasma
immersion ion implantation and deposition (PIII&D) technique. They analyzed that
deposited carbon thin film has graded carbide interface which strengthens the film
adhesion. They found enhanced corrosion protection in both PIII treated and PIII&D
treated sets of samples. They observe implanted and carbide layer is mechanically
stronger than NiTi substrate, they concluded that PIII&D is an effective technique for
improving corrosion resistance, mechanical properties and cell viability of orthopedic
nickel titanium shape memory alloy[29].
Tan et al used plasma source ion implantation (PSII) technique to improve surface
properties of NiTi shape memory alloy, they implanted different doses of oxygen ions
5×1016, 1×1017 and 3×1017 ions/cm−2. They investigated the pitting and corrosion
behavior of prepared and untreated samples by cyclic potentiodynamic polarization test.
Their results revealed that corrosion resistance depends upon heat treatment and ion
implantation: they found maximum corrosion resistance in the sample implanted with
oxygen ion dose 1×1017ions/cm2 with Af = 21oC. They concluded that oxygen ion
implanted nickel-titanium samples possess better corrosion resistance in Hanks
solution[30].
There are some applications if nitinol other than as implant material: Lee et al assumed
that boron implanted Ni-Ti alloy has potential to develop better nitinol root canal
instrument for outstanding cutting properties, they modified the surface of nitinol with
boron ion beam of energy 110KeV, the ion dose was chosen 4.8×1017ions/cm2. They
found considerably enhanced surface hardness in boron implanted samples as compare to
untreated sample, they concluded that ion implanted nitinol is harder than stainless
steel[31].
28
Corrosion resistance and nickel ion leakage from the surface of nitinol is a matter of great
concern for biomedical applications, therefore a big fraction of researchers working on
nitinol tried to resolve corrosion and ion leakage issues by various methods: Saugo et al
tried anodisation of nickel titanium alloy to hinder ion leakage and to improve in-vivo
corrosion. They found that anodisation process considerably reduce the nickel ion
leakage and this process also enhances the titanium content in the outermost surface as
TiO2, which consequently improve anticorrosion performance in ringer lactate
solution[32].
Flamini et al also attempted to resolve the same problem of stability of nitinol in chloride
containing environment as human blood plasma contain high fraction of chlorine and
other corrosive species: Flemini et al modified the surface of nitinol by self-assembled
alkylsilane compounds (propyltrichlorosilane (C3H7SiCl3) and then the coating of doped
polypyrrole. They observed good adherence between polypyrrole and underlying
alkylsilane film, they concluded that organic coating is promising for anti-corrosive
protective treatment of Ni-Ti shape memory alloy[33].
2.5 Processing of magnesium and magnesium alloys for biomedical
applications.
Magnesium is a wonderful metal for biomedical application owing to excellent
biocompatibility and remarkable mechanical properties, moreover, biodegradability
provides an opportunity to avoid complications of second surgery for the removal of the
implant from the body. The young modulus of permanent implants is approximately 10
times greater than the bone i.e. 100-200 G Pa for permanent implant while 10-30G Pa for
bone, the mismatch of mechanical properties between implant material and adjacent bone
causes serious clinical issues[34]. Rapid biodegradability is one of the major issue
associated with magnesium, different allying and surface modification techniques are
being applied to tailor in-vivo degradation rate of magnesium.
29
Waksman et al planed a study to investigate safety and efficacy of biodegradable
magnesium stents, they randomly deployed magnesium alloy or stainless steel stents in
coronary arteries of domestic and mini pigs, and they sacrificed domestic pigs after two
days (on the third day) or 28 days, mini pigs were sacrificed after three months,
interesting results were obtained: they observed signs of degradation after 28 days, they
found no evidence of stent particle embolization, inflammation or thrombosis, at 28 days
and 3 months they observed significantly less neointimal area in comparison to stainless
steel stent area, they concluded that magnesium alloy stents are safe to use in-vivo [35].
Wang et al investigated degradation of magnesium alloy AZ 31 in simulated body fluid
(SBF) and Hanks solution, they found significantly reduced degradation rate of
magnesium alloy after mechanical processing, they specified that hot rolling yields
improved corrosion protection, while their subsequent treatment does not improve
corrosion resistance further [36].
Lock et al explored the application of magnesium alloy as ureteral stent: they investigated
the affectivity of magnesium and magnesium alloy as antibacterial and biodegradable
ureteral stent, they demonstrated the decreased viability of Escherichia colibacterial and
reduced colony forming units after 3 days of incubation in artificial solution. They
concluded that antibacterial properties coupled with biodegradation in artificial urine
present an alternative approach to design next generation ureteral stents [37].
A coating over the surface of magnesium and its alloys could be promising to reduce the
rate of biodegradation, fluoride coating is a conventional method for magnesium base
materials. Zhang et al treated cardiovascular Mg-Nd-Zn-Zr alloy by immersion in
hydrofluoric acid, they observed the formation of 1.5 micro meter thick layer of
magnesium fluoride after immersion. The surface roughness was increased and zeta
potential was found to shift more negative value, they also found more hydrophilicity
after surface treatment by static contact angle technique. Cell viability investigation
revealed that encouraging results [38].
Another attempt was made to improve corrosion properties of magnesium base material
using coating technique by Gu et al: they deposited silane coating on magnesium alloy by
electrodeposition, the fabricated samples were studied by various techniques i.e. scanning
30
electron microscopy, Fourier transformed infrared spectroscopy, contact angle study and
biocompatibility. They found the coatings deposited at -2.0 volts are more corrosion
resistant in comparison to other silane coatings, moreover the silane coatings produced
significantly increased biocompatibility as tested by cell viability test, reduced hemolysis
rate and platelet adhesion, they concluded that silane coating provides better corrosion
protection and other related biomedical properties [39].
Liu et al also attempted to fabricate corrosion resistant coating over the surface of the
magnesium based material, they fabricated Mg-Mn-Ce coating by simple one-step
electrodeposition method. They employed energy-dispersive X-ray spectroscopy (EDX)
Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR),
and X-ray photoelectron spectroscopy (XPS) techniques were employed to analyze the
surface properties of fabricated samples. The contact angle study revealed maximum
contact angle was 159.8o. Potentiodynamic polarization test and electrochemical
impedance spectroscopy revealed that the prepared surface more corrosion resistant in an
aqueous solution of NaCl, Na2SO4, NaClO3, and NaNO3.The authors concluded that
investigated method is effective rapid and low cost method for industrial fabrication of
super hydrophobic, anti-corrosive surfaces [40].
Another attempt was made to improve corrosion protection of magnesium based alloy as
the rapid corrosion is the major problem associated with orthopedic and cardiovascular
application of magnesium and its alloys. Huo et al applied chemical conversion treatment
and electroless Ni plating to improve corrosion properties of AZ 91D magnesium alloy.
They applied potentiodynamic polarization test to investigate the variation in corrosion
properties of prepared samples, they observed improved corrosion resistance of
magnesium alloy in 3.5 wt% of NaCl solution at pH 7.0, and scanning electron
microscopy indicated porous topographic structures which provide advantage adsorption
prior to electroless nickel coating. They concluded that corrosion resistance of untreated
samples was limited it improved by surface treatment of AZ 91D samples[41].
31
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investigation on corrosion and hardness of ion implanted AISI 316L stainless steel.
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[21] Zou JX, Zhang KM, Hao SZ, Dong C, Grosdidier T. Mechanisms of hardening, wear
and corrosion improvement of 316L stainless steel by low energy high current pulsed
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[22] Kheirkhah M, Fathi M, Salimijazi HR, Razavi M. Surface modification of stainless
steel implants using nanostructured forsterite (Mg2SiO4) coating for biomaterial
applications. Surface and Coatings Technology 2015;276:580-6.
[23] Shih C-C, Shih C-M, Su Y-Y, Su LHJ, Chang M-S, Lin S-J. Effect of surface oxide
properties on corrosion resistance of 316L stainless steel for biomedical applications.
Corrosion Science 2004;46:427-41.
[24] Shabalovskaya S, Anderegg J, Van Humbeeck J. Critical overview of Nitinol
surfaces and their modifications for medical applications. Acta Biomaterialia 2008;4:447-
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[25] Gill P, Musaramthota V, Munroe N, Datye A, Dua R, Haider W, et al. Surface
modification of Ni–Ti alloys for stent application after magnetoelectropolishing.
Materials Science and Engineering: C 2015;50:37-44.
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thrombogenicity of nitinol stents—In vitro evaluation of different surface modifications
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[27] Maleki-ghaleh HK-a, J; Sadeghpour-motlagh, M; Shakeri, M S; Masoudfar, S; et al.
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: Materials in Medicine; Dec 2014;25:2605-17.
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alkylsilanes and polypyrrole. Materials Science and Engineering: C 2014;44:317-25.
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35
Chapter 3
Experimental details and characterization techniques
The experiments are performed using 2MV pelletron accelerator in Accelerator lab,
CASP GC University Lahore, different parameters e.g. energy, nature and charge states
of bombarded ions are chosen for the different experiment. The chosen parameters are
determined by nature of the hypothesized final product and its potential applications.
Sample characterization techniques are also determined by the potential applications and
presumed impact of different ion implantation [1-3]. This chapter contains the details of
the pelletron accelerator, sample preparation, and characterization techniques.
3. Introduction to pelletron Accelerator
Pelletron accelerator is a type of electrostatic accelerator, this type of accelerator is based
upon Van de Graf charging system, and it can have a terminal voltage ranges from
500KV to 25 MV, the corresponding energy of charged particles ranges from few MeV
to several hundred MeV.
3.1 Working principle of Pelletron accelerator
In pelletron accelerator pre accelerated (few KeV) charged particles are further
accelerated to very high energy (several hundred MeV) using an electrostatic field, the
high potential difference between the plates is developed by mechanical transportation of
charges from one plate to another. The charges are transported using chains repetitively
running from one end to other end, the charging belts pick charges from one plate and
accumulate the charges on other. The process does not involve the rubbing but the belt
picks the charges by induction (the detail is discussed in the section of charging system).
3.2. Working and different parts of Pelletron accelerator
The pelletron accelerator is made up of various essential components, some important
components are listed below.
Ion sources
36
Switching magnets
Faraday cups
Accelerating tank
Stripping system
focusing systems
Beam lines
End stations
Ion sources
There are several ion sources being used for production and injection of the ions to
accelerating system of an accelerator. The choice of ion source depends upon nature of
charge particle needed.
SNICS ion source
SNICS stands for the source of a negative ion by cesium sputtering, SNICS ion is used
when ions from solid state species are needed. This is a negative ion source based on
sputtering of the solid source by cesium. Cesium is heated in an oven, the heated cesium
vapors move from oven to area between the cathode and hot ionizing surface. Some
cesium ions condensed on cooled cathode while some Cs atoms get ionized by a hot
ionizing surface. The ionized Cs ions accelerate toward the cathode and sputter the
cathode material, the sputtered negative ions are then focused and pre-accelerated by the
potential between the cathode and ionizing surface.
37
Fig1: schematic diagram of SNICS ion source1.
Switching magnet and Faraday Cup
The switching magnets are electromagnets they inject the ions coming from source to
beamline, the magnetic field of switching magnet is controlled by coil current, the
magnetic field, in turn, determines the bending radius or bending angle.
The ion beam coming from ion source contains a variety of species e.g. sputtered ion may
have multiple charges, multiple clusters (bi atomic, triatomic etc.) and contamination,
therefore it is necessary to monitor the beam. The beam current is monitored by Faraday
cups. This is metallic cup it measures species of charged particle hitting it in the vacuum.
38
Fig2: Faraday Cup(NEC Model FC50)2
When a charged particle collides with the Faraday cup it gains a net charge the cup then
discharged to measure the current equivalent to incoming charged particles.
Accelerating tank
The accelerating tank is a major part of the electrostatic accelerator, multiple processes
including the main process of acceleration of charged particles take place in this part of
the accelerator. This part contains following components.
Charging system
Stripping system
Sulfur hexafluoride gas
In the electrostatic accelerator, a stable electrostatic field is provided to electrodes in
order to accelerate the charged particles. The high potential difference is developed inside
the accelerating tank with the help of charging chains. The charging chains are made up
of metallic pellets connected to each other with the help of insulating material like nylon.
The charging chains pick up charges from one end by induction process and leave the
charges to other end. Negatively charged inductor pushes electrons off by induction
process, the pellets are in contact with a grounded drive pulley, and therefore the
39
electrons are grounded. The positively charged metal pellets then move to the high
potential terminal there the pellets pass through the negatively biased suppressor, this
electrode prevents arcing. After the metallic pellets leave suppressor the negative charges
flow to pellets leaving a net positive charge on the pulley. This way a high potential is
developed between two ends by continues mechanical transportation of charges.
Fig3: charging chains (NEC pelletron accelerator)3
A gas stripping system is introduced in the way of charged particles to reverse the
polarity of incoming ions, the incoming negatively charged ions are twice accelerated by
same electrostatic field by stripping process, gaseous or solid state stripping is used to
strip some electrons from negative ions, after removal of few electrons, the negative ion
is converted into positive ion. The stripped ion after changing polarity once again gets an
electrostatic push, consequently doubling its energy. Gas stripping is normally done by
nitrogen or argon gas, in our experiments we used nitrogen gas for striping.
40
Fig4: schematic of the charging system in pelletron accelerator4
Sulfur hexafluoride gas is introduced in accelerating tank to prevent corona discharge,
this is an electrical discharge in surrounding gases due to high terminal voltage, although
the accelerating tank is vacuumed using turbomolecular pumps up to 10-8 pa the chances
of corona discharge are still there due to a high voltage about 2MaV.
41
Fig5: schematic diagram of accelerating tank5
Focusing system
A highly energetic beam of positively charged ions emerges from accelerating tank, the
ion beam needs to be focused. Quadrupole magnet is introduced in the way of the ion
beam to focus the beam line. Quadrupole magnet is also known as Q magnets, as the
speed of accelerated ions in an accelerator is very high, therefore magnetic deflection is
more effective than electrostatic deflection. Maxwell equations show that it is impossible
for a quadrupole magnet to focus the beam of charged particles in both the axes (x-
pinching and y-pinching) simultaneously.
F = E+ q (V×B) (Lorentz equation)
The quadrupole magnets are of two types: F quadrupole magnet, they focus the ion beam
in the x-axis (horizontally) while defocusing vertically. D quadrupole magnet focuses
vertically but defocuses horizontally.
42
Fig6: Schematic of quadrupole magnet
Fig7: magnetic field line of quadrupole magnet and direction of Lorentz forces6
43
Beam Lines
The ion beam is once again deflected to a specific beam line in the assembly of multiple
beam lines with the help switching magnets. The beam lines lead to the end station,
where the beam of charged particles is utilized, before the end station the beam is once
again monitored by the Faraday cup just before entering to end station chamber.
Fig8: diagram of pelletron accelerator
3.3Characterization techniques
The fabricated samples were characterized using following techniques
X-ray diffractometer (XRD)
Scanning electron microscope (SEM)
Raman spectroscopy
Contact angle study
Cell viability study
Hardness testing
Electrochemical corrosion study
Particle induced x-ray emission study (PIXE)
44
3.3.1 X-ray diffractometer (XRD)
The materials exhibit unique diffraction pattern, therefore diffraction pattern is the
fingerprint for material identification. English physicists W.H. Bragg developed a
relationship to explain why cleavage planes reflect the beam of X-rays when they are
incident at a specific angle.
2d sinθ = nλ (1)
Where d= interatomic layer
n= integer for constructive interference
λ= wavelength of incident X –ray beam
θ = scattering angle
Fig9: schematic of x-ray reflection from different crystal planes7
This is called Braggs law, this law is basic working principle of X-diffraction
identification of crystal system.
45
The XRD technique can reveal following information about an unknown crystal
Crystal system of a material
Interatomic spacing
Size and shape of crystallite
Internal stress of crystallite
Orientation of single crystal or grain.
Construction and working
A typical XRD contains following part
X-ray tube
This is an evacuated tube, contain a copper anode and an electron source usually a heated
filament, the emitted electrons are accelerated in an electrostatic field between the copper
anode and a cathode. Bremsstrahlung radiations are produced when accelerated electrons
are suddenly stopped by the metal target. The incident electron has sufficient energy to
eject the inner shell electron out from the target, consequently, the electron from outer
shells will jump to inner shell releasing energy in the form of X-rays. These x-rays are
then used for diffraction from unknown crystal system.
Transducers produce electrical signals when they are exposed to incoming radiation, the
transducers are used as a detector. They count the number of diffracted x-ray photon, the
number of counts is equal to the intensity of the diffracted beam of x-ray at a specific
angle.
Goniometer
X-ray goniometer is a device that is used to record the direction of x-rays after diffraction
from the specimen. An x-ray detector is mounted on goniometer such a way the
goniometer can move to a certain position where the necessary conditions of diffraction
are fulfilled.
46
Fig10: Schematic of a typical XRD operation8
3.3.2Scanning electron microscope (SEM)
SEM is an electron microscopy used to study the surfaces of objects. This microscopy
technique uses a beam of the focused electron to scan the surface of a material and to
produce an image of the scanned material.
Working of SEM
Electron gun produce beam of electrons, the beam of electrons is focused by using
electromagnetic lenses. High energy backscattered electrons and low energy secondary
electrons are ejected from the surface of the specimen, by the interaction of bombarded
electrons from the electron gun. The secondary and backscattered electrons reveal
information about the sample. The signal obtained from the specimen contains the details
of topography, size, and composition of the specimen under study. The diffracted
backscattered electrons contain the information about crystal system of the specimen.
Primary and secondary backscattered electrons carry the topographical information. The
47
x-rays emitted by the specimen after the interaction with incident electrons carry the
compositional evidence.
Fig11: different types of species emitted from sample carries different information9
Different part of SEM
A type SEM comprises of basic following parts
Electron gun
Electromagnetic lenses
Sample holder/ stage
Vacuum system
Detectors
Requirements for SEM operation
There are some requirements for accurate operation of SEM.
Vacuum system
Cooling system
48
Vibration free ground
Power supply
Advantages
Little or no sample preparation is required
It is a nondestructive technique
The image collection is rapid
A detailed three dimensional image is obtained
The magnification ranges from 20x to 30000x
The technique is user friendly: easy to operate and harmless
Very small and big samples up to 10cm can be analyzed
Disadvantages
Only solid samples can be analyzed.
Samples more than 40 mm thick cannot be studied.
The device operate in high vacuum 10-6 torr.
The only samples which are stable in vacuum chamber are possible to examine.
Wet samples are not likely to be examined
Samples needs to be electrically conducting, insulating samples are coated with
conducting material.
49
Fig12: Schematic diagram of SEM10
3.3.3Raman spectroscopy
Interaction of incident monochromatic light with molecules changes the frequency, every
molecule interact with incoming light differently, therefore by the shift of frequency the
nature of molecule can be determined
Fig13: schematic of typical Raman scattering by a molecule
There are two types of scattering
Elastic scattering
50
Elastic scattering also called Rayleigh scattering. In this type of scattering energy and
wavelength remain conserved only the direction changes. Elastic scattering also called
Raman scattering. In this type of scattering the scattered photon have
diminished/increased energy. Energy and wave length does not remain conserve.
Stokes and Anti-Stokes scattering
In stokes scattering, scattered photon have lesser energy than the incident photon. The
final state is higher in energy than the initial state. In case of anti-stokes scattering, the
scattered photon have higher energy than the incident photon. The final electronic state is
lower in energy.
Operation of Raman spectrometer
The sample is illuminated with a beam of monochromatic light. The incident light
scattered by the sample, a very small fraction of light scattered in-elastically (Raman
scattering), typically one part out of one million. The scattered light from the sample is
filtered and directed to the spectrometer, where it is analyzed. The molecules in the
sample absorb only a specific frequency of incoming laser light, which gives the
fingerprints of molecules. The Raman spectrum is plotted by varying frequency of
incident light and intensity of scattered light of a particular frequency.
Fig 14: schematic of Raman spectroscopy analysis11
51
3.3.4Biocompatibility study
Biocompatibility is the property of material being compatible with the biological system,
for a clinical implant material biocompatibility refers to the performance of implant
material inside a biological system such that it does not trigger immunological response
and does not release toxic materials. Biocompatibility is a general term in a broad sense a
biocompatible material does not harm the user. The implant materials and devices need to
be tested for their biocompatibility property. Biocompatibility of a candidate biomaterial
is measured by cell viability assay.
Fig15: lay out of cell culture hood12
Cell viability
Cell viability refers to no of living or dead cell in a particular sample or on the scale of a
total number of cells. Cell viability assay measures how healthy the cells are after
interaction with a particular chemical or a biomaterial. MTT assay is an authentic
indicator of cellular activities. The MTT assay is based upon the reduction of yellow
water-soluble tetrazonium dye.
52
Fig16: different steps involved in cell viability study13
Assay protocol
1. Separate the media from cultures.
2. Put 50 µL of serum-free media and 50 µL of MTT.
3. Incubate the plate at 37.5°C for 3 hrs.
4. Then add 150 µL of MTT solvent to each well.
5. Wrap plate in foil and shake it with shaker for about 15 min.
6. Read the plate before 1 hour.
3.3.5 Bioactivity study
Along with biocompatibility, another related biological property is bioactivity, bioactivity
of a biomedical implant refers to the activity of that material inside a biological system.
Bioactivity of an implant material is often evaluated by simulated body fluid (SBF). SBF
was first introduced by Kokubo and his colleague [4, 5]. The fabricated samples are
immersed in SBF for seven days usually, at 37 0C. The ionic species in SBF get
precipitated over the surface of the immersed sample to form a bone-like structure called
hydroxyapatite. The bone binding ability of an orthopedic implant material is also
53
assessed by its ability to form apatite layer over its surface while immersed in SBF under
physiological conditions.
Table1: Ionic concentrations of the simulated body fluid and human blood plasma
Ion Simulated body fluid
(SBF)
(mmol/dm3)
Human blood plasma
(mmol/dm3)
1 Na+ 142.0 142.0
2 K+ 5.0 5.0
3 Mg2+ 1.5 1.5
4 Ca2+ 2.5 2.5
5 Cl- 147.8 103.0
6 HCO3- 4.2 27.0
7 HPO42- 1.0 1.0
8 SO42- 0.5 0.5
3.4 Stopping range of ions in matter (SRIM)
The range of implanted ions in target lattice is an important parameter. It depends upon
multiple factors such as nature of target material, nature of incoming ions and energy of
ions. The incident atom performs multiple elastic collisions with the target atom. The
momentum is transferred from bombarded ion to target atoms during the process of
elastic collision. Sometimes the transferred energy is sufficient enough to overcome the
binding energy of target atom, then a surface atom may be ejected from target surface
called the sputtered atom.
The energy of incident ion is lost in the target lattice by two phenomena. Nuclear energy
loss and electronic energy loss, the equation for energy transferred is given below.
54
.
𝐸transferred = 4𝑀1𝑀2/(𝑀1
+ 𝑀2) 𝐸𝑡 𝑠𝑖𝑛2 𝛼/2 (2)
Where M1= mass of ion
M2 = the mass target atom
α = angle of deviation
Et= total energy of ion
The electronic energy loss takes place due to inelastic repulsive interactions between the
electrons of bombarded ions and the electrons of target atoms. The nuclear energy loss
takes place in discrete steps by elastic energy transfer. The range of bombarded ion may
be defined as the path length of the single atom in a target material.
𝑅𝑎𝑛𝑔𝑒 = ∫𝑑𝐸
𝑑𝐸/𝑑𝑥
𝐸0
𝐸𝑚𝑎𝑥 (3)
Schematic diagram of sputtering by ion matter interaction (Source: Nastasi et at., 1996)
SRIM is a software package to simulate the ion matter interaction. This package was first
introduced in 1985. TRIM (transport of ions in matter) is the core of SRIM package. This
package is helpful to ion implantation community, it helps the experimentalists and the
55
researchers of other disciplines in a number of ways[6]. The TRIM is based upon Monte
Carlo method of random sampling to obtain numerical outcomes. The software package
uses two basic approximations. Firstly it uses the analytical formula to obtain atom-atom
collision secondly it uses the concept of free flight path between the consecutive
collisions. Monte Carlo method has some advantages over the analytical methods which
are based upon transport theory. The. Monte Carlo method can be used for
comprehensive calculations of ion-atom interaction. The main limitation of Monte Carlo
method is that it is a time-consuming method. Therefore there is a divergence between
computer time and statistical precision. The issue is resolved considerably by sacrificing
little accuracy, the TRIM user has the option to bypass the approximation and calculate
the problem with the highest accuracy. The software package uses some specific
assumption for calculations. The ions are assumed to change their path during binary
collisions and follow straight free-flight-path. It is assumed that the ions losses their
energy as a result of nuclear and electronic scattering. The energy loss during nuclear
scattering is discrete and continuous energy loss by electronic interactions. The nuclear
and electronic energy losses are assumed to be independent of each other.
𝑑𝐸
𝑑𝑥= (
𝑑𝐸
𝑑𝑥)
𝑛𝑢𝑐𝑙𝑒𝑎𝑟+ (
𝑑𝐸
𝑑𝑥)
𝑒𝑙𝑒𝑐𝑡𝑟𝑜𝑛𝑖𝑐(4)
The target is assumed non-crystalline and the atoms are located at random positions,
therefore the anisotropic properties are ignored. This method allows a wide range of
energy calculation typically from 0.1keV/u to several MeV/u. Energy-dependent free-
flight-path is introduced to deal with the calculations that involve high energy particles.
The developed formulism is applicable to all ion-target combinations. The program
provides the information about penetration depth of ion in target lattice at a particular
energy, damage events, recoiled atoms, vacancies and interstitials etc.
Sample preparation
Cutting, grinding and polishing of stainless steel samples
The samples of stainless steel 306 are cut into cylindrical shape with the help of diamond
wheel cutter. The length of the samples was 1cm and the diameter of the samples was 1
56
cm. The samples were grinded with the help of different grits of silicon carbide paper
such as 320, 500, 1000, 1500, 2000, 2500 and 3000 respectively. We continuously
monitored the scratches of samples with the help of an optical microscope. Different
micron of diamond was used to have a mirror like shine, minimize surface scratches. The
samples were cleaned subsequently in an ultrasonic bath in de-ionized water and then
acetone.
Cutting, grinding and polishing of nitinol samples
Four NiTi wire (2 mm diameter)samples of 6cm each are polished using SiC paper of
1000 grit size, then polished using metal polish paste and are subsequently cleaned by
an ultrasonic bath in de-ionized water and acetone. Samples are annealed at 450oC for 2
hrs. This temperature is chosen from earlier studies which show more Ni release from the
samples annealed greater than this temperature [7].
References
[1] Roeder RK. Chapter 3 - Mechanical Characterization of Biomaterials A2 -
Bandyopadhyay, Amit. In: Bose S, editor. Characterization of Biomaterials. Oxford:
Academic Press; 2013. p. 49-104.
[2] Wang H, Chu PK. Chapter 4 - Surface Characterization of Biomaterials A2 -
Bandyopadhyay, Amit. In: Bose S, editor. Characterization of Biomaterials. Oxford:
Academic Press; 2013. p. 105-74.
57
[3] Morra M, Cassinelli C. Biomaterials surface characterization and modification. The
International journal of artificial organs 2006;29:824-33.
[4] Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity?
Biomaterials 2006;27:2907-15.
[5] Cho S-B, Nakanishi K, Kokubo T, Soga N, Ohtsuki C, Nakamura T, et al.
Dependence of Apatite Formation on Silica Gel on Its Structure: Effect of Heat
Treatment. Journal of the American Ceramic Society 1995;78:1769-74.
[6] Ziegler JF, Ziegler MD, Biersack JP. SRIM – The stopping and range of ions in
matter (2010). Nuclear Instruments and Methods in Physics Research Section B: Beam
Interactions with Materials and Atoms 2010;268:1818-23.
[7] Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T. Solutions able to
reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W3. Journal of
biomedical materials research 1990;24:721-34.
References to figures
58
Chapter 4
Results and Discussions
4. Ion implantation in stainless steel and nitinol
Stainless steel and nickel-titanium alloys (nitinol) are important bio-material. These
materials are currently being used for various biomedical applications. The significance
and associated problems with these materials have been discussed in previous chapters
(section.1 and section. 2). Some major problems associated with stainless steel and
nitinol were considered to study, i.e. in-vivo stability (corrosion protection): these
materials often corrode in physiological environment and release toxic ions e.g. nickel,
chromium, etc.[1]. The leaked ions cause allergy, disorders in bone growth, cell death
and some other undesired effects. Therefore it is necessary to study the factor affecting
ion release and corrosion properties. The incubation of Hydroxyapatite (HA) over the
surface of candidate biomaterial is an important factor, this factor determines the in-vivo
behavior of implant material: An apatite layer gets deposited over the surface of implant
material inside the human body. This apatite layer bonds between implant material and
neighboring bones. In-vivo apatite forming-ability of a candidate biomaterial can be
evaluated in-vitro by using simulated body fluid (SBF). In-vitro incubation of apatite
layer determines bio-activity and bone bonding ability of material under investigation [2].
Cell viability of a candidate biomedical material is also an important concern: cell
viability of a material refers to the degree of survival and to increase in number of cells,
the interaction of material surface to alive cells may cause cell death which leads to a
decreased percentage of alive cells, which refers to decreased cell viability. Sometimes
the interaction does not cause cell death or very less fatality to cells, the interaction may
also lead to increase in the number of cells, which is called cell proliferation or increased
cell viability (more than 100%). Minimum and maximum cell viability of a material for
safe use is not defined. Threshold cell viability depends upon the application of material,
some researchers claim that good cell viability is more than 50%. In our experiments, we
have investigated the variation in cell viability in treated samples.
59
Some mechanical properties are also very important for orthopedic implant material, in
our experiments we investigated the variation of surface hardness after the treatment of
samples.
Surface wettability of a material is another important biomedical property. Some studies
claimed that biocompatibility of a material depends upon hydrophilicity (contact angle)
[3]. Researchers are attempting to study the relationship between surface wettability and
bio-compatibility [4, 5], in present work the relationship between hydrophilicity and bio-
compatibility hydrogen ion implanted samples is also studied.
4.1 Effect of nitrogen ion implantation in stainless steel
Stainless steel 316L is the most commonly used orthopedic material[6, 7]. Stainless steel
is chosen for orthopedic and some other applications e.g. artificial heart valve, medical
needles, medical syringes orthodontic wires, catheters etc. Because of its
biocompatibility, mechanical properties, and low cost. Nitrogen is a wonderful element it
is the fourth most abundant element in the human body and its compounds are basic
building blocks of biological system e.g. nitrogen is the major component of amino acid
and amino acid make up the proteins. Because of its biological importance, it is
hypothesized that nitrogen ions in the near-surface region of orthopedic implants may
yield suitable effects e.g. enhanced bonding ability, improved stability and cell response
to the surface. Literature also reports enhanced corrosion protection by nitrogen ion
implantation [8, 9]. The enhanced corrosion protection by N ion implantation is due to
the formation of nitrides and oxynitrides in ion implanted region. The oxynitride layer
prevents the exposure of surface to reactive ions (cl, O2 etc.). The metallic nitrides have
exceptional electrochemical properties, particularly chemical stability making metallic
nitride a potential candidate to be used as electrode in lithium-ion batteries [8, 10].
Stainless steel 306 is implanted with various doses of nitrogen ions using a 2MV
pelletron accelerator for the improvement of its surface biomedical properties. The
samples are characterized for mechanical, biomedical and chemical properties using
various techniques.
60
4.1.2 Earlier work
The metallic materials are being employed for diverse biomedical applications in various
fields of biomedical science. The examples of metallic materials as biomaterials includes;
the mesh of stainless steel or nickel alloy as coronary heart stent, stainless steel or cobalt
as artificial femoral head (artificial hip) for total hip replacement, stainless steel and
titanium as bone plates and screws etc. Therefore there is an earnest need to improve the
relevant properties of candidate biomaterials [11-14] . Stainless steel (SS) is an important
alloy for many biomedical applications because SS is stable (appropriate corrosion
resistant), bioactive and has good mechanical properties, but there are always some
associated deficiencies with every material. The deficiencies of SS includes toxic ion (Ni,
Cr) release from the surface and mismatch of mechanical properties with bone (hardness,
tensile) etc. [15-18]. Due to the mismatch of mechanical properties of bone and implant
material, an unwanted effect is produced, which is called stress shielding effect, due to
the effect the bone does not share the mechanical stress, consequently, leading to the
reduction in bone density. Some mechanical properties of femur bone and stainless steel
are tabulated below.
Table1: comparison of different properties of bone and stainless steel
Properties Femur bone Stainless steel
1 Density (g/cm3) 1.6-1.7 8
2 Tensile strength (M Pa) 90-130 680-750
3 Compressive strength (M Pa) 130-200 500
4 Hardness (vicker) 50-100 155
Biocompatibility and bioactivity are two different properties, for a material
biocompatibility refers to the degree of friendliness, being harmonious to body
functioning and not stimulating any unwanted in-vivo response, while the bioactivity
refers to being beneficial to biological system or triggering an in-vivo positive response.
The bioactivity and biocompatibility are two fundamental properties required to use a
material as a biomaterial. The bioactivity of a material surface is determined by cellular
response to the
61
surface, the cellular response to a particular surface is estimated by the exposure of the
surface to a specific type of cells. Size, shape and number of alive cells determine the
behavior of the surface to exposed surface after a specific time.
Different surface modification techniques are being employed to improve cellular
response to surface of metallic materials and some related properties [18, 19]. Metals and
alloys are being treated in variety of ways [20, 21] in order to tune some required surface
properties. Researchers are working to modify the material surfaces for improved
properties by plasma immersion ion implantation and thin film fabrication, nano pores or
micro/nano surface roughness fabrication etc [22-25].
Jiang et al. had shown uniform apatite growth on porous nitinol substrate [24, 26]. Kawai
et al prepared porous Ti metal treated with H2SO4/HCl mixed acid solution and/or given
heat treatment, they concluded maximum bone ingrowth on fully treated samples and it
was attributed to accumulation of positive charges on the surface. Kawai et al reported
similar results in another study where they produce micrometer surface roughness by
H2SO4/HCl and heat treatment at 600C, they also prepared nanometer roughness by
NaOH followed by HCl and then the samples were heat treated both of these sample
showed good apatite firming ability in SBF and in-vivo. These results were credited to
concentration of positive charges on the surface [27, 28].
The biocompatibility of an orthopedic implant can be evaluated by in-vitro exposure of
samples; SBF is a wonderful solution for the in-vitro test. Incubation and proliferation of
hydroxyapatite in SBF on a surface determines its biocompatibility and efficacy as
biomedical implant.
We have implanted nitrogen ions to produce roughness on the surface of stainless steel
samples. As a result of dual effects of surface roughness and presence of nitrogen ions on
the surface we observed significantly improved results.
62
4.1.3 Nitrogen Ion implantation
Nitrogen ions are produced in SNICS ion source by sputtering of nitrogen source. The
produced ions are pre accelerated in SNICS source then out of the cluster of many
species
(e.g. impurities, monoatomic, bi-atomic etc. ions of N and the species with different
charge state q=1, 2, 3 etc.) singly charged monoatomic nitrogen ions are chosen to
implant in target lattice. The remaining unwanted species are filtered out by deflecting
them to some angle using an electromagnet. The pre-accelerated chosen ions are further
accelerated in accelerating tank under an electrostatic field strength of 250 kV.
The energy corresponding to given voltage is estimated by the following equation.
E = (q+1) V
The selected ions are implanted on four stainless steel samples at a constant energy of
500keV and one sample is studied as the un-implanted sample. Mono-atomic singly
charged (q=1) nitrogen ions are implanted into stainless steel lattice at constant energy.
The number of implanted ions is varied in different samples. The detail of ion dose is
given in table1.
The ion dose was calculated using equation
Dose/sec = (I × 6.3 × 1018) ÷ beam size
Dose = [(I × 6.3 × 1018) ÷ beam size] × time of exposure
Where I = beam current
Beam size = 2cm × 2cm = 4cm2
Table 2: ion dose for four samples
Samples Dose (number of ions/cm2)
NSS1 3.6×1013
NSS2 2.3×1014
NSS3 4.5×1014
NSS 4 2.8×1015
63
4.1.4 Immersion in simulated body fluid
Four ion implanted samples and one untreated sample is immersed in Kokubos
simulated body fluid for 7days at 37.5 oC to incubate a layer of hydroxyapatite[29], the
extent of incubation of hydroxyapatite determines bioactivity of samples[23]. The
immersed samples were then analyzed by various techniques.
4.1.5 Results and Discussions
4.1.5.1 Raman Spectroscopy profiles Raman spectroscopy is employed to analyze the chemical nature of species incubated
over the surface of samples after immersion in SBF. Total of six samples are chosen for
Raman spectroscopy: four ion implanted samples after immersion in SBF, one un-
implanted sample after immersion in SBF and one un-implanted sample without
immersion in SBF.
Raman spectroscopy is performed using DXR Nicolet Thermo scientific. Raman Spectra
of HA over the surface of samples under investigation is shown in Fig: 1. each sample is
investigated by Raman spectroscopy from three different points. It is observed that
pristine sample (un-implanted without immersion in SBF) does not show any
representative peak of HA. The Raman peaks are observed in all the remaining five
samples but with different peak intensities. The Raman peak positions are compared with
literature [30, 31]. It is found that all the peak positions represents the composition of
HA. The peak positions from the literature are tabulated below.
Table3: different peak positions of HA groups
Sr.no Raman Peak position (cm−1 ) bond
1 422 to 454 υ2 (PO4)3-
2 568 to 617 υ 4 (PO4)3-
3 815 to 921 C-C stretching
4 957 to 962 υ 1 (PO4)3-
5 1003 to 1005, 1006 to1055 υ 3 (PO4)3
6 1065 to 1071 CO32-
7 3572–3575 O-H stretching
64
From the Raman spectra of different samples it is observed that all the finger prints of
HA are present in prepared samples, different modes of vibrations of (PO4)3- are observed
at peak positions 918 cm-1,410 cm-1, 1045cm-1 and 615cm-1 , the mentioned peak
positions represents υ1, υ2, υ3 and υ4 of (PO4)3- respectively. CO3 bond is observed at
1170 cm-1, amide III peak is also found at 1295 cm-1. A prominent peak is observed at
770cm-1, Hadrich et al attributed this peak to hexagonal structure of stoichiometric HA
[32]. Moreover the sharp and distinct peaks represents ordered and fine growth of HA.
From the spectra of different samples it is clear that HA has incubated in all the prepared
sample and untreated sample, but the magnitude of incubation is different in different
samples, the intensity of HA peaks characterizes the magnitude of incubated HA, the
spectrum shows increased incubation by increasing ion dose, as maximum peak intensity
is observed in maximum ion dose sample and minimum peak intensity is found in least
ion dose sample. Therefore it is concluded that presence of nitrogen ions in near surface
region of SS facilitate the growth of HA over the surface.
65
0 500 1000 1500 2000 2500 3000 3500
-100
0
100
200
300
400
500
600
Inte
nsity
Raman Shift(cm-1)
Sample4
Sample4
Sample3
Sample2
Sample1
UnEx
pristine
Pristine
Conterol10
12ion/cm
2
1013
ion/cm2
1014
ion/cm2
1015
ion/cm2
PO
-3 4v 2
PO
4-3v 4
PO
4-3v 1 P
O4-3
v 3C
O-2 3
amid
eIII C
H2w
ag
Fig1: Raman Spectra of incubated HA
4.1.5.2 XRD studies
XRD study of prepared samples is performed to analyze the nature of incubated
crystalline species over the surface of ion implanted and untreated SS samples and to
study the effect of N ion implantation on the crystallinity of incubated species. XRD
profiles are shown in Fig: 2.
The typical HA XRD peak positions are HA 210 at 2θ=28o, HA 211 at 2θ=32o, HA 300
at 2θ=35o HA 113 at 2θ=45o, HA 213 at 2θ=49o approximately [33-35].
The XRD patterns specify the presence of typical HA peaks in highest N ion implanted
sample [210], [211], [300] at 2θ position= 28o, 31.6o and 34.5o respectively the next two
peaks at 2θ position= 43o and 500 are common in both hydroxyapatite and stainless
steel[22, 36], these are the positions at which both hydroxyapatite and stainless steel
profile exhibits peaks.
66
The XRD profiles shows absence of hydroxyapatite peaks in control sample and low ion
dose samples (sample 1, 2 and 3) except the peak at [300] which gradually evolves with
increasing ion dose, and the maximum intensity of this peak can be observed for greatest
ion dose 1×1015. From the profiles we can also observe origination of [211] and [210]
peaks in greater ion dose samples [21].
The results suggests that the HA growth is proportional to number of N ions, maximum
growth is observed in highest (1015ions/cm2) ion dose sample and minimum or no growth
is found in controlled and minimum ion dose sample (sample1) while sample 2 and 3
exhibits evolutionary behavior between un-implanted and heavily implanted samples .
20 30 40 50 60 70 80
-200
0
200
400
600
800
1000
1200
1400
1600
1800
Inte
nsity
(a.u
)
(degree)
Unexposed
sample
NSS1
NSS2
NSS 3
NSS 4
HA(210)
HA(211)
HA [300]
SS (110)
HA (113)
SS (200)
HA (213)
Fig 2: XRD profiles of incubated HA
4.1.5.2.1 Estimation of range of ions in material lattice (SRIM study)
The nitrogen ions are implanted into stainless steel lattice at 500KeV energy. This
energy is chosen to force the ions to stay at the surface of samples so that the implanted
ions could interact chemically with ionic species present in SBF, the range of implanted
ions are estimated by SRIM shown in Fig 2a . Implanted nitrogen ion resides within 0.5
µm (500 nm) of target depth as calculated using SRIM. These ions produce porosity,
surface roughness and amorphization. The amorphous surfaces are more efficient for
67
hydroxyapatite settlement [37]. Liu et al [38] prepared hydrogenated amorphous surface
of silicon and they found improved bioactivity along with evolution of hydroxyapatite
peaks in XRD spectrum. They also established that the improved biocompatibility is not
only due to surface amorphization or hydrogen implantation only but this is due to
combined effect of Hydrogenated amorphous surface.
Fig2a: SRIM profile of depth of N ions in target substrate
4.1.5.3 SEM Results
Scanning electron microscopy study is performed to investigate topography of prepared
samples after immersion in SBF and variation in extent of growth of HA on sample
surface by varying N ion dose. Moreover the percentage of area covered by incubated
HA is also an interesting feature to study, it determines the response of modified surface
for bone growth.
SEM images of four prepared samples (sample1, sample2, sample3 and sample4) are
shown in Figures 3 and 4. Figure 3 shows the images of ion modified SS surface, while
Figure 4 shows the images of ion modified surfaces after immersion in SBF. The impact
of bombarded ions on the sample surface is visible in the form of holes, surface
roughness and other surface imperfections.
Fig 4 depicts the growth of hydroxyapatite in all the four samples, but the surface area in
sample 1 and sample 2 are found largely uncovered which indicates restricted growth of
hydroxyapatite, SEM micrograph of sample 1 shows discontinued, localized
precipitated hydroxyapatite at specific regions only, likewise SEM profile of sample 2
68
represents similar restricted growth of hydroxyapatite but the growth is found enhanced
as compare to sample1. SEM topography of Sample 3 shows that almost all the surface
area is covered with hydroxyapatite but the density of growth is different in different
regions.Finally, SEM profile of sample 4 shows fully covered surface area with a uniform
layer of hydroxyapatite.
This clearly confirms encouraging effects of N ion implantation for hydroxyapatite
growth on the surface. SEM results are consistent with XRD, Raman spectroscopy and
MTT study of as prepared samples.
Fig 3: SEM Micrographs before immersion in SBF
69
Fig 4: SEM Micrographs of NSS1, NSS2, NSS3 and NSS4 respectively after immersion
in SBF.
4.1.5.4 in- vitro Cell Viability Studies
Biocompatibility of all produced nitrogen implanted stainless steel samples is
investigated by using cultured human oral fibroblasts (P4) and cellular response is also
observed by using standard MTT assay protocol. Samples with 5 mm in height and 1cm
in diameter are prepared and sterilized by using autoclave (15 min at 121°C/ 15 psi).
Similarly, cells are cultured in DMEM (Dulbecco's Modified Eagle's Medium) media
supplemented with 10 % of FCF serum (dye for protein staining), 1% penicillin /
streptomycin, 1% glutamine (Sigma Aldrich UK). Moreover, cells are allowed to
confluent (100%) over the surface of tissue cultured plate and are detached by using
trypsin EDTA (Sigma Aldrich, UK). Cells are seeded into wells of 24 well plates
containing test sample seeding density of 1.25 x 104 cells / ml. A material and non-
material control (positive and negative) are introduced for the direct comparison
respectively. Both material and cells are incubated at 37°C in a 5 % CO2 atmosphere for
24 hrs.
However, for quantitative measurement MTT assay is performed individually on all
prepared samples. 0.1 ml of MTT solution are aseptically added to each well and are left
for incubation at 37°C for 4 hrs. Cells are than lysed with Isopropanol. The intensity of
colored solution is measured by using a photo spectrometer at a wavelength of 570 nm.
Fig: 6 shows percentage cell growth versus N ion dose in different samples.
70
0.0 2.0x1015
4.0x1015
6.0x1015
8.0x1015
1.0x1016
50
100
150
200
250
300
Ce
ll vi
ab
ility
%
Ion Dose (ions/cm2)
Fig 5: Cell viability VS ion dose graph
Fig 6: Histogram representation of Cell viability in different samples
Human oral fibroblasts (passage 4) are used to study in-vitro cellular response to the
sample surface by varying N ion dose. Cell growth versus ion dose is shown in Fig:6
Percentage cell viability, morphology and cell proliferation are studied in comparison of
a control sample (SS sample 1) after 24 hours of incubation at 37 o C; all the samples are
found positive as the minimum observed cell viability is 60% in sample 2. We observe
decreased cell viability in sample 1 and sample 2 as compared to unexposed sample
(Control SS sample) then a slight increase in sample 3, 95%, 60% and 83% respectively.
A drastic increase in cell growth approximately 300% is observed for ion dose of 1015
ions/cm2. Our quantitative result agrees with Rizwan et-al[39] up to the limit of their
maximum implanted ions 1014ions/cm2, they attributed reduced cytocompatibility to
surface roughness. It has been reported the effect of surface roughness on hydrophilicity
71
[40] and it is generally claimed the decrease in hydrophilicity with increasing roughness.
It is also well known from the literature that hydrophilicity is proportional to
bioactivity[41]. Our results support this assumption. Figure (a) and figure (b) shows
proportional variation in hydrophilicity and cell viability, which supports earlier
literature.
4.1.5.5 Contact Angle studies
Contact angle of all the samples are evaluated at three different points using Goniometer
Model 100-00 (220).UK. The average of three measurements is plotted in Fig 7 against
ion dose for each sample.
From the figure, we can conclude that contact angle of all the samples is less than 90o and
therefore all the samples are generally hydrophilic. It is observed that hydrophilicity
changes with ion doses. The ion bombardment on the surface produces roughness which
strongly affects the wetting property. The figure also indicates that the untreated sample
which is the smoothest is most hydrophilic. Contact angle increases in implanted samples
up to the sample 2 (ion dose=1013ion/cm2) which is the least wetting (this sample was
found to be least biocompatible as well in MTT study). After this dose limit the next dose
is 1014ion/cm2, this is the number of ions which is sufficient to create a mono atomic layer
on the surface of the material. The larger number tend to create bigger damage area (the
damage of multiple incoming ions combines to produce a single bigger damaged area as
seen in SEM profiles), the damage size after a certain limit is a local smoother region
which may reduce the hydrophobic effect
0.0 2.0x1014
4.0x1014
6.0x1014
8.0x1014
1.0x1015
73
74
75
76
77
78
79
cont
act a
ngle
(de
gree
)
ion dose (ion/cm2)
72
Fig 7: contact angle VS ion dose
Many authors reported that hydrophilicity can be affected by the roughness of the
surface, Kubiak et al demonstrated that contact angle was strongly correlated with surface
roughness [42] they plotted graphs between contact angle and surface roughness, the
graph
shows a hump like symmetry in which medium rough surfaces are least hydrophobic,
which was also reported by Bikerman in 1949, they reported that rough solids surfaces
are comparatively hydrophobic[41]. Kittu et al also reported that the surfaces become
more hydrophilic as roughness decreases [43].
4.1.5.6 Hardness Results Variation in micro-hardness by ion implantation is shown in Fig 8 against N ion dose.
Micro-hardness of samples is measured using Vickers hardness tester at a maximum load
of 200g. The figure shows that the least hard sample is the control sample while the
hardest among all the samples is sample 1. Sample 2 and sample 3 are less hard than the
sample 1, the hardness then increases in sample 4 as compared to sample 2 and sample 3.
It is known that ion implantation in substrate lattice generates defects and amorphization.
It is known that the amorphous surfaces are generally harder than ordered surfaces. The
hardness is increased due to the presence of the defects which are produced by ion
implantation which prevent further lattice movements and dislocations which is a cause
of surface hardening, another reason of improved hardness is the formation of nitride
phase which tends to harden the surface[44, 45].
73
0.0 2.0x1014
4.0x1014
6.0x1014
8.0x1014
1.0x1015
280
285
290
295
300
305
310
Hard
ness
(HV)
ion dose (ion/cm2)
Control
NSS1
NSS2
NSS3
NSS4
Fig 8: Hardness VS ion dose
4.2 Effect of Hydrogen ion implantation in stainless steel
Effect of hydrogen ion implantation is being investigated for various application:
researchers working in semiconductor field are trying to use H- ion beam for exfoliation,
the changing parameters of H ion implantation leads to different results. Hydrogen ion
implantation is also being applied for resistivity tailoring and to modify mechanical
properties. We have hypothesized that incorporation of H- ions in the surface of the
biomedical implant may yield improved hydrophilicity and biocompatibility due to
greater chances of H-bonding. The effect of hydrogen ion implantation on surface
wettability and biocompatibility of stainless steel is investigated. Hydrogen ions are
implanted in near surface of stainless steel to facilitate hydrogen bonding at different
doses with constant energy of 500KeV.
4.2.1 Introduction
Orthopedic biomaterials are important class of materials; because of their use in various
applications such as, bone plates for healing of broken bones, artificial hip joints for
replacement of damaged joints, intramedullary nails to maintain alignment and position
of bone and to share load of bone, bone defects filler, bone graft, spine, orthodontics
wires etc[46, 47].
74
An appropriate set of biological and mechanical properties is required for proper
functioning and safety of these materials to be used in the biological environment.
Existing materials and alloys do not cater the necessary requirement properly. Therefore
researchers are looking for new and/or modified material to exploit suitable properties
which would take care of human body requirements [48-50]. Stainless steel titanium and
its alloys, cobalt chromium alloys, nickel titanium alloy and some polymers are being
used at present [51-53], because of their unique properties but they have shortcoming for
desired purposes. Researchers are currently working on two approaches for achieving
desired properties. They are trying to fabricate new class of materials by alloying while
another class of material scientist harvesting unique interfacial properties.
In addition to suitable mechanical properties, appropriate biological properties are also
important. Surface wettability is an important property, it is believed that hydrophilic
materials are generally biocompatible [54, 55]. Surface hydrophilicity and hydroxyapatite
precipitation depend upon H-bonding due to partial electrostatic attractive forces between
less electronegative H and more electronegative OH group (as OH present is both water
molecule and HA molecules). Thus we assumed that H ion incorporation in the near-
surface region of an orthopedic implant will enhance the chances of H-bonding, thereby
improving hydrophilicity and hydroxyapatite precipitation.
We have implanted different doses of 500KeV H-ion on stainless steel and prepared
samples are then characterized for wettability, biocompatibility and bioactivity.
4.2.2 Ion implantation.
Stainless steel-306 samples are bombarded with H ion in evacuated target chamber of
2MV Pelletron accelerator (Accelerator Lab, CASP-GCU Lahore). Mono-atomic singly
charged (q=1) H ions are selected for experiment. The chosen ions are accelerated to 500
KeV of energy. According to SRIM calculation H ions of given energy may penetrate
within 2.48µm depth. These H ion are implanted in all samples for various ion doses
(1012, 1013, 1014 and 1015 ions/cm2) in HSS1, HSS2, HSS3 and HSS4 samples
respectively.
75
4.2.3 Immersion in simulated body fluid
Simulated body Fluid (SBF) is prepared according to Kokubos protocol [23]. The ion
implanted samples are cleaned with DI water and acetone. Then these are dried and are
immersed in 25ml of SBF at 37.50C for eight days. After eight days of immersion
samples are removed from SBF and then are oven dried to remove water contents.
4.2.4 Results and Discussions
4.2.4.1 Contact Angle Studies
Contact angle (CA) of all the samples are evaluated at three different points using
Goniometer Model 100-00 (220).UK. All the three readings of the same sample are
approximately equal with minor error is found to be ± 0.1 degree. The average of three
measurements is plotted in Figure 9 against ion dose.
It is assumed that greater H-bonding by H ion implantation will result smaller CA
consequently [56], the wettability profile of treated samples reveals variation in contact
angle as expected. The figure depicts an exponentially decreasing relation of contact
angle with respect to ion dose variation. Maximum CA is observed in untreated sample
which is 74o. Then it is decreased continuously with increasing number of ions/cm2,
(69.1o, 67.8o. 64.54o and 62o) for HSS1, HSS2, HSS3 and HSS4 samples respectively.
Impact of energetic H-ions with sample surface produces craters, surface roughness,
amorphization and other lattice imperfections. Surface roughness is also an important
parameter which impacts the surface wettability, as reported by some studies. Kubiak et-
al [57] measured variation in apparent contact angle with respect to average roughness
and they concluded that CA with stainless steel surface decreases up to a certain limit of
roughness, then it increases for more irregular surfaces. Kittu et al [43] performed
experiments on different materials and they also observed that the magnitude of contact
angle decreased by increasing surface roughness. More over their graphs demonstrated
that after a certain limit of surface roughness the contact angle increases, and they
showed concave up type of graphs in their study. In our experiment, the H ions are
smaller in size, and at energy 500 KeV they penetrate in the lattice without significantly
damaging the surface lattice, but if the ion dose is increased beyond a certain limit the
surface damage is profound [58] , because of ion recoiled ion and knocked out atoms.
76
Therefore along with chemical interactions of H- ions surface roughness is an additional
factor to improve surface hydrophilicity[59].
Fig:contact angle versus H+ ion dose
4.2.4.2 Raman Spectroscopy
In- vitro bioactivity of a material is usually evaluated by SBF [60]. Raman spectroscopy
is performed to investigate the nature of incubated species on prepared surfaces after 8
days of immersion of samples in SBF and to draw comparison between untreated sample
and (different doses of H) ion implanted samples and to have a quantitative assessment of
precipitated species. Raman spectroscopy is performed using DXR Nicolet Thermo
scientific as mentioned previously. Figure 10 shows Raman spectra of four treated
samples and one untreated sample. Raman profiles reveal the presence of all the
representative peaks of HA in untreated and prepared samples , υ1, υ2, υ3 and υ4 of PO4
group of HA are present at 910 cm-1,390 cm-1, 1050 cm-1 and 610 cm-1 respectively, CO3
bond is observed at 1170 cm-1 [30]. It is found that peak intensity of precipitated HA is
proportional to H ion dose given to the surface of sample. Growth of HA in untreated
sample is lowest while maximum growth in highest ion implanted sample HSS4 after the
same time of immersion in equal quantity of SBF. Improved growth of HA is attributed
0.0 2.0x1014
4.0x1014
6.0x1014
8.0x1014
1.0x1015
62
64
66
68
70
72
74
co
nta
ct a
ng
le (
de
gre
e)
H ion dose/cm2
Control Sample
HSS1
HSS2
HSS3
HSS4
77
to presence greater number of H ions in near surface region which enhances the chances
of H- bonding.
3600 3200 2800 2400 2000 1600 1200 800 400
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
Inte
nsity(a
.u)
Raman Shift (cm-1)
Control
HSS1
HSS2
HSS3
HSS4
Fig 10: Raman spectroscopy of sample surfaces after immersion in SBF
4.2.4.3 Mass of incubated species
Effect of H rich surface on HA incubation is also determined by the mass of incubated
species. The samples are weighed before immersion and after immersion (after drying)
using Ohaus Analytical Plus instrument (reliability 0.01mg) to determine the mass gained
by sample surface after immersion in SBF. The measurements are mentioned in table and
mass of incubated species is plotted against H ion dose in Figure 12. The Fig shows
increase in mass of HA by H ion implantation. Figure 3a also shows a macroscopic view
of the enhanced growth of HA by H ion implantation.
Fig 11: Macroscopic view of sample surface after immersion in SBF
78
0.0 2.0x1015
4.0x1015
6.0x1015
8.0x1015
1.0x1016
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
ma
ss o
f in
cub
ate
d H
A(g
m)
Hydrogen ions (ion/cm2)
ControlHSS1
HSS2
HSS3
HSS4
Fig 12: Mass of incubated HA versus H+ ion dose
Table4: Mass of sample before and after immersion in SBF after drying
Sample mass of Sample
before immersion
(gm)
mass of Sample
after immersion
and subsequent
drying (gm)
Difference/ mass
of incubated
species(mg)
1 untreated Sample 3.0082 3.0082 0
2 HSS-1 2.1452 2.1452 0
3 HSS-2 2.3658 2.3660 0.2
4 HSS-3 2.2653 2.2657 0.4
5 HSS-4 2.5880 2.5885 0.5
4.2.4.4 in- vitro Cell Viability Studies
Biocompatibility of all ion implanted stainless steel samples is investigated by using
cultured human oral fibroblasts (passage 4) and cellular response is also observed by
using standard MTT assay protocol. Samples with 5mm in height and 1cm in diameter
are prepared and sterilized by using autoclave (15 min at 121°C/ 15 psi). Similarly, cells
are cultured in DMEM media supplemented with 5 % of FCF serum, 1% penicillin /
streptomycin, 1% glutamine and 0.2% amphotericin (Sigma Aldrich UK). Moreover,
cells are allowed to confluent (100%) over the surface of tissue cultured plate and
79
detached by using trypsin EDTA (Sigma Aldrich, UK). Cells are seeded into wells of 24
well plate containing test sample seeding density of 2.0 x 104 cells / ml. A material and
non-material control (positive) and (negative) are introduced for the direct comparison
respectively. The cells are incubated at 37°C in a 5 % CO2 atmosphere for 24 hrs.
However, for quantitative measurement MTT assay is performed individually on all
prepared samples. 1 ml of MTT solution is aseptically added to each well and left for
incubation at 37°C for 40 min. Cells are then lysed with Isopropanol. The intensity of
colored solution was measured by using a photo spectrometer at a wavelength of 570 nm.
Cell viability of untreated sample and hydrogen ions implanted samples is studied
quantitatively with standard MTT assay protocol. Fig 13 shows percentage cell growth
versus H implanted samples with respect to positive reference (100%). All the treated
samples show cell viability more than 100%, maximum cell viability is observed about
192% in HSS1 likewise HSS2 and HSS3 also exhibit comparable cell viability of 170%
and 177% respectively, the untreated sample exhibits cell viability 141%. Least cell
viability is observed for highest H ion dose (1015 ions/cm2) of HSS4 which is 122.2%. H
implantation in near surface region makes the surface friendlier for hydrogen bonding
which consequently enhances hydrophilicity, HA precipitation and cell viability. Some
studies suggests that appropriate hydrophilic surfaces are often biocompatible [40, 61]
Surface topography is an important factor which determine biomedical response of
surface. Many studies has been conducted to understand the relation of protein
absorbance, cyto- compatibility and other cell responses with surface roughness [62-64].
HSS4 contains roughest surface among the entire sample under observation because it is
most heavily bombarded with H ions. Zareidoost et al claimed higher surface roughness
produce lower cyto- toxicity and better biocompatibility [65], a typical spindle like shape
of oral fibroblasts in all the treated samples is observed and it is observed that in treated
samples the cells are able to grow relatively longer [66, 67].
80
Fig 13: Bar graph of cell growth in different samples
4.4 Effect of carbon ion implantation in nitinol lattice
Several studies reveals that carbon ion implantation in material lattice improves corrosion
resistance and some mechanical properties of material [68, 69], in the first experiment
carbon ions were implanted in nitinol, the main objective of experiment was to study
variation in ion leakage by varying implanted ion dose.
4.4.1 Overview
Nitinol has been admired for super elasticity and shape memory effects. These two
properties make this alloy useful for number of biomedical applications such as
vascular stent, dental implant and bone fixation plates in orthopedics. But there are
some serious issues with this material and one of them is in vivo corrosion and toxic
Nickel release [70]. The released nickel causes many undesired effects like allergy, in
vivo cell death and disordered bone growth [71]. Several experiments were performed to
passivate the surface of this alloy and to build a barrier to prevent Ni escape from the
surface. Liu et al[72] studied the effects of multilayered Ti/TiN or single layered TiN
films on corrosion resistance of NiTi alloy in artificial saliva. Yeung et al [73]
treated NiTi alloy with nitrogen and oxygen using plasma immersion ion
implantation(PII I) and they concluded that nitrided samples were better for orthopedics
because they observed better degree of cell proliferation and lesser nickel ion release
0
50
100
150
200
250
HSS 1 HSS 2 HSS 3 HSS 4 Positive
Cel
l gro
wth
in p
erce
nta
ge
Hydrogen Ions Implanted Stainless Steel Samples Cell Viability Results with MTT Assay
81
from the surface. Zhao at al [74] also selected PIII to fabricate graded surface layers
of TiN and TiC on NiTi alloy. They observed consistent hydrophilicity but improved
surface roughness, improved corrosion resistance and better cell adhesion and
proliferation. Pogrebnjak et al [75] studied the effect of high dose implantation on nitinol.
They found increase in wear resistance, nano-hardness and corrosion resistance. Bulk
properties like pseudo plasticity and shape memory effect remained invariant. Green et al
[76] amorphized the surface of nitinol by N+ ion implantation and by controlled shot
peening. They found N+ ion implanted Ni-Ti contained a TiN phase within the surface
which reduced wetting. Lee et al implanted nitinol surfaces with high dose of boron up to
1017ions/cm2, they concluded that surface hardness of boron implanted nitinol exceeds
the surface hardness of stainless steel[77].
In this paper both ion implantation and oxidation layer coating are employed to
modify the surface of NiTi alloy. The samples are then analyzed using Fourier
transformed infrared spectroscopy (FTIR),x-ray diffraction (XRD), proton induced x-
ray emission (PIXE), hardness test and electrochemical corrosion potential.
4.4.2 Ion implantation and heat treatment of samples.
An oxidation layer is developed on the samples after annealing in muffle furnace. Out of
four prepared samples oxidation layer of one sample is removed by polishing the sample
using metal polish paste and subsequent cleaning in acetone. Ions are implanted using
Pelletron accelerator, having terminal voltage capacity 2MV in accelerator lab of GC
University (CASP) Lahore. Pelletron accelerator facility (2MV) is used for ion
implantation. Four samples are exposed with a beam of C + ions having 0.75MeV energy
for different intervals of time. The dose of each sample is calculated by using current of
the beam and exposure time. Sample 1, sample 2, sample 3 and the sample without
oxidation layer exposed to 2.4×1012, 8×1013, 8.32×1013and 3.32×1014ions /cm2
respectively.
4.4.2.1 Immersion of samples in SBF and Sample Preparation for PIXE Analysis SBF is prepared as proposed by Kokubo and his colleagues[78]. C+ implanted NiTi wires
are immersed in SBF for 150 days in order to provide enough time for ion leakage.
82
Residue in the SBF is dried at 35oC. This powder is then analyzed for presence of Ni ions
and remaining number of Calcium and Phosphate ions.
4.4.3 Results and Discussion
4.4.3.1 XRD Analysis X-Ray diffraction studies are performed to study the impact of ion implantation and
oxidation layer on crystallinity of nitinol surface. XRD profiles of four fabricated
samples are shown in figure 14, starting from least dose (≈2.4×1012ion/cm2) to maximum
(≈3.3×1014ion/cm2) and a profile of nitinol wire without oxidation layer and implanted
with the same dose (≈3.3×1014ion/cm2).
Maximum crystallinity has shown by the sample with minimum ion dose and by the
sample without oxidation layer. Crystallinity decreases (amorphization) by increasing the
dose. The fourth sample which is without oxidation layer and treated with maximum dose
has crystallinity comparable to the sample with least dose. Amorphization produced in
samples is due to hammering effects of implanted ions[79].
20 30 40 50 60 70 80
0
100
200
300
400
500
inte
nsi
ty(a
u)
2theta(degree)
2.4*1012
ions/cm2
8*1013
ions/cm2
3.3*1014
ions/cm2
3.3*1014
ions/cm2
witout oxidation layer
Fig 14: XRD Spectra of four Samples with doses
We know that the atomic radius of carbon (≈70pm) is smaller than both Ni (≈124pm) and
Ti (≈170pm). Substitutional settlement of C+ ions cannot increase the unit cell volume.
As peak shifting in samples 2 and 3 toward left indicates the increased volume of unit cell
that is due to interstitial accumulation of C+ Invariant peak position of sample 4 is evident
83
of lesser penetration due to harder surface. Peak shift in samples 2 and 3 also shows the
presence of stresses in the lattice due to penetration of ions. Oxidation layer and
implanted C+ are responsible for surface amorphization.
4.4.3.2 FTIR Analysis FTIR Analysis Fourier transformed infrared (FTIR) analysis is performed to analyze the
nature of functional groups accumulated on the substrate. Fig 15 shows the FTIR profiles
of different samples. Samples 1, 2 and 3 show phosphate absorbance peaks at wave
number approximately of 1000cm-1[80]. The fourth sample without oxidation layer does
not carry any representative peak of hydroxyapatite. These results can be confirmed from
PIXE analysis as well[81], which is given in next section.
500 1000 1500 2000 2500 3000
98
100
102
104
106
108
110
112
Inte
nsi
ty (
a.u
)
Wave Number (cm�-1)
2.4*1012
ions/cm2
8*1013
ions/cm2
3.3*1014
ions/cm2
3.3*1014
ions/cm2
without oxidation layer
Fig 15: FTIR spectra of four samples, revealing hydroxyapatite incubation on three
sample except the one without oxidation layer.
4.4. 3.3 PIXE Analysis
Proton/Particle induced X-Ray emission (PIXE) analysis is performed to estimate the
number of nickel ion released in simulated body fluid (SBF) and to calculate the number
of phosphate and calcium radicals left in SBF after 150-day incubation of hydroxyapatite
on as prepared samples. Remaining calcium and phosphate ions in SBF can reveal the
deposited calcium and phosphate ions on prepared samples. Proton beam of 3.8MeV
energy is used for PIXE analysis. Fig 16 shows normalized nickel release in SBF with
respect to ions/cm2. Ions release from the sample 4 is maximum with the same dose of
84
ions as sample 3. Nickel release decreases by decreasing of implanted ion dose.
Implanted ions in nitinol produce dual effect on the surface of the substrate: firstly they
cause surface damage by producing void and channels resulting in an increased surface
area; secondly, the amorphization is also achieved by implanted ion. SRIM calculations
show that at 0.75MeV energy one C+ ion can produce about 722 vacancies in the lattice.
Damage produced on the surface provides additional degrees of freedom for ion leakage.
The competing factor surface amorphization can prevent ion escape from the lattice. Fig.
16 shows the competition of both mentioned factors compete up to a limit then the ion
escape factor becomes more dominant as the dose is increased from a certain limit
(9×1013ions/cm2), which results in a steeper graph. Other positive effects of C+
implantation and oxidation layer are clear from Fig. 6 that calcium and phosphate
incubation is greater on heavily implanted species. Least number of Ca and P species is
found in the solution in which sample 3 is immersed which conversely indicates
maximum deposition. Minimum apatite growth and maximum nickel release are
observed by the same sample without oxidation layer.
3.0x1013
6.0x1013
9.0x1013
1.2x1014
1.5x1014
0.00000
0.00001
0.00002
0.00003
0.00004
0.00005
0.00006
0.00007
no
rma
lize
d n
ickle
io
n r
ele
ase
ions/cm2
without oxidation layer
Fig16: PIXE study of Ni ion release, Normalized graph Nickel release versus ion/cm2
85
Fig17: PIXE spectra of four samples
0.0 3.0x1013
6.0x1013
9.0x1013
1.2x1014
1.5x1014
0.00
0.01
0.02
0.03
0.04
0.05
0.06
no
rma
lize
d r
em
ain
ing
co
nce
ntr
atio
n
of C
a a
nd
P s
pe
cie
sin
SB
F
ion/cm2
p
Ca
witout oxidation layer
Fig 18: PIXE study of Ca and P ions transferred from SBF to substrates, Normalized
graph between remaining Ca and P ions in SBF versus ion/cm2
4.4. 3.4 Corrosion potential and passivation time
Four samples are chosen to study the behavior of fabricated samples toward corrosion
tendency. Sample1 (dose=2.4×1012ions/cm2), sample 2 (dose=8×1013 ions/cm2), un-
annealed sample (dose=3.3×1013ions/cm2), and the sample without oxidation layer
(dose=3.3×1013ions/cm2). The corrosion potential versus time graph of these four
samples is shown in Fig 19 Sample 1with minimum implanted carbon ions has maximum
initial corrosion potential among all the other that is more than +6VSCE. The corrosion
potential then increases with time toward a saturation point that is approximately 12VSCE
86
for Sample 2 with greater number of implanted ions (8×1013ions/cm2) and
correspondingly has greater surface damage due to greater ion exposure. The sample is
initially more reactive in ringer lactate solution as compared to sample1.This is due to
larger exposed surface area. The surface area is increased due to voids and damages
produced on the surface by C+ ion bombardment and consequently greater local corrosion
on wider surface area. Similar initial behavior is observed for sample3 with maximum
number of implanted ions, maximum damage and largest number of voids resulting in
highest initial activity. Right after the initial activity on the surface the passivation starts
and all the samples attain the saturation point. These three samples show the same linear
behavior with time. The fourth sample whose oxidation layer is removed exhibits
peculiar behavior starting from +ve potential= +2VSCE and stays on the same value
throughout the observation. There is no increase in corrosion potential, no saturation
value and no passivation like sample1, Sample2 and sample 3. This shows that oxidation
layer in other three samples is responsible for initializing and catalyzing the passivation
and lesser damage depth in sample4.Three samples are initially active and the activity
decreases as the vacant sites (voids due to damage) seal themselves with oxidation
species resulting in greater corrosion potential. No oxidation layer in fourth sample and
lesser penetration depth due to harder surface which does not appreciate passivation [82].
Fig 19: Corrosion potential versus Time, graph showing passivation with time.
87
4.4.3.5 Hardness Test
Micro-hardness of samples is measured using Vickers hardness tester at a maximum load
of 200g. The hardness profile shows a linear increase in hardness by increasing
ions/cm2except the sample without oxidation layer. The slope of the graph is steeper
initially up to 9 x1013ions then the slop decreases. In other words, less increase in
hardness per unit ion occurs after 9 x1013 ions/cm2. Similar hardness behavior is also
reported by Naveed et al [45]. They found proton irradiated un-annealed nitinol gets
harder with greater rate per unit ion initially and then lesser increase for higher doses, that
is due to increased surface damage, wear and tear.
0.0 3.0x1013
6.0x1013
9.0x1013
1.2x1014
1.5x1014
200
220
240
260
280
300
320
340
360
380
400
420
440
ha
rdn
ess
ions/cm2
without oxidation layer
Fig 20: Hardness profile of fabricated samples (Hardness VS C+ ions/cm2)
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Chapter 5
5. Conclusion and future work
Several experiments are performed to improve the performance of currently used,
conventional biomedical materials (stainless steel, nickel-titanium alloy) and potential
candidate materials (iron and magnesium) for future biodegradable implant application.
The modified materials are tested for their potential applications and related issues, such
as surface modified nickel titanium alloy is tested for nickel ion release in simulated body
fluid because the toxic ion escape from the surface of nickel titanium alloy is an issue.
The modified stainless steel samples are characterized corrosion, cell viability and
wettability properties because the stability and improved hydroxyapatite incubation is
desired for stainless steel.
Out of all the performed experiments only three experiments are reported in presented
thesis and to the international journals. Some of the experiments are in the pipeline
(under the process of write up and characterization), and many of the experiments are not
found suitable to report due to adverse effects of ion implantations.
5.1 Conclusions In the first experiment singly charged, monoatomic nitrogen ions are accelerated under
250 KV potential and implanted in stainless steel surface subsequently. The effect of
nitrogen ion implantation on the surface of stainless steel is investigated using different
techniques. XRD profiles show improved incubation of HA over the surface in higher ion
dose samples. Raman spectroscopy describes greater peak intensity of HA functional
groups by greater ion implantation, XRD and Raman spectroscopy results are confirmed
by SEM profiles, these profiles show greater surface area of samples is covered with HA
in greater ion implanted samples. Human oral fibroblasts response shows enhanced cell
viability by ion implantation. Therefore improved biomedical properties are observed in
prepared sample.
In the second experiment, stainless steel surfaces are modified using an energetic beam of
H ions with the help of particle accelerator by using various ion doses. The effect of
increasing ion dose is observed on different properties of stainless steel.
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It is observed that:
Hydrophilicity of surface is found increasing with H ion dose.
Greater HA incubation is observed on treated samples.
Treated surfaces are found more compatible for cell growth as observed in cell viability
profiles of samples.
Nickel titanium alloy was modified by accelerated carbon ion implantation. Singly
charged monoatomic carbon ions were accelerated under 0.375 MV of potential, 0.75
MeV ions are implanted in nickel titanium alloy. The prepared samples are evaluated for
stability (corrosion resistance), hydroxyapatite incubation, hardness and nickel ion
release in simulated body fluid. Hydroxyapatite incubation and passivation (increase
in corrosion potential VSCE) is not observed at all in the sample without oxidation
layer. Maximum nickel release is also observed from the same sample.
Increasing ion dose in the samples produces following effects
Lesser crystallinity and increased volume of the unit cell which is attributed to the
interstitial accumulation of C +ions in the lattice.
Greater initial reactivity in ringer lactate solution, but all the samples saturate at
same Level of corrosion potential (VSCE= 12V) after the same interval of time
(≈450seconds).
Ni release and calcium phosphate incubation increase insignificantly up to
8×1013ions/cm2 and then increase per unit ion
FTIR and PIXE analysis are in agreement to confirm no incubation of
hydroxyapatite in the sample without oxidation layer.
Hardness increases with greater rate up to 8×1013ions/cm2 and then lesser increase
/ion and hardness is maximum for the sample without oxidation layer.
5.2 Future work
Biodegradable materials both polymeric and metallic are being considered as potential
candidate for biomedical applications. The advantage to use biodegradable material is to
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avoid the second surgery, the second surgery is normally performed to remove implanted
material. The elimination of a surgery from the therapeutic process may potentially
reduce the risk, complications and cost of the process. Therefore biodegradable materials
may be considered for future studies.
Iron and magnesium are the favorite materials of researchers due to their generic property
of biodegradability. But there are issues with both iron and magnesium, the issues include
rapid degradability of magnesium and gentle degradability of magnesium, the problem
may be attempted to cater in different ways.
Some new materials may be searched for the purpose.
Some composites may be prepared to have desired set of properties.
Surface modification techniques may be applied to tailor the surface properties.
Ion implantation is a versatile technique to tailor the surface properties.
List of Publications
Muhammad Ahsan Shafique, G. Murtaza, Shahzad Saadat, Muhammad K H
Uddin & Riaz Ahmad. Improved cell viability and hydroxyapatite growth on
nitrogen ion-implanted surfaces.Radiation Effects and Defects in Solids 172, 7-8,
(2017)
Muhammad Ahsan Shafique, G Murtaza, S. Saadat, Z. ZAHEER, M.
Shahnawaz, M. KH UDDIN, RIAZ AHMAD.STUDY OF NICKEL ION
RELEASE IN SIMULATED BODY FLUID FROM C+-IMPLANTED NICKEL
TITANIUM ALLOY, Surface Review and Letters. 1650045(2016).
Muhammad A Shafique, R Ahmad, Ihtesham Ur Rehman: Study of wettability
and cell viability of H implanted stainless steel. Mater. Res. Express 5 036509
(2018).
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MA Shafique, SA Shah, M Nafees, K Rasheed, R Ahmad. Effect of doping
concentration on absorbance, structural, and magnetic properties of cobalt-doped
ZnO nano-crystallites. International Nano Letters 2 (1), 31.
SA Shah, A Majeed, MA Shafique, K Rashid, SU Awan. Cell viability study of
thermo-responsive core–shell superparamagnetic nanoparticles for multimodal
cancer therapy. Applied Nanoscience 4 (2), 227-232.
M Nafees, W Liaqut, S Ali, MA Shafique. Synthesis of ZnO/Al: ZnO
nanomaterial: structural and band gap variation in ZnO nanomaterial by Al
doping. Applied Nanoscience 3 (1), 49-55(2013).
M Nafees, S Ali, S Idrees, K Rashid, MA Shafique. A simple microwave assists
aqueous route to synthesis CuS nanoparticles and further aggregation to spherical
shape Applied Nanoscience 3 (2), 119-124 (2013).
G Murtaza, R Ahmad, MS Rashid, M Hassan, A Hussnain, MA.
Shafique.Structural and magnetic studies on Zr doped ZnO diluted magnetic
semiconductor. Current Applied Physics 14 (2), 176-181 (2014)
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