chapter 1: introduction 7 chapter 2: objectives and scope
TRANSCRIPT
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INDEX
ABSTRACT ................................................................................................. 3
Resumen ................................................................................................... 3
RESUM ...................................................................................................... 3
ACKNOWLEDGEMENTS ................................................................................ 5
Chapter 1: INTRODUCTION .................................................................. 7
Chapter 2: OBJECTIVES AND SCOPE ..................................................... 9
Chapter 3: BACKGROUND ................................................................... 11
3.1. Biomaterials ............................................................................... 11
Biocompatibility .................................................................... 11
Historical development ........................................................... 12
Biological response ................................................................ 13
3.2. Titanium as a biomaterial ............................................................. 14
Physicochemical properties ..................................................... 15
Titanium and dental implants .................................................. 16
3.3. Infection .................................................................................... 17
Bacterial biofilm .................................................................... 18
Antifouling coatings ............................................................... 18
AMPs ................................................................................... 21
Chapter 4: MATERIALS AND METHODS ............................................... 22
4.1. Overview ................................................................................... 22
4.2. Sample preparation ..................................................................... 23
Cutting ................................................................................ 24
Mounting .............................................................................. 24
Grinding and polishing ........................................................... 25
Ultrasonic cleaning ................................................................ 27
4.3. Plasma surface activation ............................................................. 29
4.4. Amine-Terminated PEG Electrodeposition ....................................... 30
Circuit diagram ..................................................................... 30
Electroplating conditions ........................................................ 34
Physicochemical Characterization ............................................ 36
4.5. Functionalization with AMPs: Magainin II and Lactoferrin 1-11 .......... 39
Physicochemical characterization and comparative AMP ............. 42
4.6. Biological characterization ............................................................ 42
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Cytotoxicity .......................................................................... 42
Bacterial adhesion ................................................................. 43
Fluorescence......................................................................... 43
Chapter 5: RESULTS AND DISCUSSION ............................................... 45
5.1. Electrodeposition characterization ................................................. 45
Experimental set-up and faced setbacks ................................... 45
Resistance variation............................................................... 49
Roughness ........................................................................... 50
Thickness ............................................................................. 52
Covered area ........................................................................ 56
Free amines presence ............................................................ 57
Contact angle ....................................................................... 58
ATR-FTIR ............................................................................. 59
XPS ..................................................................................... 62
SEM .................................................................................. 63
5.2. Cytotoxicity ................................................................................ 65
5.3. Adhesion assays ......................................................................... 65
5.4. Fluorescence .............................................................................. 68
Chapter 6: CONCLUSIONS ................................................................... 70
Chapter 7: REFERENCES ..................................................................... 72
ANNEX 1: Citotoxicity assay protocol ........................................................... 78
ANNEX 2: Bacteria assay protocol ............................................................... 80
ANNEX 3: Budget ..................................................................................... 81
ANNEX 4: Enviromental impact................................................................... 86
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ABSTRACT
Dental implants are commonly used as a replacement of native tooth. However,
bacterial infection can be decisive in the success or failure of the implant.
Therefore, with the aim of reducing the risk of infection, an antifouling coating has
been developed. Polyethylene glycol (PEG) has been electrodeposited on to
titanium surface and carried to study. PEG-amine electrodeposition has been
optimized after surface characterization and bacterial assays.
As a complement after electrodeposition, samples have been treated with two
different antimicrobial peptides, Lactoferrin 1-11 and Magainin-2, by means of two
bonding methodologies.
RESUMEN
Hoy en día es muy común el uso de implantes dentales como un reemplazo de la
dentadura natural. Sin embargo, las infecciones bacterianas constituyen un factor
decisivo en el éxito o fracaso del implante.
Por este motivo, se ha desarrollado un tratamiento antifouling. Se ha
electrodepositado Polietileglicol (PEG) en superficies de titanio. La
electrodeposición de PEG-amina ha sido caracterizada y sometida a ensayos de
bacterias, con la consiguiente optimización del recubrimiento.
Complementando el tratamiento, tras la electrodeposición de PEG-amina, las
muestras fueron tratadas con dos péptidos antimicrobianos distintos, Lactoferrina
1-11 and Magainina-2, por medio de dos metodologías de enlace.
RESUM
Avui dia és molt comú l'ús d'implants dentals com a reemplaçament de la
dentadura natural. No obstant això, les infeccions bacterianes constitueixen un
factor decisiu en cuant a l'èxit o fracàs de l'implant.
Per aquest motiu, s'ha desenvolupat un tractament antifouling. S’ha
electrodepositat Polietileglicol (PEG) en superfícies de titani. La electrodeposició de
PEG-amina ha estat caracteritzada i sotmesa a assajos de bacteris, amb la
consegüent optimització del recobriment.
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Complementant el tractament, després de l’electrodeposició de PEG-amina, les
mostres van ser tractades amb dos pèptids antimicrobians diferents, Lactoferrina
1-11 and Magainina-2, per mitjà de dues metodologies d'enllaç.
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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ACKNOWLEDGEMENTS
I wish to express my sincere thanks to Dr. Daniel Rodríguez, my TFG tutor, for trusting me, for giving
me the freedom to make decisions and providing me with all the necessary facilities for the research.
I place on record, my sincere thank you to Judit Buxadera, for the encouragement and attention who
has shown, for holding my hand when I was in trouble and giving me the keys to see always the see
the glass half full.
I am extremely grateful to the BIBITE members for the support shown, for making me feeling at home
and welcoming me always with a smile.
I take this opportunity to express gratitude to Elia Vidal and Quim, who helped me when I thought
there was no options in the crossroad.
I am also grateful to Zhitong, because of those afternoons waiting for me until I finished my work
before leaving.
I also place on record, my sense of gratitude to my parents, for supporting me, standing my stress
moments and encouraging me in the distance; without them I definitely would not have been able to
take part in this venture.
To sum up, I would also like to thank those who direct or indirectly have helped me in the fulfillment of
this project.
Thank you everyone.
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CHAPTER 1:
INTRODUCTION
Currently, tooth loss is a common problem which can be solved by means of dental
implants. Success or failure of implants depends on the health of the person
receiving it and the tissues in the mouth, a proper osseointegration and the
biocompatibility of the biomaterial used for the implant, being bacterial infection
the most extend cause of failure. In this way, by decreasing the risk of infection,
it will also decrease the probability of failure of the implant.
Regarding biomaterials, one of the most important properties in terms of reducing
the rate of infection is the reduction of bacterial adhesion. In this stage,
Polyethylene glycol (PEG) is an antifouling polymer which contributes positively to
this purpose. By using the adequate methods, an immobilization of this molecule
to a metal surface (Ti in our case) is able to present a high rate of inhibition in the
adsorption of proteins. Likewise, the immobilization of antimicrobial peptides
(AMPs), which are part of the innate immune response found among all classes of
life can help to improve its effectivity.
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CHAPTER 2:
OBJECTIVES
AND SCOPE
In this study, amino-terminated PEG (PEG-amine) has been immobilized on
titanium surfaces by means of electrodeposition. Process optimization has been
studied for pulsed and non-pulsed regime, electrodeposition time and voltage. The
immobilization procedure and chemical bonding condition have been characterized
for the amine-functionalized PEG surfaces. The characterization methods
employed include Interferometry, contact angle, Attenuated Total Reflectance, X-
ray Photoelectron Spectroscopy, Scanning Electron Microscope and Ultraviolet-
Visible spectrometry.
Complementing the treatment, two different antimicrobial peptides (AMPs) have
been brought to assessment, Lactoferrin 1-11 (LF1-11) and Magainin-2 (MG-2),
by means of two different methodologies. Thus, amine-functionalized PEG surfaces
have been treated by either crosslinking with N-Succinimidyl 3- Maleimide-
Propionate (SMP) or carboxylic acid activation with EDC and sulfo-NHS to bond the
AMP to the PEG.
Afterward, the antifouling treatment and a combination of both methods, PEG-
amine electrodeposition and AMPs, have been tested. In this way, biological
response in hFFs human cells, bacterial adhesion of Staphylococcus Aureus,
fluorescence and different methods of characterization as for PEG-amine
electrodeposition have been studied.
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CHAPTER 3:
BACKGROUND
3.1. Biomaterials
A variety of devices and materials are used in the treatment of disease or injury.
There are several definitions of “biomaterial”. One of the most commonly used is:
“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’’
American National Institute of Health, 2005
Biomaterials can be metals, ceramics, polymers, glasses, and composite materials.
Such materials are used as molded or machined parts, coatings, fibers, films,
foams and fabrics.
Biomaterials can arise from nature or be synthesized in the laboratory using a
variety of chemical approaches. They are used in different functions either
biopassive, like being used for a heart valve, or bioactive with a more interactive
functionality such as hydroxy-apatite coated hip implants. Biomaterials are also
used every day in dental applications, surgery, and drug delivery [4].
Biocompatibility
A complementary definition needed to understand an important aspect of
biomaterials is that of "biocompatibility":
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“Biocompatibility is the ability of a material to perform with an appropriate host
response in a specific application”
Williams, 1987
Since the primordial requirement when choosing a biomaterial is the approbation
of the body, the implant is expected to converge with the tissues, presenting as
less as possible inflammation or toxicity, which would affect to the quality of life
of the patient or even could finish in the failure of the implant.
Historical development
The field of biomaterials is not as new as it could seem. The first biomaterials used
were gold and ivory for replacements of cranial defects. This was as early as 4000
years back, when the Egyptians and Romans used linen for sutures, gold and iron
for dental applications and wood for toe replacement but with very little knowledge
about the problem of corrosion.
Figure 1: Biomaterials for human application [4]
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Celluloid was the first man-made plastic used for cranial defects and polymethyl
methacrylate (PMMA) was one of the first polymers accepted since World War II.
Furthermore, Nylon, Teflon, silicone, stainless steel and titanium were some of the
other materials which were put into use after World War II [1][4].
Nowadays, with the availability of better diagnostic tools and surgical procedures
as well as advancements in the knowledge on materials, medical devices have
assumed widely acclaimed significance. Thus, bioimplants are regularly used in
dentistry, orthopedics, plastic and reconstructive surgery, ophthalmology,
cardiovascular surgery, neurosurgery, immunology, experimental surgery and
veterinary medicine.
Biological response
When characterizing a biomaterial, paying attention to the biological response is
highly important. The implantation of a material implies a microscale reaction
between its surface and the environment cells, whereas the interaction of the
system determines the viability of the medical device.
Host reactions following insertion of the medical device, as shown in figure 3,
comprise [1][2]:
Injury. The first deed after the implantation bring out an injury to tissues
or organs, which apart from stimulating innate immunity, it also leads to
thrombus genesis involving activation of the coagulation systems through
blood-material interactions and finally, a provisional matrix formation.
Acute inflammation, which is able to take hours or up to a week depending
on the level of injury caused surrounding the implant.
Chronic inflammation. The continuous inflammatory stimulus results into
a chronic inflammation. It is differentiated by the action of monocytes,
lymphocytes macrophages within the proliferation of blood vessels and
connective tissue.
Figure 2: World's first prosthetic limb found on 3,000-year-old Egyptian mummy [64]
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Granulation tissue development. The intervention of macrophages,
fibroblasts proliferation and vascular endothelial cells in the new healing
tissue are identified, reaching the peak of healing inflammation, within the
creation of granulation tissue.
Fibrous capsule development and foreign body reaction. Granulation
tissue ends up in a fibrous capsule formation which is drawn apart from the
biomaterial by the cellular components of the foreign body giant cell
formation. The foreign body reaction, might keep on at the tissue-implant
interface for the lifetime of the implant.
3.2. Titanium as a biomaterial
First trials of using titanium as a biomaterial in implant devices date back to the
1930s. The degree of tolerance was similar to stainless steels and cobalt alloys.
Its better biocompatibility, high corrosion resistance and mechanical resistance,
ensures a good load transmission and mechanical stiffness close to bone
throughout the timeline of the implant.
Commercially pure titanium (Ti cp) is one of the most common titanium base
implant biomaterials, together with Ti-6Al-4V (ELI) alloy. Determined as
biologically inert when being implanted into host bodies, it is highly tolerated by
the human tissues and tend to remain against to adverse reactions [40].
Figure 3: Sequence of events in the host
following material implantation. Adapted. [2]
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Physicochemical properties
Titanium is an allotropic metal (with more than one crystalline structure)
presenting a hexagonal 𝛼 phase (HCP) at room temperature and a cubic β phase
(BCC) further up 1155K (882ºC).
The main alloying elements influencing its mechanical properties are N, C, O, Fe
and H (table 1). Its biocompatibility is caused by the formation of an oxide film,
TiO2, over its surface. This oxide is a robust and stable coat that grows
spontaneously in contact with air and prevents the diffusion of the oxygen from
the atmosphere. Therefore, the oxide coating gives corrosion resistance to
titanium.
Table 1: Chemical composition of Ti commercially pure (ISO 5832-2).
Ti cp
Chemical composition
N C O Fe H Ti
Grade 1 0.03 0.10 0.18 0.20 0.0125
Balance
(0.989 -
0.996)
Grade 2 0.03 0.10 0.25 0.30 0.0125
Grade 3 0.05 0.10 0.35 0.30 0.0125
Grade 4 0.05 0.10 0.40 0.50 0.0125
Besides, titanium is very light with a density of 4.5 g/cm3, a high melting point 1998K (1725ºC), a low thermal and electrical conductivity which means it is a
good conductor of heat and electricity.
Table 2: Mechanical properties of Ti cp grade 2 (International titanium association,
2005 [52].
Strength
Ultimate
Tensile
Strenght,
UTS (MPa)
0.2% Yield
Strenght, YS (MPa)
Percent
elongation,
%EL
Minimum 345 275 20
Typical 438 352 28
In figure 4, ideal properties for a hard tissue replacement biomaterial are given.
Comparing with data placed in table 2 and preceding paragraphs, there is a clear
compatibility of Ti (and specially Ti cp grade 2) for hard tissue replacement.
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Titanium and dental implants
Dental implants have been commonly employed to replace missing teeth for the
last 40 years, but in recent years their use has been increasily widespread. A dental
implant works as an artificial tooth root that is attached to the jaw so as to hold
an artificial tooth. They are usually made from titanium or a titanium alloy.
Figure 4: Desired conditions for ideal hard tissue replacement [31]
Figure 5: Natural tooth vs Endosteal
implant [15]
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Two kinds of implants do exist:
Endosteal, surgically implanted directly into the jawbone. This is the most
generalized dental implant. Each implant holds one or more prosthetic
teeth. It is usually employed as an alternative for patients with bridges or
removable dentures. (fig.5)
Subperiosteal, on the peak of the jaw through the gum tissue, holding
directly the prosthesis. They are used in those cases with poor quality
and/or quantity of the jawbone.
Since the more used are endosteal implants, this study is focused in this type of
implants [13][14].
3.3. Infection
Although the application of dental implants has been improving since its
beginnings, there is still needed a huge body of research until getting dental
implants similar to natural dentition. In present state of knowledge, the most
susceptible element to infection is the periodontal tissue (the tissues surrounding
the implant).
Native tooth root surface is attached by fibers forming the periodontal ligament;
dental implants do not have fiber attachment though (fig.6). In place of fibers,
there is a tight tissue adhesion that makes easier that an inflammation becomes
bigger once appeared [55].
The inflammatory response is called periimplantitis (periodontitis for natural teeth)
and it is due to the growth of anaerobic organisms with the subsequent risk of
destruction of the bone and soft tissues supporting the tooth. Periodontitis causes
a resorption of the gum, receding from the implant and forming infected pustules.
This phenomeno may led to in a bone loss and failure of the dental implant [34].
Figure 6: (A) Natural bone. The gum tissue
fibers are attached to the tooth root surface.
(B) Dental implant. There is no fiber
attachment to the implant surface. [55]
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Bacterial biofilm
It is considered that more of 95% of bacteria existing in the Earth are in biofilms.
A biofilm is a well-organized, cooperating community of microorganisms formed
under liquid conditions, for example, dental plaque.
If bacterial plaque biofilm is accumulated, the gingival tissues will become inflamed
and as said before, inflamed tissue is less resistant to bacteria or food particles
entering into the space created. Then, a close circuit starts and bacteria have the
potential to destroy tissue and bone unable to be self-repaired.
Staphylococcus aureus
A strain of Staphylococcus aureus has been studied in multiple assays in this research. It is a kind of gram positive anaerobe bacterium responsible of a multitude of diseases in humans.
It can be found in skin, mucus, membranes and the respiratory track and among the several diseases that it can cause, there are staphylococcal food poisoning, staphylococcal scalded skin syndrome, skin and soft tissue infections, septic arthritis, bacteremia or endocarditis [23].
Antifouling coatings
Antifouling surfaces are those that are able to be resistant to protein adsorption or
cell adhesion. Thus, antifouling coatings are used to prevent bacterial biofilm
formation pursuant to a biomolecules layer which are commonly proteins.
Figure 8: Staphylococcus aureus [51]
Figure 7: Bacterial biofilm formation on biomedical implant.
Adapted from Jiri Gallo, et al. (2014)
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PEG
Figure 9: Different polymers used in implantology [31]
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Poly-(ethylene glycol), PEG, is a synthetic polyether with hydrophilic properties
that inhibits the adsorption of proteins with mass weight (Mw) <100,000. If it had
a higher molecular weight it would pass to be called Poly-(oxy-ethylene), PEO. The
immobilization of PEG on a metal surface is an important step in making metal
surfaces biofunctional and also the bonding manner used to adhere PEG to the
implant surface.
The polymer used in this project is Poly(ethylene glycol) bis(3-aminopropyl) terminated (H2NCH2CH2CH2(CH2CH2O)nCH2CH2CH2NH2), which is a PEG
previously functionalized with a repeated chain and two amine terminals (NH2-PEG-NH2) (fig.10).
Both -NH2 terminals are free to react with the oxide layer of titanium (also
pretreated by plasma surface activation), paying attention to have a basic pH
solution.
Figure 10: Poly(ethylene glycol) bis(amine) [Sigma Aldrich]
Figure 11: Chemical bonding manner of the implant surface after plasma and PEG electrodeposition.
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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Apart from being hydrophilic, PEG is very soluble in water so that it acts such as a
soluble protein with a similar molecular mass but 5-10 times bigger. This property
confers the difficulty of protein adhesion and reduces its antigenicity (Ramón et
al., 2009).
AMPs
Antimicrobial peptides (AMPs) are an essential part of innate immunity that been
evolving throughout history in most living organisms trying to combat microbial
battle. An AMP are known as host defense peptides, due to marked activity they
do against bacteria, fungi, virus and parasites [3].
Then, by combining antimicrobial peptides with other treatments of biomaterials
in the implants branch, it would be possible to reduce the risk of infections and
improve even more the implants whose surface is already been modified with
satisfactory results.
As mentioned, in this study two different AMPs have been tested: Lactoferrin 1-11
(LF1-11) and Magainin-2 (MG-2).
Lactoferrin 1-11
Lactoferrin 1-11, hLF1-11 (GRRRRSVQWCA), is an AMP derived from the N
terminus of human lactoferrin, which shows several modulatory actions within
monocytes, intensifying their actions in innate immune response. hLF1-11 is
obtained from human lactoferrin [16].
Magainin-2
Magainin-2, MG-2 (GIGKFLHSAKKFGKAFVGEIMNS), is an 𝛼-helical AMP, which
means that forms toroidal pores such as groups in order to disrupt bacterial membranes. These natural peptides compete against lots of bacteria and fungi and helps inducing osmotic lysis of protozoa. MG-2 peptides are obtained from the skin
of the African clawed frog Xenopus laevis [65].
Figure 12: Xenopus laevis frog [19]
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CHAPTER 4:
MATERIALS AND
METHODS
4.1. Overview
In figure 13, a global sight in an orderly way of the contents and proceedings
effected this study can be observed.
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4.2. Sample preparation
Quality control of titanium surface and processing is highly important, above all
concerning its use in biomedical implants. The smoother the surface is, the more
security will be that the properties and behavior are caused because of the material
nature and not because of the surface porosity. This is why a proper metallography
of titanium plays an important role [5].
Ti (Grade 2)
Metallographic preparation
"Ti" samples carried to analysis
(used as control) Plasma surface activation
"Ti-plasma" samples carried to analysis PEG-amine
electrodeposition (method optimized in this
study)
"Ti-PEG" samples carried to analysis
Functionalization with AMPs
Method 1: Crosslinking
Lactoferrin 1-11
"Ti-LF1" samples carried to analysis
Magainin II
"Ti- MG1" samples carried to analysis
Method 2: NHS-EDC
Lactoferrin 1-11
"Ti-LF2" samples carried to analysis
Magainin II
"Ti- MG2" samples carried to analysis
Figure 13: Global sight of this study
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In the whole study, titanium commercially pure titanium grade 2 was used because
of its consideration in any application whereon formability and corrosion resistance
are important.
Cutting
Samples were cut from a 2 meter-long titanium bar of 10mm diameter. They were
cut with a thickness of 2 mm.
Mounting
The next step was mounting the samples in order to obtain a bigger structure
which fitted the polishing machine. Hot compression mounting with phenolic resin
(MultiFast bakelite) took place in a STRUERS LaboPress-3 machine onto 5
samples/montage.
Table 3: Parameters applied in compression mounting
Heating time (min) Cooling time (min) Force (N) Temperature
(ºC)
5 3 20 (4,5 lbs) 180
Figure 14: Ti sample before
metallographic preparation
Figure 15: Samples already mounted in the Bakelite
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Grinding and polishing
A methodic procedure was pursued, as shown in table 4, with a polishing machine
BUEHLER (Phoenix 4000, USA) (fig.15) that gave pretty good results, obtaining a
mirroring effect and reducing the mean roughness to a few 10-20nm. By this way,
posterior experiments would be done in similar conditions avoiding the different
surface textures as result of the cutting process. In its defect, either from
availability or from machine breakdown, a substitute polishing machine STRUERS
(Rotopol-31, Denmark)(fig.16) was used without mixing in the experiments the
samples obtained so as to avoid little differences in the surface affecting to the
result interpretation.
Table 4: Grinding and polishing parameters pursued
Cloth
Grain
size
(m)
Sense of
rotation
Speed
(rpm) Lubricant Force (N)
Time
(min)
Number of
iterations
(1 new
cloth/iteration)
P600 25 antiparallel
rotation 150 H2O
31,14
(7 lbs) 10 1
Figure 17: polishing machine BUEHLER (Phoenix 4000, USA)
Figure 16: STRUERS (Rotopol-31, Denmark)
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Cloth
Grain
size
(m)
Sense of
rotation
Speed
(rpm) Lubricant Force (N)
Time
(min)
Number of
iterations
(1 new
cloth/iteration)
P800 22 antiparallel
rotation 150 H2O
31,14
(7 lbs) 10 2
P1200 14 antiparallel
rotation 100 H2O
26,70
(6 lbs) 7 3
P2400 10 antiparallel
rotation 100 H2O
22,24
(5 lbs) 4,5 3
Velvet
(reusa
ble)
1 antiparallel
rotation 100
OP-S +
H2O
(distille
d)
13,34
(3 lbs) 15 1
As seen in table 4 above, in the final steps 250 ml of dripping OP-S (colloidal
silica) were applied together with distilled water, the last one applied every 60
seconds. If this final step was not made, the surface of the titanium sample would
exhibit a high scratched aspect (fig.18).
In this study, a total of 300 samples have been used in the different tests.
After being polished, samples were taken out from the bakelite by means of a saw,
as shown in figure 19.
Figure 18: Samples waiting for the OP-S last polishing step.
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Ultrasonic cleaning
The usefulness of ultrasounds for cleaning depends greatly on the nature of the
material to be cleaned. In this work, with the aim of avoiding contaminants or rest
of products in the titanium surface, a succession of parameters were applied after
each polishing procedure, as shown in table 5.
Figure 19: Saw of Bakelite
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Table 5: Ultrasonic cleaning methodology
Step Product Time (min) Function
1 Cyclohexane 10
Hydrophobic molecule which removes
rests of hydrophobic molecules and
contaminants, such as fats
2 Isopropanol 10
Organic molecule that works as a
solvent for waxes vegetable oils,
natural and synthetic resins
3 H2O (distilled) 10 It detaches either rests of the products
used before and others substances
4 Ethanol 10
Organic molecule useful in solving
polar and nonpolar substances (oils,
lubricants, etc.)
5 Acetone 10
Organic molecule which helps in
removing organic substances,
withdrawing them from the titanium
surface
The ultrasonic cleaning bath used was a SELECTA (4L cap., Barcelona)(fig.20).
Figure 20: Ultrasonic cleaning bath
Figure 21: Samples after metallographic preparation
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4.3. Plasma surface activation
With plasma surface activation, non-reactive or non-wettable surfaces can be
modified to obtain better reactive surfaces [44].
The plasma equipment used in the research was a Plasma System Femto (DIENER
Electronic, Germany) (fig.22), low pressure and temperature with a radiofrequency plasma formation (13,56 MHz). Likewise, the parameters applied figure at table 6 (Judit Buxadera, et al., 2014) [10].
Table 6: Conditions in the wall cavity of the plasma equipment.
Part of the
process Parameters
Pumping down 0,1 mbar, 1-3 min
Gas supply Argon, 5 min 0,4mbar
(10% deviation)
Plasma
100W, 0,4 mbar, 10
min (7-8
samples/process)
Venting 1 min, up to 999 mbar
Samples were put on petri dishes in the center of the reaction chamber (fig.23),
always in the same position, after having taken a reference point since the first plasma surface activation was made.
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4.4. Amine-Terminated PEG Electrodeposition
Taking Yuta Tanaka, et al. studies (2007-2010) as a reference, an antifouling
coating has been developed by the immobilization of Amine-Terminated PEG on
titanium surface with electrodeposition [56][57][58][59].
For monitoring a proper conductivity of titanium, electrochemical impedance
Spectroscopy (EIS) controls were made before and after each test, to check good electrical contact and dismissing any electrolyte leakage into the sample support
(D. Rodríguez Rius, 1999)[50] [37]. Avoiding leaks was very important because there was an iron spring putting pressure on the sample at the back, which would
shorcircuit the electrical circuit due to its lower resistivity.
Circuit diagram
Figure 23: Plasma
equipment
Figure 22: Plasma reaction chamber while working
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The scheme of the electrodeposition process and chemical reactions that took place is next (fig.24):
Electrolyte
The ionic solution was made by dissolving both PEG-amine and NaCl with distilled
H2O as a solvent.
The solution contained 2% PEG-amine and 0.3M of NaCl in 150ml H2O:
150𝑚𝑙 𝐻2𝑂 ∙ 2 𝑔 𝑃𝐸𝐺𝑎𝑚𝑖𝑛𝑒
100𝑚𝑙= 3𝑔 𝑃𝐸𝐺𝑎𝑚𝑖𝑛𝑒
150𝑚𝑙 𝐻2𝑂 ∙ 0.3𝑚𝑜𝑙
1000𝑚𝑙∙
58.443𝑔
1𝑚𝑜𝑙= 2.629𝑔 𝑁𝑎𝐶𝑙
Electrodes
Connecting the electric circuit up to the electrolyte there were three electrodes:
Counter electrode. The anode of the reaction consisted in a platinum
electrode.
Figure 24: Scheme of the
electrodeposition procedure. Adapted [36]
Patricia Carrasco Pividal
- 32 -
Working electrode. Samples worked as the anode in the electrolytic cell.
They were connected to the circuit by means of a holder (fig.26). Holder
and samples were screwed with a nut (fig.25).
Reference electrode. A saturated calomel electrode (SCE BAS Inc. Japan)
was used. The basic electrochemical reaction taking place in the electrode
is:
𝐻𝑔2𝐶𝑙2 + 2𝑒− <=> 2𝐻𝑔 + 2𝐶𝑙−
This electrode gives a known and constant voltage of 0.24V versus the
Normal Hydrogen Electrode (NHE), which is the potential of a platinum
electrode in 1N acid solution at standard conditions.
Figure 25: Support
Figure 265: Nut
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
- 33 -
Additionally, as the reaction of oxidation anode-cathode is light sensitive, it was
covered with aluminum foil to protect it from the room light.
Potentiostat
The voltage source employed was a potentiostat PARSTAT 2273 (Princeton Applied
Research, UK).
Figure 27: Set up
Figure 28: Potentiostat while operating
Patricia Carrasco Pividal
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Electroplating conditions
Initial testing
The first experiment of PEG-amine electrodeposition was done with 30 samples.
The potential before electrodeposition was 0.5V and once achieved a voltage was
applied with a constant sweep rate of -0.1V/s and kept at a continuous regime of
-5.0 VSCE vs counter electrode (Tanaka et al., 2007) [58]. This requires a 5V
command from de potentiostat.
As observed in Adriana Carreter studies (2014), after doing electrodeposition times
of 2, 5 and 120 minutes, the most optimal time was 5 minutes, as used in Tanaka
et al. research. Therefore, the electrodeposition time applied was 5 minutes.
Optimization of the Electrodeposition Parameters
Looking for an optimal electrodeposition procedure, pulsed vs continuous regime
were contrasted.
So as to adjust the potentiostat to the parameters desired in the experimental
design (DOE), it was necessary to put in a potential divider, a passive linear circuit
that produced an output voltage 10 times its input (fig. 29).
The resulting circuit can be seen either physical and schematically in figures 29
and 30.
Figure 29: External view of the potential divider
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
- 35 -
Final test equipment
On the occasion of a breakdown in the potentiostat, the final tests with the optimal
conditions applied had to be done with a kind of an alternative potentiostat. It
consisted of 3 electronic devices which were: a function generator 3314A (Hewlett
Packard, USA), an oscilloscope DSO1052B (Agilent Technologies), and a
multimeter 3800 (METEX, Germany).
Since the function generator produced a determined potential being influenced by
its internal resistance, reading a desired voltage value in the screen did not claim
that this was the real difference of voltage experimented in our circuit. Therefore,
The multimeter allowed to control the medium voltage and the oscilloscope let
know the maximum and minimal values of the square wave coming from the
function generator. The time of pulse was regulated with the frequency.
Figure 30: Schematic of the
resulting circuit
Figure 32: Controlling
an equilibrated
medium voltage
Figure 32: Generator indicating 1.725V offset, when it was actually giving 0V.
Patricia Carrasco Pividal
- 36 -
The difficulty of this manner of working eradicated in the high required control
during experiments, since the resistance did not change just when using a new
sample, but also during electrodeposition. By covering the sample, the resistance
was increased little by little (with a reduction of the voltage and the risk of inverting
the sense of intensity), so it was necessary a strict control of the voltage by paying
attention to the different devices. If this control would not have been erected, the
voltage had been changing in a different way with each sample and the results of
posterior experiments would not have been believable.
However, an advantage of this method was that the real value of voltage was
evaluated asserting that it was given properly. With a conventional potentiostat,
especially when demanding a short duration of pulses, there is the risk that the
device does not have enough time to arrive to the voltage wanted when it has to
be changed again.
The all-together assemblage is showed in figure 33.
Physicochemical Characterization
White-light Interferometry
Interferometry techniques were used to measure roughness and thickness and to
know with certainly that there were no rests of substances when the case arose.
The interferometer utilized was a Wyko 9300NT (Veeco, Germany) through white
light with Vertical Scanning interferometry (VSI) and the software Wyko Vision 32
(Veeco).
The following table shows the parameters applied to measurements:
Figure 33: The alternative potentiostat while testing
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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Table 7: Interferometry set up parameters.
Parameters
Type VSI
Magnification 50.0 X
Size 640x480
sampling 198 nm
Terms removed Tilt option
Contact angle measurement
The wettability of the samples was determined by Contact Angle (CA) measurement. The apparatus used was a Contact Angle SCA20 (DataPhysics,
Germany) and the software SCA20 (Dataphysics).
Two drops were made in each sample, 1µL/drop, with a speed of 2µL/s. The fluid used was high-purity water.
Figure 34: Interferometer analyzing a
sample with the 50x lens
Patricia Carrasco Pividal
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ATR-FTIR
With the aim of contrasting the presence of expected chemical bonding, Attenuated
Total Reflectance (ATR), which is a method of Fourier Transform Infrared
Spectroscopy (FTIR), has been resorted.
FTIR spectrometer was a Nicolet 6700 (Thermo Scientific, Wisconsin-USA) and the
software OMNIC.
ATR experimental set up had 512 scans, 4. Resolution (data spacing 1.98cm-1) and
the estimated time of each collection was 33 min.
XPS
X-ray photoelectron spectroscopy (XPS) technique was made by means of a SPECS
system and a XR50 anodic source of MG, working at 150W and a detector Phoibos
MCD-9 (D8 Advance SPECS, Germany) at the Research Center of nanoEngineering
(CRnE, UPC.BarcelonaTECH).
X-Ray spectrum was registered with a 25eV pass energy in 0.1 intervalsand a
pressure under 10-9mbar.
Figure 35: FITR spectrometer
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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4.5. Functionalization with AMPs: Magainin II and Lactoferrin 1-11
AMP immobilization was performed pursuing two different treatments, which differ
in the chemical bond methodology to Ti-PEG samples.
Peptides
As mentioned, two different peptides have been used: Magainin II and Lactoferrin
1-11. For the second methodology, the peptides sequences were defined with an
extra C (Cysteine) at the extreme of the chain (table 8).
Table 8: Peptides used in this study.
Peptide Name
code
Sequence Molecular mass
(g/mol)
Magainin II MG1 GIGKFLHSAKKFGKAFVGEIMNS 2508.45
MG2 CGIGKFLHSAKKFGKAFVGEIMNS 2611.75
Lactoferrin
1-11
LF1 GRRRRSVQWCA 1416.30
LF2 CGRRRRSVQWCA 1518.45
Stock solution preparation
Both peptides were purchased from GeneScript (CA, USA). On reception, these
were 1mg peptide aliquots, whereas the concentration wanted was 1mM. It was
therefore necessary to calculate the Phosphate Buffered Saline (PBS) volume
required (pH=6.5) for each peptide:
MG1: 1𝑚𝑔 ∙1𝑔
103∙
1𝑚𝑜𝑙
2508.45𝑔∙
103𝑚𝑚𝑜𝑙
1𝑚𝑜𝑙∙
1𝐿
1𝑚𝑚𝑜𝑙∙
106𝜇𝐿
1𝐿= 398.65𝜇𝐿
MG2: 1𝑚𝑔 ∙1𝑔
103∙
1𝑚𝑜𝑙
2611.75𝑔∙
103𝑚𝑚𝑜𝑙
1𝑚𝑜𝑙∙
1𝐿
1𝑚𝑚𝑜𝑙∙
106𝜇𝐿
1𝐿= 382.88𝜇𝐿
LF1: 1𝑚𝑔 ∙1𝑔
103∙
1𝑚𝑜𝑙
1416.30𝑔∙
103𝑚𝑚𝑜𝑙
1𝑚𝑜𝑙∙
1𝐿
1𝑚𝑚𝑜𝑙∙
106𝜇𝐿
1𝐿= 706.06𝜇𝐿
LF2: 1𝑚𝑔 ∙1𝑔
103∙
1𝑚𝑜𝑙
1518.45𝑔∙
103𝑚𝑚𝑜𝑙
1𝑚𝑜𝑙∙
1𝐿
1𝑚𝑚𝑜𝑙∙
106𝜇𝐿
1𝐿= 658.56𝜇𝐿
Onze prepared, these solutions were kept in freezer (-20ºC) until use.
Patricia Carrasco Pividal
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METHODOLOGY 1:
NHS – EDC (MG1/LF1)
The first method of bonding consisted in the carboxylic acid activation with EDC
(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride) and Sulfo-NHS
(N-hydroxysulfosuccinimide). C-terminal union occurred by means of carboxylic
acid with primary amines.
Products required:
NHS
EDC
MG1 and LF1 peptides
PBS pH=6
PBS pH=8
Procedure:
1) NHS-ester activation. An activation solution containing NHS, EDC,
peptide and PBS (pH=6.5) was prepared and mixed for 15 minutes.
1mL solution:
1𝑚𝑙 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 ∙200𝜇𝑚𝑜𝑙
1000𝑚𝑙∙
1𝑚𝑚𝑜𝑙
1000𝜇𝑚𝑜𝑙 𝑝𝑒𝑝𝑡𝑖𝑑𝑒∙
1000𝑚𝑙
1𝑚𝑚𝑜𝑙 𝑝𝑒𝑝𝑡𝑖𝑑𝑒∙
= 0.2𝑚𝑙 = 200 𝜇𝐿 𝑝𝑒𝑝𝑡𝑖𝑑𝑒
-For MG1:
0.2mM peptide 0.4mmol COO- 4mmol EDC
1𝑚𝑙 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 ∙4𝑚𝑚𝑜𝑙 𝐸𝐷𝐶
1000𝑚𝑙 ∙
1000𝑚𝑙
100𝑚𝑚𝑜𝑙 𝐸𝐷𝐶= 0.04𝑚𝑙 = 40 𝜇𝐿 𝐸𝐷𝐶
𝑉𝑁𝐻𝑆 = 𝑉𝐸𝐷𝐶 = 40 𝜇𝐿 𝑁𝐻𝑆
1𝑚𝐿 𝑎𝑐𝑡. 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = 200 𝜇𝐿 𝑀𝐺1 + 40 𝜇𝐿 𝐸𝐷𝐶 + 40 𝜇𝐿 𝑁𝐻𝑆 + 720 𝜇𝐿 𝑃𝐵𝑆 6.5
-For LF1:
0.2mM peptide 0.2mmol COO- 2mmol EDC
1𝑚𝑙 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 ∙2𝑚𝑚𝑜𝑙 𝐸𝐷𝐶
1000𝑚𝑙 ∙
1000𝑚𝑙
100𝑚𝑚𝑜𝑙 𝐸𝐷𝐶= 0.02𝑚𝑙 = 20 𝜇𝐿 𝐸𝐷𝐶
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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𝑉𝑁𝐻𝑆 = 𝑉𝐸𝐷𝐶 = 20 𝜇𝐿 𝑁𝐻𝑆
1𝑚𝐿 𝑎𝑐𝑡. 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = 200 𝜇𝐿 𝐿𝐹1 + 20 𝜇𝐿 𝐸𝐷𝐶 + 20 𝜇𝐿 𝑁𝐻𝑆 + 760 𝜇𝐿 𝑃𝐵𝑆 6.5
2) Mix 1mL activation solution with 1mL PBS (pH=8).
3) Amine reaction. The solution was added to the samples and left
reacting during 2 hours at room temperature.
METHODOLOGY 2:
Crosslinking tiol-amine (MG2/LF2)
The second way of bonding used was crosslinking with N-Succinimidyl 3-
Maleimide-Propionate (SMP), in which N-terminal bonding was done by means of
the terminal cysteine.
Products required:
SMP
DMF
PBS pH=6
Acetone
Distilled water
Ethanol
Nitrogen (optional)
Procedure:
1) Samples were put in a glass beaker, previously cleaned and dried.
2) 10mL SMP were added at a 2mg/m concentration (7.5mM) in N, N-
Dimetilformamide (DMF).
3) 1h mixing.
4) Samples were washed with DMF (x3), acetone (x1), distilled water (x10),
ethanol (x3), acetone (x3) and dried with nitrogen.
5) Samples were put into a well plate.
6) Peptide was added at a 100µM concentration in PBS pH=6.5 (1/10 dilution
from the stock solution) let reacting at least 2 hours.
Patricia Carrasco Pividal
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Physicochemical characterization and comparative AMP
The procedure adopted was the same as physicochemical characterization of PEG-
amine, in section 4.4.3. .
4.6. Biological characterization
Biological characterization of coatings was made by means of cytotoxicity, bacterial
adhesion and fluorescence tests. All the experimental brought to study counted
with 3 samples for each condition so as to do posterior statistical tests.
Cytotoxicity
With the aim of evaluating the toxicity of the samples, the activity of lactate
dehydrogenase (LDH) enzyme was measured. The presence of this enzyme would
indicate that the cells releasing it were damaged.
As the covering is thought to be in contact with the human tissue, human foreskin
fibroblasts (hFFs) were employed.
Annex 2 shows the Cytotoxicity assay protocol.
Figure 36: Neubauer chamber used for cell counting
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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Bacterial adhesion
Bacterial adhesion tests took three days. The methodology applied is given in
Annex 2.
Figure 37 shows the aspect of the plates just before doing the bacteria counting.
Fluorescence
Fluorescence is known as an easy method to detect dead-alive bacteria. In this
technique, two pictures are compared: a red-black and a green-black image. Green
area shows bacteria presence in the surface and red area shows how many of them
have already dead.
Methodology
Just as for bacterial adhesion, inoculum preparation is required.
An overnight after the inoculum preparation, 0.2abs inoculum was prepared and
samples were put in immersion for 24h.
Bacterial dying was done with the LIVE/DEAD® BacLight Bacterial Viability kit and
after 15 min they were analyzed by fluorescence microscopy.
Figure 37: Bacterial plates
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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CHAPTER 5:
RESULTS AND
DISCUSSION
5.1. Electrodeposition characterization
Experimental set-up and faced setbacks
When it came to carrying through with electrodeposition, different handicaps had
to be confronted in a problem solution pattern. Thus, adjustments and
alternatives were applied so as to achieve the expected aim in the diverse facts.
Mounting hindrances
Some sides of samples had to be manually polished because of cutting
defects. The outgoing hampered the entrance of samples in the 10mm
diameter hole of the support.
There was a loss of PEG effectiveness. After around 25-30 electrodeposition
tests, the electrolyte was replaced.
Patricia Carrasco Pividal
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Sometimes the support did not fit properly and water went inside (fig.38).
But if water got inside during electrodeposition, the sample was not taken
as valid and it was eliminate of the test (Tests were not do again with the
same sample because it already had rests of PEG). This problem was
controlled with an O-ring seal, Teflon or/and another sample pattern for
gaining thickness.
To control the entrance of water, EIS tests were made before each
electrodeposition test. The electrical impedance had to be similar to figure 39 to
be considered as valid.
Figure 39: Electrical impedance valid control
On the other hand, figure 40, shows an example of a control in which there was
not a proper electrical impedance corresponding titanium. This meant there was
0
10
20
30
40
50
60
70
80
90
0
50000
100000
150000
200000
250000
300000
ph
ase
of
Z(d
eg)
|Z|
(Oh
ms)
Frequency (Hz)
EIS - sample 41
|Z| (Ohms) phase of Z (deg)
Figure 38: support state because of water entrance
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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an entrance of water in the support and the electrical impedance given was coming
from the iron spring.
Figure 40: Electrical impedance improper control
Optimization obstacles
Optimization of the Electrodeposition Parameters
After a review of articles related to electrodeposition optimization in metals and
others relative to our polyether, significant parameters were put together in a
Design Of Experiments (DOE), done by means of the software Minitab.
However, while treating to apply the conditions to the software of the potentiostat,
PowerSuite, it was found out that, although according to the manual the
potentiostat was able to provide them (up to 20mµs/pulse and ±10V), depending
on the electrochemical technique these ranges were reduced. Thus the software
could not gave the required requisites ([1,5]ms/pulse and [-5,+5] V).
All of the software possibilities were studied. The most similar are shown on table
10 with their limitations.
Table 9: Different electrochemical techniques of PowerSuite
Electrochemical
technique Option Features and limitations
POWER CORR 1 Galvanic Step Valid if it worked with V instead of A
0
20
40
60
80
100
120
140
160
0
200
400
600
800
1000
1200
1400
1600
1800
ph
ase
of
Z (d
eg)
|Z|
(Oh
ms)
Frequency (Hz)
EIS - sample 66
|Z| (Ohms) phase of Z (deg)
Patricia Carrasco Pividal
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Electrochemical
technique Option Features and limitations
2
Potentiostatic (+iterations)
[V1(t0, t1)+ V2(t1, t2)]· n iterations
If (1+1+1)ms3ms/iterationat least 3points/process
3𝑝𝑜𝑖𝑛𝑡𝑠
𝑝𝑟𝑜𝑐𝑒𝑠𝑠·
1
1𝑚𝑠·
1000𝑚𝑠
1𝑠·
60𝑠
1𝑚= 180000
𝑝𝑜𝑖𝑛𝑡𝑠
𝑚𝑖𝑛· 5𝑚𝑖𝑛 =
900000𝑝𝑜𝑖𝑛𝑡𝑠
𝑝𝑟𝑜𝑐𝑒𝑠𝑠 𝑜𝑓 300 𝑚𝑖𝑛
So it was necessary to do 300000 iterations but the
maxima iterations admitted were 100.
POWER PULSE 3
Recurrent Potential Pulses LIMITED
t ≥ 4ms
-2V < V < +2V
POWER STEP 4
2 steps (+iterations) 2 steps
-10V < V < +10V
t1 ≥ 4ms, t2 ≥ 4ms
Same problem as in Potentiostatic POWER CORR
This is why a potential divider was added and the technique applied was Recurrent
Potential Pulses POWER PULSE. The potential divider is shown on figures 29 and
30, section 4.4.1.
The conditions of the design of experiments (DOE) figure on table 11.
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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Table 10: DOE parameters
Design Regime V01
(V)
V12
(V)
T01
(s)
T12
(s)
Total time (s)
[(T01 + T12) · N iterations
in pulsed regime]
d1 continuous 5 - - - 300
d2 pulsed 2.5 5 4 4 300
d3 pulsed 2.5 5 8 8 300
d4 pulsed 0 5 4 4 300
d5 pulsed 0 5 8 8 300
d6 pulsed 0 5 6 6 300
d7 pulsed -2.5 5 4 4 300
d8 pulsed -2.5 5 8 8 300
In the optimization procedure a total of 120 valid samples were required (plus
controls). Therefore, 15 samples of each condition were carried to analysis to
different techniques of characterization.
Resistance variation
As mentioned, EIS controls were done before each electrodeposition test, but also
after. By this way, the variation of the electric impedance was measured in order
to see how titanium impedance changed after PEG electrodeposition.
Patricia Carrasco Pividal
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Figure 41: |z| variation pre-/post- PEG electrodeposition
As observed in figures 41 and 42, there were no significant variations.
Figure 42: phase of z variation pre-/post- PEG electrodeposition
Roughness
Ti and Ti-Plasma controls samples showed no changes in their roughness (just as
expected).
Samples roughness was measured before and after the electrodeposition
procedure so as to see how changed their value from Ti to Ti-PEG.
Tests showed a minimal dissimilarity. This could mean it was a so slight cover and
as it was transparent it was penetrated by the interferometer light. In case that
this would happened, the interferometer was measuring again the Ti surface.
0
50000
100000
150000
200000
250000
300000
|Z|
(Oh
ms)
Frequency (Hz)
Pre and post PEG electrodeposition comparison
Pre-electrodeposition Post- electrodeposition
0
10
20
30
40
50
60
70
80
90
ph
ase
of
Z (d
eg)
Frequency (Hz)
Pre and post PEG electrodeposition comparison
Pre-electrodeposition Post- electrodeposition
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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In addition, little bubbles were detected indicating tha PEG electrodeposition was
not uniform, what would support the idea that the Ti–PEG surface was not as flat
as Ti surface (fig.43).
With the aim of having the answer, a highly light carbon coating (only 3 shots)
was applied so as to give opacity.
Table 11: Ti and Ti-PEG roughness
Surface statistics Ti Ti- PEG Ti-PEG
(carbon coating)
Mean roughness,
Ra (nm) 13.19 15.07 34.02
Root Mean Square
roughness, Rq
(nm)
18.37 19.25 42
As indicated in table 12, results showed a higher roughness but this could be
because predictions were right or because the carbon shots had left rests in the
surface. To clarify, a Ti sample was treated also with carbon shots and the results
were convincing (table 13). The carbon coating worked, and PEG provided Ti
samples a mean of an approximately 20 nm higher roughness.
Figure 43: Ti-PEG surface
Patricia Carrasco Pividal
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Table 12: Ti roughness
Surface
statistics Ti
Ti
(carbon coating)
Ra (nm) 9.21 9.66
Rq (nm) 13.11 13.22
Thickness
With the purpose of knowing the PEG coating thickness, it was thought that the
best and more economical option would be covering part of the sample with an
impermeable layer in order to avoid the contact of this part with the electrolyte.
Once finished the electrodeposition procedure, it would be retired and the resting
step could be measured.
Different options were considered. Among them, the most important are given
below:
1. Sterilization ribbon.
PROS: It had been used in others studies of the department and not excessive
rests were observed in the surface after being retired.
CONTRAS: It is NOT permeable.
REJECTED
2. Paint or varnish
PROS: It was impermeable.
CONTRAS: When retiring it would be unavoidable the contact in the adjoining
within the PEG coating (Since PEG electrodeposition coating is expected to have
a very small scale), distorting results.
REJECTED
3. White glue
Thinking about possible alternatives, the idea of using white glue appeared from
of its use in handicrafts and the knowledge that it did not used to leave rests
in some materials when being retired.
PROS: It was impermeable, easy to retire and apparently did not leave rests.
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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With the aim of confirm it would not leave rests, Ti samples roughness was
tested with the interferometer (fig.46) and a half part of the samples was
covered later (fig.44).
Afterward, two facts of interest were verified:
The interferometer arrived to focus the step in spite of the height difference
(fig.45).
- After taking away the white glue with laboratory pliers, roughness of the
part previously coated was measured again (fig.47).
Figure 44: Sample covered
with white blue in a half part
Figure 45: Titanium-white glue step
Patricia Carrasco Pividal
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A comparative view of the sample roughness and the aspect of the surface
before and after the procedure is given below:
According to results it seemed that a white glue coating would be a good option.
However, it was detected a loss of consistance in electrodeposition cheks. It
was sensitive to the electrolyte solution.
Figure 47: Ti surface before the blue coating input
Figure 46: Ti surface after removing the coating
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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CONTRAS: the covering dissolved when contacting the solution.
REJECTED
4. White glue + varnish
As the cover was perfect for the titanium surface, even not for the electrolyte,
the idea of putting a cover to the cover growth up. Like this, varnish would act
as a shield versus the electrolyte.
Thus, a commercial nail polish was applied on the white glue coating (fig.48)
After doing some tests, satisfactory results were confirmed.
VALID METHOD
As mentioned, samples were doubly covered and submitted to PEG
electrodeposition. Later, coatings were removed the height of the step Ti vs Ti-
PEG was measured (fig.49). Six equidistant values were taken from each sample.
Figure 48: Ti samples doubly covered (white
glue + nail polish)
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Finally, the thickness of each condition in the design of experiments was obtained
(fig.50).
Covered area
The covered are of the different optimization parameters presented different
aspect. The distribution of the coating is given in figure 51.
d1 d2 d3 d4
Thickness
Figure 49: Ti vs Ti-PEG step
0
50
100
150
200
250
300
350
d1 d2 d3 d4 d5 d6 d7 d8
nm
THICKNESS
Figure 50: tickness of DOE parameters
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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d5 d6 d7 d8
Figure 51: Covered area
Free amines presence
The presence of free amines in the Ti-PEG samples is a good indicator for posterior
treatments in the surface. As in this study a posterior AMP treatment took place,
we opted to do this control test.
The test was very simple and it was done by means of the Nynhidrine kit. If the
fluid containing the sample turned blue, there was presence of free amines in the
surface (fig.53).
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Contact angle
The wettability presented for each electrodeposition condition displayed a different
game of contact angles. “d4” showed the smaller contact angle, versus “d1”
(continuous pulse) and “d6”. The rest of conditions present a range of 25-45º
(fig.54).
0
10
20
30
40
50
60
70
80
90
d1 d2 d3 d4 d5 d6 d7 d8
Θ(º
)
Water contact angle
Figure 53: Free amine survey
Figure 54: Water contact angle of Ti-PEG.
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ATR-FTIR
By means of ATR-FTIR, the presence of chemical bonds has been contrasted.
First experiment
The first experiment, whose conditions were taken from precedent studies showed
a characteristic peak that indicated the presence of PEG at 1045.31cm-1
corresponding to O-C-O stretching together with a light peak at 2920cm-1
attributed to C-H groups. No presence of amine groups were detected though
(fig.55).
Electrodeposition otimization
The conditions brought to study in electrodeposition optimization, showed chemical
several bonding and stretching peaks at wavenumbers (fig.56). Their band
assignation is given in table 14.
Figure 55: ATR-FTIR spectra of Ti-PEG ("d1" condition).
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Table 13: Band assignation of Ti-PEG samples
Wave numbers (cm-1)
C-H bonding 842, 961
C-O-C stretching 1060, 1113, 1147
C -C stretching 1237, 1282, 1340, 1365
C-H wagging 1337, 1342
C-C stretching 1237, 1282, 1340, 1365
C-H wagging 1337, 1342
C-H scisssoring 1511, 1540
C-N streching 1566, 1575
N-H bonding 1646, 1650
C-H streching 2813, 2856, 2915
After determining d6 condition as the most optimal, Ti-PEG samples obtained from
both devices were compared (fig.57). The final experiment was done with the
alternative device, which showed highly notorious peaks (fig.58).
Figure 56: ATR-FTIR DOE spectra (Ti-PEG samples)
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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Figure 58: Ti-PEG sample from the final experiment.
Figure 57: Ti-PEG sample with "d6" condition obtained from the potentiostat (red)
and the alternative device (blue)
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To clear up, a comparison of the initial versus the final conditions from PEG
optimization is given in figure 59. An obvious improvement of the procedure is
observed.
XPS
The first experiment was analyzed by means of XPS (table 15). Results showed a higher amount of carbon, nitrogen and sulphur was detected on the samples
coated with AMPs, sign of a successful immobilization process.
Table 14: Atomic concentration of the control and treated samples
C 1s O 1s N 1s Ti 2p S 2p
Ti 23.4±1 59.6±1 0.6±0 16.3±0 0.0±0.0
PEG-amine 28.1±1 47.1±1 0.9±0 34.5±0 0.0±0.0
LF crosslinker 49.1±7 33.1±6 11.9±1 5.2±2 0.7±0.0
MG crosslinker 51.4±6 32.8±5 9.6±1 5.6±1 0.5±0.0
LF (EDC/NHS) 51.4±6 33.7±3 8.3±1 6.3±1 0.2±0.0
MG (EDC/NHS) 50.2±9 35.9±5 7.4±1 6.2±4 0.1±0.0
Figure 59: Initial vs final conditions("d6" condition obtained from the potentiostat
(red) and from the alternative device (blue)).
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SEM
PEG adhesion was observed with SEM technique (fig.60). Pictures were taken in a
500x scale.
SEM analysis showed that:
- d1, d3 and d4 parameters showed a lighter presence of PEG.
- d6 and d7 presented a higher covering amount.
- d4, d5 and d8 showed an irregular surface aspect.
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5.2. Cytotoxicity
Cytotoxicity assays were realized and showed a 100% of success (fig.61). All the
samples had a cell survival rate higher than 80%, demonstrating the
biocompatibility of the coating in vitro. Some samples even surpassed the rate
considered as maximum survival.
5.3. Adhesion assays
Four bacterial adhesion assays were done. Two of them with the initial conditions,
a huge assay with the optimization parameters and the last one applying the most
optimal conditions.
First condition assays
Assays showed a decrease on the bacterial adhesion with the presence of PEG-
amine. This antibacterial effect was further increased with the presence of the
AMP. In this stage, a similar behavior was observed between MG-2 and LF1-11
(fig.62).
Figure 61: Cell cytotoxicity.*
* Samples indicated with the same symbol have no statistically significant differences (p>0.05)
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PEG- electrodeposition optimization assay
Bacterial adhesion was the decisive factor in PEG-electrodeposition optimization (fig.63).
A large assay took place and the results cleared up what was the best option with the help
of the design of experiments with the software Minitab.
Figure 62: First condition material adhesion.*
* Samples indicated with the same symbol have no statistically significant differences (p>0.05)
Figure 63: Bacterial adhesion of electrodeposition optimization.*
* Samples indicated with the same symbol have no statistically significant differences (p>0.05).
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Figure 64 shows the optimization results. The optimal parameters given were V1
=0V and a T1=6ms, coinciding with “d6” condition.
Final assay
A final assay was made with a clearly bacteria reduction in relation to controls (Ti
and Ti-Plasma) (fig.65).
The effectivity of PEG optimization was as big that showed comparative numbers
with those samples treated with AMP.
Figure 64: DOE analysis
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Figure 65: Bacterial adhesion with the optimized electrodeposition parameter *
* Samples indicated with the same symbol have no statistically significant
differences (p>0.05)
5.4. Fluorescence
Fluorescence test were done showing a significant mortal rate of bacteria in
comparison to controls. MG1 and MG2 presented the best value (fig.66).
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Figure 66: Staphylococcus aureus Fluorescence
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CHAPTER 6:
CONCLUSIONS
Titanium surface has been functionalized with a PEG-amine antifouling coating by
an electrodeposition method.
Electrodeposition optimization has been studied by means of surface
characterization and bacterial assays. Results have been analyzed within a design
of experiments. The optimal condition obtained has been a pulsed regime with a
voltage on of 5V and voltage off of 0V, 6ms/pulse time duration.
The presence of chemical bonds has been contrasted with the starting condition.
Spectral peaks have improved reducing its transmittance, and new characteristic
peaks have appeared.
This PEG coating has been used as a stage for the immobilization of AMP as
antibacterial agents. The bonding of AMP to PEG-amine has been successfully
established with both SMP and EDC/NHS.
The coatings showed a total biocompatibility, with a cell survival higher of 80% in
all cases. There has also been a notorious decrease on the bacterial adhesion.
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CHAPTER 7:
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ANNEX 1: CITOTOXICITY ASSAY PROTOCOL
Before starting the test, cell culture media (DMEM) must be prepared by mixing
the components in table 9.
Table 15: DMEM
Substance Quantity
FBS 5 ml
HEPES 1 ml
Antibiotic
(Strep/Pen) 500 µl
L-glutamine 500 µl
Primary DMEM 43 ml
Procedure:
Step 1: Sample immersion in DNEM
1) Sample sterilization (10 min UV).
2) 2 washes with sterile PBS
3) Change the samples onto a 48 holes well plate.
4) Add 480 L of DMEM to each sample
5) Put the well plate in the incubator (37º) for 72h for releasing soluble
compound which could contain.
Step 2: Cell plating
1) Cell counting in a Neubauer chamber (fig.36) and diluting up to the desired
concentration. For cell counting, 10 µl of the cell solution were put in each
chamber square and cell counted by means of a microscopy.
2) Row hFFs on a 96 holes well plate, 5000 cells/100 µl DNEM.
3) Let it in the incubator (37ºC) for 24h.
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Step 3: Cell-sample Interaction
1) 1:2, 1:10, 1:100 and 1:1000 dilutions of cell interacting media were
prepared. If the coating had had a cytotoxic effect, dilutions would have
allowed to known the initial concentration since there is cytotoxicity.
2) 100µl of each dilution were added in each of the cell containing hole and
put in incubator (37ºC) for 24h.
Step 4: Cell lysis
Cell lysis with M-PER (Mammalian Protein Extraction Reagent) took place.
100µl of M-PER where put in each hole with the aim of breaking the cell
membrane, spreading cell contents to media.
Step 5: LDH activity measurement
1) Cytotoxicity Detection Kit (LDH) Roche (Germany) was required.
2) 5.74ml of tetrazolium salt solution were mixed with 127µl catalyzer.
3) 100 µl of the mixture were put in each hole with cell lysis.
4) After 10 min, a prepared solution from the kit containing chlorhydric acid
was added. This acid changed the solution pH, denaturing the protein
and stopping the reaction.
Step 6: Absorbance measurement
1) Samples absorbance was measured with an UV spectrometer, 492nm
wavelength.
2) The survival percentage was calculate by means of the operation:
survival% =Abssample − AbsC-
AbsC+ − AbsC-
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ANNEX 2: BACTERIA ASSAY PROTOCOL
First day (in the afternoon)
Inoculum preparation. Bacterial caught from a S. aureus plate stocked at the
fridge. They were put in a falcon with 5 ml media (BHI + distilled H2O) and put in
incubator (37ºC).
Second day (at middday)
-Samples were sterilized with ethanol
-Inoculum was prepared with a 0.2 absorbance (abs)
-Sample inmersion in 0.2 abs inoculum
-After 2h, plates (BHI + agar) were sowed up to 4 dilutions.
Third day (13-14h later)
Bacteria counting. The dilution that used to be counted was d3.
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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ANNEX 3: BUDGET
The budget of the study, including materials, devices and workers, is given below.
The salary of a junior engineer is stablished as 30.00 €/h.
Table 1. Titanium cp2 cost
Quantity Prize Cost
Titanium cp 2
(10mm diameter)
(300samples)·0.002m 368.45€/u 221.07€
Table 2. Metallographic preparation.
Quantity Prize Cost
Cutting 6h 46.00€/h 276.00€
P800 20u 1.29€/u 25.80€
P1200 30u 1.29€/u 38.70€
P2400 40u 1.88€/u 75.20€
Velvet 2u 5.80€/u 11.60€
OP-S 1L 161€/L 161€
Polishing machine 35h 30.00€/h 1050.00€
Staff 40h 30.00€/h 1200.00€
TOTAL 2838.30 €
Table 3. Cleaning
Quantity Prize Cost
Acetone 1.00L 16.78€/L 16.48€
Ethanol 1.00L 10.95€/L 10.95€
Water 5.00L 0.08€/L 0.4€
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Staff 6h 30.00€/h 180.00€
TOTAL 207.83 €
Table 4. Surface treatments
Quantity Prize Cost
Plasma cleaning Diener 8h 20.00€/h 120.00€
PEG-amine 25g 80€/5g 0.75€
NaCl 0.5g 120.00€/kg 0.20€
KCl 0,05 48.30€/L 0.05€
Peptides 4 124.45€/u 73.20€
Ar gass bottle 1u. 283.50€ 283.50€
Technician 25h 42.00€/h 840.00€
Staff 240h 30.00€/h 7200.00€
TOTAL 10330.33€
Table 5. Physicochemical characterization
Quantity Prize Cost
Interferometer 45h 35.00€/h 1575.00€
Interferometer Technician 2h 42,00€/h 84,00€
Contact Angle 1h 20.00€/h 20,00€
SEM 3h 72.76€/h 218.28€
FTIR 2h 35.00€/h 140.00€
TIR Technician 1.5h 42.00€/h 42.00€
ATR 8h 35.00€/h 140.00€
ATR technician 1h 42.00€/h 42.00€
XPS 2 samples 100€/sample 200.00€
staff 20h 30.00€/h 960.00€
TOTAL 2252.56€
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Table 6. Biological characterization
Quantity Prize Cost
PBS 2u 1.156€/u. 3.12€
DMEM 0.425l 40.40€/l 1.72€
HEPES (1M) 0.001l 1104.50€/l 1.10€
FBS 0.005l 88.40€/l 0.44€
Sodium piruvate 0.0005l 84.20€/l 0.04€
Penicillin /Streptomicine 0.0005l 125.00€/l 0.06€
L-glutamine 0.0005l 105.20€/l 0.05€
Tripsine 0.002l 69.28€/l 0.14€
BSA liofilitzated 0.5g 5.94€/g 2.97€
M-PER 0.0048l 783.88€/l 3.76€
culture 8h 10.00€/h 80.00€
S.aureus 1strain 45.00€/strain 45.00€
Medium 128g 0.0944€/g 12.08€
Agar 128g 0.0944€/g 12.08€
Bacteria hood 30h 10.00€/h 300.00€
Kit FITC 1/10Kit 550.00€/Kit 55.00€
Fluorescence microscope 4h 60€/h 240.00€
Cytotoxicity Detection Kit 1/40Kit 600,70€/Kit 15.02€
Spectrophotometer 0,25h 7,64€/h 1.91€
Staff 80h 30,00€/h 2400.00€
TOTAL 3174.364€
Table 7. Secondary staff costs
Quantity Prize Cost
Laboratory use 2000.00€
Bibliographic reference
research
120h 30.00€/h 3600.00€
Analysis of results 100h 30.00€/h 3000.00€
Memory writing 130h 30.00€/h 3900.00€
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TOTAL 12500.00 €
Table 8. Total costs
Category Cost Total
Titanium cp2 costs 221,07€
Metallographic preparation costs 2838.30€
Cleaning costs 207.83€
Surface treatment costs 10330.33€
Physicochemical characterization costs 2252,56€
Biological characterization costs 3174,36€
Secondary costs 12500,00€
Subtotal 31,524.55 €
I.V.A (21%) 6,620.15 €
TOTAL I.V.A. 38,144.22 €
Surface functionalization of titanium with antifouling and inhibition of bacterial adhesion treatment in the human body
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ANNEX 4: ENVIROMENTAL IMPACT
The environmental impact of the study has been taken into account, especially in
metallographic preparation of samples. Chemicals have been used as less as
possible and, if required, they were used in the hood.
The use of lab coat is obligatory in the laboratory so as to avoid the outgoing of
substances.
Organic solvents, such as ethanol and acetone, have to be carefully treated.
There is a laboratory protocol of chemical wasting. Wastes have to be stocked hermetically and properly labeled with name and data of the first use. ECOCAT is
the enterprise that manages the remains. Every six months wastes are retired and treated by the same enterprise
Comparing to other coating methods, the techniques employed in this study have
a low energy consumption.