Hemocompatibility and antimicrobial analysis of coated and uncoated silk fibroin fibers

CYPRUS INTERNATIONAL UNIVERSITY
INSTITUTE OF GRADUATE STUDIES AND RESEARCH
Department of Bioengineering
HEMOCOMPATIBILITY AND ANTIMICROBIAL
ANALYSIS OF COATED AND UNCOATED SILK
FIBROIN FIBERS
MSc THESIS
OBI FELIX CHIBUZO
NICOSIA - 2019

CYPRUS INTERNATIONAL UNIVERSITY
INSTITUTE OF GRADUATE STUDIES AND RESEARCH
HEMOCOMPATIBILITY AND ANTIMICROBIAL
ANALYSIS OF COATED AND UNCOATED SILK
FIBROIN FIBERS
MSc THESIS
OBI FELIX CHIBUZO
Supervisor
Assoc. Prof. Dr. Hatice Erkurt
NICOSIA – 2019

THESIS APPROVAL CERTIFICATE
The thesis study of Bioengineering Department graduate student (Felix Chibuzo Obi) with student
number 21800054 titled “Hemocompatibility and Antimicrobial Analysis of Coated and
Uncoated Silk Fibroin Fibers” has been approved with unanimity by the jury and has been
accepted as a Master of Science Thesis.
Thesis Defense Date:
Jury Members Signature
1) Thesis Supervisor ……………....
Assoc. Prof. Dr. Hatice Erkurt
2) Member .......................
Assoc. Prof. Dr. Doğa Kavaz
3) Member .......................
Asst. Prof. Dr. Ertan Akün
Prof. Dr. Tahir ÇELİK
Director of the Institute
Cyprus International University
Institute of Graduate Studies and Research

DECLARATION
Name and Surname: Obi Felix Chibuzo
Title of Dissertation: Hemocompatibility and Antimicrobial Analysis of Coated and
Uncoated Silk Fibroin Fibers
Supervisor(s): Assoc. Prof. Dr. Hatice Erkurt
Year: 2019
I hereby declare that all information in this document has been obtained and presented in
accordance with academic rules and ethical conduct. I also declare that, as required by
these rules and conduct, I have fully cited and referenced all material and results that are
not original to this work.
I hereby declare that the Cyprus International University, Institute of Graduate Studies
and Research is allowed to store and make available electronically the present
Dissertation.
Date:
Signature: __________________

i
ACKNOWLEDGEMENT
Firstly, I want to return all the glory to Almighty God for a dream come true, His grace,
sustenance and provision made the completion of this work possible.
I also want to specially thank my supervisor and Head of Department-Assoc. Prof. Dr
Hatice Erkurt for all her support, encouragement and assistance from the onset of this
project. This work couldn’t have been successful without her. I also want to thank the
following persons who assisted us in the Laboratory work, Lab Assistant Huzeifa Umar,
Lab Assistant Peyman Ince, Lab Technician Nurten Asina, and Lab Assistant Sylvia
Kpange. You Guys are wonderful. It was nice meeting and working with you. A big thanks
to you all.
I wouldn’t forget to thank my friends and colleagues, whom I wouldn’t be able to mention
all for their support, encouragement and assistance, Francis, Confidence, Convenant, Tayo
and a lot of others. You guys are the best, I love you all.
I am forever grateful to my Parents-Chief and Lolo Livinus Obi who opened the door of
education to me and also my siblings, Collins, Victor, Elias, Augustine, ThankGod, Lynda
and Annex for all the supports and encouragements. I love you all, you mean the world to
me.
I will also not forget to thank the management and staff of Biyomik laboratory for allowing
us use their laboratory to carry out the hemocombatility analysis done this research, this
research wouldn’t have been possible without them. Thank you.
Above all, I return all the glory to God Almighty for making these project a success.

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ABSTRACT
Silk fibroin is a natural biomaterial created by Spiders; the larva or caterpillar of
domesticated silk moth (silk worm), Bombyx mori; other moth gene such as Antheraea,
Gonometa, Samia, Cricula; and other numerious insects. Silk fibroins are widely use in
Tissue Enginnering and also has applications in medical devices and biological products.
The main aim of this work is carry out surface modification on silk fibroin fibers by
coating them with a polymer-Tetra (ethylene glycol) dimethacrylate solution. Coating was
carried out by repeating dipping and drying the silk fibroin fibers in the coating solution.
The coated and uncoated silk fibroin fibers were observed under an light microscope to
determine the difference in diameter of the coated and uncoated silk fibroin fibers.
Hemocompatibility test was carried out on both coated and uncoated silk fibroin fibers.
Characterizations of the coated silk fibroin fibers was done by SEM and antimicrobial
analysis. The silk fibroin fibers demonstrate effective antimicrobial capability against a
broad range of six selected microbes namely Pseudomonas aeruginosa (ATCC 27853),
Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 28923),
Escherichia coli (ATCC 25922), Bacillus cereus (ATCC 10876) and Candida albicans
(ATCC 90028) as examined by the antimicrobial susceptibility tests. Results showed that
effective antimicrobial activities are exhibiting higher inhibition ratios. This research
work aims to find out if coating silk fibroin fibers with Tetra (ethylene glycol)
dimethacrylate with make them a better biomaterial when applied in Tissue Engineering
and it other applications in biomedical engineering.
Keywords: Coated Silk Fibroin Fibers; Tetra (ethylene glycol) Dimethacrylate;
Hemocompatibility; Antimicrobial Activity; Tissue Engineering.

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TABLE OF CONTENTS
ACKNOWLEDGMENTS …………………………………..…………. I
ABSTRACT ……………………………………………………………… II
TABLE OF CONTENTS ………………………………..……………… III
LIST OF TABLES ……………………………………………………… V
LIST OF FIGURES……………………………………………………….. VI
CHAPTER 1: INTRODUCTION
1.1 Aim of the study ……………………………………………………... 3
1.2 Objective of the study ……………………………………………... 3
1.3 Scope of the study ……………………………………………... 4
CHAPTER 2: LITERATURE REVIEW
2.1 Surface modification and functionalization of silk fibroin fibers……… 5
2.2 Prospect of surface modification and functionalization of silk fibroin fibers 5
2.2.1 Surface modification by Physical techniques………………......... 6
2.2.2 Surface modification by Chemical approach….......………………. 10
2.3 Surface modification for biomaterial applications…………………….. 11
2.4 Biocompatibility of silk fibroin……………………………………….. 13
2.5 Biodegredability of silk fibroin………………….…………………...... 13

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CHAPTER 3: MATERIALS AND METHODS
3.1 Materials………………………………………………………………. 15
3.2 Preparation of the uncoated silk fibroin fibers………………………… 16
3.2.1 Preparation of the coated silk fibroin fibers…………………..…. 16
3.3 Sterilization of coated and uncoated silk fibroin fibers……………….. 17
3.4 Measurement of the diameter of the coated and uncoated silk fibroin fibers. 17
3.5 Hemocompatibility test procedure……………………………………… 17
3.6 Antimicrobial susceptibility test procedure…………………………….. 18
3.6.1 Preparation of the coated and uncoated silk knitted fibroin fibers. 18
3.6.2 Disc diffussion method……………………....……………………. 19
3.8 Characterization of the coated silk fibroin fibers………………………. 20
3.7.1 Scanning Electron Microscopy Analysis……..…………………. 20
CHAPTER 4: RESULTS AND DISCUSSIONS
4.1 Thickness of the uncoated fibers…………………….………………… 21
4.2 Thickness of the coated fibers ………………………...………………. 22
4.3 Hemocompatibility test results……………………….…………………. 23
4.4 Antimicrobial susceptibility test results………………..……………… 43
4.5 Scanning Electron Microscopy (SEM) analysis………………..……… 46
4.6 Application of the study……………………...........………………… 49
CHAPTER 5: CONCLUSION AND RECOMMENDATIONS

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4.1 Conclusion…………………………………………………………….. 50
4.2 Recommendations…………………………………………………….. 50
REFERENCES………………………………………………………….. 51

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LISTS OF TABLES
Table 4.1: Thickness of the Uncoated Fibers………...……………….….. 21
Table 4.2: Thickness of the Coated Fibers ………………………..……... 22
Table 4.3: Showing Normal Standard Clotting Time……….……………. 23
Table 4.4: Hemocompatibility Test Results for 10cm of Uncoated Fiber…. 24
Table 4.5: Hemocompatibility Test Results for 10cm of Coated Fiber……. 24
Table 4.5: Hemocompatibility Test Results for 12cm of Uncoated Fiber…. 30
Table 4.7: Hemocompatibility Test Results for 12cm of Coated Fiber….... 30
Table 4.8: Hemocompatibility Test Results for 14cm of Uncoated Fibe…. 36
Table 4.9: Hemocompatibility Test Results for 14cm of Coated Fiber…... 36
Table 4.10: Antimicrobial Susceptibility Test Results…………….……… 45

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LISTS OF FIGURES
Figure 1.1: Bombyx mori silkworm and cocoon................………………… 1
Figure 2.1: Scanning electron micrograph of irradiated silk fiber surface… 7
Figure 2.2: SEM image of silk fibroin fibers showing SF6 Plasma tends… 10
Figure 3.1: Silk fibroin fibers................…………...............………………. 15
Figure 3.2: Coating and drying of silk fibroin fibers………...............……. 16
Figure 3.3: Inverted light microscope………...............………………........ 17
Figure 3.4: Photo of a Centrifuge and a Coagulation Machine…......……. 18
Figure 4.1: Image showing the five dimensions taken on uncoated fibers.. 21
Figure 4.2: Image showing the five dimensions taken on coated fibers….. 22
Figure 4.3: Graph of PTSec test results for 10cm uncoated fibers……….. 25
Figure 4.4: Graph of PTSec test results for 10cm coated fibers………....... 25
Figure 4.5: Graph of INR test results for 10cm uncoated fibers………...... 26
Figure 4.6: Graph of INR test results for 10cm coated fibers………...…... 27
Figure 4.7: Graph of PT% test results for 10cm uncoated fibers…….…... 28
Figure 4.8: Graph of PT% test results for 10cm coated fibers………..…... 29
Figure 4.9: Graph of PTSec test results for 12cm uncoated fibers………... 31
Figure 4.10: Graph of PTSec test results for 12cm coated fibers……….... 31
Figure 4.11: Graph of INR test results for 12cm uncoated fibers….......…. 32
Figure 4.12: Graph of INR test results for 12cm coated fibers…………... 33
Figure 4.13: Graph of PT% test results for 12cm uncoated fibers………..… 34
Figure 4.14: Graph of PT% test results for 12cm coated fibers……….….... 35
Figure 4.15: Graph of PTSec test results for 14cm uncoated fibers……….. 37
Figure 4.16: Graph of PTSec test results for 14cm coated fibers…….…... 37
Figure 4.17: Graph of INR test results for 14cm uncoated fibers…………. 38
Figure 4.18: Graph of INR test results for 14cm coated fibers……….…... 39
Figure 4.19: Graph of PT% test results for 14cm uncoated fibers………... 40
Figure 4.20: Graph of PT% test results for 14cm coated fibers……….….. 41

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Figure 4.21: Antimicrobial zones of inhibition for uncoated fibers ……….. 44
Figure 4.22: Antimicrobial zones of inhibition for coated fibers ………..…. 44
Figure 4.23: Graph showing zones of inhibition for uncoated and costed fibers. 45
Figure 4.24: SEM micrograph of 50µm uncoated fiber at X300 magnification.. 46
Figure 4.25: SEM micrograph of 50µm coated fiber at X300 magnification... 47
Figure 4.26: SEM micrograph of 20µm uncoated fiber at X800 magnification.. 47
Figure 4.27: SEM micrograph of 20µm coated fiber at X800 magnification.... 48
Figure 4.28: SEM micrograph of 10µm uncoated fiber at X1400 magnification. 48
Figure 4.29: SEM micrograph of 10µm coated fiber at X1400 magnification.. 49

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CHAPTER 1
INTRODUCTION
Fibroin is present in Silk as an insoluble protein. It is secrited by Spiders; the larva or
caterpillar of domesticated silk moth (silk worm), Bombyx mori; more also some moth
gene such as Antheraea, Gonometa, Samia, Cricula; and other numerious insects. Silk is
been synthesized in the glands of these organisms by specialized epithelial cells
Figure 1.1: Bombyx mori silkworm and cocoon (Charu and David, 2007)
The natural state of silk consist mainly of two proteins, sericin and fibroin, two singular
filaments of fibroin are coated with a glue-like layer of sericin called brins.
Layers of antiparallel beta sheets make up fibroin protein. Its stucture is mostly made up
of sequence of recurrent amino acid (Gly-Ser-Gly-Ala)n. The glycine and alanine content
anables the tight packing of the sheets and these contribute to silk’s rigid structure and
tensile strength. Silk fibroin has found application in many area such as Biomedical
Engineering and the Textile Industry as a result of a its stiffness and toughness
combination. Silk fibroin is an excellent Biomaterial as a result of its remarkable
mechanical properties.
Regenerated Silk Solution has recently be use to form different biomaterials like films and
sponges which have medical applicatios (Charu and David, 2007). The surface properties

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of silk can be altered to immobilize cellular growth, these is acheive by chemically
modifying them through amiono chain reaction. Silk have been successfully modified
through molecular engineering sequnces like cell recognition. Silk biodegradability has
been found to be closely associated with corresponding matter of b-sheet crystallinity and
it processing mode. These b-sheet structure of silk permits the the tight packaging of piled
sheets of hydrogen bonded anti-parallel chains of the protein. Huge hydrophobics domains
interspaced with small-scale hydophobic domains are responsible for silk assambly, these
also contributed to the resiliency and strength of silk fibers. Cells as well as cell lines have
been affluently grown on different silk biomaterials in vitro and in vivo with appreciative
biological outcome. Tissue engineers have successfully used silk scaffolds in wound
healing and to engineer bone, cartilage, tendon and ligaments tissues.
Scientist have fully explored silks obtained from silkworm and orb-weaving spiders to get
a better grip of the properties and processing mechanism of this proteins that makes them
suitable biomaterials. Silks obtain from orb-weaving spiders and silkworm and has been
found to have excellent mechanical properties, biocompatibility, biodegradability,
morphological flexibility, are environmental stable and can be easily modify to
immobilize growth factors. Tissue engineers incorporate the physical, chemical and
biological clues of silk to fabricate scaffolds that encourages cell migration, cell adhesion
and cell differentiation. Biomaterial should have appropriate degradation pace to allow
the newly formed tissue to deposit its extracellular matrix (ECM) and regenerate the
needed tissue. Additionaly, biomaterials should have the appropriate mechanical property
that supports the functional tissue development. Biomaterials should also be
biocompatible and produce only appropriate inmune response in the host. For centuries,
silk fibers gotten from B. mori have been successfully be use as suture materials, these
has lead to more investigation of silk and a biomaterial. Structural differences in silk of
same of different species leads to functional differences, these is mainly associated to the
differences in the primary amino acid sequence, the processing and environmental factors.

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Silk are biocompatible, biodegredable, have excellent mechinical properties, are
govornable to organic or aqueous solvent processing and can easily be modified with
chemicals to make them suitable for a wide range of applications in biomedical
engineering.
1.1 Aim of the Study
This Research is aimed at the following;
1. Determining the Hemocompatibilty of coated and uncoated silk fibroin.
2. Determining the difference in the diameter of coated silk fibroin to uncoated silk
fibroin.
3. Determining the susceptibility of coated and uncoated silk fibroin to a wide
spectrum of microbes.
4. Comparing the inhibition activities of microbes on coated silk fibroin and uncoated
silk fibroin.
5. Determining which microorganism is more sensitive to coated and/or uncoated
silk fibroin.
6. Providing a point of reference for other researchers on the subject in the future.
1.2 Objective of the Study
In this research work, pure silk fibroin fibers synthesized from silkworm were coated with
Tetra (ethylene glycol) dimethacrylate. Hecompatibility test was carried out on both
coated and uncoated silk fibroin fibers.
In the second phase of these research work, the coated and uncoated silk fibroin fibers
were used to carry out antimicrobial susceptibility test to measure and compare the various
zone of inhibition. The wide spectrum of microorganisms used are namely Escherichia
coli (ATCC 25922), Candida albicans (ATCC 90028), Staphylococcus aureus (ATCC

4
25923), Enterococcus faecalis (ATCC 29212), Bacillus cereus (ATCC 10876), and
Pseudomonas aeruginosa (ATCC 27853).
These research work intends to find out if coating silk fibroin with tetra (ethylene glycol)
dimethacrylate has any effect on its hemocompatibility and will also study in details the
antimicrobial properties of the Silk fibroin by running some antimicrobial test on the fibers
using the above mentioned microbes and bacteria. These research finding will be of
paramount importance to biomedical and tissue engineers as it will help them better
understand the biocompatibility of silk fibroin in fabricating Scaffolds.
1.3 Scope of the Study
The present study was limited to determining the hemocompatibilty of coated and
uncoated silk fibroin fiber and determing and comparing the antimicrobial properties of
coated and uncoated silk fibroin fibers. The coagulation mechine used in the
hemocompatibilty analysis only gave values for Prothrombin Time (seconds),
International Normalized Ratio (in seconds) and Prothombin Time Percentage, it couldn’t
give values for Activated Partial Thromboplastin Time (APTT). A far better analysis
would have been possible if it could give all four values listed above. The study was also
limited by insufficient blood samples, the hemocompatibilty analyis took a longer time
because we had to wait to get more blood samples to continue with the analysis. Different
patient blood samples were used in the hemocompatibilty analyses, a better result will
have been obtained if same blood samples were used as this will keep every other factor
constant.

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CHAPTER 2
LITERATURE REVIEW
2.1 Surface Functionazition and Modification of Silk Fibroin Fibers
Silk is an excellent biomaterial in the medical field due to its remarkable properties.
However, photo-induced aging, wrinkling, degradation caused by microorganism,
deformation and even biocompatibility problems of silk has pose a challenge in some
advance application. To overcome this shortcomings and to meet the huge demands of
smart natural biomaterials, technology has use a range of functional materials to remodel
the surface and functionality of silk fibroin fibers in the last decade.
Surface modifications of silk may not only conquer these limitations but will also increase
it end-use performance. Guohong et al. (2011) did an extensive research on the
methodologies, main ideas, and treatment methods for the surface functionalization and
remodeling of silk fibroin considering their enhanced properties and possible applications.
This Chinese scientist were motivated by the success of previous studies and believed that
the successful modification of silk product will give rise to an assuring future in the both
textile industry as well as in biomedical engineering. Stated below is a review of some of
their findings.
2.2 Prospects of Surface Modification and Functionalization of Silk Fibroin Fibers
Base on methods and techniques, surface functionalization and modification of silk fibroin
fibers can be classified into three, modification by physical methods; modification through
chemical approaches and functionalization using nano materials.
Base on application, surface functionalization and modification of silk fibroin fibers can
be classified into two, surface modifications for biomaterials applications and surface
pretreatment for improving dyeing and finishing performance. In this review, we will only

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discus modification by physical methods; modification through chemical approaches and
modifications for biomaterials applications.
2.2.1 Surface Modification by Physical Methods
This technique involves the modification of the surface of a material through Ultra Violet
(UV) treatment, Gas treatment or Plasma treatment.
Dielectric barrier discharge principle is use on UV lamps to intense and almost
monochromatic light in the spectrum region of ultraviolet vacuum tube. The multiple
bonds in silk fibroin fibers have electron pairs like nitrogen or oxygen; these electron pairs
are nonbonding, these bonds strongly absorb light with wavelength less than 200nm. The
molecular chains on the surfaces of silk fibroin fibers are easily broken by high-energy
photons forming free radicals and microspores on the surface of the fiber. Atmospheric
oxygen reacts with these radicals to form highly excited oxygen. These excited oxygen
further interacts with the fiber surface forming oxygen that contain polar groups like
carbonyl (C=O) and hydroxyl (OH) groups. Wickability and wettability of silk fibroin
fibers are enhance by chemical and physical modification of the fiber surface without
affecting the bulk properties. Silk fibroin fiber surfaces modified with UV irradiation have
been found to improve adhesion and enhance wettability with little losses in weight,
strength and crystallinity. Increasing the hydrophilicity of silk matrix materials will make
them more beneficial for cell growth and cell adhesion thus making them more suitable
biomaterials.

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Figure 2.1:. SEM images of (a) opposite (not irradiated); (b) irradiated surface of silk
fiber with monochromatic (172 nm) excimer lamp (for 10 min), (Guohong Li et al, 2012).
Surface modification through Gas depends on the species of gas used; this technique of
surface modification is capable of imparting different properties to silk fibroin fibers.
Ozone which is a powerful oxidizing agent affects raw and degummed silk fabrics
(Guohong et al, 2011). Treating silk fibroin fibers with ozone affects their pliability and
yellowness index, this is because of the oxidation of amino acid residues, mostly glycine,
tyrosine, serine and alanine. Guohong et al. (2011) find out in their research that treating
silk fibroin fibers with Ozone gas reduces the tyrosine content by over 40%. The colour
formed comes from the generation of 3, 4-dihydroxy phenylalanine, chromophoric
products which contain carbonyl groups majorly from tyrosine, and formkynurenine from
tryptophan and cystine. The generation of new amino groups and sudden burst of silk
macromolecules leads to loss breaking strength and elongation, and the removal of
gaseous products (i.e. carbon dioxide, aldehyde, ammonia, nitrates and ketones) leads to
weight loss. For over 20 years, liquid ammonia has been used to pretreat cotton fibers to
stabilize it shape and improve it softness, this apparently lead to the decline in the dyeing
rate of cottons. Interestingly, treating silk fibroin fibers with hundred percent ammonia-
gas at atmospheric pressure showed more successful improvement of its softness with
little or no change in crystallinity and no decline in apparent dyeing rate.

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Silk fibroin surfaces can also be modified through technique known as Plasma Treatment.
Plasma in this context comprises of a mixture of electrons, positively and negatively
charged particles, molecules and neutral atoms. Plasma has the ability to conduct
electricity and the electrons equilibrium and positively and negatively charged ions makes
it a neutral medium. When activated, physical and chemical reactions are initiated by the
electrons and photons through physical bombardment and plasma polymerization.
Plasma treatment is relatively environmental friendly; it also has the advantage of
modifying only the outer atomic layers of materials without obstructing the bulk
properties, lower chemical is consumed and no waste water is produced thus makes it
perfect for textile industries. The amazing part of plasma surface modification is that we
can use various gases to synthesize plasma, such as carbon dioxide, fluorine, oxygen,
argon, SF6, water vapor, nitrogen, air and helium CF4.
Silk fibroin fibers absorb water instantly, thus the angle of contact for untreated silk
fibroin fabric is taken as 0°. The hydrophobicity of silk fibroin fibers has been improved
with Hexafluoropropene (C3F6) plasma (Shen and Dai, 2007). The angle of contact
increased from 0° to 119° after one minute of treatment. Fluorine-containing groups, like
as -CF, and -CF3, -CF2 were detected to be the major contribution factor to the
hydrophobic for remodeled fibers. To prevent plasma polymerization giving unwanted
fluorinated surface films, sulfur hexafluoride is use as the origin of fluorine atoms. The
CFx/F ratio in SF6 is fundamentally zero thus polymerization does not take place. Silk
fibroin fiber treated with SF6 plasma has their water angle of contact increased from 0°
up to 145°, with a little amount of thin film and increase roughness when compare to the
surface of untreated silk fibroin fiber. (see Figure 2.2 below). X-ray Photoelectron
Spectroscopy finds out distinct signals of CF, CF2 and CF3 groups but did not detect any
sulfur signals. It was concluded from the results that only F radicals are accountable for
the noticed surface chemical changes. The rise in roughness of treated fabrics most likely
be because of amalgamation of etching by high energy species produced in SF6 plasma,

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materials accumulation on the sample surface, and the substitution of hydrophilic species
by fluorine containing moieties. These factors enhance the hydrophobicity of the silk
fibroin fiber.
The smooth morphology of silk fibroin fiber makes it unable to hold water. Modifying the
surface of SFF with Oxygen plasma does not only increase the roughness of the fiber
surface but will also introduce polar groups (-OH, -NH2, -C=O, -COOH) to the surface
layers (Molina et al, 2005). Both outcomes could improve the hydrophilicity of silk fibroin
fibers. Treated silk fibroin fibers have better resistance to pigment bleeding relative to
untreated ones. Also treated silk fibroin fibers has been found to produce deeper and more
vividly colored images when use on inkjet printers than untreated silk fibroin fibers.
Treating the surfaces of silk fibroin fibers with Argon and Oxygen gas plasma before
silver plating them may increase the silver particle deposited on the surfaces; this is due
to the introduction of hydrophilic groups to the fiber surfaces. Silk fibroin fiber surfaces
can be functionalized with siloxane groups with the formation of plasma polymer film by
treating them with low temperature plasma D4 ([(CH3)2SiO]4). Highly improved inter-
woven resistant, good water repellent, soft, and wrinkle resistant silk fibers can be derived
from such treatment. There are presently not too many commercial applications of plasma
treatment despite the numerous improvements in silk fibroin fibers using plasma treatment
in the laboratory. These is mainly due to two reasons: scale-up problem for industrial
application; reduction in the life time of the treated SFF thus not meeting industrial
standards in term of resistance to washing, perspiration, light and other conditions.

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Figure 2.2: SEM images of a) Untreated b) SF6 plasma treated silk. (Guohong et al,
2011)
2.2.2 Surface Modification by Chemical Approach.
SFF amino acids have major peptide chains as well as side chains, these chains contain
many active groups we can utilize sites for active modification. Researches done on
chemical modification of silks are intended to remodel its chemical, mechanical and
physical properties. Chemical surface modification is typically achieved through two
methods namely: Grafting copolymerization techniques and surface modification using
chemical agents.
Grafting copolymerization techniques involves the use of vinyl monomers such as 2-
hydroxyethyl methacrylate (HEMA) and diethylene glycol dimethacrylate (EGDMA-2)
to effectively treat silk by increasing silk weight and improving their silk performance
characteristics, such as crease recovery, photo yellowing resistance, oil and water
repellency, color fastness and chemical resistance (Das et al, 2001). Graft
copolymerization has the slightest degradation of the initial silk properties. The extent of
grafting and the kind of grafting monomer used are responsible for specific properties of
the grafted silk.
In Surface modification through the use chemical agents, remodeling agents like acid
anhydrides and polycarboxylic acids reacts with the primary amino acids in silk to

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assemble chemical bonds that enhance silk properties such as thermal stability, water
repellency, and fire retardancy with very little effect on the silk’s mechanical properties
(Guan et al, 2009). These chemical agents can as well be used as a linkage bridge to graft
other materials to silk fibroin fibers, this can endow the silk with totally new properties.
An example of such linkage bridge is 1, 2, 3, 4-butanetetracarboxylic acid, which can
bond hydroxylfunctionalized organophosphorus oligomers on silk fibroin fibers.
Modifying silk fibroin fibers with HFPO greatly enhance its flame retardancy. To graft
polysaccharide Chitosan on silk fibroin fiber, acid anhydrides and Polycarboxylic acids
can also be used as linkage bridges. The dye ability and antibacterial potential of silk
fibroin is increase by this modification. The resilience and handle properties of silk fibroin
fibers have been improved by treating it with Epoxide using (EPSIA) silicone-containing
epoxy crosslinking as an agent. Fiber hydrophobicity of silk fibroin fibers also increases
when the different amino acid side chains in it reacts with Isocyanates. The acid and alkali
resistence of this modified silk fibroin fibers also been enhanced.
Despite the success of chemical surface modification of silk fibroin fibers, modification
to increase its performance and quality while preserving it desired stylistic properties is
very strenuous.
2.3 Surface Modification for Biomaterials Applications.
The biodegradability and easy handling of silk fibroin fiber has made it a potential
biomaterial. Silk fibroin fibers have successfully be used to engineer tendon and ligaments
in recent years. The use of silk fibroin fibers as 3-dimension scaffolds for anterior cruciate
ligament was first studied by Kaplan and Altman. They found out that twisted scaffolds
have mechanical properties very similar to that of human anterior cruciate ligament.
(Altman et al, 2002). They however, noticed that the twisted fibers scaffolds have little
internal pores and this disallow the in growth of connective tissues. Chen et al. (2008)
made a porous ligament scaffolds that provides mechanical strength and has internal
connective pores, they achieved this by incorporating knitted silk fibers and a collagen

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matrix. Their work shows that scaffolds obtain by incorporating knitted silk with collagen
sponge has excellent biocompatibility, optimal internal space and good mechanical
strength. The medial collateral ligaments of rabbit amended with silk-collagen scaffolds
were found to be tougher than that of silk treated medial collateral ligaments after twelve
weeks of operation, this is as a result of the collagen matrix in the internal space of silk-
collagen scaffolds allows neoligament regeneration by regulating the matrix gene
expression. This suggests that silk-collagen scaffolds have a favourable application for
tendon and ligament tissue engineering. Due to the challenge of inflammatory reactions
in silk fibroin application, many researches have been carried out to improve the
biocompatibility of silk fibroin fibers. A potentially effective method of integrating silk
with other functional materials is surface modification. Silk fibroin fibers modify with
immobilized Arg–Gly–Asp (RGD) peptide were found to be more beneficial for human
tenocyte adhesion, differentiation and proliferation than unmodified silk fiber. RGD
modified silk fibroin fiber may likely to ease tendon–bone healing, this has wide
applications in tendon tissue engineering (Kardestuncer et al, 2006). To minimize the
unfavourable immune response due to sericin, sericin-free silk fibroin fibers were treated
with gelatin using ordihydroguaiaretic acid as a cross linker to biomimic the indigenous
structure of native silk fibroin fibers for application in ligament tissue engineering.
The swelling and mechanical properties of silk fibroin fibers remodeled with gelatin were
found important with no inflammatory reaction relative to native silk fibroin fibers after
four weeks' subcutaneous implantation in rats (Liu et al, 2007). As cell culture substrate,
silk fibroin fibers modified with RGD (arginineglycine-aspartic acid) will significantly
enhance anterior cruciate ligament fibroblasts attachment and spreading, human bone
marrow stromal cells, and even collagen matrix production in vitro (Chen et al, 2003).
RGD modified silk scaffolds also encourages the deposition of calcium and a more
organized extracellular matrix. The phosphorylcholine groups on silk fibroin fibers
grafted with 2-methacryloyloxyethyl phosphorylcholine serves as a biocompatible
surface. This research find out a decline in the number of adhering platelets to MPC-

13
grafted silk fibroin fibers as compared to that of unmodified silk fibroin fibers (Peng et
al, 2006). Calcium phosphate coated silk fibroin fiber, an example of inorganic–organic
composites can be prepared by sol–gel processing combined with ultraviolet (UV)
irradiation after dipping and withdrawing the silk fibroin fiber from the sol solution.
Calcium phosphate coated silk fibroin fibers are not toxic to the cells just like uncoated
silk fibroin fibers. Coated silk fibroin fibers were also found to have a hemostatic effect
which make them potential wound dressing material (Kaneko et al, 2009).
Chemical and physical properties of substrate surfaces are important in their interaction
with cells. Researchers are utilizing the various surface modification techniques to adjust
cell response to silk substrates and also to improve the cell adhesion of silk substrates,
thus enabling them to adapt to more specific needs of cell lines. More research on the
surface modification of silk fibroin fibers need to be carried out.
2.4 Biocompatibility of Silk Fibroin
Biocompatibility is the ability of a biomaterial to achieve a suitable host response in a
particular application (Williams, 1999). Biocompatibility assesses the degree and time
frame of the adverse changes in homeostatic mechanisms that govern the host response.
Biocompatibilty predicts whether a biomaterial poses a potential harm to the host
(Aramwit, 2014). The biocompatibility of a material is generally indicated by low and
non-sustained inflammatory response, low thrombosis formation and little or no
cytotoxicity (Bailey, 2013). Silk fibroin has been to know to be biocompatiple with the
human blood. We hope to arcentain these claim by the end of our Study.
2.5 Biodegredability of Silk Fibroin
Biodegradability of a Biomaterial is the ability of the Biomaterial to decompose over time
as a result of biological activities without causing an inappropriate host response. Since
silk fibroin has been widely used as a Biomaterial, its degredability is obviously impottant
to Biomedical Engineers.

14
Recently, natural and synthetic polymers have been investigated for pharmaceutical and
medical applications. Natural polymers like silk fibroin, collagen and chitosan has been
found more advategeous than synthetic ones due to their favourable properties which
includes biodegradability, bioresorbability and good biocompatibility. The chemical and
physical properties of these natural polymer can easily be modified to get desirable
degradability. Silk fibroin among the other natural polymer is a more suitable option for
biomaterial as scaffolds in biomedical application because of its controllable
biodegradability, non-cytotoxicity, high tensile strength, low antigenicity and
noninflammatory characteristics (Stitzel J at el, 2006).

15
CHAPTER 3
MATERIALS AND METHODS
3.1 Materials
The silk fibroin fibers were bought from the market. The reagents use in this study are of
high purity and standard based on the track record of the vendors they were purchased
from. Tetra (ethylene glycol) dimethacrylate from Aldrich Chemical Company, Inc.
Milwaukee WI 53233 USA were used for this analysis. They coagulation machine
(Coatron M2) and centrifuging machine use in these study are from Biyomik Laboratory,
Lefkosa. Light microscope from the bioengineering department laboratory of Cyprus
International University was also use in these study. Sterillazation of the silk fibroin fibers
used in this study was done using ultra voilet (uv) radiation in the microbiology laboratory
of Cyprus International University. Blood samples used in this study are real patients
blood samples from Biyomik Laboratory, Lefkosa. Aparatus used in this study include
petridish, spetular, scissors, rular, test tubes etc. All the glass wares used were properly
washed and afterward left to dry in a room temperature. This research was carried out
utililsing the laboratoties in Cyprus International University, facilities in Biyomik
Laboratory were also utilised.
Figure 3.1: Silk fibroin fibers

16
3.2 Preparation of the Uncoated Silk Fibroin Fibers
Two strands of the fibers were twisted to make a single strand, three strands were also
twisted to form a single strand, this was done to give varieties in the dimension of the
fibers. When a reasonble quantity was obtained, they were sterelised using uv radiation in
microbiolody laboratory of Cyprus International University. The dimension of the fibers
were measured using a Light microscope and these fibers were use in this study.
3.2.1 Preparation of the Coated silk Fibroin Fibers
Some of these uncoated fibers were coated by repeatedly dipping them in Tetra (ethylene
glycol) dimethacrylate solution. These uncoated fibers were dipped into the coating
solution for 30 minutes and dried for 10 minutes and put back into the coating solution for
30 minutes and dried for 10 minutes repeatedly. The drying was done at room temperature.
These procedure was carried out about 8 times in a day and was repeated for about 10
days. Samples are then taken for sterillization and their dimestion is been measured with
a Light microcope and considerable difference was noticed. The coated fibers were also
used in these study. Figure 3.2 below shows the drying of the coated silk fibroin fibers.
Figure 3.2: Drying of the coated silk fibroin fibers

17
3.3 Sterilization of Coated and Uncoated Silk Fibroin Fibers
Basic sterilization process were carried out on coated and uncoated silk fibroin fibers
before every test and analysis throughout the research.
Sterilization were done at the microbiology laboratory of Cyprus International University
using UV radiation.
3.4 Measurement of the Diameter of the Coated and Uncoated Silk Fibroin Fibers
The coated and uncoated silk fibroin fibers were taken to Microbiology laboratory of
Cyprus International University were their diameter was measured with a light
microscope. Five different measurements were taken on each sample, the average
calculated and the difference between the diameter of both samples were noted.
Figure 3.3: Light microscope
3.5 Hemocompatibility Test Procedure
Coated and uncoated fibers were cut into lengths of 10 cm, 12 cm and 14 cm. Each length
were then cut into smaller pieces and put into separate blood samples in a test tube and
the fibers are allowed to absorb the blood for some time. The blood are then centrifuged
to separate the plasma from the blood cells and then put into a Coagulation machine for
analysis. 5 minutes centrifuging time was used in this study. Prothrombin Time (Seconds),

18
International Normalized Ratio (in Seconds) and Prothrombin Time Percentage were
tested. Blood sample are then removed from Coagulation machine, the test tubes are
shaked and allow for another absorbtion time and the process is repeated. In this study, 10
minutes, 20 minutes, 45 minutes and 90 minutes absorbtion was used.
a b
Figure 3.4: a) Centrifuge b) Coagulation Machine
3.6 Antimicrobial Susceptibility Test Procedure
3.6.1 Preparation of the Coated and Uncoated Knitted Fibers
Silk fibroin fibers were hand knitted into circular form using the magic circle knitting
method. Twelve (12) of these knitted silk fibroin fibers were used in this research. After
knitting them, they were sterilized using UV radiation. Six of these knitted fibers were
then coated with Tetra (ethylene glycol) dimethacrylate by repeatedly dipping them in the
coating solution. After coating and drying them, they were again sterilized. Six coated
knitted silk fibroin fibers and six uncoated knitted silk fibroin fibers were used to carry
out antimicrobial susceptibility test in this research.

19
3.6.2 Disc Diffussion Method
Antimicrobial susceptibility tests are conducted to determine the in vitro activity of an
antimicrobial material against certain microbial species. The antimicrobial susceptibility
tests in this research was carried out with six microorganisms, five of which were bacteria
(3 gram positive and 2 gram negative) and one a yeast. They are namely Staphylococcus
aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Bacillus cereus (ATCC
10876), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), and
Candida albicans (ATCC 90028) respectively. Mueller Hinton agar was used instead of
other agar such as nutrient agar, chocolate agar, blood agar, and so on based on Clinical
and Laboratory Standards Institute’s (CLSI) (2012) for carrying out antimicrobial
susceptibility testing.
Firstly, the solid agar medium of Mueller Hinton Agar was made following the
manufacturer’s guideline of 38.0 g/L and then sterilized by autoclaving at 121o
C for 15
minutes. Afterwards it is poured into the petri dishes at a depth of approximately 4mm
and when cool enough, it is placed in the refrigerator and left overnight. Nutrient broth of
all the microrganisms to be used were prepared to a standard of 0.5 McFarland
(approximately 106 cells/ml). The agar plate is then labelled and using a sterile cotton
swab on its surface to inoculate it with 10 μL of Nutrient broth containing a specific
microorganism.
Already prepared silk fibroin fibers were carefully placed in the middle of the innoculated
agar. When all of the samples have been placed in the culture media inoculated with the
different microorganisms. They are placed at room temperature for 10 minutes and then
incubated upside down at 37±0.1°C for 24 hours. After 24 hours results were readily
visible.

20
In these Research, a pre-study was done and afterward, the experiments was reconducted
with all samples in seperate petridish.
3.8 Characterization of Coated Silk Fibroin Fibers
3.8.1 Scanning Electron Microscopy Analysis
Scanning electron microscope (SEM) is a type of electron microscope that uses focused
beam of electrons to scan and produce the surface images of samples. The electrons are
made to interact with the atoms in the sample, producing different signals that give us
information about the sample's surface composition and topography.
Scanning Electron Microscopy analysis was done at Cyprus International University by
using a SEM Model Jsm- 6510 model at an acceleration voltage 10kV.

21
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1 Thickness of the Uncoated Fibers
Five dimensions of the thickness of the uncoated fiber were taken with an inverted light
microscope. Table 4.1 below shows the values that were obtained.
Table 4.1: Thickness of the uncoated fibers
Measurement M1 M2 M3 M4 M5
Thickness (µm) 236.8 347.6 350.8 231.2 282.7
Figure 4.1 below is a photo gotten from the light microscope. It shows the five dimensions
taken for the uncoated fibers.
Figure 4.1: Five dimensions of the thickness of the uncoated fibers

22
The Average of the thickness of the uncoated fibers is calculated as shown below;
236.8µm +347.6µm +350.8µm+231.2µm +282.7µm = ±289.82 µm
5
4.2 Thickness of the coated fibers
Five dimensions of the thickness of the coated fiber were taken with an inverted light
microscope. Table 4.2 below shows the values that were obtained.
Table 4.2: Thickness of the coated fibers
Measurement M1 M2 M3 M4 M5
Thickness (µm) 474.2 310.4 446.9 398.0 407.7
Figure 4.2 below is a photo gotten from the inverted light microscope. It shows the five
dimensions taken for the coated fibers.
Figure 4.2: Five dimensions of the thickness of the coated fibers

23
The Average of the thickness of the coated fibers is calculated as shown below;
474.2µm +310.4µm +446.9µm+398.0µm +407.7µm = ±407.44µm
5
Difference in thickness of the coated to uncoated fiber = 407.44-289.82=117.62 µm
4.3 Hemocompatibility Test Results
Invitro Blood Coagulation Test
This test and analysis were done to determine the anticoagulant activity of the coated and
uncoated silk fibroin fibers. Certain markers were taken to be able to check the anti-
coagulant activity of these fibers, which are the prothrombin time (PT), international
normalized ratio (INR) and prothrombine time percentage. Ethical and proper permissions
were taken from the Laboratory and head of the department of blood coagulation before
samples of blood were used.
These were used to determine the hemocompatibility of coated and uncoated silks fibroin
fibers through the different clotting times that were analyzed. Table 4.3 below gives the
normal standard clotting time or values for a healthy person without thinning drugs.
Table 4.3: Normal standard clotting time for a healthy person without thinning drugs
PTSec INR PT%
11.5-15 0.80-1.20 70-120
The tables and graphs below shows the Hemocompatibility test results obtained.

24
Table 4.4: PTSec, INR and PT% results for 10cm of uncoated fibers with five different
blood samples at different absorption time.
Initial Values Values after
10mins
Values after
20mins
Values after
45mins
Values after
90mins
Blood Sample 1
PTSec
INR
PT%
13.3sec
1.05INR
92%
13.0sec
1.02INR
97%
12.8sec
1.00INR
100%
12.7sec
0.99INR
102%
13.5sec
1.07INR
89%
Blood Sample 2
PTSec
INR
PT%
29.3sec
2.93INR
26%
32.4sec
3.34INR
23%
32.4sec
3.34INR
22%
33.5sec
3.49INR
22%
33.0sec
3.43INR
22%
Blood Sample 3
PTSec
INR
PT%
13.3sec
1.05INR
92%
13.2sec
1.04INR
94%
13.2sec
1.04INR
94%
12.5sec
0.97INR
105%
13.6sec
1.08INR
88%
Blood Sample 4
PTSec
INR
PT%
15.0sec
1.23INR
73%
16.3sec
1.37INR
62%
16.5sec
1.39INR
61%
16.8sec
1.42INR
59%
17.3sec
1.48INR
56%
Blood Sample 5
PTSec
INR
PT%
22.4sec
2.07INR
38%
21.5sec
1.96INR
40%
21.7sec
1.99INR
40%
22.3sec
2.06INR
38%
22.1sec
2.03INR
38%
Table 4.5: PTSec, INR and PT% results for 10cm of coated fibers with five different
10mins
Values after
20mins
Values after
45mins
Values after
90mins
Blood Sample 1
PTSec
INR
PT%
12.7sec
0.99INR
102%
13.6sec
1.08INR
88%
13.3sec
1.04INR
94%
13.2sec
0.04INR
94%
13.4sec
1.06INR
91%
Blood Sample 2
PTSec
INR
PT%
25.1sec
2.40INR
32%
24.5sec
2.33INR
33%
24.9sec
2.38INR
32%
24.4sec
2.31INR
33%
24.3sec
2.30INR
34%
Blood Sample 3
PTSec
INR
PT%
13.3sec
1.05INR
92%
13.0sec
1.02INR
97%
12.9sec
1.01INR
98%
13.2sec
1.04INR
94%
13.3sec
1.05INR
92%
Blood Sample 4
PTSec
INR
PT%
16.6sec
1.40INR
61%
17.0sec
1.45INR
58%
16.4sec
1.38INR
62%
17.4sec
1.49INR
59%
17.4sec
1.49INR
56%
Blood Sample 5
PTSec
INR
PT%
14.6sec
1.19INR
76%
14.5sec
1.18INR
77%
14.4sec
1.17INR
78%
14.6sec
1.19INR
76%
15.0sec
1.23INR
73%

25
Figure 4.3: Graph of PTSec test results for 10cm uncoated silk fibers at different
absorption time.
Figure 4.4: Graph of PTSec test results for 10cm coated silk fibers at different absorption
time.
0
5
10
15
20
25
30
35
40
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins
0
5
10
15
20
25
30
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

26
The PTSec results for 10cm of the uncoated silk fibroin fibers for blood samples 1, 3 and
4 fell within the standard range of a healthy person (11.5-15), that of blood sample 5 was
a little bit out of range while that of blood sample 2 was very much out of range, this will
must likely be that the patient with blood sample 2 has some health conditions that affects
the blood clotting time. Blood sample 4 gave the best PTSec results for 10cm of the
uncoated silk fibroin fibers as it gave an increase in PTSec with increase in absorption
time.
For 10cm of the coated silk fibroin fibers, the PTSec of blood sample 1,3, and 4 fell within
the standard range of a healthy person, blood sample 4 is a little out of range while blood
sample 2 is verry much out of range. The coated silk fibroin fibers also gave higher PTSec
values than the uncoated silk fibroin fibers, these indicates that the coated silk fibroin
fibers are more biocompatible with the blood samples than the uncoated silk fibroin fibers.
Figure 4.5: Graph of INR test results for 10cm uncoated silk fibers at different absorption
time.
0
0,5
1
1,5
2
2,5
3
3,5
4
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

27
Figure 4.6: Graph of INR test results for 10cm coated silk fibers at different absorption
time.
The INR results for 10cm of the uncoated silk fibroin fibers for blood samples 1, 3 and 4
fell within the standard range of a healthy person (0.80-1.20), that of blood blood sample
5 was a little bit out of range while that of blood sample 2 was very much out of range,
this will must likely be that the patient with blood sample 2 has some health conditions
that affects the blood clotting time. Blood sample 4 gave the best INR results for 10cm of
the uncoated silk fibroin fibers as it gave an increase in INR with increase in absorption
time.
For 10cm of the coated silk fibroin fibers, the INR result for blood sample 1,3, and 5 fell
within the standard range of a healthy person, blood sample 4 is a little out of range while
blood sample 2 is verry much out of range. The coated silk fibroin fibers also gave higher
PTSec values than the uncoated silk fibroin fibers, these indicates that the coated silk
0
0,5
1
1,5
2
2,5
3
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
Initial values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

28
fibroin fibers are more biocompatible with the blood samples than the uncoated silk
fibroin fibers.
Figure 4.7: Graph of PT% test results for 10cm uncoated silk fibers at different absorption
time.
0
20
40
60
80
100
120
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

29
Figure 4.8: Graph of PT% test results for 10cm coated silk fibers at different absorption
time.
The PT% results for 10cm of the uncoated silk fibroin fibers for blood samples 1, 2 and 3
fell within the standard range of a healthy person (70-120), that of blood sample 4 was a
little bit below the range while that of blood sample 5 was very much below the range, it
is must likely be that the patient with blood sample 5 has some health conditions that
affects the blood clotting time. Blood sample 1 gave the best PT% results for 10cm of the
uncoated silk fibroin fibers as it gave an increase in PT% with increase in absorption time.
For 10cm of the coated silk fibroin fibers, the PT% result for blood sample 1,3, and 5 fell
within the standard range of a healthy person, blood sample 4 is a little bit below the range
while blood sample 2 was very much out of range. The coated silk fibroin fibers also gave
higher PT% values than the uncoated silk fibroin fibers, these indicates that the coated
silk fibroin fibers are more biocompatible with the blood samples than the uncoated silk
fibroin fibers.
0
20
40
60
80
100
120
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

30
blood samples at different absorption time
10mins
Values after
20mins
Values after
45mins
Values after
90mins
Blood Sample 1
PTSec
INR
PT%
15.2sec
1.25INR
71%
15.0sec
1.23INR
73%
14.8sec
1.21INR
74%
15.5sec
1.28INR
68%
16.1sec
1.35NR
64%
Blood Sample 2
PTSec
INR
PT%
15.6sec
1.25INR
68%
15.6sec
1.29INR
68%
15.8sec
1.31INR
66%
17.0sec
1.45INR
58%
16.2sec
1.36INR
63%
Blood Sample 3
PTSec
INR
PT%
11.3sec
0.85INR
135%
11.6sec
0.88INR
126%
11.6sec
0.88INR
126%
12.6sec
0.98INR
104%
12.8sec
0.98INR
110%
Blood Sample 4
PTSec
INR
PT%
12.8sec
1.00INR
100%
12.6sec
0.98INR
104%
12.9sec
1.01INR
98%
13.8sec
1.10INR
85%
13.5sec
1.07INR
89%
Blood Sample 5
PTSec
INR
PT%
25sec
2.39INR
32%
24.8sec
2.36INR
33%
24.4sec
2.31INR
33%
26.6sec
2.56INR
30%
26.1sec
2.52INR
33%
10mins
Values after
20mins
Values after
45mins
Values after
90mins
Blood Sample 1
PTSec
INR
PT%
14.1sec
1.13INR
82%
14.0sec
1.12INR
83%
14.1sec
1.05INR
92%
14.5sec
1.18INR
77%
15sec
1.20NR
No Plasma
Blood Sample 2
PTSec
INR
PT%
17.6sec
1.51INR
55%
18.7sec
1.64INR
50%
19.2sec
1.69INR
48%
20.6sec
1.86INR
43%
20.4sec
1.83INR
43%
Blood Sample 3
PTSec
INR
PT%
16.6sec
1.40INR
61%
17.6sec
1.51INR
55%
18.0sec
1.56INR
53%
18.7sec
1.64NR
50%
18.9sec
1.66INR
49%
Blood Sample 4
PTSec
INR
PT%
23.5sec
2.20INR
35%
24.3sec
2.30INR
34%
24.0sec
2.26INR
34%
25.7sec
2.47INR
31%
26.0sec
2.51INR
31%
Blood Sample 5
PTSec
INR
PT%
13.9sec
1.11INR
84%
14.0sec
1.21INR
83%
13.8sec
1.10INR
85%
14.3sec
1.15INR
80%
14.2sec
1.14INR
81%

31
absorption time.
Figure 4.10: Graph of PTSec test results for 12cm coated silk fibers at different
absorption time.
0
5
10
15
20
25
30
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after20mins
Values after 45mins
Values after 60mins
0
5
10
15
20
25
30
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

32
The PTSec results for 12cm of the uncoated silk fibroin fibers for blood samples 1, 2, 3
and 4 fell within the standard range of a healthy person (11.5-15), that of blood sample 5
was very much out of range, this will must likely be that the patient with blood sample 5
has some health conditions that affects the blood clotting time. Blood sample 3 gave the
best PTSec results for 12cm of the uncoated silk fibroin fibers as it gave an increase in
PTSec with increase in absorption time.
For 12cm of the coated silk fibroin fibers, the PTSec of blood sample 1 and 2 fell within
the standard range of a healthy person, blood sample 2 and 3 is a little out of range while
blood sample 4 is verry much out of range. Blood sample 3 gave the best PTSec results
for 12cm of the coated silk fibroin fibers as it gave an increase in PTSec values with
increase in absorption time. The coated silk fibroin fibers also gave higher PTSec values
than the uncoated silk fibroin fibers, these indicates that the coated silk fibroin fibers are
more biocompatible with the blood samples than the uncoated silk fibroin fibers.
Figure 4.11: Graph of INR test results for 12cm uncoated silk fibers at different
absorption time.
0
0,5
1
1,5
2
2,5
3
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 60mins

33
time.
The INR results for 12cm of the uncoated silk fibroin fibers for blood samples 1 and 4 fell
within the standard range of a healthy person (0.80-1.20), that of blood blood sample 1
and 2 were a little bit out of range while that of blood sample 5 was very much out of
range, this will must likely be that the patient with blood sample 5 has some health
conditions that affects the blood clotting time. Blood sample 3 gave the best INR results
for 12cm of the uncoated silk fibroin fibers as it gave an increase in INR with increase in
absorption time.
For 12cm of the coated silk fibroin fibers, the INR result for blood sample 1 and 5 fell
within the standard range of a healthy person, blood sample 2 and 3 were a little out of
range while blood sample 4 was very much out of range. Blood sample 1 gave the best
INR results for 12cm of the uncoated silk fibroin fibers as it results were both within the
range of a healthy person and also increases in with increase in absorption time. The
coated silk fibroin fibers generally gave higher INR values than the uncoated silk fibroin
0
0,5
1
1,5
2
2,5
3
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

34
fibers, these indicates that the coated silk fibroin fibers are more biocompatible with the
blood samples than the uncoated silk fibroin fibers.
Figure 4.13: Graph of PT% test results for 12cm uncoated silk fibers at different
absorption time.
0
20
40
60
80
100
120
140
160
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 60mins

35
Figure 4.14: Graph of PT% test results for 12cm coated silk fibers at different
absorption time.
The PT% results for 12cm of the uncoated silk fibroin fibers for blood samples 1, 3 and 4
fell within the standard range of a healthy person (70-120), that of blood sample 2 was a
little bit below the range while that of blood sample 5 was very much below the range, it
is must likely be that the patient with blood sample 5 has some health conditions that
affects the blood clotting time.
For 12cm of the coated silk fibroin fibers, the PT% result for blood sample 1 and 5 fell
within the standard range of a healthy person, while blood sample 2, 3 and 4 were very
much below the range. Blood sample 1 also didn’t give any PT% value after 90 minutes
because there was no plasma left in it. The coated silk fibroin fibers also gave higher PT%
values than the uncoated silk fibroin fibers, these indicates that the coated silk fibroin
fibers are more biocompatible with the blood samples than the uncoated silk fibroin fibers.
0
10
20
30
40
50
60
70
80
90
100
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

36
10mins
Values after
20mins
Values after
45mins
Values after
90mins
Blood Sample 1
PTSec
INR
PT%
13.1sec
0.98INR
103%
13.0sec
0.97INR
105%
12.7sec
0.95INR
110%
12.6sec
0.94INR
112%
12.5sec
0.93NR
113%
Blood Sample 2
PTSec
INR
PT%
12.8sec
0.98INR
108%
12.6sec
0.94INR
112%
12.5sec
0.93INR
114%
13.3sec
1.00INR
100%
13.5sec
1.10INR
110%
Blood Sample 3
PTSec
INR
PT%
13.2sec
0.99INR
101%
12.7sec
0.95INR
110%
12.6sec
0.94INR
112%
13.0sec
0.97NR
105%
13.1sec
0.98INR
110%
Blood Sample 4
PTSec
INR
PT%
13.9sec
1.06INR
91%
13.7sec
1.04INR
94%
13.3sec
1.00INR
100%
14.2sec
1.09INR
87%
14.9sec
1.10INR
89%
Blood Sample 5
PTSec
INR
PT%
14.8sec
1.14INR
80%
15.4sec
1.20INR
74%
14.4sec
1.11INR
85%
15.5ec
1.21INR
73%
14.9sec
1.30INR
70%
10mins
Values after
20mins
Values after
45mins
Values after
90mins
Blood Sample 1
PTSec
INR
PT%
14.2sec
1.09INR
87%
14.3sec
1.10INR
86%
13.9sec
1.06INR
91%
14.5sec
1.12INR
83%
14.6sec
1.14NR
87%
Blood Sample 2
PTSec
INR
PT%
12.9sec
0.96INR
107%
29.0sec
0.93INR
114%
11.9sec
0.87INR
129%
12.7sec
0.95INR
110%
13.0sec
1.02INR
100%
Blood Sample 3
PTSec
INR
PT%
13.7sec
1.04INR
94%
13.1sec
0.98INR
103%
12.2sec
0.90INR
121%
12.8sec
0.96NR
108%
13.0sec
0.99INR
108%
Blood Sample 4
PTSec
INR
PT%
12.6sec
0.94INR
112%
12.3sec
0.91INR
119%
11.4sec
0.83INR
144%
12.0sec
0.88INR
126%
12.3sec
0.90INR
120%
Blood Sample 5
PTSec
INR
PT%
13.5sec
1.02INR
97%
31.7ec
1.00INR
100%
12.3sec
1.91INR
119%
12.7ec
1.95INR
110%
13.0sec
1.00INR
112%

37
absorption time.
Figure 4.16: Graph of PTSec test results for 14cm coated silk fibers at different
absorption time.
The PTSec results for 14cm of the uncoated silk fibroin fibers for blood samples 1, 2, 3,
4 and 5 fell within the standard range of a healthy person (11.5-15).
0
2
4
6
8
10
12
14
16
18
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins
0
2
4
6
8
10
12
14
16
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

38
For 14cm of the coated silk fibroin fibers, the PTSec of blood sample 1, 2, 3, 4 and 5 also
fell within the standard range of a healthy person.
The coated silk fibroin fibers gave higher PTSec values than the uncoated silk fibroin
fibers, these indicates that the coated silk fibroin fibers are more biocompatible with the
blood samples than the uncoated silk fibroin fibers.
There are also irregular increase and decrease in PTSec values but to a greater extent, there
were gradual increases in PTSec values with increase in absorption time.
Figure 4.17: Graph of INR test results for 14cm uncoated silk fibers at different
absorption time.
0
0,2
0,4
0,6
0,8
1
1,2
1,4
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

39
time.
The INR results for 14cm of the uncoated silk fibroin fibers for blood samples 1, 2, 3, 4
and 5 fell within the standard range of a healthy person (0.80-1.20).
For 14cm of the coated silk fibroin fibers, the INR of blood sample 1, 2, 3, 4 and 5 also
The coated silk fibroin fibers gave higher INR values than the uncoated silk fibroin fibers,
these indicates that the coated silk fibroin fibers are more biocompatible with the blood
samples than the uncoated silk fibroin fibers.
0
0,2
0,4
0,6
0,8
1
1,2
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

40
There are also irregular increase and decrease in INR values but to a greater extent, there
were gradual increases in INR values with increase in absorption time.
Figure 4.19: Graph of PT% test results for 14cm uncoated silk fibers at different
absorption time.
0
20
40
60
80
100
120
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

41
Figure 4.20: Graph of PT% test results for 14cm coated silk fibers at different
absorption time.
The PT% results for 14cm of the uncoated silk fibroin fibers for blood samples 1, 2, 3, 4
and 5 fell within the standard range of a healthy person (70-120). Blood sample 1 gave
the best PT% results as there were increase in PT% values with increase in absorption
time.
For 14cm of the coated silk fibroin fibers, the PT% of blood sample 1, 2, 3, 4 and 5 also
The coated silk fibroin fibers gave higher PT% values than the uncoated silk fibroin fibers,
these indicates that the coated silk fibroin fibers are more biocompatible with the blood
samples than the uncoated silk fibroin fibers.
0
20
40
60
80
100
120
140
160
Blood Sample
1
Blood Sample
2
Blood Sample
3
Blood Sample
4
Blood Sample
5
Initial Values
Values after 10mins
Values after 20mins
Values after 45mins
Values after 90mins

42
There are also irregular increase and decrease in INR values for the uncoated silk fibroin
fibers but to a greater extent, there were gradual increases in INR values with increase in
absorption time.
The coated and uncoated silk fibroin fibers were evaluated for their blood compatibility,
using the hemocompatibility assays such as PT, INR and PT%.
There are ramdom changes in the values of PT, INR and PT%, this could be as a result of
using different blood samples and some of this samples could be lacking some clotting
factors such as vitamin K. Generally, there was an increase in PT, INR and PT% values
as the absorption time increases.
PT% ranges from 70 -120%. As the PT percentage approaches 120 and above, it shows a
great blood coagulation activity, and vice versa and drugs are needed to help reverse the
situation.
The Normal range of PTSec ranges from 11.5-15 seconds. Any increase in the time
indicates an intake of an anti-coagulant drug or food that helps enhances the
anticoagulation activity.
An increase in INR result above 0.80-1.20 based on the standard values from few
laboratories indicates anticoagulation activity, also its value is dependent on the PT sec,
PT percentage and APTT values. Patients with an increased values of PT and APTT,
following a decreased PT percentage will have a high INR result which indicates poor
blood clotting and those with low PT and APTT following an increase in PT percentage
will show a reduced INR result which indicates blood coagulation activity.

43
These results shows that the silk fibroin fibers use in the study are highly biocompatible
with the blood because of their activity in the blood causing enhanced anticoagulant
activity.
4.4 Antimicrobial Susceptibility Test Results
The pre-study results after 24 hours were good. Both the coated and uncoated silk fibroin
were all susceptible to all the microorganisms, they all showed zones of inhibition after
incubation.
The main experiments results after 24 hours were even better. As in the pre-study, both
the coated and uncoated silk fibroin showed zones of inhibition. Silk fibroin fibers alone
and silk fibroin fibers coated with Tetra (ethylene glycol) dimethacrylate solution
exhibited excellent antibacterial susceptibility. However, the coated silk fibroin fibers
generally showed a larger zone of inhibition against all the microorganisms than the
uncoated silk fibroin fibers.
Figure 4.21 and 4.22 below shows the susceptibility of the uncoated and coated silk fibroin
fibers to the following microorganism in order.
A. Escherichia coli (ATCC 25922) (Gram-negative)
B. Enterococcus faecalis (ATCC 29212) (Gram-positive)
C. Bacillus cereus (ATCC 10876) (Gram-positive)
D. Pseudomonas aeruginosa (ATCC 27853) (Gram-negative)
E. Staphylococcus aureus (ATCC 25923) (Gram-positive)
F. Candida albicans (ATCC 90028). (Yeast)

44
Figure 4.21: Antimicrobial zone of inhibition for uncoated silk fibroin fibers
Figure 4.22: Antimicrobial zone of inhibition for coated silk fibroin fibers

45
The Table 4.10 below shows the diameter of the area of inhibition obtain for the
antimocrobial susceptibility test for both the uncoated and coated fibers.
Table 4.10: Diameter of the area of inhibition measure from the antimicrobial
susceptibility test results
Microorganism Uncoated Silk Fibrion Fibers(mm) Coated Silk Fibroin Fibers(mm)
Escherichia coli 19 30
Enterococcus faecalis 16 29
Bacillus cereus 25 37
Pseudomonas aeruginosa 40 50
Staphylococcus aureus 36 51
Candida albicans 20 38
Figure 4.23 below gives a graphical representation of the different zones of inhibition for
both coated and uncoated silk fibroin fibers for all the microorganisms.
Figure 4.23: Graph of the diameter of the the zones of inhibition for uncoated
and coated silk fibers on the different microorganisms
0
10
20
30
40
50
60
Uncoated Silk Fibrion
Fibers(mm)
Coated Silk Fibroin
Fibers(mm)

46
4.5 Scanning Electron Microscopy (SEM) Analysis
Scanning Electron Microscopy (SEM) was adopted to analyze the sample's
surface topography and composition. The Scanning Electron Microscopy analysis was
done at Cyprus International University by using a SEM Model JSM-6610 at an
acceleration voltage 15kV.
The results for the SEM at a magnification of X300 shows the two seperate streads of silk
fibers that were twisted. At a magnification of X800 at 11mm, the seperate streads of silk
fibers were more clearly seen and the coating of the fibers with the coating solution also
visible.
At higher magnifications of X1400, the biodegradability of the coated silk fibroin fibers
could be seen, these shows how the biomaterial will degrade with time. SEM results
clearly showed the surface topograpy and composition of the coated silk fibroin fibers.
Figure 4.24-2.29 below show the SEM micrograph of the coated fibers at different μm
and at different magnifications. The figures show a detailed information of the
surface topography and composition of the coated fibers.
Figure 4.24: SEM migcrogragh of uncoated silk fibers of 50µm at X300 magnification.

47
Figure 4.25: SEM migcrogragh of coated silk fibers of 50µm at X300 magnification.
Figure 4.26: SEM migcrogragh of uncoated silk fibers of 20µm at X800 magnification.

48
Figure 4.28: SEM migcrogragh of uncoated silk fibers of 10µm at X1400
magnification.

49
4.7 Applications of the Study
This research can be potentially applied in Tissue Engineering, drug delivering systems,
wound healing and the pharmacautical industry. Scaffold built from this coated silk fibers
will be a great relief to millions of patients worldwide who have a defeative organ or tissue
or have lost vital organs and tissues. Patients will have a close to a normal life and will be
save from stigmazation and pain.

50
CHAPTER 5
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion
The results obtained from this research affirmed that surface modification of silk fibroin
fibers with Tetra (ethylene glycol) dimethacrylate enhances its biocompatibility and
antimicrobial properties. This research also showed that silk fibroin fibers are
biocompatible and have antimicrobial properties on their own but modifying them with
other substances will enhance these properties and make them more suitable for different
biomedical applications.
5.2 Recommendations
In this research, different blood samples were used in the hemocompatibbility study, better
results will be obtain if the same blood sample were used as this will keep other factors
constant.
Before applying the results of this reserch, extensive in vivo analysis of biomaterial must
be carried out and appropraite approval obtained.

51
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