NairnMD_0310_std

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Evaluation of Selected Contemporary Biomaterials and
Surface Treatments for Soft Tissue Repair Prosthesis
A Thesis submitted by
Michael Douglas Nairn BSc
In fulfilment of the requirements for the degree of Doctor of Philosophy at Heriot-
Watt University, Edinburgh, United Kingdom
School of Textiles and Design March 2010
“This copy of the thesis has been supplied on condition that anyone who consults it is
understood to recognise that the copyright rests with its author and that no quotation
from the thesis and no information derived from it may be published without the prior
written consent of the author or of the University (as may be appropriate).”

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Abstract
The aim of this project was to determine the best materials and surface treatments for
soft tissue repair and to enhance our understanding of material / cell interactions by
comparing the response of human cells growing on a selection of currently approved
and novel biomaterials. This study focused on comparing the materials and also
investigated the effect of modifying the surfaces using gas plasma and other
treatments with the aim of enhancing cell growth. In addition, chitosan was studied
to examine the reported bacteriostatic effect and promotion of human cell growth.
Chitosan has many properties but this research focused on its reported acceleration of
wound healing haemostatic and bacteriostatic properties. To examine the
bacteriostatic properties of chitosan, a number of experimental designs were used.
The bacteriostatic study led onto a selection of means to incorporate chitosan
into/onto some of the biomaterials being tested.
A selection of biomaterials were examined for their ability to support tissue growth in
native and surface modified forms (plasma treatment/ chitosan treatment). Cells were
seeded on the samples and the growth of the cells was measured at weekly intervals.
The outcome of this research was that the optimal material for soft tissue repair was
found to be polyurethane with an ammonia plasma treatment. This can be made into
a mesh prosthesis for hernia repair and can be coated with chitosan to inhibit bacterial
colonisation if required.

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Acknowledgements
I would like to thank Alex Fotheringham for encouraging me and for believing in me.
I would like to thank my family for their love and support. I would like to thank my
friends in Galashiels for making life fun when I was there. I‟d also like to thank my
friends at Riccarton for the same reason. I would like to thank Stuart Wallace,
Vikash Agrawal, Rodger Spark, Margaret Robson, Andrew McCulloch and all the
others that helped with my work in Galashiels. I would like to thank Prof Brian
Austin and Dawn Austin for their help in the microbiology lab. I would like to thank
Lei Zhang for his tutorial on tissue culture. I would like to thank Anderson
Caledonia for their help with sterilising my samples. I would also like to thank
Solvay and Rodenburg Biopolymers for their materials. In general I want to thank
everybody that has helped me along my way. I would like to make a special thank
you for Yifan Wu who has shown extraordinary patience, caring and understanding
while I have been working on this project.
Correspondence and Requests for materials should be addressed to:
Michael D Nairn
10 Park Terrace
Stirling
FK8 2JT

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Contents
Evaluation of Selected Contemporary Biomaterials and Surface
Treatments for Soft Tissue Repair Prosthesis .............................................i
Abstract.......................................................................................................ii
Acknowledgements ...................................................................................iii
Contents.....................................................................................................iv
Definitions ................................................................................................vii
Chapter 1 – Introduction............................................................................. 1
1.1 Research Aims ........................................................................................................1
1.2 Current Situation.....................................................................................................2
1.2.1 Hernia repair.....................................................................................................3
1.2.2 Complications ..................................................................................................6
1.3 Prosthesis Related Infections ..................................................................................8
1.3.1 Incorporation of antimicrobials into medical prostheses .................................9
1.4 Tissue Engineering..................................................................................................9
1.5 Reasons for Improvement .....................................................................................11
1.5.1 Niche ..............................................................................................................11
Chapter 2 - Material Review .................................................................... 13
2.1 Potential Materials ................................................................................................14
2.1.1 Material selection...........................................................................................14
2.1.2 Chosen Materials............................................................................................23
2.2 Potential Treatments for Materials........................................................................24
2.2.1 Chitosan/ chitin coating .................................................................................24
2.2.2 Low Pressure Plasma Treatment....................................................................25
2.2.3 Hyaluronic acid..............................................................................................27

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2.2.4 Laser pitting ...................................................................................................27
2.2.5 Micro-grooves................................................................................................27
2.2.6 Chosen treatments for materials.....................................................................28
Chapter 3 - Methodology.......................................................................... 29
3.1 Examination of Chitosan as a Bacteriostat ...........................................................29
3.1.1 Materials.........................................................................................................29
3.1.2 Methods..........................................................................................................34
3.2 Production of Biomaterial Samples ......................................................................47
3.2.1 Extrusion ........................................................................................................47
3.2.2 Film Casting...................................................................................................52
3.2.3 Plasma Treatment...........................................................................................53
3.3 Sample Characterisation .......................................................................................59
3.3.1 Differential scanning calorimetry (DSC) Analysis........................................59
3.3.2 SEM Analysis ................................................................................................59
3.4 Tissue Culture Study.............................................................................................62
3.4.1 Methodology ..................................................................................................62
3.4.2 Experimental Work........................................................................................65
Chapter 4 - Results ................................................................................... 72
4.1 Examination of Chitosan as a Bacteriostat ...........................................................72
4.2 Production of Biomaterial Samples ......................................................................97
4.3 Sample Characterisation .......................................................................................97
4.3.1 Differential scanning calorimetry (DSC) Analysis........................................97
4.3.2 SEM Analysis ..............................................................................................101
4.4 Tissue Culture Study...........................................................................................115
Chapter 5 – Discussion........................................................................... 139
5.1 Examination of Chitosan as a Bacteriostat .........................................................140

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5.2 Production of Biomaterial Samples ....................................................................144
5.3 Sample Characterisation .....................................................................................144
5.3.1 Differential scanning calorimetry (DSC) Analysis......................................144
5.2.2 SEM Analysis ..............................................................................................144
5.4 Tissue culture study.............................................................................................146
Conclusions...............................................................................................................153
Appendix ................................................................................................ 156
A. Examination of Chitosan as a Bacteriostat........................................................156
A.1 Details of media used for the examination of chitosan as a bacteriostat........156
A.2 Unedited graph from Method 2c....................................................................158
A.3 Data from Method 4b.....................................................................................159
B. PLA pore size data ............................................................................................178
C. Tissue culture study...........................................................................................184
C.1 Tissue culture study raw data....................................................................184
C.2 Fluorescence images .................................................................................207
References .............................................................................................. 303

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Definitions
Aliphatic
“Pertaining to any member of one of the two major groups of organic compounds,
those with branched or chain structure.”
(Dorland, 2009)
Alloplast
“An inert foreign body used for implantation into tissue.”
(Dorland, 2009).
Antibiotic
“Antibiotics are a class of natural and synthetic compounds that are able
selectively and at low concentrations to destroy or inhibit the growth of other
organisms, especially microorganisms.”
(Oxford Dictionary of Biochemistry and Molecular Biology, 2000).
Ångström
A unit of length equal to one hundred-millionth of a centimetre (10-10
meter).
(The Oxford Dictionary, Thesaurus and Wordpower Guide, 2001)
Apoptosis
“Cell death as a result of an intracellular “suicide” programme. It is a normal
and essential event during development generally and within the immune system.
Apoptosis does not lead to lysis of cells and thus avoids damage to neighbouring
tissue. Alt. Programmed cell death.”
(Henderson's Dictionary of Biology, 2008)

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Biocompatibility
“The ability of a material to perform with an appropriate host response in a
specific application”
(Definitions in Biomaterials, 1986).
This is the preferred definition, commonly referred to as the Williams definition of
biocompatibility (The Williams Dictionary of Biomaterials, 1999).
“Comparison of the tissue response produced through the close association of the
implanted candidate material to its implant site within the host animal to that
tissue response recognised and established as suitable with control materials”
(ASTM International, 2008). This is a specific definition as it refers solely to
implanted devices and the local tissue response.
Biomimetic material
“Any material that is structurally or chemically analogous to a component of
plant or animal tissue and which can be incorporated into any product whose use
is based on the characteristics of that tissue component.”
(The Williams Dictionary of Biomaterials, 1999)
Bactericidal
“Causing the death of bacteria.”
Bacteriostatic
“Inhibiting growth but not killing bacteria.”

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Chitin
“Insoluble, linear polysaccharide forming the principal constituent of arthropod
exoskeletons and found in some plants, particularly fungi.”
(Dorland, 2009)
Cytotoxic
“Attacking or destroying cells.”
Extrusion
“To shape a material such as metal or plastic by forcing it through a die. “
(The Oxford Dictionary, Thesaurus and Wordpower Guide, 2001)
Fibroblast
“Flattened, irregular-shaped connective tissue cell, ubiquitous in fibrous
connective tissue. It secretes components of the extracellular matrix, including
type 1 collagen and hyaluronic acid.”
Fistula
“An abnormal passage between two internal organs or from an internal organ to
the body surface.”
(Dorland, 2009)
Granuloma
“Inflammatory tissue nodule containing proliferating lymphocytes, fibroblasts,
giant cells and epithelioid cells, which forms in response to chronic infection or
persistence of antigen.”
(Roitt and Delves, 1994).

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Hernia
“Protrusion of a portion of an organ or tissue through an abdominal opening.”
(Dorland, 2009)
Abdominal hernia
“One through the abdominal wall, either a congenital defect or a complication of
pregnancy or a surgical incision.”
(Dorland, 2009)
Diaphragmatic hernia
“Hernia through the diaphragm.”
(Dorland, 2009)
Incisional hernia
“One through an old abdominal incision.”
(Dorland, 2009)
Inguinal hernia
“Hernia into the inguinal canal.”
(Dorland, 2009)
Cystocele
“Hernial protrusion of the urinary bladder, usually through the vaginal wall.”
(Dorland, 2009)
Enterocele
“An enterocele is essentially a vaginal hernia in which the peritoneal sac
containing a portion of the small bowel extends into the rectovaginal space
between the posterior surface of the vagina and the anterior surface of the
rectum.”
(Diagnosing and Treating an Enterocele, 1999).

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Rectocele
“Hernial protrusion of part of the rectum into the vagina.”
(Dorland, 2009)
Mechanotransduction
“Mechanotransduction refers to the many mechanisms by which cells convert
mechanical stimulus into chemical activity.”
(Katsumi et al., 2004, Liu et al., 1996).
Nosocomial
“Hospital acquired, in relation to infections.”
Osteoblast
“Bone forming cell that secretes the bone matrix.”
Parastomal
“Para- indicating beside or near. Stoma (stomal) mouth-like opening,
particularly an incised opening which is kept open for drainage or other
purpose.”
(Dorland, 2009)
Plasma (gas)
“Plasma, the 4
th
state of matter, is a partially ionised gas containing ions,
electrons, atoms and neutral species.”
(Palmers, 1999)

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Prolapse
“1. ptosis; the falling down, downward placement, of a part of the viscus. 2. To
undergo such displacement.”
(Dorland, 2009)
Uterine;
“Downward displacement of the uterus so that the cervix is within the vaginal
orifice (first degree prolapse), the cervix is outside the orifice (second degree
prolapse), or the entire uterus is outside the orifice (third degree prolapse).”
(Dorland, 2009)
Pelvic Floor
Fig i (Stanford University, 2008)
“The pelvic floor or pelvic diaphragm is composed of muscle fibres of the levator
ani, the coccygeus and associated connective tissue which span the area
underneath the pelvis. The pelvic diaphragm is a muscular partition formed by
the levators ani and coccygei, with which may be included the parietal pelvic
fascia on their upper and lower aspects. The pelvic floor separates the pelvic
cavity above from the perineal region (including perineum) below.”
(Stanford University, 2008)

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Kegel exercises
“A Kegel exercise, named after Dr. Arnold Kegel, consists of contracting and
relaxing the muscles which form part of the pelvic floor (sometimes called the
"Kegel muscles").”
(Wikipedia, 2008).
Seroma
“A seroma is a pocket of clear serous fluid that sometimes develops in the body
after surgery. When small blood vessels are ruptured, blood plasma can seep out;
inflammation caused by dying injured cells also contributes to the fluid.”
(Roitt and Delves, 1994)
Somatic
“Adjective of soma. Soma; The body: The body of an animal or plant excluding
the germ cells.”
(20th Century Dictionary, 1983)
Stability
“Ability of a substance or material to resist chemical change”
(The Williams Dictionary of Biomaterials, 1999)
Thrombogenicity
“Property of a material which induces and/or promotes the formation of a
thrombus”
(Definitions in Biomaterials, 1986)
Thrombus
“A stationary blood clot along the wall of a blood vessel, frequently causing
vascular obstruction.”
(Dorland, 2009)

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Chapter 1 – Introduction
1.1 Research Aims
To review the advantages and disadvantages of materials used in soft tissue
repair and to review potentially alternative materials.
To investigate in depth a limited selection of these alternative materials.
To investigate the value of gas plasma treatment on the ability of these
materials to support tissue growth in vitro.
To investigate the reported benefits of using chitosan in relation to medical
device applications
One aim of this project was to perform an in depth study into surgical devices used
for soft tissue repair (e.g. hernias and prolapses)
The next aim was to evaluate a selection of materials chosen because they are in
common use or because they have potential as surgical biomaterials and to explain
their advantages and disadvantages, review the potential alternative materials and
attempt to demonstrate the efficacy of some alternative materials / surface treatments
as tissue scaffolds.
The third aim was to examine how a small selection of surface treatments (gas
plasma and chitosan coating) affects their properties as tissue scaffolds (Angelova
and Hunkeler, 1999, Chandra and Rustgi, 1998, Guidoin et al., 2000).
By examining how well fibroblasts grew on these materials, biomaterials can be
developed that will become incorporated into healthy tissue rather than “scar plates”
thus avoiding the negative consequences and therefore this work sought to clarify the
potential of a selection of biomaterials based on their ability to support tissue growth
in vitro.

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In addition to this core body of work, this project aimed to examine the role chitosan
can play in biomaterials. By examining the bacteriostatic effect of chitosan and
techniques to incorporate chitosan into biomaterials, it was postulated that the
biomaterials would incorporate the benefits of containing chitosan, while retaining
the properties of the material the chitosan is combined with.
1.2 Current Situation
Polymers are a promising class of biomaterials that can be engineered to meet
specific end-use requirements (Angelova and Hunkeler, 1999). They can be selected
according to key “device” characteristics such as mechanical resistance,
degradability, permeability, solubility and transparency but the currently available
polymers need to be improved by altering their surface and bulk properties.
There are many examples of materials that have been used for medical implants that
have elicited undesired responses. Current mesh prostheses are made of
polypropylene (PP), polyethylene terephthalate (PET) or polytetrafluoroethylene
(PTFE), though all of them reveal some disadvantages (Klinge et al., 2002a). The
extended implantation of alloplastic material in the flexible frame of muscles and
fascial tissue is known to cause specific mesh-related complications like restriction of
the abdominal wall mobility (McLanahan et al., 1997, Vestweber et al., 1997),
induction of intra-abdominal adhesions with erosion of adjacent organs or
consecutive fistula formation (Schneider et al., 1979, Fitzgerald and Walton, 1996),
to the bladder (Houdelette et al., 1991, Gray et al., 1994, Hume and Bour, 1996),
bowel (DeGuzman et al., 1995, Kaufman et al., 1981, Soler et al., 1993, Miller and
Junger, 1997), blood vessels (Schumpelick and Kingsnorth, 1999) and ductus
deferens (Silich and McSherry, 1996). Next to an unavoidable inflammatory foreign
body reaction (FBR) the prosthesis usually is embedded into a fibrous scar plate,
which is responsible for a considerable shrinkage of the mesh area of about 40%
(Amid, 1997, Meddings et al., 1993).

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Hernias and prolapses are caused by a weakness or defect in the supportive tissues
that contain the bodily organs (Morris-Stiff and Hughes, 1998). A hernia (also called
a rupture), is a general term referring to a protrusion of a tissue through the wall of
the cavity in which it is normally contained. In more specific terms, hernia is usually
used to describe a protrusion of the abdominal contents through the abdominal wall.
This is usually treated surgically by the implantation of a polypropylene mesh over
the defective part of the abdominal wall.
A prolapse is a type of hernia that occurs exclusively in women (Creighton and
Lawton, 1998). It is characterised as a failure in the pelvic floor, causing the descent
of the uterus. This often presents itself as stress incontinence or in more severe cases,
the uterus can descend so far that it protrudes through the vagina. Treatments for
prolapses can range from pessaries (which act to provide internal support for the
uterus), the implantation of a „sling‟ to support the urethra, to hysterectomies (the
complete removal of the uterus and ovaries)
1.2.1 Hernia repair
Abdominal Hernia
Abdominal wall hernia repairs are performed over 990,000 times each year in the
USA, which makes it second only to cataract procedures, the most common surgical
procedure performed in the USA (Rutkow, 1997).
Although surgical techniques in hernia surgery have improved, recurrence used to be
a common complication (Engelsman et al., 2007). Therefore, the idea of increasing
the strength of the abdominal wall by implanting a mesh was explored with the
introduction of a polypropylene (PP) mesh in 1962 by Uscher (Uscher, 1962).
The strength of the abdominal wall depends on the collagen fascia layers, which are
the structures to be replaced by a mesh (Engelsman et al., 2007). From a mechanical
point of view, abdominal wall implants should become an integral part of the

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abdominal wall. This requires complete incorporation of the mesh into the fascial
margins of the defect. In the repair of abdominal wall defects, surgical meshes can
either be placed fully intra-abdominally (on the surface of the peritoneal lining) or in
between different anatomical layers of the abdominal wall. In both situations, the
aim of the treatment is to consolidate a musculo-fascial defect without tension on the
surrounding tissues.
The most common mesh material used for hernia repair is still polypropylene (PP),
although there are alternatives. Trostle et al (Trostle, 1994) mentions polypropylene,
expanded polytetrafluoroethylene (ePTFE) polyethylene terephthalate polyglactin
910 (PET) and polyglycolic acid (PGA). These materials vary from rigid strong non-
absorbables like PP, to moderately strong very pliable absorbables like PGA.
Vaginal Vault Prolapse
Women face an 11% lifetime risk of surgery for pelvic organ prolapse or urinary
incontinence (Olsen et al., 1997). Prolapse and prolapse related conditions account
for nearly a quarter of women waiting for routine gynaecological surgery (Creighton
and Lawton, 1998). The condition is rarely life threatening but can cause
considerable discomfort and stress. Patients with pelvic prolapses commonly have a
general state of „pelvic relaxation‟, with stress incontinence and some degree of
vaginal prolapse coexisting in many patients (Cespedes, 2002). These prolapse
conditions include urethral hypermobility, cystocele, rectocele, enterocele and uterine
prolapse.
The pelvic floor acts as a support for the pelvic organs and a prolapse occurs when
this support fails due to a weakness in the musculo-fiberous tissue (Creighton and
Lawton, 1998). The main support for the pelvic viscera is provided by a group of
muscles collectively known as the levator ani (Cespedes, 2002). An intact pelvic
floor allows the pelvic and abdominal viscera to „rest‟ on the levator ani, significantly
reducing the tension on the fascia and supporting ligaments. The pelvic ligaments are
not true ligaments and are simply condensations of endopelvic fascia covering the

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pelvic structures. The vagina can be anatomically divided into the proximal, middle
and distal regions. The proximal segment is also called the vault or cuff and is
stabilised by the cardinal and uterosacral ligaments. Uterine and vault prolapse are
associated with damage to these supportive structures.
Treatments recommended for the different types of prolapse depend on the severity
of the condition. Preventative treatments include strengthening of pelvic floor
muscles using Kegel exercises (Visco and Figuers, 1998). Cespedes (Cespedes, 2002)
mentions that in mild cases of asymptomatic prolapse in which no other procedures
are anticipated, the patient will not require surgery. For the elderly patient with
severe total vault prolapse who no longer desires sexual intercourse or in whom a
short procedure is required because of medical conditions, a vaginal closure or
colpocleisis can be performed.
Common techniques available for uterine and vaginal suspension (transvaginal
procedure) require drawing each side of the fault together causing restriction of
movement. The concept of tension free surgery (the use of a mesh) avoids the need
to draw the two sides together and leads to improved wellbeing for the patient with
little to no restriction on their movement but the complications must be addressed.
For the repair of vaginal vault prolapses, one of the popular techniques is to suspend
the vaginal vault by attaching it to the sacrum using a mesh or cadaverous fascia.
This procedure is ideal for young women with severe vault prolapses wishing to
retain their fertility or wishing to maintain their sexual activities. Transabdominal
suspension using a mesh or cadaverous fascia is a relatively morbid procedure with
results comparable to a transvaginal procedure (Nichols, 1991, Kovac and
Cruikshank, 1993).
There are other techniques involving permanent suturing of the uterus to alternative
support structures, but they are not much better (Cespedes, 2002). The choice of

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technique is a difficult one and can dramatically affect the quality of life for the
patient.
1.2.2 Complications
To reduce complications, first one must analyse them and the mechanisms behind
them. Klosterhalfen et al (Klosterhalfen et al., 1998) report that while there are
undisputed advantages to using polypropylene meshes, reports of complications after
implantation are increasing. Serious complications such as perforation and fistula
formation are rare but minor and local complaints such as seromas, misfeelings and
decreased abdominal wall mobility are observed in about half of the patients. A
recent paper (Steele et al., 2003) showed complications in 36% of patients from a
population of 58 patients requiring Parastomal hernia repair, with complications
including recurrence (26%), surgical bowel obstruction (9%), prolapse (3%), wound
infection (3%), fistula (3%) and mesh erosion (2%). No patient required extirpation
of the mesh. Of the 15 patients with recurrence, 7 underwent successful repair for an
overall success rate of 86%.
Morris-Stiff et. al. (Morris-Stiff and Hughes, 1998) mention that despite the reported
low tissue reactivity and long term maintenance of tensile strength associated with PP
mesh, they had seen four patients in whom these properties failed during long term
follow up of forty patients in a single unit. The four patients included three with
dense adhesions (one with severe infection) and one with primary mesh failure, all
requiring re-operation. It is mentioned that complications of non-infected wounds are
notably absent from current literature (1998) and suggests that these complications
may occur more often than is reported. The reasons proposed are short periods of
follow up, a lack of association between the complications and the mesh or reluctance
to report them.
PP and PET fibre meshes can cause tissue damage including; reduced mobility,
severe adhesion formation causing bowel obstruction, subsequent erosion and
formation of fistulas when placed in direct contact with the intestine and the

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incorporation of the prosthesis into a fibrous scar plate which in turn can cause the
mesh to shrink up to 40% (U.Klinge et al., 2002, Law and Ellis, 1988, Klinge et al.,
2002b). Therefore, its application is avoided when the mesh may be in direct contact
with the intestines.
In addition to problems with adhesion, when polymeric biomaterials are implanted
within the body, the immune system responds. This reaction is caused by a foreign
body reaction (Coleman et al., 1974, Marchant and Anderson, 1986, Marchant et al.,
1986). Foreign body reactions are characterised by an initial acute inflammatory
reaction. A chronic granulomatous (see Granuloma in Chapter 1) tissue reaction may
persist, even after encapsulation has occurred. The foreign body reaction seems to be
induced by continuous chemical or mechanical stimuli arising from the biomaterial
implants (Coleman et al., 1974). Morphological analysis of this reaction reveals the
presence of a large number of macrophages, which generally attempt to phagocytose
the material. Usually the foreign body is much larger than individual macrophages
and is not easily degraded. Some of the macrophages then merge their cytoplasm to
become multinucleated giant cells also called foreign body giant cells. If the foreign
body cannot be degraded by phagocytes, granulation tissue is formed to isolate the
implant from the rest of the body tissues. The foreign body reaction may be assessed
in a semi-quantitative way by the enumeration of inflammatory cells, namely,
polymorphonuclear leukocytes (PMN) and activated macrophages or giant cells
found either at the surface of the implanted biomaterials in the inflammatory
exudative fluid elicited by implants (Coleman et al., 1974, Marchant and Anderson,
1986, Marchant et al., 1986).
The contribution of phagocytic cells to the foreign body reaction may involve two
closely related mechanisms (Vaudaux et al., 1994). In the first, the neutrophils or
macrophages phagocytose the smaller fragments of the biodegraded or corroded
metallic or plastic implants. These fragments cannot be degraded further and they
may persist intracellularly in the neutrophils or macrophage for a prolonged period of
time or may be ingested by other phagocytes if cell death does occur. In the second
reaction also called “frustrated phagocytosis,” phagocytic cells are confronted with

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foreign particles such as nylon wool, glass, cotton, polysulfone fibres, polystyrene or
polypropylene materials too large to be ingested (Henson, 1971, Johnston and
Lehmeyer, 1976, Klock and Bainton, 1976, Wright and Gallin, 1979, Yanai and
Quie, 1981). Phagocytes coming into contact with this non-phagocytosable foreign
material become permanently activated in a way similar to phagocytes containing the
smaller fragments of non-degradable foreign particles; each kind of phagocyte may
separately or in concert secrete or passively release several important inflammatory
mediators (Coleman et al., 1974, Marchant et al., 1986), including acidic or neutral
hydrolases, activated complement components, tumour necrosis factor (TNF),
interleukins, prostaglandins, plasminogen activator and coagulation factors (Vaudaux
et al., 1994). The respective roles and the relative importance of these secreted
factors in the control and maintenance of acute and chronic phase of the
inflammatory response to implants are not yet well defined (Baggiolini, 1982,
Coleman et al., 1974, Gallin, 1984).
1.3 Prosthesis Related Infections
Infections are one of the most frequent and serious complications associated with
indwelling medical devices (Vaudaux et al., 1994).
Infections of biomaterial applications, including surgical meshes, are especially
troublesome as a biofilms can be formed on the mesh. Biofilms are formed when
micro-organisms colonise a surface and excrete a polysaccharide matrix. Micro-
organisms in this biofilm are protected against the host immune response and
antimicrobial attack (An and Friedman, 1998, Zimmerli et al., 1984). The body
continues to try to clear the microorganisms and this ends up causing damage to the
surrounding tissue. This will often lead to major complications which can be
potentially life-threatening and will in the majority of cases result in removal of the
mesh (Costerton et al., 1999). Bacteria look for a permanent surface to bind to as it
affords them greater protection against the body‟s immune system, so a non-
permanent implant should circumvent that problem.

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1.3.1 Incorporation of antimicrobials into medical prostheses
To avoid the problem of biomaterial related infections, one can try to incorporate
antibiotic or bacteriostatic compounds into the material. Most published data for
antimicrobial textiles and fibres are generated by placing a fabric on an inoculated
nutrient agar plate and measuring the inhibition zone (stanford.edu, 2008). This
procedure depends on diffusion of the antimicrobial agent in the agar. Further work
is usually required to discover the mechanism of the antimicrobial properties. This is
required if one wishes to discover if the compound being tested is bacteriocidal or
bacteriostatic. The difference between bacteriocides and bacteriostats is subtle.
Antibiotics have been widely used and antibiotic pathogens have developed as a
result, but the inhibition of growth using bacteriostats is less common and could be
used as a prophylactic alternative to antibiotics. With an appropriate antimicrobial
incorporated into a biomaterial, it is anticipated that this would significantly reduce
the chances of post operative infection and potential biofilm production
1.4 Tissue Engineering
The desired effect of any tissue engineering is to restore, maintain or improve the
function of human tissues.
The tissue engineering paradigm is to isolate specific cells through a small biopsy
from a patient, to grow them on a three-dimensional biomimetic scaffold under
precisely controlled culture conditions, to deliver the construct to the desired site in
the patient‟s body and to direct new tissue formation into the scaffold that can be
degraded over time (Lee and Mooney, 2001). Tissue engineering (TE) merges many
aspects of engineering and life sciences, aiming towards the primary understanding of
cell functions and the advancement of biological substitutes (Wiria et al., 2007).
Degradable materials are less susceptible to infection and intend to cause less of a
foreign body response (Badylak et al., 2001). However, the lack of strength over
time is a concern for certain clinical applications where adequate tensile properties
are necessary and required.

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“Tissue engineering concepts of producing a lattice for the ingrowth of cells in vivo
to lay down the appropriate matrix have been used very successfully for the skin and
for the repair of the facia in hernias. The approach used by researchers has been to
assume that cells and their accompanying matrix need a scaffold to enter, adhere to
and proliferate in an ordered manner. The three features of the tissue-engineered
scaffold are the overall architecture and porosity, the fibre morphology and the
surface chemistry. The use of knitted polyester meshes with pore sizes many orders
of magnitude larger than the repair matrix requires can result in a tissue response that
is inadequate. Pore sizes of between 10-50 µm and overall porosity of 85-90% with a
multifilament fibre yarn with fibre diameters of 1-10 µm appear to be the most ideal
for tissue ingrowth.” (Minns, 1999)
Other papers claim slightly different values for the “optimal” pore size. In a recent
paper, the author conducted a study where he developed polycaprolactone scaffolds
with varied pore sizes using a centrifugation method and therefore studied how
different pore sizes suit different applications. The scaffold section with 380–405 µm
pore size showed better cell growth for chondrocytes and osteoblasts, while the
scaffold section with 186–200 µm pore size was better for fibroblasts‟ growth. The
scaffold section with 290–310 µm pore size showed faster, new bone formation than
those of other pore sizes (Oh et al., 2007).
In cartilage tissue remodelling in response to mechanical forces, (Grodzinski et al.,
2000) Grodzinski, mentions recent studies which suggest that mechanotransduction is
critically important in vivo in the cell mediated feedback between physical stimuli
and the resulting macroscopic biomechanical properties of the tissue. This should be
an important consideration when selecting materials, especially degradable materials
intended to regenerate damaged tissue.
Another important consideration, often overlooked, is how the regenerating tissue
reacts with the prosthesis. The material used should elicit no negative effects on the
growing cells. This can be difficult to measure, but testing the cells for Heat Shock
Protein (an indicator of macrophage stress) (Henze et al., 1996) and produced by

11
other cells growing in a stressful environment) is potentially an effective way of
quantifying cellular stress. A simpler approach may be to measure how well cells
grow on a sample. This approach may not be so accurate, but should indicate a
cellular preference for a particular material/ surface that can then be followed up by
more elaborate testing.
1.5 Reasons for Improvement
These papers indicate the need for a new type of mesh implant for the repair of
incisional hernias and vaginal prolapses. In discussion with Dr Fotheringham (PhD
Supervisor) and Dr Browning (Gynaecologist), it became apparent that instead of
trying to produce a new permanent implant, the market would soon be ready for an
absorbable implant that could encourage the patient‟s tissue to repair the fault and
then dissolve so there is no surface for bacteria to adhere to and the problem
adhesions and encapsulation would be avoided, as these are a host response to a
foreign material placed within the body. With permanent implants, the immune
system takes the material as a threat and when bacteria bind the implant and bind to
it, this amplifies the problem. By having an implant that is constantly dissolving, the
problem of the macrophages trying to engulf the entire implant is avoided. Instead,
the immune system is able to encapsulate the small fragments of dissolving material.
The bacteria do not have a permanent surface to adhere to which will reduce the
chance of infection at the implant site in the long term. With a permanent implant,
even if the surgery is performed perfectly, the chance of infection at the implant site
is still dramatically increased.
1.5.1 Niche
There is demand in the medical profession for a new generation of medical implants.
They are looking for implants for repairing hernias and prolapses that will avoid the
problems that current mesh technologies cannot. This review is one of several
indicating the problems associated with the current permanent meshes on the market.

12
The ultimate solution would be a re-absorbable implant that would encourage new
tissue to grow over the implant to eventually replace it, one that would not antagonise
the immune system and inhibit bacterial growth/ adhesion. An implant that satisfies
these parameters would find many applications.
Therefore a strategy would be to take the body as a template and try to mimic the
body‟s original structure. The main obstacle to determining the characteristics
required for such a design is the fact that there is little research into the mechanical
properties of the pelvic floor and even less on how the body subconsciously controls
it. An implant could be designed to mimic the mechanical properties, but little will
be known about how successful it will be without the necessary somatic control.
With this in mind, it would be sensible to make sure that this implant will exceed
requirements.

13
Chapter 2 - Material Review
In the process of selecting materials, there are several requirements a biomaterial
must meet. The most important of these is biocompatibility. It must not illicit an
undesired response when placed within the body. The ideal material should be;
biocompatible
stable
biomimetic
The material should maintain strength as long as required. It should have strength and
bear load in a manner homologous with the tissue it is to emulate but it should not be
so strong that it restricts the mobility of the patient, or damage surrounding tissue
under stress.
In the case of biodegradables for soft tissue repair, one is looking for a material that
will transfer load from the device to the tissue as it is regenerating (Grodzinski et al.,
2000) so that the repaired tissue will be strong enough when the material has
degraded.
In addition, the device should not be prohibitively expensive. Therefore if the
medical device cost is kept to a reasonable level, it will be a viable option for more
patients and be better placed to compete with its competing products.
The choice of material(s) is of vital importance to the success of an implant but there
are so many aspects that need to be examined. The ideal implant should inhibit
adverse reactions and bacterial growth/attachment yet promote healthy, controlled
tissue regeneration. Unless cloned tissue is used, there is little chance there will be a
single material that can emulate the native tissue, therefore a combination of
materials and treatments may be necessary.

14
The materials used in this project shall be a selection of materials that fit three
criteria. They will be either 1. Currently approved materials, 2. Available novel
materials and 3. Modifications of these materials.
2.1 Potential Materials
The materials selected for this study shall be selected for the following reasons; they
should be either currently used as medical prostheses (in the case of the permanent
materials), or potentially suitable for medical use (in the case of the resorbable
materials) and they should be available to the researcher.
There are two objectives for this study. One is to evaluate a range of permanent and
degradable materials as scaffolds for tissue regeneration. The other aim is to evaluate
a selection of surface treatments for their ability to enhance biocompatibility and
tissue regeneration whilst maintaining their bulk properties.
2.1.1 Material selection
Polymers used as biomaterials can be naturally occurring, synthetic or a combination
of both. (Angelova and Hunkeler, 1999)
Naturally derived polymers are abundant and usually biodegradable (Chandra and
Rustgi, 1998). Their principal disadvantage lies in the development of reproducible
production methods, because their structural complexity often renders modification
and purification difficult. Additionally, significant batch-to-batch variations occur
because of their „biopreparation‟ in living organisms (plants, crustaceans) (Angelova
and Hunkeler, 1999) .
Synthetic polymers are available in a wide variety of compositions with readily
adjusted properties. Processing, copolymerization and blending provide
simultaneous means of optimizing a polymer‟s mechanical characteristics and its

15
diffusive and biological properties. The primary difficulty is the general lack of
biocompatibility of the majority of synthetic materials, although poly (ethylene
oxide) (PEO) and poly (lactic-co-glycolic acid) are notable exceptions. Synthetic
polymers are therefore often associated with inflammatory reactions, which limit
their use to solid, unmoving, impermeable devices (Angelova and Hunkeler, 1999).
With these considerations in mind the next stage is to narrow the field of prospective
materials through a process of elimination.
In „functional assessment and tissue response of short- and long-term absorbable
surgical meshes‟ (Klinge et al., 2001) it is mentioned that while non-absorbable
devices usually tend to produce fistulas in direct contact with the bowels, the
interposition of short-term absorbable meshes result in large incisional hernias in
almost all cases. The study investigated the functional and histological consequences
of a short-term polyglactin 910 (Vycryl®, loss of 50% of its mechanical stability
within three weeks) and a long-term absorbable mesh polylactide (LTS, preserved
>50% of its stability for over one year). The PG-mesh initially revealed a
pronounced inflammatory reaction and a significantly increased formation of
connective tissue in the interface mesh/recipient tissues correlated to an increased
stiffness of the abdominal wall compared to the sham-group (The sham-group
consists of incisions sutured together with no implanted mesh). However, a loss of
mechanical stability and an increase in elasticity could be detected three weeks after
implantation, which may be explained by the rapid absorption of the mesh material.
In contrast to PG, the LTS mesh indicated a decreased but persisting inflammatory
reactions in the interface mesh/recipient tissues and significantly reduced induction of
connective tissue. Although the formation of scar tissue was diminished compared to
PG, the LTS mesh preserved its mechanical stability after 180 days. The results
indicate that the frequent development of incisional hernias with short-term
absorbable meshes (PG) might be due to the decreased mechanical stability and
dilation of the newly formed connective tissue after 2-3 weeks. Moreover extensive
scar tissue formation may promote adhesion formation.

16
To decide which of the many biomaterials to study, one must make out a list of
potential materials and weigh up the criteria for and against. This will not be a
complete list, as there are many exotic biomaterials being developed and therefore it
will contain materials that are readily available.
Natural Polymers
Proteins and protein based polymers
Collagen
Collagen is expensive and suffers from large batch-to-batch and source-to-source
variations typical of natural extracts (Angelova and Hunkeler, 1999). Collagen
would be an ideal material if complications such as variation and potential for disease
transfer could be circumvented. In addition, tissue sources that have origins from
other humans or animals remain problematic mainly due to immunogenic responses
by the patients (Shin et al., 2003).
Koob (Koob and Hernandez, 2002) published research data on the modification of
native collagen to produce re-synthesised collagen fibre. The outcome of this work
was a biologically based tendon bio-prosthesis with mechanical properties equivalent
to native tendon. Ultimate tensile strength of the NDGA cross-linked fibre was
greater than that of native tendon, while the elastic modulus and strain at failure were
comparable to those of tendon fibres.

17
Polysaccharides and derivatives
Chitin / Chitosan
Chitin is one of the most abundant natural amino-polysaccharides and is estimated to
be produced annually almost as much as cellulose. Its immunogenicity is low, in
spite of the presence of nitrogen (Majeti and Kumar, 2000). Its purity can vary as a
result of its origin (e.g. (crab shell chitin = low purity, Squid chitin = higher purity.)
It can also vary in molecular weight (e.g. squid chitin = high molecular weight) and
these factors can affect the properties of the chitin. Another variable for chitin is the
degree of deacetylation. Chitosan is a deacetylated form of chitin and by varying the
degree of deacetylation, its biodegradability and solubility can be modified. Chitosan
biodegrades hydrolytically and this is enhanced by the presence of lysozyme (Lee et
al., 1995). The susceptibility to lysozyme of chitin derivatives is controlled by the
degree of acetylation at the C2-position and/or by the introduction of various
substituents at the 6-0-position of the N-acetylglucosamine residue (Nishimura et al.,
1985).
Chitosan has many possible applications, but the applications of most interest for this
study are its tissue culture properties and its bacteriostatic effect. Chitosan has some
level of antimicrobial activity and fibres made from chitosan are available in the
marketplace (stanford.edu, 2008). Coatings of chitosan on conventional fibres or
films appear to be a more realistic prospect for development of this material
(Broughton et al., 2001).
Fig 2.1 - Chemical formula of chitosan in Haworth‟s projection (Murúg, 2007).

18
Chitosan In relation to medicine
In a paper by Hwang (Hwang et al., 2000) it is mentioned that nitric oxide (NO)
contributes towards cytotoxicity in cell proliferation during inflammation of wound
healing. NO is a highly reactive free radical and is employed by the immune system
to respond to inflammatory agents such as LPS (lipopolysaccharide derived from
bacterial cell walls) and interferon-gamma that activate macrophages and stimulate
them to produce NO. Chitin and chitosan show a significant inhibitory effect on NO
production by the activated macrophages. This would help explain the beneficial role
that chitin and the deacetylated derivatives have on wound healing.
Deacetylated chitin derivatives such as 70% deacetylated chitin (DAC-70) and 30%
deacetylated chitin (DAC-30) have potent immunological activities for activation of
peritoneal macrophages in vivo, suppression of Meth-A tumour cells in syngenic
BALB/c mice and stimulation of non-specific host resistance against Escherichia coli
infection in mice (Nishimura et al., 1984). Chitin and chitosan are also effective for
the protection of host against infection with Candida albicans and Staphylococcus
aureus and against growth of Ehrlich and Sarcoma 180 ascites tumour (Suzuki et al.,
1982, Suzuki et al., 1984). All deacetylated derivatives of chitin are reported to
enhance the activity of natural killer (NK) cells as well (Nishimura et al., 1985).
Because chitin and its deacetylated derivatives do not provoke an unfavourable
immunological response, chitin derivatives have been suggested for bandages,
sutures and other items placed in the human body (Brown, 1999) although purity will
be an issue in these applications (Broughton et al., 2001).
One issue with using chitin and chitosan for medical devices is the difficulty in
producing useable fibres. The poor tensile strength of chitosan fibres, especially in
the wet state, is a key deficiency (Notin et al., 2006). This is part of the reason why
there are so few products using chitin or chitosan on the market with the exception of
wound dressings (Niekraszewicz, 2005, Ong et al., 2008). This is being addressed by
scientists working on novel extrusion techniques or via the use of additives during
extrusion (Notin et al., 2006, Qin et al., 2002).

19
Synthetic Polymers
Polyanhydrides
Polyanhydrides are a group of polymers with two sites in the repeating unit
susceptible to hydrolysis (Angelova and Hunkeler, 1999). Polyanhydrides are useful
materials for drug delivery. The degradation rates can be altered with changes in the
polymer backbone. Aliphatic polyanhydrides degrade within a few days while
aromatic polyanhydrides can degrade slowly over a period of several years.
Aliphatic polyesters
Almost the only high molecular weight compounds shown to be biodegradable are
the aliphatic polyesters. The reason for this is the extremely hydrolysable backbone
found in these polyesters (Angelova and Hunkeler, 1999).
Poly-ε-Caprolactone (PCL)
Poly(ε-caprolactone) (PCL) has been studied as a substrate for biodegradation and as
a matrix in controlled-release systems for drugs and its slow rate of degradation in
vivo makes it suitable for devices with longer working lifetimes (1–2 years) (Chandra
and Rustgi, 1998).
This material is primarily being developed as a bone substitute for use in
maxillofacial reconstructive surgery. However, it could be adapted to other areas
where bioabsorbable composite materials may be used (Corden et al., 2000).
In vitro biocompatibility of both the in situ polymerised PCL and commercially
available PCL (Solvay‟s CAPA 6400) material has been assessed using osteoblasts
derived from human craniofacial bone cells. The material is highly biocompatible
with these cells which will attach and spread on both the PCL types.

20
The main factor influencing cell behaviour seems to be the surface topography of the
polymer samples (Corden et al., 2000). A tendency of cells to group, showing zones
with more cellular density, was observed on PCL films, although these nuclei of
growth disappeared when cultures reached confluence (Serrano et al., 2005).
Polyglycolic acid
Polyglycolic acid or PGA is the simplest linear aliphatic polyester, with repeat units –
OCH2CO- and is a readily degradable highly crystalline polymer used for sutures and
other implantable devices (The Williams Dictionary of Biomaterials, 1999).
The advantage of poly-glycolic acid is the degradability by simple hydrolysis of the
ester backbone in aqueous environments such as body fluids. Furthermore, the
degradation products are ultimately metabolized to carbon dioxide and water or are
excreted via the kidney (Chandra and Rustgi, 1998).
Although poly-glycolic acid is a commonly used biomaterial in medical devices, it is
a short term resorbable polymer which eliminates it as a structural component of a
tissue repair mesh for connective tissue, although it is often used as a copolymer to
increase the degradation rate.
Poly-l-lactic acid
PLA is a relatively hydrophobic linear aliphatic polyester, with repeat units
OCHCH3CO. PLA has similar properties to polyglycolic acid except that
degradation occurs more slowly. PLA exists in two stereoregular forms, D-PLA and
L-PLA and in the racemic D,L-PLA (The Williams Dictionary of Biomaterials,
1999).
Polymeric scaffolds including synthetic materials such as poly(L-lactic acid) have
attracted significant interest in the tissue engineering community as a consequence of

21
their biocompatibility, ease of processing into three-dimensional structures, their
established safety as suture materials and the versatility that they offer for producing
chemically defined substrates for graft matrices (Kanczler et al., 2007).
Aromatic polyesters
Polyethylene terephthalate is an aromatic polyester (aromatic polyesters are often just
termed polyester). The sample used in this project was donated by Vascutek Ltd in
the form of an arterial prosthesis and has therefore been tested to ensure its
biocompatibility and anti-thrombogenicity.
Aliphatic-aromatic polyesters
Solanyl Flexibilitis component (or Eastar Bio GP copolyester)
Aliphatic-aromatic co-polyester the name of Solanyl®
is derived from Solanum
Tuberos. The polymer is made from by-products of potato processing, the potato
peels (Rodenburg Biopolymers, 2004).
Having seen this material, the Flexibilitis grade appears to have very good
mechanical properties and it would be interesting to find out how human cells react to
it. For the purpose of this work, it shall be referred to as Solanyl.
Polypropylene
Polypropylene is a thermoplastic homopolymer, made by the chemical industry and
used in a wide variety of applications including medical devices such as Marlex®
which is a commercially available hernia repair patch. Polypropylene has many
advantages and disadvantages but the material has a long history in medical devices
and therefore it is important as a control. Experiments were conducted in this thesis
to determine if tissue response could be improved.

22
PTFE (Polytetrafluoroethylene) (Teflon®
)
PTFE is a chemically inert homopolymer, with a very low coefficient of friction
(Young and Lovell, 1991) and as such, has found numerous applications in
biomedical devices. PTFE is commonly used in vascular grafts and tendon repair,
both applications where low friction and hydrophobicity are an advantage, but this is
a disadvantage when looking for cell adhesion and tissue regeneration. Therefore
this material is not ideal for this study as it is so hydrophobic.
Thermoplastic polyurethanes
Polyurethanes are a large family of polymers in which urethane bonds are formed in
the backbone of molecule chains by the reaction between a polyol and an isocyanate
and can be either thermoplastic or thermosets.
Among synthetic materials, polyurethanes have been considered to be the most
suitable material in various biomedical applications, which is connected to their
biocompatibility, biodegradability and controlled microstructure and properties
(Corneillie et al., 1998). They also have excellent mechanical properties which
makes them well suited to biomedical applications.
Carbon fibre
Carbon fibre initially appears to be a very suitable material as described by R.J.
Minns (Minns, 1999). In his paper, Tissue engineered Synthetic Scaffolds for Tissue
repair– a textile approach to implant design he states that individual carbon fibres
appear to present an attractive surface, morphologically and chemically, to the
attachment of fibroblasts which eventually produce a collagenous framework within
the implant scaffold at the sites desired.
During questioning at the MedTex conference in 2003 (Bolton, UK), when R.J.
Minns was presenting, Royston Dawber raised an issue, mentioning that he was
aware of an autopsy on a 60 year old woman who had died and it was discovered that

23
a carbon fibre from a tendon repair had migrated through her body and been
discovered in her brain. This news was enough to discount this material as a
potential prosthesis for this project. Incidentally, there have not been any recent
papers proposing the use of carbon fibre for soft tissue repair.
2.1.2 Chosen Materials
Polypropylene
Polypropylene was chosen partially because it is widely used for medical prostheses,
therefore would act as a reference material. In addition it would be of value to see if
altering its surface properties would improve the tissue reaction.
Polyurethane
Chosen for its biocompatibility, biodegradability and mechanical properties.
Polyester (Vascutek)
This material is an example of a vascular prosthesis. As it is currently used in
surgery, examining the way cells proliferate on this material and how the cells react
to the material is of great interest. This was used as the gold standard control and to
demonstrate how well cells should grow on a biomaterial. It was also examined to
see if the cell material interaction can be improved by plasma treatment.
Poly-ε-Caprolactone 6400
Chosen because it is biocompatible, flexible, biodegradable and has a large body of
published work relating to medical use.

24
Solanyl
Chosen for its mechanical properties, biodegradability and lack of published work.
Poly lactic acid
Chosen because it is biocompatible, biodegradable and has a large body of published
work relating to medical use.
In addition to the reasons stated above, another selection criterion for these materials
was that they were available in sufficient quantities to perform this research project.
2.2 Potential Treatments for Materials
2.2.1 Chitosan/ chitin coating
As mentioned earlier by R. Broughton (Broughton et al., 2001) coatings of chitosan
on conventional fibres or films appears to be a more realistic prospect for
development of this material although development of a fibre would be very useful.
To make this idea a commercial reality, a method needs to be developed of applying
a uniform coating of chitosan to a material. There is a concept of spray application
that could have many applications in the medical sphere. Some materials may need
surface alteration to make the material wetable before any lasting chitosan coating
can be applied. In a preliminary study into the effect of chitosan-coated material on
MRSA and Staphylococcus epidermis, the coated polypropylene performed poorly,
whilst the chitosan-coated cotton cloth had an observable effect. This indicated that
the material needed to be wetable for the chitosan coating to adhere sufficiently to the
material to be useful (Method 3c in the results section).
Another potential approach to incorporating chitosan to a polymer would be to use
gas plasma to cross link the chitosan to the polymer surface. It would be interesting
to compare the various methods of chitosan coating.

25
2.2.2 Low Pressure Plasma Treatment
Low pressure plasma treatment can be used to alter a materials hydrophobicity /
hydrophilicity, sterilise materials without the problems associated with other methods
and to erode the surface to enhance roughness of a material (Palmers, 1999). This
can be achieved in a reproducible manner by ionising the gas in a controlled and
qualitative way within a vacuum vessel (pumped down to a pressure in the range of
10-2
to 10-3
mbar). The gas is ionised with the help of a high frequency generator.
The highly reactive particles react with the surface of the substrate. The gas used can
be altered, the power used and length of exposure can be altered to promote the
desired effect (ablation, crosslinking, activation or deposition). The formed reactive
particles react in a direct way with the surface without damaging the bulk properties
of the treated material as the surface modification is limited to the outermost 10 to
1000A (Ångström) of the substrate.
The lifetime of the treated polymer surface can be a concern. A disadvantage of
polymer surface treatments is that the modified surfaces undergo surface
restructuring with time (Yang et al., 2002) owing to the mobility of the polymer chain
in the amorphous regions (Murakami et al., 1998, Kim et al., 2003), which is driven
by thermodynamic need to lower the overall interfacial energy of the system
(Koberstein et al., 1998).
Oxygen Plasma
Oxygen plasma treatment is an effective means of enhancing the hydrophilicity of a
polymer‟s surface. This enables polymers that would normally be unsuitable for
tissue growth to be able to support the attachment of cells. According to Van-Kooten
(van-Kooten et al., 2004), the improved wettability of oxygen plasma treated
materials was related to improved cell proliferation, increased fibronectin surface
coverage and increased expression of adhesion related proteins.
There also appear to be other advantages to oxygen plasma treatment. In adhesion of
Pseudomonas aeruginosa strains to untreated and oxygen-plasma treated poly (vinyl

26
chloride) (PVC) from endotracheal intubation devices by K. Triandafillu
(Triandafillu et al., 2003) they mention that oxygen plasma treatment has a beneficial
effect against the bacterial colonization of a Oxygen plasma treated PVC, reporting a
70% reduction in adhering bacteria although they concede that this reduction is
however unlikely to be sufficient to prevent P. aeruginosa colonization of
endotracheal intubation devices.
This would be an attractive surface treatment to examine, as it appears to yield
promising results. Unfortunately technical problems conspired to make this
treatment unavailable for the majority of the materials.
Argon Plasma
Argon is an inert gas, so while it will ablate the surface of the polymer and improve
the hydrophilicity, it will not create a functional group on the surface of the polymer.
Ammonia Plasma
It is hypothesised that plasma treatment with ammonia would improve tissue growth
along a biomaterial more than argon plasma treatment. This was suggested as
ammonia is made of nitrogen and hydrogen, which are the building blocks of proteins
(Proteins are built from amino acids and amino acids are so called because they
contain an amine group (NH2)). Therefore it was suggested that a material presenting
nitrogen and hydrogen on its surface would mimic a protein and therefore encourage
cell binding and greatly enhance its biocompatibility.
Fluorine Treatment
Fluorine is the most electronegative and reactive of all elements (Fessenden and
Fessenden, 1990). Treatment of polyester with a solution of fluoropolymer
(polyvinylidene fluoride) has been shown to reduce thrombogenicity (Maini, 1999).

27
Due to this lower thrombogenicity, this biomaterial is now used for vascular
prostheses with a diameter of 6mm.
2.2.3 Hyaluronic acid
In a paper by D. Girotto (Girotto, 2003) it is reported that the re-differentiation
capabilities of human articular and chick embryo sternal chondrocytes were evaluated
by culture on HYAFF-11 and its sulphate derivative, HYAFF-11-S, polymers derived
from the benzyl esterification of hyaluronate. Initial results showed that the HYAFF-
11-S material promoted the highest rate of chondrocytes proliferation.
2.2.4 Laser pitting
Prof Duncan Hand at Heriot-Watt University in Edinburgh developed a technique
using lasers to introduce pits of controllable size into a material (Fotheringham et al.,
2004). It was thought that this would be useful for encouraging cells to grow on the
proposed implant. This was discussed and while material could be pitted for tissue
culture study, the technology was prohibitively expensive and slow in its current
incarnation.
2.2.5 Micro-grooves
In a paper by E.T. den Braber (Braber, 1996), planar and micro-textured silicon
substrata were produced and made suitable for cell culture by radio frequency glow
discharge treatment and media were produced with grooves with widths of 2μm, 5μm
and 10μm and depth of 0.5μm. Cell counts proved that neither the presence of the
surface grooves nor the dimensions of the grooves had an effect on cell proliferation,
although cells grown on the 2μm and 5μm wide grooves were elongated and aligned
parallel to the surface grooves. It was also shown that cells on the 10 µm grooves
were almost comparable with the control with no grooves. Finally, it was also
observed that cells on the micro-textured substrates were capable of spanning the
surface grooves.

28
It was also mentioned that these results contradict the work reported by Green (Green
et al., 1994) and Ricci (Ricci, 1994). It goes on to mention that a response to surface
topography is dependent on cell type, which would account for the discrepancies
between this and other studies.
2.2.6 Chosen treatments for materials
Given more time and resources, one could compare all of these surface treatments
and develop treatment combinations but unfortunately, only a few treatments could
be analysed, due to the aforementioned limitations.
The treatments chosen were plasma treatment and chitosan coating and a
combination of plasma treatment and chitosan coating.

29
Chapter 3 - Methodology
The experimental studies can be split into two distinct groups. The first is the
examination of chitosan as a bacteriostat and the second is the production and testing
of biomaterial samples.
3.1 Examination of Chitosan as a Bacteriostat
This series of experiments was designed to examine the bacteriostatic effects of
chitosan on common hospital bacteria. The bacteria chosen were methicillin resistant
Staphylococcus aureus (MRSA 9551) and Staphylococcus epidermis (Staphylococcus
epidermis).
3.1.1 Materials
Nutrient agar (NA)
Nutrient broth (NB)
Plate Count Agar (PCA)
Petri dishes (~10 cm)
Culture bottles (~25ml)
Inoculation loop
Bunsen burner
Scissors
Tweezers
Ethanol (100%)
Distilled Water
Methylene blue
Acetic Acid (2M)
Sodium Hydroxide (2M)

30
Chitosan (Purisan squid chitosan – high molecular weight)
Cotton cloth (unbleached)
Neubauer Improved Haemocytometer (Vol = 1/400 ml per small square)
Incubator (37 C & 20 C)
Autoclave
Gilson pipettes (20μl – 1ml)
Chitosan Materials (Various Production Methods)
All chitosan work was performed using Purisan™ PB-103 squid chitosan, high
molecular weight from Sigma Aldrich (made by Technology Resource International
Corporation). The 2M acetic acid was made from glacial acetic acid (reagent grade,
Acacia).
All of these samples were autoclaved (sterilised) prior to use in the experiment, at
121 C for 15 minutes unless stated otherwise. This produced some discolouration in
the chitosan coated cotton cloth and the chitosan film sample and it also softened the
film sample, making it supple rather than the rigid film that it was before autoclaving.
Chitosan Gel
Chitosan samples were prepared by dissolving 1g, 0.1g, 0.01g or 0.001g (+/–
0.0001g) of chitosan in 10 mls acetic acid (2M, pH5). The 1g sample was so thick it
needed heating to 70 ºC to fully dissolve.
Chitosan Suspension
To 1g, 0.1g or 0.01g chitosan was added to 10mls of distilled water. The chitosan did
not dissolve and thus needed constant agitation to keep the chitosan powder in
suspension.

31
Chitosan Film
Chitosan was dissolved in acetic acid (2M, pH5) and the acid was allowed to
evaporate, leaving a film of chitosan (and traces of un-evaporated acetic acid). No
attempt was made to remove acetic acid residues. For materials coated in chitosan,
materials were dipped in 0.1% (w/v) chitosan in acetic acid solution and then allowed
to hang dry in a fume cupboard for 12 hrs.
Chitosan Coated Cotton cloth
The chitosan-coated cotton cloth was made by dipping woven cotton cloth (made at
Heriot-Watt University) first in chitosan solution (0.1g chitosan dissolved in 100mls
acetic acid (2M, pH5)) and then transferred into a NaOH bath (0.1M pH 13 in excess)
to neutralise the acid and precipitate the chitosan and then the excess chitosan was
squeezed out of the material using a glass rod on a glass plate. The samples were
then washed under cold water and hung on an aluminium bar to dry at ~20 C (room
temperature) for 24hrs.
The control was cotton cloth treated in acetic acid without the chitosan and
neutralised in sodium hydroxide and washed in water then dried in the same way.
Chitosan Fibre
Attempts were made to try to produce useable chitosan fibres but these were not
entirely successful, although this could yield more success with a suitable investment
of time. Initial attempts yielded some success but within the project there was neither
the time nor more importantly, the equipment available to yield useful results.
1g of chitosan was added to 20mls dilute acetic acid (2M, pH5) and mixed using a
glass rod. This was then left for half an hour to dissolve. The resulting thick gel was
then filtered through a Buchner funnel and extruded using a syringe with a 1ml
pipette tip attached into a 2M NaOH bath. The fibre was then collected from the
NaOH bath and dried on a glass rod.

32
Culture Media
Standard Nutrient Agar (NA) plates
This process was scaled to make the required quantity of NA plates. To make 5 NA
plates (containing approx 20mls of agar each), 2.8g NA powder and 100mls distilled
water were measured out. The NA powder was added to the distilled water in a glass
bottle and swirled to mix. A cap was placed on the bottle (loosely, to prevent the
bottle exploding inside the autoclave) and autoclaved in a Dixons Vario 2228
autoclave at 121 C for 15 minutes. When the autoclaved solution was cool enough to
handle, the solution was removed from the autoclave and Swirled until no
concentration haze was observable at the bottom of the bottle. The mixture was then
allowed to cool to ~ 60ºC. When the solution had cooled, approx 20mls of NA
solution was poured onto each Petri dish (10 cm) and then allowed to set. The NA
plates were then left for 24 hours at around 20ºC before use to remove excess
moisture. As a rule, more NA plates were produced than were required to allow for
unforeseen circumstances.
Chitosan NA plates
This process was scaled to make the required quantity of chitosan NA plates. To
make 3 NA plates containing each acetic acid solution (containing approx 20mls of
agar each), 2.8g NA powder and 90mls distilled water were measured out into 5
different bottles. 5 bottles of 10 mls acetic acid were prepared with varying
quantities of chitosan powder added to each of the 5 bottles (1g, 0.1g, 0.1g, 0.001g
chitosan or no chitosan for the control). The 5 bottles were swirled to mix. A cap
was placed on each bottle (loosely, to prevent the bottle exploding inside the
autoclave) and autoclaved in a Dixons Vario 2228 autoclave at 121 C for 15 minutes.
When the autoclaved solutions were cool enough to handle, the solutions were
removed from the autoclave and Swirled until no concentration haze was observable
at the bottom of the bottle. The mixtures were then allowed to cool to ~ 60ºC. When
the solutions had cooled, approx 20mls of each solution was poured onto 3 Petri
dishes and then allowed to set. The plates were then left for 24 hours at around 20ºC
before use to remove excess moisture.

33
Standard Nutrient Broth (NB)
This process was scaled to make the required quantity of NB. To make 10 bottles of
NB (containing 10mls of NB each), 2.5g NB powder and 100mls distilled water were
measured out. The NB powder was added to the distilled water in a glass bottle and
swirled to mix. A cap was placed on the bottle (loosely, to prevent the bottle
exploding inside the autoclave) and autoclaved in a Dixons Vario 2228 autoclave at
121 C for 15 minutes. When the autoclaved solution was cool enough to handle, the
solution was removed from the autoclave and Swirled until no concentration haze
was observable at the bottom of the bottle. The mixture was then allowed to cool to
~ 60ºC. When the solution had cooled, 10mls of NB solution was dispensed into 10
sterilised 25ml Culture bottles (universal bottles or universals). The NB bottles were
then allowed to cool to room temperature (20ºC).
Chitosan NB
This process was scaled to make the required quantity of chitosan NB. To make 3 NB
universals containing each acetic acid solution (containing approx 10mls of broth
each), 5g NB powder and 200mls distilled water were added to a bottle. The bottle
was swirled to mix. 5 bottles of 10 mls of distilled water were prepared with varying
quantities of chitosan powder added to each (1g, 0.1g, 0.1g, 0.001g chitosan or no
chitosan for the control). A cap was placed on each bottle (the NB solution, the
chitosan suspensions, the control and 15 universals) with the caps attached loosely (to
prevent the bottles exploding inside the autoclave) and autoclaved in a Dixons Vario
2228 autoclave at 121 C for 15 minutes. When the autoclaved solutions were cool
enough to handle, the solutions were removed from the autoclave and the NB
solution was swirled until no concentration haze was observable at the bottom. The
NB solution, the chitosan suspensions and the 15 universals were then allowed to
cool to ~ 60ºC. When the solutions had cooled, 9mls of NB solution was dispensed
into each of the 15 universals. 1 ml of each chitosan suspension was added to 3
universals (vortexing the suspensions prior to extracting the suspension using a
vortex mixer). 1ml of distilled water was added to the 3 remaining universals
(vortexing the water prior to extracting the suspension for consistency). The NB
mixtures were allowed to cool to room temperature (20ºC) prior to use.

34
Chitosan Film Plates
As for standard NA plates but with chitosan film added to plate after inoculation with
bacteria. Any air pockets under chitosan film were squeezed out.
Chitosan Coated Material Plates
As for chitosan film plates, but with the chitosan film having a material embedded
(cotton cloth or polypropylene mesh).
3.1.2 Methods
Examination of Chitosan as a Bacteriostat
Methodology
The experimental methods for the chitosan study were derived from discussions with
academic staff after an extensive review of the available literature. The methods
were designed primarily to examine the bacteriostatic effect of chitosan in relation to
hospital pathogens and evolved into a study that examined how the quantity of
chitosan available and the form in which the chitosan was presented affected the
bacteria.
Cell Count
Using the Gilson 20µl pipette, take 10µl of cells. Stain cells using methylene blue
(10µl methylene blue to 10µl cell suspension). Place methylene blue stained cells on
Haemocytometer (improved Neubauer BS748, depth 0.01mm, 1/400mm2
) and place
cover slip on top of the drop of cells. Place Haemocytometer on microscope. Count
cells in 10 random squares. Cells are counted when in the middle of the square (not
touching the lines) and when in contact with the bottom and left sides of the square.
Cells touching the top and left sides are excluded from the cell count figure. Get the
average of the 10 cell counts. Divide the average by 16, then multiply by 4. multiply
that figure by 106
and you have the cells per ml.

35
Method 1 - Chitosan dissolved in dilute acetic acid incorporated into nutrient agar
Vs MRSA 9551 and Staphylococcus epidermis
This experiment was designed to examine the growth of MRSA 9551 and
Staphylococcus epidermis on nutrient agar plates containing chitosan gel.
Obtain cultures of MRSA 9551 and Staphylococcus epidermis and culture the
Staphylococcus epidermis on nutrient agar and the MRSA 9551 on DST agar for 24
hrs. Dissolve 1g chitosan in 10mls acetic acid (2M). Dissolve 0.1g chitosan in 10mls
acetic acid (2M). Add 1g chitosan in 10mls acetic acid (2M) to 90mls nutrient agar.
Add 0.1g chitosan in 10mls acetic acid (2M) to 90mls nutrient agar. Add 10mls
acetic acid (2M) to 90mls nutrient agar. Prepare 100 ml nutrient agar. Prepare 2x
10mls 0.9% saline solution. Autoclave the prepared nutrient agars and saline
solutions at 121ºC for 15 minutes. Shake (swirl) autoclaved agars well without
producing bubbles, allow to cool to 40-50ºC and pour into Petri dishes
(approximately 20mls each) and allow to cool to room temperature.
Take the 24 hour culture of MRSA and inoculate an inoculation loop full of MRSA
into 10mls 0.9% saline and use a vortex mixer to ensure through mixing. Take the 24
hour culture of MRSA and inoculate an inoculation loop full of MRSA into 10mls
0.9% saline and use a vortex mixer to ensure through mixing. Perform cell count of
the saline inoculums using Neubauer improved haemocytometer. Add 100mls of
MRSA inoculum to the control (NA), the control containing acetic acid (NA + acetic
acid), the NA + 1g chitosan in 10 ml acetic acid, NA + 0.1g chitosan in 10 ml acetic
acid and spread the inoculum across the plates with sterile glass beads. Add 100mls
of Staphylococcus epidermis inoculum to the control (NA), the control containing
acetic acid (NA + acetic acid), the NA + 1g chitosan in 10 ml acetic acid, NA + 0.1g
chitosan in 10 ml acetic acid and spread the inoculum across the plates with sterile
glass beads. Inoculate 2 NA plates, one with MRSA and the other with
Staphylococcus epidermis and spread the inoculum across the plate with sterile glass
beads, then add a 1cm square of chitosan film to each. Incubate at 37 C for 48hrs
and then examine for signs of growth.

36
Method 1b - Modified method
This method is a modified version of method 1. By neutralising the acetic acid
control and using only the 1g chitosan and 0.1g chitosan samples, all of the agar
plates would be solid enough to inoculate. In addition, the plates were inoculated
using a sterile swab of saline inoculum instead of an inoculation loop (to increase the
quantity of inoculum).
Obtain cultures of MRSA 9551 and Staphylococcus epidermis and culture
Staphylococcus epidermis on nutrient agar and MRSA 9551 on DST agar for 24 hrs.
Dissolve 1g chitosan in 10mls acetic acid (2M). Dissolve 0.1g chitosan in 10mls
acetic acid (2M). Add 1g chitosan in 10mls acetic acid (2M) to 90mls nutrient agar.
Add 0.1g chitosan in 10mls acetic acid (2M) to 90mls nutrient agar. Add 10mls
acetic acid (2M) (neutralised to pH 7 using NaOH) to 90mls nutrient agar. Prepare
100 ml nutrient agar. Autoclave the prepared nutrient agars and saline solutions at
121ºC for 15 minutes. Shake (swirl) autoclaved agars well without producing
bubbles, allow to cool to 40-50ºC and pour into Petri dishes (approximately 20mls
each) and allow to cool to room temperature.
Take a sterile swab and dip it in sterile saline solution (0.9%) then take a swab of
MRSA and inoculate the control (NA) making sure to cover the entire plate. Repeat
this process for the control containing acetic acid (NA + acetic acid), the NA + 1g
chitosan in 10 ml acetic acid, NA + 0.1g chitosan in 10 ml acetic acid. Take a sterile
swab and dip it in sterile saline solution (0.9%) then take a swab of Staphylococcus
epidermis and inoculate the control (NA) making sure to cover the entire plate.
repeat this process for the control containing acetic acid (NA + acetic acid), the NA +
1g chitosan in 10 ml acetic acid, NA + 0.1g chitosan in 10 ml acetic acid. Take a
sterile swab and dip it in sterile saline solution (0.9%) then take a swab of MRSA and
inoculate a nutrient agar making sure to cover the entire plate and then add a 1cm
square of chitosan film. Take a sterile swab and dip it in sterile saline solution
(0.9%) then take a swab of MRSA and inoculate a nutrient agar making sure to cover
the entire plate and then add a 1cm square of chitosan film. Incubate at 37 C for
48hrs and then examine for signs of growth.

37
Method 2 - Chitosan dissolved in acetic acid added to nutrient broth Vs MRSA 9551
and Staphylococcus epidermis
Method 2 was redesigned so that the experiment would be performed using nutrient
broth and measuring the growth of the bacteria spectrophotometrically using a LKB
Biochrom Ultrospec II. With this study, all of the samples and the control contained
acetic acid.
Obtain cultures of MRSA 9551 and Staphylococcus epidermis and culture on
Staphylococcus epidermis nutrient agar and MRSA 9551 on DST agar for 24 hrs.
Add 1g chitosan to 10mls acetic acid (2M). Add 0.1g chitosan to 10mls acetic acid
(2M). Add 0.01g chitosan to 10mls acetic acid (2M). Make 100 ml nutrient broth
(2.5g nutrient broth powder + 100mls distilled water). Make 4 x 110 ml chitosan
nutrient broth (2.5g nutrient broth powder + 100mls distilled water) +; (1g chitosan +
10 ml acetic acid (2M)), (0.1g chitosan + 10 ml acetic acid (2M)), (0.01g chitosan +
10 ml acetic acid (2M)) and (10 ml acetic acid (2M)). Autoclave the prepared
nutrient broth and 0.9% saline solution and 10 glass culture bottles (~25ml) at 121ºC
for 15 minutes. Shake (swirl) autoclaved broth well and allow to cool to room
temperature. Dispense 2 (x10mls) of each media into a universal (2x NB, 2x NB
+10mls acetic acid (2M), 2x NB +10mls acetic acid (2M) + 1g chitosan, 2x NB
+10mls acetic acid (2M) + 0.1g chitosan and 2x NB +10mls acetic acid (2M)+ 0.01g
chitosan).
hour culture of Staphylococcus epidermis and inoculate an inoculation loop full of
MRSA into 10mls 0.9% saline and use a vortex mixer to ensure through mixing.
Perform cell count of the saline inoculums using Neubauer improved
haemocytometer. Add 0.5ml of MRSA to one of each of the nutrient broth. Add
0.5mls of Staphylococcus epidermis one of each of the nutrient broth. Incubate at
37 C. After 2 hours take 1 ml of bacterial broth from each culture and add each
sample to a 1ml spectrophotometry curvette. Measure the absorbance of the samples

38
at AD550nm. Examine every 2 hours for 8 hours using the spectrophotometer and
then once after 24 hours.
Method 2b - Modified method - Chitosan added to nutrient broth Vs MRSA 9551
In this method, the chitosan powder was not dissolved in acetic acid. Instead, it was
suspended in distilled water. This was to study how colloidal chitosan affected
bacterial growth and to remove any effect the pH may have on bacterial growth.
Prepare 1g chitosan in 10mls distilled water. Prepare 0.1g chitosan in10mls distilled
water. Prepare 0.01g chitosan in 10mls distilled water. Make 5 x 110 ml nutrient
(2.5g nutrient broth powder + 100mls distilled water +; (1g chitosan + 10mls distilled
water), (0.1g chitosan + 10mls distilled water), (0.01g chitosan + 10mls distilled
water) and Prepare 100 ml nutrient broth. Autoclave the prepared nutrient broth and
0.9% saline solution and 12 glass culture bottles (~25ml) at 121ºC for 15 minutes.
Shake (swirl) autoclaved broth well and allow to cool to room temperature. Dispense
3 (x10mls) of each media into a universal vortexing each time to resuspend chitosan
(3x NB, 3x NB + 1g chitosan, 3x NB + 0.1g chitosan and 3x NB + 0.01g chitosan).
Perform cell count of the saline inoculums using Neubauer improved
haemocytometer. Add 0.5ml of MRSA to one of each of the nutrient broth. Add
0.5mls of Staphylococcus epidermis one of each of the nutrient broth. Incubate all of
the samples (including the sterile controls) at 37 C and examine every hour for 4
hours using spectrophotometer at AD550nm and then once after 24 hours.

39
Method 2c - Modified method - Chitosan added to nutrient broth Vs MRSA 9551
This method is a further refinement of method 2b. In method 2b the nutrient broths
were stationary when in the incubator. This method includes the use of a platform
shaker to encourage the chitosan powder to remain in suspension while in the
incubator. The platform shaker agitated the chitosan powder into suspension
therefore it was necessary to let the chitosan powder to settle before
spectrophotometer readings to prevent the chitosan suspension from influencing the
absorbance readings. An absorption wavelength of 550nm was used for the
spectrophotometer as is it the optimal wavelength for bacterial turbidity readings.
Prepare 1g chitosan in 10mls distilled water. Prepare 0.1g chitosan in10mls distilled
water. Prepare 0.01g chitosan in 10mls distilled water. Prepare 5 x 110ml nutrient
(2.5g nutrient broth powder + 100mls distilled water +; (1g chitosan + 10mls distilled
water), (0.1g chitosan + 10mls distilled water), (0.01g chitosan + 10mls distilled
water) and Prepare 100ml nutrient broth. Autoclave the prepared nutrient broth and
0.9% saline solution and 8 glass culture bottles (~25ml) at 121ºC for 15 minutes.
Shake (swirl) autoclaved broth well allow to cool to room temperature. Dispense 3
(x10mls) of each media into a universal (2x NB, 2x NB + 1g chitosan, 2x NB + 0.1g
chitosan and 2x NB + 0.01g chitosan).
Add 0.2ml of MRSA to one of each of the nutrient broth. Add 0.2mls of
Staphylococcus epidermis one of each of the nutrient broth. Incubate all of the
samples (including the sterile controls) at 37 C on a platform shaker and examine
every hour for 4 hours using spectrophotometer at AD550nm and then once after 24

40
hours (allow the chitosan suspension to settle ~15mins to before taking
spectrophotometer readings).
Method 3 - Testing of Chitosan treatment of Cotton cloth
This experiment was designed to examine the efficacy of chitosan coatings on a
material (cotton cloth) to inhibit bacterial growth of MRSA 9551 and Staphylococcus
epidermis. This method is a development of the chitosan film sample tested in
method 1.
Staphylococcus epidermis and MRSA 9551 in nutrient broth for 24 hrs. Prepare 120
ml nutrient agar and 60mls 0.9% saline solution. Autoclave the prepared nutrient
agars, saline solution and cotton cloth samples at 121ºC for 15 minutes. Shake
(swirl) autoclaved agars well without producing bubbles, allow to cool to 40-50ºC
and pour into 6 Petri dishes (approximately 20mls each) then allow to cool to room
temperature.
MRSA and inoculate a nutrient agar making sure to cover the entire plate. Take a
sterile swab and dip it in sterile saline solution (0.9%) then take a swab of MRSA and
inoculate a nutrient agar making sure to cover the entire plate, then add a 1cm square
of untreated cotton cloth. Take a sterile swab and dip it in sterile saline solution
(0.9%) then take a swab of MRSA and inoculate a nutrient agar making sure to cover
the entire plate, then add a 1cm square of chitosan coated cotton cloth. Take a sterile
swab and dip it in sterile saline solution (0.9%) then take a swab of MRSA and
inoculate a nutrient agar making sure to cover the entire plate. Take a sterile swab
and dip it in sterile saline solution (0.9%) then take a swab of Staphylococcus
epidermis and inoculate a nutrient agar making sure to cover the entire plate, then add
a 1cm square of untreated cotton cloth. Take a sterile swab and dip it in sterile saline
solution (0.9%) then take a swab of Staphylococcus epidermis and inoculate a
nutrient agar making sure to cover the entire plate, then add a 1cm square of chitosan

41
coated cotton cloth. Incubate at 25 C for 72 hrs and then examine for signs of
growth.
Method 3b - Modified method - Testing of Chitosan treatment of Cotton cloth
This method is a refined version of method 3. The samples were covered in
aluminium foil to maintain the sterility while cooling down from the autoclave cycle
and instead of using a swab to inoculate the agar plates, 20 µl of inoculum was used
to standardise the quantity of bacteria on each agar plate.
ml nutrient agar. Autoclave the prepared nutrient agars, saline solution and cotton
cloth samples at 121ºC for 15 minutes. Cotton cloth samples were wrapped in
aluminium foil during the autoclave cycle. Shake (swirl) autoclaved agars well
without producing bubbles, allow to cool to 40-50ºC and pour into 6 Petri dishes
(approximately 20mls each) and allow to cool to room temperature.
Dispense 20µl of MRSA broth onto a nutrient agar and spread around the NA using a
Bunsen sterilised inoculation loop making sure to cover the entire plate, then add a
1cm square of untreated cotton cloth using Bunsen sterilised tweezers and squeeze
out any air bubbles under the samples. Dispense 20µl of MRSA broth onto a nutrient
agar and spread around the NA using a Bunsen sterilised inoculation loop making
sure to cover the entire plate, then add a 1cm square of chitosan coated cotton cloth
using Bunsen sterilised tweezers and squeeze out any air bubbles under the samples.
Dispense 20µl of Staphylococcus epidermis broth onto a nutrient agar and spread
around the NA using a Bunsen sterilised inoculation loop making sure to cover the
entire plate, then add a 1cm square of untreated cotton cloth using Bunsen sterilised
tweezers and squeeze out any air bubbles under the samples. Dispense 20µl of
Staphylococcus epidermis broth onto a nutrient agar and spread around the NA using
a Bunsen sterilised inoculation loop making sure to cover the entire plate, then add a
1cm square of chitosan coated cotton cloth using Bunsen sterilised tweezers and

42
squeeze out any air bubbles under the samples. Incubate at 25 C for 72 hrs and then
examine for signs of growth.
Method 3c - Modified method - Testing of Chitosan treatment of Cotton cloth &
polypropylene
This method is a modified version of method 3. The method is the same as for
method 3 with the addition of a chitosan coated polypropylene mesh. In addition, the
samples were placed in glass bottles (with lids) to prevent the moisture in the
autoclave from effecting the chitosan coating and to maintain the sample sterility
until they were used.
ml nutrient agar and 100mls 0.9% saline solution. Autoclave the prepared nutrient
agars, saline solution and cotton cloth samples at 121ºC for 15 minutes. Cotton cloth
and polypropylene samples were placed in “universal” bottles during the autoclave
cycle to keep them dry (as the samples were dry after the autoclave, they would need
moistening with 0.9% saline so they would adhere to the agar). Shake (swirl)
autoclaved agars well without producing bubbles, allow to cool to 40-50ºC and pour
into 6 Petri dishes (approximately 20mls each) and allow to cool to room
temperature.
MRSA and inoculate a nutrient agar making sure to cover the entire plate, then add a
2cm square of untreated cotton cloth (sterilised) using Bunsen sterilised tweezers and
squeeze out any air bubbles under the samples. Take a sterile swab and dip it in
sterile saline solution (0.9%) then take a swab of MRSA and inoculate a nutrient agar
making sure to cover the entire plate, then add a 2cm square of chitosan coated cotton
cloth (sterilised) using Bunsen sterilised tweezers and squeeze out any air bubbles
under the samples. Take a sterile swab and dip it in sterile saline solution (0.9%)
then take a swab of MRSA and inoculate a nutrient agar making sure to cover the

43
entire plate, then add a 2cm square of chitosan coated cotton cloth (non-sterilised)
using Bunsen sterilised tweezers and squeeze out any air bubbles under the samples.
MRSA and inoculate a nutrient agar making sure to cover the entire plate, then add a
2cm square of chitosan coated polypropylene mesh (sterilised) using Bunsen
sterilised tweezers and squeeze out any air bubbles under the samples.
Staphylococcus epidermis and inoculate a nutrient agar making sure to cover the
entire plate, then add a 2cm square of untreated cotton cloth (sterilised) using Bunsen
sterilised tweezers and squeeze out any air bubbles under the samples. Take a sterile
swab and dip it in sterile saline solution (0.9%) then take a swab of Staphylococcus
epidermis and inoculate a nutrient agar making sure to cover the entire plate, then add
a 2cm square of chitosan coated cotton cloth (sterilised) using Bunsen sterilised
tweezers and squeeze out any air bubbles under the samples. Take a sterile swab and
dip it in sterile saline solution (0.9%) then take a swab of Staphylococcus epidermis
and inoculate a nutrient agar making sure to cover the entire plate, then add a 2cm
square of chitosan coated cotton cloth (non-sterilised) using Bunsen sterilised
tweezers and squeeze out any air bubbles under the samples. Take a sterile swab and
dip it in sterile saline solution (0.9%) then take a swab of Staphylococcus epidermis
and inoculate a nutrient agar making sure to cover the entire plate, then add a 2cm
square of chitosan coated polypropylene mesh (sterilised) using Bunsen sterilised
tweezers and squeeze out any air bubbles under the samples. Incubate at 25 C for 72
hrs and then examine for signs of growth.
Method 4 - Chitosan suspended in nutrient broth Vs MRSA
Prepare 3 litres of plate count agar and 2 litres of 0.9% saline solution. Sterilise the
plate count agar and 0.9% saline solution in the autoclave at 121ºC for 15 minutes.
Shake (swirl) autoclaved agars well without producing bubbles, allow to cool to 40-
50ºC and pour into and poured into 200 Petri dishes (approximately 20mls each) and
allow to cool to room temperature. Once the plate count agars have cooled, store for
1 week to dry out a little (so that when they are inoculated, there isn‟t excess

44
moisture enabling the bacteria to spread). Obtain culture of MRSA 9551 and culture
on nutrient agar for 24 hrs. Accurately weigh out 1g chitosan and add to 10 ml of
distilled water in to a “universal” bottle (25ml). Accurately weigh out 0.8g chitosan
and add to 10 ml of distilled water in to a “universal” bottle (25ml). Accurately
weigh out 0.6g chitosan and add to 10 ml of distilled water in to a “universal” bottle
(25ml). Accurately weigh out 0.4g chitosan and add to 10 ml of distilled water in to
a “universal” bottle (25ml). Accurately weigh out 0.2g chitosan and add to 10 ml of
distilled water in to a “universal” bottle (25ml). Sterilise 200 „Universals‟. Dispense
9mls of 0.9% saline solution into 150 „universals‟. Dispense 10 ml of distilled water
in to a “universal” bottle (25ml) (the control). Prepare 100mls of nutrient broth.
Prepare 3x 10mls 0.9% saline solution in “universal” bottles (25ml). Sterilise the
chitosan samples, nutrient broths and saline solution in saline in the autoclave at
121ºC for 15 minutes. Take 1ml of the chitosan/ distilled water mixture (mix by
pipetting 3x first) and add to 9mls of nutrient broth).
into 10mls 0.9% saline and use a vortex mixer to ensure through mixing. Perform
cell count of the saline inoculum using Neubauer improved haemocytometer. Add
200mls of MRSA inoculum to the control (NB + 1ml distilled water), the NB + 1g
chitosan, NB + 0.1g chitosan, NB + 0.01g chitosan. Incubate at 37 C for 48hrs on a
platform shaker and then examine for signs of growth. Take 1ml of each sample and
add to 9mls 0.9% saline, vortex mix, then take 1ml of the inoculated saline and
inoculate it into 9mls 0.9% saline. Repeat a further 5 times for 10-6
dilution and 7
times for 10-8
dilution. The 48hr samples should be diluted to 10-8
and 10-6
, 10-7
&
10-8
samples should be used to inoculate plate count agars. (100ul per plate count
agar, spread across the plate count agar using sterile glass beads). Return cultures to
platform shaker in 37ºC incubator after the dilutions have been performed. The 72hr
Samples should be taken and diluted to 10-8
and 10-6
, 10-7
& 10-8
samples should be
used to inoculate plate count agars. (100ul per plate count agar, spread across the
plate count agar using sterile glass beads). Return cultures to platform shaker in 37ºC
incubator after the dilutions have been performed. The plate count agars had the
colonies counted 24 - 48 hours after inoculation and the results were noted.

45
Method 4b - Modified method
This method is a refined version of method 4. The concentrations of chitosan (and
the control) are performed in triplicate (e.g. control 1, control 2 and control 3). In
addition, the dilutions performed have been expanded to 10-9
on certain days in order
to have plate count agars containing countable numbers of colonies.
Prepare 4 litres of plate count agar. Sterilise the plate count agar in the autoclave at
121ºC for 15 minutes. Shake (swirl) autoclaved agars well without producing
bubbles, allow to cool to 40-50ºC and pour into and poured into 200 Petri dishes
(approximately 20mls each) and allow to cool to room temperature. Once the plate
count agars have cooled, store for 1 week to dry out a little (so that when they are
inoculated, there isn‟t excess moisture enabling the bacteria to spread). Obtain
culture of MRSA 9551 and culture on nutrient agar for 24 hrs. Accurately weigh out
1g chitosan and add to 10 ml of distilled water in to a “universal” bottle (25ml).
Accurately weigh out 0.8g chitosan and add to 10 ml of distilled water in to a
“universal” bottle (25ml). Accurately weigh out 0.6g chitosan and add to 10 ml of
distilled water in to a “universal” bottle (25ml). Accurately weigh out 0.4g chitosan
and add to 10 ml of distilled water in to a “universal” bottle (25ml). Accurately
weigh out 0.2g chitosan and add to 10 ml of distilled water in to a “universal” bottle
(25ml). Dispense 10 ml of distilled water in to a “universal” bottle (25ml) (the
control). Sterilise 200 „Universals‟ and prepare 2 litres of sterile 0.9% saline
solution. Dispense 9mls of 0.9% saline solution into 150 „universals‟. Prepare
100mls of nutrient broth. Prepare 3x 10mls 0.9% saline solution in “universal”
bottles (25ml). Sterilise the chitosan samples, nutrient broths and saline solutions in
the autoclave at 121ºC for 15 minutes. Take 1ml of the chitosan/ distilled water
mixture (mix by pipetting 3x first) and add to 9mls of nutrient broth).
into 10mls 0.9% saline and use a vortex mixer to ensure through mixing. Add
100mls of MRSA inoculum to the control (NB + 1ml distilled water), the NB + 0.1g
chitosan, NB + 0.08g chitosan, NB + 0.06g chitosan, NB + 0.04g chitosan, NB +
0.02g chitosan (Perform this stage in triplicate). Perform cell count of the saline

46
inoculum using Neubauer improved haemocytometer. Incubate at 37 C for 48hrs on
a platform shaker and then examine for signs of growth.
Dilution procedure;
take 1ml of each sample and add to 9mls 0.9% saline, vortex mix, then take 1ml of
the inoculated saline and inoculate it into 9mls 0.9% saline. Repeat a further 5 times
for 10-6
dilution and a further 7 times for 10-9
dilution, ensuring to vortex mix each
dilution.
Samples should be taken and diluted to 10-8 after 48 hours and 10-6
- 10-8
samples
used to inoculate plate count agars. (100ul per plate count agar, spread across the
plate count agar using sterile glass beads). Once dilutions are performed, wash the
universals and repeat step 11 and 12 so that the salines are ready for the next day.
Samples should be taken and diluted to 10-9 after 72 hrs and 10-7
- 10-9
samples used
to inoculate plate count agars (100ul per plate count agar, spread across the plate
count agar using sterile glass beads). Once dilutions are performed, wash the
universals and repeat step 11 and 12 so that the salines are ready for the next day.
Samples should be taken and diluted to 10-8 after 96 hours and 10-6
- 10-8
samples
should be used to inoculate plate count agars. (100ul per plate count agar, spread
across the plate count agar using sterile glass beads). The plate count agars should
have the colonies counted ~48 hours after inoculation and the results should be noted.

47
3.2 Production of Biomaterial Samples
Table 3.1 Sample summary.
Table 3.1 illustrates the source and production methods used to produce the samples
used in the experiments.
3.2.1 Extrusion
Polypropylene tape
Materials
Polypropylene pellets MFI-19 (borealis polypropylene)
ESL vertical extruder (model 250)
Material
Details
Poly-ε-
Caprolactone
6400
Solanyl Polylacticacid Polyester Polypropylene Tuftane
Polyurethane
Source Solvay Rodenburg
Biopolymers
CargillDow Vascutek Borealis
polypropylene
Lord
Corporation
Grade 6400 Flexibilitis N/A VP1200K
Virgingrade
N/A N/A
Methodof
fabrication
Extrusion Extrusion Filmcasting-
dissolvedin
dichloromethane
(DCM)
Extrusion
followedby
knitting
Extrusion extrusion
MeltingPoint
(°C)
62.5 112.5 168 257.5 151 149.5
Extrusion
Temperature
(°C)
76 125@280psi N/A N/A 235 N/A
Tape/sample
width(mm)
1.33 0.97 1.13 2 1.4 1
Tape/sample
thickness
(mm)
0.09 0.19 0.01 0.9 0.16 0.05
Additional
notes
Handdrawn
over47°Croller
Unableto
extrudeauseful
tape,therefore
preparedasa
film
Obtainedinthe
formofpre
fabricated
vasculargraft
Obtainedasa
prefabricated
sheet

48
Fig 3.2 (Younes et al., 2009) - Diagram of ESL vertical extruder illustrating the extruder screw, die head
(in green), the air quench chamber and winding apparatus. The barrel heaters are divided into zones so
that the temperature of the molten polymer can be controlled from where it enters the extruder screw
through to the die head. The extruder screw forces the polymer through the barrel, increasing the pressure
of the molten polymer until it reaches the die head.

49
Method
The polypropylene tape was produced with the following extruder settings.
Extruder
Zone 1 Zone 2 Zone 3
180°C 180°C 185°C
Pump Die Head
193°C Zone 1 Zone 2
208°C 211°C
Melt Extruder screw speed
212°C 19.6-18.7 rpm
Pre pump pressure Die Head Pressure
769-860psi 514psi
Metering Pump Air Quench Winder
4.1rpm 23% 3rpm
Polymer Draw Frame
Roller No1 Roller No2 Roller No4
34mpm 80mpm 158mpm
80ºC 80ºC 80ºC
Table 3.3 polypropylene extrusion parameters. These setting were determined by Stewart Wallace, the
extrusion technician at Heriot-Watt University.
Solanyl
Materials
Solanyl Flexibilitis pellets
ESL Laboratory Extrusion, Melt Spinning and Draw Equipment. Labspin 892

50
Fig 3.4 ESL Laboratory Extrusion, Melt Spinning and Draw Equipment. Labspin 892.
Method
The Solanyl tape was produced with the following extruder settings
Material Details
Source Rodenburg Biopolymers
Grade Flexibilitis
Method of fabrication Extrusion
Melting Point (°C) 119.2
Extrusion Temperature (°C) 125 @280psi
Tape / sample width (mm) 0.97
Tape / sample thickness (mm) 0.19
Table 3.5 Solanyl extrusion parameters. These setting were determined by Stewart Wallace, the extrusion
technician at Heriot-Watt University.

51
Solanyl + 2% Chitosan Powder (W/W)
This was extruded as per Solanyl but was mixed with chitosan powder at 2% w/w
prior to extrusion.
Poly-ε-caprolactone 6400 tape
Materials
Solvay poly-ε-caprolactone
Bradford University Research Ltd. Small Scale Ram Extruder
Fig. 3.6 Bradford University Research Ltd. Small Scale Ram Extruder

52
Method
The poly-ε-caprolactone tape was produced with the following extruder settings.
Extruder
Zone 1 Zone 2 Zone 3
80°C 80°C 80°C
Pump Die Head
105°C Zone 1 Zone 2
105°C 105°C
Melt Extruder screw speed
105°C 19.6-18.7 rpm
Pre pump pressure Die Head Pressure
769-860psi 514psi
Metering Pump Quench Tank Winder
1.5rpm 10.8% 3rpm
Table 3.7 poly-ε-caprolactone extrusion parameters. These setting were determined by Stewart Wallace,
the extrusion technician at Heriot-Watt University.
3.2.2 Film Casting
PLA film
Perform all work using Dichloromethane in a fume cupboard. 1g of PLA (Cargill
Dow) fibre is placed in a 200ml Pyrex glass beaker. Add 30mls Dichloromethane
(DCM) (Acros Organics). Wait for the PLA to dissolve completely. Pour solution
on glass sheet and place in rack for glass plates. Wait for the DCM to evaporate
(takes about 4 hours but can be left longer). Collect the film. Place the film in an
airtight bag and squeeze out any air and store it at room temperature in the bag until
required.

53
3.2.3 Plasma Treatment
Materials
Polypropylene
Tuftane polyurethane
Polyester (Vascutek polyester VP1200K™)
Poly-ε-Caprolactone 6400 tape
Solanyl
PLA
Plasma treatment at Riccarton campus (Nanotech)
Equipment
Argon gas
Ammonia gas
Pirani 10 Pressure gauge
Thruline Watt meter (model 43, Biro Electronic Corporation, Cleveland Ohio)
Parallel plate plasma equipment (pressure chamber parallel plates and purge
system by Nanotech, model PE250, serial 115)
Vacuum pump
RF generator (solid state power generator, Eni Powersystems Inc, model OEM-6,
serial 729)
Fume cupboard (to vent the spent gases)
Silane calibrated flow meter to be used for argon gas (therefore actual gas flow
rate = output reading x [flow factor for new gas/flow factor for the calibrated gas]
= output reading x 1.4 [1.4 is the argon conversion factor] /0.4 [0.4 is the silane
conversion factor])
Ammonia calibrated flow meter

54
Method
Recommended settings
Pressure 10-1
Torr
Power 50-100W
Zero flow (for silane calibrated flow meter) registers as 0.5cc (therefore all flow
readings will be compensated for by removing the 0.5cc
Recommended gas flow (valves open) is 20cc
Electrode gap 2.5cm
Safety checks
Check the cooling water for the RF unit is running. Check the RF power is off when
the chamber is open. For Argon treatment - Set the regulator on the gas cylinder to a
maximum of 5 bar.
Procedure
Before first run (warm up)
Before any treatment takes place, the following need to be performed to prepare the
equipment (argon gas is the vent/purge gas)
Close the plasma chamber and turn on the vacuum pump. Flush the system with
argon gas to purge out any other gases (open the needle valve and turn on the electric
valve). Set flow meter to 20 cubic centimetres (cc). Adjust the pressure to
recommended levels (10-1
Torr). Turn on RF and tune for 0 reflected power (by
adjusting input and load controls) and record forward power. Turn power off. Turn
gas off. Vent gas.

55
For Argon treatment
Fume cupboard should be checked to make sure it is on before anything else to vent
any waste gases. Open argon cylinder (5 bar max). Turn on vacuum pump. Purge
gas lines and plasma chamber with Argon. Turn on water-cooling for RF generator.
Turn on the rest of the equipment (gauges, valves). Perform dummy run to ensure
RF generator and gas flow are set to desired specifications. Pump out the chamber to
about 10-1
Torr (open the valve to the pump) and periodically vent the chamber with
argon (will partially release the vacuum) and repeat at least 5 times to ensure air has
been removed (displaced by the argon). Pump down chamber for trial treatment to
10-1
Torr (no sample). Adjust gas flow (for treatment gas) until the pressure within
the chamber is 20-1
Torr and record the gas flow. Turn on the RF generator and
adjust the power to desired level. Check the Watt meter and adjust settings until
there is 0 reflected power (all the power is going forward). Equipment should be set
now for your samples so, close the valve to the pump and fill the chamber with argon
to return the pressure to atmospheric pressure. Place samples on lower plate. Pump
down chamber for treatment to 10-1
Torr. Open the treatment gas valve (the flow rate
is already set). When ready, turn on the RF generator (power level already set) and
administer RF power for a measured time (for the treatment used, the time is 1
minute). When time has expired, turn off the RF generator. Vent the chamber to
atmospheric pressure (close the pump valve and admit argon to the chamber to
relieve the vacuum).
For Ammonia gas treatment
Prior to commencing the ammonia gas line needs to be vented with argon (as the gas
line is shared with other gases) the rest of the procedure is the same as for argon,
except for the addition of step 15.
For potentially toxic or malodorous treatment gases, add more vent/ pump down
cycles after step 14 to remove treatment gas completely from the chamber prior to
relieving the pressure to atmospheric pressure and opening the treatment chamber.
Flow rates (excess gas used for both gases)

56
Argon mean flow rate = 28.35cc
Ammonia mean flow rate = 9.8cc
(Operating pressures were the same = 20-1
Torr)
RF time = 1minute
Fig. 3.8
Nanotech plasma chamber

57
Fig. 3.9
View of the plasma chamber during warm up showing the high energy plasma
Europlasma Plasma Treatment
Equipment
Argon Gas
Oxygen Gas
Europlasma Surface Treatment CD400PC MHz System
The following settings were used (settings were stored as file mike2)
Gas Flow 0.4 SLM (standard Litres per Minute)
Power 300W
RF Time 5Mins
Pressure 200Mtorr

58
Method
Place sample to be treated in the plasma chamber. Load configuration file “mike2”
and allow the process to run. Collect and store sample in an airtight bag at room
temperature.
Fig. 3.10
Europlasma plasma treatment machine showing the computerised controls on the left hand
side and the plasma chamber on the right hand side

59
3.3 Sample Characterisation
3.3.1 Differential scanning calorimetry (DSC) Analysis
All standard materials (untreated) were analysed by DSC (Mettler DSC 12E). This
was done to determine the melting point. Samples were placed in aluminium
crucibles and heated. The temperature increased at 5ºC per minute.
3.3.2 SEM Analysis
The electron microscope was used to examine the standard materials and plasma
treated materials to determine if there was any observable physical change to the
material surface due to plasma treatment.
The materials first needed to be splutter coated for 60 seconds using a Polaron sc7620
splutter coater before being examined in a Hitachi S-530 scanning electron
microscope.
Method
Instrument Switch On
Turn on the cooling water about 2 full turns (tap marked blue). Switch on the power
at the wall (LOW, WARM UP and STOP lamps will glow red). Move (lower) EVAC
POWER lever to on position (up). Press the EVAC button on console (LOW and
WARM UP lamps will glow red). Wait for 20 minutes until HIGH lamp is lit green.
Sample Preparation
Samples are prepared by placing them on SEM stubs (1cm aluminium disks with a
female thread on their base corresponding to the SEM sample mount) in the Polaron
splutter coater to coat them with a fine film of platinum, so the microscope can see
the surface.

60
Fig. 3.11 - Polaron sc7620 splutter coater
Fig. 3.12 - Hitachi S-530 scanning electron microscope

61
Introducing Samples to Column
Press AIR button. Wait until hear an audible hiss. Open the sliding drawer. Screw
the sample stub on. Close the sliding drawer and hold. Press EVAC button (pump
will kick in). Wait (around 2 minutes) until HIGH lamp is lit green.
Image Formation
Move (lower) DISPLAY lever to on position (up). Wait until ACC VOLTAGE
READY lamp is lit steady red (not flashing). Switch on ACC VOLTAGE (normally
5 or 10 kV). Press the left-most SCANNING SPEED button (TV rate, 0). FOCUS
control: switch to AUTO and press COARSE button to produce image. Flick WFM
switch (under concealing panel) down. Adjust FILAMENT knob clockwise (to about
2 o'clock position) until trace at maximum height position on screen [if necessary use
MANUAL CONTRAST BRIGHTNESS to make trace visible on screen). Press the
left-most SCANNING SPEED button to restore image. Press ABC button twice
under AUTO condition to optimise brightness and contrast. To suit eye, B and C can
be controlled by switching to MANUAL and rotating lower B and C knobs. Use
AUTO (coarse / fine) or MANUAL control to adjust image focus. Move around
sample at low magnification to locate position of interest. Adjust magnification to
required level, focusing as required for image quality.
Instrument Shutdown
Reduce magnification to lowest. Turn ACC VOLTAGE off. Wait for about 1
minute, then move (lower) DISPLAY lever to off position (down). Press AIR button,
await audible hiss. Remove sample. Close drawer, press EVAC button, wait until
HIGH lit green. Depress STOP button and wait until LOW and STOP lamps lit red.
Move (lower) EVAC POWER lever to off position (down). Wait for around 20
minutes. Switch off instrument at wall. Turn off cooling water.

62
Analysis of Pore Size of PLA Sample
The PLA pore size was determined by selecting SEM image representative of the
PLA SEM images and measuring the dimensions of each pore (the horizontal and
vertical), measuring the area of the pores using a ruler and calculating the percentage
of pores in relation to the area of the image.
3.4 Tissue Culture Study
3.4.1 Methodology
This experimental method was derived after reading through research papers and
observing a gap in the research. Many papers extolled the benefits of a particular
material or examined explanted devices from human or animal subjects. The primary
aim of this study was to conduct a basic study to evaluate a range of materials on a
quantitative level. In addition to the standard materials, modified materials were
included so that the modifications could be evaluated directly with the standard
materials. This study was designed to be as simple and as controlled as possible.
Capillary tubes were used to act as ballast to prevent the samples from floating.
Background
This experiment was designed to evaluate a range of materials for their ability to
support human cell growth. This was a simple experiment that used MRC-5 cells to
determine which material / surface treatment was optimal. Initially, Human foetal
fibroblasts were going to be used but the cells from the supplier were at the end of
their passage limit and subsequently died very quickly. The cells were seeded
directly onto the test material with no additional materials used to encourage
attachment (e.g. Matrigel). Gelatine was tested as a means to improve cell
attachment but it was discarded as it would influence the results.

63
Preparation
The samples needed mounting for the tissue culture study so that the samples would
sink when placed in the tissue culture media. Glass capillary tubes were chosen as
they would provide the necessary ballast to ensure the samples remained submerged.
The samples were then sterilised at Anderson Caledonia using ethylene gas.
Ethylene gas was chosen as it did not involve high temperatures that could melt some
of the polymers with low melting points.
Samples Preparation
Materials
10 cm soda glass capillary tubes
Glass cutter
Paperclip
70% Ethanol
Sterilisation Bags
Samples
Polypropylene
Argon plasma treated polypropylene (Nanotech)
Ammonia plasma treated polypropylene (Nanotech)
Polypropylene coated in chitosan
Argon plasma treated polypropylene coated in chitosan (Nanotech)
Ammonia plasma treated polypropylene coated in chitosan (Nanotech)
Tuftane polyurethane
Argon plasma treated Tuftane polyurethane (Nanotech)
Ammonia plasma treated Tuftane polyurethane (Nanotech)
Argon plasma treated Tuftane polyurethane coated in chitosan (Nanotech)
Argon Plasma treated Tuftane polyurethane (Europlasma)

64
Oxygen plasma Treated Tuftane polyurethane (Europlasma)
Vascutek polyester
Argon plasma treated Vascutek polyester (Nanotech)
Ammonia plasma treated Vascutek polyester (Nanotech)
Poly lactic acid
Argon plasma treated poly lactic acid (Nanotech)
Ammonia plasma treated poly lactic acid (Nanotech)
Argon plasma treated poly-ε-caprolactone 6400 (Nanotech)
Ammonia plasma treated poly-ε-caprolactone 6400 (Nanotech)
Solanyl
Argon plasma treated Solanyl (Nanotech)
Ammonia plasma treated Solanyl (Nanotech)
Solanyl extruded with 2% chitosan (w/w)
Solanyl coated in chitosan
Argon plasma treated Solanyl coated in chitosan (Nanotech)
Ammonia plasma treated Solanyl coated in chitosan (Nanotech)
Method
Cut capillary tubes into 3cm lengths using a glass cutter. Cut samples into 4cm
lengths. Insert into the capillary tubes using a paperclip to poke the ends in. The
samples were prepared in excess (19 of each sample + one un-mounted for analysis in
SEM). Rinse samples 5 times with 70% ethanol and then placed in gas sterilisation
bags. Sterilise samples at Anderson Caledonia (ethylene gas sterilised).

65
Fig 3.13 - Demonstration of how the biomaterial sample was mounted to the capillary tube.
3.4.2 Experimental Work
For this experiment, the samples were seeded with a small drop of MRC-5 cells and
the samples were then inoculated over 29 days.
Materials
Trypsin (10x Concentration) 100ml. Invitrogen
Fetal Bovine Serum, certified (heat inactivated) Origin U.S. Invitrogen
Performance, mycoplasma, virus bacteriophage and endotoxin tested
Culture Medium - McCoy‟s 5a + 2mM Glutamine
Gilson Pipettes - P20, p200, p1000 and p10 ml Pipettes
Phosphate Buffered Saline Tablets
25cm3
Iwaki®
Culture Flasks (Non-Treated, Hydrophobic surface)
Centrifuge
Tissue Culture Incubator (37ºC, 5%CO2)
-80˚C freezer
Media (modified minimal essential eagles medium) = 500mls minimal essential
eagles medium + 50ml FBS + 11ml l-glutamine +5.5 ml NEAA
L-lysine
NEAA (nonessential amino acids)

66
DMSO (Dimethyl sulfoxide)
Trypsin
FBS (Fetal bovine serum)
PBS (Phosphate buffered saline)
Flasks
Cryogenic storage tubes
2um filters
Centrifuge tubes
Pipettes & tips
Water bath
LaminAir hood
Biocide ZF (Spray Disinfectant for Incubators and Sterile Cabinets in Cell
Culture Area)
Ethanol
10% Chloros
Haemocytometer- improved Neubauer BS748, depth 0.01mm, 1/400mm2
Samples
Sterile tweezers
Iwaki®
25ml tissue culture flasks (both treated and untreated)
Liquid N2
Liquid N2 Storage
Centrifuge
Water bath
Molecular Probes “live or dead” viability/cytotoxicity kit (L-3224) (Invitrogen)
o Contains Calcein AM and Ethidium Homodimer-1
Human Foetal Lung Fibroblasts (http://www.ecacc.org.uk/)

67
Cell Line Name MRC-5
ECACC No. 97112601
Cell Line Description Established from normal lung tissue of a 14 week old male
foetus. The cells undergo between 60-70 population
doublings before senescence. The virus susceptibility of this
line is similar to WI-38. This cell line is supplied on a
standing order basis.
Species Human
Tissue Lung, foetal
Morphology Fibroblast
Sub Culture Routine Split sub-confluent cultures (70-80%) 1:3 to 1:6 i.e. seeding
at 2-4 x 10,000 cells/cm using 0.25% Trypsin or
Trypsin/EDTA; CO2; 37C.
Culture Medium EMEM (EBSS) + 2mM Glutamine + 1% Non Essential
Amino Acids (NEAA) + 10% Foetal Bovine Serum (FBS).
Karyotype 2n = 46, diploid
Depositor Dr P Jacobs, NIBSC, London
Country UK
References (Jacobs et al., 1970)
Table 3.14 Summary of MRC-5 cells
Method
Preparation
To ensure the experiment would not have any glitches, the planning stage was vital to
make the experiment as controlled as possible. The most important factor was to
ensure the cells were all in the same condition / passage number and consumables
were available when required.
Standard methods
Preparation for any work in the LaminAir hood (Heraeus HS12)
Turn on hood 30mins prior to work to stabilize air flow. Clean the LaminAir hood
with Biocide ZF. Clean the LaminAir hood with Ethanol. Then sterilise the
LaminAir hood with UV. Clean everything with Ethanol before placing in the
LaminAir hood.

68
Procedure for Thawing Cells
Warm up media and place in suitably labelled tissue culture flask. Remove the
chosen cells from the liquid nitrogen storage. Place the cryo vial in the 37ºC water
bath for ~ 1minute. Before the pellet is completely thawed, remove the vial, clean the
vial (with biocide ZF) and pace in the LaminAir hood. Immediately empty the
contents of the vial into 1ml of the pre-warmed media. Place the media and cells in a
centrifuge tube and down at 2000rpm for 4mins at 30°C (Heraeus Megafuge 1.0R).
Pour off the media and Re-suspend the cell pellet in 2mls of fresh media. Empty the
re-suspended cells in a tissue culture flask containing 8mls of pre-warmed media.
Attach the flask cap loosely and place in the incubator.
Passage procedure
Warm up media, Trypsin and PBS (no Ca2+
or Mg2+
) to 37°C for ~30mins before use
in a Grant OLS200 water bath. Examine cells carefully. If cells are ~70% confluent,
then proceed with passage. If there is contamination, dispose of the cells. If the cells
are less than ~70% confluent but the media has turned orange/yellow, change media.
If passage is required, dispose of old media. Use PBS (no Ca2+
or Mg2+
) to wash
cells once (use pipette (~10mls) then dispose of PBS. Add 2mls of Trypsin and place
in incubator for 2mins at 37°C. Once cells detach (Trypsin = orange), give the flask
a tap against the side of a hard object to dislodge the cells from the bottom of the
flask. Check cells on the microscope (Axiovert 25). They should be rounded and
floating freely in the media. If any cells remain attached to the bottom of the flask,
give the flask an additional tap. Add 2mls media (10% FBS) to neutralize the
Trypsin. Put in centrifuge tube and spin down at 2000rpm for 4mins at 30°C.
Dispose of media + Trypsin. Add 8mls media. Re-suspend cell pellet in new flask.
Check cells under inverted microscope. Place cells in incubator (37ºC, 5% CO2).
Cell count procedure
Dispose of old media. Use PBS (no Ca2+
or Mg2+
) to wash cells once (use pipette
(~10mls) then dispose of PBS. Add 2mls of Trypsin and place in incubator for 2mins
at 37°C. Once cells detach (Trypsin = orange), give the flask a tap against the side of

69
a hard object to dislodge the cells from the bottom of the flask. Check cells. They
should be rounded and floating freely in the media. If any cells remain attached to the
bottom of the flask, give the flask an additional tap. Add 2mls media (10% FBS) to
neutralize the Trypsin. Take 10µl of cells. Stain cells using methylene blue (10ul
methylene blue to 10ul cell suspension). Place methylene blue stained cells on
Haemocytometer (improved Neubauer BS748, depth 0.01mm, 1/400mm2
) and place
cover slip on top of the drop of cells. Place Haemocytometer on microscope (Zeiss
Axiovert 25). Count cells in 10 random squares. Cells are counted when in the
middle of the square (not touching the lines) and when in contact with the bottom and
left sides of the square. Cells touching the top and left sides are excluded from the
cell count figure. Obtain the average of the 10 cell counts. Divide the average by 16
and then multiply by 4. Multiply that figure by 106
and you have the cells per ml.
Procedure for Freezing Cells (cryogenic storage)
Take cells after step 9 of passage procedure and wash the cells with media
(containing FBS). Centrifuge cells again as per step 9 of passage procedure. Re-
suspend cells in freezing medium (10% DMSO, 20% FBS and 70% standard media).
DMSO is filter sterilized using a 2um filter. Dispense cells into cryo tubes. When
freezing, do it slowly (1hr @ 4°C, 1hr @ -20°C and 1hr @ -80°C then place in liquid
nitrogen).
Procedure for fluorescence staining of the samples
Remove the LIVE/DEAD reagent stock solutions from the freezer and allow them to
warm to room temperature. Add 20µL of the supplied 2mM EthD-1 stock solution to
10ml of sterile, tissue culture–grade D-PBS, vortexing to ensure thorough mixing.
This gives an approximately 4µM EthD-1 solution. Combine the reagents by
transferring 5µL of the supplied 4 mM calcein AM stock solution (Component A) to
the 10mL EthD-1 solution. Vortex the resulting solution to ensure thorough mixing.
The resulting approximately 2µM calcein AM and 4µM EthD-1 working solution is
then added directly to cells. Note that aqueous solutions of calcein AM are
susceptible to hydrolysis. Aqueous working solutions should therefore be used

70
within one day. Cut the sample off the capillary tube mounting and place sample in a
Petri dish. Add 100–150µl of the combined LIVE/DEAD assay reagents, using
optimized concentrations, to the surface of the sample. Incubate the cells for 30–45
minutes at room temperature. Following incubation, add about 10µL of the fresh
LIVE/DEAD reagent solution or D-PBS to a clean microscope slide. Using fine-
tipped forceps, carefully (but quickly) invert and mount the sample on the
microscope slide. Place the slide on the on the Leica DMIRE2 confocal microscope.
Set the microscope to 500nm for the Calcein stain and 550nm for the ethidium stain.
View the labelled samples under the fluorescence microscope.
Experimental Technique
Before the experiment could commence, the cells from the ECACC needed to be
grown to sufficient quantities to supply the entire experiment. To do this, the cells
were initially split into 4 flasks (1-4) and then frozen. Each batch were then grown
and split and the passages were recorded as n, n.x, n.x.y, n.x.y.z so the vials could be
easily traced back to the original split. This also made it easier to ensure the cells
used for the experiment were all from the same passage stage. It was essential to
ensure the cells in the experiment were from the same passage number, as non-
immortalised cells in culture only have a finite number of passages before they die
and the cells health varies with passage level. This was one level of continuity built
into the experiment.
Before the start of each run, the cells to be used were resuscitated from cryogenic
storage and given time to recover and reach ~ 70% confluences. The cells were then
Trypsinised, centrifuged and re-suspended in 1ml of media.
The samples were inoculated with 20µl of cell suspension and left for ~10 minutes in
an empty flask before media was added, to give the cells a chance to attach to the
substrate without the media washing them off. After the 10mins, 10mls of media was
added and the C 5% CO2) with loosened
caps.

71
Fig. 3.15 - Illustration of biomaterial inoculation.
After inoculation, a cell count was performed. Sample groups were started in three
groups per month, two days apart. Growth along the sample was measured using a
photocopy of a ruler (Helix shatter proof) with millimetre markings on acetate after 7
days, 14 days 21 days and 29 days. The same acetate ruler was used throughout the
experiment (the acetate copy was compared to the original ruler to ensure the
gradations were accurate). At the end of the 29 day study period, the samples were
removed from the capillary tube mounting and placed in Petri dishes. The samples
were then stained with Molecular Probes “live or dead” viability/cytotoxicity kit (L-
3224) and examined on the Leica DMIRE2 confocal microscope to confirm the level
of growth.

72
Chapter 4 - Results
The chitosan study was devised to test the reported antimicrobial effects of chitosan.
The experiments were designed to present chitosan to MRSA and Staphylococcus
epidermis and examine how effective chitosan is against the opportunistic pathogens.
In addition, examining how the bacteriostatic effect varied with the quantity of
chitosan presented to the bacteria would clarify how varying the chitosan quantity
would alter growth. Through the development of the experimental design, the
experiment evolved. Performing studies where the chitosan was presented to the
bacteria in different forms, while not directly comparable with each other added an
interesting dimension to the study.
Vs MRSA 9551 and Staphylococcus epidermis.
Method 1 was the preliminary study designed to evaluate the efficacy of chitosan as a
bacteriostat.
Many of the chitosan (+ acetic acid) plates did not solidify enough to inoculate. This
included the acetic acid control, the 0.001g & 0.01g chitosan samples and some of
the 0.1g & 1g chitosan samples. This was due to the acetic acid hydrolysing the agar
and destroying the structure of the polysaccharide.

73
Cell counts MRSA 9551 S. epidermis
1 54 7
2 41 12
3 51 7
4 43 6
5 48 8
6 44 21
7 36 15
8 42 10
9 46 21
10 37 9
Average 44.2 11.6
Table 4.1 - Cell count data for MRSA and S. epidermis inoculum - haemocytometer count (volume of
square = 1/400ml)
Therefore to reach bacteria per ml;
((Average cells per box) x 4) x (10-6
) = Cells per ml
(44.2 x 4) x 10-6
= 1.77 x 109
bacteria per ml for MRSA
(11.6 x 4) x 10-6
= 4.64 x 108
bacteria per ml for S. epidermis

74
MRSA 9551 S. epidermis
Control 1 (NA) 1 small cream colony (less than
1mm)
5 small yellow colonies (less
than 1mm)
Control 2 (NA) 1 small yellow colony (less than
1mm) not cream (like MRSA)
therefore contamination
1 small orange colony (under
agar) not cream (like MRSA)
therefore contamination
NA + Chitosan Film 29 small colonies less than
0.5mm in diameter, all in one
location around initial streak. No
growth anywhere near the film
No colonies
NA + 1g Chitosan in 10
ml acetic acid (1)
No growth No growth
ml acetic acid (2)
No growth No growth
ml acetic acid (3)
No growth No growth
NA + 0.1g Chitosan in
10 ml acetic acid
No growth No growth
Table 4.2 - Results after 48 hrs
In this experiment, growth was low and the control containing acetic acid did not
solidify due to hydrolysis of the agar therefore this experiment was revised.
Method 1b - Modified method – Chitosan dissolved in dilute acetic acid
incorporated into nutrient agar Vs MRSA 9551 and Staphylococcus epidermis
Method 1b is a modified version of method 1.
The NA control was kept and instead of the NA + acetic acid control, NA + acetic
acid neutralised to pH 7 (using NaOH and a corning pH meter 215) was used. The
NA + 1g chitosan in 10 ml acetic acid and NA + 0.1g chitosan in 10 ml acetic acid
agar plates were re-used after re-sterilisation (as there was no previous growth).
In addition, instead of diluting the bacteria, each plate was inoculated from the saline
inoculum with a sterile swab.

75
The chitosan film was also included in this experiment. The NA plates with the
chitosan film were inoculated before adding the film, so the growth could be
examined to see if the bacteria would grow up to, under or over the film.
After inoculation, the plates were incubated for 48hrs at 37 C
MRSA 9551 S. epidermis
NA control Good growth. A lawn grew from
where the plate was inoculated.
No contamination.
Good growth. A lawn grew from
No contamination.
NA + neutralised acetic
acid control
No contamination.
No contamination.
Chitosan film on NA Bacterial growth surrounding
film. No growth on film.
Bacterial growth surrounding
film. No growth on film.
1g Chitosan + 1ml acetic
acid in NA
No growth, bacteria still present.
The plate appears no different
from when inoculated.
(Bacteriostatic effect)
The plate appears no different
from when inoculated.
0.1g Chitosan + 1ml
acetic acid in NA
Table 4.3 - Results after 48 hrs
This experiment produced interesting data for how the bacteria reacted in the
presence of chitosan film, but the data for the nutrient agar containing chitosan was
less clear. The acetic acid control still did not provide a suitable control for the
chitosan samples (as the chitosan could not be neutralised without the chitosan
precipitating and not mixing with the agar).
With this in mind, Nutrient broth appeared to be a better choice than nutrient agar as
this would avoid the problem of agar hydrolysis.

76
Three quantities of chitosan powder were weighed out; 1g, 0.1g and 0.01g (+ /-
0.001g). These samples were added to 10 ml acetic acid (2M), forming a range of
solutions designed to avoid the problem of agar hydrolysis by substituting nutrient
agar for nutrient broth. This method uses spectrophotometry to measure the turbidity
(cloudiness resulting from the bacterial growth) to produce quantitative data. The
samples were inoculated using a standard inoculum (an inoculation loop of bacteria
mixed in a saline solution, and then counted).
Cell counts MRSA 9551 S. epidermis
1 34 20
2 12 25
3 36 30
4 38 18
5 38 33
6 54 22
7 44 34
8 40 41
9 45 30
10 57 32
Mean 39.8 28.5
Table 4.4 - Cell count data for MRSA and S. epidermis inoculum.
Haemocytometer Vol = 1/400 ml per small square (visible through microscope)
Therefore for;
MRSA = (39.8 * 4) * (106
) = 1.59 x 109
bacteria per ml
S. epidermis = (28.5 * 4) * (106
) = 1.14 x 109
bacteria per ml
The broths were then inoculated with 0.5mls of the bacterial dilutions (either MRSA
9551 or S. epidermis).

77
Cell density was measured spectrophotometrically for Staphylococcal species using a
wavelength of 550nm. Samples were measured every 2 hrs to examine for changes
in growth. After 2hrs there was growth in the NB control, but there was no growth in
the acetic acid control. The pH was inhibiting bacterial growth. The experimental
design would have to change so that the pH was no longer an issue.
Method 2b - Modified method - Chitosan added to nutrient broth Vs MRSA 9551
This experiment was redesigned so the chitosan would not be dissolved into solution.
This would determine if chitosan in suspension would elicit the desired bacteriostatic
effect. Three quantities of chitosan powder were weighed out; 1g, 0.1g and 0.01g (+
/- 0.001g). These samples were added to 10 ml distilled water, forming a range of
suspensions (the powder did not remain in suspension long before settling out and
therefore needed constant agitation).
1ml of the suspension was added to each of the chitosan nutrient broths (therefore the
1g becomes 0.1g, although it is still referred to as 1g) and 1ml of distilled water was
added to the control.
The samples were inoculated directly from an inoculation loop to increase the
quantity of bacteria present in each broth. After inoculation, the samples were placed
on a shelf in a 37ºC incubator.

78
Sample Contents AD550nm (11.30 -
11.45am) (+/- 0.007)
Control (ref) NB 0.001
Control 0.01g NB + 1ml (0.01g chitosan /10mls distilled water) 0.007
Control 0.1g NB + 1ml (0.1g chitosan /10mls distilled water) -0.005
Control 1g NB + 1ml (1g chitosan /10mls distilled water) 0.065
S. epidermis
Control
NB + 0.5mls S. epidermis broth -0.005
MRSA Control NB + 0.5mls MRSA 9551 broth -0.005
S. epidermis
0.01g
NB + 1ml (0.01g chitosan /10mls distilled water) +
0.5mls S. epidermis broth
-0.004
S. epidermis 0.1g NB + 1ml (0.1g chitosan /10mls distilled water) +
0.007
S. epidermis 1g NB + 1ml (1g chitosan /10mls distilled water) +
0.077
MRSA 0.01g NB + 1ml (0.01g chitosan /10mls distilled water) +
0.5mls MRSA 9551 broth
-0.006
MRSA 0.1g NB + 1ml (0.1g chitosan /10mls distilled water) +
-0.004
MRSA 1g NB + 1ml (1g chitosan /10mls distilled water) +
0.089
Table 4.5 - Absorbance reading at Time 0
Sample AD550nm
Control (ref) 0.000
Control 0.01g -0.004
Control 0.1g 0.002
Control 1g 0.007
S. epidermis Control -0.005
MRSA Control -0.007
S. epidermis 0.01g -0.004
S. epidermis 0.1g 0.011
S. epidermis 1g -0.004
MRSA 0.01g -0.005
MRSA 0.1g -0.002
MRSA 1g 0.025
Table 4.6 - Absorbance reading at 1hr

79
Sample AD550nm
Control (ref) 0.006 reset to 0.000
Control 0.01g -0.004
Control 0.1g -0.003
Control 1g 0.001
S. epidermis Control -0.002
MRSA Control -0.010
S. epidermis 0.01g -0.004
S. epidermis 0.1g 0.004
S. epidermis 1g 0.130
MRSA 0.01g -0.015
MRSA 0.1g -0.008
MRSA 1g 0.102
Table 4.7 - Absorbance reading at 2hrs
3 hrs
This experiment was abandoned as there was no growth in the controls.
Method 2c - Modified method - Chitosan added to nutrient broth Vs MRSA 9551
This is a modified version of method 2b. In this method, the nutrient broths were
kept on a platform shaker to keep the chitosan powder in suspension.

80
Sample Name Description
Reference Control (NB)
S. epidermis NB + 0.5mls S. epidermis broth
S. epidermis 0.01g NB + 1ml(0.01g chitosan /10mls distilled water) + 0.5mls S.
epidermis broth
S. epidermis 0.1g NB + 1ml(0.1g chitosan /10mls distilled water) + 0.5mls S.
epidermis broth
S. epidermis 1g NB + 1ml(1g chitosan /10mls distilled water) + 0.5mls S. epidermis
broth
MRSA NB + 0.5mls MRSA 9551 broth
MRSA 0.01g NB + 1ml(0.01g chitosan /10mls distilled water) + 0.5mls MRSA
9551 broth
MRSA 0.1g NB + 1ml(0.1g chitosan /10mls distilled water) + 0.5mls MRSA
9551 broth
MRSA 1g NB + 1ml(1g chitosan /10mls distilled water) + 0.5mls MRSA
9551 broth
Table 4.8 – Sample descriptions.
Time 0
Samples inoculated and absorbance measured (AD550nm).
The samples were left for 15mins to allow the chitosan suspension to settle before
taking spectrophotometer readings.
Reference = NB.
Sample AD550nm
Reference -0.001 (+/- 0.005)
S. epidermis 0.000 (+/- 0.005)
S. epidermis 0.01g -0.010 (+/- 0.004)
S. epidermis 0.1g 0.000 (+/- 0.004)
S. epidermis 1g 0.135 (+/- 0.004)
MRSA -0.010 (+/- 0.001)
MRSA 0.01g -0.016 (+/- 0.002)
MRSA 0.1g -0.004 (+/- 0.003)
MRSA 1g 0.113
Table 4.9 - Absorbance reading at Time 0

81
Sample AD550nm
Reference 0.000 (+/- 0.004)
S. epidermis 0.000 (+/- 0.004)
S. epidermis 0.01g -0.001 (+/- 0.003)
S. epidermis 0.1g 0.000 (+/- 0.002)
S. epidermis 1g 0.113
MRSA 0.006 (+/- 0.002)
MRSA 0.01g -0.006 (+/- 0.004)
MRSA 0.1g 0.001 (+/- 0.003)
MRSA 1g 0.125
Table 4.10 - Absorbance reading at 1hr
Sample AD550nm
Reference 0.000 (+/- 0.001)
S. epidermis 0.005 (+/- 0.002)
S. epidermis 0.01g 0.003 (+/- 0.001)
S. epidermis 0.1g 0.006 (+/- 0.002)
S. epidermis 1g 0.070 (+/- 0.001)
MRSA 0.016 (+/- 0.004)
MRSA 0.01g 0.005 (+/- 0.001)
MRSA 0.1g 0.007 (+/- 0.001)
MRSA 1g 0.080 (+/- 0.003)
Sample AD550nm
Reference 0.000 (+/- 0.001)
S. epidermis 0.020 (+/- 0.002)
S. epidermis 0.01g 0.008 (+/- 0.001)
S. epidermis 0.1g 0.008 (+/- 0.001)
S. epidermis 1g 0.017 (+/- 0.002)
MRSA 0.160 (+/- 0.002)
MRSA 0.01g 0.148 (+/- 0.002)
MRSA 0.1g 0.147
MRSA 1g 0.174 (+/- 0.002)

82
After 25 hrs the samples were taken out of the 37 C incubator and shaken to re-
suspend the bacteria and chitosan. In the 1g chitosan and S. epidermis sample, It was
noticed the chitosan powder (which settles quite quickly (~5-10 minutes) was looking
bigger (particle size). When it settled, it was less dense and of greater volume. It
appears to have agglutinated with the S. epidermis. With this observation, it was
decided to examine the other samples closely. It appeared that it had the same effect
on the other S. epidermis samples, but not with the MRSA 9551 samples. This was
an unexpected outcome as S. epidermis and MRSA 9551 are closely related. It was
decided to continue with taking the spectrophotometer readings and see what
differences that revealed.
Sample AD550nm
Reference 0.000 (+/- 0.001)
S. epidermis 0.450 (+/- 0.001)
S. epidermis 0.01g 0.502 (+/- 0.001)
S. epidermis 0.1g 0.429 (+/- 0.001)
S. epidermis 1g 0.113 (+/- 0.002)
MRSA 0.558
MRSA 0.01g 0.510 (+/- 0.001)
MRSA 0.1g 0.466 (+/- 0.002)
MRSA 1g 0.549 (+/- 0.001)

83
chitosan Vs MRSA 9551 & S.epidermis excluding 1g chitosan sample
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
00:20.0 01:15.0 02:15.0 04:00.0 26:10.0
time (hours)
AD550
Reference (Nutrient Broth)
MRSA 9551 Control
MRSA 9551 0.01
MRSA 9551 0.1
S.epidermis Control
S.epidermis 0.01
S.epidermis 0.1
Fig. 4.14 - Absorbance readings for MRSA and S. epidermis

84
Fig. 4.14 has the values for NB + 1ml (1g chitosan/10mls distilled water), NB + 1ml
(1g chitosan/10mls distilled water) + S. epidermis and the NB + 1ml (1g
chitosan/10mls distilled water) + MRSA removed. This was because the high level
of chitosan was distorting the absorbance readings. The original graph is in appendix
A.2, Method 2c. This graph shows two results. The chitosan appears to have a
bacteriostatic effect on MRSA as shown by the reduced growth of the samples with
0.01g of chitosan and 0.1g of chitosan. This bacteriostatic effect also appears to be
related to the quantity of chitosan present. The S. epidermis does not appear to
demonstrate reduced growth in the presence of chitosan.
For this study, two types of cotton cloth were used, chitosan gel coated and untreated.
Hypothesis; inoculate NA plate so that a bacterial lawn will develop and place cotton
cloth on top (treated or untreated) and observe for signs of inhibition around edge of
material.
After 24 hours the cotton cloth plates were examined but there was insufficient
bacterial growth on the agar plates.
Method 3b - Modified method - Testing of Chitosan treatment of Cotton cloth
After the poor growth in method 3, the nutrient agar plates were inoculated using a
20µl pipette and the inoculum was spread around the agar using an inoculation loop
in an attempt to produce better bacterial lawn growth.
There were zones of inhibition around the chitosan treated samples, but the bacterial
growth was poor, so the experiment was repeated with fresh bacteria and fresh
samples.

85
Repeat of method 3b
Observation
MRSA 9551 Cotton cloth control little growth, but up to edge of material
MRSA 9551 Chitosan treated cotton
cloth
not very good growth, but still observable zone of
inhibition
S. epidermis Cotton cloth control Reasonable growth up to the edge of the material
S. epidermis Chitosan treated cotton
cloth
Good growth. Observable zone of inhibition on three
sides of the treated sample
Table 4.15 – chitosan treated cotton cloth results
There were zones of inhibition around the chitosan treated samples, but the bacterial
growth was poor, so it was decided to repeat the experiment with fresh bacteria.
Method 3c - Modified method - Testing of Chitosan treatment of Cotton cloth
Method 3c is a modified version of method 3 as it contains a slightly expanded range
of test samples. Some of the treated cotton cloth was kept un-sterilised to see if the
high-pressure, high temperature steam has any effect on the bacteriostatic effect of
the chitosan.
The chitosan did not adhere to the polypropylene as well as it did on the cotton cloth
(polypropylene mesh is multifilament and knitted into an open structure and is not
very wetable. The cotton cloth is a natural fibre woven structure and it is
hydrophilic).

86
Sample Observation
MRSA cotton cloth control
(sterilised)
Good lawn growth up to and under cotton cloth
sample
MRSA Chitosan treated cotton cloth
(sterilised)
Good lawn growth. Zone of inhibition between
0.5mm and 3mm
MRSA Chitosan treated cotton cloth
(not sterilised)
0.5mm and 7mm.
MRSA Chitosan treated
polypropylene (sterilised)
Good lawn growth. Possible zone of inhibition less
than 0.5mm.
Sample Observation
S. epidermis cotton cloth control
(sterilised)
Strong lawn growth up to and under cotton cloth
sample
cloth (sterilised)
0.5mm and 3mm.
cloth (not sterilised)
0.2mm and 1.5mm.
S. epidermis Chitosan treated
polypropylene (sterilised)
Good lawn growth. No zone of inhibition.
Table 4.16 – Chitosan coated cotton cloth and polypropylene results
The poor performance of the chitosan coated polypropylene could be due to the
chitosan film failing to adhere to the hydrophobic polypropylene.
Method 3c (Modified method - Testing of chitosan treatment of cotton cloth) yielded
some interesting data relating to the coating of cotton cloth with chitosan solution,
with zones of inhibition observed. The polypropylene coated with chitosan had little
to no observable effect on the bacteria. This study was derived partially to examine
the potential for chitosan coatings for medical applications. One application could be
as an anti infective coating for medical devices, but this could find a use as a spray-
able coating for textiles within a hospital environment by reducing the ability of
opportunistic pathogens to thrive on the clothes worn by hospital personnel and the
soft furnishings found within a hospital environment, reducing potential transmission
vectors. These applications are worthy of further study to determine their efficacy for
the hospital environment.

87
This method continues on from method 2c, but instead of using spectrophotometry to
measure the bacterial growth, plate count agars were used along with serial dilutions
of the broths at set time points to produce clearer data on the effect of chitosan in
suspension in different quantities.
In this study, the chitosan samples are referred to as 0.01g, 0.008g, 0.006g, 0.004g,
0.002g and 0g. These refer to the w/v of chitosan in NB (where 0g is the control).
A plate count was done to find out how concentrated the inoculum was using a
haemocytometer.
Cell (area on haemocytometer) count (number of cells)
1 5
2 7
3 6
4 7
5 9
6 9
7 13
8 8
9 8
10 4
Mean 7.6
Table 4.17 - Cell count data for MRSA inoculum.
To convert this to cells per ml;
7.6 x (4x10-6
) = 3.04x107
cells per ml in the initial inoculum
After 48 hours, the nutrient broths were observed. Upon observation, it was clear
that the broths displayed some degree of variation in their visual appearance. As a

88
result, broths that appeared to be either more turbid or less turbid than the other
broths of a certain chitosan concentration were not used for plate count purposes.
Sample Sample No. Observation
0.01g Chitosan 1 more turbid than the other two
0.008g Chitosan 2 less turbid than the other two
0.006g Chitosan - no variance
0.004g Chitosan 3 little less turbid than the other two
0.002 g Chitosan 2, 3 2 was more turbid, 3 was less turbid, 1 was
in the middle
0g Chitosan (control) - no variance
Table 4.18 - Samples discounted on the basis of a difference in the broth appearance.
Sample Sample No.
0.01g Chitosan 3
0.008g Chitosan 3
0.006g Chitosan 2
0.004g Chitosan 2
0.002 g Chitosan 1
0g Chitosan (control) 1
Table 4.19 - Samples used for the initial dilution
Dilution
Sample 10-6 10-7 10-8
Control (0g) tmtc 110 9
0.002g 0 0 4
0.004g tmtc 181 20
0.006g 461 64 6
0.008g 180 86 -
0.01g tmtc 111 0
Table 4.20 - Results from 48 hours. Tmtc – too many to count

89
The results from 48 hrs shows an unusual result for 0.002g and it is likely to be
caused by human error.
Dilution
Sample 10-6 10-7 10-8
Control (0g) ng 1049 ng
0.002g 3 ng ng
0.004g ng ng ng
0.006g ng ng ng
0.008g ng ng ng
0.01g ng ng ng
Table 4.21 - Results from 72 hours. Ng – no growth
This result was unexpected. The control, only one PCA grew and showed an
abundance of bacteria. The 0.002g, only one plate grew and showed a marked
decrease in culture density. There are four possible explanations for this. 1. Problem
with the culture media, 2. Human error or 3. (discounting the control) that the viable
bacterial population had decreased to such a level, that the dilutions did not contain
enough bacteria or 4. Phenomena as yet not understood.
Further to this unexpected result, the broth cultures used the day before were kept and
stored in the 20ºC incubator in case of such problems. When they were examined,
they had changed appearance. Some of the broths had almost lost their turbidity,
whilst others had a clear section at the top of the broth as if the media had separated.
Realising the relevance of this, the results were noted so they could be correlated to
the findings.

90
Sample Repeat Observation
Control (0g) 1 Medium turbidity. Can see through
0.002g 1 The lower 6/7th of the broth = very turbid, can‟t see
through. Top 1/7th, very clear. Upon disturbance, turbid
layer settles back, leaving the clear top 1/7th
0.002g 3 Medium turbidity. Can see through
0.006g 2 A little more turbid. Can see through
0.008g 1 0.008 1 and 0.008 3 look identical. Both are very turbid.
Top 1/7th is not clear, but appears to be starting to clear.
0.008g 3 0.008 1 and 0.008 3 look identical. Both are very turbid.
Top 1/7th is not clear, but appears to be starting to clear.
0.01g 1 very Turbid – opaque
0.01g 3 Quite turbid. Can still see through
Table 4.22 - Observations of broth appearance
In discussion with a colleague it was explained that MRSA changes from Gram +ve
to Gram –ve when a colony reaches a certain age. Further studies will be performed
with fresh cultures of MRSA.

91
Method 4b - Modified method - Chitosan suspended in nutrient broth Vs MRSA
Method 4 was modified to include a more appropriate selection of dilutions for
inoculating the plate count agar.
The graphs on the next few pages show growth rates over time of MRSA challenged
by chitosan in varying quantities.
The raw data has been excluded from the results section. The full results can be
found in appendix A.3, method 4b.

92
06/10/03 Chitosan Vs MRSA study
0
50,000,000,000
100,000,000,000
150,000,000,000
200,000,000,000
250,000,000,000
300,000,000,000
350,000,000,000
400,000,000,000
0 1 2 3
Day
CellsPerml
Control
0.02g of Chitosan in 10mls of media
Fig. 4.23 - Graph of average growth of MRSA Vs Chitosan.

93
Looking at fig. 4.23 there is one sample that shows improved growth over the
control, and the other 4 indicating lower growth than the control. The 0.1g sample is
clearly showing improved growth and the reasons for this are unknown. The initial
inoculum for this experiment was approximately 3.85 x 107
cells (1.93 x 108
cells per
ml), and the maximum viable number of cells in the control during the experiment
was approximately 2.69 x 1011
cells per ml.
Looking at the samples between 0.02g and 0.08g, we see two interesting features.
Firstly, none of these samples contain bacterial growth greater than the control.
Secondly, the growth rate is considerably slowed. The slow growth could be due to
the bacteriostatic effect of chitosan inhibiting the slowing the growth of the bacteria.
With a slowed growth of the bacteria, one may assume that the bacteria would still
reach the abundance found in the control, but what we see is that the bacteria decline
at lower abundance than the control. This might suggest that chitosan increases the
auto toxic effect of the bacteria, preventing them reaching the numbers of the control
and causing them to die at lower bacterial concentrations.
With the 0.1g sample out growing the control, it could be that there was an error in
the experiment or that chitosan is most effective at a certain concentration and may
even be metabolised by the bacteria when out with that concentration.
No graph could be produced for the chitosan and MRSA data from 20/10/03. This
was due to the sample dilutions not falling within the countable range.

94
0
500,000,000
1,000,000,000
1,500,000,000
2,000,000,000
2,500,000,000
3,000,000,000
3,500,000,000
0 1 2 3
Day
CellsPerml
Control

95
Looking at fig 4.24 we can see that all of the chitosan samples show lower growth
than the control. The initial inoculum for the 05/11/03 experiment was
approximately 9.76x 106
cells (4.88x 107
cells per ml), and the maximum viable
number of cells in the control during the experiment was approximately 3.09 x 109
cells per ml.
Looking at this graph, we see that none of the samples containing chitosan develop
the same number of bacteria as the control, although the 0.08g sample comes near
and the 0.1g sample shows the lowest growth. The only obvious difference between
this experiment is the concentration of the inoculum, which is ~1/4 the concentration
used in the 06/10/08 study. It would therefore appear that the efficacy of chitosan is
related in some manner to the quantity of bacteria used for the initial inoculation.

96
0
2,000,000,000
4,000,000,000
6,000,000,000
8,000,000,000
10,000,000,000
12,000,000,000
14,000,000,000
16,000,000,000
18,000,000,000
0 1 2 3
Days
CellsPerml
Control

97
Fig 4.25 does not show the same trend as the previous two graphs (fig 4.23 and 4.24).
The initial inoculum for this experiment was approximately 1.88 x 107
cells (9.40 x
107
cells per ml), and the maximum viable number of cells in the control during the
experiment was approximately 5.33 x 109
cells per ml.
This data does not appear to correlate with the two previous graphs. It could be
human error but it is more likely that there is some phenomenon occurring that is as
yet unknown. The only known variable is that this experiment contained half the
inoculum of the 06/10/03 experiment and double the inoculum of the 05/11/03
experiment. The question as to whether this is a factor in the variation seen in the
results can only be addressed by further study.
The polyurethane and polyester were fabricated externally (commercially available
materials). These samples were cut into sample sizes and plasma treated. The
polypropylene, poly-ε-caprolactone and Solanyl were extruded at Heriot-Watt
University as described in the methodology section. The PLA was to be extruded but
no useable tape could be produced, therefore it was cast as a film instead.
The plasma treatment was to be performed on the Europlasma equipment using
argon, oxygen and ammonia, but due to a technical fault with the equipment,
alternative equipment was used (the Nanotech equipment). Only argon and ammonia
gas were available for the Nanotech plasma equipment. As some samples had been
treated on the Europlasma equipment, they were included in the tissue culture study.
The DSC images display the energy required to increase the temperature of the
sample over a range of temperatures versus time. The dips and spikes in the energy

98
profile correlate to the polymer sample proceeding through different phases. The
lowest dip is the melting point of the polymer.
Fig. 4.26 - Polypropylene DSC. Melting point 151.4ºC
Fig. 4.27 - Polyurethane DSC. Melting point 149.5ºC

99
Fig. 4.28 - Polyester DSC. Melting point 257.5ºC
Fig. 4.29 - Polycaprolactone DSC. Melting point 62.5ºC

100
Fig. 4.30 - Solanyl Flexibilitis DSC. Melting point 112.5ºC
Fig. 4.31 - Poly-L-Lactic Acid DSC. Melting point 168.0ºC

101
4.3.2 SEM Analysis
The samples were analysed by scanning electron microscope to examine the surface
for change after plasma treatment and to illustrate differences in surface morphology.
Some of the materials have a very plain surface but they are included to illustrate the
difference in the surfaces of the biomaterials.
Artefacts are visible in some of the SEM images (dust ect).
Polypropylene
Fig. 4.32 - Polypropylene control. Very plain surface with few surface grooves produced during
extrusion

102
Fig. 4.33 - Argon Plasma Treated Polypropylene (Nanotech). At this magnification, there is no visual
difference between this polypropylene and the control polypropylene.
Fig. 4.34 - Ammonia Plasma Treated Polypropylene (Nanotech). At this magnification, there is no visual
difference between this polypropylene and the control polypropylene.
Fig 4.35 - Spherical cap shapes of water on untreated
fibre surface.
Source (Wei et al., 2004)
Fig 4. 36 - Growth and coalescence of water
droplets on plasma treated PP fibre surface.
Source (Wei et al., 2004)
Fig 4.35 is an environmental SEM image of untreated polypropylene and Fig 4.36 is
an environmental SEM image of oxygen plasma treated polypropylene. These
images illustrate that although there is no visual difference in the surface, the
hydrophilicity of the sample in fig. 4.36 is greater than the sample in fig. 4.35.

103
Tuftane Polyurethane
Fig. 4.37 - Tuftane Polyurethane. This material has a very smooth surface.
Fig. 4.38 - Argon Plasma Treated Tuftane Polyurethane (Nanotech). At this magnification, there is no
visual difference between this polyurethane and the control polyurethane.

104
Fig. 4.39 - Ammonia Plasma Treated Tuftane (Nanotech). At this magnification, there is no visual
difference between this polyurethane and the control polyurethane.
Fig. 4.40 - Argon Plasma Treated Tuftane Polyurethane (Europlasma). At this magnification, there is no
visual difference between this polyurethane and the control polyurethane.

105
Fig. 4.41 - Oxygen Plasma Treated Tuftane Polyurethane (Europlasma). At this magnification, there is
no visual difference between this polyurethane and the control polyurethane.
Vascutek polyester
Fig. 4.42 - Vascutek Polyester control. The structure of this material is very different to the other
materials therefore no direct comparisons may be made between this material and the others.

106
Fig. 4.43 - Argon Plasma Treated Vascutek polyester (Nanotech). At this magnification, there is no
visual difference between this polyester and the control polyester.
Fig. 4.44 - Ammonia Plasma Treated Vascutek polyester (Nanotech). At this magnification, there is no
visual difference between this polyester and the control polyester.

107
Fig. 4.45 - Poly-ε-Caprolactone 6400 control. This material displays a grooved surface.
Fig. 4.46 - Argon Plasma Treated Poly-ε-Caprolactone 6400 (Nanotech). At this magnification, there is
no visual difference between this Poly-ε-Caprolactone and the control Poly-ε-Caprolactone.

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Fig. 4.47 - Ammonia Plasma Treated Poly-ε-Caprolactone 6400 (Nanotech). At this magnification, there
is no visual difference between this Poly-ε-Caprolactone and the control Poly-ε-Caprolactone.
Solanyl
Fig. 4.48 - Solanyl Control. This material has a very smooth surface.

109
Fig. 4.49 - Argon Plasma Treated Solanyl (Nanotech). At this magnification, there is no visual
difference between this Solanyl and the control Solanyl.
Fig. 4.50 - Ammonia Plasma Treated Solanyl (Nanotech). At this magnification, there is no visual
difference between this Solanyl and the control Solanyl.

110
Fig. 4.51 - Solanyl extruded with 2% chitosan (w/w). The fine bumps in this image were interpreted as
chitosan powder
Fig. 4.52 - Solanyl extruded with 2% chitosan (w/w). The fine bumps can be seen more clearly in this
image.

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Fig. 4.52 shows the chitosan powder incorporated into the Solanyl tape.
Poly-l-lactic acid
Fig. 4.53 - Poly-l-lactic acid Control. The highly porous structure can be seen in this image. It is
assumed that this structure is due to the solvent casting method of production (Chun et al., 2000).
Fig. 4.54 - Argon Plasma Treated Poly-l-lactic acid (Nanotech). At this magnification, there is no visual
difference between this Poly-l-lactic acid and the control Poly-l-lactic acid.

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Fig. 4.55 - Ammonia Plasma Treated Poly-l-lactic acid (Nanotech). At this magnification, there is no
visual difference between this Poly-l-lactic acid and the control Poly-l-lactic acid.
Analysis of Pore Size of PLA Sample
With the PLA displaying a highly porous structure, measurement of the pore size was
performed. The following images were used in the measurements as they were
deemed representative of the PLA pore size. The pore sizes were calculated
assuming the pores were circular. The area of the pores was calculated using the
equation (4.1).
Area of a circle = π x Diameter Equation (4.1)
The measurements were converted to scale using the scale bars in the SEM images.
For pores where only half was visible in the image, the area was halved.

113
Fig. 4.56 This image is the first of the PLA images to be measured and therefore will be referred to as
PLA 1.
Fig. 4.57 This image is the second of the PLA images to be measured and therefore will be referred to as
PLA 2.

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Fig. 4.58 This image is the third of the PLA images to be measured and therefore will be referred to as
PLA 3.
Pore Size Measurements of Figures 4.55 – 4.57
The PLA images were printed out and the dimensions of the pores were measured
using a ruler. The scale bar in the images was used to convert the measurements
from cm to µm. The complete measurement data can be found in appendix B.
PLA 1 PLA 2 PLA 3
Average pore area (µm2
) 7.04 7.44 12.22
Total area of pores in image (µm2
) 563.03 402.03 464.37
Percentage porosity 55.80% 39.84% 46.02%
Average area of pores for the three images (µm2
) 8.90
Average percentage of pores for the three images 47.22%
Table 4.59 Summary of PLA pore size measurements.

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The result of these measurements was that the PLA had pores between 0.13µm2
to
39.58 µm2
, with the average pore size at 3.31 µm2
and a percentage area of pores of
17.4%.
4.4 Tissue Culture Study
The following images help illustrate the difficulty with measuring the growth
accurately. The use of cellular stains was avoided to prevent potential detrimental
effects on cell growth.
The next 2 pages show photographs of the tissue culture samples through a
microscope. These photographs are for illustration only.
Fig. 4.60
Polypropylene
Fig. 4.61
Polyurethane
Fig. 4.62
Polyurethane

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Fig. 4.63
Polyester
Fig. 4.64
Polyester
Fig. 4.65
Poly-ε-caprolactone
Fig. 4.66
Solanyl

117
Fig. 4.67
Poly-L-Lactic acid
Fig. 4.68
Poly-L-Lactic acid
Figures 4.60 through to 4.68 illustrate what was seen down the microscope when
measuring the MRC-5 cell growth along the samples. These images are a mixture of
demonstrating the material as seen through the microscope combined with attempts
to photograph the cells growing on the material clearly. These images were taken
using a 35mm SLR with a microscope mount.
When the samples were examined weekly, measurements of growth along the
samples were recorded. In addition, cells could sometimes be seen growing on the
glass sample support or the tissue culture flask. This was recorded and the data can
be seen in appendix C.2. The colour of the media was recorded to provide supporting
evidence for the growth measurements. This can also be seen in appendix C.2.
All of the materials tested comprised a control, an argon treated material and an
ammonia treated material. For some materials, other treatments were included. Both
Solanyl and polypropylene were treated with a chitosan solution both with and
without plasma treatment. In addition, some one off treatments were tested.

118
These treatments were; argon treated polyurethane (on the Europlasma machine),
oxygen plasma treated polyurethane (on the Europlasma machine), chitosan powder
sprinkled on to a sample of polyurethane prior to argon plasma treatment (Nanotech)
and Solanyl extruded with 2% chitosan powder (w/w).
The oxygen and argon samples were created using the Europlasma equipment. The
equipment failed shortly after these treatments and therefore alternative equipment
was used for the other samples. The argon treatment provided a comparison between
the two different plasma treatment machines, while the oxygen plasma treatment can
only be directly related to the Europlasma treated argon sample and the control. The
chitosan powder sprinkled on to a sample of polyurethane prior to argon plasma
treatment sample was made to determine whether plasma could be used to attach
powders to surfaces and to compare this method with the addition of chitosan film. It
was then used in the study to determine if the chitosan powder would persist on the
material and to see what effect it might have on cell growth.
The following pages show graphs that chart the growth of the cells along the samples
over a period of 29 days. Each sample was replicated 9 times, although not every
sample produced a clear result. See the appendix C.1 for further details.

119
Polypropylene Data
0
5
10
15
20
25
30
0 8 15 22 29
Day
AverageGrowth(mm)
Polypropylene
Polypropylene Argon treated (Nanotech)
Polypropylene Ammonia treated (Nanotech)
Polypropylene Chitosan treated
Polypropylene Argon and Chitosan treated (Nanotech)
Polypropylene Ammonia and Chitosan treated (Nanotech)
Fig. 4.69 - Graph of average growth of fibroblasts on the polypropylene samples.

120
Fig 4.69 shows the growth of MRC-5 cells along the polypropylene samples.
Looking at the first 15 days of growth, it is clear that the untreated polypropylene
performs the worst for supporting initial growth of the cells. The majority of the
other treatments seem to perform better, supporting 2-2.5mm of growth on the
samples (polypropylene argon treated, polypropylene chitosan treated, polypropylene
argon chitosan treated and polypropylene ammonia chitosan treated). The best
material over the initial 15 days was the polypropylene ammonia treated. This data
indicates that any of the treated materials performs better than native polypropylene
for supporting growth over 15 days.
Over the next 15 days, the data shows a change in the growth rate of he MRC-5 cells
on the samples. The polypropylene samples with gas plasma treatment and chitosan
coating maintain a steady growth rate but perform poorly when compared to the other
samples. The unmodified polypropylene displays a sharp increase in growth up to
day 22 and then displays no further growth over the remaining 7 days. The
polypropylene samples with single treatments (chitosan, argon and ammonia) display
sustained growth, out performing the native polypropylene, although the chitosan
coating growth rate appears to be tailing off over the last 7 days. The slowing of the
chitosan coated polypropylene sample could in part be due to the hydrophobic
polypropylene on which the coating was applied. The chitosan coating may be
partial, with sections of no coating. The plasma treated samples show sustained
growth, due in part to a consistent surface modification which enhances the
polypropylene hydrophilicity.

121
Polyurethane Data
0
5
10
15
20
25
30
0 8 15 22 29
Day
AverageGrowth(mm)
Polyurethane
Polyurethane Argon treated (Nanotech)
Polyurethane Ammonia treated (Nanotech)
Polyurethane with chitosan powder prior to argon treatment (Nanotech)
Polyurethane Argon treated in (Europlasma)
Polyurethane Oxygen treated (Europlasma)
Fig. 4.70 - Graph of average growth of fibroblasts on the polyurethane samples.

122
Fig. 4.70 shows the growth of MRC-5 cells along the Polyurethane samples. This
selection of treatments includes plasma treatment from two different plasma
treatment machines; therefore they shall be differentiated by manufacturer of the
control equipment (Nanotech and Europlasma). Looking at the first 15 days of
growth, it is clear that the untreated polyurethane is out-performed by the modified
polyurethane samples. The oxygen plasma treated polyurethane (Europlasma) and
the polyurethane sprinkled with chitosan powder prior to argon treatment were both
displaying greater growth rates than the untreated polyurethane over the first 15 days.
The two Nanotech treated samples and the Europlasma argon sample perform very
well, although the difference in growth rate between the two argon treatments is
interesting, with the Nanotech sample encouraging twice the growth of the
Europlasma samples. This could be partially due to differences in the plasma
chamber. The Nanotech chamber was far smaller than the Europlasma chamber and
as a direct result, although the gas was in excess, and the other parameters were
matched as closely as possible, the distance between the plates (between which the
RF frequency was discharged) was far smaller, therefore producing a more focused
plasma discharge. This hypothesis will need to be confirmed in a later study.
In the latter 14 days of the study it can be seen that the control sample and the oxygen
plasma treated sample perform similarly, while the argon and chitosan sample and
the Europlasma argon treated sample are outperformed by the control. This was an
unexpected outcome.
In contrast, the two samples treated on the Nanotech equipment, the argon and
ammonia samples dramatically outperform the control, with the ammonia again
performing the best with an average growth ~15mm greater than the control. The
two Nanotech samples also perform considerably better than the polypropylene
samples with the corresponding treatments. The results also show that the
polyurethane control outperforms the polypropylene control.

123
Vascutek Polyester Data
0
5
10
15
20
25
30
0 8 15 22 29
Day
AverageGrowth(mm)
Vascutek Polyester
Vascutek Polyester Argon treated (Nanotech)
Vascutek Polyester Ammonia treated (Nanotech)
Fig. 4.71 - Graph of average growth of fibroblasts on the polyester samples.

124
Fig. 4.71 shows the growth of MRC-5 cells along the Vascutek samples. This graph
shows the control material out performing both of the plasma treated samples. This
material is used as a vascular prosthesis and when adding cells to the samples, the
cells could be seen to wick into the sample very quickly, demonstrating the
hydrophilicity. By plasma treating the samples, it is possible that the hydrophilicity
was reduced resulting in lower growth on the samples. Although the plasma treated
samples do not perform as well as the control, it can still be seen that the ammonia
plasma treated sample performs better than the argon plasma treated sample, although
for the first 15 days, the growth rate is similar.

125
Poly-L-Lactic Acid Data
0
5
10
15
20
25
30
0 8 15 22 29
Day
AverageGrowth(mm)
Polylactic Acid Polylactic Acid Argon treated (Nanotech)
Polylactic Acid Ammonia treated (Nanotech)
Fig. 4.72 - Graph of average growth of fibroblasts on the polylactic acid samples.
Fig. 4.72 shows the growth of MRC-5 cells along the poly-l-lactic acid samples. The
data also shows the control performing better than the plasma treated samples and

126
therefore, like the polyester, these plasma treatments are not stimulating tissue
growth.

127
Poly-e-Caprolactone Data
0
5
10
15
20
25
30
0 8 15 22 29
Day
AverageGrowth(mm)
Polycaprolactone Polycaprolactone Argon treated (Nanotech)
Polycaprolactone Ammonia treated (Nanotech)
Fig. 4.73 - Graph of average growth of fibroblasts on the poly-ε-caprolactone samples.

128
Fig. 4.73 shows the growth of MRC-5 cells along the polycaprolactone samples.
With this collection of samples, they all perform similarly over the first 15 days. It is
only in the last 15 days where there is an obvious difference in growth. The order of
ammonia plasma, argon plasma then control can be seen and while the ammonia
performs best, the argon plasma treatment is only marginally better than the control.
It can also be seen that the argon plasma does not appear to encourage further growth
after 15 days, suggesting that the cells are having difficulty growing on this substrate,
and are performing poorly on the control which indicates a decline.

129
Solanyl Flexibilitis Data
0
5
10
15
20
25
30
0 8 15 22 29
Day
AverageGrowth(mm)
Solanyl Flexibilitis
Solanyl Flexibilitis Argon treated (Nanotech)
Solanyl Flexibilitis Ammonia treated (Nanotech)
Solanyl Flexibilitis extruded with 2% Chitosan powder
Solanyl Flexibilitis Chitosan treated
Solanyl Flexibilitis Argon and Chitosan treated (Nanotech)
Solanyl Flexibilitis Ammonia and Chitosan treated (Nanotech)
Fig. 4.74 - Graph of average growth of fibroblasts on the Solanyl samples.

130
Fig. 4.74 shows the growth of MRC-5 cells along the Solanyl samples. This sample
collection contains a few variations, so the standard three materials shall be looked at
first.
Over the first 15 days, the control and the ammonia plasma samples show steady
growth (the ammonia picks up after day 8) but it takes until day 15 for the argon
plasma samples to show growth. Over the next 15 days, the control and ammonia
plasma samples show continued and steady growth but the argon plasma samples
show a burst of growth then a decline. These three samples perform similarly to the
PLA samples, with growth around the 5mm point and like the PLA, the two basic
plasma treatments do not perform as well as the control. The main difference
between the Solanyl control and the PLA is that the PLA control performed better.
The additional treatments with the exception of Solanyl with chitosan coating (no
plasma) promote better tissue growth than the control. The Solanyl with argon
plasma and chitosan coating and the Solanyl with ammonia plasma and chitosan
coating perform similarly except for the last seven days, where the ammonia and
chitosan samples show a sharp increase in cell growth. The Solanyl with 2%
chitosan does not show noticeable growth for the first 15 days, and then it shows a
sharp increase in growth for the last 15 days. This is an interesting finding and could
be related to the fact that the chitosan is embedded in the polymer. The sharp
increase in growth may occur as the surface of the polymer erodes slightly, exposing
the chitosan to the cells.

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Control Sample Data
0
5
10
15
20
25
30
0 8 15 22 29
Day
AverageGrowth(mm)
Polypropylene Polyurethane Vascutek Polyester
Polylactic Acid Polycaprolactone Solanyl Flexibilitis
Fig. 4.75 - Graph of average growth of fibroblasts on the control samples.

132
Fig 4.75 shows the average growth of the control materials over 29 days. Over the
first 15 days, there are three distinct groups. The first group showing very little
growth includes polypropylene, polyurethane and polycaprolactone. The second
group with growth average growth around 4mm includes Vascutek polyester and
Solanyl. The last sample is the only sample with growth above 5mm in the first 15
days is the PLA. At day 22, all but the polycaprolactone, group around the 5mm of
growth, but over the last 7 days, the growth changes for most of the samples. The
polycaprolactone shows no growth. The Solanyl continues to encourage steady
growth. The polypropylene shows no further growth after day 22. The polyurethane
and polylactic acid produce better growth and the Vascutek polyester samples
encourage a steady increase in growth rate indicating that it is the best standard
material for supporting tissue growth. This is not a surprising finding as this material
is commercially available but also because it is the only material with a knitted
structure.

133
Argon Treated Samples Data
0
5
10
15
20
25
30
0 8 15 22 29
Day
AverageGrowth(mm)
Polypropylene Argon treated (Nanotech) Polyurethane Argon treated (Nanotech)
Vascutek Polyester Argon treated (Nanotech) Polylactic Acid Argon treated (Nanotech)
Polycaprolactone Argon treated (Nanotech) Solanyl Flexibilitis Argon treated (Nanotech)
Fig. 4.76 - Graph of average growth of fibroblasts on the argon plasma treated samples.

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Fig 4.76 shows the argon treated samples. In relation to fig 4.75, it shows that the
argon treatment of polyurethane and polypropylene were a success, as they supported
greater growth than the best control material. It also shows how the argon treatment
reduced growth compared to the controls, with the exception of polycaprolactone,
where the average growth is approximately the same.

135
Ammonia treated Samples Data
0
5
10
15
20
25
30
0 8 15 22 29
Day
AverageGrowth(mm)
Polypropylene Ammonia treated (Nanotech)
Polyurethane Ammonia treated (Nanotech)
Vascutek Polyester Ammonia treated (Nanotech)
Polylactic Acid Ammonia treated (Nanotech)
Polycaprolactone Ammonia treated (Nanotech)
Solanyl Flexibilitis Ammonia treated (Nanotech)
Fig. 4.77 - Graph of average growth of fibroblasts on the ammonia plasma treated samples.

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Fig 4.77 primarily shows that the ammonia plasma was the best treatment for the
polypropylene and polyurethane. The average growth supported on the
polypropylene is almost as much as the average growth on the control Vascutek
polyester. This is double the average growth measured on the polypropylene control.
The ammonia treated polyurethane supports the highest average cell growth of all the
samples tested, improving the average growth by around 15mm over the
polyurethane control, and almost 10mm over the Vascutek polyester control.

137
Sample
Average
growth After
29 Days (mm)
Standard
Deviation
Polyurethane Ammonia treated 25.3 8.8
Polyurethane Argon treated 18.0 14.5
Vascutek Polyester 15.9 12.7
Polypropylene Ammonia treated 14.9 14.4
Solanyl Flexibilitis Ammonia and Chitosan treated 12.3 14.9
Polypropylene Argon treated 12.0 14.4
Polyurethane Oxygen treated in (Europlasma) 11.3 14.5
Polyurethane 10.3 12.9
Solanyl Flexibilitis extruded with 2% Chitosan powder 10.0 15.5
Poly-l-lactic acid 10.0 15.0
Polypropylene Chitosan treated 9.6 13.0
Polyurethane with Chitosan powder prior to argon treatment 8.4 13.0
Poly-ε-caprolactone Ammonia treated 8.3 13.6
Solanyl Flexibilitis Argon and Chitosan treated 7.6 11.9
Polyurethane Argon treated in (Europlasma) 7.3 13.0
Polypropylene 6.7 13.2
Solanyl Flexibilitis 6.4 12.0
Polypropylene Ammonia and Chitosan treated 6.2 11.0
Vascutek Polyester Ammonia treated 5.9 9.8
Solanyl Flexibilitis Ammonia treated 5.0 12.2
Poly-l-lactic acid Argon treated 5.0 12.2
Poly-l-lactic acid Ammonia treated 5.0 12.2
Polypropylene Argon and Chitosan treated 3.6 9.9
Solanyl Flexibilitis Chitosan treated 3.3 6.3
Solanyl Flexibilitis Argon treated 3.2 7.8
Vascutek Polyester Argon treated 2.0 3.2
Poly-ε-caprolactone Argon treated 1.1 3.0
Poly-ε-caprolactone 0.7 2.0
Table 4.78 - Average cell growth of samples after 29 days arranged in descending order
As can be seen by the tissue culture data, ammonia treated polypropylene is the
fourth best material, with untreated Vascutek polyester performing marginally better.

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The top two performers are the plasma treated polyurethanes. The ammonia treated
polyurethane comes out top, with an average growth figure 7mm greater than the
argon treated polyurethane.
When looking the data, one can see a trend where the ammonia plasma treatment out
performs argon treatment, with the exception of the PLA where they both perform the
same. This was as hypothesised, as the ammonia will present nitrogen and hydrogen
on the surface, much like proteins.
Samples were examined on day 30 using a Leica confocal microscope. Cells were
stained using an Invitrogen live/ dead cytotoxicity test (containing ethidium
homodimer and calcein AM cellular stains). These images were to be used primarily
as conformation of optical microscope measurements and to gain an insight into the
quality of cell growth. The fluorescence images were not consistent across the
selection of samples and some of them indicated that the cells had been ripped off
during the preparation of the samples for fluorescence microscopy.
Fig 4.79 Polypropylene 2 sample from 24/09/2005 illustrating an abrupt termination of cells due to the
cells ripping off the sample during sample preparation.
The data from the fluorescence imaging can be found in appendix C.2.

139
Chapter 5 – Discussion
This project aimed to;
review the advantages and disadvantages of materials used in soft tissue
repair and to review potentially alternative materials.
investigate the reported benefits of using chitosan in relation to medical
device applications.
investigate in depth a limited selection of alternative materials.
investigate the value of gas plasma treatment on the ability of these materials
to support tissue growth in vitro.
This research set out to investigate the current state of biomaterials used for soft
tissue repair. Current mesh prostheses made of polypropylene (PP), polyethylene
terephthalate (PET) or polytetrafluoroethylene (PTFE) have proven themselves
invaluable for the repair of soft tissue defects but they can often lead to complications
such as restriction of the abdominal wall mobility, intra-abdominal adhesions with
erosion of adjacent organs (or consecutive fistula formation) and inflammatory
foreign body reaction where the prosthesis is embedded into a fibrous scar plate
causing shrinkage of the mesh area (~40%). They can also provide a surface which
bacteria can colonise causing persistent infections that can sometimes only be cleared
by the removal of the prosthesis. Polypropylene meshes, which have been in use
since 1962, are still the most common material for hernia repair (Morris-Stiff and
Hughes, 1998) due to their perceived long term maintenance of tensile strength and
low tissue reactivity although in a study conducted in 1998 a failure rate of 10% was
recorded (4 out of 40 patients in a single unit) and therefore it was concluded that the
complications associated with polypropylene meshes are under reported. While these
complications are rarely life threatening, they highlight the need for further research
into these devices. Degradable biomaterials used (e.g. polyglycolic acid) can also
cause complications such as the recurrence of the hernia due to failure of the device
and inflammatory reactions caused by rapid degradation of the material.

140
It is hypothesised that complications associated with medical devices are associated
with an inability to assimilate with the host tissue, therefore by improving host tissue
regeneration, complications will be reduced.
To address the issue of prosthesis related infections, there needs to be a material that
could inhibit bacterial growth that was suitable for use within the human body.
Chitosan (& chitin) have been reported to inhibit bacterial growth and fungal growth
whilst enhancing human tissue growth (and many other attributes). It was therefore
hypothesised that by using chitosan as a coating or incorporating chitosan into a
biomaterial, tissue regeneration would be enhanced and prosthesis related infections
would be reduced.
From these two hypothesises two lines of research became evident.
To examine the efficacy of chitosan as a bacteriostat.
To examine a range of biomaterials for their ability to encourage fibroblast
growth and see if fibroblast growth could be improved by modifying the
surface of the biomaterial.
As a result of the reports of chitosan as a bacteriostat in the literature, this study was
devised to determine the efficacy of chitosan against a common and prolific
opportunistic pathogen. Staphylococcus epidermis was also included in the early
study but MRSA was the most relevant candidate for testing the efficacy of chitosan.
MRSA is gram positive and resistant to certain antibiotics. It is also one of the most
problematic infections to clear when acquired in a hospital environment and can be
life threatening and therefore it is the most interesting bacterium to test the
bacteriostatic claims reported for chitosan.
The means of testing the bacteriostatic effect of chitosan followed two distinct paths.
One was to examine the ability of chitosan to inhibit growth on a surface and the

141
other was designed to determine a quantifiable effect on MRSA (and to a lesser
extent S. epidermis) in broth culture by varying the quantity of chitosan in the broth.
The methods employed were of an evolutionary nature in that deficiencies in one
method were addressed in the next.
Vs MRSA 9551 and Staphylococcus epidermis.
Method 1 was the beginning of the method development where initial investigations
into both aspects of this study started producing preliminary data but the methods
diverged as the requirements of each method were developed. From there, method 3
examined the inhibition of growth around chitosan film or materials coated in
chitosan film while methods 2 and 4 examined the growth of MRSA in solutions
containing chitosan.
The data for Method 2 suggested a bacteriostatic relationship between chitosan and
MRSA. This experiment illustrates the bacteriostatic effect of chitosan against
MRSA (although the data is less clear for S.epidermis). This experiment shows the
first four hours of bacterial growth in the presence of chitosan in detail, although after
the first 4 hours, there is a 22 hour gap between measurements. The data clearly
shows the lag phase and logarithmic growth phase but not the stationary phase or
death phase. This experiment may have benefited from hourly measurements over
the 26 hour period, but with the resources available, this was not possible.
Method 3 was designed to expand on the early investigation in method 1 of chitosan
film. The cotton cloth coated with the chitosan solution demonstrated an observable
zone of inhibition. The polypropylene coated with chitosan solution was not so
effective. The result for polypropylene coated in chitosan against MRSA produced a

142
partial zone of inhibition. When tested with S. epidermis, no zone of inhibition was
observed.
Method 3 examined the effect of a chitosan coated material on an inoculated agar
plate. Although the chitosan coating was not pH neutralised, the results indicate a
zone of inhibition around most of the treated samples. These methods indicate that
chitosan coating may be a viable means of producing materials which inhibit
bacterial growth with the added advantage that it is easy to apply. This application
could be used in both medical devices and on textiles used within the hospital
environment although further research to optimise this coating is recommended.
Method 4b was the final evolution of methods 2 and 4. This method yielded a large
quantity of data on the efficacy of chitosan in suspension Vs MRSA. These results
suggest that the bacteriostatic effect varies with the quantity of chitosan presented to
the bacteria but it does not appear, from this data, to be a direct relationship.
The studies in method 4b suggest that there is a large variation in the efficacy of
chitosan powder as a bacteriostat against MRSA. There are two potential
explanations for the results in the 06/10/03 and 19/01/05 studies. First, the efficacy
of chitosan may be related to the concentration of bacteria. Second, the initial
measurements were taken after 24 hours, and it is possible that the control may have
already finished the exponential phase and stationary phase and started on the death
phase before the first measurements were taken. In future, it would be interesting to
measure the samples every 4 hours in the first 24 hours to see if the extra resolution
proves this hypothesis to be the case.
Examination of Chitosan as a Bacteriostat Summary
The chitosan study set out to examine the efficacy of chitosan as a bacteriostat. To
that end, it has been observed that chitosan does have a bacteriostatic effect. In film

143
form the chitosan produced a clear zone of inhibition against MRSA and in
suspension it caused inhibition of growth for the majority of samples tested. In terms
of producing quantitative data, the methods developed towards the end of the study
could still not elucidate a relationship between the quantity of chitosan suspension
and the effect on MRSA.
This study indicates that when chitosan is applied to a material as a film, it has a
bacteriostatic effect against MRSA and when it is used as a suspension it can produce
a bacteriostatic effect although it is a variable one. As the main application of
chitosan in this project is as a coating, this data suggests it will impart bacteriostatic
properties to the material it is applied to.
It has been reported that among other properties that chitosan is an effective
bacteriostat, but this study indicated the need for further studies to clarify the
susceptibility of a wide range of pathogens to chitosan. Chitosan is a difficult
material to study in these experiments as it is not readily soluble except in acidic
solutions and is therefore difficult to study a neutral environment. Chitosan has an
effect on bacteria as reported in the literature, but the results of this study found that
the results varied greatly depending on how the chitosan was presented and the
concentration of the inoculum.
There are many sources of chitosan including crustacea and fungi and many different
degrees of deacetylation (chitosan is stated as being greater than 70% deacetylated).
There are also many modifications of chitosan, including water soluble chitosan but
this study used the same chitosan throughout this study and the tissue culture study
(high molecular weight, high purity squid chitosan). Chitosan derived from different
organisms and different deacetylation techniques may demonstrate different levels of
bacterial growth inhibition. With all of the reported benefits of using chitosan,
research will continue and further applications will be developed. Until experimental
analysis reveals a better chitosan for inhibiting bacterial growth, the high purity squid
chitosan will be useful as a bacteriostatic coating for medical prostheses to inhibit
post-operative infections.

144
The biomaterial samples used in this research were produced using a variety of
production methods. This variety of production methods introduced differences in
morphology of the biomaterials. This factor limits the conclusions that can be drawn
between biopolymers used in this research although with these differences noted,
careful comparisons may be made. The two samples with the greatest difference in
surface morphology were the PLA and the polyester. The polyester and PLA were
included because they were so different from the extruded samples and because they
were expected to outperform the extruded samples due to increased cell adhesion.
The DSC analysis provided the melting points of the polymers. Due to the low
melting point of the poly-ε-caprolactone, the samples could not be sterilised using an
autoclave (121ºC for 15 minutes) so ethylene gas sterilisation was used instead.
5.2.2 SEM Analysis
The SEM analysis of the samples showed a degree of variation in the surfaces of the
different polymers. Some of the polymers had a very smooth surface (Solanyl and
polyurethane), some had minor grooving from the extrusion process (polypropylene
and poly-ε-caprolactone) and two materials had very different surfaces (polyester and
polylactic acid). These differences affect the growth of cells on the materials
therefore conclusions from the tissue culture study should only be made with these
differences in mind. Plasma treatment made no observable change to the surfaces of
the biomaterials, but as seen in fig. 4.35 and 4.36, plasma treatment has a great effect
on the hydrophilicity/ hydrophobicity of a polymer.
Looking at the Solanyl containing 2% chitosan (w/w), particles of chitosan powder
can be seen on the surface of the sample. It appears from fig. 4.52 that the chitosan
powder was not evenly distributed through the Solanyl. This should not be of great

145
importance for this study, but should be addressed if this material is to be developed
further.
The PLA images illustrate the porous nature of the film cast using DCM evaporation.
The pore sizes were measured and compared to the area of the images used in the
pore size measurements.
Average area of pores for the three images (µm2
) 8.90
Average percentage of pores for the three images 47.22%
Table 5.1 summary of the pore size measurements.
The PLA shows a highly porous structure although the average pore size and
percentage porosity are both lower than the optimal sizes/ percentages quoted by Oh
(186–200 µm pore size (Oh et al., 2007)) and Minns (10-50 µm pore size overall
porosity of 85-90% (Minns, 1999)).

146
5.4 Tissue culture study
This project aimed to determine which biomaterial or surface treatment yielded the
greatest fibroblast growth and to test a selection of surface treatments to see if they
can be used to improve current biomaterials. The theory behind this was that by
testing biomaterials for their ability to support fibroblast growth, a logical foundation
is created for the design and optimisation of soft tissue repair prostheses. This was
achieved by inoculating the biomaterial samples at one end with a 20µl drop of
MRC-5 cells and measuring their growth along the 30mm strip of material. As the
samples could not all be tested at once, the samples were tested in mixed groups of
three treatments of three materials in triplicate and each sample was tested 9 times.
Therefore 243 samples were tested in total (27 different samples tested 9 times each),
each tested over a 29 day period.
Prior to testing the biomaterials with MRC-5 cells, the materials were washed in 70%
ethanol, ethylene gas sterilised and examined using SEM to observe any differences
between the materials and to observe any differences in the surfaces as a result of
modification. When examining the data in chapter 4.1, the surface topography can be
seen. By examining these SEM images, it can be seen that the surface topography is
different for each material. The difference is modest between polypropylene, Tuftane
and Solanyl and polycaprolactone. With the PLA and polyester samples the surface
topography is quite different. This would have an effect on how well the MRC-5
cells grew on the substrate therefore comparisons should be made only with these
differences in mind. No difference was observed in the SEM images between the
plasma treated samples and the standard materials, although the Solanyl containing
2% chitosan had slight bumps on the surface due to the incorporation of the chitosan
powder.
The tissue culture study performed well as a comparison between the six different
materials and produced useful data on how the plasma treatment affected tissue
growth on the different materials. In addition to evaluating plasma treatment,

147
selections of chitosan based treatments were tested but these were primarily
explorative in their nature (to examine the viability of such treatments).
Polypropylene, the most commonly used biomaterial in surgery, made a useful
reference point for studying biomaterials. It was also the starting point for examining
modifications that can enhance biocompatibility. Three treatments were shown to
improve fibroblast growth on polypropylene (argon plasma, ammonia plasma and
chitosan coating with no prior plasma treatment). Argon and chitosan treated
polypropylene and ammonia and chitosan treated polypropylene both demonstrate
inferior tissue growth compared to the control after 29 days, although growth is faster
over the first 15 days, therefore any benefit derived from these treatments is
transitory.
The polyurethane data indicates that three treatments produced enhanced growth over
the unmodified material, the ammonia treatment, the argon treatment and the oxygen
treatment, although all of the treatments show enhanced growth over the first 15
days. This reflects what is seen for the polypropylene data. The two samples of
polyurethane treated with argon plasma on different plasma treatment equipment
produce radically different results. The polyurethane treated with argon on the
Europlasma equipment and the polyurethane treated with argon on the Nanotech
equipment were used to illustrate that although the equipment was different, the
effect was the same but as can be seen by the data, they produced quite different
results. This was quite unexpected and will need to be investigated further. Shortly
after treating the polyurethane with argon and oxygen on the Europlasma equipment,
the plasma equipment became faulty, so other samples treated on the Europlasma
equipment were discarded and the plasma treatment was performed in the older
Nanotech equipment. Although the Europlasma argon and ammonia treatments
performed poorly compared to the equivalent Nanotech samples, the oxygen plasma
treated sample showed improved growth over the standard material. This suggests
that if the sample was treated with oxygen plasma on the Nanotech equipment, the
oxygen plasma may have performed very well. Oxygen plasma could not be

148
produced on the Nanotech equipment as there was no oxygen gas available at the
time of plasma treatment.
The Vascutek polyester data clearly shows that argon and ammonia plasma treatment
does not improve fibroblast growth. What is interesting about the data is that the
ammonia plasma still outperforms the argon plasma.
The PLA was predicted to perform well, considering it had a porous surface. It
performed better than untreated polypropylene, untreated poly-ε-caprolactone and
untreated Solanyl. The untreated PLA does not promote as much growth as the
untreated Vascutek polyester, although it does outperform the untreated
polypropylene. None of the plasma treatments improved growth on the PLA.
The untreated poly-ε-caprolactone performed poorly. Argon treatment had little
effect compared to the control. Ammonia treatment promoted approximately thirteen
times the growth of the poly-ε-caprolactone control. This is the greatest
improvement over the control recorded. In addition, ammonia treatment of poly-ε-
caprolactone promoted growth slightly greater than the polypropylene control.
The data for Solanyl indicated that the argon, ammonia and chitosan treatment
(without plasma pre-treatment) produce products that are inferior to the control
material. In contrast to the polypropylene samples, the argon and ammonia pre-
treated Solanyl coated in chitosan both perform better than the control material, with
the ammonia and chitosan treated Solanyl promoting twice the growth of the control.
The Solanyl containing 2% chitosan performed well; therefore this method of
incorporating chitosan in degradable biomaterials requires further study to determine
the best ratio of chitosan to polymer and to examine which other degradable
biopolymers can benefit from the addition of chitosan.
The data for the control samples reveals that three of the tested materials support
greater fibroblast growth than polypropylene (Vascutek polyester, polyurethane and

149
poly lactic acid) but it is clear that plasma treatment can be used to produce a better
surface for fibroblast growth. With the plasma treatment, the samples were
considered a success if they supported cell growth greater than the untreated material.
They were a greater success if they encouraged cell growth beyond the untreated
material and produced growth greater than polypropylene (the benchmark).

150
Average Growth Of Permanent Materials In Relation To Polypropylene
0
5
10
15
20
25
30P
olyurethane
A
m
m
onia
treated
P
olyurethane
A
rgon
treated
V
ascutek
P
olyester
P
olypropylene
A
m
m
onia
treated
P
olypropylene
A
rgon
treated
P
olyurethane
O
xygen
treated
in
(E
uroplasm
a)
P
olyurethane
P
olypropylene
C
hitosan
treated
P
olyurethane
w
ith
C
hitosan
pow
derpriorto
argon
treatm
ent
P
olyurethane
A
rgon
treated
in
(E
uroplasm
a)
P
olypropylene
P
olypropylene
A
m
m
onia
and
C
hitosan
treated
V
ascutek
P
olyesterA
m
m
onia
treated
P
olypropylene
A
rgon
and
C
hitosan
treated
V
ascutek
P
olyesterA
rgon
treated
AverageGrowthOver29Days(mm)
Graph 5.2 - Summary of fibroblast growth on permanent materials. Polypropylene is shown in black as it is the control. The untreated materials are shown in green.
The yellow bars are samples where the treatment improved fibroblast growth over the untreated material. Blue bars are samples where the surface treatments reduced
fibroblast growth compared to the untreated samples.

151
Graph 5.3 - Summary of fibroblast growth on resorbable materials. Polypropylene is shown in black as it is the control. The untreated materials are shown in green.
The yellow bars are samples where the treatment improved fibroblast growth over the untreated material. Blue bars are samples where the surface treatments reduced
fibroblast growth compared to the untreated samples.

152
The argon and ammonia treated polyurethane were clearly the most successful
treatments, but growth on ammonia plasma treated polypropylene was more than
twice the growth of standard polypropylene. Ammonia and chitosan treated Solanyl
produced the best growth on a degradable material, although there is only 2.3mm
difference in average growth between this and Solanyl containing 2% chitosan which
would make both materials viable options for degradable prostheses. The Ammonia
and chitosan treated Solanyl would also impart a bacteriostatic effect due to the
chitosan film therefore inhibiting prosthesis related infections. The Solanyl
containing 2% chitosan may also impart this protection, although further study would
be required to prove this. The oxygen plasma treated polyurethane (Europlasma)
does not perform as well as the argon plasma treated polyurethane (Nanotech) or the
ammonia plasma treated polyurethane (Nanotech, but when the poor performance of
the Europlasma treated argon sample is taken into account (an average growth of 10.7
difference between the Europlasma and Nanotech argon treated samples) it can be
suggested that oxygen plasma could perform better if produced on the Nanotech
equipment.

153
Conclusions
It is clear from the data that chitosan is affecting the growth of MRSA, with chitosan
film producing observable zones of inhibition against MRSA, although there is not a
direct relationship between the quantity of chitosan powder in solution and the effect
on growth. This project achieved its aim to examine the bacteriostatic effect but
further work will be required to find a direct relationship. Medical applications may
include implanted devices and textiles used in the hospital environment (e.g. soft
furnishings, nurses‟ uniforms and doctors‟ coats).
The tissue culture study completed the objective of comparing a range of biomaterials
and surface treatments in a consistent and unbiased manner, producing interesting
results. It is also clear from the data that gas plasma treatment can improve fibroblast
growth on some of the biomaterials.
Ammonia and chitosan treated Solanyl and Solanyl containing 2% chitosan proved to
be the best degradable biomaterials tested. These materials should be tested in vivo
for their ability to repair soft tissue defects. The ammonia and chitosan treated
Solanyl may also be tested as repair prostheses for non-sterile tissue repair, perhaps
as a suture material as it should inhibit infections associated with such wound
closures. If the Solanyl containing 2% chitosan proves to be effective as a
bacteriostat, it too may be suitable for this application.
The polyurethane sample treated with ammonia plasma appears to be an interesting
candidate for further study as the only material with 100% survival of cells in culture
and the best growth measurements over the 29 days. This material requires further
study to determine its efficacy in vivo and to develop the best design to support the
load of abdominal and pelvic floor contents. The next stage for this material is to
design mesh prosthesis for animal trials, so the efficacy can be determined in vivo.

154
Other materials performed well, with ammonia treated polypropylene yielding a great
improvement in cell growth over untreated polypropylene. As polypropylene is
already a widely used material for soft tissue repair, it would be feasible to produce a
new polypropylene prosthesis with ammonia treatment. This would provide a
prosthesis with the handling and mechanical characteristics that surgeons are familiar
with but with the benefit of improved incorporation within the patient.
As hypothesised, the ammonia plasma consistently performs better than the argon
plasma (with the exception of polylactic acid, where it performs the same as argon).
Further work will be needed to determine if the ammonia does deposit NH groups as
hypothesised.
Further study is required into the use of gas plasma, considering the difference in
results from polyurethane samples treated with the same gas using different
equipment. With further testing and optimisation, the Europlasma equipment could
produce results equivalent to the Nanotech equipment by altering the gap between the
charged plates. The Europlasma has advantages over the Nanotech equipment in that
it is computer controlled and should therefore be able to produce more consistent
results.
Recommendations for further study of gas plasma include;
Examining a greater range of gases for their ability to enhance cell
proliferation on biomaterials.
Examine the efficacy of gas plasma treatments for a broader range of
biomaterials gases for their ability to enhance cell proliferation on
biomaterials.
Examining different parameters within the plasma chamber to optimise gas
plasma treatments for enhancing cell proliferation on biomaterials.
Examine the efficacy of atmospheric plasma treatment as an alternative to low
pressure plasma treatment for coating biomaterials

155
Recommendations for further developing the methods used to examine chitosan as a
bacteriostat include;
Measuring the growth of the bacteria in the presence of chitosan (in
suspension) at one to two hour periods during the first 24 to 48 hours.
Testing the bacteriostatic effect of chitosan against a greater range of
pathogens.
An investigation into the molecular basis behind the bacteriostatic effect so it
can be optimised and then verified.
Testing the efficacy of chitosan derived from different sources, with different
molecular weights to determine which chitosan has the greatest effect on
bacterial growth.
Recommendations for developing optimal soft tissue repair prosthesis include an in
vivo study, where the response of the immune system can be taken into account. This
would include testing the selected biomaterials in a range of morphologies as well as
a range of treatments as the morphology of the implant will have a great effect on the
response of host tissue to the medical device. Novel production techniques may
allow the production of materials with biomimetic structures that may enhance
biocompatibility.
In conclusion, it is the recommendation of this study that the optimal material tested
in this project was ammonia plasma treated polyurethane. The next stage of
development would be to develop prototype prosthesis and perform in vivo testing to
gather data on tissue regeneration and immune response.

156
Appendix
Items in the appendix consist of supporting material of considerable length or
additional data that would interrupt the flow of the thesis.
A. Examination of Chitosan as a Bacteriostat
A.1 Details of media used for the examination of chitosan as a bacteriostat

157
Nutrient agar (NA)
pH 7.4 approx
Oxoid code cm3
lot 01036886
2.8g per 100mls distilled water
formula [per litre]
lab-lemco powder [Oxoid L29] 1g
Yeast extract [Oxoid L20] 2g
Peptone [Oxoid L37] 5g
Sodium chloride 5g
Agar No 3 [Oxoid L13] 15g
Nutrient broth (NB)
pH 7.5 +/- 0.2
Oxoid code cm67
lot 10559702
2.5g per 100mls distilled water
formula [per litre]
lab-lemco powder [Oxoid L29] 10g
Peptone [Oxoid L37] 10g
Sodium chloride 5g
Plate Count Agar (PCA)
A medium for the enumeration of viable organisms on milk and dairy
products
pH 7.0 approx
Oxoid code CM325
formula [per litre]
Yeast extract [Oxoid L21] 2.5g
Tryptone [Oxoid L42] 5g
Dextrose 1g
Agar No1 [Oxoid L11] 9g
Table A.1

158
A.2 Unedited graph from Method 2c
Chitosan Vs S.epidermis & MRSA 9551
-0.100
0.000
0.100
0.200
0.300
0.400
0.500
0.600
00:20.0 01:15.0 02:15.0 04:00.0 26:10.0
Time
AD550
Reference (Nutrient Broth)
MRSA 9551 Control
MRSA 9551 1
MRSA 9551 0.1
MRSA 9551 0.01
S.epidermis Control
S.epidermis 1
S.epidermis 0.1
S.epidermis 0.01
Fig. A.2
Unedited graph for method 2c including 1g chitosan suspension reading.

159
A.3 Data from Method 4b
Method 4b
07/10/03 Data
Table A.3
ChitosanandMRSAdata
06/10/03
initialinoculum
cellcountmean
48.2
cellsperml
1.93x10-8
cellsin200µlinoculum
3.86 x10-7
Resultsfrom07/10/2003
cellcountsfromplatecount
agars(bydilution) cellspermlforthesevalues(bydilution)
Averageofcellsper
mlvalues
10-6
10-7
10-8
10-6
10-7
10-8
c1
c2 962 9.62x10-10
c3 44092 4.41x10-11
2.69x10-11
0.02-1 3504 3.50x10-11
0.02-2 593 290 5.93 x10-9
2.90x10-10
0.02-3 457 4.57x10-10
1.08x10-11
0.04-1 248 2.48x10-10
0.04-2
0.04-3 1572 1.57x10-11
9.10x10-10
0.06-1 412 4.12x10-10

160
0.06-2 29 2.90x10-9
0.06-3
2.21x10-10
0.08-1 352 3.52x10-10
0.08-2 351 3.51x10-10
0.08-3 649 6.49x10-10
4.51x10-10
0.1-1
0.1-2 3044 3.04x10-11
0.1-3
3.04x10-11
Averageofcellsper
mlvalues
10-6
10-7
10-8
10-6
10-7
10-8
c1 1016 1.02x10-11
c2 407 4.07x10-10
c3 3992 3.99x10-11
1.81x10-11
0.02-1 3364 3.36x10-11
0.02-2 1616 1.62x10-11
0.02-3 2216 2.22x10-11
2.40x10-11
0.04-1 305 3.05x10-10
0.04-2 2276 2.28x10-11
0.04-3 105 1.05x10-10

161
8.95x10-10
0.06-1 1920 1.92x10-11
0.06-2 1800 1.80x10-11
0.06-3 1788 1.79x10-11
1.84x10-11
0.08-1 958 9.58x10-10
0.08-2 788 7.88x10-10
0.08-3 3200 3.20x10-11
1.65x10-11
0.1-1 2340 2.34x10-11
0.1-2 6636 6.64x10-11
0.1-3 2416 2.42x10-11
3.80x10-11
Averageofcellsper
mlvalues
10-6
10-7
10-8
10-6
10-7
10-8
c1 2610 2.61x10-11
c2 1990 1.99x10-11
c3 563 5.63x10-10
1.72x10-11
0.02-1 1610 1.61x10-11
0.02-2 1618 1.62x10-11
0.02-3 1380 1.38x10-11
1.54x10-11
0.04-1 842 8.42x10-10

162
0.04-2 1262 1.26x10-11
0.04-3 1484 1.48x10-11
1.20x10-11
0.06-1 1996 2.00x10-11
0.06-2 2050 2.05x10-11
0.06-3 1760 1.76x10-11
1.94x10-11
0.08-1 3094 3.09x10-11
0.08-2 2766 2.77x10-11
0.08-3 1182 1.18x10-11
2.35x10-11
0.1-1 2644 2.64x10-11
0.1-2 0
0.1-3 2228 2.23x10-11
1.62x10-11
20/10/03 Data
Table A.4
Chitosan and MRSA data 20/10/03
initial inoculum
cell count mean 20.6
cells per ml
8.24 x10-7
cells in 200µl inoculum
1.65 x10-7
Each culture for this experimental
run was plated out two times (a & b)
Results from 21/10/03
cell counts from plate
count agars (by
cells per ml for these values (by
dilution)
Average of cells
per ml values

163
dilution)
10-7
10-8
10-9
10-7
10-8
10-9
c1a 1 1 1.00 x10-7
1.00 x10-8
c1b 3 3.00 x10-8
c2a 85 11 8.50 x10-8
1.10 x10-9
c2b 65 6 1 6.50 x10-8
6.00 x10-8
1.00 x10-9
c3a 60 11 3 6.00 x10-8
1.10 x10-9
3.00 x10-9
c3b 82 10 8.20 x10-8
1.00 x10-9
8.56 x10-8
0.002-1a 32 1 3.20 x10-8
1.00 x10-8
0.002-1b 51 3 5.10 x10-8
3.00 x10-8
0.002-2a 64 4 1 6.40 x10-8
4.00 x10-8
1.00 x10-9
0.002-2b 68 2 1 6.80 x10-8
2.00 x10-8
1.00 x10-9
0.002-3a 12 2 1.20 x10-9
2.00 x10-9
0.002-3b
6.96 x10-8
0.004-1a 55 5 2 5.50 x10-8
5.00 x10-8
2.00 x10-9
0.004-1b 93 6 1 9.30 x10-8
6.00 x10-8
1.00 x10-9
0.004-2a 104 10 1.04 x10-9
1.00 x10-9
0.004-2b 142 14 2 1.42 x10-9
1.40 x10-9
2.00 x10-9
0.004-3a 130 8 1.30 x10-9
8.00 x10-8
0.004-3b 118 3 1.18 x10-9
3.00 x10-8
1.07 x10-9
0.006-1a 131 21 1.31 x10-9
2.10 x10-9
0.006-1b 92 16 9.20 x10-8
1.60 x10-9
0.006-2a 103 19 1.03 x10-9
1.90 x10-9
0.006-2b 95 11 9.50 x10-8
1.10 x10-9

164
0.006-3a 71 7.10 x10-8
0.006-3b 78 7.80 x10-8
1.24 x10-9
0.008-1a 46 65 4.60 x10-8
6.50 x10-9
0.008-1b 11 0.00 1.10 x10-9
0.008-2a 16 16 1.60 x10-8
1.60 x10-9
0.008-2b 28 9 2.80 x10-8
9.00 x10-8
0.008-3a 198 12 3 1.98 x10-9
1.20 x10-9
3.00 x10-9
0.008-3b 180 9 2 1.80 x10-9
9.00 x10-8
2.00 x10-9
1.56 x10-9
0.01-1a 0 10 1.00 x10-9
0.01-1b 42 11 4.20 x10-8
1.10 x10-9
0.01-2a 74 14 7.40 x10-8
1.40 x10-9
0.01-2b 62 8 6.20 x10-8
8.00 x10-8
0.01-3a 1 9 1.00 x10-7
9.00 x10-8
0.01-3b 0 0
7.77 x10-8
count agars (by
dilution)
dilution)
Average of cells
per ml values
10-7
10-8
10-9
10-7
10-8
10-9
c1a 67 7 1 6.70 x10-8
7.00 x10-8
1.00 x10-9
c1b 18 9 1.80 x10-8
9.00 x10-8
c2a 6 6 6.00 x10-7
6.00 x10-8
c2b 30 1 3.00 x10-8
1.00 x10-9
c3a 1 1.00 x10-7
c3b 24 2.40 x10-8
5.15 x10-8

165
0.002-1a 5 15 5.00 x10-8
1.50 x10-10
0.002-1b 10 1.00 x10-8
0.002-2a 362 3.62 x10-9
0.002-2b 248 2.48 x10-9
0.002-3a 179 1.79 x10-9
0.002-3b 156 1.56 x10-9
3.58 x10-9
0.004-1a
0.004-1b
0.004-2a
0.004-2b
0.004-3a
0.004-3b
0.006-1a
0.006-1b
0.006-2a 39 3.90 x10-8
0.006-2b 38 3.80 x10-8
0.006-3a 59 5.90 x10-8
0.006-3b 48 4.80 x10-8
4.60 x10-8
0.008-1a
0.008-1b
0.008-2a
0.008-2b

166
0.008-3a
0.008-3b
0.01-1a
0.01-1b
0.01-2a
0.01-2b
0.01-3a
0.01-3b
count agars (by
dilution)
dilution)
Average of cells
per ml values
10-6
10 -7
10 -8
10-6
10 -7
10 -8
c1a 70 10 7.00 x10-7
1.00 x10-8
c1b 49 3 2 4.90 x10-7
3.00 x10-7
2.00 x10-8
c2a 148 11 2 1.48 x10-8
1.10 x10-8
2.00 x10-8
c2b 199 16 3 1.99 x10-8
1.60 x10-8
3.00 x10-8
c3a 88 6 8.80 x10-7
6.00 x10-7
c3b 77 3 1 7.70 x10-7
3.00 x10-7
1.00 x10-8
1.20 x10-8
0.002-1a 95 6 9.50 x10-7
6.00 x10-7
0.002-1b 119 6 1.19 x10-8
6.00 x10-7
0.002-2a 108 8 1.08 x10-8
8.00 x10-7
0.002-2b 187 5 1 1.87 x10-8
5.00 x10-7
1.00 x10-8
0.002-3a 35 2 3.50 x10-7
2.00 x10-7
0.002-3b 49 5 4.90 x10-7
5.00 x10-7
7.79 x10-7

167
0.004-1a 82 2 8.20 x10-7
2.00 x10-7
0.004-1b 149 1 1.49 x10-8
1.00 x10-7
0.004-2a 215 14 2.15 x10-8
1.40 x10-8
0.004-2b 192 12 3 1.92 x10-8
1.20 x10-8
3.00 x10-8
0.004-3a 441 30 1 4.41 x10-8
3.00 x10-8
1.00 x10-8
0.004-3b 366 35 3.66 x10-8
3.50 x10-8
1.99 x10-8
0.006-1a 49 9 4.90 x10-7
9.00 x10-7
0.006-1b 42 11 4.20 x10-7
1.10 x10-8
0.006-2a 393 56 3 3.93 x10-8
5.60 x10-8
3.00 x10-8
0.006-2b 457 67 2 4.57 x10-8
6.70 x10-8
2.00 x10-8
0.006-3a 102 8 3 1.02 x10-8
8.00 x10-7
3.00 x10-8
0.006-3b 94 1 3 9.40 x10-7
1.00 x10-7
3.00 x10-8
2.35 x10-8
0.008-1a 207 12 1 2.07 x10-8
1.20 x10-8
1.00 x10-8
0.008-1b 234 16 2.34 x10-8
1.60 x10-8
0.008-2a 829 106 4 8.29 x10-8
1.06 x10-9
4.00 x10-8
0.008-2b 816 75 13 8.16 x10-8
7.50 x10-8
1.30 x10-9
0.008-3a 39 1 3.90 x10-7
1.00 x10-7
0.008-3b 118 4 1.18 x10-8
4.00 x10-7
4.12 x10-8
0.01-1a 306 1 2 3.06 x10-8
1.00 x10-7
2.00 x10-8
0.01-1b 298 13 2 2.98 x10-8
1.30 x10-8
2.00 x10-8
0.01-2a 65 31 3 6.50 x10-7
3.10 x10-8
3.00 x10-8
0.01-2b 177 24 3 1.77 x10-8
2.40 x10-8
3.00 x10-8

168
0.01-3a 110 48 5 1.10 x10-8
4.80 x10-8
5.00 x10-8
0.01-3b 98 40 9.80 x10-7
4.00 x10-8
2.43 x10-8
Large portions of the data for this study were uncountable; therefore there is no graph
for these data.
04/11/03 Data
Table A.5
initial inoculum
cells per ml
4.88 x10-7
9.76 x10-6
Each culture for this experimental
run was plated out two times (a & b)
count agars (by
dilution)
dilution)
Average of cells
per ml values
10-7
10-8
10-9
10-7
10-8
10-9
c1a 10 11 1 1.00 x10-8
1.10 x10-9
1.00 x10-9
c1b 50 35 5.00 x10-8
3.50 x10-10
c2a 24 4 2 2.40 x10-8
4.00 x10-8
2.00 x10-9
c2b 26 6 1 2.60 x10-8
6.00 x10-8
1.00 x10-9
c3a 68 13 6.80 x10-8
1.30 x10-9
c3b 103 11 1.03 x10-9
1.10 x10-9
3.09 x10-9
0.02 - 1a 80 2 8.00 x10-8
2.00 x10-8
0.02 - 1b 54 5.40 x10-8
0.02 - 2a 64 8 3 6.40 x10-8
8.00 x10-8
3.00 x10-9
0.02 - 2b 87 10 1 8.70 x10-8
1.00 x10-9
1.00 x10-9
0.02 - 3a 60 7 6.00 x10-8
7.00 x10-8

169
0.02 - 3b 66 8
6.60 x
10-8
8.00 x
10-8
8.93 x10-8
0.04 - 1a 27 5 2.70 x10-8
5.00 x10-8
0.04 - 1b 40 6 4.00 x10-8
6.00 x10-8
0.04 - 2a 257 33 1 2.57 x10-9
3.30 x10-9
1.00 x10-9
0.04 - 2b 181 32 2 1.81 x10-9
3.20 x10-9
2.00 x10-9
0.04 - 3a 149 17 1.49 x10-9
1.70 x10-9
0.04 - 3b 168 24 3 1.68 x10-9
2.40 x10-9
3.00 x10-9
1.73 x10-9
0.06 - 1a 62 2 6.20 x10-8
2.00 x10-8
0.06 - 1b 41 8 4.10 x10-8
8.00 x10-8
0.06 - 2a 4 2 4.00 x10-7
2.00 x10-8
0.06 - 2b 24 2 2.40 x10-8
2.00 x10-9
0.06 - 3a 79 3 2 7.90 x10-8
3.00 x10-8
2.00 x10-9
0.06 - 3b 93 8 2 9.30 x10-8
8.00 x10-8
2.00 x10-9
8.09 x10-8
0.08 - 1a 196 21 2 1.96 x10-9
2.10 x10-9
2.00 x10-9
0.08 - 1b 212 26 2.12 x10-9
2.60 x10-9
0.08 - 2a 59 5 5.90 x10-8
5.00 x10-8
0.08 - 2b 78 4 3 7.80 x10-8
4.00 x10-8
3.00 x10-9
0.08 - 3a 346 40 4 3.46 x10-9
4.00 x10-9
4.00 x10-9
0.08 - 3b 350 33 3.50 x10-9
3.30 x10-9
2.29 x10-9
0.1 - 1a 83 2 8.30 x10-8
2.00 x10-8

170
0.1 - 1b 7 2 7.00 x10-7
2.00 x10-8
0.1 - 2a 14 3 1.40 x10-8
3.00 x10-8
0.1 - 2b 45 7 1 4.50 x10-8
7.00 x10-8
1.00 x10-9
0.1 - 3a 90 4 9.00 x10-8
4.00 x10-8
0.1 - 3b 95 8 1 9.50 x10-8
8.00 x10-8
1.00 x10-9
5.67 x10-8
count agars (by
dilution)
dilution)
Average of cells
per ml values
10-6
10 -7
10 -8
10-6
10 -7
10 -8
c1a 25 4 0.00 2.50 x10-8
4.00 x10-8
c1b 837 40 5 8.37 x10-8
4.00 x10-8
5.00 x10-8
c2a 693 3 6.93 x10-8
3.00 x10-8
c2b 580 76 8 5.80 x10-8
7.60 x10-8
8.00 x10-8
c3a 79 4 7.90 x10-8
4.00 x10-8
c3b 73 15 7.30 x10-8
1.50 x10-9
6.91 x10-8
0.02 - 1a 501 51 4 5.01 x10-8
5.10 x10-8
4.00 x10-8
0.02 - 1b 405 52 2 4.05 x10-8
5.20 x10-8
2.00 x10-8
0.02 - 2a 280 36 2.80 x10-9
3.60 x10-9
0.02 - 2b 246 27 2.46 x10-9
2.70 x10-9
0.02 - 3a 66 14 6.60 x10-8
1.40 x10-9
0.02 - 3b 651 60 6.51 x10-8
6.00 x10-8
1.24 x10-9
0.04 - 1a 59 5 5.90 x10-7
5.00 x10-7
0.04 - 1b 4 4.00 x10-7
0.04 - 2a 159 11 1.59 x10-9
1.10 x10-9
0.04 - 2b 149 9 1.49 x10-9
9.00 x10-8

171
0.04 - 3a 3408 328 24 3.41 x10-9
3.28 x10-9
2.40 x10-9
0.04 - 3b 2984 321 26 2.98 x10-9
3.21 x10-9
2.60 x10-9
1.78 x10-9
0.06 - 1a 208 32 2 2.08 x10-8
3.20 x10-8
2.00 x10-8
0.06 - 1b 186 60 6 1.86 x10-8
6.00 x10-8
6.00 x10-8
0.06 - 2a 724 96 8 7.24 x10-8
9.60 x10-8
8.00 x10-8
0.06 - 2b 43 14 4.30 x10-8
1.40 x10-9
0.06 - 3a 104 10 1.04 x10-9
1.00 x10-9
0.06 - 3b 117 12 1.17 x10-9
1.20 x10-9
7.23 x10-8
0.08 - 1a 330 38 3.30 x10-9
3.80 x10-9
0.08 - 1b 371 44 3.71 x10-9
4.40 x10-9
0.08 - 2a 95 10 9.50 x10-8
1.00 x10-9
0.08 - 2b 118 12 1.18 x10-9
1.20 x10-9
0.08 - 3a 403 42 4.03 x10-9
4.20 x10-9
0.08 - 3b 399 39 3.99 x10-9
3.90 x10-9
2.97 x10-9
0.1 - 1a 506 23 8 5.06 x10-8
2.30 x10-8
8.00 x10-8
0.1 - 1b 58 9 5.80 x10-8
9.00 x10-8
0.1 - 2a 183 17 4 1.83 x10-8
1.70 x10-8
4.00 x10-8
0.1 - 2b 120 16 3 1.20 x10-8
1.60 x10-8
3.00 x10-8
0.1 - 3a 92 11 2 9.20 x10-7
1.10 x10-8
2.00 x10-8
0.1 - 3b 125 15 3 1.25 x10-8
1.50 x10-8
3.00 x10-8
3.13 x10-8
count agars (by
dilution)
dilution)
Average of cells
per ml values

172
10-6
10 -7
10 -8
10-6
10 -7
10 -8
c1a 79 21 7.90 x10-7
2.10 x10-8
c1b 23 2.30 x10-8
c2a 89 8.90 x10-8
c2b 91 9.10 x10-8
c3a 183 1.83 x10-9
c3b 185 1.85 x10-9
1.14 x10-9
0.02 - 1a 256 34 2.56 x10-8
3.40 x10-8
0.02 - 1b 284 36 2.84 x10-8
3.60 x10-8
0.02 - 2a 297 2.97 x10-9
0.02 - 2b 291 2.91 x10-9
0.02 - 3a 538 40 5.38 x10-8
4.00 x10-8
0.02 - 3b 536 58 5.36 x10-8
5.80 x10-8
9.17 x10-8
0.04 - 1a 508 47 5.08 x10-8
4.70 x10-8
0.04 - 1b 535 56 5.35 x10-8
5.60 x10-8
0.04 - 2a 222 16 2.22 x10-8
1.60 x10-8
0.04 - 2b 36 11 3.60 x10-7
1.10 x10-8
0.04 - 3a 693 100 6.93 x10-8
1.00 x10-9
0.04 - 3b 778 106 7.78 x10-8
1.06 x10-9
5.11 x10-8
0.06 - 1a 565 5.65 x10-8
0.06 - 1b 363 3.63 x10-8
0.06 - 2a 314 25 3.14 x10-8
2.50 x10-8
0.06 - 2b 297 15 2.97 x10-8
1.50 x10-8

173
0.06 - 3a 30 0.00 3.00 x10-8
0.06 - 3b 41 0.00 4.10 x10-8
2.65 x10-8
0.08 - 1a 477 23 4.77 x10-8
2.30 x10-8
0.08 - 1b 350 19 3.50 x10-8
1.90 x10-8
0.08 - 2a 190 1.90 x10-9
0.08 - 2b 101 1.01 x10-9
0.08 - 3a 992 79 9.92 x10-8
7.90 x10-8
0.08 - 3b 673 62 6.73 x10-8
6.20 x10-8
7.23 x10-8
0.1 - 1a 588 71 5.88 x10-8
7.10 x10-8
0.1 - 1b 708 90 7.08 x10-8
9.00 x10-8
0.1 - 2a 50 5.00 x10-8
0.1 - 2b 657 47 6.57 x10-8
4.70 x10-8
0.1 - 3a 543 60 5.43 x10-8
6.00 x10-8
0.1 - 3b 394 50 3.94 x10-8
5.00 x10-8
5.97 x10-8
18/01/05 Data
initial inoculum
cells per ml
9.40 x10-7
1.88 x10-7
Results from the 19th Jan 05
count agars (by
dilution)
dilution)
Average of cells
per ml values
10-6
10 -7
10 -8
10-6
10 -7
10 -8
c1 262 16 2.62 x10-9
1.60 x10-9

174
c2 105 1.05 x10-10
c3 572 62 5.72 x10-9
6.20 x10-9
5.33 x10-9
0.02 - 1 365 59 3.65 x10-9
5.90 x10-9
0.02 - 2 139 1.39 x10-10
0.02 - 3 36 3.60 x10-9
6.76 x10-9
0.04 - 1 179 1.79 x10-10
0.04 - 2 904 9.04 x10-9
0.04 - 3 166 1.66 x10-10
1.45 x10-10
0.06 - 1 423 47 4.23 x10-9
4.70 x10-9
0.06 - 2 145 1.45 x10-10
0.06 - 3 155 1.55 x10-10
9.73 x10-9
0.08 - 1 5560 5.56 x10-9
0.08 - 2 268 36 2.68 x10-9
3.60 x10-9
0.08 - 3 2984 2.98 x10-9
3.71 x10-9
0.1 - 1 151 1.51 x10-10
0.1 - 2 173 1.73 x10-10
0.1 - 3 376 118 3.76 x10-9
1.18 x10-10
1.20 x10-10
Results from the 20th
cell counts from plate cells per ml for these values (by Average of cells

175
count agars (by
dilution)
dilution) per ml values
10-7
10-8
10-9
10-7
10-8
10-9
c1 117 15 1 1.17 x10-9
1.50 x10-9
1.00 x10-9
c2 68 10 6.80 x10-9
1.00 x10-10
c3 120 16 1 1.20 x10-9
1.60 x10-9
1.00 x10-9
3.03 x10-9
0.02 - 1 34 3.40 x10-10
0.02 - 2 92 2 9.20 x10-9
2.00 x10-9
0.02 - 3 40 6 4.00 x10-9
6.00 x10-9
1.10 x10-10
0.04 - 1 53 16 5.30 x10-9
1.60 x10-10
0.04 - 2 300 13 3.00 x10-10
1.30 x10-10
0.04 - 3 75 15 7.50 x10-9
1.50 x10-10
1.45 x10-10
0.06 - 1 447 54 8 4.47 x10-9
5.40 x10-9
8.00 x10-9
0.06 - 2
0.06 - 3 507 5.07 x10-9
5.74 x10-9
0.08 - 1 48 8 4.80 x10-9
8.00 x10-9
0.08 - 2 32 3.20 x10-9
0.08 - 3 78 9 7.80 x10-9
9.00 x10-9
6.56 x10-9
0.1 - 1 471 57 6 4.71 x10-9
5.70 x10-9
6.00 x10-9
0.1 - 2 547 90 5 5.47 x10-9
9.00 x10-9
5.00 x10-9

176
0.1 - 3 69 5 6.90 x10-9
5.00 x10-9
5.97 x10-9
Results from the 23rd
count agars (by
dilution)
dilution)
Average of cells
per ml values
10-6
10 -7
10 -8
10-6
10 -7
10 -8
c1 17 1.70 x10-8
c2 89 11 8.90 x10-8
1.10 x10-9
c3 22 3 2.20 x10-8
3.00 x10-8
5.36 x10-8
0.02 - 1 60 6.00 x10-9
0.02 - 2 285 34 2.85 x10-9
3.40 x10-9
0.02 - 3
4.08 x10-9
0.04 - 1 24 2.40 x10-9
0.04 - 2 387 3.87 x10-10
0.04 - 3 66 6.60 x10-9
1.59 x10-10
0.06 - 1 56 5.60 x10-9
0.06 - 2 111 6 1.11 x10-9
6.00 x10-8
0.06 - 3 35 3.50 x10-9
2.70 x10-9
0.08 - 1 66 6.60 x10-9
0.08 - 2 51 5.10 x10-9
0.08 - 3 36 3.60 x10-9
5.10 x10-9

177
0.1 - 1 80 4 8.00 x10-8
4.00 x10-8
0.1 - 2 39 4 3.90 x10-8
4.00 x10-8
0.1 - 3 135 12 1.35 x10-9
1.20 x10-9
7.57 x10-8

178
B. PLA pore size data
PLA pore size measurement data
Table B.1 pore size measurement from fig 4.24 (PLA 1)
Area of picture
Width of printed out
PLA image (cm)
Real width of PLA
image measured (µm)
Height of printed out
PLA image (cm)
Real height of PLA
image measured
(µm)
20.3 31.72 20.36 31.81
Real area (µm2
)= 1009.05
pore size measurements
Diameter of pore on
printout (cm)
Real pore Diameter
(µm)
Area of pore (pie x
(Diameter )) (µm2
)
0.9 1.41 4.42
1.2 1.88 5.89
1.5 2.34 7.36
1.1 1.72 5.40
0.9 1.41 4.42
0.8 1.25 3.93
1 1.56 4.91
3.8 5.94 18.65
1.3 2.03 6.38
2 3.13 9.82
0.7 1.09 3.44
0.8 1.25 3.93
1.4 2.19 6.87
0.7 1.09 3.44
4.3 6.72 21.11
2.7 4.22 13.25
2.5 3.91 12.27
1 1.56 4.91
2.7 4.22 13.25
1.5 2.34 7.36
0.7 1.09 3.44
0.6 0.94 2.95
0.6 0.94 2.95
3.3 5.16 16.20
1.5 2.34 7.36
0.7 1.09 3.44
0.6 0.94 2.95
1 1.56 4.91
0.8 1.25 3.93
0.8 1.25 3.93

179
2.1 3.28 10.31
2 3.13 9.82
1.5 2.34 7.36
2.1 3.28 10.31
0.6 0.94 2.95
2 3.13 9.82
1.8 2.81 8.84
1 1.56 4.91
1.7 2.66 8.34
2 3.13 9.82
0.7 1.09 3.44
0.6 0.94 2.95
2 3.13 9.82
1.3 2.03 6.38
1.8 2.81 8.84
3.2 5.00 15.71
2.5 3.91 12.27
1.3 2.03 6.38
1 1.56 4.91
1.8 2.81 8.84
1.1 1.72 5.40
1.3 2.03 6.38
0.7 1.09 3.44
2 3.13 9.82
2.3 3.59 11.29
0.7 1.09 3.44
0.6 0.94 2.95
1.7 2.66 8.34
0.5 0.78 2.45
0.4 0.63 1.96
0.9 1.41 4.42
1.4 2.19 6.87
0.9 1.41 4.42
1.3 2.03 6.38
0.7 1.09 3.44
2 3.13 9.82
1.9 2.97 9.33
1 1.56 4.91
1.5 2.34 7.36
0.7 1.09 3.44
0.9 1.41 4.42
1.3 2.03 6.38
0.9 1.41 4.42
2.4 3.75 11.78
2 3.13 9.82
0.7 1.09 3.44
2.4 3.75 11.78
1 1.56 4.91

180
1.7 2.66 8.34
1.4 2.19 6.87
Average Pore area
(µm2
) 7.04
Total area of pores
(µm2
) 563.03
Percentage pores (%) 55.80
St Dev of pore area 3.88

181
Table B.2 pore size measurement from fig 4.25 (PLA 2)
Area of picture
PLA image (cm)
Real width of PLA
PLA image (cm)
Real height of PLA
image measured
(µm)
20.3 31.72 20.36 31.81
Real area (µm2
)= 1009.05
Diameter of pore on
printout (cm)
Real pore Diameter
(µm)
Area of pore (pie x
(Diameter )) (µm2
)
2.7 4.22 13.25
2.8 4.38 13.74
1.8 2.81 8.84
2.2 3.44 10.80
2 3.13 9.82
0.9 1.41 4.42
0.8 1.25 3.93
2.5 3.91 12.27
1.9 2.97 9.33
1.8 2.81 8.84
1.2 1.88 5.89
1 1.56 4.91
0.7 1.09 3.44
0.9 1.41 4.42
1.1 1.72 5.40
2.9 4.53 14.24
0.6 0.94 2.95
0.5 0.78 2.45
1.2 1.88 5.89
3.6 5.63 17.67
0.8 1.25 3.93
3.3 5.16 16.20
0.9 1.41 4.42
1 1.56 4.91
1.2 1.88 5.89
0.5 0.78 2.45
0.5 0.78 2.45
0.6 0.94 2.95
0.4 0.63 1.96
2 3.13 9.82
4.1 6.41 20.13
1.7 2.66 8.34
1 1.56 4.91
0.6 0.94 2.95

182
0.5 0.78 2.45
0.7 1.09 3.44
1 1.56 4.91
1.1 1.72 5.40
1 1.56 4.91
0.8 1.25 3.93
4.3 6.72 21.11
1.3 2.03 6.38
0.7 1.09 3.44
0.9 1.41 4.42
1 1.56 4.91
0.6 0.94 2.95
0.9 1.41 4.42
0.7 1.09 3.44
0.5 0.78 2.45
2.1 3.28 10.31
5.5 8.59 27.00
1.5 2.34 7.36
1.5 2.34 7.36
3.6 5.63 17.67
Average Pore
area(µm2
) 7.44
Total area of pores
(µm2
) 402.03

183
TableB.3 pore size measurement from fig 4.26 (PLA 3)
Area of picture
PLA image (cm)
Real width of PLA
PLA image (cm)
Real height of PLA
image measured
(µm)
20.3 31.72 20.36 31.81
Real area (µm2
)= 1009.05
Diameter of pore on
printout (cm)
Real pore Diameter
(µm)
Area of pore (pie x
(Diameter )) (µm2
)
2.5 3.91 12.27
1.7 2.66 8.34
1.3 2.03 6.38
1.3 2.03 6.38
1.3 2.03 6.38
1.8 2.81 8.84
1.9 2.97 9.33
0.9 1.41 4.42
2 3.13 9.82
4.7 7.34 23.07
1.2 1.88 5.89
1.8 2.81 8.84
1.3 2.03 6.38
6.1 9.53 29.94
2 3.13 9.82
0.6 0.94 2.95
5.5 8.59 27.00
3.2 5.00 15.71
5 7.81 24.54
7.2 11.25 35.34
1.2 1.88 5.89
1.5 2.34 7.36
2.5 3.91 12.27
1.1 1.72 5.40
1.2 1.88 5.89
5.9 9.22 28.96
1.2 1.88 5.89
0.6 0.94 2.95
1.2 1.88 5.89
3 4.69 14.73
2.1 3.28 10.31
1.4 2.19 6.87
2.5 3.91 12.27
1.1 1.72 5.40
2.5 3.91 12.27

184
1.8 2.81 8.84
5.8 9.06 28.47
4.7 7.34 23.07
Average Pore
area(µm2
) 12.22
Total area of pores
(µm2
) 464.37
C. Tissue culture study
C.1 Tissue culture study raw data
Assumptions
All of the samples measure 30mm in length. Each sample was tested 9 times. All
plasma treatment work was performed on the Riccarton apparatus unless stated
otherwise.
Key
Grey table cells, couldn‟t be seen clearly enough to measure. Media Red = no
change in the pH of the media. This means that the media is not being metabolised
much/ at all by viable cells. Media Orange = slight change in the pH of the media.
This means that the media is being metabolised by cells in the flask. 21/08/2005, no
data for inoculum. An oversight due to late working hours and heavy work load.

185
Polypropylene Data
Inoculation
/cell count
day (cells
per ml) Cell growth up biomaterial strip (mm)
Day 0 8 15 22 29
Date 20/06/2006 28/06/2006 05/07/2006 12/07/2006 19/07/2006
Polypropylene 1 140000 0.00 0.00 0.00 0.00
Polypropylene 2 140000 0.00 0.00 0.00 0.00
Polypropylene 3 140000 0.00 0.00 0.00 0.00
Polypropylene treated with
Argon plasma 1 140000 0.00 0.00 0.00 0.00
Argon plasma 2 140000 0.00 0.00 0.00 0.00
Argon plasma 3 140000 0.00 0.00 0.00 0.00
Ammonia plasma 1 140000 0.00 0.00 0.00 0.00
Ammonia plasma 2 140000 0.00 0.00 0.00 0.00
Ammonia plasma 3 140000 0.00 0.00 0.00 0.00
Polypropylene coated with
0.1% Chitosan solution 1 140000 0.00 0.00 0.00 0.00
Argon plasma then coated with
Polypropylene treated with 140000 0.00 0.00 0.00 0.00

186
0.1% Chitosan solution 2
Ammonia plasma then coated
with 0.1% Chitosan solution 1 140000 0.00 0.00 0.00 0.00
with 0.1% Chitosan solution 2 140000
Day 0 8 15 22 29
Date 18/07/2006 26/07/2006 02/08/2006 09/08/2006 16/08/2006
Polypropylene 1 330000 0.00 0.00 0.00 0.00
Polypropylene 2 330000 0.00 0.00 0.00 0.00
Polypropylene 3 330000 0.00 0.00 0.00 0.00
Argon plasma 1 330000 0.00 16.00 19.00 24.00
Argon plasma 2 330000 0.75 7.00 15.50 24.00
Argon plasma 3 330000 0.00 0.00 0.00 0.00
Ammonia plasma 1 330000 1.50 10.00 16.00 28.00
Ammonia plasma 2 330000 5.00 8.00 15.00 26.00
Ammonia plasma 3 330000 1.00 24.00 15.00 20.00

187
Day 0 8 15 22 29
Date 25/08/2006 02/09/2006 09/09/2006 16/09/2006 23/09/2006
Polypropylene 1 190000 0.00 0.00 0.00 0.00
Polypropylene 2 190000 0.00 0.00 30.00 30.00
Polypropylene 3 190000 0.00 3.13 30.00 30.00
Argon plasma 1 190000 0.00 0.00 0.00 0.00
Argon plasma 2 190000 0.00 2.00 30.00 30.00
Argon plasma 3 190000 0.00 0.00 12.00 30.00

188
Ammonia plasma 1 190000 3.00 0.50 20.00 30.00
Ammonia plasma 2 190000 0.00 0.00 0.00 0.00
Ammonia plasma 3 190000 1.50 4.00 5.00 30.00

189
Polyurethane Data
Day 0 8 15 22 29
Date 14/07/2005 22/07/2005 29/07/2005 05/08/2005 12/08/2005
Tuftane® Polyurethane 1 170000 0.00 0.00 0.00 0.00
Tuftane® Polyurethane treated
with Argon plasma 1 170000 5.20 24.00 30.00 30.00
with Ammonia plasma 1 170000 0.00 0.00 30.00 26.00
Tuftane® Polyurethane dusted
with Chitosan powder then
treated with Argon plasma 1 170000 2.50 6.00 30.00 30.00
Day 0 8 15 22 29
Date 21/08/2005 29/08/2005 05/09/2005 12/09/2005 19/09/2005
Tuftane® Polyurethane 1 0.00 0.00 0.00 0.00

190
with Argon plasma 1 7.75 30.00 30.00 30.00
with Ammonia plasma 1 0.00 30.00 30.00 30.00
treated with Argon plasma 1 0.00 30.00 30.00 30.00
with Argon plasma on the
Europlasma machine 1 14.00 30.00 30.00 30.00

191
with Oxygen plasma on the
Day 0 8 15 22 29
Date 25/08/2005 02/09/2005 09/09/2005 16/09/2005 23/09/2005

192
Europlasma machine 1 240000 0.00 0.00 0.00 0.00

193
Polyester Data
Day 0 8 15 22 29
Date 16/07/2005 24/07/2005 31/07/2005 07/08/2005 14/08/2005
Vascutek Polyester 1 100000 3.00 3.00 4.20 6.00
Vascutek Polyester treated with
Argon plasma 1 100000 0.00 0.00 0.00 0.00
Argon plasma 2 100000 4.00 5.00 6.00 6.00
Argon plasma 3 100000 0.00 0.00 0.00 0.00
Ammonia plasma 1 100000 0.00 0.00 0.00 0.00
Ammonia plasma 2 100000 0.00 0.00 0.00 0.00
Ammonia plasma 3 100000 0.00 0.00 0.00 0.00
Day 0 8 15 22 29
Date 03/08/2005 11/08/2005 18/08/2005 25/08/2005 01/09/2005
Argon plasma 1 235000 0.00 0.00 2.50 4.00
Argon plasma 2 235000 0.00 0.00 0.00 0.00
Argon plasma 3 235000 0.00 0.00 0.00 0.00
Ammonia plasma 1 235000 0.00 0.00 0.00 0.00

194
Ammonia plasma 2 235000 0.00 0.00 0.00 0.00
Ammonia plasma 3 235000 0.00 0.00 3.00 7.20
Day 0 8 15 22 29
Date 21/08/2005 29/08/2005 05/09/2005 12/09/2005 19/09/2005
Vascutek Polyester 1 2.75 3.50 8.00 30.00
Argon plasma 1 0.00 0.00 0.00 0.00
Argon plasma 2 0.00 0.00 0.00 0.00
Argon plasma 3 3.00 5.50 6.50 8.00
Ammonia plasma 1 4.00 7.00 6.00 9.00
Ammonia plasma 2 0.00 0.00 5.75 7.00
Ammonia plasma 3 4.00 5.20 30.00 30.00
Poly-ε-caprolactone Data
Day 0 8 15 22 29
Date 14/07/2005 20/07/2005 29/07/2005 05/08/2005 12/08/2005
Poly-ε-caprolactone 6400 1 170000 0.00 0.00 0.00 0.00

195
Poly-ε-caprolactone 6400
treated with Ammonia plasma 1 170000 0.00 2.00 4.00 6.00
Day 0 8 15 22 29
Date 18/07/2005 26/07/2005 02/08/2005 09/08/2005 16/08/2005

196
Day 0 8 15 22 29
Date 25/08/2005 02/09/2005 09/09/2005 16/09/2005 23/09/2005
treated with Argon plasma 2 240000
treated with Argon plasma 3 240000
treated with Ammonia plasma 2 240000
Solanyl Flexibilitis Data
Day 0 8 15 22 29
Date 20/06/2005 28/06/2005 05/07/2005 12/07/2005 19/07/2005
Solanyl Flexibilitis 1 140000 0.00 0.00 0.00 0.00
Solanyl Flexibilitis treated with
Argon plasma 1 140000 0.00 0.00 0.00 0.00
Argon plasma 2 140000 0.00 0.00 0.00 0.00
Argon plasma 3 140000 0.00 0.00 0.00 0.00

197
Ammonia plasma 1 140000 0.00 0.00 0.00 0.00
Ammonia plasma 2 140000 0.00 0.00 0.00 0.00
Ammonia plasma 3 140000 0.00 0.00 0.00 0.00
Solanyl Flexibilitis extruded
with 2% Chitosan powder
(w/w) 1 140000 0.00 0.00 0.00 0.00
(w/w) 2 140000 0.50 0.00 0.00 0.00
(w/w) 3 140000 0.00 0.00 0.00 0.00
Solanyl Flexibilitis coated with

198
Day 0 8 15 22 29
Date 03/08/2005 11/08/2005 18/08/2005 25/08/2005 01/09/2005
Argon plasma 1 235000 0.00 0.00 0.00 0.00
Argon plasma 2 235000 0.00 0.50 27.00 19.00
Argon plasma 3 235000 0.00 0.00 0.00 0.00
Ammonia plasma 1 235000 0.00 0.00 0.00 0.00
Ammonia plasma 2 235000 2.00 16.00 24.00 30.00
Ammonia plasma 3 235000 0.00 0.00 0.00 0.00
(w/w) 1 235000 0.00 0.00 15.00 30.00
(w/w) 2 235000 0.00 1.75 15.00 30.00
(w/w) 3 235000 0.00 0.00 0.00 0.00

199
Day 0 8 15 22 29
Date 25/08/2005 02/09/2005 09/09/2005 16/09/2005 23/09/2005
Solanyl Flexibilitis 3 190000 n/a n/a n/a n/a
Argon plasma 1 190000 n/a n/a n/a n/a

200
Ammonia plasma 1 190000 n/a n/a n/a n/a
(w/w) 1 190000 n/a n/a n/a n/a
(w/w) 2 190000 n/a n/a n/a n/a
(w/w) 3 190000 n/a n/a n/a n/a

201
Poly-L-Lactic acid Data
Day 0 8 15 22 29
Date 16/07/2005 24/07/2005 31/07/2005 07/08/2005 14/08/2005
Poly-L-Lactic acid 1 100000 0.00 17.50 17.50 30.00
Poly-L-Lactic acid treated with
Argon plasma 1 100000 0.00 0.00 0.00 0.00
Argon plasma 2 100000 0.00 0.00 0.00 0.00
Argon plasma 3 100000 6.00 30.00 30.00 30.00
Ammonia plasma 1 100000 0.00 2.50 0.00 0.00
Ammonia plasma 2 100000 0.00 0.00 0.00 0.00
Ammonia plasma 3 100000 3.00 3.00 30.00 30.00
Day 0 8 15 22 29
Date 21/08/2005 29/08/2005 05/09/2005 12/09/2005 19/09/2005
Poly-L-Lactic acid 1 Can‟t
measure.
Cells
growing on
Cell
attachment,
can't
measure.
Cell
attachment,
can't
measure.
Cell
attachment
at both
ends. Can't

202
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
remained
red
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube. Media
turned
orange
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
turned
orange
measure,
probably
all the
way. Cells
growing
on
capillary
tube +
cells
growing
on tissue
culture
flask.
Media
turned
orange
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
turned
orange
Cell
attachment,
can't
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube. Media
turned
orange
Cell
attachment,
can't
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
turned
orange
Cell
attachment
at both
ends. Can't
measure,
probably
all the
way. +
Cells
growing
on
capillary
tube +
cells
growing
on tissue
culture
flask.
Media
turned
orange
measure.
Cell
attachment,
Cell
attachment,
Cell
attachment

203
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
remained
red
can't
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube. Media
turned
orange
can't
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
turned
orange
at both
ends. Can't
measure,
probably
all the
way. +
Cells
growing
on
capillary
tube +
cells
growing
on tissue
culture
flask.
Media
turned
orange
Argon plasma 1
Can‟t
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
turned
orange
Cell
attachment,
can't
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube. Media
turned
orange
Cell
attachment,
can't
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
turned
orange
Cell
attachment
at both
ends. Can't
measure,
probably
all the
way. +
Cells
growing
on
capillary
tube +
cells
growing
on tissue
culture
flask.
Media
turned
orange

204
Argon plasma 2
0 0 0 0
Argon plasma 3
Can‟t
measure
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
remained
red
Few Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube. Media
turned
orange.
Cell
attachment,
can't
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
turned
orange
Cell
attachment
at one end,
can't
measure +
cells
growing
on
capillary
tube +
cells
growing
on tissue
culture
flask.
Media
turned
orange
Ammonia plasma 1
Cell
attachment.
No cells
growing on
capillary
tube or
tissue
culture
flask.
Media
remained
red
Cell
attachment,
cells
growing on
capillary
tube. Media
turned
orange
Cell
attachment,
can't
measure.
Cells
growing on
capillary
tube + cells
growing on
tissue
culture
flask.
Media
turned
orange
Cell
attachment
at one end,
possibly
the other,
can't
measure +
cells
growing
on
capillary
tube +
cells
growing
on tissue
culture
flask.
Media
turned

205
orange
Ammonia plasma 2
0 0 0 0
Ammonia plasma 3
Cell
attachment.
No cells
growing on
capillary
tube or
tissue
culture
flask.
Media
remained
red
Cell
attachment,
cells
growing on
capillary
tube + cells
growing on
tissue
culture
flask.
Media
turned
orange
Cell
attachment,
can't
measure,
cells
growing on
capillary
tube + cells
growing on
tissue
culture
flask.
Media
turned
orange
Cell
attachment
at one end,
possibly
the other,
can't
measure +
cells
growing
on
capillary
tube +
cells
growing
on tissue
culture
flask.
Media
turned
orange
Day 0 8 15 22 29
Date 25/08/2005 02/09/2005 09/09/2005 16/09/2005 23/09/2005
Poly-L-Lactic acid 1 240000 Few cells
growing
on
capillary
tube.
sample
came off
Cell
attachment.
Free
floating.
Possibly
confluent.
Cells
growing on
tissue
culture
flask, +
cells
Cell
attachment,
can't
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
Can‟t
measure.
(Free
floating.
Both ends
have cells.
Few cells
growing
on tissue
culture
flask, +
cells

206
growing on
capillary
tube. Media
turned
orange
tube.
Media
turned
orange
growing
on
capillary
tube.
Media
turned
orange
Poly-L-Lactic acid 2 240000 Few Cells
growing
on tissue
culture
flask. No
cells
growing
on
capillary
tube.
Cell
attachment,
free
floating.
Cells
growing on
capillary
tube. Media
remained
red
Cell
attachment,
can't
measure.
Cells
growing on
tissue
culture
flask, +
cells
growing on
capillary
tube.
Media
turned
orange
Cells on
one end
contracting
sample.
Can't
measure.
Probably
not on
other end.
Cells
growing
on tissue
culture
flask.
Media
turned
orange
Poly-L-Lactic acid 3 240000 0 0 0 0
Argon plasma 1
240000 0 0 0 0
Argon plasma 2
240000 0 0 0 0
Argon plasma 3
240000 0 0 0 0
Ammonia plasma 1
240000 0 0 0 0
Ammonia plasma 2
240000 0 0 0 0
Ammonia plasma 3
240000 0 0 0 0

207
C.2 Fluorescence images
Below, a list of tissue culture samples with the measurements from the last day of
measuring growth along the sample, with corresponding fluorescence images and
descriptions from the last day of each sample. Where there are no fluorescence
images, there was either nothing to see, or no good image could be obtained.
Sample Growth measured on
previous day and
observations
Notes recorded
during
fluorescence
measurement
Fluorescence microscopy images
(and image number)
Day 0 29 30
Date 20/06/2005 19/07/2005 20/07/2005
Polypropylene 1 0.0 mm no visible cells No image
Polypropylene +
Argon 1
0.0 mm no visible cells
No image
Polypropylene +
Argon 2
No image
Polypropylene +
Argon 3
No image
Polypropylene +
ammonia 1
No image
Polypropylene +
ammonia 2
No image
Polypropylene +
ammonia 3
No image
Polypropylene +
Chitosan 1
No image

208
Polypropylene +
Chitosan 2
2.8 mm + cells
growing on capillary
tube
2.1
2.2
2.3
Polypropylene +
Chitosan 3
5 mm + cells
tube
3.1

210
Polypropylene +
Argon + Chitosan 1
0.0 mm
1.1
1.2
Polypropylene +
Argon + Chitosan 2
0.0 mm
2.1
2.2
Polypropylene + 0.0 mm no visible cells No image

211
Argon + Chitosan 3
Polypropylene +
ammonia + Chitosan
1
No image
Polypropylene +
ammonia + Chitosan
2
No image
Polypropylene +
ammonia + Chitosan
3
No image

212
Day 0 29.0
Date 20/06/2005 19/07/2005 20/07/2005
Solanyl 1 0.0 mm no visible cells No image
Solanyl 2 0.0 mm no visible cells No image
Solanyl 3 0.0 mm no visible cells
3.1
3.2
Solanyl + Argon 1 0.0 mm no visible cells No image
Solanyl + ammonia 1 0.0 mm no visible cells No image
Solanyl 2% Chitosan
1
0.0 mm no visible cells No image
Solanyl 2% Chitosan
2
Solanyl 2% Chitosan 0.0 mm no visible cells No image

213
3
Solanyl + Chitosan 1 0.0 mm no visible cells No image
Solanyl + Chitosan 2 0.0 mm no visible cells No image
Solanyl + Chitosan 3 2 mm + cells
tube
no visible cells No image
Solanyl + Argon +
Chitosan 1
Solanyl + Argon +
Chitosan 2
Solanyl + Argon +
Chitosan 3
Solanyl + ammonia +
Chitosan 1
Solanyl + ammonia +
Chitosan 2
No image
Solanyl + ammonia +
Chitosan 3
No image

214
Day 0.0 29.0 31.0
Date 14/07/2005 12/08/2005 14/08/2005
Polyurethane 1 0 mm Media
remained red
no visible cells
No image
Polyurethane 2 14 mm + cells
tube. Media turned
orange.
2 pictures
3.1
3.2
remained red
no visible cells
No image
Polyurethane +
Argon 1
30 mm + cells
tube + cells growing
on tissue culture
flask. Media turned
orange.
4 pictures
1.1

215
1.2
1.3
1.4
Polyurethane +
Argon 2
30 mm + cells
tube + cells
growing on tissue
culture flask. Media
turned orange.
5 pictures
2.1

217
Polyurethane +
Argon 3
10 mm + cells
tube + cells
growing on tissue
turned orange.
cells 1/4 way
up
3.1
3.2
Polyurethane +
ammonia 1
26 mm + cells
tube + cells
growing on tissue
turned orange.
1.1
Polyurethane +
ammonia 2
16 mm + cells
tube + cells
growing on tissue
turned orange.
2.1

218
2.2
2.3
2.4
Polyurethane +
ammonia 3
30 mm + cells
tube + cells
growing on tissue
turned orange.
3.1

219
3.2
3.3
Polyurethane +
Argon + Chitosan
powder 1
30 mm + cells
tube + cells
growing on tissue
turned orange.
1.1
1.2

221
Polyurethane +
Argon + Chitosan
powder 2
0 mm Media
remained red
no visible cells
2.1
Polyurethane +
Argon + Chitosan
powder 3
2.25 mm + cells
tube Media
remained red
Growth ¼
along the
length of
sample? No image

222
Day 0.0 29.0 31.0
Date 14/07/2005 12/08/2005 14/07/2005
polycaprolactone 1 0 mm Media
remained red
no visible cells
No image
remained red
no visible cells
No image
polycaprolactone 3 0.4 mm + cells
tube + cells
growing on tissue
turned orange.
one end of
sample
3.1
3.2
polycaprolactone +
Argon 1
0 mm Media
remained red
no visible cells
No image
polycaprolactone +
Argon 2
0 mm Media
remained red
Growth at both
ends.
2.1= one end
2.1

223
2.2
2.3
2.4
polycaprolactone +
Argon 3
0 mm Media
remained red
Cells span 1/3
of sample from
one end and ½
way along
sample from
other end
3.1

224
3.2
3.3
3.4
polycaprolactone +
ammonia 1
6 mm + cells growing
on capillary tube +
cells growing on
tissue culture flask.
Media turned orange.
1/4 to 1/2
1.1

225
polycaprolactone +
ammonia 2
0.0 mm Media
remained red
no visible cells
2.1
polycaprolactone +
ammonia 3
0 mm Media
remained red
1/3 to 1/2
3.1

226
Day 0.0 29.0 30.0
Date 16/07/2005 14/08/2005 15/08/2005
Polyester 1 6 mm + cells growing
on capillary tube +
cells growing on
Cells visible
all the way
along sample
or fibres
fluorescing
1.1
1.2
1.2 zoom

227
Polyester 2 11 mm dense, 30 mm
total + cells growing
on capillary tube +
cells growing on
Cells visible
all the way
along sample
or fibres
fluorescing
2.1
2.1 zoom
2.2
Polyester 3 8 mm dense, 30 mm
total + cells growing
on capillary tube +
cells growing on
Cells visible
all the way
along sample
or fibres
fluorescing
3.1

228
3.2
3.3
3.3 zoom
Polyester + Argon 1 0 mm Media
remained red
Cells visible
all the way
along sample
or fibres
fluorescing
1.1

229
1.2 zoom
Polyester + Argon 2 6 mm + cells growing
on capillary tube +
cells growing on
Cells visible
all the way
along sample
or fibres
fluorescing
2.1
2.2 zoom
remained red
Cells visible
all the way
along sample
or fibres
fluorescing
3.1

230
3.2 zoom
Polyester + ammonia
1
0 mm Media
remained red
Cells visible
all the way
along sample
or fibres
fluorescing
1.1
1.2 zoom
Polyester + ammonia
2
0 mm Media
remained red
Cells visible
all the way
along sample
or fibres
fluorescing
2.1

231
2.2 zoom
Polyester + ammonia
3
0 mm Media
remained red
Cells visible
all the way
along sample
or fibres
fluorescing
3.1
3.1 zoom
3.2

232
Day 0.0 29.0 30.0
Date 16/07/2005 14/08/2005 15/08/2005
Polylactic acid 1 Cells growing on
capillary tube +
cells growing on
tissue culture flask,
possibly all the way.
Cells visible
all the way
along sample
1.1
1.2
1.3

233
1.4
capillary tube +
cells growing on
no visible cells
No image
capillary tube +
cells growing on
0.7
3.1
3.2

234
Polylactic acid +
Argon 1
0 mm Media
remained red
no visible cells
1.1
Polylactic acid +
Argon 2
0 mm Media
remained red
no visible cells
No image
Polylactic acid +
Argon 3
Cells growing on
capillary tube +
cells growing on
Cells visible
all the way
along sample
3.1
3.2

236
Polylactic acid +
ammonia 1
Cells growing on
capillary tube. Media
remained red
no visible cells
No image
Polylactic acid +
ammonia 2
0 mm Media
remained red
no visible cells
2.1
Polylactic acid +
ammonia 3
Cells growing on
capillary tube +
cells growing on
Cells visible
all the way
along sample
3.1
3.2

238
Day 0.0 29.0 31.0
Date 18/07/2005 16/08/2005 17/08/2005
Polypropylene 1 0 mm Media
remained red
remained red
remained red
Polypropylene +
Argon 1
Cells growing on
capillary tube +
cells growing on
tissue culture flask
can't measure
improvement. Media
turned orange.
24.0
1.1
1.2
1.3

239
1.4
1.5 end
Polypropylene +
Argon 2
Cells growing on
capillary tube +
cells growing on
can't measure
improvement. Media
turned orange.
24.0mm pic22
at end, pic23 is
start end
2.1
2.2 end

240
2.3
2.4
Polypropylene +
Argon 3
0 mm Media
remained red
Cells can be
seen growing
along the edge
of the material
only.
3.1
Polypropylene +
ammonia 1
Cells growing on
capillary tube +
cells growing on
can't measure
improvement. Media
turned orange.
28.0
1.1

241
1.2
Polypropylene +
ammonia 2
Cells growing on
capillary tube +
cells growing on
can't measure
improvement. Media
turned orange.
26.0
2.1
2.2
2.3

242
Polypropylene +
ammonia 3
Cells growing on
capillary tube +
cells growing on
can't measure
improvement. Media
turned orange.
20 picture 36
3.1
Polypropylene +
Chitosan 1
0 mm. Piece of
detached film
floating in media.
Media remained red
1.1
1.2
1.3

243
1.4
Polypropylene +
Chitosan 2
Cells growing on
capillary tube +
cells growing on
can't measure
improvement. Media
turned orange.
16.0
2.1
2.2

244
2.3
2.4
Polypropylene +
Chitosan 3
0mm? Cells growing
on capillary tube +
cells growing on
Polypropylene +
Argon + Chitosan 1
0 mm Media
remained red
Sample
destroyed
No image
Polypropylene +
Argon + Chitosan 2
Cells growing on
capillary tube +
cells growing on
can't measure
improvement. Media
turned orange.
Polypropylene +
Argon + Chitosan 3
1/4 mm? Remaining
length looks empty.
cells growing on
patches along
edge
3.1

245
Polypropylene +
ammonia + Chitosan
1
Cells growing on
capillary tube + cells
growing on tissue
culture flask can't
measure
improvement. Media
turned orange.
not consistent
1.1
1.2
1.3
1.4

246
Polypropylene +
ammonia + Chitosan
2
Cells growing on
growing on tissue
culture flask can't
measure
improvement. Media
turned orange.
2.1
2.2
2.3

247
Polypropylene +
ammonia + Chitosan
3
0mm. cell debris no visible cells
No image

248
Day 0 29.0 30.0
Date 18/07/2005 16/08/2005 17/08/2005
remained red
Unclear, but
unlikely to be
cell growth
1.1
polycaprolactone 2 6 mm plus + cells
on tissue culture
flask. Media turned
orange. cell debris
Unclear, but
unlikely to be
cell growth
2.1
remained red
Unclear, but
unlikely to be
cell growth
3.1

249
polycaprolactone +
Argon 1
0 mm Media
remained red
Unclear, but
unlikely to be
cell growth
1.1
polycaprolactone +
Argon 2
Few cells growing on
growing on tissue
turned orange.
Unclear, but
unlikely to be
cell growth
2.1
polycaprolactone +
Argon 3
0 mm Media
remained red
Unclear, but
unlikely to be
cell growth
3.1
polycaprolactone +
ammonia 1
0 mm Media
remained red
Unclear, but
unlikely to be
cell growth
1.1

250
polycaprolactone +
ammonia 2
0 mm Media
remained red
Unclear, but
unlikely to be
cell growth
2.1
polycaprolactone +
ammonia 3
0 mm Media
remained red
Unclear, but
unlikely to be
cell growth
3.1

251
Day 0 29.0 30.0
Date 03/08/2005 01/09/2005 02/09/2005
Polyester 1 7.5 mm (patches of 5,
2 and .5 mm, + cells
on tissue culture
flask. Media turned
orange.
Cells visible
all the way
along sample?
(Error
reading?)
1.1
1.2
1.3

252
1.4 zoom
1.5
Polyester 2 0 mm Media
remained red
Cells visible
all the way
along sample?
(Error
reading?)
2.1
2.2

253
Polyester 3 5 - 8.75 mm + cells
on tissue culture
flask. Media turned
orange.
Cells visible
all the way
along sample
(error
reading?)
3.1
Polyester + Argon 1 4 mm. Media turned
orange.
Cells visible
all the way
along sample?
(Error
reading?)
1.1
1.2
remained red
Cells visible
all the way
along sample?
(Error
reading?)
2.1

254
2.2
remained red
Cells visible
all the way
along sample?
(Error
reading?)
3.1
Polyester + ammonia
1
Few cells growing
on tissue culture flask
Media remained red
Cells visible
all the way
along sample?
(Error
reading?)
1.1

255
1.2 zoom
Polyester + ammonia
2
0 mm Media
remained red
Cells visible
all the way
along sample?
(Error
reading?)
2.1
Polyester + ammonia
3
7.2 mm (2 one end, 5
other). Media turned
orange.
Cells visible
all the way
along sample?
(Error
reading?)
3.1
3.2

256
Day 0 29 30
Date 21/08/2005 19/09/2005 20/09/2005
remained red
no visible cells
No image
Polyurethane 2 30 mm (dense and
loose bits, 2mm d,
4mm l, 6mm d, 8mm
l, 4mm d, 2,2mm l) +
cells growing on
growing on tissue
turned orange.
no growth
beyond 1/3
(2.1 very
difficult to
visualise)
patchy, non-
confluent
No image
Polyurethane 3 24 mm (1mm dense,
6mm gap/loose, 23
mm dense or can't
see) + cells growing
on capillary tube +
cells growing on
(5 cells,
nothing else)
No image
Polyurethane +
Argon 1
30 mm (confluent) +
cells growing on
growing on tissue
turned orange.
Cells visible
all the way
along sample.
Good growth.
Confluent,
gaps where
removed from
dish
No image
Polyurethane +
Argon 2
0 mm Media
remained red
Polyurethane +
Argon 3
30 mm (confluent) +
cells growing on
growing on tissue
Good growth.
Continuous
along edge
both sides.
Plenty in
No image

257
turned orange. middle. All the
way
Polyurethane +
ammonia 1
30 mm (confluent) +
cells growing on
growing on tissue
turned orange.
Good growth.
Confluent all
the way
No image
Polyurethane +
ammonia 2
30 mm (confluent) +
cells growing on
growing on tissue
turned orange.
Gap from 6-
12mm. Good
growth. Non-
confluent.
Gaps
surrounded by
cells
No image
Polyurethane +
ammonia 3
30 mm (confluent) +
cells growing on
growing on tissue
turned orange.
Patchy growth.
Mostly along
edge. Not
dense. All the
way
No image
Polyurethane +
Argon + Chitosan
powder 1
30 mm (confluent) +
cells growing on
growing on tissue
turned orange.
Very good
growth.
Confluent all
the way, both
sides.
Excellent
No image
Polyurethane +
Argon + Chitosan
powder 2
0 mm Media
remained red
Polyurethane +
Argon + Chitosan
powder 3
0 mm (signs of dead
cells) Media
remained red
Polyurethane +
Argon @ Galashiels
1
30 mm (confluent) +
cells growing on
Cells confluent
all the way
along sample.
No image

258
growing on tissue
turned orange.
Dense
Polyurethane +
Argon @ Galashiels
2
6mm v. loose, v. little
clump of cells
growing on tissue
culture flask, no cells
tube. Media turned
orange.
Polyurethane +
Argon @ Galashiels
3
0 mm signs of dead
cells at one end
Media remained red
Polyurethane +
Argon @ Galashiels
4
0 mm Media
remained red
Polyurethane +
Argon @ Galashiels
5
30 mm (confluent) +
cells growing on
growing on tissue
turned orange.
Cells confluent
all the way
along sample.
Very dense all
the way
No image
Polyurethane +
Argon @ Galashiels
6
0 mm Media
remained red
Polyurethane +
Oxygen @ Galashiels
1
0 mm Media
remained red
Polyurethane +
Oxygen @ Galashiels
2
0 mm Media
remained red
Polyurethane +
Oxygen @ Galashiels
3
0 mm Media
remained red
Polyurethane +
Oxygen @ Galashiels
30 mm (confluent) +
cells growing on
Dense for first
5mm. Cells
No image

259
4 capillary tube + cells
growing on tissue
turned orange.
thick to 12mm.
Not very dense
to 28mm.
Confluent for
last 2mm.
Cells probably
ripped off in
places
Polyurethane +
Oxygen @ Galashiels
5
30 mm (confluent) +
cells growing on
growing on tissue
turned orange.
cells confluent
on other side
(outward
facing side,
very dense
No image
Polyurethane +
Oxygen @ Galashiels
6
0 mm Media
remained red

260
Day 0 29 30
Date 21/08/2005 19/09/2005 20/09/2005
Polyester 1 30 mm (dense for
first 6mm, cells
everywhere + cells
on tissue culture
flask. Media turned
orange.
1.2 is one end.
Other end
damaged by
removal. 1/4
from other end
is picture 1.3.
all the way
1.1
1.2
1.3

261
Polyester 2 26 mm (4mm gap
16mm from one end,
10mm from other.
5mm very dense +
cells growing on
growing on tissue
turned orange.
2.2 is one end.
Vas 2.3 is the
other end.
Very dense for
about 6mm.
2.4 is past
dense bit
2.1
2.2
2.3
2.4

262
Polyester 3 7 mm (5.5mm dense,
0.5mm less dense,
1mm other end. Rest
mostly gap with one
or two cells + cells
on tissue culture
flask. Media turned
orange.
3.1 is very
dense section.
3.2 is ~ 6mm
from end. Like
3.3 for rest.
Little
clumping 8mm
from 3.1
3.1
3.2
3.3
Polyester + Argon 1 0 mm traces of dead
cells, Media
remained red
no visible cells
1.1

263
Polyester + Argon 2 0 mm traces of dead
cells, Media
remained red
no visible cells
2.1
Polyester + Argon 3 8 mm (7.25mm one
end, 0.75mm the
other end) + cells
on tissue culture
flask. Media turned
orange.
3.1 is one end,
gap
for~10mm,
picture 3.2,
loose until
10mm from
end (picture
3.3) then
confluent and
dense until
end.
3.1
3.2

264
3.3
3.4
3.5
Polyester + ammonia
1
9mm (7mm one end,
dense, other end,
1.25mm, 5.75mm gap
then 0.75mm) + cells
growing on tissue
turned orange.
1.1 is one end.
All the way,
but mostly not
very dense,
like 1.4 &1.5.
1.3 is mid, 1.4
and 1.5 is
either side
1.1

265
1.2
1.3
1.4
1.5
Polyester + ammonia
2
7 mm. Media turned
orange
2.1 is 10mm
from dense
end, then
nothing (like
picture 2.2) to
other end.

266
2.1
2.2
Polyester + ammonia
3
30 mm ( 9mm v.
dense, dense all the
way) + cells growing
on capillary tube +
cells growing on
3.1 is one end,
like that for
5mm, then less
dense like 3.2
for 2mm, then
more dense
like 3.3 for last
5mm
3.1
3.2

268
Day 0 29 30
Date 21/08/2005 19/09/2005 20/09/2005
Polylactic acid 1 Cell attachment at
both ends. Can't
measure, probably all
the way. Cells
on tissue culture
flask. Media turned
orange
fluorescence
indicates
nothing
resembling
live cells
present
1.1
1.2
1.3

269
both ends. Can't
the way. + Cells
on tissue culture
flask. Media turned
orange
if this is cell
growth, then
its all the way ,
patchy in
places(see
picture)
2.1
2.2
2.3
2.4

270
2.5
2.6
2.7
both ends. Can't
the way. + Cells
on tissue culture
flask. Media turned
orange
Picture 3.2 =
one end, 3.4 =
the other,
picture 3.7 =
dense bit.
Cells visible
all the way
3.1

272
3.6
3.7
Polylactic acid +
Argon 1
Cell attachment at
both ends. Can't
the way. + Cells
on tissue culture
flask. Media turned
orange
All the way,
mostly on edge
(avoiding
artefacts)
picture 1.4 and
picture 1.5.
spanning
width
occasionally
1.1
1.2

273
1.3
1.4
1.5
Polylactic acid +
Argon 2
0 mm Media
remained red
1st picture is
cells between
14-18mm
(approximately
) that is all
2.1

274
Polylactic acid +
Argon 3
Cell attachment at
one end, can't
measure + cells
on tissue culture
flask. Media turned
orange
Picture 33 is
typical density.
Picture 3.2 is
1/4 way up.
Cells all the
way, but not
very dense.
One or two
gaps on the
other end.
Probably
ripped off
from removal
from the glass
3.1
3.2
3.3
Polylactic acid +
ammonia 1
Cell attachment at
one end, possibly
other, can't measure +
cells growing on
growing on tissue
turned orange
Cells grew all
the way, but
have been lost
due
mechanical
removal of
sample from
capillary tube.
1.1

275
1.2
Polylactic acid +
ammonia 2
0 mm Media
remained red
no visible cells
2.1
2.2
Polylactic acid +
ammonia 3
Cell attachment at
one end, possibly
other, can't measure +
cells growing on
growing on tissue
turned orange
Picture 3.1 is
one end,
confluent,
same all the
way.
3.1

277
Day 0 29 30
Date 25/08/2005 23/09/2005 24/09/2005
Polypropylene 1 0 or 6 mm (possibly
6mm patch 6mm
from one end, or no
cells.) Media
remained red
no visible cells
No image
Polypropylene 2 Cells attached and
growth along sample.
Can't see clearly
enough to measure. +
Cells growing on
growing on tissue
turned orange /red
There were
more cells.
Appears a lot
have been
ripped off.
Picture 2.2 is
ripped site.
5mm from one
end; 12mm is
empty/ ripped
off. Cells
remain on
10mm of other
end.
2.1
2.2
2.3

278
2.4
Polypropylene 3 Cells attached and
growth up. Can't see
clearly enough to
measure. Cells
on tissue culture
flask. Media turned
orange
Looks like
what cells
were there are
dead by now.
Similar
appearance to
pp2. No live
cells left.
No image
Polypropylene +
Argon 1
0 mm? Media
remained red
no visible cells
No image
Polypropylene +
Argon 2
Definite attachment
one end. Can't see
well enough to
measure. + Cells
on tissue culture
flask. Media turned
orange
Confluent.
Some cells still
alive. 2.1 are
live and dying
cells, 2.2 are
also live and
dying cells.
2.1

279
2.2
Polypropylene +
Argon 3
Can‟t see attachment.
Can tell tomorrow. +
Cells growing on
growing on tissue
turned orange
Confluent and
not very
populated bits.
3.1 is empty
bit. 3.2 is
better bit, 3.3
is as clear as it
gets I think.
Reasonable.
3.3 for 7mm,
virtually
empty for
13mm, like 34
for next 7mm,
last 3mm
pretty empty.
Probably
mechanical
damage from
removal of
sample from
capillary tube.
3.1
3.2
3.3

280
3.4
Polypropylene +
ammonia 1
Definite attachment
one end. Can't see
well enough to
measure. + Cells
on tissue culture
flask. Media turned
orange
Confluent.
Cells start
dying due to
length of time
required to
examine all of
the samples.
Picture 1.1
shows some
cells alive on
the other side
with a gap
from removal
from the
capillary tube.
1.1
1.2
Polypropylene +
ammonia 2
0 mm Media
remained red
no visible cells
No image

281
Polypropylene +
ammonia 3
Definite attachment
both ends. Can't see
well enough to
measure. + Cells
on tissue culture
flask. Media turned
orange
3.1 and 3.2
unclear
(external side).
Confluent but
cells dying on
external side.
Cells still alive
on inner side.
3.1
3.2
3.3
Polypropylene +
chitosan 1
Definite attachment
well enough to
measure. + Cells
on tissue culture
flask. Media turned
orange
Confluent but
for gaps where
removal from
capillary tube
caused holes.
1.1 and 1.2
1.1

282
1.2
Polypropylene +
Chitosan 2
Definite attachment
well enough to
measure. + Cells
on tissue culture
flask. Media turned
orange
Confluent in 3
bits. Looks
like rest has
been ripped
off. 2.1 and 2.2
are ripped bits.
2.1
2.2
Polypropylene +
Chitosan 3
0 mm Media
remained red
no visible cells
No image

283
Polypropylene +
Argon + Chitosan 1
Definite attachment
well enough to
measure. Cells
on tissue culture
flask. Media turned
orange /red
0 (practically)
like 1.1 and
1.2 for all. V.
poor)
1.1
1.2
Polypropylene +
Argon + Chitosan 2
0 mm (peeling
chitosan film. Think,
almost def, none
Media remained red
no visible cells
No image
Polypropylene +
Argon + Chitosan 3
0 mm. Media
remained red
no visible cells
No image
Polypropylene +
ammonia + Chitosan
1
0 mm. Media
remained red
no visible cells
No image
Polypropylene +
ammonia + Chitosan
2
0 mm. Media
remained red
no visible cells
No image

284
Polypropylene +
ammonia + Chitosan
3
Definite attachment
well enough to
measure. cells
on tissue culture
flask. Media turned
orange
just along edge
(like bottom
right of 3.1
and 3.2 except
for 2mm (3.1)
3.1
3.3

285
Day 0 29 30
Date 25/08/2005 23/09/2005 24/09/2005
Polycaprolactone 1 0. Very few cells
tube (~12 cells max).
Media remained red
no visible cells
No image
Polycaprolactone 2 Possible attachment.
Measure next day.
Cells growing on
growing on tissue
turned orange /red
Very few cells.
2.1
2.2
polycaprolactone 3 0 mm. Media
remained red
no visible cells
No image
polycaprolactone +
Argon 1
8 mm + cells growing
on capillary tube +
cells growing on
Media turned orange
10mm not very
dense. 1.1 is
end of cells,
1.2 is middle
of cells
1.1

286
1.2
polycaprolactone +
Argon 2
Contaminated
therefore discarded No image
polycaprolactone +
Argon 3
Contaminated
polycaprolactone +
ammonia 1
30 mm + cells
on tissue culture
flask. Media turned
orange
confluent all
the way
1.1
1.2
polycaprolactone +
ammonia 2 No image

287
polycaprolactone +
ammonia 3
30 mm (lots of
attachment both ends,
all the way. (Cells
on tissue culture
flask). Media turned
orange
Cells grew all
the way.
Mostly
confluent. 1.2
is healthy bit.
3.1
3.2
Solanyl 2% Chitosan
2
30 mm Lots of cells
on tissue culture
flask. Media turned
orange
16 definitely
cells, a lot
(optical
microscope)
No image
Solanyl 2% Chitosan
3
0 mm. Media
remained red
no visible cells
No image
Solanyl + Chitosan 1 Few cells growing on
growing on tissue
culture flask,
probably 0. Media
turned orange
4 or 5 clusters
of cells,
nothing
directly
measurable
1.1

288
1.2
Solanyl + Chitosan 2 Few cells growing on
capillary tube, lots of
floating cells,
probably 0. Media
turned orange
no visible cells
No image
Solanyl + Chitosan 3 17.5 mm in clusters
(6.3, 4& 4.5 mm) +
cells growing on
growing on tissue
turned orange
Clusters of
cells
No image
Solanyl + Argon +
Chitosan 1
Nothing visible.
Media remained red
no visible cells
No image
Solanyl + Argon +
Chitosan 2
4 mm in clusters (1.5,
0.5, 2 mm) + cells
tube. Media turned
orange
Clusters of
cells
2.1

289
2.2
Solanyl + Argon +
Chitosan 3
26 mm in two
clusters (8, 2mm gap
then 18 mm) + cells
on tissue culture
flask. Media turned
orange
Cells visible
all the way
along sample.
3.1
3.2
Solanyl + ammonia
+ Chitosan 1
12-30 mm + cells
on tissue culture
flask. Media turned
orange
Cells visible
all the way
along sample
with little gaps
probably from
removal from
glass tube) No image

290
Solanyl + ammonia
+ Chitosan 2
30 mm, lots of cells
on tissue culture
flask. Media turned
orange
Cells visible
all the way
along sample
with little gaps
probably from
removal from
glass tube)
2.0
Solanyl + ammonia
+ Chitosan 2
30 mm lots of cells
on tissue culture
flask. Media turned
orange
Cells visible
all the way
along sample
with little gaps
probably from
removal from
glass tube) No image

291
Day 0 29 30
Date 25/08/2005 23/09/2005 24/09/2005 No image
Solanyl 1 Sample detached at
one end. Cells
growing all the way
along sample. Cells
at both ends, + cells
on tissue culture flask
Media remained red
Confluent for
all but last part
of sample. Still
plenty of cells,
just not all the
way. Possibly
ripped off.
No image
Solanyl 2 Cells present on
detached end, not
much else + cells
on tissue culture
flask. Media
remained red
no visible cells
No image
Solanyl 3 Contaminated
Solanyl + Argon 1 Contaminated
therefore discarded
No image
therefore discarded
No image
Solanyl + ammonia
1
Contaminated
therefore discarded
No image
Solanyl + ammonia
2
Contaminated
therefore discarded
No image
Solanyl + ammonia
3
Contaminated
therefore discarded
No image
Solanyl 2% Chitosan Contaminated No image

292
1 therefore discarded
Solanyl 2% Chitosan
2
Contaminated
therefore discarded
No image
Solanyl 2% Chitosan
3
Contaminated
therefore discarded
No image
Solanyl + Chitosan 1 4mm gap, 2mm,
2mm gap, 2mm,
2mm gap, 2mm.
4mm other end +
cells growing on
growing on tissue
culture flask . Media
turned orange
Picture 1.1 is
dead bit, 1.2 is
part of
confluent bit.
1.1
1.2
Solanyl + Chitosan 2 0 mm. Media
remained red
Solanyl + Chitosan 3 0 mm. Media
remained red
Solanyl + Argon +
Chitosan 1
0 mm. Media has
remained red

293
Solanyl + Argon +
Chitosan 2
8 mm (8mm dense,
then can't see well
enough. Probably a
lot more) + cells
on tissue culture
flask. Media turned
orange /red
Mostly
confluent (2.1
is loose bit, 2.2
is confluent
bit.
2.1
2.2
Solanyl + Argon +
Chitosan 3
30 mm confluent all
the way. Cells
on tissue culture
flask. Media turned
orange /red.
Confluent all
the way.
No image
Solanyl + ammonia
+ Chitosan 1
0 mm. Media
remained red
No visible
cells.
No image
Solanyl + ammonia
+ Chitosan 2
30 mm (confluent all
the way I think,
pretty sure) + cells
on tissue culture
flask. Media is
orange/ red.
3x2mm gaps
in confluence.
Still cells.
Probably due
to removal.
(Picture 2.2,
cells conf)
No image
sol + ammonia + 0 mm. Media no visible cells No image

295
Day 0 29 30
Date 25/08/2005 23/09/2005 24/09/2005
Polyurethane 1 25 mm (5 mm gap,
2mm from one end.
Rest is confluent.
Cells growing on
growing on tissue
turned orange
1.1 is one end.
Patchy like 1.1
for 4mm, gap
for next
10mm, patchy
for last 16mm.
Confluent and
loose bits like
1.2, 1.3 & 1.4
1.1
1.2
1.3

296
1.4
Polyurethane 2 0 mm. Media
remained red
no visible cells
No image
Polyurethane 3 0 mm. Media
remained red
no visible cells
No image
Polyurethane +
Argon 1
2mm + cells growing
on capillary tube, no
cells growing on
Media remained red
2mm
1.1
Polyurethane +
Argon 2
0 mm. Media
remained red
no visible cells
No image
Polyurethane +
Argon 3
the way.) Cells
on tissue culture
flask. Media turned
orange
Confluent
except for rips.
Live and dying
bits. 3.1 has
both, 3.2 is
dying bit, 3.3
is clear bit
3.1

297
3.2
3.2
3.4
Polyurethane +
ammonia 1
5.25 mm (4mm
confluent. Other end,
2mm gap, 0.75mm,
0.5mm gap, 0.5mm)
+ cells growing on
growing on tissue
turned orange
1.1 is isolated
patch at one
end. Like 1.2
for first 6mm
other end.
Cells only 1/3
way up width.
Probably the
strip was tight
1.1

298
against the
tube
prohibiting
further
spreading.
1.2
Polyurethane +
ammonia 2
the way.) cells
on tissue culture
flask. Media turned
orange
All the way.
Mostly
confluent.
Gaps both
sides, but cells
on both sides.
All the way.
2.2 is gap/
ripped bit 2.1
2.2
Polyurethane +
ammonia 3
the way.) + Cells
on tissue culture
flask. Media turned
orange
confluent for
first 7mm,
4mm ripped
but with cells,
2mm
confluent,
14mm mostly
empty/ few
cells
3.1

299
3.2
Polyurethane +
Argon + Chitosan
powder 1
Can‟t measure
accurately. Plenty of
cells, but some ripped
off from moving
flask. + Cells
on tissue culture
flask. Media turned
orange
Mostly empty.
Either poor
growth or lost
due to ripping.
1.1 is best bit;
the rest is
almost empty/
scattered.
4x2mm one at
each end, 1.1
and other
dying bit.
Mostly empty.
Rest like 1.2 at
best.
1.1
1.2
Polyurethane +
Argon + Chitosan
powder 2
0 mm. Media
remained red
Polyurethane +
Argon + Chitosan
powder 3
0 mm. Media
remained red
Polyurethane +
Argon @ Galashiels
0 mm. Media
remained red

300
1
Polyurethane +
Argon @ Galashiels
2
0 mm. Media
remained red
Polyurethane +
Argon @ Galashiels
3
0 mm (little cells
tube & cells growing
on tissue culture
flask). Media
remained red
Polyurethane +
Oxygen @ Galashiels
1
12 mm + (12mm
confluent/ patchy.
6mm gap, then can't
tell. Plenty on other
end.) + Cells growing
on capillary tube +
cells growing on
Media turned orange
4.5mm
confluent to
not (1.1 is
bloody
unclear.), 6mm
form other end
is 3mm
cluster. Poorly
stained.
1.1
Polyurethane +
Oxygen @ Galashiels
2
0 mm (2-3cells on
flask, little + cells
tube. Media turned
orange
no visible cells
No image
Polyurethane +
Oxygen @ Galashiels
3
30 mm (all the way
confluent except
2mm patchy 2mm
from one end). Media
turned orange
28 (2mm gap
2mm from one
end. Some
cells still alive
8hrs after
staining)
3.1

302
Day 0 29 30
Date 25/08/2005 23/09/2005 24/09/2005
Polylactic acid 1 Can‟t measure. (Free
floating. Both ends
have cells. Few cells
growing on tissue
culture flask, + cells
tube. Media turned
orange
confluent all
the way
No image
Polylactic acid 2 Cells on one end
contracting sample.
Can't measure.
Probably not on other
end. Cells growing
on tissue culture
flask. Media turned
orange
11mm.
Confluent to
not dense. 1.1
is healthy bit,
1.2 where cells
have started
dying
No image
Polylactic acid 3 0 mm. Media
remained red
Polylactic acid +
Argon 1
0 mm. Media
remained red
Polylactic acid +
Argon 2
0 mm. Media
remained red
Polylactic acid +
Argon 3
0 mm. Media
remained red
Polylactic acid +
ammonia 1
0 mm. Media
remained red
Polylactic acid +
ammonia 2
0 mm. Media
remained red
Polylactic acid +
ammonia 3
0 mm. Media
remained red

303
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