1

An Investigation into the Effects of

Sterilisation on Poly(caprolactone) for

In Vitro and In Vivo Medical Use

A Dissertation Submitted to the University of Manchester for the Degree of Master of

Science by Research in the Faculty of Engineering and Physical Sciences

2013

Adam Philip Strange

School of Materials

2

Declaration

The author declares that no portion of the work referred to in the dissertation has been

submitted in support of an application for another degree or qualification of this or any

other university or other institute of learning.

Copyright

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administrative purposes.

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Presentation of Dissertations and with the consent of the author.

3

CONTENTS

Declaration ................................................................................................................................................... 2

Copyright ...................................................................................................................................................... 2

Contents ............................................................................................................................................................. 3

Abstract .............................................................................................................................................................. 4

Chapter 1 Introduction, Literature Review and Aims ..................................................................... 5

1.1 New Introduction ............................................................................................................................... 5

1.2 Electrospinning ................................................................................................................................... 8

1.3 Polymers for Electrospinning ..................................................................................................... 13

1.4 Sterilisation ........................................................................................................................................ 18

Chapter 2 : Materials and Methods ....................................................................................................... 30

Chapter 3 : Results ....................................................................................................................................... 36

3.1 Fourier-Transform Infrared Spectroscopy ............................................................................ 36

3.2 Differential Scanning Calorimetry ............................................................................................. 39

3.3 Tensile Testing .................................................................................................................................. 41

3.4 Cell Culture ......................................................................................................................................... 44

3.5 Gel Permeation Chromatography .............................................................................................. 46

3.6 Water Contact Angle ....................................................................................................................... 47

3.7 Scanning Electron Microscopy .................................................................................................... 49

Chapter 4 : Discussion and Conclusion ................................................................................................ 54

Appendix A – FTIR Data ............................................................................................................................. 66

Appendix B – GPC Data .............................................................................................................................. 71

Appendix C – Contact Angle Data ........................................................................................................... 73

Chapter 5 : References ............................................................................................................................... 74

Word Count: 17,126

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Abstract

The requirements on any implanted biomaterial are strict. Amongst these, the

biomaterial must be sterile. In order to achieve this, there are several methods which

are recognised as being suitable for sterilisation, amongst which are gamma irradiation

and ethylene oxide. Poly(caprolactone) is a polymer which is regularly used as a

biomaterial; it is safe, readily available, has good handling properties and is

biocompatible. This study sought to investigate the effects of two lab based sterilisation

methods, ethanol and ultraviolet irradiation, and compare them to the effects of

sterilisation by gamma irradiation and ethylene oxide sterilisation. Significant changes

in physical behaviour, such as mechanical properties and cell culture response were

reported. There were minimal gross chemical affects reported. No one sterilisation

method could fully replicate the effects of another, but in some areas suitable

replacements could be found; ethylene oxide, for example, was found to be a poor

scaffold in terms of cell culture. Overall, it was decided that papers which report the

effects of one type of sterilisation on PCL can only be used in certain circumstances to

decide the effects of another type of sterilisation. The important factor is to look at

which particular tests are being run to determine whether or not a particular

sterilisation method is suitable.

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Chapter 1 Introduction, Literature Review and Aims

1.1 Introduction

Any non-living device or construct which comes into contact with the body is known as

a biomaterial. They range from mundane low-risk disposable items, for example

plasters and bandages (usually known as Class I), to complexly engineered long-term

devices such as total hip replacements and cardiac pacemakers (Class III.) Typically,

these biomaterials can be grouped into several classes, depending on the properties of

the role they must fulfil, and the dangers posed to patients should issues arise. The

clinical need for safe and effective implanted biomaterials (Classes II and III) worldwide

cannot be underestimated; billions of dollars each year is spent on surgeries and after-

care of patients with similarly large amounts of money being spent on researching new

and improved biomaterials. This research translates into a moderate number of new

devices being approved for full clinical use each year; the Food and Drug Administration

(FDA) in the United States permitted 47 devices in 2012, its highest number in recent

years (FDA 2012). Similar numbers of devices have been approved by the Medicines

and Healthcare Regulatory Agency (MHRA, the regulatory body of the United Kingdom)

per year, with 7774 devices permitted for “full clinical use” as of June 2013 (MHRA

2013). Patient confidence is maintained by regulatory bodies such as these, and the

rigorous protocols they enforce to ensure the highest levels of safety for all involved.

Mistakes, errors and negligence however, have reciprocally large effect on the

effectiveness of the devices and on patient confidence. The breast implant scandal

caused by the French company Poly Implant Prothèse (PIP), who used non-medical

grade silicone is an important reminder of this; despite no recorded illnesses due to the

implants, the fact that the high standards were actively ignored caused mass panic and

concern. Strong regulation and adherence to the regulations ensures that patients,

clinicians and scientists are all protected.

To that end, detailed investigations are carried out on all parts of an implant, so that

their properties and behaviour can be well characterised. Mechanical properties,

6

chemical makeup, and degradation are all topics that are commonly investigated,

However recently a smaller, but by no means less important, number of papers look at

more niche topics, chief amongst which is the effects of sterilisation on an implant.

If any modern biomaterial will find its way into clinical use, then it is essential to make

sure that it is safe and will perform effectively; clinical trials are the usual testing

methods that is used (Olbrich et al. 2007; Ebersole et al. 2012). Testing before clinical

use is common-place; it stands to reason that any experiment in vitro should seek to

emulate the conditions in vivo. With polymers, sterilisation can change the percentage

crystallinity, molecular weight and mechanical properties of the polymers due to

effected chemical changes (Narkis 1984; Cottam et al. 2009; Rogers 2005). The effects

on metals are not as well characterised, but sterilisation is generally understood to have

the chance to form surface oxides or allow defects to expand in vacancy sites (Adrian

and Gross 1979; Hamilton 2013; Rogers 2005). Methods have been developed for fluid

sterilisation, although these tend to use sterilisation techniques which are similar to

solids (Rogers 2005; Salmisuo and Petterson 2012) If any biomaterial would be

translated from the lab to clinical use, it would only be through rigorous testing of all of

its properties and functionality post-sterilisation (Cottam et al. 2009; Bosworth, Gibb,

and Downes 2012). It is important to note that any sterilisation method could

potentially have a significant effect on the mechanical and chemical properties of the

biomaterial; the effects of sterilisation are an area that is still not well characterised

(Cottam et al. 2009).

Focusing on manufacturing techniques that have been used to make implantable

scaffolds, there are several key methods, for example extrusion, rapid prototyping, salt

leaching, and electrospinning (Pham, Sharma, and Mikos 2006; Weir et al. 2003; Sabir,

Xu, and Li 2009). For the purpose of this research, the focus will be on electrospinning,

and the electrospinning of poly(ε-caprolactone)(PCL) in particular, although in addition

poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA) are also used. All three of

these polymers are biodegradable and easily electrospun (Li et al. 2002; Aghdam et al.

2011). Electrospinning is a technique by which a solvated polymer is expelled from a

needle, passing through an electrical field before being deposited as fibres onto a

collector (Reneker et al. 2000; Doshi and Reneker 2011; Yang et al. 2005). The original

7

experiments with the technique for widespread clinical use gave rise to aligned fibres if

a rapidly rotating collector was used, encouraging use for tendons, muscles and other

naturally aligned materials (Bhardwaj and Kundu 2010; J.-F. Liu and He 2010; Kumbar

et al. 2008). More advanced techniques have allowed for needless electrospinning and

two pole air gap electrospinning; giving rise to highly aligned fibres whilst allowing for

3D cell culture to occur (Doshi and Reneker 2011; Jha et al. 2011).

In choosing the correct polymer to make, it is important to link in handling

characteristics, polymer behaviour during use, and degradation reactions (Seretoudi,

Bikiaris, and Panayiotou 2002; Doshi and Reneker 2011; Li et al. 2002; Cipitria et al.

2011). If the electrospun polymer is to be used in vivo in any way, as would be expected

in the context of this research, then the decision must also include regulatory approval

and biocompatibility studies.

Modern biomaterials are almost exclusively implanted with cells incorporated into the

overall structure; the scaffold must allow for good culture conditions for cells, especially

if the culture is to be considered for in vivo work (Hemmrich et al. 2005; Kumbar et al.

2008; Li et al. 2002). To this end, experiments often use cell infiltration and

proliferation assays in order to determine cell viability on the scaffold. (Liao et al. 2006).

A scaffold which promotes cell viability is more likely to be successful than a scaffold

which actively hinders cell growth and proliferation (Olbrich et al. 2007; Cipitria et al.

2011).

This work will incorporate all of the aspects and themes above, and hopes to provide

more information with regards to sterilisation and how it can affect scaffolds, with a

potential knock-on effect for in vivo use for biomaterials. Analysis will be performed on

all levels, with a specific interest into the physcio-chemical effects on the polymer of

sterilisation.

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1.2 Electrospinning

1.2.1 The Process of Electrospinning

Electrospinning in medicine and life sciences has developed over centuries as an

efficient process which can mimic the extra-cellular matrix (ECM) structure in vitro;

often an implanted biomaterial will try to either mimic or replace previous tissues

(Cipitria et al. 2011; Liao et al. 2006). The ultrafine fibres with nanotopographical

features allows for natural adherence of cells onto the scaffold, disproportionally high surface

area:volume ratios allows for a high cell density, with pore sizes comparable with that found

in the ECM (Kumbar et al. 2008; Aghdam et al. 2011). The polymer is ejected from the

needle, and receives a high voltage, causing a highly randomised jet (the Taylor cone) to

be attracted to the grounded collector plate (Qin and Wu 2011; Croisier et al. 2012).

Figure 1.1 - Generic Electrospinning Diagram –this shows an average electrospinning rig. The pump

ejects a polymer at a set rate through the syringe, which is then passed through an electrical current

before being collected after having travelled a certain distance.

1.2.2 Historical Uses of Electrospinning

One of the principle discoverers of electricity and magnetism, William Gilbert, first

reported the techniques and science behind electrospinning and electrospraying in the

late 16th/early 17th centuries (Gilbert 1600). Few records of further work survive,

although Gilbert’s work helped shape our understanding of electricity and charge; he

reports that amber (which is naturally electrostatic) exerted a force against a droplet of

water, that was proportional to the distance between the two objects (Tucker 2012).

9

Later, the prodigious scientist Robert Hooke, theorised that the “cell” structures he had

discovered in biological entities could be replicated by a ductile “glutinous composition”

(Tucker 2012). Further work on this topic was reported at what would later become the

University of Manchester; the researchers’ stated aims being to discover a ductile and

malleable organic substance to be drawn into fibres for manufacturing; nitrocellulose

(fulfilling the requirements) was discovered by accident within a decade (The

Manchester Guardian 1840; Tucker 2012). Although the number of uses, papers and

patents involving electrospinning has increased significantly since the turn of the 21st

century, the first recorded mention of electrospinning as a technique was in 1901, with

further work performed in one of the last German-USSR collaborations before their

conflict in the Second World War, the end result being a successful electrospinning

process in 1938 (Tucker 2012) .

Continuing throughout and beyond the Second World War, and doubtless spurred on by

novel research in what would eventually become the first synthetic biomaterial (PLGA),

electrospinning was repeatedly suggested as a technique which would vilify Hooke’s

suggestion of a replaceable cell structure (Tucker 2012). Codified mathematical

modelling into the fluid flow was set by Geoffrey Taylor, for whom the Taylor cone in

electrospinning was named. It was not until the 1990s that the nanotechnological

aspects of electrospinning were promoted, with the name of electrospinning entering

(relatively) common parlance in scientific circles (Cooley 1902; Kumbar et al. 2008; Qin

and Wu 2011; Cipitria et al. 2011; Tucker 2012). After several years and some

convoluted research, it was shown that there was definite promise for electrospinning

as a ‘novel’ technique for tissue engineering; a field that was at the time captivating the

imaginations of the Press, public and scientists as a whole, mostly with regards to

famous mainstream media publications such as ‘Vacanti’s Ear’ (Li et al. 2002; Matthews

2002) As explained above, the similarity of the electrospun fibres to collagen in the ECM

allowed for a level of biomimicry that stimulated research into skin, vessels, bone,

cartilage and neural areas of regeneration (Hutmacher and Cool 2007). Since the mid-

1990s, research into electrospinning has expanded exponentially; literature searches

show tens of thousands of papers within the last 15 years. For reasons of brevity and

readability, only key review and research papers from recent years will be included

here.

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Electrospinning was not, however the only nanomesh/nanofibre construction technique

present at the time; there was considerable research into self-assembly and phase

separation – both of which had already shown some successes with regards to in vitro

testing (Cipitria et al. 2011). What set electrospinning apart was the natural formulation

of a high surface:volume area, which was useful for both cells and drug delivery

processes, and a level of naturally variable porosity, essential for mass transport and

macromolecular exchange (Szentivanyi et al. 2011; Vaz et al. 2005; Bianco et al. 2011;

Baati et al. 2012; Deitzel et al. 2001). Another advantage is allowing a co-

electrospinning of various agents, both natural and synthetic. Previous concepts that

have been suggested include the addition of core-sheath nanofibres, which would allow

for a phase transition effect for electrospinning (Luo et al. 2012).

At a glance, one can see a relatively common reporting in papers of this conjoined

technique; PLGA co-spun with PCL, gelatine and collagen, whilst PCL has been found

with all of the above, and Ca-P ceramics, further showing the flexibility of the technique

(Cipitria et al. 2011; K. Kim et al. 2004; Dahlin, Kasper, and Mikos 2011; Subbiah et al.

2005).

1.2.3 Electrospinning Parameters

Analysis of the literature shows several general and widely agreed upon characteristics

of electrospinning; these which often act as constraints for the electrospinning process.

(Thompson et al. 2007; K. Kim et al. 2003). The most fundamental issue with

electrospinning is the low turnover and volume production that can occur (low rates at

approximately 2 kg to higher of 30 kg have been reported); a significantly lower mass

when compared with other materials (Fang et al. 2010). Production values are often

limited on the basis of lower density for most electrospin polymers and therefore low

weight of the polymers, when compared with other materials such as metals or

ceramics (Rnjak-Kovacina and Weiss 2011; Vaquette and Cooper-White 2011).

Secondly, processing control is limited by a reliance on uncontrolled variables during

fibre deposition, often caused by the dynamic interactions in the polymer jet

(Thompson et al. 2007). This can lead to bead formation, and investigations into

limiting such interactions have often proved to be inconclusive (Thompson et al. 2007;

Yarin and Zussman 2004).

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For example, in work with electrospinning hyaluronic acid ,it was found that increasing

air-flow around the polymer jet lowered bead formation and increased the percentage

of nanofibres that were spun (Wang et al. 2005). Other investigations have suggested

that low surface tension is the primary case of bead formation, and that by controlling

the solution concentration and adding ionic salts, the effect of bead formation can be

reduced or eliminated (Y. Liu et al. 2008). During cell seeding experiments, it was found

that bead artefact formation occasionally assisted in cell culture; it was hypothesised

that the beads allowed for a larger surface area for cell attachment (Deitzel et al. 2001;

Wang et al. 2005).

Fibre diameter ranges are often highly unpredictable at both a given location and across

the electrospun mesh as a whole (Thompson et al. 2007). One paper reports wide

variations in dry fibre diameter; from between 1.15 μm and 6.65 μm (Fridrikh et al.

2003). This variation can hinder detailed characterisation of an electrospun scaffold;

changes in fibre diameter after sterilisation could be absolute (that is, a certain value of

diameter is gained or loss due to chemical changes) or it could be dependent on the

original diameter of the fibre (a percentage change which could be an increase due to

swelling, or decrease due to chemical scission) (Thompson et al. 2007; Bhardwaj and

Kundu 2010; Subbiah et al. 2005). Polymer solution rheology is often dependent on the

complicated interactions between the polymer and the solvent (Dahlin, Kasper, and

Mikos 2011; Thompson et al. 2007).

Although well characterised, it is important to note that the interactions between the

chemicals and the physical machinery in electrospinning can limit the ease of formation

of patterning; this can then limit the level of cell attachment which is possible (Fatih-

Canbolat et al. 2011; Schenke-Layland et al. 2011; Thompson et al. 2007). Whilst

randomly formed fibrous mats provide a weak level of biomimicry which can assist in

cell proliferation, this work will focus on connective tissues, requiring aligned fibres

(Kumbar et al. 2008; Skotak et al. 2011; Fatih-Canbolat et al. 2011).

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Table 1.1 Problems Facing Electrospinning

Problem Effect Comment Solution

Low turnover

(compared with

other material

techniques)

Must repeat

manufacturing

several times

Could lead to

inconsistencies in

the final product

Larger-scale

electrospinning

machines, effective

use of samples

Undefined variables

(especially surface

tension)

Uneven fibre

distribution, bead

formation

Final product may

have varied surface

properties

Increase air-flow to

raise surface

tension

Fibre diameter Uneven fibre size

and distribution,

variable mechanical

properties

Final product could

have an overly

complex surface,

unneeded when

aligned fibres are

required

Controlled chamber

environment, small

needle-collector

distance

As with all physical manufacturing, there are some input parameters that are often used

to provide a basis for electrospinning. Again, as above, there are several different

standards of parameters, but thirteen stand out across many papers. These are

volumetric charge density, initial elongation viscosity, distance from nozzle to collector,

solvent vapour pressure, initial polymer concentration, density, surface tension

(between the polymer/solvent and air), electrical potential, relaxation time, humidity of

solvent vapour in air, perturbation frequency, jet radius (at the initial, intermediate and

final timepoints) (Thompson et al. 2007; K. Kim et al. 2003; Ekaputra et al. 2011;

Bhardwaj and Kundu 2010; Rnjak-Kovacina and Weiss 2011; Sundararaghavan and

Burdick 2011; Cipitria et al. 2011). These parameters are sometimes reported,

sometimes changed or sometimes ignored, depending on the level of detail of the

investigation, and the importance that electrospinning has been given. There are

reasonable limitations to the amount of alteration that each parameter can be subjected

to: polymer concentration, electric charge and the end-to-end distance between the

nozzle and the collector comprise three parameters that could be simply changed.

13

Other parameters often require advanced electrospinning machines to be measured or

changed (Thompson et al. 2007). Again, it may be considered that precise

measurements of every parameter has no practical value, especially when considering

clinical work (Aghdam et al. 2011).

Polymer concentration concentrations are rarely lower than 5% or higher than 30% for

practical purposes in terms of electrospinning (Thompson et al. 2007). Concentrations

lower than 5% often contain too little polymer for an effective electrospun surface,

whilst an excess of 30% polymer is normally to viscous and cannot be deflected by the

electrical field in electrospinning (Thompson et al. 2007)

As a process the use of electrospinning generally aims to form a single randomised

mesh of nanofibres, with no particular order or control over bead formation, nanofibre

diameter, length or any other physical property (Thompson et al, 2007; Bhardwaj et al,

2010; Kim et al, 2004; Vaz et al, 2005). A polymer, then, can be electrospun successfully

into a mat or into aligned fibres but a higher degree of control is not currently available.

1.3 Polymers for Electrospinning

The selection of a polymer for electrospinning for use as a biomaterial is widely

considered to be highly important, and there are many different properties that the

polymer must possess. For this particular research, the polymer must be biocompatible,

easily degradable, with well-characterised mechanical and chemical properties. In

addition, the aim of this work is not to discover a new material or improve on the

properties of already available materials, so the polymer selected must currently be

used clinically with a high level of success, and hold statutory approval from bodies

such as the FDA or a CE mark.

To this end, PCL has been selected for the electrospining. PCL is a ubiquitous polymer,

as has often been used in biomaterials and regenerative medicine. It is semi-crystalline

and has often been used to make degradable and absorbable implants and sutures for

surgical use. A level of elastic response is granted to an implant due to the semi-

crystalline nature of the polymer, essential if the implant has to be compressed or

forced into a particular area (Cipitria et al. 2011).

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PCL is a suitable polymer for electrospinning, and with the correct concentration of

solution, can easily allow for directed and aligned nanofibre construction, through a

process of chain entanglements (Qin and Wu 2011). It has been noted several times that

an essential parameter in the electrospinning process of PCL is in fact the choice and use

of the solvent. For high levels of biocompatibility, suitably solvent nature and low

temperature and ease of volatilisation, 1,1,1,3,3,3hexafluro-2-propanol (HFIP) has often

been cited as the correct choice of solvent for electrospinning PCL; despite the highly

toxic nature and carcinogenic nature of HFIP, volatilisation can rapidly occur, leaving

HFIP a safe polymer to use (Szentivanyi et al. 2011; Subbiah et al. 2005; Liao et al. 2006;

Kidoaki, Kwon, and Matsuda 2005; Fatih-Canbolat et al. 2011). Cell viability has been

suggested to be higher with HFIP as the solvent than any other; this indicates a

preference for HFIP when cell work is to be carried out, despite some solvents showing

a better overall profile of results from electrospinning (Fatih-Canbolat et al. 2011;

Szentivanyi et al. 2011; S. J. Kim et al. 2010; Cipitria et al. 2011; Bhardwaj and Kundu

2010).

With the increase into research of electrospun PCL, there has been a significant increase

in the number of applications in the regenerative medical field. Having received FDA

approval for many years already as sutures, PCL products are often seen as more

attractive when looking into clinical trials for different areas. If a commonly found

preparation of PCL is used for the new biomaterial product, then this can often present

a slightly shorter route through pre-clinical testing, both in vitro and in vivo (Sun et al.

2010). Most modern uses now require a coating or post-manufacture

modification/processing for added effect, often focusing on adsorption of proteins and

other biomolecules onto the surface (Cipitria et al. 2011). Studies have shown the use of

collagen, laminin, heparin, and gelatine, amongst others, to be successful in allowing a

good level of surface interaction between the implant and native biochemical factors in

situ (Liao et al. 2006; Dahlin, Kasper, and Mikos 2011; Ma, Mao, and Gao 2007). Coatings

can also have a direct effect on the growth of the tissues around the implant; neurite

growth after laminin deposition has been cited as a notable effect (Ji et al. 2013; Cipitria

et al. 2011; Sundararaghavan and Burdick 2011; Sun et al. 2010).

15

It is not always, however, practical to coat an implant before testing; depending on the

deposition technique used, the natural porosity of PCL (a good characteristic of the

polymer) may be lost due to a large sized coating for example (Liao et al. 2006; Jha et al.

2011; Vaz et al. 2005; Kumbar et al. 2008).

When an electrospun PCL-derived implant is needed for tissues with an inherent

directional order to them (e.g. tendons, ligaments and nerves), aligned electrospinning

can be used to form electrospun fibres in a certain direction (Yang et al. 2005). Various

techniques have been designed to accomplish this, often the collecting mandrel is

rotated at a high enough speed (the minimum has been reported to be anywhere in

between the region of 500 and 1000 RPM) to cause deposition of the fibres in an aligned

pattern (Kidoaki, Kwon, and Matsuda 2005; Fatih-Canbolat et al. 2011; Seretoudi,

Bikiaris, and Panayiotou 2002).

1.3.1 Characterisation Techniques for PCL Analysis

With all the possible variables in the electrospinning process of PCL, it is important that

the final product is suitably characterised, so that the specific properties of that material

can be factored in to any analysis, especially if the material will have a clinical use (Vaz

et al. 2005; Lasprilla et al. 2010).

Often, the most important characterisation factor that a material might have is its

resistance to mechanical load. A material will be subjected to mechanical forces during

the implantation surgery and during tissue growth (Cottam et al. 2009; Liao et al. 2006).

PCL implants must resist any load and not lose their function; uniaxial tensile testing is

often employed to find the average maximum loads that scaffolds can endure. Results

from this testing will often take the form of a stress-strain curve, from which Young’s

modulus (E), yield stress (σs), yield strain (ε), fracture deformation stress (σf), fracture

deformation strain (εf) can be obtained.

For high load applications involving large scaffolds (skeletal applications in particular),

an energy release per volume through failure (the integration of a stress-strain curve)

may be helpful (Cipitria et al. 2011; Levenberg et al. 2005). The standard testing

16

technique does have some drawbacks however. With regards to clinical use one of the

chief criticisms that could be levelled at traditional uniaxial tensile testing for PCL is the

use of standard polymeric testing conditions (Seretoudi, Bikiaris, and Panayiotou 2002;

Qin and Wu 2011).

50% humidity and room temperature (taken as between 23-25°C) as standard testing

conditions do allow for relatively easy repeatability, but fail to replicate physiological

conditions to which any biomaterial would be subjected. Proposed solutions to this

include raising the testing environment to 37°C (this would cause an increase in PCL

stretching) or conditioning the PCL in a standardised culture medium before tensile

testing. (Cipitria et al. 2011; Seretoudi, Bikiaris, and Panayiotou 2002). The selections of

media for conditioning a scaffold could also be specific to the eventual clinical role of the

scaffold, with the conditioning being used under a similar rationale as conditioning for

cell seeding – a more biomimetic environment allows for results that can better model

physiological conditions, so can be referred to easier when looking at in vivo and in vitro

testing.

Chemical composition is a significant factor in the behaviour of any material, and

directly links into almost all other properties that a material might have (Hemmrich et

al. 2005; Masson et al. 1997; Sabir, Xu, and Li 2009). An effective but occasionally

destructive method of testing chemical composition is to use ATR Fourier Transform

Infrared Spectroscopy (FT-IR), which can detect the molecular weight of a polymer

sample, as well as show if any additives to the electrospinning process have being

incorporated into the scaffold (Cipitria et al. 2011). It has been shown that different

processes techniques and inclusion of additives to a sample can alter the chemical

structure and therefor the sample’s behaviour; residual ethanol from lab sterilisation

and cleaning, for example, can lead to a change in behaviour of PCL and could also affect

any cells that were being seeded onto the scaffold. (Vaquette and Cooper-White 2011;

Yun et al. 2004; Cipitria et al. 2011). FT-IR requires the use of an imaging crystal to be

placed close to a sample, with a depth of analysis that allows for the imaging of the

whole electrospun fibre, not just the surface, as is common with other classes of

materials (Cipitria et al. 2011). However, the requirement for such close proximity of

the imaging crystal to the fibres often crushes them, rending the sample destroyed

17

(Aghdam et al. 2011). Penetration of the FT-IR evanescent wave is approximately 1 μm,

which is sufficient to measure the electrospun mats (Aghdam et al. 2011)

The percentage or level of crystallinity of PCL will greatly affect the tensile strength of

any scaffold (Qin and Wu 2011). Analysis of this is done by means of differential

scanning calorimetry (DSC), where a sample is heated and the fluctuation in thermal

energy through the sample is graphed (Qin and Wu 2011; Bianco et al. 2011).

The crystallinity of electrospun PCL can be influenced by the presence of residual

solvent, with a higher solvent boiling point being proportional to a higher glass

transition temperature for PCL (Qin and Wu 2011).

As seen below, radiation can have the effect of either increasing or decreasing

crystallinity of a sample, due to chain scission and free radical reactions (Masson et al.

1997; Cottam et al. 2009). Stretching the polymer after it has been electrospun, as could

occur during collecting or testing, can also increase crystallinity (Cipitria et al. 2011). As

it is possible to control the percentage level of crystallinity (up until a point), the

increased mechanical properties that occur with increased crystallinity can be tailored

to each specific application.

The hydrophobic or hydrophilic properties of the electrospun PCL can be inferred by

measuring the contact angle of a droplet of water on the surface – a higher angle shows

a higher level of hydrophobicity. Reports for PCL vary; films appear to have a lower

contact angle than electrospun nanofibre meshes, with values from 105° to 129° being

suggested (Cipitria et al. 2011; Ekaputra et al. 2011). However rough the consensus,

PCL is clearly not a wildly hydrophilic polymer; the creation of a PCL composite with

gelatine or collagen greatly increases the hydrophilicity – in large concentrations, the

contact drops to approximately 0° (Liao et al. 2006; Sabir, Xu, and Li 2009; Pham,

Sharma, and Mikos 2006; Cipitria et al. 2011). Chemically, PCL is a relatively simple

structure, comprising sigma and pi bonds between C, H and O; this can be seen in Figure

1.2. This structure makes PCL a good candidate for analysis by x-ray photoelectron

spectroscopy (XPS); the small size of samples commonly present leads however, to

semi-quantitative analysis, often done to confirm or deny whether a new substrate was

included or excluded during the electrospinning process (Cipitria et al. 2011). Analysis

18

previously performed showed that XPS can detect residual OH groups, which will be a

sign of scaffold alteration after sterilisation and cleaning with ethanol (Prabhakaran

2008; Martins et al. 2009).

Figure 1.2 Chemical Structure of poly(ε-caprolactone)

1.4 Sterilisation

Infection during surgery has always been a major issue in medicine, with new

techniques and procedures being developed to help with infection prevention (Sabir,

Xu, and Li 2009). Prophylactic and reactive treatments are often not always used

correctly, with infection around implant surgery sites causing large immediate

problems (the infection itself) as well as directly contributing to the failure of any

implanted biomaterial (Dellinger et al. 2005; Sabir, Xu, and Li 2009). Key to preventing

infection is correctly ensuring sterilisation of biomaterials that are used (Cottam et al.

2009). Standardised requirements for clinical sterilisation have been produced and

used for at least a decade (Cottam et al. 2009). There are several methods that have

regulatory approval for clinical use, and are commonly used as such: steam sterilisation

(autoclaving), ethylene oxide (EO), gamma irradiation, depyrogenation (dry heat), and

filtration (Rogers 2005). More exotic methods, such as argon/hydrogen plasma and

electron beam sterilisation also exist, and several more besides, but are not often used

due to their deleterious effects on materials, and may not have universal regulatory

approval (Tretinnikov, Ogata, and Ikada 1998; Brétagnol et al. 2008).

Although very effective, the use of these techniques at regulatory level requires a large

amount of experience and is an expensive and time consuming process (Masson et al.

1997; Cottam et al. 2009). It is more usual, therefore, for in vitro testing and other

19

laboratory work to be carried out using samples that have been sterilised using ethanol

and/or UV light (Bosworth, Gibb, and Downes 2012; Huang et al. 2003; Pham, Sharma,

and Mikos 2006)Whilst far quicker and easier to use than clinical methods, allowing

more work to be carried out, it means that published results may only be relevant for

samples that have used laboratory-level sterilisation.

As this work will be using PCL, it is important to take the physical and chemical

characteristics of the polymer into consideration when choosing which sterilisation

methods to use. Due to the low melting point of PCL (a bulk sample would be expected

to melt at 60°C, with high surface area:volume designs having a melting point at around

55°C) it is obvious that some of the previously mentioned techniques will not be

suitable (Sun et al. 2010; Rogers 2005).

Procedures involving heat as their primary method for sterilisation (dry heat and

autoclaving) are immediately ruled out, as these often function at over 120°C, which

higher than the melting point of PCL which is 60°C (Rogers 2005). A filtration technique

would only be suitable for sterilising a solution, and is not applicable in this situation.

Plasma and electron beam sterilisation, whilst valid techniques, are comparatively

complicated to use, whilst not being too common; other methods can be equally

effective without over-complicating their use (Brétagnol et al. 2008; Tretinnikov, Ogata,

and Ikada 1998). The remaining two methods suitable for this work, ethylene oxide and

gamma irradiation have a proven track record in biomaterials science, being widely

used and are considered ‘effective’ (Rogers 2005; Gorna and Gogolewski 2003; Narkis

1984; Benson 2002). Key to the work undertaken in this thesis, the technique would

ideally not significantly or appreciably degrade, damage or in some way alter the nature

of the biomaterial, and certainly not to a level where the material function was impacted

(Cottam et al. 2009; Masson et al. 1997; Rogers 2005; Cipitria et al. 2011). There must

also be a level of trust in the technique, backed up by scientific research, that allows for

regulatory and professional approval (Rogers 2005).

20

Table 1.2 Showing Key Information For Sterilisation – collation of information found in this review.

Sterilisation

Method

Main Uses Advantages Disadvantages Key

Reference

Gamma Polymers

Thin metals

Quick product

turnaround

High penetration

Bulk processing

High initial cost

Inherent safety

issues

(Rogers

2005)

EO Simple

macroscale

structures

High diffusivity

High

permeability

Toxic residue (Rogers

2005)

Autoclave Heat resistant

polymers/

metals

Low cost

Quick

Simple process

Few materials resist

heat & moisture

(Rogers

2005)

Dry Heat Most metals

Ceramics

Only used when

other processes

are unavailable

Expensive

Very slow process

Limited materials

(Rogers

2005)

Plasma Some

polymers

Rapid

sterilisation

Change in surface

properties

(Szentivanyi

et al. 2011)

Electron

Beam

Thin polymers Precision

sterilisation

Expensive

Little regulatory

approval

(Tretinnikov

, Ogata, and

Ikada 1998)

For a sterilisation technique to be considered effective, however, it must meet certain

requirements. These can be condensed into the following: first and foremost, it must kill

off all potential pathogens, lest it not fulfil its primary purpose (Rogers 2005). The

method must also be rapid (some techniques, such as radiation at various wavelengths,

can be performed on a conveyer belt in a matter of minutes) and must be compatible

with securing packaging methods to prevent reinfection (Rogers 2005). If possible, a

method must also seek to destroy the pathogen in an attempt to lessen an inflammatory

response in vivo, rather than just simply killing the organisms (Rogers 2005).

21

Techniques which aim to kill or disable organisms may not necessarily be synonymous

with sterilisation, as a material may only be considered sterile if, after statistically

significant sampling of products, it is reasonable to assume the material is 100% free of

active organisms (Rogers 2005). Laboratory based techniques, such as

washing/immersing in ethanol are more properly considered cleaning techniques, as

there is not a high degree of certainty regarding 100% killing of organisms (Rogers

2005). Resistance to sterilisation is an issue that has often been noted, and techniques

are always reviewed to prevent for the formation of sterilisation-resistant lineages of

microbes (Rogers 2005). Although primarily bacterial resistance to gamma or chemical

sterilisation has caused concern, resistance has been found in fungi, viruses and prions

(Rogers 2005). Thankfully, other techniques can be used or combined in tandem to

ensure adequate elimination of resistant strains; heated moisture methods are often

thought to be the most effective in dealing with resistance to sterilisation (Tretinnikov,

Ogata, and Ikada 1998; Brétagnol et al. 2008; Rogers 2005). Any living pathogens which

remain on the sample after sterilisation form what is termed the “bioburden” of the

sample, and a percentage of the sterilised batches would be tested for bioburden,

primarily to ensure that the sterilisation has worked effectively. Should a statistically

significant number of samples within a batch show an unacceptable level of bioburden,

it is likely that the whole batch would be re-sterilised, and a possible recall of previously

distributed products may even occur. A brief overview of the different types of

sterilisation may be found in Table 1.2, above.

1.4.1 Gamma Irradiation

Gamma radiation is a high frequency (10 EHz) ionising form of radiation, containing a

high level of energy per ejected photon. The small wavelength (10 pm) allows an

extremely high level of penetration by the wave-particle, and the ionisation will easily

disrupt cellular DNA leading to apoptosis and necrosis in mammalian cells. When

artificially generated, the dose and path of the rays can be precisely controlled. This

allows for contrasting uses such as wide-scale imaging of shipping containers at ports,

to highly focused “gamma knives” used in some cancer surgeries. More commonly,

gamma rays can be used to prevent the spoiling of fresh food and for sterilisation of

medical equipment. For hospital sterilisation and other small-scale sterilisation

22

procedures, caesium-137 is used as the isotope in a semi-portable sterilisation unit. For

large scale sterilisation, as well as some chemotherapy application, cobalt-60 is used

(Rogers 2005).

Gamma irradiation for medical-grade sterilisation has found polymer products as its

niche. In terms of commonly used polymers for medical use, only poly(proplyne)(PP)

and poly(tetrafluoroethylene)(PTFE) are considered not to be suitable for gamma

irradiation, due to the amount of degradation to the chemical structure that can occur.

(Rogers 2005). When any polymer is subjected to high energy radiation, such as during

gamma sterilisation, two competing processes occur. Firstly, scission of the chemical

bonds occurs, lowering the mechanical strength and elastic modulus of the polymer,

weakening it overall (Benson 2002; Rogers 2005). Secondly however, polymer

strengthening can occur during high energy bombardment, due to the formation of

radical groups that can lead to cross-linking (Benson 2002; Rogers 2005). This cross-

linking results in a higher level of mechanical properties for the polymer (Rogers 2005;

Benson 2002). Due to the conflicting nature of cross-linking and scission reactions,

predicting the change in mechanical properties after irradiation is notoriously difficult,

and is often determined experimentally (Narkis 1984; Rogers 2005; Masson et al. 1997).

When comparing and contrasting polymer behaviour under sterilisation, it is common

to report the ratio between how often cross-linking and scission occurs (Rogers 2005).

Whilst this may not necessarily determine the exact properties of the altered material, it

has shown to be an effective tool for estimating the changes (Rogers 2005) It is also

important to note that the change in mechanical properties may not always be

undesirable, or that an undesirable change would be to weaken the polymer; depending

on the context, strengthening the polymer may increase the mechanical properties to an

unsuitable level (Brétagnol et al. 2008; Tretinnikov, Ogata, and Ikada 1998). Simple

manufacturing techniques can be used to buffer a minor change in mechanical

properties. Generally, polymers can be protected from small changes by increasing the

cross-linking percentage or increasing the curing temperature , as appropriate for the

material (Rogers 2005). This cannot be considered a perfect solution, however. Such

techniques would only work on a number of polymers.

23

The amount of radiation that is given in each irradiation of a sample (the dose) is tightly

regulated, and follows ISO protocols. Such doses are measured in Greys (Gy), with one

Gy being the absorption of one Joule per kilogram of material. ISO 13409, the accepted

standard for irradiating medical devices, along with ISO 11137 calls for a 25 kGy dose

for each product as a starting level (Rogers 2005; Cottam et al. 2009). Although this

could be excessive or inadequate for some products, a vast array of research into the

remaining pathogens (the measure of bioburden) provided statistical evidence for

promoting 25 kGy as a good starting point; each type of product should, of course, be

checked to ensure that no pathogens remain (Benson 2002; Cottam et al. 2009). For a

device that may need a higher dose than the standard 25 kGy or has unusual properties,

further investigation is performed. Fractional doses of the 25 kGy are given to each

device, and analysis for bioburden can be used to suggest the required level of

irradiation, in accordance with ISO 11137 (Rogers 2005). Due to the high penetration of

gamma radiation, however, it is unusual for any change to the standard protocol to be

needed, in the context of medical devices (Rogers 2005; Cottam et al. 2009).

Damage from irradiation on polymers has been documented, and is often seen as a

drawback to this method (Narkis 1984; Brétagnol et al. 2008). For biomaterials that do

not need their properties to be kept at precise levels, this may not be a problem (Rogers

2005; Benson 2002; Cottam et al. 2009). However, there are many implants that require

that level of precision, and any significant deviation from this could cause clinical and

regulatory issues (Rogers 2005). Drug eluting stents, for example, must release their

drug at a specific rate for a specific amount of time, often with the stent degrading at a

constant rate to allow this to happen, and ensuring a zero-order release (Olbrich et al.

2007; Rogers 2005). Damage from radiation has been shown to alter the

physiochemical properties of several polymers, so in cases where this damage is not

acceptable, alternative sterilisation methods should be used (Benson 2002; Rogers

2005; Cottam et al. 2009; Brétagnol et al. 2008).

In terms of the effects of gamma irradiation on PCL, there is a small amount of literature

available, although this can be conflicting at times. Analysis of PCL films suggests that

radiation has a clear effect on samples in terms of their degradation with regards to

mass (Cottam et al. 2009).

24

Untreated samples would degrade at a slower rate, and showed a more even and

smooth microscopic structure, when compared with samples irradiated at 25 kGy

(Cottam et al. 2009). Qualitative and semi-quantitative changes in physical structure

between irradiated and non-irradiated samples were mirrored with an increase in

tensile stress strength (Cottam et al. 2009). Possible reasons for this centred on an

increase in crystallinity, as measured by DSC analysis (Cottam et al. 2009; Benson

2002). Small changes in crystallinity, and other physiochemical measurements have

often been suggested as having a disproportionate effect on mechanical properties

(Xing, Zha, and Yang 2010; Masson et al. 1997). However, a more recent analysis of

electrospun PCL has shown gamma irradiation to effect a massive loss in mechanical

properties, with 15 kGy (below the ISO standard) being reported as producing a 61%

loss in mechanical strength (Bosworth, Gibb, and Downes 2012).

Unusually, however, there was not a reciprocal loss in maximum elongation as would

perhaps have been expected. (Bosworth, Gibb, and Downes 2012). Although the reasons

for this are not speculated upon in the paper, directional differences in the crosslinking

to scission ratio, brought on by different bonding energies, could explain a change of

this type. Comparable findings were also seen in GPC analysis, showing a reduction in

molecular mass in the samples with the highest degree of irradiation of between 12-

33%, which would again point to gamma radiation causing chain scission effects

(Bosworth, Gibb, and Downes 2012). Further evidence is given when detailed

investigations into GPC show the number average molecular weight (Mn) decreasing

(indicative of scission) but the weight average molecular weight (Mw) increasing

marginally, suggesting that cross-linking is occurring in some areas. (Cottam et al.

2009). Similar occurrences, with similar implications draw, were also reported in

experiments with PCL nanospheres (Masson et al. 1997)

Whilst the physical and mechanical changes in PCL are important, it is essential to

remember that the future aim of most experiments is to head towards a stage where

these scaffolds can be implanted as medical devices. Several papers have reported no

significant changes in cell culture or viability rates after sterilisation (Cottam et al. 2009;

Bosworth, Gibb, and Downes 2012). Gamma irradiation was often compared with

25

ethanol (commonly used for lab sterilisation/cleaning), and flow cytometery results did

not show any significant changes in cell attachment (Cottam et al. 2009).

Although no results have been reported for virgin samples (there is always a need for

sterilisation at some level of a sample), ethanol and gamma irradiated samples both

allow cells to orientate and elongate in the direction of the fibres, suggesting that there

was not any loss in cell guidance (Cottam et al. 2009). Additionally, sample which were

sterilised both by irradiation and ethanol did not show any difference from when the

samples were sterilised individually (Bosworth, Gibb, and Downes 2012).

1.4.2 Ethylene Oxide

Whilst gamma irradiation is perfectly suited to sterilising liquids and thin solids,

autoclaving can rapidly sterilise heat resistant materials, ethylene oxide has found

wide-ranging popularity in hospitals, especially with polymer products (Rogers 2005).

Ethylene oxide is an aromatic ether compound, with little overall charge. It is modestly

reactive, with a very high chemical volatility, ensuring that it is gaseous at easily

accessible temperatures (with a boiling point of ~10°C), increasing its availability

(Rogers 2005).

Sterilisation with EO is a comparatively complex technique after looking at gamma

irradiation, and, whilst highly dangerous to use, there are none of the inherent superior

risks that inevitably come from using radiation (Rogers 2005). Gaseous EO is pumped

into the sterilisation chamber at a reasonably cool temperature of anywhere between

50 – 60°C, which can then penetrate the vast majority of samples; although thick and

complex patterns, like woven mats, are often sterilised with caution (Rogers 2005).

At least one research group has suggested that EO is unable to fully penetrate complex

shapes, ager finding residual microbes on an EO-sterilised product, but not one

sterilised by autoclaving (Yoon et al. 2012).

The temperature used has proven to be an effective one, as most polymers will have

higher melting points (Rogers 2005). It should be observed though, that the large group

of biomaterials that are designed to react at physiological conditions, especially

temperature, would be categorically unsuitable for sterilisation in this manner.

26

Damage, destruction and compromise that generally follow autoclaving and irradiation

are often absent when EO is used, meaning that it remains the preferred sterilisation

method when dealing with biomaterials that must have specifically tailored properties

(Rogers 2005).

Drug eluting stents have been previously discussed as a class of biomaterial for which

controlled properties are paramount, and it is in cases like this that EO is invariably

used (Rogers 2005).

The process of sterilisation starts with the formation of a vacuum around in the

sterilisation chamber, where the samples sit. Samples are subjected to a high

percentage of humidification, which has been shown to have a synergistic effect with EO

for sterilisation. (Smith et al. 1988; Brétagnol et al. 2008). When there is insufficient

humidification, then the sterilisation processes does not always occur successfully

(Rogers 2005). Regulations require the use of a high degree of humidity, in some areas a

value of approaching 100% humidity is required. (Rogers 2005). Cyclical steam pulsing

is used to increase the temperature and humidity in the sterilisation chamber; constant

heat and humidity system were found to be too slow and inefficient to allow for the

rapid sterilisation process for which EO is often used (Rogers 2005; Brétagnol et al.

2008). Next, EO is added at either 50-70°C for 3-5 hours or room temperature for 12

hours (although this is rare, given that EO is often used as a rapid process), or

depending on precisely what equipment is being sterilised (Rogers 2005). Metal

products are ready to be used immediately, whilst polymers require a degassing

process afterwards (Rogers 2005).

Even though there is no radiation involved and few reports of damage to the sterilised

products, this does not mean that EO is a flawless sterilisation method. On the contrary,

there are a vast number of safety issues that need to be considered. EO is highly toxic,

teratogenic and carcinogenic, whilst also being flammable and explosive (Rogers 2005).

Toxic gases that are produced during the breakdown of EO, such as ethylene

chlorohydrin and ethylene glycol, much be removed by degassing and air washes, along

with EO, which is highly toxic itself (Rogers 2005; Brétagnol et al. 2008). As a result,

high levels of safety precautions must be taken, which adds to the time and cost of EO

sterilisation (Rogers 2005). Whilst high energy gamma irradiation takes moments,

27

products that are to be sterilised using EO often take several days to thoroughly

perform (Brétagnol et al. 2008). Non-metal samples must be further degassed, for a

suggested time of at least 12-24 hours (Rogers 2005).

Despite the added complications that EO can cause, it is a testament to the effectiveness

and many advantages that EO is still used as a preferred method.

The process by which EO sterilises is remarkably similar to a radiation process, leading

EO to be classed as a radiomimetic poison (Rogers 2005). In a similar method to

alkylating antineoplastic agents used in cancer medication, EO attaches an alkyl group

to nitrogen 7 in the purine ring on guanine (Lopes, Oliveira, and Oliveira-Brett 2013;

Kovalenko et al. 2012; Rogers 2005). Using anti-cancer medication such as cysplatin and

temozolomide as examples, it is known that alkylation of double strand DNA (dsDNA)

immediately begins to interfere with the activities of the cells (Kovalenko et al. 2012;

Lopes, Oliveira, and Oliveira-Brett 2013). Condensation of dsDNA soon follows, and cell

replication is disrupted to the point of apoptosis and necrosis (Lopes, Oliveira, and

Oliveira-Brett 2013). Whilst the effectiveness of alkylating antineoplastic agents and

pseudo-alkylating antineoplastic agents on mammalian cells and the organism at whole

has been known for close to a century (mustard gas used in World War I is an example),

there have also been some studies to show that the agents are also effective against

bacterial species (Escalante 2011; Jugg et al. 2013). Since anti-cancer medication targets

rapidly dividing cells, it is reasonable to assume that bacteria and other rapidly

proliferating organisms are targeted in the same way, and killed by alkylation.

There have been very few published investigations into the effects of EO on PCL; those

that have been published do not look often at PCL as an electrospun fibre. However,

implications can be gleaned from the papers, with caveats as to their whole relevance.

Extruded PCL was found to have a slightly higher thermographic profile under DSC

analysis after sterilisation, when compared to pre-sterilisation results, with a melting

temperature increase of 2K being reported (Weir et al. 2003). Whilst a very small

change, this increase in melting temperature was thought to be caused by annealing,

and therefore is unlikely to occur at the low temperatures to which electrospun PCL will

be exposed (Cipitria et al. 2011; Subbiah et al. 2005; Weir et al. 2003). Interestingly,

there was no statistically significant change in molecular weight reported after GPC

28

analysis, contrasting with the gamma radiation findings (Weir et al. 2003; Cottam et al.

2009). Again divergent with the gamma radiation results, mechanical strength though

tensile testing did not give different results before and after sterilisation; though the

samples tested were highly dissimilar to electrospun PCL (Weir et al. 2003).

Evidently, further investigation and a larger body of research into this area is needed

before conclusive results can be drawn.

Similarly, there is little conclusive evidence on the effects of EO sterilisation on

electrospun PCL with regards to cell culture. Papers published that use EO have shown

there to be little influence on the material (Curran, Tang, and Hunt 2009; See et al.

2012). One paper, using PCL and PCL doped with PLGA suggested that there was a high

degree of cell attachment even when EO sterilisation had occurred; hMSCs were

successfully cultured with no issues reported (Curran, Tang, and Hunt 2009).

All surfaces (pure PCL and PCL doped with up to 30% PLGA) maintained viable levels of

cell attachment, and the sterilisation process did not detrimentally affect cell

proliferation, which remained at the expected level (Curran, Tang, and Hunt 2009). A

hydrogel culture system, exposed to higher levels of EO sterilisation than normal, due to

their thickness, also reported no unusual results in cell culture which could be

attributed to EO sterilisation (See et al. 2012). It is reasonable to assume that, with

regards to cell culture, both EO and gamma radiation have similar effects on cell culture;

the sterilisation method may alter the PCL on a molecular level, but the cell attachment

features (normally the orientated nanofibres) remain mostly unaltered.

29

1.5 Aims and Objectives

The aims of this investigation are twofold:

1. To investigate the effects of certain types of sterilisation on electrospun PCL.

a. Namely, the clinical sterilisation methods will be gamma sterilisation at

15 kGy, 25 kGy, 50 kGy, and ethylene oxide.

b. The laboratory or non-clinical sterilisation methods will be ultra violet

and ethanol.

c. There will be a control, known as virgin, such that data obtained on the

sterilised samples can be compared against native untreated PCL.

2. To decide which method(s) of laboratory sterilisation can replicate the effects of

clinical sterilisation without incurring the cost and delays necessary in the latter.

a. Once a comparison between sterilisation methods has been established,

scientists working on electrospun PCL can interpret the published

literature and ensure that accurate comparisons are drawn between the

different types of sterilisation.

30

Chapter 2 : Materials and Methods

2.1 Commercial Electrospun Fibres

PCL fibres from The Stellenbosch Nanofibre Company, South Africa, have been used as

the main source of samples for experimentation. These fibres were made to a standard

commensurate to established good manufacturing practice (GMP), and have been made

on a large commercial scale. It is expected that this source of fibres would be used if an

implant was to be designed and used clinically. The PCL fibres were manufactured to

achieve submicron fibres in A3-sized aligned mats. The original PCL crystals were

Purabsorb PC12 (Purac) at 1.2 dl/g. Average fibre diameter on manufacture was given

as 665nm, although with a large variation, recorded as being 49%. It is important to

note that all sample mats were originally rinsed in ethanol in distilled water, and then

dried, as part of the manufacturing process. This was in addition to any sterilisation that

would later take place.

2.2 Preparation of Electrospun Fibres

In addition to the commercial fibres above, PCL was also made in the lab under the

following condtions.120 kDa PCL (Sigma) was dissolved in HFIP (Hexafluoro-2-

propanol, from Sigma) for 24 hours using a magnetic stirrer, to form a 10% w/v

solution. The solution was loaded into a 5ml syringe and placed in the electrospinning

chamber. The chamber was prepared by placing a rotating earthed mandrel (of

diameter 120mm and width 1.4mm) 20 cm from the needle tip. The electrospinning

process is under computer-control, and is set to the following conditions: needle

diameter 12.21mm, solution flow rate 1ml/h, solution volume 0.25 ml, RPM of mandrel

600, voltage applied 20kV, electrical current 4mA. This allowed for a working length of

approximately 30cm of aligned fibres to be spun. Fibres were removed by cutting

around the edge of mandrel with a scalpel, allowing the fibres to be removed with the

minimal amount of damage and contact. Fibres were stored in a cool, dry area in a Petri

dish until needed.

31

2.3 Ethanol Sterilisation

Fibres were cut using a scalpel to between 1-2cm (as needed for further use), and fibre

ends that had been potentially damaged during handling were discarded. Each

individual short fibre strip was placed in an Epindorf.

Ethanol solutions were prepared at 50%, 70%, 90% and 100% v:v concentrations.

1.5ml of 50% ethanol was added to each Epindorf in non-sterile conditions, and the

fibres were left for 24 hours. The 50% ethanol was changed for 70% ethanol under

sterile conditions, in a laminar flow hood. The same process was followed for 90% and

100% ethanol solutions. The 100% ethanol was removed under sterile conditions, and

washed with PBS (phosphate buffered saline, pH 7.4, from PAA Labs). 1.5ml of PBS was

added, and left for 25 hours, after which time the PBS was removed and the fibres were

left to air-dry in a sterile environment for at least 48 hours. The fibres were then stored

in sterile conditions to be used in subsequent experiments.

2.4 UV Sterilisation

In a laminar flow hood, a bed of tissue paper was securely taped with autoclave tape to

the floor of the hood. Fibres were flattened without stretching and taped to the tissue

paper, preventing movement due to air flow. Scalpels and forceps were also placed in

the hood, and UV radiation was applied for 30 minutes. The samples were turned over

and taped to the tissue, and irradiated for 30 minutes. The tape, tissue paper and any

damaged sections of fibres were discarded. Fibres were cut according to need under

sterile conditions, and stored in a Petri dish or similar, before being used in subsequent

experiments. It is important to note that “sterile” with regards to EtOH and UV

sterilisation means that a sample has had ethanol or ultraviolet radiation applied

(respectively) and is sterile for the purposes of lab-based experiments. As reported

earlier, such techniques are not acceptable or permitted for medical or clinical use.

32

2.5 Gamma Sterilisation

PCL samples were sent to an external company (Isotron, UK) and sterilised using

gamma radiation at three set doses: 15kGy, 25kGy and 50kGy. 15kGy was chosen as the

standard minimum for most materials, with the two higher doses present to ensure full

sterilisation and penetration of the PCL. For ease of labelling, the doses are hereafter

referred to as 15G, 25G and 50G where appropriate. Fibres were kept in sterile

conditions after returning, and were used in subsequent experiments. Samples of PCL

fibres at each level of irradiation were tested for their residual bioburden.

2.6 Ethylene Oxide Sterilisation

PCL samples were sent to an external company (Isotron, UK) and sterilised using a

standard EO sterilisation protocol. The sterilised samples were given sufficient time to

degass, with the intention that no EO would be present in or on the PCL. Samples were

kept in sterile conditions, and were used in subsequent experiments. It is important to

note that “sterile” with regards to EO and gamma sterilisation means that a sample has

gone through a certified sterilisation procedure with recognised levels of deactivation

or destruction of pathogens.

2.7 Scanning Electron Microscopy

Fibre lengths of 1cm with duplicate repeats were used. Samples were fixed in

glutaraldehyde (if cells were present) and dried using a serial ethanol dilution and

critical point drying with bis(trimethylsilyl)amine (otherwise hexamethyldisilazane,

HDMS). Fibres were mounted onto SEM stubs, and sputter coated with gold. SEM

imaging was performed with a working distance of 8 mm and 5 keV acceleration.

Representative images were taken for analysis. Manual and automated measuring of

fibre length and width was possible. Fibres previously seeded with cells were also

imaged (see Chapter 2 Section 9)

2.8 Diffraction Scanning Calorimetry

Fibre lengths of 1cm, with triplicate repeats were used. Fibre samples were placed and

sealed in aluminium pans, with their weights recorded. Analysis was performed on a

DSC Q100 machine (TA Instruments), with ramp heating at 10°C/minute to an end

temperature of 100°C. Data analysis was performed using the program Universal

33

Analysis 2000 (TA Instruments), with percentage crystallinity being recorded from

curve integration (ΔH = 135.44 J/g).

2.9 Fourier Transform – Infrared Spectroscopy

Fibre lengths of 1cm, with triplicate repeats were used. Samples were prepared and

placed on a Smart Orbit stage (Thermo Scientific) and analysed using a Nicolet 5700

spectrometer (Thermo Scientific). Background spectra were obtained at 32 scans at the

beginning and end of testing. Each fibre was sampled using 16 scans under standard

settings. In addition, spectra of HFIP, ethanol at the given concentrations for ethanol

sterilisation, distilled water and PBS were tested at 32 scans, so that any aberrant peaks

in the PCL spectra could be identified. Data was exported and analysed using Excel

(Microsoft Office).

2.10 Tensile Testing

Fibre lengths of 2cm with five repeats were used. Samples were mounted onto a paper

window, with the ends secured and a clear 1cm of fibre available for testing. The width

was measured using a micrometer and callipers, and the long ends of the window cut, so

only the fibres were loaded. The testing was performed on an Instron machine under

standard and regulated conditions of 23°C and 50% humidity. Samples were allowed to

equilibrate for at least 12 hours before testing. The properties of maximum stress,

Young’s modulus, maximum strain and break displacement could be analysed.

Testing was performed under computer control, with the following conditions:

extension rate of 5mm/minute, full scale load of 100g, grip distance 15mm, and 1N load

cell. Data was exported and analysed using Excel.

2.11 Cell Seeding

L929 mouse fibroblasts (Sigma Aldrich) were cultured using Dulbecco's Modified Eagle

Medium (DMEM)(Invitrogen), modified by the addition of 2mM l-glutamine and 10%

Foetal Bovine Serum (FBS). The fibroblasts were seeded onto sterilised PCL samples

secured to CellCrown Scaffdex (Sigma Aldrich) at a density of 50,000 cells/cm2.

Scaffdexes were placed on ultra-low attachment 24-well cell culture plates (Costar).

Cells were incubated at 37°C and ppCO2 5%.

34

An Alamar blue assay was utilised in order to assess cell metabolic activity on the

scaffolds. 5 mg of Resazurin sodium salt (Sigma) was dissolved in 40 ml of PBS and

filter-sterilised to make the Alamar blue solution. The solution was stored in the dark at

4 ºC when not in use. Each PCL sterilisation condition was repeated in quadruplicate, at

1, 3 and 7 days. Cells were also seeded onto the cell plate, without a scaffold, as a

control. A plate reader (Origin?) was used to analyse the colour change caused by the

degradation of the Alamar blue due to the metabolic rate of the cells. Data from this was

exported and analysed using Excel.

Samples were dehydrated at 1, 3 and 7 days for SEM imaging. Fixation using 1.5%

gluteraldehyde at 4°C for 30 minutes was performed, and dehydration using an ethanol

series of 50%, 70%, 90% and 100% concentration for 2x3 minutes.

Final dehydration using hexamethyldisilazane (HDMS) took place in a fume hood over

24 hours to ensure HDMS evaporation. Each sample was mounted on an SEM stub, and

sputter-coated with gold, before being imaged.

2.12 Gel Permeation Chromatography

Two samples of 2cm PCL strips per condition (4cm for each of the seven PCL

conditions) were rapidly and fully dissolved in 2ml of tetrahydrofuran (THF)(0.2% w/v,

Fisher) and injected into a GPC system (Applied Chromatography Systems) to

determine the average molecular weight (Mw) of PCL, and to note any changes from the

virgin samples.

All pieces of data obtained were compared to the reference Mw provided by polystyrene.

Due to the low volume of PCL-THF, the solute did not undergo filtration before injection

into the GPC system. Results were calculated using PSS GPC software, with the data

exported and further analysed using Excel.

2.13 Water Contact Angle

Several 2cm strips of PCL for each sterilisation condition were taped to microscope

slides and placed in a contact angle measuring machine (Kruss DSA100) . 10 sets of 5μl

droplets of ionised water were dropped onto the PCL, and their angles measured and

recorded by the Drop Shape Analysis software package. This data was then exported,

35

analysed and graphed in Excel. Photographs were also taken using Drop Shape Analysis,

using the camera already built-in to the measuring device.

36

Chapter 3 : Results

The results reported here were chosen from a large bank of collected results, as they are

the clearest and most representative set of results available. Where applicable,

additional results (or their precursors or successors before/after analysis is performed)

are reported as smaller images below the main data

3.1 Fourier-Transform Infrared Spectroscopy

FT-IR was performed on samples in order to determine the functional groups present in

the PCL after manufacturing, and to notice any changes which occur as a result of the

laboratory and clinically permitted sterilisation procedures. Virgin, ethanol and UV

samples, along with ethylene oxide and gamma sterilised samples were analysed at

least three times, in accordance with the protocol laid out in Chapter 2 Section 9. If the

processes applied to the sample resulted in a change in chemical structure, then this

would be observable in the transmission and absorbance spectra. Representative and

aggregate results of each criterion are presented.

37

Figure 3.1 Combined Graph for UV, Ethanol and Virgin – whilst %absorbance may differ between

samples, there are no troughs which show a specific chemical change, n =3. A: 1160 cm-1 tertiary alcohol

C-O. B: 1750-1000 cm-1 C-C and C=O bonds. C: 2300-2200 cm-1 CO2. D: 2850-3000 cm-1 C-H stretch.

38

Figure 3.2Averaged Spectra Data for all Samples – mean average values for each data point for each

condition were calculated, and plotted onto a single graph, n = 3

Given the almost-perfect alignment of the spectra on top of each other, it is clear that

there has been no disenable chemical changes (Figure 3.3). The only are of significant

difference is the low intensity trough at 2300-2200 cm-1 at annotation C (Figure 3.1)

which is indicative of carbon dioxide. This almost certainly atmospheric and respiratory

CO2, and an artefact of the experimentation process, and was present in the spectra of

all but two of the samples analysed (Appendix A Virgin 1and EtOH 1).

39

3.2 Differential Scanning Calorimetry

DSC analysis was performed on samples of PCL to determining the melting point and

percentage crystallinity of the polymer. The analysis was performed according to the

method set out in Chapter 2 Section 8.

Investigation into the DSC data did not show a statistically significant difference

between the melting points of virgin, ethanol and UV samples (P>0.05) and it can be

seen that any difference between the data is very marginal. The % crystallisation values

are more conclusive, and there is a clear difference between each set of samples, with a

7.2% difference between the ethanol and UV samples. There is a marked difference

between the crystallisation data for the lab sterilisation (virgin, ethanol and UV) and the

clinically acceptable methods (gamma and ethylene oxide), the biggest difference being

26.0% (between UV and 50G).

There was very little observable change in melting point, the average melting point for

all samples was 57.9°C; no sample showed a change greater than ±3°C.

Virgin UV Ethanol

Sample Melting

Point %

Crystallisation Sample

Melting Point

% Crystallisation

Sample Melting

Point %

Crystallisation

1 59.72 38.39 1 60.12 41.20 1 60.72 33.19

2 59.35 34.99 2 59.57 40.46 2 59.71 29.29

3 58.97 37.62 3 59.99 39.07 3 60.35 36.77

Avg 59.35 37.00 Avg 59.89 40.24 Avg 60.26 33.08

40

Figure 3.3 DSC of All Samples – the two testing results, melting point and % crystallisation, are

displayed next to each other for ease of reference, n =3.

41

3.3 Tensile Testing

Uniaxial tensile testing was performed to observe the change (if any) on the mechanical

properties of sterilised PCL samples, when compared to unsterilised PCL. The average

values for the Young’s modulus, ultimate tensile stress, and strain were calculated.

Samples were prepared and tested using the protocol outlined in Chapter 2 Section 10.

An example graph, showing how the following values were calculated can be found in

Appendix C.

Figure 3.4 Average Young’s Modulus – this was calculated using the equation E = σε from the raw data

and compared with the general equation of a line (y =mx + c) on the stress -strain curve plotted from the

raw data . Averaged data is reported in MPa for comparison, n =5.

78.50

48.01

97.31

69.82

29.27 33.50

48.14

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

Yo

un

g's

Mo

du

lus

(MP

a)

Young's Modulus of Samples

Virgin EtOH UV 15G 25G 50G EO

42

Figure 3.5 Ultimate Tensile Strength –raw data plotted on a stress-strain graph was analysed to find

the highest point on the curve, and this data was recorded and averaged, n =5.

8.72 8.51

10.32

6.92

10.22

5.85

10.72

0.00

2.00

4.00

6.00

8.00

10.00

12.00

UTS

(M

Pa)

Ultimate Tensile Strength of Samples

Virgin EtOH UV 15G 25G 50G EO

43

Figure 3.6 Strain – the starting dimensions of all of the samples was recorded, and compared against

the final elongation value , at maximum load of 100g and extension of 5mm/minute. This was then put

into the engineering strain equation (ε =l 0/l) and the average data was reported here, n =5

There has been a clear change in the mechanical properties of the PCL after a

sterilisation technique was used, when compared to the virgin samples. In terms of

Young’s modulus, the low radiation sterilisation methods (UV and 15G) have the least

effect on the PCL, with UV being the only method which caused an increase in the

Young’s modulus (Figure 3.5). Ultimate tensile strength changes are harder to

characterise, but ethanol is the only sterilisation method with a statistically insignificant

change in values (Figure 3.6 and 3.7). 15G and 50G caused drops in ultimate tensile

stress, with UV, 25G and EO all increasing the ultimate tensile strength to around similar

values (Figure 3.6). The averaged strain values remained somewhat constant with

ethanol, UV and 15G having very similar figures to the virgin sample (Figure 3.7). Every

type of sterilisation showed a marked difference in at least one of the testing categories.

0.31 0.32

0.28 0.30

0.40

0.19

0.43

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Stra

in

Strain of Samples

Virgin EtOH UV 15G 25G 50G EO

44

3.4 Cell Culture

Mouse L929 fibroblasts were cultured on scaffolds on Scaffdex, as set out in Chapter 2

Section 11. Some cells had Alamar Blue added to them, and samples were passed

through a fluorescence plate reader.

Upon the analysis of the fluorescence graph, there are several clear features which

present themselves. Firstly, with the exception of ethylene oxide, the use of any PCL

scaffold can increase cell growth to a higher level than the tissue plastic control. At day

7, the 50G sample managed to culture 5x the number of cells than the control, with all

but one of the other samples at least doubling the number of cells present (ethanol was

the exception, showing a ~60% increase over the control number) (Figure 3.7).

Ethylene oxide however, consistently performed worse than the control, decreasing the

number of cells by 30% one day after seeding (Figure 3.7).

45

Figure 3.7 Fluorescence Data – 9 averages per day per sample of fluorescence data were taken, and

plotted against each other.

46

3.5 Gel Permeation Chromatography

Gel permeation chromatography was performed in accordance with the procedure laid

out in Chapter 2 Section 12. The molecular mass of the sample has a relation to the

amount of chain scission and cross-linking that occurred.

Figure 3.8 GPC – averaged data was plotted from the molecular weight of that samples that was

reported by the equipment.

The molecular weight of the PCL after sterilisation generally falls, with the higher

energy gamma causing sharper falls in molecular weight (Figure 3.9). UV was the only

method which showed a significant increase in molecular weight, with ethylene oxide

also showing an increase, but within the error range of the virgin sample (Figure 3.9)

47

3.6 Water Contact Angle

The contact angle of water on PCL after different sterilisation conditions was measured

in accordance with the protocol in Chapter 2 Section 13.

Figure 3.9Photograph of Water on Virgin PCL – this image shows the time at which the contact angle

would be recorded; the moment when the water hits the PCL.

48

Figure 3.10 Water Contact Angle – averaged data from 10 left and 10 right angles per PCL condition.

Sterilising PCL had a clear effect on the hydrophilicity of the PCL, with all samples

becoming more hydrophilic (Figure 3.12). The contact angle of water on ethanol

sterilised PCL was significantly lower than all other samples (Figure 3.12). The range of

contact angles for each sample can be seen in Appendix D; in some cases the angles are

relatively similar (15G) and in other there is a wide variation (virgin and 50G). Figure

3.11 is a photograph of the drop of water at the point of making contact with the PCL.

49

3.7 Scanning Electron Microscopy

Micrographs were taken of PCL at several set magnification factors. Samples selected for

SEM imaging can be split broadly into two categories – one set of micrographs shows

the effects (if any) on the microstructure of the PCL after the sterilisation techniques

outlined in Chapter 2 Sections 2-5 are applied. The other set of samples imaged show

the growth of cells on the various types of PCL sterilisation.

Post-sterilisation low magnification images showed few noticeable changes to the

morphology, and certainly nothing that looked to cause significant problems in further

experiments (Figure 3.14). Higher magnification images showed some damage to the

structure was present in UV-sterilised samples, which appears to suggest that

mechanical strength will be altered (Figure 3.15, D).

Day 1 of seeding showed that virgin, ethanol and 15G had low amount of cell growth,

but that UV-sterilised PCL was already showing good levels of cell spreading, without

major colonies appearing to form (Figure 3.16). By Day 7, however, there is almost

complete coverage of cells on all imaged scaffolds, for virgin, ethanol and UV. The PCL

fibres themselves are hidden below the cell sheet, without only some fibres visible

through cracks in the cell coverage (Figure 3.17).

50

Figure 3.11 SEM of Scaffolds Post-Sterilisation – as can be seen in the micrographs above, there are

few morphological changes at this magnification. A: virgin; B: UV sterilised; C:EtOH.

51

Figure 3.12 SEM of Scaffolds Post-Sterilisation – a higher magnification shows that there is some

damage done to the fibres after sterilisation; some are broken, others appear dried -out and brittle (D).

A: ethanol sterilised; B: UV sterilised; C: ethanol sterilised; D, UV sterilised.

52

Figure 3.13SEM Images of Day 1 Post-Seeded Scaffolds - all images were taken of scaffolds after 1

day of cell seeding. All cells were seeded at a density of 50,000 cells/cm2. There is a noticeable

difference in cell number between the different samples. A: virgin scaffold, 610x; B: ethanol sterilised;

C: UV sterilised; D:15G sterilised.

53

Figure 3.14 SEM of Day 7 Post-Seeded Scaffolds - all images were taken of scaffolds after 7 days of

cell seeding. All cells were seeded at a density of 50,000 cells/cm2. There is no apparently noticeable

difference in cell number between the different samples, as almost total coverage has occurred. A:

virgin scaffold; B: ethanol sterilised; C: UV sterilised.

54

Chapter 4 : Discussion and Conclusion

In the course of this research, six different conditions of PCL were tested against the

virgin samples and the results of the various experiments are recorded in Chapter 3:

Results. As mentioned in Chapter 1: Literature Review and Chapter 2: Materials and

Methods, each of the six sterilisation methods were chosen primarily for being known

to not cause immediate and severe damage to PCL (Rogers 2005). For example, had an

autoclave or dry-heat been used, then the high temperatures needed for effective

sterilisation (<100°C) would have irrevocably damaged the structure of PCL (Bosworth,

Gibb, and Downes 2012; Rogers 2005). Having seen that the protocols for the lab-based

methods of ethanol and UV sterilisation, and for industry-standard gamma irradiation

did not involve heating, these were chosen as the methods to be used (ISO 2002; Rogers

2005). In addition, a revised process for low-temperature ethylene oxide sterilisation

has recently gained popularity. Previously, ethylene oxide needed temperatures

comparable to the melting point of PCL, and without extensive tests on PCL, the high

temperatures would have had uncertain effects. However, the lower temperature

ethylene oxide sterilisation does not approach the melting point of PCL but remains a

permitted system for sterilisation.

The experiments which were performed seek to determine the effects of sterilisation on

PCL, and how reliable the cheaper and easier lab-based methods are in comparison to

the clinically approved ones. By extension, if there are similarities between the two

different classes of sterilisation, then the data published for lab-based sterilisation can

also be thought to approximate the behaviour of PCL for commercial sterilisation (for

which data is not as prevalent.) It is expected that there will be situations where one

sterilisation method is similar to another in a specific category (tensile strength or

melting point, for example); it is unlikely that there is one sterilisation method which

wholly mirrors the effects of another method.

55

4.1 Physiochemical Changes

The hierarchal nature of PCL provides many benefits to its physical and chemical

properties, but also creates a significant weakness. If the fibres are significantly

damaged, then flaws in the macrostructure will propagate, and PCL will start to behave

differently.

The changes could affect the structure and biocompatibility of the scaffold (due to

topological differences or the addition or removal of certain chemical groups) or the

degradation rate and products of the PCL. Altering the chain length, crystallinity or

crosslinking density will change the polymer behaviour, such stiffness, strength or

melting point. To determine the extent of these morphological or chemical changes, a

battery of experiments were performed.

Firstly, FT-IR was performed to determine the overall chemical structure of the PCL.

The virgin scaffold provided a control to show the spectra of PCL after it has been

commercially electrospun. There are several observable troughs in all of the spectra in

Figures 3.1 – 3.3 typical of PCL.

Intense troughs at around 1160 cm-1 (Figure 3.1, notation A) are indicative of a tertiary

alcohol (C-O), and troughs at 1750-1000 cm-1 show C-C and C=O bonds, with the trough

(Figure 3.1, notation B) showing the main absorbance spectra, and the smaller peaks to

the right showing scissoring, bending and stretching of the two bonds. Troughs at 2850-

3000 cm-1 (Figure 3.1, notation D) show the alkane C-H stretch which is also expected.

There is an extremely faint curve on the ethanol spectra (Figure 3.1 EtOH) which may

suggest that some ethanol is conserved through the sterilisation/cleaning process and

did not evaporate away. However, the curve is extremely shallow and the absorbance

percentage falls below that of the virgin spectra (Figure 3.1 Virgin). The only difference

in Figure 3.3 appears to be the change in absorbance percentages and not the presence

or absence of peaks themselves. It can be concluded, therefore, that there does not

appear to be a chemical change between each tested samples. Several conclusions can

be drawn from this. If the electrospun virgin PCL is a suitable substrate for cell growth,

as PCL is, then there should not be issues with cell growth from a chemistry perspective

56

(Aghdam et al. 2011; Qin and Wu 2011). Secondly, since there are no changes to the

chemistry, then it is reasonable to assume that the degradation products and behaviour

will remain constant. This will prove beneficial as chemical changes could alter the

degradation products or rate of degradation which in turn may affect cell viability

(Hutmacher and Cool 2007; Weir et al. 2003). Given the similarity between all of the

spectra, any one sterilisation method could easily replicate the chemical effects of

sterilisation for another method, assuming similar protocols as set out in Chapter 2 are

followed.

As the overall chemical makeup of the PCL was found to have not been altered, it was

next decided that an examination into the chemical layout and structure of the PCL

would give a clearer insight into sterilisation had affected it. The high energy radiation

used in gamma sterilisation is enough to cause both chain scission and cross-linking,

which often occur concurrently and in different ratios (Benson 2002; Gorna and

Gogolewski 2003). DSC analysis of the melting points showed very small rises in

temperatures for ethanol, UV and ethylene oxide, with ethylene oxide showing an

increase in melting point of 1.56°C (Figure 3.4). The gamma sterilisation caused a

decrease in melting point, with 15G and 25G giving almost identical results (Figure 3.4).

The difference in 50G could be due to the fact that the higher gamma levels often

require more than one pass through the sterilisation equipment, which could have

altered the ratio of cross-linking and scission. Virgin, ethanol and UV all seem to have

very similar chances of reproducing the effects of gamma irradiation or sterilisation by

ethylene oxide.

Changes in percentage crystallisation are clearer to see. All of the gamma sterilisation

methods show an increase in the region on 20% in crystallisation, along with ethylene

oxide. Ethanol also showed a moderate increase and UV a moderate decrease. Gamma

irradiation no doubt caused large-scale alterations to the amorphous phase of PCL,

changing it into a crystalline phase (Figure 3.4).

Both ethanol and ethylene oxide are highly invasive techniques; they are the only ones

used which involve chemical use and this may go some way to explain the changes in

crystallinity. UV also uses radiation as a sterilisation method, but uses lower energy

gamma radiation, and is there for considerably weaker (Benson 2002; Rogers 2005).

57

Given that for most systems the energy required for making bonds is generally higher

than breaking them (primarily due to entropy), crystalline phases may be removed and

restructured in favour of amorphous regions, therefore giving a lower percentage

crystallinity for UV than for the virgin sample (Figure 3.4)(Narkis 1984). The values of

all gamma conditions and ethylene oxide were over 50% for percentage crystallisation,

showing that the PCL had moved from the amorphous-region dominance shown in

virgin PCL (37% crystallinity) to crystalline-region dominance. Unfortunately, none of

the lab-based methods of sterilisation are able to replicate the percentage crystallinity

of PCL after sterilisation with the other methods.

Having determined from DSC that there were significant changes occurring in the

structure of the PCL after sterilisation, GPC was performed to see if any insight could be

gained into the ratio of chain scission to cross linking. A lower molecular weight is

indicative of chain scission, which is often accompanied by high energy processes

(Cottam et al. 2009). All of the gamma sterilisation methods showed a modest decrease

in molecular weight, up to 10% with the 50G sample and successive falls in average

molecular weight for each of the gamma doses (Figure 3.9), showing a move towards

overall chain scission. Shorter chains will generally melt at a lower temperature, and

reviewing the data from the DSC shows that the gamma sterilised PCL had lower

melting points than the virgin sample (Figure 3.4). Remembering that PCL is a polymer,

it is quite possible that the scissioned ends from the gamma areas re-polymerised and

formed tightly packed crystalline regions, explaining the major change in percentage

crystallinity seen in the DSC data (Figure 3.4) Ethanol shows no change, as it did with

the melting point on the DSC, and only minimal changes with the percentage

crystallinity (Figures 3.4 and 3.9). UV showed a 20% increase in molecular weight,

suggesting that cross-linking was by far the dominant effect here (Figure 3.9). This is

again consistent with the percentage crystallinity data, which showed a decrease in

crystallisation for UV, contrary to the large increases in the gamma values (Figure 3.4).

A change average molecular weight would also likely have an effect on the mechanical

properties of PCL. Virgin PCL can make a reasonable approximation of the effects of the

clinical-grader sterilisation, however if some sterilisation is needed, then ethanol will

also work.

58

A different angle of approach was to look at the hydrophobicity and hydrophilicity of

the PCL, and to observe any changes in the contact angle of water on PCL. PCL is an

averagely hydrophobic surface with low surface energy (Weir et al. 2003). All

sterilisation methods lowered the contact angle of PCL, making it more hydrophilic

(Figure 3.12). UV, all gamma and ethylene oxide all decreased the contact angle by 2-

6%, whilst ethanol had a significant effect, reducing the contact angle by 14°.

It is interesting to note that ethanol is a highly hydrophilic and hydroscopic substance,

and it is possible that there was some modification or absorbency of ethanol onto the

PCL.

If there was surface modification, and not straight absorbency of ethanol, this would not

necessarily be picked up during FT-IR due to PCL containing bound –OH groups. None

of changes in PCL alters PCL enough to be considered hydrophilic(a contact angle of 90°

is generally the threshold for hydrophilicity) but there has clearly been alteration

which would favour cell attachment (Huang et al. 2003; Pham, Sharma, and Mikos 2006;

Cipitria et al. 2011). 15G is the clinical-grade sterilisation method that is most similar to

the virgin sample, and UV is the most similar to 15G, although the differences between

all of the clinical sterilisation methods are minimal.

Having looked at the changes that were in the chemistry of PCL, the next set of

experiments sought to determine the effects of sterilisation at the macroscopic level in

the form of mechanical and tensile testing. When implanting a biomaterial in vivo it is

essential that the biomaterial can withstand the mechanical load placed on it, whilst not

interfering with the normal function of the body; this is especially true in terms of

regenerative medicine, where failure can be disastrous for the body’s recovery (Kohane

and Langer 2008; Olbrich et al. 2007; Kumbar et al. 2008). However, it is also difficult to

model exactly how the material will behave; often in vivo animal tests are the only way

to perform the experiments, but an element of scalability is needed to predict how the

material will work in humans (Sun et al. 2010).

From the outset, it was likely that there would be an effect on the mechanical

properties; sterilisation had affected the outcome of most of the other tests so far, and

changes in mechanical properties were easier to predict than the experiments

59

previously done. Average molecular weight, as tested using GPC, changed for all but one

of the conditions (ethanol) and so changes in tensile testing were predicted (Figure 3.9).

When tensile testing is performed, one of the commonly cited values is that of Young’s

modulus, the ratio of stress to strain in a material, whilst the material is in a state which

obeys Hooke’s law. Young’s modulus can easily be compared between materials of all

classes, and often provides a baseline against which materials can be judged against

each other.

In all but one case, the Young’s modulus fell considerably when compared to the virgin

sample, this ranged from 8.68 – 49.23 MPa, representing a drop to 37% of the virgin

sample’s original strength for the 25G sample (Figure 3.5).

Interestingly, 50G had a higher average Young’s modulus than 25G despite the dose of

radiation for 50G being twice that of 25G, and 50G having a higher overall percentage

crystallinity. Ethanol and ethylene oxide again showed very similar results, suggesting

that their physical methods of sterilisation, along with chemical similarity, had a

comparable effect on PCL (Figure 3.5). For a second time, UV defied the trend by

increasing in Young’s modulus by 18.81 MPa, similar to the GPC result (Figure 3.5 and

3.9).

Ultimate tensile strength is the maximum stress that the material can take at failure. In

the context of PCL, a ductile fibrous material, it is assumed that the ultimate tensile

strength will be at the point when the first fibre fails. Examination of the stress-strain

curves shows that in some cases the material carried on deforming for a long time after

the ultimate tensile strength point (the apex of the graph) had been met. UV was shown

to have very similar ultimate tensile strengths as 25G and ethylene oxide (10.32 MPa

compared to 10.22 and 10.72 MPa, respectively), all of which were higher than virgin

and ethanol (8.72 and 8.51 MPa) (Figure 3.6). 15G and 50G were both lower than the

virgin sample, with 50G again having the lowest value, in keeping with the prediction

that multiple doses of gamma radiation will weaken the structure (Rogers 2005).

Strain is the maximum elongation that a sample can take before failure, and the

maximum strain point is often closely related to the ultimate tensile strength as they

both peak when all the fibres are bearing their maximum load. Although the data shown

60

is the strain at failure and not the strain at the maximum load point, the two graphs of

strain and ultimate tensile strength are reasonably similar (Figures 3.6 and 3.7). Again,

50G has the lowest value, but this time it is only 25G and ethylene oxide that have

higher values than the virgin sample (0.4 and 0.43, compared with 0.31), with ethanol,

UV and 15G have comparable strains to virgin. The 50G value (0.19) is significantly

lower than all of the other values, and is almost certainly related to the high degree of

crystallinity that 50G exhibited. Changes in the mechanical properties of UV scaffolds,

as predicted by the changes in morphology under SEM imaging (Figure 3.15 D) were

borne out by the mechanical testing data. However, the results counter-intuitively

showed an increase or no change in mechanical strength, rather than a decrease which

would have been expected by the apparent damage seen (Figure 3.15).

It is possible that the shrinking that seemed to occur as a result of UV radiation helped

to remove flaws present in the structure, whist at the same time lowering the amount of

force placed on the cross-sectional area.

Disappointingly, it is difficult to draw any overarching conclusions from the mechanical

properties. The data shown finds itself in agreement with another study, whose authors

also observed the difficulties in drawing any conclusions as (specifically for the gamma

samples) whilst there is a trend in GPC and DSC data, this is not borne out in the tensile

testing data (Bosworth, Gibb, and Downes 2012). A lack of a dose-dependent pattern

hampers the possibility of finding a perfect method of reproducing sterilisation effects.

15G seems to be the closest to the mechanical properties of the virgin sample, and

ethanol sterilisation will do a sufficient job of representing 15G in terms of mechanical

properties.

4.2 Cellular Response

Whilst learning how the PCL scaffolds physically behave after sterilisation is important,

the essential tests that had to be performed involved learning how the scaffold would

react to cells. It is the responses of the cells to the different types of sterilisation which

will ultimately decide whether or not a certain sterilisation method can be used

clinically. Expanding on that slightly in the context of this research, a lab-based

sterilisation method would be expected to replicate the effects of clinically permissible

61

sterilisation methods on cells. Conceivably, an excellent cell response could ameliorate a

poor performance in physical characteristics by a sterilisation method; the proviso

naturally being that none of the physical performance categories could fall below a

minimal level for implantation. From the outset, there was a focus on the negative

changes that could be made after ethanol and ethylene oxide sterilisation; it was already

known that cell culture with scaffolds is generally more effective than on tissue culture

plastic. Gamma and UV irradiation do not leave the sterilised object with any lasting

radiation, and the only changes in cell response would be indirect; changes to the

morphology of the PCL fibres could alter a cell response. Ethanol and ethylene oxide

were considered to be more interesting subjects for cell work; any absorption of the

chemical into the fibres may not have been picked up in very small quantities by the FT-

IR (especially in the middle of the fibre bundles), but there was enough evidence from

FT-IR to show that no adsorption occurred.

Unfortunately, not all of the conditions for every sample were able to be analysed due to

the failure of the SEM filament. Enough images were made for the virgin, ethanol and UV

samples at all stages; however the only 15G ones available are of quite low quality and

only show the results of cell seeding. No images were able to be taken for the 25G, 50G

and ethylene oxide samples at any stage. Day 3 cell culture scaffolds, along with a

control group of scaffolds in media without cells, were also prepared, but were unable

to be imaged due to the aforementioned technical issues.

Sterilisation was shown to have little effect on the microscale of the fibres when viewed

under a low-power SEM (Figure 3.14). Imperfections in the fibres, thick flat ribbons of

PCL, can be clearly seen. These could have affected the mechanical properties of the

sample, and might have caused changes in cell response. However, the fibres appear to

be aligned and ordered (Figure 3.14). A closer examination of the scaffolds, but still

within the micron level, showed a level of disorganisation in the scaffold, and damage to

some of the UV-sterilised fires (Figure 3.15 C, D).

Cell culture a Day 1 showed vary varied results (Figure 3.16). The very disorganised

PCL structure seen mostly in Figure 3.16 A showed the effect that the cell culture media

can have on a fibre bundle. The only scaffold which showed noticeable cell growth was

UV, which also has a very organised fibre structure with a very high percentage of

62

aligned fibres. Whilst the correlation and causation effect of this is not known, it is

reasonable to assume that the ECM organised by the cells had an effect on pulling the

fibres to organise them.

Also, as an orientated structure tends to provide a more effective cell scaffold, it could

be that the cells responded more positively to the high degree of orientation. If the

images of 15G had been clearer at a higher magnification that was achievable, it is

possible that some of the previous questions could have been answered (Figure 3.16 D).

It appears that the fibres are orientated, but with the amount of cell growth is not

visible. Regardless of the cause of the initially stunted cell growth, a cell sheet formed on

all of the three samples imaged by Day 7 (Figure 3.17).

It can be surmised that the cracks seen in all of the cell sheets was caused by the

destructive dehydration preparation process necessary for SEM imaging, which would

have caused the cells to shrink and decrease in size. This resulted in an organised

fracturing process of the cell sheet, which the pieces taking on a jigsaw-like quality.

From the evidence shown, it is clear that regardless of the sterilisation method used, the

cells can eventually go on to form a cell sheet across the scaffold. Although the rate of

cell growth appeared to have differed, neither ethanol nor UV had a lasting effect on cell

growth.

The only way to get implied data on cell growth for all sterilisation conditions is to use

the florescence data to draw conclusions. The same density of cells was cultured onto

scaffolds for florescence data as it was for SEM imaging. The results for Day 1 seem to

conflict with SEM data obtained, which shows a reasonably similar level of cell growth

for virgin, ethanol and UV (Figure 3.8). 50G and ethylene oxide had halting starts, with

both numbers being lower than the tissue culture plastic control; 536 and 178

compared with 619 (Figure 3.8). At Day 3, all of the gamma sterilisation methods had

very similar results (1621, 1439 and 1503), with 50G making a significant

improvement. The cell growth on ethanol declined significantly (the only time in this

experiment), losing over half of the cells (942 down to 403), whilst virgin and UV

samples also decreased, but only slightly, and the control had a very modest increase

(Figure 3.8). Ethylene oxide continued to perform very poorly, almost half as bad as the

63

ethanol result. By the end of the Day 7 results, the data had spread out significantly.

Ethanol and UV were still worse than the virgin sample, the latter of which was found to

be a good match for 15G and 25G (virgin was 2239 with the gamma samples at 2627

and 2915). 50G was surprisingly high, with a florescence of 1000 greater than the

second placed sterilisation technique, 25G. A possible reason for this, along with some

unusual performances in the mechanical testing section could possibly be put down to

the multiple passes that were needed for that high level of gamma sterilisation.

Ethylene oxide, however, proved to be significantly cytotoxic, with the control being

twice as effective for cell culture. Following from this experiment, ethylene oxide should

be more closely investigated to show possibility of any ethylene oxide being retained

after sterilisation of the scaffold.

There are several reasons for the effectiveness of some of the gamma techniques, but

the most compelling appears to be the effectiveness of gamma irradiation as a

sterilisation method.

The presence of bacteria and other harmful pathogens will damage the seeded cells, and

as a consequence stunt their growth. As gamma irritation is a highly effective in

disabling pathogens, the near-sterile fibres should have provided an ideal environment

for cell growth. This would explain why 50G, which needs multiple passes for

sterilisation, would be more effective than a single dose of gamma radiation. Another

explanation for cell growth on gamma-irritated scaffolds suggested that a rougher

surface improved the attachment of the cells (Cottam et al. 2009; Bosworth, Gibb, and

Downes 2012). Whilst feasible, no experiments were performed here to look at the

surface texture. Changes to tensile properties, the only other area in which the

sterilisation methods had a significant effect, also provide a possible explanation; cells

may react better to changes in stiffness. It is likely however, that changes in one area are

not enough to greatly affect the cell number overall; the effects are multi-factorial.

64

4.3 Further Work and Conclusions

Future work should look at the effects that ethylene oxide can have on cell growth, as a

priority, x-ray photoelectron spectroscopy (XPS) can also be used to detect surface-

chemistry changes. Trying to find a pattern for the varying mechanical properties and

any effects this might have on cellular behaviour is also important. Repeats of cell

culture work and SEM imaging, outside the scope of this work, would help with some of

the conclusion drawn here.

No changes could be seen in FT-IR and GPC, showing that there are no gross changes in

the chemistry of PCL. Alterations at the boundary between physical and chemical

effects, such as in the crystallinity and melting temperature shown in DSC, suggest that

any changes in chemistry are subtle, and not on the larger scale that FT-IR and GPC can

detect. Significant differences in the physical response to mechanical load and water

contact angle again show that sterilisation affects how the material will behave, with

significant differences being seen. Changes to cell response are extreme when looked at

from the quantitative level that fluorescence allows.

Qualitatively, from the few SEM images that were obtained, the cells form a large sheet

(after time) and generally perform equally.

In terms of choosing which lab based sterilisation method to use to replicate each

clinically-based technique, there are some conclusions which can be drawn. For gross

chemical structure any method can substitute for another (FT-IR). In terms of molecular

weight, virgin and ethanol can give good approximations for gamma and ethylene oxide

(GPC). The melting point of PCL is unlikely to have to be replicated, but UV makes a

good substitute for ethylene oxide, and virgin and ethanol are suitable for gamma (DSC).

Crystallinity cannot be suitable replicated by a lab-based sterilisation method, and any

papers or experiments which reply on percentage crystallinity of PCL will have to be

looked at dubiously if conclusions between ethanol and virgin against gamma and

ethylene oxide are drawn (DSC). When looking at hydrophilicity, UV should be used for

gamma and ethylene oxide (water contact angle).

65

Decreases in the stiffness caused by radiation are hard to replicate, although virgin

should work for a 15kGy dose (Young’s modulus.) Ultimate tensile strength did not

follow a pattern, but UV could be suitably used for 15kGy and 50kGy. Strain is also hard

to replicate properly, virgin, ethanol or UV could work as a stand-in for 15kGy. Concrete

results from cell culture are hard to get, but avoiding ethylene oxide due to cytotoxicity

is important. Concerns about over-proliferation of cells may mean that the highest level

of irradiation, 50kGy should also not be used. 50kGy does not follow a clear trend with

respect to the doses at 15kGy and 25kGy, and it is conceivable that this was an errant or

anolmols sample. The limitations of this study prevented an entire repeat of a sterilised

sample, but this should be investigated as a matter of some urgency in another study.

Summing up the data, it is clear to see that the type of sterilisation does have some

effect on PCL in certain areas, but predicting patterns and the conclusions that can be

drawn will need much more work than for which this study allows.

66

Appendix A – FTIR Data

FTIR Spectra of all Samples – each PCL condition was tested, and the results listed here, n=3

67

68

69

70

71

Appendix B – GPC Data

GPC Graphed data – the peaks which gave the data in Figure 3.9 are reported here.

72

Appendix C – Stress Strain Curve

An Example Stress Strain Curve – this is the stress-strain data obtained from virgin fibres.

Calculations in Chapter 3 Section 3 Tensile Testing were made using figures in the following areas. A:

Young’s modulus. B: Ultimate tensile strength. C: strain

0

2

4

6

8

10

12

14

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Stre

ss (

MP

a)

Strain

Stress vs Strain of Virgin PCL Samples

1

2

3

4

5

B

A

C

73

APPENDIX D – CONTACT ANGLE DATA

All Water Contact Angles

74

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