The Use of Electrospinning to Generate Polyvinyl Alcohol Nanofibers in Order to Replicate Collagen Fiber

Orientations in Bone

Third Year Project

Varman Perinpanathan

April 2014

100640263

Supervisor: Dr Asa Barber

With an increasing ageing population and a shortage of bone donors there is a need to identify non-

traditional methods to replicate bone for medical purposes. With the increase in technology and the

materials available researchers have been looking for other solutions and nanotechnology and especially

nanofibers. The objective for many researchers now is to incorporate these technologies into bone in a

sustainable and reliable method. The aim of this project is to examine and understand the role of collagen

and the hierarchical components within bone and to generate polyvinyl alcohol nanofiber alignments to

replicate them via electrospinning.

School of Engineering and Materials Science

Engineering/Materials

Third Year Project

DEN318

April 2014

DECLARATION

This Report Entitled:

The Use of Electrospinning to Generate Polyvinyl Alcohol Nanofibers in Order to Replicate Collagen Fiber

Orientations in Bone

Was composed by myself and based on my own work. The work of others has been fully

acknowledged within the text and referenced, this includes the use of figures and tables.

This report has not been submitted for any other qualification.

NAME Varman Perinpanathan

Signed

Date

1

Contents

1. Introduction 5

2. Understanding Bone and Collagen 5

2.1.Collagen’s Structural Integrity 6

2.2.Relative Diameters 6

2.3.Stress-strain Relationships 6

2.3.1. Tensile Strength Relative to Nanofibers 7

2.4.The Organic Matrix 7

3. The Role of Nanofibers 8

3.1.Technique and Practicality 9

3.2.Current Uses 9

3.3.The Usefulness of Polymers 9

4. Electrospinning and its Applications 9

4.1.A Brief History 9

4.2.The Taylor Cone 10

4.3.Applications in the Medical Field 11

5. Polyvinyl Alcohol 11

5.1.Suitable Spinning Parameters 13

5.1.1. Voltage 13

5.1.2. Distance 13

6. Biocompatibility 14

6.1. Important Considerations 14

6.2.Test Methods for Synthetic Polymers 14

6.3.The Importance of Mechanical Testing 15

6.4.Host-tissue Response 15

7. Scanning Electron Microscopy 16

7.1.Key Components 16

8. Fabrication and Characterisation of Poly(Vinyl Alcohol)/alginate blends, Shahidul Islam,

Mohammad Rezaul Karim 18

8.1.Replicating Anisotropy 19

8.2.The Importance of Micro-crack Dissipation 20

9. Tensile Testing of Bone Explants 20

9.1.1. Explants from the Transverse Direction 21

9.1.2. Explants from the Longitudinal Direction 22

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9.1.3. Explants Taken from 45 Degrees 23

9.2. Indications of Structure 24

9.3.Lamellae Organisation 25

10.Nanofiber Orientations 25

10.1. What Tensile Testing Will Simulate 26

11.Materials & Methods 27

11.1. Solution Preparation 27

11.2. Electrospinner Settings 29

12.Results 40

13.Discussion 42

13.1. Experimental Difficulties 42

13.2. Findings 45

13.3. Future Work 45

14. Acknowledgements 46

15.References 46

List of Figures

Figure 1: Molecular Mechanics of Mineralised Collagen Fibrils in Bone, Nair, Gautieri, Chang, Buehler............................................................................................................................................................8Figure 2 The transition between slow and rapid acceleration of the polymeric solution that occurs in electrospinning, The New Zealand Institute for Plant and Food Research Ltd, Joanna Gatford, 8 September 2008..........................................................................................................................................11Figure 3 An SEM image showing the magnification of electrospun PVA at a magnification of 10916x, from reference 24.........................................................................................................................12Figure 4 Testing for Biocompatible Polymers, see reference 25..........................................................14Figure 5 A schematic of a Scanning Electron Microscope.....................................................................17Figure 6 SEM images of electrospun PVA nanofibers prepared by using different PVA solution concentrations of (a) 8 wt.%, (b) 10 wt.%, and (c) 12 wt.%, see reference 15....................................18Figure 7 Stress–strain curve of electrospun PVA/Alg blend nanofibers with various volume ratios of 10 wt.% of PVA to 2 wt.% of Alginate solutions of (a) 100/0, (b) 80/20, and (c) 60/40 10, see reference 15.................................................................................................................................................19Figure 8Stress-strain relationships of 3 samples of transverse bone explants...................................21Figure 9 Stress-strain relationships of three longitudinal bone explants..............................................22Figure 10 Stress-strain relationships between 3 bone explants taken at 45 to the direction of long bone...............................................................................................................................................................23Figure 11 Variation in the alignment of collagen in turkey tendon, see reference 13.........................26Figure 12 Preparation of 10% concentration PVA (80ml) solution on a heated magnetic stirrer......28Figure 13 8% concentration PVA (80ml)..................................................................................................29Figure 14 Needle and dye system with plate collector system, PVA nanofibers collected on the Al foil..................................................................................................................................................................31

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Figure 15 ES control for high voltage, initially 15kV was used which was gradually increased to approximately 22.5kV..................................................................................................................................32Figure 16 The use of the 55mm diameter roller with foil collector.........................................................33Figure 17 The PHD Infusion 2000 with syringe containing the PVA polymeric solution....................34Figure 18 Initial foil frame used to collect fibre orientations...................................................................36Figure 19 Smaller frames used to collect fibers that could occur in random initial alignments along the roller........................................................................................................................................................37Figure 20 55cm roller under 22.4kV..........................................................................................................38Figure 21 The hierarchical structure of bone from whole bone to mineralised collagen, http://newscenter.lbl.gov/news-releases/2011/08/29/the-brittleness-of-aging-bones-%E2%80%93-more-than-a-loss-of-bone-mass/...............................................................................................................39Figure 22 Optically microscoped nanofibers produced from 10% PVA solution collected on the plate system.................................................................................................................................................40Figure 23 Optically microscoped nanofibers produced from 10% PVA solution collected on the roller system.................................................................................................................................................41Figure 24 Incidences of the PVA polymer leaking at the needle and tube interface before being drawn into the needle..................................................................................................................................43Figure 25 Methods used in order to prevent leakages of the solution occurring................................44

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1. Introduction

‘The economic impact of musculoskeletal conditions in the United States represents $126

billion’[33]. Bone is an extremely complex composite of different proteins, polypeptides,

extracellular matrix, blood vessels etc. Unlike other tissue it also consists of a very unique

regeneration biology and fracture repair which leaves it devoid of scarring and therefore

allows normal function to resume after trauma or fracture. Replicating this process entirely

would be a challenge and the ability of any artificial tissue or organ to completely imitate

the role of the host’s tissue seamlessly is a challenge. However, there are simpler ways to

replicate bone regarding its mechanical and functional roles rather than its biological

composition. With the polyvinyl alcohol that is intended to replicate bone, it is ideal for it to

provide a balance and successful incorporation in order to the normal biological functions

of bone to occur around it.[3] The host tissue and the artificial polymer interface will consist

of varying interactions that will consist of understanding the dynamic environment that

bone consists of. The most important consideration regarding the role of the polyvinyl

alcohol is the stresses that it can mimic regarding collagen and its role in many of the

forces that it has to avoid such as shear, torsional, tensional etc.[2]

2. Understanding Bone and Collagen

As with any technology that is introduced into the body, there is a requirement to

understand the host tissue, in this case, cortical bone. The mechanical properties of bone

are determined by its structure. Within mammals and birds, the stiffness of bone is dictated

by its fibrous protein collagen content. The vast majority of these bones are hollow and

consist of a cortical external layer with a cancellous or intramedullary component which

does very little to support the structure mechanically; it aids with blood circulation and

other circulatory responsibilities. Water is an important mechanical component of bone

particularly in the organic components of bone.[14] The inorganic components rely heavily

on the mineralisation of collagen fibrils by varieties of calcium phosphate.[3] Calcium

phosphate crystals exist in varying sizes with differing morphologies. The crystals are very

reactive due to a high surface area to volume ratio. Due to the impurity of the crystals that

can occur from this high reactivity, carbonate will replace the phosphate groups at bone

extremities reducing the crystallinity of the area.[14]

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2.1. Collagen’s Structural Integrity

Collagen Levels of structure and alignment have a significant role to play in the function of

bone in any given location of the body whether it is regarding the resistance of tension,

compression and shear or torsional forces. It is a structural protein and for this reason

collagen is the most abundant protein in the body and accounts for 30% of the body’s total

protein content. Of this protein content, 90% consist of collagen.[11] The remaining 9-10%

of proteins are classified as non-collagenous (NCPs). Within the body the tissues that

provide the most structural support are the tendons and bones and other connective

tissue. Collagen is a group of twenty related proteins that form triple helices i.e.

tropcollagen is a common example that exists as the most basic structural sub-unit of all

forms of collagen.[14]The triple-helix strands of collagen have the capability and

propensity to bind together to form structures on a micro-scale that lead onto become

bone and tendon on a macro-scale. Bone has a hierarchical structure that makes it very

stable; this is because of the interactions between the organic and inorganic compositions

of bone i.e. the interactions between the extracellular matrix and the mineralised fibrils.

Bone is also under constant dynamic remineralisation and remodelling via the interactions

with osteoclasts and osteoblasts, this is necessary for healthy bone tissue.[13]

2.2. Relative Diameters

Following on from the hierarchical structure of bone, there are two distinct forms of bone,

these consist of lamellar and woven bone. The collagen in woven bone generally consists

of diameters of approximately 0.1 – 2.5 μm and have a very random orientation.[13] The

random arrangements lead to high mineralisation and in most cases is very porous.

Lamellar bone is more ordered in its arrangement and organised into sheets of collagen

fibres (lamellae). The fibrils lie in planes as the mineralisation occurs. The fibrils exist in

aggregated bundles of approximately 30-100 μm across. Each of these bundles consists

of a constant fibril orientation but this will vary from one bundle to the next. One further

level up in the hierarchical structure of bone is the compact or cortical bone.[13] [14]

2.3. Stress-strain Relationships

The stress-strain relationship of bone will not be absolute, this is because bone is

anisotropic structurally and therefore mechanically i.e. its properties that are measured in

different directions will vary. [13][14][19] Fracture will usually occur in stiff materials, the

material properties are dominating factors along with the geometries of the material. The

6

tensile strength is usually very easily to calculate, it is usually the load at which the

specimen breaks. Another component of mechanical testing for bone is fracture

mechanics. They are often carried out in tension. Fracture mechanics occur before the

initial yield strength of any material and not just bone. Bone however, will show variations

in the elastic deformation stage depending on the orientation of the fibres and the water

content within them.[14]

2.3.1. Tensile Strength Relative to Nanofibers

Asran et al prepared PVA nanofibers with PVA/Type I collagen and composites with

hydroxyapatite nanoparticles with electrospinning techniques. PVA has recently become a

popular choice of polymer for scaffold applications due to the mechanical properties it

possesses along with promising repair properties when combined with the correct

bioactive materials necessary for bone such as HA. Their findings indicated the promise of

using PVA within a scaffold, the morphology of their PVA nanofibers at 15% concentration

have no beads and are uniform fibers. The tensile strength of the nanofibers consisted of

0.22 +/- 0.09MPa with an elastic modulus of 2.67 +/- 0.78MPa. Bone is built up

mineralised collagen fibrils[13] with a complex structure that can be described in upto

seven hierarchical levels of organisation. The organic framework consists of a fibrous

structure with individual triple-helical modules. These molecules are arranged within long

fibrils, they are generally parallel to the long bone axis.[14]

2.4. The Organic Matrix

Bone mainly consists of an organic matrix that has been mineralised by the deposition of

calcium phosphate. Bone is a hierarchical structure that is also a composite material; this

provides it with a unique structure along with mechanical properties that cannot be found

in any other tissue in the body. Olszta et al proposed that intrafibrallar mineralisation of

collagen is achieved during bone formation, their findings have indicated that there is a

precise relationship between the hydroxyapatite and collagen fibrils in bone.[9] Collagen

has an amino acid sequence of –(glyicine-proline-hydroxyproline)- therefore allowing the

structure to form into a triple helical structure. These structures are also known as

tropocollagen molecules.[9] The Extracellular matrix of bone is mainly composed of Type I

collagen, the mechanical structure and tensile strength of bone itself is due to the collagen

forming translational modifications within the collagen and tropocollagen fibers, these

phenomena mainly consist of glycolysation and the formation of crosslinks etc. between

7

the individual fibers therefore providing a platform where bone structures contain a good

integrity when forces are applied in one or more directions.[8]

Figure 1: Molecular Mechanics of Mineralised Collagen Fibrils in Bone, Nair, Gautieri,

Chang, Buehler

The above figure indicates the microstructure and hierarchical organisation of bone and

the collagen and mineralisation that occurs. It is important to note the fibre patterns and

the fibre in the fourth image; this is the approximate diameter that nanofibres can be

electrospun.

3. The Role of Nanofibers

3.1. Technique and Practicality

Nanofibers constitute of fibers produced at diameters less than 100-500nm.[1] The most

common technique used to fabricate these fibers is electrospinning. Nanotechnology itself

has been present in research into materials science from as early as the 1960s.

Electrospinning involves the drawing of a molten state fluid, most commonly a polymeric

solution, undergoing a charge resulting in the solution to be forced out of a needle or a die

system. This causes continual stretching until the polymeric fiber is collected on charged

plate down a concentration gradient.[4] The polymeric jet that is formed will not break up

due to certain entanglements within the solution prevent any breaks in the fiber that is

formed. The process also relies on the external conditions of the system cooling and

solidifying the jet to form a fibrous structure. This also causes the solvent that is used to

evaporate leaving behind the desired polymer in, ideally, in a nanofibrous form collected

on the plate.[1][2] Electrospinning is one the key methods of attaining diameters of

8

polymers tothis type of scale practically and economically. The main parameters that

influence the properties of the polymers that are produced as fibres are; applied voltage,

feed rate of the solution, the conductive properties on the collector plate, the length or

height between the electrospinning needle and the collector and the type of collector itself

i.e. roller vs. plate.[2] Other properties within the experimental procedure will depend on

the atmospheric or ambient conditions, these include humidity; which will affect solvent

evaporation, viscosity of the polymeric solution; which will affect the density and fiber

diameter collected.[1]

3.2. Current Uses

Nanofibers already have a number of uses with medical applications such as

bioenegineering and tissue engineering; nanofibers can be used as wound dressings,

scaffolds loaded with collagen and other bone morphogenic proteins to enhance cell

proliferation at damaged or disease sites in patients, in wound dressings, tissue

engineering, and in pharmaceutical systems such as drug delivery.[2]

3.3. The Usefulness of Polymers

Polymers consist of repeating sub units, formed with a covalently bonded carbon

backbone which provides polymer molecules with the ability to slide over one another and

also due to their relatively low density, the fibers can be formed into shapes and

formations that show a high complexity while exhibiting a high strength. [1][2]The fibers

also tend to consist of a very large surface area to volume ratio.[2]

4. Electrospinning and its Applications

4.1. A Brief History

Essentially, electrospinning is the electrostatic generation and drawing of nanofibers from

a polymeric solution. The earliest recording interaction between a liquid an electrostatic

attraction was made by William Gilbert in 1600.[18] It was not until 1914 where John

Zeleny where the first mathematical models surrounding the behaviour of electrostatic

forces when he studied glycerine in a high electric field. Fifty years later, Sir Geoffrey

Ingram managed to produce the first theoretical underpinning of the mathematical

background behind the cone that forms when the fluid droplet is exposed to the effects of

the electrostatic field used in electrospinning.[18] This gave rise to the Taylor Cone.

Antonin Formhals was the first researcher to patent electrospinning as a technique and

9

established experimental set ups and procedures for the production of polymer fibres and

the first to use a variety of collection methods such as the rotating drum method.[17] His

patents existed from 1934 to 1944. [18]

4.2. The Taylor Cone

The Taylor Cone formation arises from the electrically conductive capability of the liquid

when exposed to a field of electrical current, deformation occurs; this is a result of the

surface tension. The increase in voltage at this point past a threshold a jet forms which is

the beginning of the electrospraying part of the process. The polymer solution that is

drawn through the needle where this process occurs then reaches an earthed collector.

Two standard electrospinning setups are used; vertical and horizontal, the options to

researchers depend on the strength of the current and the distance the fibers are required

to be spun. The external conditions are composed of mainly atmospheric and room

temperatures in order for the solvents to evaporate leaving the thin fibres behind.[17]

Conditions also require ventilation as the polymeric solutions can emit harmful vapours

during the process. Reneker refined the descriptions surrounding the interplay between

shapes, surfaces and fluid rheology and these interactions with electrical charge.

Fundamentally, without charge, the process cannot occur. The charge is carried by ions

which flow faster through the fluid.[7] The fluid itself is neutral and within the system the

ratio of positive to negative ions is 1:1. Ions tend to be diffusive and repulsive and

therefore the electrical potential tends to be the same all over the liquid.[16]

10

Figure 2 The transition between slow and rapid acceleration of the polymeric solution that

occurs in electrospinning, The New Zealand Institute for Plant and Food Research Ltd,

Joanna Gatford, 8 September 2008

Figure 2 shows the transition between slow acceleration and a rapid acceleration that is

present in the electrospinning procedure as a result of the variation between ohmic and

convective current flows outside of the nozzle or spinning tip. This process is present in all

electrospinning procedures and is key for the generation of nanofibers via the rapid

acceleration onto the collector.

4.3. Applications in the Medical Field

With the emergence of electrospinning and its production of nanofibers, researchers

became increasingly interested in the potential of fibre fabrication in nanotechnology.

Electrospinning is a solid technique that can generate fibers for many applications given

the right parameters. The applications have potentials that range from aerospace to

medical materials; it is particularly useful in medical applications because the diameter of

the particles can replicate the naturally occurring fibrous molecules that exist in the body.

[6]

5. Polyvinyl Alcohol

Poly(vinyl alcohol) (PVA) has a range of biomedical purposes due to its solubility with

water and its low cytotoxicity. PVA is unique among polymers as it does not require a large 11

scale polymerisation reaction in order to link its monomers (polyvinyl acetate) into the

polymer chain. The reaction that is required is polyvinyl acetate in the presence of

methanol and sodium hydroxide catalyst.[26] Polymeric fibers in general can be

electrospun easily if the experimental parameters such as voltage and concentration are

adequately kept to within the potential of obtaining fibers that can carry out their intended

design function. PVA has high chemical stability and inertness and normal temperature

and also has good mechanical properties. One property that PVA has that been identified

by Zhang et al is that PVA can be formed into mats which is the result of the fibers being

randomly deposited, which as a result, leads to a larger surface area than normally

collected woven structures and a smaller pore size. This capability of PVA in an

electrospun form has increased its potential to another variety of applications particularly

to do with porous structures such as bone which is likely to increase surface cohesion

between the artificial structures and host cells.[10][11]

Figure 3 An SEM image showing the magnification of electrospun PVA at a magnification

of 10916x, from reference 24

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5.1. Suitable Spinning Parameters

PVA itself in preparation for electrospinning usually requires temperatures around 90-98

degrees Celsius within water bath conditions and continuous stirring for two or more hours

in the form of a rotating viscometer as Zhang et al have demonstrated in their findings. Li

et al have used conditions ranging from 6-9% PVA concentration dissolved in de-ionized

water and gently stirred at 90 degrees Celsius for 2 hours to achieve a homogenous

solution ready for electrospinning. Li et al also varied other parameters including the

distance of the nozzle from the collector from 260mm to 380cm in equal increments, the

voltage used varied between three levels of 17.5kV, 20kV and 22.5kV. The parameters

that were kept constant were the feed rate (0.2ml/h), the diameter of the tip of the needle

(0.5mm) and the collector used (flat metal plate covered in aluminium foil). Their findings

indicated that at lower concentrations of PVA to de-ionized water the PVA fibers formed

beads in their structure which indicated that the process that had occurred was more

electrospraying in nature. This is phenomena occurs because the viscoelastic properties

are not strong enough to overcome the coloumbic force and therefore results in broken

and smaller jets of PVA. At higher concentrations, their findings resulted in resulted in the

gradual disappearance of the beads and a more elongated fiber structure occurring

instead. Li et al had explained that due to the increasing viscoelastic properties the charge

could not break up the solution into smaller droplets during the process due to the

increased amount of intertwining and entanglement of the fibers.

5.1.1. Voltage

The spinning voltage used by Li et al had indicated that with a lower voltage had produced

fibers that were more uniform in terms of fiber diameter with narrower distributions along

the collector whereas the increased voltages had resulted in a more varied distribution of

diameters. This phenomena has been explained in two parts: the increased voltage results

in a greater electrostatic force on the solution which, theoretically, should result in thinner,

more uniform fibers; however, the greater acceleration draws out more per second.[7]

5.1.2. Distance

The collection distances were 260-380mm in equal increments of 40mm. Li et al have

shown in their findings that the morphology have changed with increasing the distance; the

fibers were found to be thinner when the collection plate was at its maximum distance.

13

This resulted in a more time for the fiber to decelerate to a lower velocity while being

drawn out over a greater distance resulting in a thinning of the jet.

6. Biocompatibility

6.1. Important Considerations

A general overview of the importance of safety of any medically related implant device is

established, ‘biomaterials include a broad range of materials that must meet stringent and

diverse requirements to be acceptable for use in the body and to meet the needs of

specific devices’ Michael N. Helmus. Polymers that are considered to be biomaterials fall

under two categories: non-biodegradable and biodegradable, however, both must fulfil the

major parameters of implant and biomaterial design such that they are not non-toxic, non-

carcinogenic and non-mutagenic along with extrinsic properties that make the material

easily fabricated and sterile.[35] Further intrinsic properties of biodegradable polymer

consist of the ability to remain mechanically adept while it undergoes chemical, physical,

mechanical and biological external forces.

6.2. Test Methods for Synthetic Polymers

The table below identifies the type of test methods that synthetic polymers will have to

undergo in order to identify specific experimental data that can be further drawn upon.

Type of Test Methods Response

Chemical Analysis Chromatography

Hydrolytic stability

IR spectroscopy

Oxidative stability

Degradation is possible

Measurement of purity,

molecular distribution

Physical Analysis Solution Rheology

Intrinsic Viscosity

Identification of molecular

weight

Mechanical Stress/strain properties

Viscoelastic properties

Ultimate polymer strength

Viscoelastic flow

Biological In Vitro and Ex Vivo

properties

Determining

biocompatibility

Figure 4 Testing for Biocompatible Polymers, see reference 25

One of the key properties of an implantable polymer is its hydrolytic stability i.e. its ability

to maintain physical integrity when exposed to an aqueous environment.[25] Hydrolysis is

14

a double decomposition reaction that requires the input of a water molecule for every bond

to be broken. It is the single step that causes degradation given the right conditions; water

ionises at a greater rate when the internal body conditions alters from pH 7 to acidic or

alkaline conditions.[27]

6.3. The importance of mechanical testing

The examination of mechanical properties is required to find a comparison and identify

similarities between materials that will be at the interface between implantation; in this

case, bone and PVA. The stress-strain test is the most widely used and recognised

mechanical test in the world, it is useful from a mechanical viewpoint as it provides both

the strength of material along with its toughness.[25] The stress-strain curve itself provides

other possible observations such as the modulus of elasticity, stress at the material’s yield

and the percentage strain at rupture or failure. Stress itself is determined as ‘the elastic

resistance of intermolecular bonds in a material to being elongated, loads cause

elongation, which then causes stresses. Three principal strains and stresses include

tension, compression and shear’. Strain is defined as ‘the deformation or change in

dimensions and/or shape caused by a load on any structural material’.[33]

6.4. Host-tissue Response

The use of this PVA orientation technology has the potential to be used in cortical bone

grafts, as a result the mechanical properties in vivo is an important for the responses

within the body, the interactions at graft interfaces and the host tissue functionality that

may result in stress-shielding because of Wolff’s Law. [30] An ideal scenario for a bone

graft, regardless of the material involved is the aspects of the environment with the

mechanical properties at the bone-graft junction. While cortical bone is relatively simple to

replicate, the stable fixation may not be. [31] The complications of any artificial material is

due to the constant dynamic nature of bone and its remodelling processes which maintains

the competence of bone to its mechanical needs.[32]

Due the nature of bone and its remodelling processes that it constantly undergoes, bone

grafts tend to be unsuitable for young individual in particular young adults and children.

Bone grafts have been used in trauma treatment but with the ageing population in hand,

there is an increased need in grafts and bone replacement within the elderly demographic

in order to maintain quality of life. Bone remodelling and fracture healing in elderly people

has a decreased proliferative activity of osteogenic precursor cells and reduced maturation

15

of osteoblast precursors.[33] Along with these reasons have been attributed to local and

systemic decline in altered receptor levels leading to an inability for osteonic processes to

occur at the optimum rate. Furthermore, a drop in growth factor secretion and an alteration

of the extracellular matrix within bone are also determining factors in the mechanical

properties in bone in the elderly.[34]

7. Scanning Electron Microscopy

Scanning Electron Microscopy (SEM) is an imaging technique that uses focussed beams

of electrons in order to generate images around 300,000 times the magnification.

Electrons are negatively charged particles and unlike photons, which are used in normal

light microscopy, they require electromagnetic forces to guide them. SEM has the potential

to identify the depth of the field approximately 300 times the depth when compared to light

microscopy resulting in the images obtained being 3-dimensional. The composition of

specimens is more precise for the user to analysis. This is particularly important in the

study of nanofiber composition because of the variation in orientations that can occur

allowing for careful microanalysis of the sample.

7.1. Key Components

A scanning electron microscope usually consists of three parts: the microscope column;

this includes the electron beam gun to the sample at the base of the column; a

computational device that utilises the data from the sample and electron beam and allows

composition of an image; extra equipment that allows for data processing.

16

Figure 5 A schematic of a Scanning Electron Microscope

The electron gun consists of a thermionic gun that produces thermo-electrons that are

accelerated by voltages between a filament and an anode which produces the electron

beam itself. The compromise between voltage and working life of the filament used will

determine the clarity of the image. The column is the tool that focuses the sample using

the beam that the gun has already generated. This procedure usually generates a

backscatter of electrons. The lenses used consist of a condenser lens that identifies and

allows the user to manually adjust the intensity of the electron beam reaching the sample

while a secondary lens brings the beam into focus on the sample. The use of two different

lenses allows for scanning into two or more directions. The backscatter is detected by a

backscattered electron detector (BSD). The incidence of backscatter is entirely dependent

on the sample and its features and results in image creation via the detector. The

important feature of the sample is that it is kept under vacuum under a coating of gold in

order for the attenuation of the electron beam to be at its highest resolution and to avoid

the unwanted scattering of electrons. [20]

17

8. Fabrication and Characterisation of Poly(Vinyl Alcohol)/alginate blend nanofibers

by electrospinning method, MD. Shahidul Islam, Mohammad Rezaul Karim

Figure 6 SEM images of electrospun PVA nanofibers prepared by using different PVA

solution concentrations of (a) 8 wt.%, (b) 10 wt.%, and (c) 12 wt.%, see reference 15

Figure 6 also indicates the importance of the concentration of the polymeric solution and

how this affects nanofiber diameter, as the images indicate, the higher the concentration of

PVA, the greater the diameter of the nanofiber that is produced. A change of concentration

from 8 to 12 wt% can result in an increase in diameter by approximately 100%.

The study provided details on the variations in mechanical structure that exists between

various concentrations and blends of PVA to Alginate compositions, their findings

indicated that the stress-strain measurements between PVA and that of Alginate resulted

in a greater tensile strength due to increases in the interaction between molecules

compared to pure PVA. The above image taken from the study indicates the depth and

details of the sample which otherwise would not be possible without scanning electron

microscopy.

18

Figure 7 Stress–strain curve of electrospun PVA/Alg blend nanofibers with various volume

ratios of 10 wt.% of PVA to 2 wt.% of Alginate solutions of (a) 100/0, (b) 80/20, and (c)

60/40 10, see reference 15

The figure above indicates their findings; the blend of PVA/Alg where the highest PVA

concentration indicated the most elongation, and failure at a higher strain, whereas the

PVA/Alg in 60:40 ratio failed at almost a 20% lower strain.[15] The variation exists as a

result of the hydrogen bonding that exists as the intermolecular interaction between the

Alginate solution and PVA. This interaction, to some extent, mimics the composition of

bone. This is because the PVA itself is less stiff than the composite fibers of PVA with

added Alginate; this suggests that the stiffness is a result of the hydrogen bonding and

intermolecular forces that arise. This variation in composition is indicative of the results

expected to see in pure PVA nanofiber layers that have orientation as a dependent

variable. It is also indicative of the properties in bone in varying directions.[14]

8.1. Replicating Anisotropy

Bone in differing orientations display similar properties i.e. the characteristics of bone that

is perpendicular to the direction of the fibrallar bundles will display properties that are

similar to that displayed by the PVA/Alg blend with the highest concentration of Alg, this is

relative to the characteristics of bone when the forces are applied along the fibrallar

bundles will display similar stiffness and rupture as the pure PVA nanofiber. This 19

phenomenon exists due to the interactions between different tissues that exist within the

different orientations in the cortical layer of bone which this study is trying to replicate.

8.2. The Importance of Micro-crack Dissipation

Essentially the interaction between the inorganic (bone mineral) and the organic (matrix) is

the varying factor between the explants and this interaction between each composition is

being tensile tested. With respect to the long bone axis, the fibrillar bundles are generally

parallel to this direction. [2]Functionally, this is due to bone being good in avoiding

compression. This is especially useful in the cortical layer of the femur which is the largest

bone in the body and is exposed to a large amount of compression. Furthermore, the bone

fibrils are composed with a lateral aggregation and stagger which leads to the formation of

mineral platelets into sheets.[14] As fibrils bundle up into lamellae, the lamellae consist of

a ‘plywood structure’ and results in a twisting mechanism which branches out resulting in

the ability of bone to avoid shear and stress in multiple directions.[12]

9. Tensile Testing of Bone Explants [37]

A study taken from Queen Mary University London used three different types of explants

from a cortical region of a bovine femur, the explants were taken at three varying angles:

longitudinally, transversely and diagonally to the direction of the cortical bone. The

specimens were tensile tested under a 0.01% tension test at 100Hz. The user manually

stopped the test if the specimen is displaced by 20% or more or if the force drops by more

than 50%. The specimen is removed and stored. 9 samples in total underwent tensile

testing; three longitudinal, three from each direction. Bone stiffness depends on fibre

orientation; the stress-strain relationship within the explants will indicate this.[37] Micro-

cracking patterns also depend on the loading orientation, generally, cracking across

lamellae and fibrallar patterns will likely result in crack deflection pathways. Due to the

variation in structure of the cortical bone there will tend to be a ‘diffusion’ of the cracking as

energy tends to be dissipated between the variations between the different component

structures of bone i.e. moving from the matrix to the inorganic structures. The general

hypothesis that can be taken into account is that the longitudinal explants will be able to

deform more elastically and indicate higher yield strength than the transverse explants

while the explants taken at 45 degrees will show a mix of characteristics.

9.1.1 Explants from the Transverse Direction

20

0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.0040

5

10

15

20

25

30

Explants Taken from the Transverse Direction

Tensile Extension (mm/mm)

Stre

ss (M

Pa)

Figure 8Stress-strain relationships of 3 samples of transverse bone explants

Firstly, regarding the explants from the transverse direction, all two of the three samples

have displayed a yield and buckle followed by relatively plateau region. These samples are

indicated by the red and green lines. The blue sample showed similar stress yield to the

green, however, beyond yielding the sample seemed to have failed and showed no signs

of buckling. The red sample did show similar patterns of similar yielding and buckling

followed by a plateau however a much lower yield, at approximately 14MPa as opposed to

27MPa.

9.1.2 Explants Taken from the Longitudinal Direction21

0 0.0002 0.0004 0.0006 0.0008 0.001 0.0012 0.0014 0.0016 0.0018 0.0020

10

20

30

40

50

60

70

80

90

Explants Taken from the Longitudinal Direction

Tensile strain (Extension) (mm/mm)

Stre

ss (M

Pa)

Figure 9 Stress-strain relationships of three longitudinal bone explants

The longitudinal explants show that a much more elastic deformation has occurred until a

tensile strain of 0.001. All three samples show the same relatively linear relationship

between tensile strain and stress. At this initial point, the sample indicated by the red line

has displayed a severe drop in strain from approximately 75MPa to 40MPa. The other two

samples did not display this phenomenon and continued to display yield strength until

eventually rupturing completely.

9.1.3. Explants Taken from the 45 Degree Direction

22

0 0.0005 0.001 0.0015 0.002 0.0025 0.0030

5

10

15

20

25

30

35

40

Explants Taken from the 45° Direction

Tensile strain (Extension) (mm/mm)

Stre

ss (M

Pa)

Figure 10 Stress-strain relationships between 3 bone explants taken at 45 to the direction

of long bone

The explants taken from cortical bone at 45° showed varying results between samples.

Each showed relative linear elastic deformation up to a tensile strain of 0.0006. Beyond

this strain each sample displayed different phenomena; the sample indicated by the blue

line showed a typical yield and buckle phenomena followed by a plateau. This shows

similarities between some transverse explant data that has been obtained with similar yield

strength at a similar tensile strain. The sample indicated by the red line showed signs of

yielding but after this initial yield the buckling did not seem to occur and the stress-strain

relationship managed to increase to a secondary yield of approximately 35MPa at a tensile

strain greater than 0.0026, this is then followed by rupture. The sample indicated by the

green line showed a dramatic drop in Stress at a Strain of approximately 0.0006 from

24MPa to 16MPa followed by a phenomena indicated by a longitudinal sample. This

sample then indicated slight buckling until failure at a strain of 0.0015 which is significantly

less than the other two samples which failed much closer to a strain of 0.0028.

Three parameters that can determine the strength of the bone explants is reflected on the

curve, firstly, the load that the bone can sustain before rupture, secondly, the deformation

it can sustain, and lastly the energy stored before it fails.[36] These parameters will 23

invariably change according to the type of explant used as a result of the collagen

alignment; stiffer fibers are unlikely to deform relative to fibers that orientated in the

direction of the matrix.

9.2. Indications of Structure

The results indicate that anisotropy of the specimens has resulted in a variation in the

mechanical properties of the three types of explants used in the investigation. The

explants taken at 45° showed the most variety in results in terms of different structural

phenomena that may or may not have occurred along with the most varying results in

terms of plastic deformation and yield strength. The variations between the three different

samples potentially show the differences in crack propagation that can occur within the

same explant; the architectural properties of bone in this direction contain an equal amount

of mechanical loading on the organic and inorganic components of the cortical bone. As

discussed earlier, crack propagation tends to occur along the matrix components and not

across lamellae, because of the orientation, the propagation of cracks and the dissipation

of energy are both as likely to occur as each other. The sample indicated by the red line of

the explant taken at 45° shows that crack propagation has shifted from the matrix to the

lamellae structures at approximately 0.001 causing an increase in stress therefore

resulting in a stress-strain relationship that would otherwise be indicated by longitudinal

explant i.e. yielding followed by a necking region and ultimately rupture. The sample

indicated by the green line has shown a drop in stress which possibly identifies a break in

the cortical lamellae and possibly more than one bundle leading to the matrix to bear the

tensile strain at this point, plastic deformation has occurred. The matrix takes over and

begins to elastically deform until it yields, buckles and plateaus which is the common

phenomenon displayed by the transverse explants. This shift in stress at these points

indicates that the architecture of bone within the 45° explants shows the ability for the

crack to propagate along the lamellae via the matrix.

The differences between the longitudinal and transverse explants are evident in the

results; with the exception of one sample, the longitudinal explants show the most elastic

deformation followed by a yield and rupture; the green sample shows a yield strength of

around 77MPa while the sample indicated by the blue line indicated a yield strength

around 95MPa and tensile strain of 0.0018 at rupture. Firstly, in contrast to the

longitudinal explants the transverse explants did not show clean ruptures, instead, two of

the samples plateaued until strains of 0.003 and 0.003725. This shows that the

24

longitudinal samples are stiffer than the transverse samples. This is due to the architecture

of the lamellae, which is being isolated in the longitudinal explants, and the organic matrix,

which is isolated in the transverse explants.

9.3. Lamellae Organisation

The lamellae, due to their organisation in order to cope with compression and other forces

require stiffness. After the elastic deformation that they undergo and due to their isotropic

nature, they will indicate a region of strain hardening followed by a rupture, as one

lamellae bundle fails, the remainder will tend to follow. The transverse explants have an

architecture that consists of the matrix bearing the tensile strain. The organic structure will

result in less catastrophic failure and rupture due to the lower stiffness in this region of the

bone, the cracks will generally dissipate and will not able to propagate along the lamellae,

this is indicated by the initial elastic deformation followed by the buckling where the energy

is released and where the stress plateaus.

10. Nanofiber Orientations

The general aim of obtaining nanofibers in various layer orientations is to replicate the

behaviour of bone regarding its composition and how this composition results in a stress-

strain relationship that will display more of an elastic deformation between the fiber layers,

which is the ideal scenario especially within cortical bone types, rather than a rupture,

which is likely to be present along stiff fiber axes. The general hypothesis of the tensile

testing would be that the fibers with orientation of 30° as the secondary layer will result in

the afore mentioned deformation as opposed to more plastic deformation and rupture that

may be exhibited by the single layer nanofiber sample. The variation in the fiber orientation

is to replicate the mechanically anisotropic nature displayed by bone as displayed in the

findings in the samples that have been tested above especially the 45°. The general aim of

the layers is to dissipate as much energy as possible and enable loading to occur at a

higher strain within the nanofiber in order to mimic anisotropy.

10.1. What Tensile Testing Will Simulate

25

The nature of the tensile test foes not account for fatigue fracture in bone, this type of

fracture and failure method usually occurs in most people as a result of continuous motion

from the muscular system due to the repetitions at a strain that is less than where the

bone’s tensile strength will occur. Fatigue can result in a lower modulus of elasticity and

lower stress than the sample’s original characteristics.[14]

Figure 11 Variation in the alignment of collagen in turkey tendon, see reference 13

The anisotropy of collagen in particular is highlighted in the above schematic from The

Material Bone: Structure-Mechanical Function Relations, S. Weiner and H.D. Wagner,

Departments of Structural Biology and Materials Interfaces, Annual Reviews Material

Science. It says the differing fiber orientations of collagen fiber based on its specific roles

within the body that include, connective tissue such as tendon, ligaments and other

structures that require mechanical anisotropy.

26

The variation in characteristics and behaviours exhibited by bone in the explant testing

carried out at Queen Mary University of London showed the various directions that existed

in fibril orientation within similar regions of cortical bone. A study carried out by Alejandro

A. Espinoza Orias BSc MSc Regarding the Anisotropy that exists in cortical bone which

results in its adaptation that bone has as a living tissue to different mechanical loading

conditions. Bone displays properties such as elastic behaviour that is stronger than

plastics with a much better ductility than ceramics. [21] These characteristics are quite

clearly indicated in the testing by QMUL when the only variable is the direction of tensile

testing. Bone mineral plays a key role in the elastic anisotropy of bone; the preferred

orientation of bone aligns in the same direction of the bones principal loading, i.e. along

the primary collagen alignment. Another factor that determines the toughness of bone is

the hydration of the collagen matrix with osteoid water, this has a plasticising effect of the

collagen molecules [21] which would allow the bone to reduce crack propagation and

increase the dissipation of energy as the water within the mineral composition acts as a

shock absorber or buffer.

11. Materials & Methods

11.1. Solution Preparation

Poly(vinyl alcohol) solution is prepared using water bath conditions at 6%, 8% and 10%

concentrations with distilled water as the solvent. Despite the use of distilled water as a

substance, solution preparation is carried in a fume cupboard using the correct utensils i.e.

spatulas to measure the weight of PVA and weighing scales for the amount of water

needed for the solvent to produce the relevant concentrations. The solutions are mixed at

a viscometer setting of 2000rpm. The temperature for optimal water bath conditions is 150

degrees Celsius. The water bath conditions are essential for PVA solution preparation as

any direct heat that is applied will cause the distilled water to evaporate particularly for

lower concetrations. The volume of water in the water bath also relates to the time needed

for the solution to be prepared.

27

Figure 12 Preparation of 10% concentration PVA (80ml) solution on a heated magnetic

stirrer

28

Figure 13 8% concentration PVA (80ml)

11.2. Electrospinner Settings

The electrospinner within Nanoforce consists of 3 main components; the PHD 2000

Infusion system which provides the steady input of polymeric PVA solution to the needle;

the casing which contains the needle stand and the circuit with external voltage controls;

and the collector which is either a plate or a roller.

The electro-spinner is set to the parameters of a feed-rate of 0.2ml/hour. However this can

be initially at a higher setting of 0.75 – 1.0ml/hr in order to provide an initial pressure that

allows the solution to flow optimally through the needle and die system. The distance

29

between the needle and the roller is set at 15cm which is used to collect aligned fibres as

opposed to the aluminium plate that collects un-aligned fibres.

The voltages that have been used consisted of 15-22.5kV in order to generate the optimal

balance between Ohmic and connective current flow.

These are the extrinsic conditions of the procedure. The parameters that cannot be

controlled are factors such as humidity within the lab space at a given time, the humidity

plays a part in the evaporation of the solvent, increased humidity levels can result in more

viscous fibers being formed whereas a dryer lab condition can result breaks in the

nanofibers as the solvent is evaporated off too early. The most typical values for humidity

throughout the experimental procedures have consisted between 32-45%. However, it was

not uncommon to experience humidity of 60-65%.

30

Figure 14 Needle and dye system with plate collector system, PVA nanofibers collected on

the Al foil

31

Figure 15 ES control for high voltage, initially 15kV was used which was gradually

increased to approximately 22.5kV

32

Figure 16 The use of the 55mm diameter roller with foil collector

33

Figure 17 The PHD Infusion 2000 with syringe containing the PVA polymeric solution

34

The method used to collect aligned fibres at approximately 30° requires square paper

frames to collect an initial layer of nanofibers and which is then removed and positioned

using a protractor. The machine then spins a secondary layer at this different orientation.

This is made easy using frames displayed in figures 18 and 19. Initially, frames made out

of card were used to try and attain fiber orientations; however, this method left was not as

effective as foil frames because of the lack of charge. The card was also difficult to

conform to the curvature of the roller when rotated 30 degrees because of its stiffness in

its original direction.

35

Figure 18 Initial foil frame used to collect fibre orientations

36

Figure 19 Smaller frames used to collect fibers that could occur in random initial

alignments along the roller

37

Figure 20 55cm roller under 22.4kV

38

Figure 21 The hierarchical structure of bone from whole bone to mineralised collagen,

http://newscenter.lbl.gov/news-releases/2011/08/29/the-brittleness-of-aging-bones-

%E2%80%93-more-than-a-loss-of-bone-mass/

Figure 21 indicates how the PVA nanofiber layers are aimed to be aligned as highlighted

by Fiber arrays.

The samples that are collected on aluminium foil will be imaged using the SEM and any

fibers that are collected on glass slides will be optically microscoped.

The frame also provides a platform for tensile tested to occur and easy placement in the

grips of the tester. The tester provides stress-strain curves for data analysis. The data

obtained will allow for comparison with other reports regarding PVA and bone stress

analysis.

39

12. Results

Figure 22 Optically microscoped nanofibers produced from 10% PVA solution collected on

the plate system

40

Figure 23 Optically microscoped nanofibers produced from 10% PVA solution collected on

the roller system

Figures 22 and 23 indicate the differences between unaligned and aligned PVA nanofibers

respectively. Both specimens were collected on glass slides and optically microscoped at

a magnification of 20x. The samples show how the use of the roller affects the nanofiber

41

density of the PVA that is collected; the unaligned fibers consist of a more densely packed

network of fibers that show random deposits. The fibers that were aligned at two layers of

30 degree orientations provided a loose, yet more unidirectional collection of nanofibers.

13. Discussion

13.1. Experimental Difficulties

Figures 22 and 23 indicate the difficulty of the electrospinning process regarding the PVA

polymer solution and the uptake into the needle due to leakages, this has resulted in the

indicated the feed rate on the PHD Infusion 2000 not being entirely accurate. The inability

of the solution to be fed into the needle had a direct impact on the fibers, if any, that are

produced. These problems existed under both of the 6% and 8% concentrations of PVA

where the solution may not have been viscous enough to flow through the needle.

The electrospinner itself required refining from the initial parameters that had been initially

set prior to the generation of fibers. The initial aim was to generate fibers using a solution

that consisted of 8% concentration of PVA to water. In the initial spinning process, this had

resulted in fibers that did not have the solvent evaporate off completely. This was

potentially a result of a combination of fluctuations in humidity in the lab along with some

parameters that were not ideal for generating nanofibers in the lab environment. The

method was gradually changed to include 6, 8 and 10% concentrations in order to identify

and generate samples that would be most visible in order to be, initially, optically

microscoped on glass slides within Nanoforce. Voltage was increased initially from 15kV to

17.5kV, this was an attempt to make the polymeric solution stretch and be submitted to

increased coulombic tensions in order to evaporate the solvent. No improvement in fiber

condition occurred at this point. Initially, when the voltage was increased to 22.4kV for 8%

concentrations no improvement in fiber quality was made, the same voltage was carried

out for 10% concentrations which yielded the most solid fibers.

6% and 10% solutions were mixed in order to see if the ratio of polymer to water was the

underlying issue. 6% provided less solid samples however, 10% provided the most stable

nanofibers.

42

Viscosity was a major determining parameter of the success of the generation of fibers. In

some instances, problems arose due to the leakages that could sometimes occur within

the machines set up which would result in not enough solution being forced through the

needle at the desired federate thus not causing the pressure required in order to generate

the surface tension at the Taylor cone.

Figure 24 Incidences of the PVA polymer leaking at the needle and tube interface before

being drawn into the needle

43

Figure 25 Methods used in order to prevent leakages of the solution occurring

Figures 24 and 25 indicate the incidences of leaks occurring with the polymeric solution

especially with 6 and 8% concentrations of viscosity; figure 25 displays a possible solution

to the problem.

Other practical difficulties included the drag under the foil frames on the roller generating

an unstable air flow on the inner edges as the roller revolved; this may have caused the

initial layer of nanofibers to not be deposited on the foil in a uniform manner which

prevented the generation of stable samples to be imaged via SEM. The use of the glass

slides was crucial in the running of the process because it allowed for the monitoring of the

process while the procedure was occurring in the lab. The main disadvantage of glass

44

slides is the difficulty in obtaining SEM images of the samples that are obtained and the

samples also cannot be mechanically tested.

13.2. Findings

Despite the difficulties in the procedure, the alignment of the nanofibers was carried out

relatively well at 10% PVA concentrations with distilled water, the voltages that were

mainly used to produce the results consisted of 22.4kV with the distance between needle

and roller set at 150mm. The roller speed used to collect the fibers shown in figure 23

consisted of 260rev/min. Judging by the patterns shown by the image, the initial layer

consisted of a unilateral deposition of the nanofibers, however, the patterns that are

formed indicate a tendency for random orientations as indicated by the wave-like patterns

that are formed on the glass slide. The secondary layer that was spun is very poorly visible

which leads to the suggestion that there is a potential repulsion between either the glass

slide and/or the initial layer of nanofibers. A possible solution that could have been used to

improve to spinning in both directions onto the slide consisted of increasing the revolution

speed of the roller in order to prevent the sway of the fiber as it is deposited onto the

charged surface. The difficulty was increasing the speed to an optimum level would be the

generation of unnecessary torque which would cause the electrospinner to undergo small

vibrations which may actually cause an increase in the incidence of leaks occurring as

mentioned previously. Humidity played an important role in the spinning process; the

higher the humidity, the more water was absorbed i.e. water acts as a plasticiser which

has the potential to reduce the tensile strength of the fibers produced.

13.3. Future Work

The generation of these nanofibers will require tensile testing in order to ensure that they

have similar mechanical properties in order for the application into bone collagen

interfaces. SEM images will also help identify the orientation factor and the thickness of

the fibers in relation to bone and of bone itself in order to provide a cohesive bonding to

the osteonic layer of bone. Other potential future applications will also potentially include

the introduction of bone morphogenic proteins and other growth factors within the fibers

that will be able to improve the adherence of the implant to and thus improve mechanical

interactions with the nanofiber and the host tissue. The extrinsic conditions regarding the

spinning process can also be kept more consistent such as humidity which has an

important role in the properties of the fibers that are generated.

45

14. Acknowledgements

I would like to thank my project supervisor, Dr Asa Barber for the guidance and support

that he has provided me with during the time I took to work on this project. I would also like

to thank the staff and PhD students at NanoForce and Nanovision who have provided me

with additional help and hands-on training with respect to the machines.

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[2] Science and Technology of Polymer Nanofibres, Andrad, Wiley, 2008

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46

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48