Combining Melt Electrowritten Scaffolds and Silk …Combining Melt Electrowritten Scaffolds and Silk...

Combining Melt Electrowritten Scaffolds and Silk Fibroin Films for Ocular Surface

Regeneration

Deanna Nicdao BSc (Chemistry)

School of Chemistry, Physics and Mechanical Engineering

Faculty of Science and Engineering

Queensland University of Technology

Submitted in fulfilment of the requirements for the degree of

Masters of Applied Science (Research), Biofabrication

2019

I

Statement of Original Authorship

Unless referenced in the customary manner, or otherwise acknowledged in the text, the work

contained in this thesis undertaken between Queensland University Technology and the Julius

Maximilian University of Würzburg has not been previously submitted to meet requirements

for an award at these or any other higher education institution. To the best of my knowledge

and belief, the thesis contains no material previously published or written by another person

except where due reference is made.

Signature:

Date: _________________________11.07.2019

QUT Verified Signature

II

Keywords

Biofabrication, corneal tissue engineering, liquid glass, melt electrowriting,

poly(ε-caprolactone), poly(vinylidene fluoride), silk fibroin

III

Preface

This submission consists of a thesis that has not previously been submitted for any degree at

any other university, and two reports attached to the end of the document, referred to as

Annexes 1 and 2, representing a 10-month project undertaken at the Julius Maximillian

University of Würzburg, which were submitted for the award of Master of Biofabrication. A

timeline detailing the completion of the entire Masters of Applied Science (Research)

Biofabrication course is included below:

University Research Stage Time Period

QUT Full-time coursework Feb 2017 – June 2017

QUT Research and writing thesis April 2017 – Feb 2018

JMU Research and writing Report 1 (Annex 1) April 2018 – August 2018

JMU Research and writing Report 2 (Annex 2) August 2018 – December 2018

IV

Acknowledgements

Queensland University of Technology

I express my gratitude to my supervisors, A/Prof. Tim Dargaville for his support and mentoring

as well as providing me this wonderful opportunity to study Biofabrication internationally. I also

thank Dr. Aurelien Forget for his knowledge and guidance during my time at QUT. I also would

like to thank Eleonore Bolle for teaching me the ropes at IHBI and CARF – it was relieving to

have someone to help with both the lab and administrative side of things in the first few months.

In addition, I thank the CARF team for allowing me to use their facilities which I very much

enjoyed. I would like to also like to thank my supervisor, Prof. Traian Chirila, and to the QEI

team, for welcoming me to their facilities and answering my many questions. I must express

my thanks to Prof. Damien Harkin for his help with cell work both in and out of the lab. And

lastly, special mention to Dr. Shuko Suzuki, who provided even more support on top of my

many questions. I would like to also thank my Australian Biofabrication colleagues, David

Pershouse, Samantha Catt, Madison Ainsworth and Martyn Steiger. It was a pleasure to go

through the classes, inductions and application processes together in the first year.

Throughout the duration of this project, I have been provided with great growth from both a

professional and academic standpoint, a priceless experience for which I am grateful overall.

Julius Maximilian University of Würzburg

First and foremost, I would like to thank my supervisor Prof. Paul Dalton for providing this

wonderful opportunity to conduct research abroad under the International Masters of

Biofabrication degree. I am eternally grateful for the valuable experiences and the people that

I have met during this degree. I extend my thanks to the MEW lab team for welcoming me to

the team. I would like to acknowledge Andrei Hrynevich, Ezgi Bakirci and Christoph Böhm for

their expertise, knowledge and guidance with my research. I thank Philipp Stahlhut and Judith

Friedlein for the SEM and EDX imaging, Jannik Mechau for the DMA analysis and Miroslav

Mirlík for the DSC analysis. I also thank the Luxenhofer group for their generosity with allowing

V

me to use their facilities. I express my gratitude towards the other Biofabrication students –

past, current and future – for the shared experiences, memories and growth. I look forward to

when we meet in the future. A special thanks to Nick Hüttner for his inspiring enthusiasm,

teamwork and encouragement. It would not have gone as smoothly without him. Last but not

least, I thank my mother and close loved ones for their unconditional love, support and faith in

my abilities.

VI

Abstract

The silk protein, fibroin, derived from the domesticated Bombyx Mori silkworm is a well-

established biomaterial. It can form thermodynamically stable materials comprised of β-sheets

with controllable thicknesses. These membranes have excellent optical properties (ca. 95%

transparency across the visible range) and are easily characterized and biochemically

functionalized due to the all-aqueous processing. Previously, these membranes have been

investigated as cell carriers for ocular surface reconstruction. However, a major limitation is

its susceptibility to tears when sutured. To resolve these issues, poly(ɛ-caprolactone) (PCL)

printed by melt electrowriting (MEW) were embedded in silk fibroin (SF) membranes for

mechanical reinforcement. Topography and suture compatibility of reinforced membranes with

PCL pore sizes coded as 100×100 μm2, 200×200 μm2 and 300×300 μm2 were analysed and

assessed. It was found that the adhesion between PCL fibres and the silk membrane were

incompatible resulting in gap formation between the two materials. To improve adhesion, O2

plasma treatment of PCL frameworks was applied prior to casting the silk membranes. In

addition, the volume of SF solution was doubled to ensure the MEW constructs were

completely covered during casting. The adhesion improved significantly however the

membranes would tear during handling. However, all SF-PCL substrates with various PCL

pore sizes were comparable for qualitative suture tests and were noted to be less fragile than

SF-only films.

Moreover, SEM and stylus profilometry revealed that the presence of PCL fibres did

indeed topographically pattern the membrane’s surface. It was hypothesized that these

topographic features could mimic the limbal palisades of Vogt in the corneoscleral-limbus

region. To observe whether cell growth was influenced by this topography, the membranes

were seeded with primary rabbit corneal epithelial cells. Cell morphology demonstrated

migratory behavior characteristic of mature limbal cells. The comparison of pores with areas

of ca. 250 μm2 versus ca. 330 μm2 showed statistical significance in cell viability. Cells

appeared to migrate along MEW frameworks, however this remained inconclusive for smaller

pore areas (ca. 120 μm2). Further investigation into a larger range of pore sizes is required for

a deepened understanding of the effect of pore size on cell behavior. Nonetheless, from these

initial investigations, it can be concluded that PCL frameworks printed using MEW provides

an alternative approach for the topographical patterning and mechanical reinforcement of the

SF membrane, advancing its potential as a corneal epithelial cell carrier.

VII

Table of Contents

Statement of Original Authorship ....................................................................................... I

Preface .................................................................................................................... III

Acknowledgements ........................................................................................................... IV

Abstract ................................................................................................................... VI

List of Abbreviations ......................................................................................................... IX

Chapter 1: Introduction and Literature Review ................................................................. 1

1.1 The Ocular Surface ...................................................................................... 1

1.1.1 Composition and Healing Process ............................................................ 1

1.1.2. Ocular Tissue Injuries and Corneal Surface Disease ............................... 3

1.2 Corneal Tissue Engineering ......................................................................... 3

1.2.1 The Role of Cell Transfer Sheets .............................................................. 3

1.2.2 Silk Fibroin Films for Corneal Epithelial Cells ............................................ 5

1.2.3 Modification of Silk Fibroin Membranes ..................................................... 7

1.2.4 Surface Topography ................................................................................. 9

1.3 Significance and Scope ............................................................................. 10

1.4 Research Objectives .................................................................................. 11

1.5 Thesis Outline ............................................................................................ 12

Chapter 2: Reinforcing Silk Fibroin Membranes with Melt Electrowriting Frameworks ........................................................................................................................................... 13

2.1 Introduction ................................................................................................ 13

2.1.1 Melt Electrowriting ................................................................................... 13

2.2 Materials and Methods ............................................................................... 15

2.2.1 Melt Electrowriter Setup .......................................................................... 15

2.2.2 Scaffold Design, Laser Cutting and Plasma Treatment ........................... 15

2.2.3 Fabrication of Films ................................................................................. 16

2.2.4 Scaffold Characterisation ........................................................................ 17

2.2.5 Qualitative Suture Test............................................................................ 17

2.3 Results and Discussion .............................................................................. 17

VIII

2.3.1 Effect of PCL fibre dimensions, pore area and pore depth on SF

membrane integrity .......................................................................................... 17

2.4 Conclusions and Limitations ...................................................................... 28

Chapter 3: Mimicking the Corneal Epithelium ................................................................ 30

3.1 Introduction ................................................................................................ 30

3.2 Materials and Methods ............................................................................... 30

3.2.1 Culture and Growth of Primary Rabbit Corneal Epithelial Cells on SF-PCL

Substrata ......................................................................................................... 30

3.2.2 Cell viability on membrane coated wells .................................................. 31

3.2.3 Imaging of Cell Culture on Free-Standing Membranes ............................ 32

3.2.4 Statistical Analysis .................................................................................. 32

3.3 Results and Discussion .............................................................................. 33

3.3.1 Comparison of Pore Sizes ...................................................................... 33

3.3.2 Quantitative Analysis .............................................................................. 33

3.3.3 Morphology of RCE Cells ........................................................................ 35

3.4 Conclusions ............................................................................................... 39

3.5 Future Directions ....................................................................................... 40

References ........................................................................................................................ 41

Appendices ....................................................................................................................... 48

Annexes ............................................................................................................................. 56

IX

List of Abbreviations

3D 3 dimensional

CLSM confocal laser scanning microscopy

CNC computerised numerical control

FDJ fibre diameter (at junction)

FDs fibre diameter (at strut)

FITC fluorescein isothiocyanate

HBSS hank’s balanced salt solution

LSCD limbal stem cell deficiency

MEW melt electrowriting

MW molecular weight

OSDs ocular surface diseases/disorders

PBS phosphate buffered saline

PCL poly(ɛ-caprolactone)

PDMS poly(dimethylsiloxane)

PEG poly(ethylene glycol)

RCE rabbit corneal epithelial (cells)

RM regenerative medicine

SCFH standard cubic feet per hour

SEM scanning electron microscopy

SF silk fibroin

TCP tissue culture plate

TE tissue engineering

TRITC tetramethylrhodamine-isothiocyanate

CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW

1

Chapter 1: Introduction and Literature Review

This following chapter begins with an introduction to the composition and healing

process of the cornea with a focus on corneal surface diseases (section 1.1). Subsequently,

a literature review will discuss silk fibroin (SF) in relation to its applicability with cellular surgery

and melt electrowriting (MEW) for corneal tissue engineering (TE). This is then followed by

the significance and scope (section 1.3), research objectives (section 1.4) and the outline of

the thesis (section 1.5).

1.1 The Ocular Surface

1.1.1 Composition and Healing Process

The ocular surface largely determines the quality of our vision. For clear vision, the

ocular surface must be healthy, smooth and homogeneously transparent1. It consists of

integral functional anatomical components - conjunctival epithelium, corneoscleral limbus,

corneal epithelium, tear film - and adjacent structures - eyelid, eyelashes, lacrimal glands 1,2.

The cornea, our window to the world, is made up of three cellular layers (Figure 1): (a) the

stratified squamous epithelium containing basal cuboidal cells, superficial wing cells and

apical squames, (b) a keratocyte containing collagenous, avascular stroma and (c) a

monolayer of endothelial cells – each of which are segregated by a specialized basement

membrane (Bowman’s and Descemet’s)3.

Figure 1. Layers of the cornea.

In addition to the cornea, the ocular surface comprises of the conjunctiva and limbus

which all work together to protect the eye from external damage and infections, provide

comfort and maintain corneal deturgescence – a relative state of hydration2. The collagen


2

interfibrillar spacing, regulated by the hydrophilic stromal proteoglycans and in turn by the

tissue state of hydration by the endothelial pump, determines the transparency of the cornea2.

The homeostasis of the corneal endothelium is the renewal of desquamating cells which are

shed from the corneal surface, shown below4:

Figure 2. Anatomical representation of limbus and corresponding migration of amplifying cells involved in the

homeostasis process. Cell division occurs within the limbus and migrate towards the cornea centre, replacing

terminal corneal ECs that shed from the corneal surface. The stroma (bottom layer) is vascularized and populated

by fibroblasts 4. Reprinted with permission under the Creative Commons Attribution License (see Appendix 7).

A study has classified this homeostasis in XYZ stages5. Component X begins at the

periphery of the cornea where the proliferation and motility of basal endothelial cells occurs

centripetally along the basement membrane, approaching the cornea’s centre. Component Y

is classified as the movement of cells from the basal layers to the superficial corneal epithelial

layers followed by the replacement of ECs which are shed from the corneal surface – depicted

as component Z5. In the literature, it is widely accepted that this self-renewal process is

maintained by undifferentiated stem cells located at the limbus4,6.


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1.1.2. Ocular Tissue Injuries and Corneal Surface Disease

Damage, whether that be acute or chronic, is inevitable with age which results in

severely damaged tissue or induces undesirable inflammatory responses. This in turn results

in the irreversible scarring of the conjunctiva and the opacification of the cornea. The spectrum

for what could be considered an ‘ocular surface disorder (or disease)’ (OSDs) is extensive,

ranging from minimally inhibiting conditions such as dry eye syndrome to ones of heavier

damage through chemical or thermal injuries, or several surgeries, which threaten vision.

Among the most severe OSDs is the limbal stem cell deficiency (LSCD) disorder in

which corneal epithelium regeneration is depleted. Limbal stem cells, also referred to as

epithelial corneal stem cells, reside in the corneoscleral limbal region. Studies have

established an association between their depletion and events that lead to visual impairment

or complete loss of vision. Moreover, animal studies have demonstrated that the more

damaged the limbal epithelium, the more the healing capacity of the ocular surface is reduced

- often a precursor to the opacification of the stroma 7-9.

Corneal disease is the second leading cause of blindness globally, frequently

associated with damage of the ocular surface. Dysfunctional corneas are currently surgically

removed and replaced with a button of corneal tissue from deceased donors, known as

keratoprosthesis10. However this tissue no longer regenerates and to maintain vision, patients

require either a second graft or an alternative option altogether11. The sourcing of such

materials is limited, as organ donation remains low, especially in developing countries such

as Brazil, Ethiopia, India, Indonesia, and the Philippines12. Restrictions arise due to religious

and cultural factors, a lack of education and the absence of eye-banking facilities11. The

transplantation of biological entities are emerging to remedy limited supplies, mainly through

autologous limbal stem cells and amniotic membranes11,13. Unfortunately, these procedures

remain inadequate with high risks of failure similar to those of donor corneal tissue, including:

unpredictable rejection rates, lack of mechanical strength and difficulties of material sourcing.

This has led to the clinical need being unmet, which in turn skewed the advancement towards

synthetic alternatives 6.

1.2 Corneal Tissue Engineering

1.2.1 The Role of Cell Transfer Sheets

The complicated management of LSCD has always, out of necessity, resorted in

surgical intervention. While minor deficiencies can be controlled through medication and


4

observation or surgical replacement of dysfunctional tissue, the replacement of epithelial stem

cells remains essential to combat their eventual depletion. Advances in stem cell biology,

along with the anatomical localization of corneal stem cells, launched the era of ‘cellular

surgery’. The basis of cellular surgery involves the harvest and ex vivo expansion cells prior

to surgery1. The first successful clinical trials of conjunctival autografts demonstrated healing

and surface stabilization in most cases, with improved visual acuity in an additional 17 cases14.

However, a major drawback, common amongst autograft transplants, is the limited amount of

available tissue that can be taken from the patient. An example being limbal autografts, where

tissue is taken from the healthy contralateral eye of the patient. This removal of tissue, even

of relatively small amounts, can seriously affect its healing ability. Furthermore, this treatment

is not available in the scenario that the damage is bilateral. Thus, allograft techniques which

harvest limbal donor tissue has been developed. Still, as previously mentioned for autologous

grafts, high rejection rates and limited tissue availability remain an issue1.

The current gold standard cell transfer sheet for the ex vivo expansion of epithelial

stem cells is human amniotic membrane (amnion)15,16. This naturally occurring biomaterial

promotes epithelial cell healing whilst inhibiting fibroblast proliferation and myofibroblast

differentiation17. The tensile strength of an amnion membrane with a thickness of ~430 μm

was found to be ~0.16 MPa with an extension length at break of ~17.33 mm and a Young’s

modulus of 0.65 MPa18. The central cornea region has a mean thickness of ~555 μm, with

respective mean thicknesses of the epithelial basement and Descemet’s membrane of ~ 55

μm and ~17 μm19. The cohesive tensile strength of normal human donor anterior and posterior

corneal regions has been measured to be 0.33 MPa and 0.19 MPa respectively20. It is clear

form these values that the biophysical properties in the cornea vary based on anatomical

region. For example, the Descemet’s membrane is similar in topography to the anterior

basement but with smaller pore sizes and hence the tighter packing of cells accounts for a

higher elastic modulus of Descemet’s membrane21. Therefore, difficulties arise when

comparing biophysical properties of native tissue with the gold standard, amnion membrane,

as its thickness also varies and in turn, mechanical properties. Additional complexity arises

due to sample variance with human donors of different age, sex and other health-associated

factors. Nonetheless, it was soon realized that amnion alone was incapable of succeeding

unless combined with transplanted limbal epithelial stem cells and/or intraoperative topical use

of mitomycin C - an anti-tumour antibiotic with potentially harmful side effects. Furthermore,

variation in donors makes it an impossible task for surgeons to assess standards of amnions

available22. The reconstruction of the ocular surface from such techniques would require

potent anti-rejection regimens to become successful. More distressing are the risks to the

patients as these treatments are intended to last their entire lifespan.


5

The emerging field of biofabrication aims to establish biocompatible materials with

desirable mechanical properties to address limited tissue availability. One promising material

for the ocular surface is SF from the domesticated (Bombyx Mori) silkworm. Its desirability

stems from its extensive use as surgical sutures, proven biocompatibility, and further

developed for various emerging biomedical applications, deeming it a potential candidate for

further ocular applications.

1.2.2 Silk Fibroin Films for Corneal Epithelial Cells

Spiders and insects have generated biopolymers with a wide range of mechanical

properties in the form of silks23. SF has been widely used in the textile and medical industries

for millennia, thus sparking interest as a biomaterial23. The optical properties, mechanical

robustness and versatility in production has made SF an ideal candidate for human epithelial

cell growth and functional organization24.

During the pupation stage of the domesticated silk moth, Bombyx Mori (family

Bombycidae), larvae produce silk cocoons which are made up of two main protein components,

fibroin and sericin. Fibroin has demonstrated to induce blood coagulation leading to its

incorporation into wound healing applications 25. The contrary component, sericin, increasingly

gains attention as a biomaterial however has not demonstrated its suitability as a strong free-

standing membrane unlike fibroin26. Recently, further viability has been demonstrated through

the attachment, growth and differentiation of human corneal epithelial progenitor cells on SF-

coated surfaces and freestanding membranes 27-30. The epithelial cell attachment to SF was

facilitated by coating with extracellular matrix proteins27.

The amount of crystalline content (i.e., secondary structure) of fibroin has been found

to greatly impact its material properties in films. A comprehensive study by Lawrence and

colleagues compared the change in thickness from dry to hydrated states of water annealed

versus methanol treated silk films31. Films were incubated in a water bath for ~ 2h and the

change in thicknesses were evaluated using non-invasive techniques, namely, Two-Photon

Excited Fluorescence and Second Harmonic Generation. It was found that films treated with

methanol underwent a statistically significant increase in thickness once hydrated

(~29 to ~46 μm). In contrast, water annealed films demonstrated no significant change

between hydrated and dehydrated states (~32 to ~33 μm). Moreover, the silk film thickness

swelling ratio (Q) was significantly greater in the methanol treated group (~1.6) than water

annealed samples (~1). Literature has found water annealed samples contain a three-fold


6

decrease in β-sheet content compared to methanol treated films32. These results thus infer

that increased β-sheet content leads to greater water absorption in silk films. This is further

supported by the higher oxygen permeability coefficient observed at 50% relative humidity of

the methanol-treated group (~5 x 10-11 mL O2 cm/s cm2 mmHg) compared to the water

annealed group (~2 x 10-11 mL O2 cm/s cm2 mmHg) after 1.5 h. It was suspected that the

difference in oxygen permeability rates between the samples is attributed to the packing

structure of the fibroin protein chains. Literature has demonstrated that if amorphous chains

are in close proximity to crystalline regions in a bulk polymer structure, chain movement is

more inhibited33. Therefore, the less ordered secondary structure of the methanol-treated

group allows for greater chain mobilisation and hence a greater capacity for both water

absorption and oxygen permeability (Figure 3).

Figure 3 Schematic representation of the secondary structure of silk films via two processing techniques: water annealing (left) or methanol immersion (right). The water annealed film contains less β-sheet content (red) to amorphous regions (purple) with an ordered secondary structure. The methanol treated film has higher β-sheet content with disordered channel-like amorphous regions which allow for the absorption of water molecules (blue).

In addition, this study also investigated the mechanical properties of the two treatment

groups (methanol-treated and water annealed) under hydrated state31. To better mimic

physiological conditions, dog-bone geometries of silk films were submerged in 0.1M PBS

solution at 37oC. Samples were pulled at 10 mm/min cross-head rate until failure. All samples

experienced failure at the neck region with both conditions exhibiting similar stress-strain

curves. The average tensile strength (n=4) of methanol treated samples (~4.0 MPa) were

greater than water annealed samples (~3.6 MPa). This indicates that a positive correlation


7

exists between the swelling ratio and tensile strength of the film, as seen in the methanol-

treated films. This increased elasticity supports the hypothesis of increased chain mobility due

to a less densely packed secondary structure of the methanol treated group. This work

demonstrated the importance of the processing technique and crystalline content (secondary

structure) of the silk film on the material properties such as swelling ratio, mechanical strength

and oxygen permeability.

1.2.3 Modification of Silk Fibroin Membranes

As stated by Suzuki et al., a viable ocular tissue-engineered construct must be

transparent, permeable to solutes, promote cell attachment and proliferation, ideally thin (2-

10 μm), and flexible yet mechanically robust enough to withstand surgical manipulation to

avoid tears or undesirable folding30. SF membranes fulfil most of these prerequisites but

improvements for cell compatibility and mechanical properties remain. Improved cell

compatibility has been observed when topographic features on the surface of the SF

membrane are present. A rough surface topography and porosity increase diffusion of oxygen,

nutrients and biomolecules, and facilitates diffusion-based waste transport. Moreover,

interconnected pore networks are favourable for intracellular communication, signalling and

spatiotemporal control34. One approach to introduce porosity involved mixing a water-soluble

polymer, poly(ethylene glycol) (PEG), with SF solution prior to casting. Here, PEG was used

as a ‘porogen’, i.e. a particulate of a specific size and shape used to create pores, in the SF

film. After the film is cast, the SF polymer chains conforms into an insoluble state. A

subsequent water wash thereby only removes the PEG, leaving pores in its place35,36. Results

showed that low molecular weight (MW) PEG porogens significantly enhanced surface

roughness and permeability of the membrane as the weight ratio of PEG to SF increased.

However, the mechanical strength and elasticity were compromised with increasing PEG

content36. This study has demonstrated that a balance exists between the porosity and

mechanical strength of the membrane by controlling the PEG to SF ratio. This ratio is used to

enhance the elasticity and hydrophilicity in films37, or improve the viscoelastic properties of SF

to produce electrospun fibres38. In addition, processing treatments in methanol37,38 or through

water annealing39 can further control the conformational transition of fibroin to form β-sheets.

Despite the introduction of topographical microstructures, it was found that human limbal

epithelial cells produced more optimal morphology and stratification on non-porous rather than

porous SF membranes40. This combined with their increased opacity and fragility deterred the

use of porous membranes as substrata for corneal cells40-42.


8

An alternative method to improve both thermal and mechanical stability is crosslinking

SF with a copolymer, including but not limited to, sodium alginate 43, cellulose 44,45, chitosan46,

poly(vinyl alcohol)47, poly(caprolactone-co-D,L-lactide)48, and PEG49. The previous study by

Suzuki et al. also cross-linked genipin, a widely used natural crosslinker for proteins derived

from the fruit Gardenia jasminoides Ellis50, with PEG-treated SF (Figure 4)30. These PEG-

treated SF membranes crosslinked with genipin were thicker (10 to 15 μm) and easier to

handle compared to uncrosslinked PEG-treated membranes. Nevertheless, they were more

fragile than untreated SF membranes (without crosslinking and PEG treatment).

A B Figure 4. Chemical structure of genipin (A) and PEG (B). PEG-treated silk membranes were crosslinked with

genipin aiming to increase mechanical stability.

Suzuki and colleagues believe that the increased fragility of such crosslinking

treatments may compromise its suitability for being routinely manufactured as freestanding

membranes30. Thus, an alternative solution to introduce more specific cell-surface anchorage

to create true focal adhesions between cells and the substrata’s surface is required.

Studies in silk composites for the specific aim of mechanical reinforcement has been

explored. A nanocomposite SF film was formed with graphene oxide through simple casting

methods in aqueous media. The presence of graphene oxide in the SF matrix was found to

reinforce the films mechanically. The films did exhibit biocompatibility however cell

proliferation was lower compared to pure SF films51. In contrast, a composite based on silk

alone, namely, silk films embedded with silk fibres was fabricated by Li and colleagues.

Microfibres absorb energy and resist compression and dispersion pressures, as found in the

human meniscus52. In this approach, SF microfibres were suspended in the aqueous SF

solution prior to casting53.


9

Figure 5. Micrographs of SF microfibres of varied lengths produced by differing temperatures under alkaline hydrolysis: (A) 60 ºC, (B) 70 ºC, (C) 80 ºC, and (D) 90 ºC52. Reprinted with permission from Springer Nature and Copyright Clearance Centre (see Appendix 7).

Microfibre-embedded scaffolds showed better resistance to compression tests. Length

of microfibres were controlled by undergoing alkaline hydrolysis at set temperatures (Figure

5). At the set temperature of 80 ºC, this method produced homogenous microfibres but also

provides less control over surface topography52.

With controllable additive manufacturing (AM) processes, physical surface

modification for topographic patterning may serve as a pathway for enhancing mechanical

robustness without negating cell growth. The electrohydrodynamic direct writing technique,

named melt electrowriting (MEW), has been recognised as a powerful tool amongst the world

of AM. The controlled deposition of fibres at the microscale has sparked substantial interest

for its incorporation into the biofabrication field. The utilisation of microfiber networks to

reinforce current biomaterials have mainly been within the musculoskeletal context which have

shown improved biomimetic stiffness and elastic properties38,54-58. Thus, further research

incorporating microfiber networks for ocular surface repair is yet to be unveiled.

1.2.4 Surface Topography

The purpose of topographical patterning is to provide contact guidance for the

alignment of cellular matrices 42. The topographic patterning of SF films have played a role in

epithelial and fibroblast adhesion, alignment, proliferation and mobility59-64. In addition, SF


10

membranes have also been found to support human limbal epithelial stem cell attachment and

proliferation relative to tissue culture28. Previous studies have used soft lithographic

techniques to introduce high resolution surface features65-67. These examples fabricated

microfluidic devices with microchannel widths from 10 to 240 μm65, micropatterned films from

silk ionic liquid solution66, and nanopatterned biosensors with controlled surface morphologies

down to 125 nm67. Until now, the use of MEW scaffolds to provide mechanical enhancement

to SF membranes is an unexplored approach. The precision provided by MEW has the

potential to generate complex surfaces to direct cellular function and matrix deposition. This

is of importance since the complex architecture and optical properties of corneal tissue is owed

to the alignment of collagen lamellae, produced by corneal fibroblast growth68-70. The

combination of these techniques is therefore especially relevant to provide a biomaterial that

is both mechanically robust and transparent to support and mimic this native tissue

environment.

1.3 Significance and Scope

For future keratoprostheses to be successful, several requirements must be met.

These corneal material substitutes are used to restore the functional visual acuity, and

sometimes alleviate associated pain with the procedure, in severely diseased or damaged

ocular surfaces. Therefore, ideally, the keratoprosthesis should be inert as to not induce an

undesired immune response from the patient. In addition, the material should be efficiently

implantable, easily examinable and allow light through to a view of the retina 11. Moreover, it

is ideal that the material is inexpensive and easily sourced1,11,13,71,72.

With the emerging MEW technique, SF membranes could be reinforced mechanically

yielding an ideal substrate for cellular surgery. This project will utilise the semi-crystalline

polyester, medical-grade poly(ɛ-caprolactone) (PCL) (Figure 6), which has been established

in a wide range of applications in TE due to its chemically tailorable properties, mechanical

robustness and biocompatible degradation products73. PCL has a Young’s modulus of 3.1

MPa, a melting temperature between 59oC and 64oC and a glass transition temperature of -

60oC 74

Figure 6. Chemical structure of PCL


11

The development of a cell transfer sheet will incorporate various patterned MEW

frameworks of PCL. The cell transfer sheet will then be tested in vitro for cell viability through

2D cell culture of rabbit corneal epithelial cells (RCE).

We hypothesize that embedding PCL constructs fabricated by MEW in SF films will

provide mechanical reinforcement compared to SF films alone for suture applications to

secure the membranes onto ocular surfaces. Therefore, this study aims to improve the current

mechanical properties of SF membranes and demonstrate a viable method for the

reinforcement of cell transfer sheets using MEW fibres.

1.4 Research Objectives

Corneal cell transfer sheets are aimed to be an ex vivo resource for corneal epithelium

to be used in the regeneration or reconstruction of the ocular surface from trauma or

abnormalities. Such a method could be used to create a device that would provide guidance

in surgery, such as suture points. The main focus of this project is to examine the potential of

MEW as a novel method to implement surface topography and mechanical enhancement of

SF membranes for corneal epithelium regeneration. To achieve the overall aim, a number of

specific objectives were defined as followed:

1. Fabrication and analysis of cell transfer sheets

2. Develop a protocol to embed PCL frameworks in SF membranes.

3. Measure the pore sizes of printed constructs to determine dimensional accuracy.

4. Analyse fractures or voids in cell transfer sheets through scanning electron

microscopy (SEM).

5. Compare the 2D cell behaviour of primary rabbit corneal epithelial (RCE) cells on

varying pore sizes in free-standing SF-PCL membranes.

The fulfilment of these objectives will provide further knowledge to answering the

primary research question; can MEW be used to implement topographic patterning and

enhance silk fibroin membranes mechanically to enable its use as a cell transfer sheet in

corneal epithelial regeneration? It is hypothesized that topographical patterning and

mechanical reinforcement will be observed, providing a step closer to the capability of SF

membranes for corneal epithelial regeneration.


12

1.5 Thesis Outline

Following this chapter, the iterative process of fabricating embedded printed constructs,

the characterization and the compatibility with surgical handling of the reinforced construct will

be discussed (Chapter 2). The comparison of cell behaviour between varying pore sizes will

be then described (Chapter 3). The conclusions, limitations and future directions will be

addressed in each of their corresponding chapters (Chapter 2 or 3).

CHAPTER 2: REINFORCING SILK FIBROIN FILMS THROUGH MEW FRAMEWORKS

13

Chapter 2: Reinforcing Silk Fibroin Membranes with Melt

Electrowriting Frameworks

2.1 Introduction

This chapter primarily details the work in optimising the fabrication protocol for casting

SF films onto MEW frameworks. A brief methods and materials section is included to outline

specifications of materials and equipment used. The optimised protocol discussed in this

chapter is the device used in chapter 3 for cell work analysis.

2.1.1 Melt Electrowriting

MEW is an AM technique in which fibres are electrostatically drawn onto a collector in

a layer-by-layer assembly, producing a 3D architecture pre-determined by G-code. The initial

patent for melt electrospinning was described by Charles Norton from MIT approved in 193675.

It was not until 1981 where the first publications on electrospinning polymer melts came to

fruition demonstrating the electrostatic deposition of fibres and Taylor cone formation76.

Figure 7. Schematic displaying mass flows observed in the MEW process. A jet is created by feeding a polymer

melt through a spinneret77. Reproduced with permission under the Creative Commons Attribution License (see

Appendix 7).

Recent advances have aided this process through programmable deposition pathway

by combining it with an x-y collector controlled by G-code. The adjustable parameters used to

control fibre deposition include: (a) distance between the tip of the spinneret and the collector,

(b) melting temperatures of the heating coil and the tip, (c) pressure, (d) collector speed, (e)

voltage of the collector plate and (f) spinneret diameter77. A Taylor cone is formed when the


14

acceleration voltage surpasses the surface tension of the fluid. The electrified polymer is then

accelerated towards the opposing collector (mass flow = dm2/dt) resulting in a deposited fibre

(mass flow = dm3/dt). An optimal fibre drag is achieved through the control of these parameters,

enabling a stabilisation between the speed at which the polymer deposits from the spinneret

and the speed of the collector A straight fibre deposition is observed at such an equilibrium

and is called the critical translation speed (CTS). When the CTS is not achieved, the fibre has

been observed to pulse resulting in such behaviours portrayed in Figure 877.

Figure 8. Fibre deposition patterns based on varied collector speed percentages of the CTS (280 + 10 mm/min):

(A) 100-110% of CTS, (B) 75-80% of CTS, (C) 30-35% and (D) 10-15%. Non-liner patterns were described as

sinusoidal meanders (B), “sidekicks” or translated coiling (C), and “figure of eight” loops (D) 77. Reproduced with

permission under the Creative Commons Attribution License (see Appendix 7).

This present study will focus on varying pore sizes, given as the free box-shaped area

between fibres, with the controllable printing parameters kept constant to avoid the fibre

deposition patterns shown above. The polymer of choice was PCL, widely used with MEW

due to its low cytotoxicity and low processing temperature78.


15

2.2 Materials and Methods

2.2.1 Melt Electrowriter Setup

A custom-built MEW printer loaded with PCL (Purasorb® PC12, MW 120, 000 g/mol)

was used to fabricate the scaffolds in this study (see Appendix 1). A spinneret (23 G) was

used to load the polymer with temperatures set to 76/86 °C and pressures set to 1.1/2.1 bar.

The voltage applied was 6.6 kV. The collector was actuated by servo motors in the x- and y-

axes to allow for computerised numerical control (CNC) using G-code in Mach3 Newfangled

Solutions software. The collector speed was coded as 800 mm/min. A working distance of 6

mm was manually set and used for all experiments. Meshes were printed once electrified

polymer jet was stabilised. A summary of the printer parameters is shown below (Table 1).

Table 1. MEW parameters used to fabricate mesh structures. T1 and T2 refers to the set temperatures at the

heating chamber and the spinneret tip respectively.

Pore size (μm)

Spinneret (G)

T1/T2 (°C) Speed (mm/min)

Pressure (bar)

Working Distance

(mm)

Voltage (kV)

100 23 76/86 800 1.1/2.1 6 6.6

200 23 76/86 800 1.1/2.1 6 6.6

300 23 76/86 800 1.1/2.1 6 6.6

2.2.2 Scaffold Design, Laser Cutting and Plasma Treatment

Mesh structures with coded pore sizes of 100×100 μm2, 200×200 μm2 and 300×300

μm2 were used for experiments presented in this thesis. This range of pore sizes were chosen

to accommodate and observe the behaviour of RCE cells on the micrometre scale in contrast

to previous studies utilising nanometre topographical patterning63. The G-code design

implemented the aforementioned pore sizes in 40 x 40 mm scaffolds, looping the alternating

0° and 90° offset angle in the x-y direction twice, resulting in a layer thickness of 4 fibres at

overlapping strut junctions. The ILS12.150D by Universal Laser SystemsTM, Inc. was then

used to laser cut meshes into circles – 15 mm in diameter to allow for shrinkage - to be placed

into Teflon® cell culture chambers and 24 well plate for subsequent cell experiments. To guide

the laser cutter, CorelDRAW© 2017 software was used to program the desired cut. Samples

were placed directly on the metal grid to prevent undesirable heat travel from the laser to other

parts of the scaffold. The placements of the scaffolds were measured and entered into the

software.


16

Table 2. Parameters for 75W CO2 (10.6 μm) Laser Settings for PCL scaffolds

Parameter Setting

Laser Power 1.5%

Movement Speed 15%

Pulses per inch (PPI) 800

Height 1.00 mm

Meshes were placed in a 12 well plate and plasma treated using PDC-32G-2 Plasma

Cleaner (Harrick Plasma©, USA) for 7 minutes per side. The chamber was flushed with

oxygen and argon gas, both were set to 10 SCFH, and plasma treatment was activated after

the pressure reached below 200 Torr. After plasma treatment, meshes were vacuum sealed

in 50 mL falcon tubes for storage.

2.2.3 Fabrication of Films

Silk fibroin was prepared at the Queensland Eye Institute, (Brisbane, Australia) from

Bombyx mori silk cocoons. Dried cocoons (2.5 g) were immersed into 1 L of boiling Na2CO3

(2.12 g/L) solution. After 1 hour, the fibroin fibres were removed and rinsed twice with HP

water at room temperature with squeezing fibres in between washes. The fibres were then

immersed in water at 60 °C for 20 minutes followed by a rinse in HP water – this step was

repeated three times to remove sericin components before allowing the material to dry

overnight in the fume hood. The dried fibroin was then dissolved in a 9.3 M LiBr solution at 60 oC for 4 hours. The solution was then loaded into a 3,500 MWCO Slide-A-Lyzer® dialysis

cassette using a syringe (18 G) attached to filters connected in series (pore sizes of 0.20 μm

and 0.80 μm). The dialysis cassette was submerged in HP water (1 L) for 72 hours, with a

water renewal at 12+ hour intervals. The obtained solution was removed by syringe and filtered

(0.20 μm and 0.80 μm). The final concentration of SF in HP water was 2.97 % (w/v). The

solution was stored at 4 °C.

Prior to casting the membrane, 5 cm diameter glass Petri dishes were pre-coated with

1 mL of 7 % (w/v) Topas® copolymer (Advanced Polymers, Frankfurt, Germany) in

cyclohexane and dried overnight. The SF solution was diluted to 1.78% with HP water, and 4

mL was casted directly onto the scaffolds placed in the Topas®-coated Petri dishes to form

membranes. Following the addition of SF solution to meshes, samples were degassed for 10

minutes to remove air bubbles from PLC frameworks. Samples were then dried overnight at

room temperature in a fan-driven oven and subsequently water annealed for 24 hours in a


17

vacuum oven at room temperature. For use in imaging and cell culture experiments, the free-

standing membranes were cut using a circular trephine blade 14 mm in diameter.

2.2.4 Scaffold Characterisation

Scanning electron microscopy (Zeiss Sigma Field Emission Scanning Electron

Microscope) equipped with a Zeiss Gemini column at an accelerating voltage of 6 kV was

conducted for morphological analysis of the adhesion between PCL and SF. Samples were

gold-coated with a layer thickness of 3-4nm (Leica EM SCD005A, Germany) prior to SEM

imaging. The pore sizes and fibre diameters were then measured using a stylus profiler

(Bruker, Dektak XT) map scan function with a stylus radius and force of 2 μm and 3 mg

respectively. Map scans of 2000 μm2 with 10 second durations and height range of up to 1

mm were obtained for both sides of the unseeded membranes, herein referred to as top and

bottom of casted membranes. Top referring to the surface exposed to air after casting and the

bottom being the side directly in contact with the petri dish when casted.

2.2.5 Qualitative Suture Test

Membranes were sutured (10-0 12” Nylon, black monofilament, EthiconTM) onto the

corneal limbus of porcine cadaver eyes (n=3); (Dissection Connection, Australia) for

qualitative assessment. Feedback was provided for the membranes’ suture compatibility

based on the following criteria:

(a) Ease of penetration for suturing

(b) Membrane durability – whether the membrane tears under tension

2.3 Results and Discussion

2.3.1 Effect of PCL fibre dimensions, pore area and pore depth on SF

membrane integrity

Microfibre frameworks were fabricated using a custom-made MEW. Analysis

techniques including light microscopy, SEM and the Dektak stylus profiler were used to

characterise the membranes. When imaging the membranes, a pattern of pore sizes was

observed with regions with smaller pores and regions with larger pores in all samples (herein

referred to as small and large pore areas respectively). This cyclic variation from the coded

designs occurs due to the repulsion of deposited fibres from charged fibres (already on the

collector) until the charge dissipates and the pore sizes align to the coded size once again.

Therefore, for clarity in this thesis, the samples are referred their designated coding sizes and


18

whether it is the top (T) or bottom (B) sides of the membrane being analysed, i.e. T100, T200

and T300 or B100, B200 and B300.

Mean averages for the smaller pore areas were 16,380.2 (+ 1 μm2), 20,407.4 (+ 1 μm2),

and 88,402.8 (+ 1 μm2) for T100, T200 and T300 respectively, showing the predicted positive

trend (Table 3). On the contrary, T100 contained a larger mean average pore dimension of

64,610.9 (+ 1 μm2) compared to 43,020.0 (+ 1 μm2) in T200. This overlap of pore areas

between T100 and T200 is due to the build-up of electrostatic charge on fibres as previously

mentioned. In contrast, T300 contained the largest pore area overall of 107,314.8 (+ 1 μm2).

Given T300 is significantly larger than the areas observed in both T100 and T200, it can

therefore be used to observe differences in cell behaviour compared to T100 and T200. The

comparison and overlap of small and large pore areas between constructs can be seen more

clearly below where T200 has pore size average within the range of T100 (Figure 9).

Figure 9. Average pore areas (μm2) of regions with small and large pore areas in T100, T200 and T300.

Nevertheless, despite the overlap between pore areas of T100 and T200 the pores

were still considered architecturally relevant as the major aim of the experiment was to reduce

membrane fragility and observe RCE cell behaviour. Thus, the constructs were used for

subsequent experiments. As described in literature77, printing instabilities with MEW increase

as scaffold size decreases due to electrostatic repulsion between fibres. This is demonstrated

in the increase of standard deviation as the coded pore size decreased.


19

Table 3. Mean average pore sizes (μm2) and fibre diameters (μm) at struts (FDs) and junctions (FDJ) of casted SF membranes

Fibre diameters were measured using the x and y profile graphs obtained through 2D

stylus profiler scans (Appendix 3). Fibre diameters were measured at two locations: (a) at

suspended struts (FDs) which were approximately 26 to 38 μm whilst fibre diameters, and (b)

at junctions (FDJ) which ranged from 38 to 59 μm. A negative correlation was observed

between FDJ and pore size, with T100 showing the largest FDJ and T300 with the lowest FDJ.

Theoretically, the smaller the pore size the higher the amount of PCL (volume) is present.

Thus, a layer of T100 would weigh more than one layer of T300. The underlying layers would

therefore support more weight in T100, leading to larger fibre diameters at FDs and FDJ (i.e.,

lower aspect ratio). This is true for FDJ values but not FDs. Here, the FDs of T100 lies

between T200 and T300. This is due to the misaligned fibre stacking indicated by the

yellow arrows in Figure 11. These fibres led to the broader peaks detected by the stylus profiler.

Additionally, this also leads to the arbitrary definition of the pore edge. Given the broad nature

of the peaks due to the casted SF membrane, the defined edge of the pore is masked by the

gradual slope (Appendix 3). In this case, to ensure consistency, pore sizes were measured

between peak maximums in these profiles.

Furthermore, FDs is observed to increase from T200 to T300. Given the layer height and

printing parameters are kept constant, the pore size and FDJ values may influence the FDs.

Here, the FDs of T200 is lower than T300 due to the its higher FDJ. Due to the behaviour of

the polymer melt in MEW, it is known that the fibre struts ‘sink’ between junctions. Therefore,

as the polymer mass flow is constant, the higher FDJ of T200 will have struts that are stretched

more between junctions, reducing the FDs. From these results between T200 and T300, FDJ

and FDs are negatively correlated. However, it has not yet been verified that a larger FDJ

correlates to a higher construct height, the z-dimensions of the construct, i.e. membrane

thickness (Figure 10) and pore depth (Table 4), must be discussed.

The thickness of each membrane was also measured using the stylus profiler. Three

points of height were selected along the obtained line scan, averaged and then plotted. The

standard deviation from these three points was also included as error bars (Figure 10). Since

the samples were mounted onto carbon tape, scans for carbon tape were obtained and are

subtracted from the averages (n=3). One exception is B300, where only two points were

Membrane Pore area (small) (μm2)

Pore Area (large) (μm2)

FDS

(μm) FDJ (μm)

T100 16380.2 64610.9 29.8 58.0 T200 20407.4 43020.0 26.3 41.8 T300 88402.8 107314.8 37.3 31.5


20

obtained from a single scan as the line plot was linear. Any points taken between these two

positions fall within the standard error (Appendix 2). All membrane thicknesses were under

100 μm. For T100, T200 and T300, the membrane thickness was found to decrease as pore

size increased. This correlates with the trend observed with FDJ values which decreased as

pore size increased. Moreover, this justifies the negative correlation between the FDJ and FDs

in T200 and T300. Thus, given the highest point of the construct is at the fibre junctions, it is

logical that T100 would have the highest thickness. This trend however, was not observed in

B100, B200 and B300. Here, B100 has the highest thickness but is followed by B300. This is

justified when observing the line plot (Appendix 2), as a steep incline is present prior to the

linear region. This demonstrates an air gap between the carbon tape and the membrane which

results in an excess measured thickness and can be considered an outlier. Furthermore, the

standard deviation of T300 almost matches its thickness (36.8 + 36.0), likely due to two

troughs and the highest peak being selected. At this scale, it is likely an air gap has led to this

deviation.

Moreover, as the scans were set to 20 seconds over 20 mm, the stylus was observed

to ‘jump’ whenever it crossed a protruding ridge of a fibre. This probing speed is too fast as

sharp ‘sawtooth’ peaks in the 2D scans was observed in the final measurements – an example

is provided (Appendix 2). Due to this, the averages obtained were from points taken at both

peaks and troughs, hence the large error bars. Realistically, if samples could be measured

without the requirement of mounting materials (i.e. carbon tape and glass slide) and combined

with an increased scan time to reduce deviation of the stylus, a more accurate thickness could

be measured. Finding suitable mounting materials and an appropriate scan speed are aims

for further investigations.


21

Figure 10. SF-PCL membrane thickness measurements measured by single stylus profiler scans by Dektak.

In a preliminary experiment, a casting volume of 2mL in a 5 cm diameter dish was

used. Once membranes dried, large fractures were evident between the PCL fibres and the

silk film at room temperature which made the membranes unsuitable for further analysis. It

was speculated that the fractures occurred due to the thickness of the stacked fibres (~80 μm)

exceeding the thickness of the silk film once dried (~30 μm). This led to de-adhered areas of

SF membrane between fibre stacks that have thinned to the extent of fracturing.

To resolve this issue, the casting volume was doubled to 4 mL yet fractures still

occurred at the interface between the SF film and PCL fibres. To further combat this issue, O2

plasma treatment was implemented. Plasma treatment has been found to increase surface

hydrophilicity of materials79. This is achieved through the introduction of polarity via

oxygenated functional groups which allows for the formation of O-containing polar functional

groups on the PCL backbone. The improved membrane quality was confirmed using stylus

profilometry, a qualitative suture test and several imaging techniques further discussed below.


22

Firstly, stereomicroscopy was used to assess fractures and observe the general

appearance of the membrane (Figure 11). At this level of magnification, no fractures were

evident, and fibres appear to be embedded within the film. Topographical ridges and grooves

created by the embedded PCL framework are more evident in T100 as indicated through the

diffraction of light resulting in dark fringes occurring along the fibres. This was further

investigated by a stylus profiler technique.

Figure 11. Stereomicroscopy images of casted SF (black arrow) membranes containing embedded PCL constructs (white arrow); T100 (left), T200 (middle), and T300 (right) with their respective small (red) and large (blue) pore areas (D-F) highlighted directly below. The yellow arrows indicate the misaligned fibre stacking in the y-direction, which impacts the FDs of T100. Scale bar = 200 μm.

As previously mentioned, FDJ was observed to be largest in T100. The 3D profiler

maps provide supporting evidence for pore depth. The stylus profiler scans assessed both top

and bottom (flipped) sides of the membranes in the dry state (Figure 12). The depths (z-axis

scale) detected by profilometry scans are tabulated below (Table 4). Assuming the height of

the PCL construct is determined by the FDJ value, it is predicted that T/B100 would

demonstrate the deepest pore depth (i.e. largest ∆z-values) with a decrease in depth to

T/B300. T/B100 supports this prediction with the largest ∆z-values for both sides – 7.9 x 10-1

mm and 9.4 x 10-3 mm respectively. However, T/B200 is an exception to this trend with the

smallest ∆z-values (i.e., shallowest pore depth) amongst all samples of 5.2 x 10-1 mm and 6.2

x 10-3 mm respectively. Thus, T/B300 has ∆z-values (6.9 x 10-1 mm/7.8 x 10-3 mm) in-between

T100/T200 and B100/B200. Given the overlapping pore dimensions between T100/200 and

the potential for undetected air bubbles being present, the accuracy of these results is

questionable. It would be best to repeat these measurements with optimised printing

parameters that better reflect the coded pore sizes. In addition, the protocol for characterising


23

membrane thickness and pore dimensions could benefit from a cross-section in SEM or non-

invasive techniques such as Two-Photon Excited Fluorescence and Second Harmonic

Generation. It was observed that T100 was the stiffest of all membranes and the edges tended

to curl away from the carbon tape. This led to air gaps existing between the carbon tape and

the dry membrane, resulting in inaccurate heights detected. This is more evident in the

B100/200/300 map scans, where large regions of red/blue indicate air gaps under the

membrane. B200 contains the largest region of red which validates the shallower pore depth

observed. Meanwhile the central regions of both B100/300 are predominantly blue, suggesting

that the sides were less adhered to the mount. In addition to finding suitable mounting

materials, it would be beneficial to use a process that either optimises or bypasses the

membrane’s adherence to carbon tape. Better yet, the non-invasive techniques previously

mentioned could examine the membranes in both a dehydrated and hydrated state. This would

provide a better understanding of how the PCL constructs would affect the swelling ratio of

the silk film.

Table 4 Pore depth calculated from difference in z-axis from Dektak scans of T100/200/300 and B100/200/300

Pore Type ∆Ztop (x10-1 mm) Bottom, ∆Zbottom (x10-3 mm) 100 7.9 9.4 200 5.2 6.2 300 6.9 7.8


24

Figure 12. Stylus profiler 3D map scans of top (as casted – left column) and bottom (flipped, underside – right column) surfaces of unseeded SF-PCL membranes: 100 μm (first row), 200 μm (middle row), 300 μm (third row) coded pore sizes.

As previously mentioned, further optimisations were carried out by doubling the

amount of SF solution and by plasma treating the PCL scaffolds prior to casting. It was found

that plasma treatment reduced the amount of crack as depicted in SEM images (Figure 13).

This was demonstrated with the circular designs (printed to a total height of two layers) as the

membrane quality is distinctly seen to improve with increased casting volume and plasma

treatment. The cross-section of both materials was imaged to assess the adhesion between

both materials by two processes: freeze fracture with liquid nitrogen, or a transverse cut using

a round-edged scalpel (Figure 14). This was completed using the optimised samples, i.e.

plasma treated and 4 mL casting volumes. Despite optimisation, both materials still

demonstrated incompatibility as prominent gaps were present between the fibres and the

casted SF membrane (Figure 15).


25

Figure 13. SEM images of membranes with circular frameworks (two layers), prepared by casting 2 mL SF solution in a 5 cm diameter dish with a non-plasma treated PCL framework (A, D) vs 2 mL casting solution with a plasma treated (pt) PCL (B, E) vs 4 mL casting solution with a plasma treated PCL (C, F). Fractures occurred most frequently in the membrane prepared with 2 mL SF solution with a non-plasma treated PCL. Plasma treatment decreased presence of fractures (E) and additional doubling the casting volume (F) rid of abrasions seen on fibre junctions in (E). Scale bars: 50 μm (A-F).

Figure 14. Cross-sectional SEM images of plasma treated SF-PCL interface, fractures via incision with a round edged scalpel (A, B) or freeze fractured after submerged in liquid nitrogen (C, D). Scale bars: 10 μm (A, B), 20 μm (C), 10 μm (D).

Despite attempts of optimisation by increasing casting volumes or plasma treatment,

several issues were not avoidable. This included the uneven distribution of the SF solution in

each pore during casting due to air bubbles trapped between fibres that were too small to be

removed by the degassing step, leading to gaps in the final membrane. Furthermore, the

fragility of the membranes in the dry state during handling still led to cracks propagating along


26

the fibre networks, in some cases. Finally, punctures were also present owing to handling the

membranes with forceps during its dry state. Thus, further optimisation through alternative

means to decrease the membrane’s fragility is essential to ensure the membrane’s suitability

for routine manufacturing.

Figure 15. SEM images displaying issues encountered with plasma treated SF-PCL membranes. Uneven casting volumes per pore (A) was observed in the initial 2 mL casting volume which was optimised by doubling (4 mL) the casting volume. Fragility of the sample is demonstrated by fractures (B) and piercings (C) of the membrane during handling. Scale bars: 200 μm (A), 100 μm (B, C).

2.3.2 Qualitative Suture Test Membranes were placed onto corneas of porcine cadaver eyes. The shape of the

membrane was maintained without any deformations during handling for all pore sizes (Figure

16). A single stitch was sutured through the membrane, corneal limbus, and back to the

membrane, and completed with a knot to assess the surgical performance of the substrates,

as indicated by the red arrows. The membranes appeared to conform well on the curvature of

the corneal surface for all pore sizes. During suturing and handling, no tears or undesirable

folding of the membrane occurred, and the suture was noted to have pierced the membrane

easily. Overall, the handling of the membranes was notably easier compared to non-reinforced

SF membranes. Thus, it can be concluded that the PCL frameworks did play a role in

mechanically reinforcing the SF membrane. In terms of the how the membrane conformed to

the corneal surface, however, remains inconclusive, as it was suspected that the porcine

cadaver eyes were cryopreserved sub-optimally due to the planar appearance of the cornea.

Literature has previously demonstrated a build-up of metabolic waste products in the aqueous

humour, for example lactate, to occur when stored in moist conditions 80. Indeed, these eyes

were packed with some physiological solutions for storage, and moisture would have led to

the flattened curvature of the samples’ corneal surfaces and their cloudy appearance.


27

Figure 16. Porcine cadaver eyes sutured with T100, T200 and T300 membranes of varying pore sizes (100, 200 and 300 μm2 pore sizes respectively). Suturing was completed by Dr. Fiona Li as indicated by the red arrows. Photographs were taken using a Canon EOS 6D digital SLR equipped with a 100 mm 1:2.8 L macro lens.

2.3.3 Future Direction: Clinically Relevant PCL Scaffold Design To determine the ideal height of the PCL construct, one design was programmed with

spokes, rings and squares at 5, 10 and 15 layers. Tears in SF films were observed at all

heights with a complete de-adherence of the central SF film region, deeming this particular

design unusable for further cell culture experiments (Figure 17). A more intact membrane was

achieved when a basic box mesh was implemented and therefore this construct was utilised

for characterisation and cell culture experiments. This initial design with a circular framework

outlining the circumference of the ocular surface serves as a proof-of-concept design that

could mechanically strengthen SF films and provide suture anchor points for surgical use

without disrupting patient’s vision.

To approach a more clinically relevant design, the issues that arose from the

embedded PCL mesh constructs in SF must be addressed. In summary, these issues are: (a)

tears in the membrane, (b) gaps between PCL fibres and SF film, and the characterisation of

(c) membrane thickness, (d) topography and (e) mechanical properties. A potential solution to

addressing the material incompatibility between PCL fibres and SF films is by implementing

an additional fabrication step – namely replica moulding through soft lithography. Here, the

PCL construct is used as a positive mould within a lens shaped cell. Once casted with an

appropriate elastomer, e.g. poly(dimethylsiloxane) (PDMS), a negative mould is formed. The

SF solution is then directly casted into this negative mould thereby producing a topographically

patterned SF-only film. This protocol bypasses common bi-material issues such as varied

rates of shrinking/swelling and the de-adherence between materials. As for improving the

characterisation of the membrane thickness and topography, suggestions include using SEM

to image the cross-section of the membrane and by optimising the probe speed of the stylus

profiler to minimise ‘sawtooth’ peaks. Additionally, smaller sample sizes (below 1x1 cm) of the

membrane, from different regions (e.g. the membrane’s centre and edges, fibre junctions and


28

struts), should be mounted onto to the carbon tape. This would minimise air gaps and

undesirable folds in the membrane during thickness scans. Finally, for quantitative mechanical

testing to take place, the tears must be avoided. A suggestion is to swell the casted PCL-SF

construct prior to delaminating it from the petri dish. Through the swelling, the SF membrane

on PCL should have a better threshold for mechanical stress, which is applied when the

sample is delaminated and handled. Alternatively, the abovementioned topographically

patterned SF-only membrane could be tested.

Figure 17. Example design for reinforcing a corneal cell sheet through PCL frameworks of varying layer thicknesses. Tears in films indicated by arrows on frameworks with 5, 10 and 15 fibre layers (A, B, C respectively) casted with SF solution (1.78 % (w/v)). Scaffolds were submerged in water for 30 min at 37 °C. Straight fibres were observed at 480 mm/min collector speed.

2.4 Conclusions and Limitations

The main intention of this work was to mechanically reinforce SF membranes and

introduce topographical patterning using 3D-printed microfibrous PCL structures. Previous

studies have attempted to reinforce SF chemically and introduce topography through a variety

of etching techniques which led to increased fragility or decreased proliferation in cell culture.

With MEW being a recently established technique for controlled fibre deposition, a novel

means of mechanical reinforcement and topographical patterning became accessible. Thus,

in this study, as a proof-of-principle, PCL frameworks were printed via MEW were embedded

into SF membranes. Fractures occurred in membranes embedded with circular constructs. An

alternative box construct was implemented to provide more stability throughout the membrane

for handling. This box construct allowed for proof-of-concept experimentation to take place

however fractures were still present. Fractures were further reduced by increasing the casting

volume of SF and applying plasma treatment. Resulting membranes were characterised using

light microscopy, SEM and stylus profilometry. The membrane thickness was found to

decrease as pore size increased for the top sides. Furthermore, the pore depth differed by two

orders of magnitude between the top sides (10-1) and the bottom sides (10-3) in all samples.

This demonstrates that the topography is realized by the evaporation of solvent. Additionally,


29

a qualitative suture test demonstrated increased mechanical stability for surgical handling.

Further cell viability will be assessed in the next chapter.

In conclusion, the clinically relevant circular design demonstrated fragility not suitable

for handling or overall routine manufacturing. However, the combination of embedding a proof-

of-concept box construct, plasma treatment and increasing casting volumes vastly improved

the transportability of the membranes whilst introducing topographical features. As mentioned,

the fabrication and characterisation protocols require improvement still. A future analysis on

the influence of pore dimensions on fracture frequency should be conducted. The experiment

should aim to use constructs with optimised printing parameters to reduce fibre misalignment

as well as cover a larger range of pore sizes for a more in-depth understanding.

CHAPTER 3: MIMICKING THE CORNEAL EPITHELIUM

30

Chapter 3: Mimicking the Corneal Epithelium

3.1 Introduction

To evolve strategies in TE, research on the impact of topographic cues on the

proliferative response of cells must be conducted. Liliensiek and colleagues have designed

nanoscale topographical features (<200 nm – 2000 nm) to mimic the anterior corneal

basement membrane63. The topographical features were etched onto a silicon master by X-ray

lithography and then were transferred to a polyurethane (Norland Optical Adhesive 61) surface

through soft lithography with PDMS. However, cell proliferation was found to decrease as

features decreased in size. From this study, the most promising feature size was within the

macroscale (2 μm). The ability to control deposition has allowed MEW to produce macroscale

pore structures. The pore sizes can thus be tailored based on specific cell size and shape.

The anatomical microstructures within the limbal region are unique to individuals, comparable

to fingerprints and are termed “conjunctivoglyphics” (“conjunctival carvings”)81.

The utilisation of MEW could allow for the embedding of such fingerprint designs into

the SF substrata. This chapter therefore investigates the cell behaviour of RCE cells in box

structure designs to mimic the topography as revealed by previous stylus profiler images in

Chapter 2. It is hypothesized that the larger macroscale features will allow for increased cell

proliferation while also providing contact guidance by mimicking the native basement

membrane. A method and materials section are included to outline specifications of materials

and equipment used. Results for cell viability and imaging of cell morphology will also be

discussed in this chapter.

3.2 Materials and Methods

3.2.1 Culture and Growth of Primary Rabbit Corneal Epithelial Cells on SF-PCL Substrata

The free-standing, reinforced membranes were fabricated and cut as mentioned in

Chapter 2. Once cut, the membranes were mounted in sterile Teflon® cell culture chambers

(Figure 18). In the case of the coated wells for the resazurin assay, plasma treated PCL

frameworks were placed in wells of a 24-well plate and SF solution was directly cast into the

wells and fabricated as described previously. The volume of SF was adjusted with the area of

well to obtain the same thickness films as the free-standing ones. The membranes placed in


31

chambers and coated wells were sterilised by submersion in 70% ethanol for 30 minutes and

washed in phosphate buffered saline (PBS) three times.

Figure 18. Protocol for mounting freestanding membranes into cell culture chambers. (A) Schematic cross-section diagram of device following assembly, (B) Upper and lower parts of device prior to assembly with silicone O-ring (red) fitted in the upper chamber, (C) Threading of chamber parts to tighten using forceps on the leverage points provided on the upper chamber, (D) Culture champers suspended in media within a standard 6-well culture plate82. Reproduced under the Copyright Clearance Centre (see Appendix 7).

Primary cultures of rabbit corneal epithelial (RCE) cells were isolated from ocular

tissues of cadaveric rabbits, cultured in 25 cm2 flasks with Green’s medium83 and

cryopreserved by Harkin’s research group. A top-seeding approach was implemented to seed

cell densities of 50 000 cells/cm2 onto free-standing membranes in chambers and 10,000

cells/cm2 onto coated wells. The Resazurin assay and replacement of medium was conducted

on day 1, 3, 7, 11 and 14.

3.2.2 Cell viability on membrane coated wells

Cell viability was assessed using a Resazurin Assay kit from Sigma Aldrich (cat. #

R7017), according to the manufacturer’s protocol. Briefly, the cells were added to a 24-well

plate at low density. The day prior to the first scheduled assay, the medium from each well

was removed and replaced with fresh medium (900 μL) as well as to an additional 3 wells not

containing cells. The following day, 10 μL of Resazurin (70 mM stock solution) was added to

each well (1:100 dilution) and incubated for 4 hours. A volume of 800 μL of medium as

subsequently removed from each well and transferred to a new 24-well plate with an additional


32

800 μL added to an empty well plate as a blank control. Cells were washed with HBSS before

replacement of medium and further incubation. The absorbance of each well was then

measured by a spectrophotometer at 570 nm (reduced) and 600 nm (oxidised). The A570 and

A600 values of the blank was deducted from the samples to compensate for background

absorbance. To assess the metabolic activity, the reduced state of resazurin, resofurin, was

calculated using the following equation:

The following table outlines the values included in the equation. The absorbance of

test wells (A) and negative controls (A’) at 600 nm and 570 nm are indicated by A600, A’600,

A570 and A’570 respectively. Table 5. Equation values for Resazurin Assay

Oxidised Reduced Molar extinction coefficient

[L mol-1 cm-1] at 570nm 80,586 155,677

Molar extinction coefficient [L mol-1 cm-1] at 600nm

117,216 14,652

3.2.3 Imaging of Cell Culture on Free-Standing Membranes

All membranes were imaged with a Zeiss Sigma Field Emission Scanning Electron

Microscope equipped with a GEMINI e-Beam column. After fixation and dehydration steps

(see Appendix 4), based on instrument availability, samples were either sputter-coated with

gold (Leica EM SCD005A, Germany) or platinum (Bio-Rad SC500) and imaged at an

accelerating voltage of 5 kV or 10 kV respectively. Moreover, confocal laser scanning

microscopy (CLSM) was employed to compliment SEM imaging. A Nikon A1R CLCM

equipped with a fluorescent probe (FITC-dextran 70 kDa) imaged the TRITC-Phalloidin

(Sigma Aldrich, Australia) stained samples prepared using the standard protocol (Appendix 5).

3.2.4 Statistical Analysis

A two-way analysis of variance (ANOVA) statistical analysis (GraphPad Prism 7) was

performed for the resazurin assay followed by post-hoc test (Tukey’s test) to analyse statistical

significance. A value of p < 0.05 was considered statistically significant.


33


3.3.1 Comparison of Pore Sizes

Fibre diameter and pore size were measured using a stylus profiler (Chapter 2).

Variation in pore sizes were consistent despite a lack of optimisation, resulting in a pattern of

small and large pores. This was more prominent in membranes with smaller pore sizes, i.e.

T100 and T200, as the electrostatic repulsion increased when fibres were deposited within

closer proximity to one another. In addition, the G-code disregards fibre sizes thus pore sizes

were smaller than coded. Moreover, once casted pores were further minimised due to the

layer of SF.

3.3.2 Quantitative Analysis

To evaluate the effect of surface topography on cell growth, SF-PCL films were

prepared by directly casting wells of a 24-well plate, and RCE cells with were seeded with a

density of 10,000 cells/cm2. The culture was maintained for 14 days, and resazurin assay was

performed on days 1, 3, 7, 11 and 14 to assess cell viability. Resazurin, a water-soluble

extracellular redox indicator, generates a soluble fluorescent pink reaction product when

reduced by living cells. This property provides a quantitative means to detect changes in cell

viability. Beneficiary aspects justifying the use of this assay include: (a) minimal cytotoxicity to

cells, (b) a straight-forward protocol, and (c) that the reduction of resazurin is directly

proportional to a wide range of viable cell numbers84.

Figure 19. Proliferation of RCE cells on BMSF-PCL T100, T200 and T300 films coated on wells of a 24-well plate. TCP and BMSF films were controls. Data represents mean average + standard mean error of a single culture over 14 days with three samples analysed for each condition.


34

A significant positive correlation was observed for all membranes up to day 7. After

day 11, positive growth curves were only observed for T200 sample, with a negative

correlation shown for the remaining groups, TCP, T100, T300 and SF (Figure 19). Notably, no

significance was found between Day 11 vs. Day 14 for any condition. As cells on the positive

control (i.e. TCP) behaved similarly to other samples, this growth trend is considered cell-

related rather than material-based. Indeed, primary cells with passage number 3 were likely

matured and no longer limbal stem cells. In addition, these cells were not co-cultured with

feeder cells (i.e. irradiated 3T3 cells) that are often used to maintain proliferative potential of

primary corneal epithelial cells. Therefore, cells would lack progenitor cell activity and undergo

migratory behaviour rather than proliferation. Nevertheless, the aim was to investigate how

the primary RCE cells will react with SF membranes with different pore sizes and following

imaging analyses will elicit further information.

A top-seeding approach was performed on silk-PCL membranes casted directly into

wells of a 24 well-plate with a density of 10,000 cells/cm2. Due to the rudimentary seeding

technique, cell density was unevenly distributed, leading to certain pores with higher densities

whilst others were left with either low or no cell densities. This is indicative with variance

between samples in day 1, with T100 and T200 showing significantly lower resazurin reduced

percentages. This would also explain the different growth rates observed after day 11.

Moreover, the presence of microtears (invisible to the eye) was not confirmed. It is possible

that cells would have fallen through such fractures after the membranes were mounted. A

suggestion for future experiments would be to examine the mounted membranes under the

microscope prior to seeding to confirm their quality.

Table 6. Tukey’s Post-Hoc Multiple Comparisons Test Comparing Column Means

Membranes 95% CI of diff. Significance Adjusted P value TCP vs. T300 -8.543 to -2.075 *** 0.0002 SF vs. T100 0.08556 to 6.553 * 0.0416 SF vs. T300 -6.552 to -0.0845 * 0.0417

T100 vs. T300 -9.872 to -3.404 **** <0.0001 T200 vs. T300 -9.328 to -2.86 **** <0.0001

The post-hoc test suggested a significance between various groups mentioned in

Table 6. The most significant were T100 vs. T300 and T200 vs. T300. From this, it is also

implied that no significance exists between T100 and T200 which is justifiable due to the

overlap of pore sizes mentioned in Chapter 2. With optimised printing, it would perhaps have

shown a significance between all pore sizes statistically. Nevertheless, at day 14, the data

suggests T300 to induce more proliferation overall. Additionally, T200 demonstrated low

proliferation on all days excluding day 14 where it exceeded other conditions.


35

3.3.3 Morphology of RCE Cells

Figure 20. Growth of RCE cells in culture. The phase-contrast images show the morphology of cells growing on (A, D, G, J, M) tissue culture plastic (control), (B, E, H, K, N) BMSF, and (C, F, I, L, O) T300 membranes, on days 1 (A, B, C), 3 (D, E, F), 7 (G, H, I), 11 (J, K, L) and 14 (M, N, O). The scale bar (200 μm) is the same for all panels.

Phase-contrast images show morphology of high-density cell populations grown on

24-well plate prior to conducting the resazurin assay (Figure 20). On day 1, the cell population

appears dense and round. Over time, more space is seen between cells which slowly appear

to flatten and elongate. This resulting flat, yet elongated morphology is attributable to


36

senescent cell behaviour. The lack of 3T3 feeder cells likely contributed to their loss of

proliferative potential. Similar findings to T300 were observed for T100 and T200 (Appendix

6).

Figure 21. Phase-contrast images of non-confluent areas of RCE cell growth after 14 days in culture. The non-central areas of wells where cell growth was not confluent is shown on (A) tissue culture plastic (control), (B) BMSF, and (C) T300 membranes. The scale bar (200 μm) is the same for all panels.

Phase contrast images were also conducted on low cell density populations (Figure

21). Minimal contact between cells is observed. After 14 days of culture, cells appear to remain

small and rounded suggesting early senescence. Some branching is evident in areas where

cells were seeded closer together (TCP and BMSF) however it appears that senescence has

stunted further proliferation. One reason being that senescence cells in turn has a senescent-

inducing effect on neighbouring cells85. The effect of no 3T3 feeder cells is perhaps seen more

prominently in these lower density populations as proliferation is less evident despite the

increased space for cell growth.


37

Figure 22. SEM images of seeded membranes of pore sizes 100, 200 and 300 μm2, showing cell-membrane and cell-cell interactions. Cells were fixed after 14 days in culture. Panel A, C and E provide macroscopic view of the membranes and B, D and F are magnified images. Scale bars: A: 200 μm; B-D: 100 μm: E: 200 μm; F: 50 μm. Note: the sample preparation required for SEM led to cell shrinkage, drying artefacts and fractures between the PCL and SF materials, as well as the cell lamellae.

Cells were seeded onto freestanding membranes via the top-seeding approach at a

density of 50,000 cells/cm2. SEM images after 14 days showed consistent cell morphology

containing lamellae in all samples, indicating migratory cell behaviour – a feature also evident

in low cell densities (Figure 22). Cells appeared to cross over fibres and accommodate pore

sizes well in all pore sizes despite the low density. Moreover, it is notable that SEM processing

possibly resulted in cell shrinkage and loss thus depicting lower cell densities once imaged.


38

Figure 23. Phalloidin staining of RCE cells seeded on SF-PCL membranes with 100 μm (A), 200 μm (B), 300 μm (C) programmed pore sizes. Scale bars = 200 μm.

To complement SEM images, actin filaments of cells on day-14 cultures were stained

with TRITC-Phalloidin and imaged using CLSM (Figure 23). Morphology of lamellae appears

to be more rounded than that observed in SEM, further revealing the detrimental effect of SEM

sample processing on cell morphologies. Moreover, in T300, cells appeared to grow from the

fibre frameworks towards the centre suggesting that pore depth of these membranes may not

be an important factor. This is not overly evident in T100 and T200, perhaps due to the

individual cell sizes occupying a larger portion of each pore. This is seen in T100 and T200

with cell density appearing higher which potentially led an over-confluent density before

migratory behaviour could be observed.


39

3.4 Conclusions

An alternative approach to providing mechanical reinforcement and contact guidance

for RCE cells on silk-PCL substrata was investigated. Previous studies for topographical

patterning have included various lithography techniques or etching with porogens whilst

mechanical reinforcement has been attempted by chemical modification, however, techniques

led to membranes which were less mechanically feasible in strength and elasticity. As MEW

becomes a more established technique in TE, a lack of studies implementing this technique

for mechanical reinforcement and topographical patterning still remain. The aim of this study

was to establish a protocol for embedding PCL microfiber box frameworks into SF membranes

to simultaneously provide mechanical enhancement accompanied by topographical patterning.

Substrata were characterised quantitatively, using Resazurin, and qualitatively through SEM

and confocal imaging.

Based on confocal images, migratory behaviour was observed to align along fibres for

T300. This was not observed in T100 and T200. One consideration being that both the cell

size and cell number per pore were higher in T100 and T200. This higher cell density

potentially led to confluency being reached before migratory behaviour could be observed.

However, SEM images slightly demonstrate lamellae to prefer alignment along fibres rather

than across pores. This suggests that pore depth may not be a significant factor compared to

x and y pore sizes.

As for the Resazurin assay, T300 showed the highest cell viability overall. T200 also

demonstrated a dramatic increase in proliferative capabilities, exceeding all conditions,

however only at day 14. Moreover, the overlap of pore sizes in T100 and T200 was statistically

evident as no significance was observed between these membranes. Thus, printing

optimisation is required to acquire a better understanding of cell behaviour in pore sizes less

than 200 μm2.

Overall, due to the rudimentary top-seeding approach and the resulting uneven

distribution of cell densities, the observations obtained remain inconclusive. A larger range of

pore sizes should be investigated however this study has provided an indication of cell viability

and morphology for pore sizes less than 300 μm2. In addition, the Resazurin assay should be

accompanied with SEM and confocal imaging of samples for each corresponding day to better

observe cell proliferation. Furthermore, alongside printing optimisation, addressing the poor

adhesion between SF and PCL proved to be the most arduous challenge which was not

overcome successfully. Once these issues are addressed, MEW has the potential to provide


40

a novel approach to the mechanical reinforcement and topographical patterning of SF

substrata.

3.5 Future Directions

a) Scaffold design Future directions include improving the scaffold design to one that is more clinically

relevant. Design considerations include printing circular frameworks that omits the

requirement of the laser cutter as well as implementing specific pores as suture anchor points

to provide guidance in surgical practice. Previous attempts have been included (Figure 17),

which utilised a circular framework however tears in the membranes resulted in these samples

being discontinued for mechanical and cell analysis.

b) Corneal Epithelial Cells A more vigorous record of the cell proliferation and migration should be documented

at a time point of every three days. This will assist in comparing whether different pore sizes

induce different cell behaviours and at what rate these behaviours occur.

c) Analysis Techniques Advanced mechanical testing should be incorporated into future experiments,

including a suture retention test for each design. This will determine whether the mechanical

reinforcement is mainly provided by the design of the construct or by presence of the PCL

framework itself.

Further staining such as DAPI could also be implemented in confocal fluorescence to also

map cell nuclei and provide a better overall picture of cell viability.

d) Improving seeding homogeneity Static seeding directly onto the membranes was employed in this study. The non-

uniform epithelial cell layer was a major limitation for the analysis of cell morphology and

behaviour. Two ways to improve this would be to increase culture time and document

behaviour of both high and low cell densities or to continuously shift the membrane. This static

approach remains inferior to current dynamic techniques mentioned in literature. A suitable

dynamic technique would be centrifugal seeding in which a cell suspension is rotated around

the membrane attached to a central needle86,87. Low speed rotations have not shown an effect

on cell morphology however seeding time can increase to up to 24 h and require higher cell

concentrations88. Despite these setbacks, it is worthwhile to implement such an approach to

allow for a more conclusive cell analysis.

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APPENDICES

48

Appendices

Appendix 1. Protocol for Fabricating Scaffolds via Melt Electrowriting

A custom-built MEW printer was utilised for the fabrication of scaffolds in this study

(Figure 24)77. A 23 G spinneret was fitted to the syringe loaded with PCL and heated to

76/86 °C. Polymer feeding was pneumatically controlled with the pressure set to 1.1/2.2 bar.

The collector to tip distance was set to 6 mm. The voltage applied was 6.6 kV, with the positive

voltage applied to the spinneret connected to the grounded aluminium collector plate.

Scaffolds were printed at ambient conditions.

Figure 24. Schematic diagram of MEW printer. The stage is controlled in the x- and y- axis programmed using

CNC programming. The printer head includes a heating ring and jacket for the spinneret tip and syringe chamber

to ensure homogenous melting of the polymer. The printer head is mounted on a manually adjustable z-axis to

adjust the distance of tip to collector77. Reproduced with permission under the Creative Commons Attribution

License (see Appendix 7).

APPENDICES

49

Appendix 2. Example Stylus Profiler Scan for measuring SF-PCL

Membrane Thickness

Figure 25. Single scans of dry SF-PCL membranes mounted onto carbon tape, completed by the Dektak stylus

profiler. Scans were 20 seconds over 4mm. A total of 3 points (shown as R and M cursors under line fit) were

obtained for each sample, including the standard (carbon tape). The average height of the carbon tape was then

subtracted from the average heights of T/B100, T/B200 and T/B300. Shown here are the B300 scan (with a linear

region between R and M cursors) and a T200 scan example exhibiting the ‘sawtooth’ peaks from fibre ridges.

APPENDICES

50

Appendix 3. Dektak Surface Analysis of Unseeded Membranes

Figure 26. X and Y profiles of 2D map of T300 scanned using the Dektak stylus profiler. The X profile displays

fibre strut peaks heights (nm) and the Y profile displays fibre junction heights (nm). Topography is also shown

through heat maps. Pore sizes were measured between peak maxima to ensure consistency.

APPENDICES

51

Appendix 4. Protocol for SEM sample preparation

The seeded T100, T200 and T300 membranes were removed from culture chambers

and rinsed 4 times with PBS. Fixation and dehydration were performed using solvents below.

The samples were then dried overnight at room temperature followed by sputter-coating prior

to SEM imaging.

Table 7. Steps for dehydration of seeded T100, T200 and T300 scaffolds for SEM imaging.

Solution Time 0.1M Cacodylate buffer rinse 10 min 0.1M Cacodylate buffer rinse 10 min 0.1M Cacodylate buffer rinse 10 min 1% OsO4 in 0.1M Cacodylate

buffer 1 hour

UHQ water rinse 10 min UHQ water rinse 10 min

50% ethanol 10 min 50% ethanol 10 min 70% ethanol 10 min 70% ethanol 10 min 90% ethanol 10 min 90% ethanol 10 min 100% ethanol 15 min 100% ethanol 15 min

Hexamethyldisilazane 30 min Hexamethyldisilazane 30 min Hexamethyldisilazane Overnight, air dry in fume hood

APPENDICES

52

Appendix 5. Protocol for TRITC-Phalloidin Staining of RCE cells

Actin filaments were stained following a standard protocol outlined below. Stained

samples were light sensitive thus the protocol was conducted with this precaution in mind by

covering the samples with aluminium foil during incubation periods and storage.

Table 8. Steps for Staining Seeded Membranes (T100, T200 and T300) with FITC-phalloidin for CLSM imaging

Solution Time PBS (with additives Ca2+ and Mg2+) 2 min PBS (with additives Ca2+ and Mg2+) 2 min PBS (with additives Ca2+ and Mg2+) 2 min

4% PFA solution 30 min PBS 2 min

0.2% Triton X-100/PBS solution 5 min, on shaker PBS 2 min PBS 2 min PBS 2 min

FITC-tagged phalloidin (actin filament, ratio 1:50) in 1% BSA/PBS

45 min, on shaker

PBS 2 min PBS 2 min

PBS, covered in foil Stored at 4 °C

APPENDICES

53

Appendix 6. Protocol for TRITC-Phalloidin Staining of RCE cells

Phase contrast images of T100 and T200 membranes showed spindle-shaped,

flattened cell morphologies attributable to senescence. Potential reasons being the lack of 3T3

feeder cells, resulting in nutrient deficient cells, or the uneven distribution of cells from the top-

seeding approach.

Figure 27 Growth of rabbit corneal epithelial (RCE) cells in culture. The phase-contrast images show the morphology of cells growing on (A, C, E, G, I) T100, and (B, D, F, H, J) T200 membranes, on days 1 (A, B), 3 (C, D), 7 (E, F), 11 (G, H) and 14 (I, J). The scale bar is the same for all panels and equals to 200 μm.

APPENDICES

54

Appendix 7. Permissions for Copyright Materials

All materials used in this thesis not generated by the author were reprinted with

permission of the copyright owners. These permissions were obtained under Creative

Commons Attributions Licenses, or via the RightsLink Copyright Clearance Centre. The

material was used either in its original form or with minor alterations, and the source was

referenced appropriately in Figure captions. The permissions for the reprinted material are

included below.

Copyright permissions for Figure 5

APPENDICES

55


ANNEXES

56

Annexes

The following two documents are two research reports of the 10-month research internship

at the University of Wuerzburg in Germany. The reports are separated into the following

annexes, entitled

Annex 1: Embedding Melt Electrowritten Fibres in Liquid Glass

and

Annex 2: Screening for Printable PVDF-Based Polymers for Melt Electrowriting

Annex 1

Embedding Melt Electrowritten Fibres in Liquid Glass

Deanna Nicdao BSc (Chemistry)

Supervisor:

Prof. Paul Dalton

Submitted in partial fulfilment of the requirements for the degree of

Master of Applied Science (Research) Biofabrication

Department for Functional Materials in Medicine and Dentistry

Julius-Maximilians-Universität Würzburg

August 2018

I

Keywords Additive manufacturing, biofabrication, melt electrowriting, poly(ε-caprolactone), liquid glass,

fused silica glass, microfluidics, microchannels

II

Abstract Recent systematic engineering advancements in melt electrowriting (MEW) include

large volume prints with micrometre resolution and the automatization of jet stabilisation for

higher throughput. A need remains for an established, scalable model with high dimensionality

for microfluidic reactors. With the continual advancements of MEW, the sacrificial templating

of MEW constructs in an appropriate matrix could achieve microfluidic models with such

scalability and high dimensionality. This work (Annex 1 and 2) investigates a novel

manufacture of microchannels for the scale-up of microfluidic reactors with a room

temperature moulding process using fused silica glass.

In this report, a suitable printing material was investigated to be used as a sacrificial

template for the moulding process. The first material chosen was poly(ε-caprolactone) (PCL)

as it is considered the gold standard for MEW. PCL fibres were embedded in a monomeric

mixture of fused silica glass (Liquid Glass) and cured under UV. Examination of the

microchannel surface under scanning electron microscopy and energy dispersive X-ray

spectroscopy found that the physical domains between silica glass and fibre were ambiguous

after curing, specifically presenting polymer leaching into the silica matrix. Optimisations with

reducing curing times and taking precaution with casting fibres were found to improve the

microchannel quality only slightly. It was hypothesized that PCL and the silica monomeric

mixture were incompatible. To confirm this, dissolution tests were conducted on PCL versus

poly(vinylidene fluoride) (PVDF), a more chemically inert material, in the slurry mixture over 1

min, 5 h an 24 h. It was confirmed that PCL was miscible in the Liquid Glass slurry and that

PVDF remained intact over 24 h. PVDF was thus chosen as a sacrificial template to fabricate

microchannels in following experiments (Annex 2). Here, a six-inlet to single-outlet microfluidic

design is introduced for future PVDF prints. The integration of MEW and moulding processes

enables and supports a higher throughput approach that could provide microfluidic models

with high dimensionality in a single-step process.

III

Table of Contents Keywords .............................................................................................................................. I

Abstract ............................................................................................................................... II

List of Abbreviations .......................................................................................................... V

1 Microfluidics: Literature Review ................................................................................ 1

1.1 From Electrons to Fluids – A Brief History of Microfluidics ......................................... 1

1.1.1 Inkjet Printing ........................................................................................................ 1

1.1.2 Integrated Circuit Technology ............................................................................... 2

1.1.3 The Miniaturisation of Analytical Systems ............................................................. 2

1.1.4 Microfluidic Development: Complex Systems ....................................................... 3

1.1.5 Microfluidic Development: Application-Oriented Systems ..................................... 3

1.2 Fundamental Laws of Microfluidics ............................................................................ 4

1.2.1 Principles of Manipulation: Traditional Microfluidics .............................................. 4

1.2.2 Principles of Manipulation: Inertial Microfluidics .................................................... 5

1.3 Microfluidics: The Challenge to 3D .......................................................................... 10

1.4 Improving Quality on the Macro-Scale ..................................................................... 12

1.5 Improving Quality on the Micro-Scale ...................................................................... 13

1.6 From Glass to Glassomer: A Brief History ............................................................... 15

2 Results and Discussion ............................................................................................ 17

2.1 Preliminary Casting of Various MEW Constructs ..................................................... 17

2.2 Reproducing the LiqGlass Protocol in Single Fibre Constructs ................................ 17

2.3 Varying Ultraviolet Curing Times of LiqGlass ........................................................... 18

2.4 Energy-Dispersive X-ray Analysis of Embedded mPCL Constructs ......................... 19

2.5 Introducing PVDF .................................................................................................... 20

2.6 Differential Scanning Calorimetry Analysis of PVDF ................................................ 20

2.7 Dissolution Tests of PVDF and mPCL ..................................................................... 21

2.8 Microfluidic Design Proposal ................................................................................... 22

IV

3 Conclusions and Future Directions ......................................................................... 25

4 Materials and Methods .............................................................................................. 26

4.1 Fabrication of Constructs ......................................................................................... 26

4.2 Imaging of Constructs .............................................................................................. 26

4.3 Thermal Properties .................................................................................................. 27

References ........................................................................................................................ 28

Appendix ........................................................................................................................... 34

V

List of Abbreviations 2D 2-Dimensional

2PP Two-Photon-Polymerisation

3D 3-Dimensional

AM Additive Manufacturing

BHF Buffered Hydrofluoric Acid

CEA Contract-Expansion Array

CLSM Confocal Laser Scanning Microscopy

CMOS Complementary Metal Oxide Semiconductor

CNC Computerised Numerical Control

DMF Digital Microfluidics

EDX Energy-Dispersive X-Ray

EWOD Electrowetting-on-dielectric

FDM Fused Deposition Modelling

FIB Focused Ion Beam

HEMA Hydroxyethyl Methacrylate

HF Hydrofluoric Acid

i3DP Inkjet 3D Printing

IC Integrated Circuitry

LOC Lab-on-chip

MEMS Microelectromechanical System

MEW Melt Electrowriting

mPCL Medical Grade Poly(ɛ-caprolactone)

NOA Norland Optical Adhesive

PEGDA Poly(ethylene glycol) di-acrylate

POC Point-of-Care diagnostics

PDMS Poly(dimethylsiloxane)

PU Polyurethane

PVA Poly(vinyl alcohol)

RM Regenerative Medicine

SEM Scanning Electron Microscopy

SLA Stereolithography Apparatus

TCP Tissue Culture Plate

TG Transglutaminase

μTAS Miniaturised Total Analysis System

1. Microfluidics: Literature Review

1

1 Microfluidics: Literature Review 1.1 From Electrons to Fluids – A Brief History of Microfluidics

Microfluidics, as suggested, is a subdiscipline of fluid mechanics within the micro- and

nanometre scale. Thus, feature sizes of this scale are characteristically used to classify a

system as a microfluidic system1. Recent advances in the additive manufacturing (AM) field

has birthed the ability to precisely manufacture feature sizes within a submicron resolution.

This development of microfluidics closely follows the history of classical microstructure

technology. The difference between the two is that the latter was revolutionized by the

scientific and commercial success of microelectronics1. The term microelectromechanical

system (MEMS) was justified in 1982 by Petersen et al.2 and later by Chen et al.3 in 1984,

describing systems which combined electrical and mechanical actuators or interrogated

components. Initially, a chip measured the acceleration of a mass moving alongside electronic

circuitry. The electronic components could be produced in complementary metal oxide

semiconductor (CMOS) technology, allowing for the creation of bulk mechanical structures

such as the accelerable masses required for these accelerometers. Sacrificial layers etched

during manufacturing allowed movement of the mass. The interrogation of this movement via

electronic circuitry resulted in the creation of the first MEMS systems. Today, the widely

researched and optimised material, silicon, remains the most commonly used material for such

systems. The relativeness of the MEMS field to microfluidics is seen through the

manufacturing techniques, such as wet or dry etching and lithography, which were almost

exclusively established for MEMS processing. In addition to the early MEMS accelerometers,

three other contributions are also worth noting for the development and diversity of

microfluidics: inkjet printing, integrated circuit technology and the miniaturisation of analytical

applications1.

1.1.1 Inkjet Printing

A key application for microfluidics is the precise deposition of miniscule amounts of

liquid. The first contributions were presented by Bassous et al.4 in 1977 and has since been a

commercial success featuring a printing head comprised mostly of silicon which acts as a

microfluidic spotting device1. The printer head contained several high-precision microscopic

nozzles, generally 10 μm in diameter, which ejected ink onto paper. Further development

focussed on the miniaturisation of individual fluidic components including pumps, mixers and

valves. An example reported in 1995 is a silicon-based bidirectional micropump illustrated in

Figure 1. This micropump was actuated by an electrostatic diaphragm with two passive check

valves fabricated by bulk micromachining technology. The pump rate was 850 μL min-1 at


2

maximum, and a back pressure of 31 000 Pa. However, due to the silicon being relatively

expensive and not being optically transparent, leading to future optical detection difficulties,

glass and polymer materials were introduced in the early 2000s as microfluidics was

established as its own research field5.

Figure 1 Silicon-based electrostatically driven diaphragm pump. Reproduced with permission6 (see Appendix).

1.1.2 Integrated Circuit Technology

The use of fluids for heat transport has been a logical solution for applications ranging

from heat pipes to high-performance graphic cards and central processing unit chips. The

prevention of overheating in integrated circuit systems remains a setback today. In 1981,

Tuckerman and Pease7 described a heat sink connected to a microfluidic channel system for

large-scale integrated circuit (IC) devices. Injecting a coolant into this channel system allowed

the heat to dissipate into the heat sink. Today, applications for microfluidics in IC technology

is limited however a microfluidic heat transfer is a viable option for high performance circuits1.

1.1.3 The Miniaturisation of Analytical Systems

The development of microfluidics as a scientific discipline has been driven by its

potential in the miniaturisation of analytic systems. The wide set of applications and the

advantages of submicron channel systems over classical macroscopic fluid handling has

attracted multidisciplinary fields from chemistry, engineering and biomedical sciences. The

first notable analytical application derived from Terry et al. in 19798. The system integrated

monolithic microvalves and an anemometric heat conductivity detector between two bonded

silicon wafers. The device was able to distinguish peaks in a multicomponent mixture of


3

nitrogen gas, n-pentane and n-hexane. This paper went largely unnoticed until the beginning

of the 1990s, where a revival of the field was sparked by the concept of miniaturised total

analysis systems (μTAS) put forward by Manz and colleagues9.

1.1.4 Microfluidic Development: Complex Systems

The emergence of polymer-based microfluidic systems began from the 2000s. One of

the most common, if not the most used material, for fabricating microfluidic systems material

is PDMS (poly(dimethyl siloxane)). Song and co-workers outlined several key pioneer

studies5. One demonstrated the integration of on-off valves, switching valves, and pumps –

entirely composed of an elastomer – fabricated through a multilayer soft lithographic

technique10. Another consisted of latching microfluidic valve structures, controlled by an on-

chip pneumatic demultiplexer11. This system contained 16 latching valves stemming from a

single input connection which routed pressure and vacuum pulses controlled by a four-bit

demultiplexer. The inclusion of standard logic gates to complex pneumatic microprocessors

led to the prediction of precise handling of fluids. Alongside the development of more complex

continuous flow systems, the manipulation of discrete microdroplets emerged, known as

digital microfluidics12-14. The electrowetting-on-dielectric principle (EWOD), where an electrical

potential is applied to manipulate fluid shape and flow, formed the basis of this technique. This

study demonstrated the dispense, transport, division and additional of droplets by applying an

electrical potential to sequential electrodes15. Digital microfluidics (DFM) thus proposes

several advantages to perform reactions over traditional counterparts including precise

dispensed volumes, encapsulation of biomolecules for monitoring, elimination of dead volume

and enhanced mixing ratios5. With these systems in place, a solid foundation was created to

prompt further investigations of the applications for various biomedical and chemical analyses

in microfluidics.

1.1.5 Microfluidic Development: Application-Oriented Systems

A foundational ideology behind microfluidics is the ability to go from an input of reagents

to an analytical output in a singular, miniaturised, and potentially automatized process. Hence,

the field is often called ‘lab-on-chip’ (LOC), biochip, or micro-total analysis system (μTAS).

The development from conventional macroscopic equipment processing in a wet lab, to a

single microscopic device has many advantages branching from the simplistic fluid dynamics

already mentioned. On top of this, the cost-effectiveness of such devices is economically, and

timely, revolutionary for several areas. Four main themes for microfluidic applications were

identified by Whitesides16: (1) national security; (2) pharmaceutical and clinical analysis; (3)

genomics; and (4) point-of-use microanalysis. Based on these applications, it is evident that


4

the microfluidic discipline requires the expertise from fields of physics, engineering, material

sciences, biochemistry, and even information technology. A main advantage in researching

this subdiscipline is that theoretical models for experimental data is relatively easy to derive.

It must be kept in mind that microfluidics still undergoes fluid mechanics and thus an

understanding on modelling fluids on the macro-scale remains relevant here1.

1.2 Fundamental Laws of Microfluidics 1.2.1 Principles of Manipulation: Traditional Microfluidics

Within microfluidic environments, small instantaneous volumes result in the diffusion-

dominated transport of mass. In other words, mixture of fluids occurs in a more controlled and

predictable manner with developed laminar flow regimes. Moreover, the large surface-area-

to-volume ratio results with homogenous thermal profiles between the device and the

contained fluid. As summarised by Chiu and colleagues17, the practical consequences of these

parameters are:

(a) Low Reynold’s numbers, where Reynolds number describes the ratio between inertial

and viscous forces in a flow and is a dimensionless number defined as:

(1)

An example for microfluidics: microchannel dimensions, , are small (~ 100 ); ~

1000 kg/m3 for water density; ~ 0.001 Pa s for viscosity; and flow velocity, ~ 0.01

m/s leads to a Reynolds number approaching 1. Meaning the flow is laminar and

predictable coupled with high velocity gradients and dominant interfacial effects which

allow for the manipulation of fluid streams in a controlled manner. This includes inertial

effects on particles without fluid turbulence and the generation of monodisperse

emulsions.

(b) Compartmentalised picolitre fluid volumes that contain single entities and concentrate

reaction products

(c) Uniform reaction conditions to control reactions in bulk or on surfaces

Based on this definition, Chiu et al. has classified two subclasses of microfluidics: single-

phase or multi-phase (depending on whether the system operates solely on a single fluid or

two or more fluidic phases in contact, for example, liquid-gas)17.

When analysing a system, a temporal component is often used, meaning ambiguity of

the defined baseline measurement risks uncertainty in the result. In microfluidics however,

temporal ambiguity is minimised due to the rapidity and uniformity of mixing events,

manifesting a platform for increased precision18 and decreased dispersity in product


5

characteristics19,20. Likewise, chemical and biological systems are mostly temperature

sensitive and variations can lead to disparate outcomes. This is avoided in planar chip

microfluidic formats, where localised hot and cold sports are uncommon17. Thus, microfluidic

tools have great potential for enhancing the detection of the proverbial needle in the haystack

by reducing these disparities creating “background noise” to elucidate signals for these

otherwise rare events.

The controllable laminar nature of flow provided by these systems ensures that particle

and fluid motion is well defined, allowing engineering for purpose. The need for high-

throughput, efficient microfluidic devices has been a major challenge since the beginning of

the field21. Rather than limiting inertia of the fluid in the system using a variety of passive (e.g.

hydrophoresis22, deterministic lateral displacement23, gravitational methods24) and active (e.g.

dielectrophoresis25-27, acoustophoresis28, magnetophoresis29) techniques, a reliance on

inertial effects may produce significantly higher throughput for minimal effort30.

1.2.2 Principles of Manipulation: Inertial Microfluidics

Inertial microfluidics has brought conventional macro-scaled methods of inertial fluidics

to be applied to an unconventional sub-micron level. The phenomenon of inertial focusing was

first described by Segré and Silberberg in 196131. For the experiment, a suspension of

randomly distributed millimetre-sized particle was flowed through a macroscopic tube (~1 cm

in diameter) and the particles (~ 1 mm in diameter) were found to migrate laterally and form

an annulus 0.6 times the radius of the tube. This demonstrated that when particles flow

through a tube with a low Reynolds number, that an additional inertial lift force is generated

lateral to the mainstream driving force of the flow32. Since then, three forces that must be

considered in inertial microfluidics was outlined by Martell and co-workers as (i) wall interaction

force, (ii) a shear gradient lift force, and (iii) a secondary-flow drag force. A brief overview of

these forces and significant factors that manipulate these forces will be discussed in this

section.

In conventional microfluidics, the inertial effect of the flow field and particles are

considered negligible whereas inertial microfluidics has coupled inertial focusing with

additional flows that occur perpendicular to the main axial flow direction. Martel and colleagues

(2014) subdivided inertial lift forces into three dominant forces that defined equilibrium

positions in fluid flow (Figure 2)30: (i) a wall interaction force (FWI) that occurs when a particle

approaches a channel wall creating constricted flow between the particle and wall resulting in

pressure build up that imparts a force perpendicular to the wall and decreases with increasing

distance from the wall; (ii) a shear gradient force (FSG) that generated from the parabolic

velocity profile of a particle in a typical microfluidic flow, resulting in the particle experiencing


6

velocities of different magnitude on either side (depicted in Figure 2b) relative to the particle’s

velocity – notably this definition is independent of particle rotation but highly dependent on

Reynolds number and position; and (iii) a relative “drag force” (FD), also referred to as Stokes’

drag, that is imparted on a particle that is the difference between the speed of the particle and

the speed of the moving fluid – this scales linearly with the particle size and the velocity of the

secondary flow, also known as Dean flow which is discussed later.

Figure 2 Schematics of the dominant inertial lift forces: (a) “wall interaction force” (FWI) lift directed away from the wall approaching zero to channel centreline due to the constricted flow between the particle and channel wall; (b) “shear gradient lift force”(FSG) directed down a shear gradient from zero at the channel centreline, the difference in velocity results in a pressure difference that imparts a force onto the particle side with highest relative velocity; and (c) “secondary-flow drag force” (FD) where a particle in a uniform flow with a low Reynolds number experiences Stokes’ drag – the relative force from the difference between the particle velocity and the fluid velocity. Reproduced with permission30 (see Appendix).

Gou et al. (2018) argues several points explaining how inertial microfluidics are

superior to conventional microfluidic devices. The advantages build upon the simplicity of both

operation and channel design – that is, by merely adjusting the flow velocity of the sample

precision can be enhanced. This means that additional features are more easily integrated,

reducing the overall cost of the device. In addition, the manipulation of inertial forces will have

little effect on the activity of biomolecules, increasing its suitability for biomedical applications.

Moreover, throughput is considerably higher compared to other methods, in turn, leading to

faster outputs and shorter experimental time33. With these advantages in mind, it would be ill-

informed to exclude the principles behind inertial microfluidics.

This phenomenon was observed in particles flowing through a straight channel at the

micro-scale which probed further investigations into how different section shapes affect the

lateral migration of particles to the dynamic equilibrium position34. The channel section is an

important factor that determines the final equilibrium position of the particles. A medium

Reynolds-number condition in a circular channels leads to particle migrating to form a Segre-

Silberberg annulus due to the symmetry of the circle (Figure 3a)35. For a square section, the

influence of the edge angle on velocity and pressure distributions must be considered. An

offset of the equilibrium position occurs where the migration of particles is corrected towards

the midpoint of each of the four channel walls (Figure 3b)33. The aspect ratio on the equilibrium

position is also shown (Figure 3c,d). If a square channel has a high/low-aspect-ratio section,

the shear gradient will be significantly less, and more favourable as an equilibrium position,


7

on the long channel wall. Switching the design to a low/high-aspect-ratio section showed no

change as the equilibrium position remained at the midpoint of the long channel wall36.

Figure 3 Schematic demonstrating the inertial focusing effect of particle equilibrium positions in channels with different shaped cross-sections: (a) circular; (b) square; (c) high-aspect-ratio rectangular and (d) low-aspect-ratio rectangular channels. Reproduced with permissions under the Creative Contributions Attributions license33 (see Appendix).

The three-dimensional dynamic process of particles is presently described by a two-

stage mathematical model proposed by Zhou37: (1) under conditions with a moderate

Reynolds number, the migration of the particle to an equilibrium position near the wall under

shear-induced inertial lift force and wall-induced inertial lift force; (2) particle migration

becomes centralised along the wall-length due to the spin-induced Saffman lift force (Table

1). This model has been investigated and demonstrated in other studies for particle focusing

dynamics (such as trapezoidal straight microchannels38 and channels with the cross-section

of an isosceles right triangle39). Straight-channel inertial microfluidics has demonstrated to

provide a simplistic sorting method for the multi-position focusing of particles.

As the inertial effects in a straight channel has been discussed, it is only logical to

discuss the theory in curved channels. In curved channels, a higher velocity flow is observed

in the central area compared to the wall areas. This results in parabolic velocity profile for the

Poiseuille flow of the main flow direction. Simply speaking, if a particle flows down a curved

channel, it will migrate from the centre to the outward line due to inertia, creating an imbalance

in the radial pressure gradient40. With a closed channel, fluid near the outward wall will

recirculate inward to meet mass conservation, due to the centrifugal pressure gradient. Thus,

creating two vortices with opposite circling rotations, lateral to the walls (Figure 4)40, a

phenomenon first proposed by Dean, known as the cross-section secondary flow41,42. In 1983,

Berger et al. calculated a mode for this model, familiar to fluid mechanics and widely used,

known as the Dean number, as expressed below43:


8

(2)

in which H is the hydraulic diameter, and R is the curvature radius of the channel. As would

increase, so would the Dean flow. The shape of the Dean flow is also related to the . If

increases, the centre of the Dean vortex will migrate towards the outer wall, with the number

of the Dean flow changing at higher velocities. The secondary flow in a Dean vortex (Figure

4)40, numerically proposed by Ookawara44 as , is an important parameter

for applied research45.

Figure 4 Dean flow and the two counter-rotating vortices perpendicular to the primary flow direction as illustrated by Di Carlo (2009). The fluid with high velocity near the channel centre will tend to continue outward to conserve mass whilst the relatively stagnant, re-circulates inward. Reprinted with permission under the Royal Society of Chemistry40 (see Appendix).

Several models have branched from this expression. Simulations included predicting

the width of the focusing band for particles of varying size were validated by experimental

data46, as well as exploring the elasto-inertial focusing particle behaviour in spiral microfluidic

channels with increasing flow rate, resulting in a six-stage process model illustrated by Xiang

et al (Figure 5)47.


9

Figure 5 Schematic diagram of the six-stage particle focusing process as proposed by Xiang et al., (2016). Each stage contains a pair of figures, each corresponding to the possible particle equilibrium positions in the horizontal plane (1) and the cross-section (3), and a fluorescence streak image (2) illustrating particle focusing behaviour overlayed with a red spectrum indicating the intensity profile of the corresponding streaks. Reprinted with permission from 47 (see Appendix).

Gou and co-workers outlined two main effects from the Dean flow for particle migration:

(1) A stirring effect is generated by the Dean flow, accelerating the lateral displacement of

particles, speeding up the migration of particles to the equilibrium position; (2) the distribution

of the particles’ equilibrium positions within the channel cross-section are influenced by the

Dean flow33. The presence of the Dean flow enhances particle mixing, and in turn focusing,

which allows for the channel length to be drastically shortened to reach the same inertial

effects observed in conventional straight-channel systems.

From the above discussion, the most important factor influencing the inertial effect is

the channel cross-section, however the final equilibrium position of particles can also be

affected by the velocity of the flow, deformation, and particle densities for example. In addition

to these factors, there are nonlinear forces including lateral force and axial drag in various

forms which can further be used to manipulate particles (Table 1)33. In contrast to the extensive

knowledge base of the characteristics listed above, the exact influence of forces such as Van-

der-Waals forces, electrostatic forces and correction fluid resistance on particles have not

been reported33. It may be that the effective distance of such forces is submicron and is

considered negligible but with mixtures of particles, with increasing complexity of biomolecules

all undergoing inertial focusing, these forces may be of significance.


10

Table 1 Summary of Lift and Drag Force Analyses. Reproduced with permissions from the Creative Contributions Attributions license33 (see Appendix).

48

49

50

51

52

1.3 Microfluidics: The Challenge to 3D The history and future direction of microfluidics has been reviewed expansively over the

past decade17,21,53,54, however the bottle-neck is still upscaling from lab bench to industry. The

commercialisation of microfabrication has been achieved through the low infrastructure costs

and manufacturing ease through the conversion of computer-assisted design (CAD) into

products scaling from a few microns to several centimetres in a single process54. The AM

market has continued to grow from USD 8.42 billion in 2017 and is expected to grow to USD

35.10 billion globally by 202355. Likewise, the microfluidics market is predicted to increase

from USD 3.6-5.7 billion in 2018 to USD 27.91 billion in 202356. The catalyst behind the

marketable success of microfluidics can be attributed to the fabrication of microfluidic devices

through soft lithography and PDMS, a technique independently developed from research

teams, Whitesides57 and Manz58 in 2001. The attraction behind this technique was mainly the

low technology and investment threshold required for microscale resolution. Even without soft

lithography, thermal aging of PDMS as an intermediate moulding material, also known as


11

double casting prototyping, is commonly used for creating microchannels (~100 μm) at

relatively low cost without the use of chemical additives59 (Figure 6). Due to the ease of

testability for new prototype devices with a production time of two days compared to over a

month for other materials requiring specialists (e.g. silicon technology), the promise of

revolutionary miniaturised devices seemed within reach. Fast forward to nearly two decades

after – PDMS is still widely used and its material properties has created a major limitation

preventing this technology from evolving from lab bench to market.

Figure 6 Scheme of PDMS double casting prototyping process, including the key thermal aging of negative PDMS mould for the fabrication of the positive PDMS product59. Reproduced with permission under the Creative Commons Attribution License (see Appendix).

The upscale of fabrication methods remain one of the major limitations in microfluidics.

The quality of microstructures relies on the material choice for the fabrication techniques, thus

the lack of standardisation in material selection and for upscaled process development must

be addressed. This is however, not a trivial challenge to change, as indicated by the prominent

use of PDMS22,60-62. To add to this dilemma, no single material is suitable for the needs of

each application. Materials such as glass63 and silicon64 have previously proven to translate

well in etching processes whilst thermoplastics are the most compatible with hot embossing

and injection moulding techniques65. These micromachining techniques have become a

popular subcategory for polymers as multi-layer structures are fabricated at low-cost, however

chip bonding is a major challenge66. Multi-layer and moulded constructs contain open

channels which require bonding covers to close channels67 and channels interconnects that

must align to reduce leakage or disruptions in laminar flow68. It is also important that the

sealing processes bonding these parts do not interfere with the surface chemistry of the

channels69. Even if these issues are resolved, the final structures are horizontally stacked 2D

layers containing low aspect ratios70. To allow for the personalisation of medical devices, the

fabrication processes must overcome quasi-2.5D structures that vary in width but not depth.

Additive manufacturing has demonstrated process flexibility, allowing for intrinsic and

complex 3D design, and has become an attractive solution to creating microchannels that vary


12

in three dimensions. This inclusion of AM for fabricating microfluidic devices can greatly

improve the durability and efficiency of production. In addition to increasing production

efficiency, several device components can be simultaneously manufactured owing to the

flexibility of material choice for the wide range of AM. One study by Chan and colleagues

developed an AM microfluidic chip containing components which were operated manually for

colorimetric assays, eliminating the need of bulky instruments71. In addition, CAD design and

AM printing of microfluidic point-of-care (POC) devices have been integrated with smartphone-

based diagnosis, introduced as iPOC3D diagnostics, for applications such as the colorimetric

detection of haemoglobin72. For such devices it is necessary to reduce user intervention in

sample processing steps to decrease error and eventually become user-friendly for future

POC device users73. Thus, automatizing device fabrication through AM is a required step to

reduce error and create high quality products.

One could argue that there are enough methods of fabrication and range of

components to begin applying microfluidic systems in a way that addresses resolution

problems rather than simply demonstrate the principles. Extensive reviews elaborating on

fabrication techniques and material choices already exist70,74-76. Rather than reiterate these

points, the following sections will outline relevant considerations to improve the quality of

microfluidic devices in three dimensions.

1.4 Improving Quality on the Macro-Scale When meeting industrial standards, material choice is a major consideration that must

not be underestimated. This includes the interactions between composite materials in a

microfluidic device and between the device and its samples. In extension, not all commodity

plastics are compatible with all assays, and materials presently used for such applications

have manufacturers that are not compelled to disclose the amount and identity of each additive

in their raw materials69. Even materials which are extensively used in lab have been found to

interfere with enzymatic reactions77. If such effects are observable on 96-well plates, then it is

only expected for these effects to be pronounced in plastic reaction chambers with larger

surface-to-volume ratios. Additional considerations include preserving any desired surface

modifications, dimensional stability of channels under high pressure – for valving,

interconnected alignments68, and precise fluid dispensing – and gas permeability – a desirable

factor for live cell culture but detrimental for product shelf life69.

The progress in 3D printing within the microfluidic field has seen success mainly in

inkjet 3D printing (i3DP), fused deposition modelling (FDM), stereolithography (SLA) and two

photon polymerisation (2PP)76. These technologies however have produced quasi-2.5D

structures, with the overall dimensional range for PDMS microfluidics being 50 to 2000 μm for


13

most 2D and 3D channels78. Combining sacrificial moulding methods with AM can provide

freedom from stringent dimension control. An interesting sacrificial material recently is sugar,

which was printed using a desktop extrusion based-printer with high efficiency and low-cost79.

By using a nozzle of 0.3 mm and varying collector speed, microchannel widths of 40 μm was

achieved with the overall microfluidic size reaching 25 × 25 × 2.2 mm. Toxic solvents were

also avoided as the sacrificial sugar structure was removed by submerging the device in

boiling water. Similarly, digitally controlled liquid dispensing or filament extrusion has been

demonstrated with aqueous and organic inks80,81, carbohydrate-based substrates82,83, eutectic

metal84, acrylonitrile butadienestyrene85, and hydrogels (e.g. gelatin and agarose)83,86 as

sacrificial moulds for microfluidic channels. The controlled deposition of sacrificial templates

has produced microchannels within the required range for microfluidic applications.

On the other hand, the choice of the substrate material is of equal importance to the

sacrificial template material to ensure high quality microfluidic features. One study tested the

compatibility of poly(vinyl alcohol) (PVA) in six different polymer matrixes: rigid and flexible

polyurethane (PU), epoxy resin, PDMS, transglutaminase (TG) enzymatically crosslinked

gelatin, Norland Optical Adhesive (NOA) 81 and PEGDA (poly(ethylene glycol) di-acrylate)87.

PVA was selected as the sacrificial template to allow the removal to be completed in an

aqueous environment87. Nozzle diameter and layer heights were varied. The smallest features

were reproducibly obtained with a nozzle diameter and layer height of 200 μm and 100 μm

respectively87. As for the matrixes, PU and epoxy resins have been demonstrated for

microfluidic systems in chemical88, biological89 and biomedical90,91 applications. In addition,

the UV-curable thiolene-based matrix, NOA 81, has shown resistance to the exposure of

organic solvents with an optical clarity similar to PDMS but without the permeability to air or

moisture92 and higher stability after surface modifications93. Matrix choice also depends on

application requirements such as hydrodynamics that allow fluid perfusion to mimic

vasculature in tissue constructs for bio-inspired applications87. Materials including PEGDA and

TG gelatin have demonstrated biocompatibility94-96. As it stands, the desired resolution of

microchannels in several cost-effective sacrificial materials is obtainable by patterning with

AM. This enables the marketability of ‘3D-printed 3D microfluidics’, but the channel profile

features, specifically the cross-section of the microchannel must be considered.

1.5 Improving Quality on the Micro-Scale The successful fabrication of a microchannel was classified by Goh and Hashimoto

(2018) in three terms: (1) full dissolution of the sacrificial mould in the matrix, revealing the

microchannel; (2) no detectable leaching in the matrix; and (3) dimensional stability and no

disintegration of the microchannel87. As discussed in the fundamental laws of microfluidics,


14

the channel cross-section is the greatest factor for influencing the inertial focusing ability of

the device34,37,40,47,90,91. Research has suggested a curved microchannel provides uniform and

predictable cell adhesion97 where as a sharp-turn creates a near-zero velocity at the turn

region – potentially beneficial for particle collection purposes98,99 however risks cell damage

and biochemical heterogeneity, such as cell signalling pathways induced by shear stress,

among cell populations100.

The sacrificial moulding of fibres provides desirable circular microchannel profiles. To

overcome quasi-2.5D structures, fibre deposition must be automated and flexible in design.

The allure for AM fibres as sacrificial templates is amplified through the fabrication of

microchannels with circular profiles from fibres that can vary in length and diameter. A recent

publication has demonstrated the ability for dimension-controlled fibre deposition of medical

grade poly(ε-caprolactone) (mPCL) using melt-electrowriting (MEW) (Figure 7)101. By varying

collector speed and polymer mass flow through pressure, fibre diameters within the range of

5 to 30 μm were precisely stacked. Furthermore, highly ordered large volume scaffold

architectures have also been fabricated using MEW102. This technique has bypassed the use

of solvents, whilst allowing for the capabilities of arbitrary design within a resolution suitable

for microfluidics.

Figure 7 Dimension-controlled fibre deposition of medical grade poly(ε-caprolactone) by varying mass flow of the polymer melt and collector speed101: (A) The jet is stabilised using an off-print sample as shown along with the scaffold and MEW head; (B) Fibre diameters were measured with variations in mass flow (air pressure) for the off-print sample; (C) Six photographed fibres (black lines) overlayed with the programmed collector path (red dashed line with blue arrows indicating direction of motion); (D) SEM image of melt electrowritten fibres, increasing from 5 to 30 μm in 5μm increments; (E) SEM images of three stacked fibres (falsely coloured) with varying diameters; and (F) stacking triplet pairs of fibres of varied sizes. Reproduced under the Creative Commons License101 (see Appendix).


15

1.6 From Glass to Glassomer: A Brief History The chosen material to fabricate microchannels must meet the high standards

previously mentioned to function as a microfluidic device. Glass is known for its smooth

surface roughness, chemical and mechanical stability and optical transparency63,103-107. The

material has evolved over centuries and currently, the most common commercially are:

borosilicates, soda-lime silicates and phosphates108. Silicates have persisted, owing to its

desirable set of properties: optical transparency, electrical insulation, chemically inert, thermal

resistance, and mechanical robustness103,104,106. To continually evolve the use of high-purity

glasses, the controlled formation of glass structures on the micro-scale must be improved.

Current techniques require high-temperature melting and casting processes which involve

hazardous chemicals103,105. Thus silica glass is notoriously difficult to structure due to its

hardness and high chemical and thermal resistance109.

In the semiconductor industry, the micromachining techniques used to pattern glass

are mainly wet and dry etching, photolithography and electron-beam lithography107. Additional

commercially available processes include the use of hydrofluoric acid (HF), buffered HF (BHF)

or reactive ion etching to etch microstructures into glass110-112. The process typically includes

a metal mask layer on a glass substrate, which the metal is then patterned using lithographic

methods. Reactive ion etching, or a HF-based solvent interacts with the exposed glass

substrate to produce microstructures. Lastly, the mask is removed107,110-113. These techniques

are well-established however comes with several disadvantages including the requirement of

clean-room facilities and equipment and the toxicity of HF which limits upscaling potential112.

These limitations, in addition to the challenges addressed in Section 1.3 must be addressed

to drive the microfluidic field forward.

It is evident that an extensive amount of literature exists proposing the potential of

glass microfluidic devices and the AM of microfluidics, thus converging these processes will

not only expedite device prototyping but also ease accessibility and empower the creativity

behind scientists as PDMS and soft lithography once did17. Printing glass has been

demonstrated through fused deposition modelling (FDM) of melted soda lime glass at 1000

ºC and a glass filament, through a manual wire feeding approach using a laser beam for

melting114,115. In both cases, however, structures resulted with high surface roughness.

Recently, Kotz and co-workers were able to demonstrate the 3D printing of silica glass with a

surface roughness of a few nanometers109. The process involves a photocurable silica

nanocomposite comprised of silica nanoparticles (~40 nm in diameter) and the monomer,

hydroxyethylmethacrylate (HEMA) that can be 3D printed and cured into a high-quality fused

silica glass with high resolution microstructures (Figure 8)109. The substrate is optically

transparent – within commercial silica glass standards – and non-porous109. Furthermore, the


16

intermediate curing step allows for the modification of the substrate prior to sintering, for

example to tailor optical properties by doping the substrate with alcoholic metal salt solutions

to create coloured glasses for optical filtering purposes117. This revolutionary nanocomposite

has allowed for the translation of glass, an extensively processed material, into the AM field

providing new micromachining opportunities especially attractive for microfluidics.

Figure 8 AM potential of fused silica glass: (a) ultraviolet-curable monomer and amorphous silica nanoparticles is combined to form a composite used in a stereolithography system. The polymerised composite undergoes a thermal debinding and sintering step (scale bar: 7 mm); (b, c) Printed and sintered glass structure examples – Karlsruhe Institute of Technology logo and pretzel respectively (scale bars: 5 mm); (d) high thermal resistance demonstration of printed fused silica glass against a flame of approx. 800 ºC (scale bar: 1 cm). Reproduced with permission from 109 (see Appendix).

The recent advances in the micromachining of LiqGlass and increased resolution of

MEW has led to these processes being chosen to fabricate microfluidic devices. The sacrificial

template will be fabricated by MEW and embedded in LiqGlass to form microchannel systems

with circular profiles within resolutions suitable for microfluidic applications. With the

convergence of these processes, a potential high-throughput, automated protocol is created

to pave the way towards true 3D-microfluidics.

2. Results and Discussion

17

2 Results and Discussion 2.1 Preliminary Casting of Various MEW Constructs Firstly, to determine the viability of the LiqGlass protocol, the compatibility of mPCL

constructs manufactured by MEW in LiqGlass, a variety of constructs were embedded and

sintered in LiqGlass by protocol at Karlsruhe Institute of Technology (KIT). The samples

included a high-volume scaffold ( mm), a tubular scaffold ( mm) and a

single fibre construct printed directly onto a glass slide. The tubular scaffold was viable (Figure

9A-C), however the cured glass for the large mesh structure cracked into fragments.

Moreover, single fibre constructs were displaced during the embedding process (Figure 9D).

With further optimisation, it is hypothesized that mPCL can be embedded in LiqGlass to form

microchannels in a transparent glass structure, with minimal air bubbles or contaminants and

no fractures.

Figure 9 Light microscopy images of a tubular (A-C) and single fibre (D) MEW structure embedded and sintered in LiqGlass. The blue dye in the tubular template illustrates the hollow microstructures (C). The displacement of the single fibre construct is displayed (D). The glass appears transparent except for air bubbles and/or particles congregates. Notably there are no apparent fractures.

2.2 Reproducing the LiqGlass Protocol in Single Fibre Constructs The curing process was replicated in our lab to optimise the LiqGlass protocol116 for

single fibre constructs. With careful, drop-wise coating of the fibres, fibre deposition appeared

to remain intact prior to curing given fibres were still visible through the uncured

nanocomposite. After curing, the fibres were no longer visible. To determine the presence of

the fibres, the casted surface was removed from the glass slide and imaged using scanning

electron microscope (SEM) to analyse the embedded surface. The surface morphology of the

substrate displayed two scenarios: (a) mPCL fibres were no longer present, leaving behind

hollow microchannels or (b) microchannels appeared to contain material (Figure 10). In both

scenarios, the channel undulated in some areas – indicating the displacement of fibres (Figure


18

10A,B), and a rupture pattern of air bubbles or fragments of material also appeared parallel to

the exterior of the microchannel walls (Figure 10C).

Figure 10 SEM images of embedded single fibre mPCL constructs in LiqGlass. Two scenarios were observed: (A, B) hollow microchannel with high aspect-ratio (scale bars: 20 μm and 200 μm respectively) or (C) microchannel containing the fibre material (scale bar: 40 μm). In both scenarios, ruptures aligning the exterior of the microchannel walls are found.

2.3 Varying Ultraviolet Curing Times of LiqGlass Non-optimal curing times were suspected to have led to the rupturing behaviour. Thus,

curing times were varied to investigate whether it influenced the microchannel quality.

Substrates appeared to be completely cured after being exposed to ultraviolet (UV) lamp (λ =

249.7 nm) for 99 s. To ensure complete curing, the sample was flipped, and the underside

was cured for an additional 99 s. The substrate appeared brittle and opaque, indicating that

the monomer underwent a high degree of polymerisation. Given this protocol intends to be

used for a two-step sacrificial moulding technique to form microchannels, the optimal partial

curing time was examined. An optimal partial curing time was investigated for times under 99

s per side. Here, the optimal time for a partially cured substrate was identified as the minimal

exposure time to reach two conditions: (a) the substrate is transparent and dry, and (b)

sacrificial fibres are visible and not displaced. The reproducible minimal time to cure was found

to be 5 s (n = 3). The occurrence of ruptures appeared to decrease with increased UV

exposure time (Figure 11). Notably, the samples were left to cool and were imaged four weeks

after curing.


19

Figure 11 Stereomicroscope images of mPCL single fibre constructs casted in LiqGlass exposed to UV (λ = 249.7 nm) at varied times. The original single fibre construct without LiqGlass depicts fibres with consistent diameter and turning points, excluding the coils (scale bars: 1000 μm). Rupturing is evident in all partially cured substrates (A-C). Ruptures are not evident in the fully cured substrate however the fibre has been substantially displaced.

2.4 Energy-Dispersive X-ray Analysis of Embedded mPCL Constructs An explanation for the more volatile rupturing behaviour in the minimal partial curing

time (5 s) is the dissolution of mPCL in the 2-hydroxyethyl methacrylate (HEMA) monomer

matrix. Therefore, it is hypothesized that mPCL leached into the uncured nanocomposite. An

energy-dispersive X-ray (EDX) analysis for carbon and silica particles was conducted to

confirm the presence of mPCL in a fully cured substrate (curing time per side > 99 s). Higher

amounts of carbon were present on the inner channel surface compared to the surrounding

chip surface. The material was suspected to have dissolved within the LiqGlass slurry,

resulting in the observed ruptures. To further confirm this, focused ion beam (FIB) etching was

used on an area which appeared to have overlapping of mPCL and liquid glass (Figure 12). It

was found that under the silica surface, a layer with more carbon was present supporting the

suspicion of mPCL leaching. Therefore, mPCL has demonstrated to not be a viable candidate

as a sacrificial template material.


20

Figure 12 SEM (A), EDX (B) and FIB (C) images of the LiqGlass microchannel surface: (A) portrays the inconsistent morphology of the inner channel with troughs and ridges with an undulated channel wall, (B) EDX analysis detects the inner channel to contain a higher trace of carbon (blue) relative to the surrounding chip surface dominated by silicon (red), (C) FIB etching was conducted on the edge where carbon appeared to seep from – additional carbon (blue) was detected beneath the layer of silicon which confirms leaching of mPCL in HEMA monomer matrix (scale bars for A, B, C respectively: 50 μm, 50 μm, 10 μm).

2.5 Introducing PVDF An alternative polymer thought to withstand the HEMA mixture was PVDF due to its’

high chemical inertness and ease of processing118. It is hypothesized that the presence of (-

CF2-) groups (shown in Figure 13A) will result in polymer-polymer interactions being favoured,

meaning more polymer-polymer interactions will occur relative to polymer-solvent interactions,

reducing the dissolution of PVDF in HEMA119.

(A) (B)

Figure 13 Chemical structures of PVDF (A) and HEMA (B).

2.6 Differential Scanning Calorimetry Analysis of PVDF A DSC analysis of PVDF determined a melting temperature of 139.19 ºC applicable

for printing (Figure 14). Printing experiments of PVDF found a temperature of 160 ºC were

required to melt the polymer. This may be due to an uneven distribution of heat of the sample

load (2 mL, packed powder) provided by the two heating rings in the printer head.


21

Nevertheless, a stable jet was achieved at 2.5 bar and 3 kV. A working distance (WD) of 4.4

+ 0.2 mm, a needle size of 22 G produced an average fibre size of 45.584 + 9.376 μm.

Figure 14 Differential scanning calorimetry of PVDF to determine the melting point temperature indicated by the arrow on heating curve (red).

2.7 Dissolution Tests of PVDF and mPCL To confirm compatibility of PVDF with the LiqGlass slurry, dissolution tests of PCL

scaffolds and PVDF fibres were conducted for 1 min, 5 h and 24 h (Figure 15). Despite the

smaller amount of PVDF present, dissolution was less evident for PVDF fibres than the PCL

scaffold. Even after 1 min, mPCL showed significant dissolution – with a substantial loss in

scaffold morphology. As hypothesized, the favouring strong polymer-polymer interactions

prevented the dissolution of PVDF. In contrast, the polymer-polymer interactions in mPCL are

mainly based on the weak Van-der-Waals interactions which can be easily overcome by

polymer-solvent interactions as observed. Thus, PVDF has demonstrated to be a more

compatible sacrificial material choice for LiqGlass casting.


22

Figure 15 Stereomicroscope images of mPCL and PVDF MEW constructs embedded and sintered in LiqGlass after submerged in uncured nanocomposite for various times. Magnified images of PVDF and mPCL after 1 min are included (D, E). A significant loss of morphology is evident in mPCL whilst PVDF fibres appear intact after 24 h (scale bars for A-C and D-E: 1000 μm and 200 μm respectively).

2.8 Microfluidic Design Proposal To assess the viability of this sacrificial moulding protocol to a relevant application, a

microfluidic design has been proposed by the collaborators at KIT. The design is based on a

miniaturised inertial reaction system for two reactants surrounded by four focusing inlets. The

two reactant inlets and four focusing inlets will merge into an outlet reaction channel (Figure

16). The four focusing inlets are hypothesized to prevent the adherence of reactants to the

outlet channel wall. Unlike conventional fabrication techniques that directly mask the CAD

model, namely soft lithography, the design must be translated into a writing path possible for

MEW.


23

Figure 16 Design of the 6-inlet microfluidic system translated towards fibre deposition for additive manufacturing. The conjoining inlets are illustrated in (A) with magnified views at two angles for clarification of how the fibres will stack (B, C). A schematic diagram for the outlet microchannel is depicted (D) where focusing inlets (FI) will surround the reactant microchannels (A and B) and translated into a fibre deposition approach (E).

The proposed design illustrates six inlets converging into a single outlet (Figure 16E).

To accommodate for the specific order of fibre stacking envisioned in the CAD model, a writing

sequence for the inlets is proposed (Figure 17).


24

Figure 17 Printing approach: (a) printing sequence of microchannel fabrication; (b) writing path for microchannels. The writing path ensures the stacking of the 6 inlets are in the desired order. All inlets will lead to a large outlet to collect the product. This will be formed using a spiral code, with overlapping fibres which will later be fused together by applying heat.

It is hypothesized that fabricating microfluidic devices using MEW will allow flexibility

in dimensions to create microchannels with round profiles that can be varied in length and

diameter with a consistent aspect ratio. By conducting these preliminary experiments, it was

shown that PCL is not a viable sacrificial material with LiqGlass but PVDF is a promising

alternative, which can lead to a variety of different microfluidic structures.

3. Conclusions and Future Directions

25

3 Conclusions and Future Directions The potential of MEW fibres for developing microfluidic chips using LiqGlass was

investigated. It was found that casting onto single fibre constructs produced microchannels

with inconsistent channel profiles and rupturing behaviour aligning the channel wall exteriors.

Microchannel quality – referring to the accuracy of transferring fibre deposition to microchannel

template – was suspected to improve with optimisation of the initial UV curing times and by

taking more precaution with the coating of fibres with the LiqGlass slurry. With varied partial

curing times and various methods of coating, the morphology and placement of microchannels

still required improvement.

Another factor potentially affecting microchannel quality were the materials being used.

Based on chemical structure of PCL, the polymer-polymer interactions (mainly Van-der-

Waals) were opposed by the polymer-solvent interactions with the HEMA monomer – justifying

the dissolution of PCL fibres prior to curing. PVDF was introduced as an alternative due to its

chemical inertness (owing to the energetically favoured fluorine-fluorine polymer-polymer

interactions) and flexible processing capabilities. To confirm this, PVDF and PCL MEW

constructs were submerged in the uncured LiqGlass matrix for 1 min, 5 h and 24 h. Dissolution

was observed in PCL scaffolds after only 1 min. PVDF fibres remained intact over 24 h.

Printing experiments achieved a stable jet at 160 ºC. A mean fibre diameter of 45.584 + 9.376

μm were produced.

Overall, these preliminary results revealed the incompatibility of PCL with the HEMA

monomer present in the liquid glass slurry. Further prototyping will therefore use PVDF as a

sacrificial material in liquid glass. Future experiments will include dimension-based printing

using PVDF, specifically printing a range of fibre diameters and fibre layers by varying collector

speed and polymer mass flow (pressure) followed by observational investigations of these

constructs after casting and sintering in LiqGlass. The proposed microfluidic reactor design

will thus implement optimal fibre diameters and layers. Resulting microchannels will then be

tested using fluorescence imaging to determine its viability as a potential microfluidic device.

4. Materials and Methods

26

4 Materials and Methods 4.1 Fabrication of Constructs Single fibre constructs were printed directly onto glass slides using custom-built MEW

printers120; parameters used are outlined in Table 2. A glass syringe was packed with polymer

(mPCL; Corbion Inc, Netherlands PURASORB PC 12, Lot# 1412000249, 03/2015, or PVDF;

Piezotech Kynar RC10.287 kindly donated by Piezotech, Pierre-Benite, France) and fitted with

nozzles (Nordson Deutschland GmbH, Germany). The heating rings were programmed for

mPCL (Bosch Rexroth AG, Germany). Polymer mass flow was controlled by a pneumatic-

based system using N2 gas. A voltage difference was supplied to the nozzle and the collector

plate with a set working distance (WD) between the spinneret tip and collector plate.

Table 2 Printing parameters for PVDF and mPCL single fibre constructs

Parameters PVDF mPCL Polymer load 1.5 + 0.5 mL 1.5 + 0.5 mL

Spinneret tip, needle length, protrusion (from printer head)

30 G, 5.02 + 0.2 mm, 1.0 + 0.2 mm

30 G, 7.28 mm, 1.0 + 0.2 mm

WD (tip to collector) 4.4 + 0.2 mm 1.5 + 0.2 mm Pressure 2.0 bar 2.0 bar Voltage 2.70 + 0.08 kV 3 kV

Temperature 160 86.6

4.2 Imaging of Constructs Prints were imaged at low resolution using a stereomicroscope (Discovery V20, Carl

Zeiss Microscopy GmbH, Germany). Fibre diameters were measured and averaged (n = 3)

using the line tool in the imaging software (Carl Zeiss, ZEN).

Embedding Constructs

The liquid glass monomer mixture was kindly donated by Dr. Bastian Rapp’s

NeptunLab based at the Institute of Microstructure Technology, Karlsruhe Institute of

Technology (KIT)116. In short, 68 % (v/v) HEMA, 7 % (v/v) tetraethyleneglycol diacrylate

(TEGDA), and 25 % (v/v) phenoxyethanol (POE) were mixed prior to dispersion. A dispersion

of 40 % (v/v) Aerosil OX50 was added to the mixture and stirred. In small increments, the

nanopowder was added and homogenized. A 0.5 (wt. %) photoinitiator DMPAP was added

and further homogenized. The slurry was degassed to remove air bubbles.

The casting protocol (Figure 18: 1-3) established by Kotz and co-workers were

reproduced116. Prior to casting, a rectangular moulding frame of silicon was cut and placed

around the print. Single fibre constructs on glass slides were casted by dropwise addition of


27

LiqGlass. Samples were cured using a UV light curing modular flood lamp system (λ = 249.7

nm; Dymax ECE 5000, United States) for various time intervals: 5s, 15s, 25s, 99s.

Imaging of Embedded Constructs

Cured samples were photographed using a stereomicroscope (Discovery V20, Carl

Zeiss Microscopy GmbH, Germany) for the evaluation of structures at low magnification. For

the morphological analysis of the inner and outer channel chip surface, SEM (Crossbeam 340,

Carl Zeiss Microscopy GmbH, Germany).

Sintering of Constructs

Samples were sintered into fused silica glass constructs at KIT, according to the

following schematic workflow (Figure 18: 4-5). In brief, thermal debinding was performed using

a high temperature drying oven (HT6/28, Carbolite, Germany). The cured ‘green parts’ were

heated from 25 ºC to 1400 ºC at a heating rate of 3 K min-1 under atmospheric conditions

(RHF17/3, Carbolite, Germany)116.

Figure 18 Schematic of LiqGlass fabrication process: (1) Amorphous silica nanopowder is dispersed into the HEMA monomer mixture; (2) LiqGlass is poured onto a template (PDMS imaged) and cured with UV light; (3) Bonding of multilayer, partially cured glass parts (if applicable); (4) Organic binder is removed by thermal debinding; (5) Powder is sintered to produce a highly transparent and dense glass body. Reprinted with permission under Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim116.

4.3 Thermal Properties A Mettler Toledo DSC1 system was used to measure the melting point (Tm) a sample

(~7 mg) of PVDF. Two cycles were conducted with a heating and cooling rate of 10 ºC/min

using a temperature range of -75 ºC to 250 ºC. The minimum of the second heating curve was

taken as the melting point.

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(107) Yuen, P. K.; Goral, V. N. Low-Cost Rapid Prototyping of Whole-Glass Microfluidic Devices. Journal of Chemical Education 2012, 89, 1288-1292. (108) 3 Common Glass Types: Properties and Applications. http://www.koppglass.com/blog/3-common-glass-types-properties-applications/ (visited on 29. July 2018). (109) Kotz, F.; Arnold, K.; Bauer, W.; Schild, D.; Keller, N.; Sachsenheimer, K.; Nargang, T. M.; Richter, C.; Helmer, D.; Rapp, B. E. Three-dimensional printing of transparent fused silica glass. Nature 2017, 544, 337. (110) Bu, M.; Melvin, T.; Ensell, G. J.; Wilkinson, J. S.; Evans, A. G. R. A new masking technology for deep glass etching and its microfluidic application. Sensors and Actuators A: Physical 2004, 115, 476-482. (111) Li, X. A., T.; Liu, Y.; Esashi, M. . Fabrication of high-density electrical feed-throughs by deep-reactive-ion etching of Pyrex glass. Journal of Microelectromechanical Systems 2002, 11, 625-630. (112) Bien, D. C. S. R., P. V.; Mitchell, S. J. N.; Gamble, H. S. Characterization of masking materials for deep glass micromachining. Journal of Micromechanics and Microengineering 2003, 13, S34. (113) Iliescu, C.; Chen, B.; Miao, J. On the wet etching of Pyrex glass. Sensors and Actuators A: Physical 2008, 143, 154-161. (114) Klein, J.; Stern, M.; Franchin, G.; Kayser, M.; Inamura, C.; Dave, S.; Weaver, J. C.; Houk, P.; Colombo, P.; Yang, M. Additive manufacturing of optically transparent glass. 3D Printing and Additive Manufacturing 2015, 2, 92-105. (115) Luo, J.; Pan, H.; Kinzel, E. C. Additive manufacturing of glass. Journal of Manufacturing Science and Engineering 2014, 136, 061024. (116) Kotz, F.; Plewa, K.; Bauer, W.; Schneider, N.; Keller, N.; Nargang, T.; Helmer, D.; Sachsenheimer, K.; Schäfer, M.; Worgull, M. Liquid glass: a facile soft replication method for structuring glass. Advanced Materials 2016, 28, 4646-4650. (117) Clasen, R. Preparation of coloured silica glasses made by sintering of particulate gels. Glastechnische Berichte 1993, 66, 299-304. (118) Inderherbergh, J. Polyvinylidene Fluoride (PVDF) Appearance, General Properties and Processing. Ferroelectrics 1991, 115, 295-302. (119) Tan, S.; Li, J.; Gao, G.; Li, H.; Zhang, Z. Synthesis of fluoropolymer containing tunable unsaturation by a controlled dehydrochlorination of P(VDF-co-CTFE) and its curing for high performance rubber applications. Journal of Materials Chemistry 2012, 22, 18496-18504. (120) Hochleitner, G.; Youssef, A.; Hrynevich, A.; Haigh Jodie, N.; Jungst, T.; Groll, J.; Dalton Paul, D.: Fibre pulsing during melt electrospinning writing. In BioNanoMaterials, 2016; Vol. 17; pp 159.

Appendices

34

Appendix All materials used in this thesis not generated by the author were reprinted with

permission of the copyright owners. These permissions were obtained under Creative

Commons Attributions Licenses, or via the RightsLink Copyright Clearance Centre. The

material was used either in its original form or with minor alterations, and the source was

referenced appropriately in Figure captions. The permissions for the reprinted material are

included below.


Appendices

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Appendices

36

Appendices

37

Copyright permission for Figure 5

Appendices

38


Appendices

39


Annex 2

Screening for Printable PVDF-Based Polymers for Melt Electrowriting

Deanna Nicdao

BSc (Chemistry)

Submitted in fulfilment of the requirements for the degree of

Master of Science

Department for Functional Materials in Medicine and Dentistry

University Hospital Würzburg

Julius-Maximilians-University Würzburg

February 2019

I

Keywords additive manufacturing, biofabrication, melt electrowriting, poly(ε-caprolactone),

poly(vinylidene fluoride)

II

Abstract In this report, the polymer-based components of melt electrowriting (MEW) printing

including the meltability, drawability and extrudability of polymers were investigated. Several

techniques were used to determine whether a material was compatible with MEW processing

based on these components. A range of poly(vinylidene fluoride) (PVDF)-based polymers

were investigated and assessed against the current gold standard material in MEW,

poly(ε-caprolactone) (PCL). Moreover, polymer blending of PCL with a PVDF-based polymer

was investigated in parallel. This was to determine whether polymer blending was a viable

approach to extend the library of compatible printing materials. The techniques used to assess

meltability, drawability and extrudability of polymer melts included micro-compounding,

rheology and dynamic mechanical analysis. The results were then compared to MEW

conditions. One aim of this report was to utilise this range of techniques to elucidate the

printability of PVDF-based polymers. The second aim is to alter the properties of an

incompatible neat polymer, i.e. 180 kg/mol PVDF, through blending with PCL to improve its

printability. Subsequent printability tests of P180:PCL blends were then compared with PCL

scaffolds using stereomicroscopy, SEM and EDX. In addition, a follow-up investigation on the

printing of a 6-inlet microfluidic design using PVDF and the embedding of PVDF fibres in a

cured silica-based nanocomposite monomer, Liquid Glass is discussed. This demonstrates

an alternative application for MEW fibres as microchannels for microfluidic devices.

Based on rheology of the neat polymers, the PVDF powder was found to have the

lowest viscosity followed by PCL and then by the neat polymer, P180. It was found that

blending P180 and PCL significantly lowered the melt viscosities for all blend compositions

compared to neat polymers. All blends performed similarly in drawability, extrudability and

printability tests. The higher molecular weight polymers (P275 and P530) and ter-PVDF

sample did not form melts during drawability and extrudability tests. SEM and EDX analysis

of the surface morphologies showed co-crystallisation in all blends. Fluorine content was found

to be dispersed in the 50:50 and PVDF-rich blend. Moreover, the embedded PP fibres in liquid

glass remained stable after curing and fluorine content was detectable along the fibre.

However, the printing of the 6-inlet design and the casting of delicate single-fibre constructs

requires optimisation. Overall, this report has shown how to utilise techniques for screening

polymers compatible for MEW and demonstrated the viability of blending PCL with P180 to

provide printable polymers.

III

Table of Contents Keywords ................................................................................................................... I

Abstract ..................................................................................................................... II

List of Abbreviations .................................................................................................. V

1. Introduction ......................................................................................................... 1

1.1 The Melt Electrowriting Process ...................................................................................... 1 1.2 Challenges .......................................................................................................................... 3

1.2.1 The MEW Process: Technological Perspective ............................................... 3

1.2.2 Polymer-based Parameters ................................................................................ 5

1.3 Establishing a Protocol ..................................................................................................... 6

1.3.1 Criteria for Material ......................................................................................................... 6 1.4 Project Outline ................................................................................................................... 7

2. Meltability of Polymers ........................................................................................ 8

2.1 Aims and Objectives ......................................................................................................... 8 2.2 Results and Discussion .................................................................................................... 8

2.2.1 Micro-Compounder ............................................................................................... 8

2.2.2 Dynamic Mechanical Analysis .......................................................................... 10

2.2.3 MEW Heating Chamber ..................................................................................... 12

2.3 Conclusions ..................................................................................................................... 13

3. Extrudability and Drawability ............................................................................ 14

3.1 Aims and Objectives ....................................................................................................... 14 3.2 Results and Discussion .................................................................................................. 14

3.2.1 Cooling Behaviour: Micro-Compounder .......................................................... 14

3.2.2 Rheology .............................................................................................................. 17

3.2.3 Extrusion and Printability with MEW ................................................................ 25

3.3 Conclusions and Future Directions .............................................................................. 30 4. PVDF as a Sacrificial Template for Microfluidics ............................................ 32

4.1 Results and Discussion .................................................................................................. 32

4.2 Conclusions and Future Directions .............................................................................. 38 5. Materials and Methods ...................................................................................... 39

5.1 Polymer Melts ................................................................................................................... 39

IV

5.2 Methods ............................................................................................................................ 39

5.2.1 Micro-Compounder ............................................................................................. 39

5.2.2 Rheometer Set-up .............................................................................................. 39

5.2.3 MEW Set-up ........................................................................................................ 39

5.2.4 Printing Parameters ........................................................................................... 40

5.2.5 Dynamic Mechanical Analysis .......................................................................... 40

5.2.6 Imaging of Constructs and Embedded Fibres ................................................ 40

5.2.7 Embedding PP Fibres in Liquid Glass ............................................................. 40

References ............................................................................................................. 42

Appendix ................................................................................................................ 46

V

List of Abbreviations 2PP Two-photon polymerisation

3D 3-dimensional

AM Additive manufacturing

CLSM Confocal laser scanning microscopy

CNC Computerised numerical control

CTS Critical translation speed

DMA Dynamic mechanical analysis

E’ Storage modulus (relative to silicon standard)

E’’ Loss modulus (relative to silicon standard)

EDX Electron-dispersive X-ray spectroscopy

FDM Fused-deposition modelling

FDJ Fibre diameter (at junction)

FDs Fibre diameter (at strut)

FTIR Fourier Transform infrared spectroscopy

G’ Storage modulus

G’’ Loss modulus

MEW Melt electrowriting

PCL Poly(ɛ-caprolactone)

P180 180 kg/mol poly(vinylidene fluoride)



PBA Poly(butylene adipate)

PMMA Poly(methyl methacrylate)

PP Poly(vinylidene fluoride) powder

PVAc Poly(vinyl acetate)

PVDF Poly(vinylidene fluoride)

PVDF-TrFE-CTFE Poly(vinylidenefluorine-trifluoroethylene-

chlorotrifluoroethylene)

RM Regenerative medicine

SEM Scanning electron microscopy

TE Tissue engineering

Tc Crystallisation temperature

Tm Melting temperature

UV Ultraviolet

WXRD Wide-Angle X-ray diffractometry

1. Introduction

1

1. Introduction 1.1 The Melt Electrowriting Process

Additive manufacturing (AM) is a layer-by-layer fabrication process using metals,

polymers and ceramics. It has a broad application field, including but not limited to, textiles,

architecture, biomaterials and tissue engineering[1]. One technique, termed melt

electrowriting (MEW), focuses on the fibre-drawing of polymer melts onto a collector plate – a

form of electrohydrodynamic jetting[2]. Through this technology, precise fibre diameters are

produced as fibres are electrostatically drawn onto a collector plate. Distinct from

electrospinning, MEW incorporates translating units by moving stages in a horizontal

Cartesian plane[3] or linearly moving rotating cylinder[4] allowing for controlled fibre deposition

to create targeted three-dimensional (3D) morphologies (Figure 1).

Figure 1 Schematic of MEW device. The polymer melt is placed in the heater chamber with a gas pressure assisted

system. In addition, the chamber is mounted on an adjustable Z-axis to set the working distance. The jet deposition

is controlled by the computer-driven collector plate in the X-Y direction. Reprinted with permission under the

Creative Commons Attribution License from [3] (see Appendix).

So far, this technology has demonstrated millimetre to micron-sized fibre diameters,

bridging technologies from low resolution, i.e. fused-deposition modelling (FDM)[5], to high

resolution, e.g. 2-photon polymerization (2PP)[6]. This resolution combined with solvent-free

processing, where solvent removal and ventilation systems are avoided, improves the

cytocompatibility of fabricated constructs. Due to this, tissue engineering (TE) applications

1. Introduction

2

have been a major focus for MEW within the past decade. As a result, the standard material

of choice has been medical-grade poly(ε-caprolactone) (PCL). In addition, PCL has

demonstrated long processability times and uniform fibres with a coefficient variation of 3%

for 15.5 μm[7]. The low melting point, semicrystalline structure and slow thermal/hydrolytic

degradation of PCL results in rapid solidification, making it an ideal material for MEW[8].

Therefore, from a processing standpoint, PCL is highly compatible with MEW.

Nevertheless, a selection of tailorable materials would broaden the applicability of

MEW. In TE, the well-known primary component of the extra-cellular matrix consists of fibrous

collagen, fibres with diameters that vary from tissue to tissue[9]. To mimic the complexity of

tissues, it is necessary to provide a library of polymers with a variety of mechanical, chemical

and biological properties. Several polymer classes have also been processed using MEW, as

mentioned in literature, including a nondegradable poly(propylene)[10], a photocurable poly(L-

lactide-co-ε-caprolactone-co-acryloyl carbonate)[11], and water-soluble poly(2-ethyl-2-

oxazoline). A recent addition to this list is the piezoelectric polymer, poly(vinylidene difluoride)

(PVDF)[12]. However, significant improvements must be made in fibre uniformity and stacking

for these materials to reach the processing capabilities of PCL. Therefore, PCL has remained

the gold standard for this technique.

Given MEW is an early technology, the optimisation of design and upscaling has been

a main focus for its advancement. This includes exploring the effects of printing parameters

including gravity[13], automatization of jet stabilisation[14] and extending the upper limits of

the construct size whilst maintaining a high resolution[15]. These studies will accelerate MEW

from a technological standpoint however, a protocol is currently not available for screening the

extrusion of polymers with MEW. To screen potential candidates, a protocol for testing their

meltability, drawability and extrudability is required. This protocol must outline the necessary

properties that will allow extrusion under MEW conditions. A similar model has been

developed for another method of AM, known as bioprinting[16], in which hydrogels are

extruded through a nozzle using a pressure-based system (Figure 2).

1. Introduction

3

Figure 2 Schematic for the assessment of bioinks: (a) to pass the initial screening, drop formation and layer

stacking must be observed, (b) rheological properties assessed are the flow initiation and yield stresses, degree of

shear thinning and recovery properties. Reprinted with permission under the Creative Common Attribution License

from [16] (see Appendix).

This protocol has been used to screen potential hydrogel candidates based on their

rheological behaviour. As MEW similarly utilises pressure to enable extrusion, it was thought

a similar protocol could be established. However, a clear distinction MEW has to bioprinting is

use of electrostatic charges to draw fibres. Therefore, the rheological properties of polymer

melts under both electrostatic- and pressure-induced traction must be understood. This will

be elaborated in the following sections; Section 1.2.1 will outline MEW parameters followed

by the relevant theoretical background of polymer melts in Section 1.2.2.

1.2 Challenges 1.2.1 The MEW Process: Technological Perspective

Studies have investigated how the printing parameters influence the jet and the fibres

and scaffolds produced to address the multi-parameter challenge[3,7,14,15]. These

parameters include the flow rate of the polymer melt, the applied potential difference between

the spinneret and collector, the feed rate onto the collector plate (i.e. collector speed) and the

printing gap (i.e. the distance between the spinneret tip and the collector plate) (Figure 3)[12].

1. Introduction

4

Figure 3 Schematic diagram of parameters affecting jet stabilisation for MEW. Reprinted with permission under

the Creative Common Attribution License from [12] (see Appendix).

The aforementioned parameters must all stabilise the jet at the chosen collector speed,

this is termed as the critical translation speed (CTS). The multi-parametric nature of MEW

printing underlines the necessity for high-throughput screening. Currently, a variety of bulk

analysis techniques are under development for MEW[12,14]. A technological review on MEW

has divided the process into three phases: (1) polymer preparation, (2) jet generation and (3)

fibre collection[17]. Phase 1 (polymer preparation) consists of preparing the polymer to reach

a threshold before extrusion in which a ‘usable liquid state with the necessary mechanical,

thermal and electrical influence’ is reached. Subsequently, Phase 2 (jet generation) describes

the extrusion of the fibre prior to making contact to the collector plate. During this time, the

environmental conditions are said to affect the thinning and solidification of the jet. Finally,

Phase 3 (fibre collection) is defined as the fabrication of well-ordered architectures signified

by the solidification of the polymer onto the collector plate. However, this process assumes

the polymer melt is successfully extruded. Thus, it can be argued that developing a thorough

protocol for Phase 1 is of equal importance to the overall MEW process and must be

developed simultaneously to advance this technology. Further information on how the

mechanical setup and chosen system parameters influence the printing process from a

component-based perspective has been elaborately described in literature[7,13,14,17] and

will therefore not be discussed further in this report.

1. Introduction

5

1.2.2 Polymer-based Parameters

The differentiation of polymer-based parameters from component-based parameters

has been introduced in literature[17]. The first and foremost polymer-based parameter that is

considered for MEW is viscosity as it largely determines the formation of the Taylor cone and

a stable jet. The degree of viscosity depends on molecular structure and molecular weight in

combination with the input of thermal energy[18]. High molecular weight polymers counteract

initial jet thinning resulting in larger fibre diameters. In contrast, polymers of low molecular

weights form thinner fibres accompanied by an inconsistent jet, referred to as jet dripping[19].

Moreover, the tacticity of the polymer is also significant as isotactic structures, characterised

by a higher degree of molecular order, result in reduced fibre diameters in comparison to

atactic polymers[19,20].

In addition to high viscosity, low conductivity is also considered a prerequisite to obtain

stable operational behaviour and viable morphological mechanical properties[2,21]. The

reason being that conductivity is a determinant for charge accumulation[22], consequently

affecting the Taylor cone formation[17]. Moreover, jet stability is compromised depending on

its tendency for electrostatically induced buckling behaviour onto the collector[23]. Polymers

with greater conductivity have been found to break within an increased electrical field, whereas

insulating materials lack the capability to initiate fibre formation[24].

An important consideration is the strong relation between the conductivity and

temperature of the working material. The input of thermal energy leading to the phase change

from solid to liquid may result in the mobilisation of charge carriers, in turn increasing the

conductivity of the polymer melt with increasing temperature[17]. In addition, the maximum

temperature settings of the machine must be kept in mind, as this eliminates the possibility of

printing polymers with too high viscosities within the capable temperature settings. From both

a component- and polymer-based perspective, the amount of thermal input is a critical factor

that influences the printing window of the material.

As seen through literature, the choice of material (Phase 1) heavily influences both the

jet formation (Phase 2) and fibre collection (Phase 3). One setback for the current list of

printable materials in MEW is their limited, or unpredictable, long-term printability. In this

report, the polymer properties of PCL will be screened and compared to the recently printed

fluoropolymer, PVDF (Figure 4).

1. Introduction

6

(a) (b) Figure 4 Chemical structure of (a) PVDF and (b) PVDF-TrFE-CTFE.

A range of PVDF polymers will be investigated, including varied molecular weights, a

branched ter-polymer, poly(vinylidenefluorine-trifluoroethylene-chlorotrifluoroethylene)

(PVDF-TrFE-CTFE), and blends of PVDF with PCL. In addition, these polymers will be

screened against the PVDF powder previously printed[12] to elucidate its printing properties.

1.3 Establishing a Protocol 1.3.1 Criteria for Material

To summarise the discussion for printable materials, three key principles emerge,

based on the MEW process and specific to the set-up available, in conjunction with the

chemical and mechanical properties previously mentioned:

1. Meltability of the Polymer: the material must homogenously melt in the syringe

chamber within the capable temperature range of the machine (< 200 ºC).

2. Extrudability/Drawability of the Polymer: the material must be extrudable with the given

spinneret diameter (< 22 G) and capable pressure range (0.5 – 6 bar).

3. Fibre Formation and Recovery: the material must form a Taylor cone and stable jet

within the capable speed range (< 10 000 mm/min) and attaches to the collector plate

for an accurate fibre deposition.

1. Introduction

7

1.4 Project Outline

This project aims to provide a basic material screening process for MEW using the standard, PCL, to compare to the printability of a range of PVDF polymers. As MEW is an early technology, the capabilities of this technology must be acknowledged to

better assess compatible materials for printing. As this technology continues to advance, so

do its applications. Therefore, it is essential to understand the process and generate a basic

approach on assessing the printability of a new material.

The discussion will follow consistently with the criteria mentioned in Section 1.3.1, with

data separated based on the objective being addressed. To assess the protocol, several

PVDF-based polymers, including PVDF blends, are compared to PCL in each chapter;

Chapter 2: Melting behaviour of the polymer will be compared by three different

techniques: (a) compounder, (b) rheometer and (c) heating chamber within the MEW

device. This will gather an understanding of whether the melting behaviour has internal

fluctuations largely based on technological components or polymers properties.

Chapter 3: Extrudability and drawability of the polymer will be assessed using

rheology and a polymer compounder. Chapter 4: This chapter will demonstrate a potential application of MEW in the

fabrication of hollow channels for microfluidics. The fibres will be embedded in a silica

nanocomposite known as ‘Liquid Glass’. Qualitative analysis techniques include

scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy

(EDX). Chapter 5: The experimental section including materials will be outlined in this

chapter.

2. Meltability of Polymers

8

2. Meltability of Polymers 2.1 Aims and Objectives

The melting behaviour of PCL against PVDF-based polymers are observed through

several techniques. Melt viscosities were quantified using a micro-compounder and a

rheometer. Qualitative evaluation of the melting process was conducted between the

rheometer and the actual MEW heating chamber. The method was applied to a range of PVDF

with varying molecular weights, PVDF:PCL blends (25%, 50% and 75% PVDF), PCL and the

copolymer PVDF-TrFE-CTFE (ter-PVDF).

2.2 Results and Discussion 2.2.1 Micro-Compounder

The micro-compounder was used to measure melt viscosities in a temperature window

within the range of printing temperatures relevant to the material. For PCL, melt viscosities

measured between 70 – 90 °C were found to negatively correlate with increasing temperature.

The viscosity decreased from 3.75 to 2.79 kPa·s. (Figure 5)

Figure 5 Melt viscosities of neat polymers over temperature: (a) Linear PVDF-based polymers measured from 180

– 200 °C, (b) branched ter-PVDF polymer measured from 165-185 °C and (c) PCL measured from 70 – 90 °C.

For the linear PVDF-based polymers, the chosen temperature window was 180 –

200°C. The higher molecular weights (P275 and P530) all exhibited a negative correlation with

increasing temperature, similar to PCL. In contrast, P180 and PP remained constant (0.46 +

0.05 kPa·s and 0.44 + 0.04 kPa·s respectively) over the whole temperature range. Ter-PVDF

was measured at a temperature range of 165 – 185 °C. It displayed the largest decrease in

viscosity with increasing temperature. However, the viscosity plateaus at 175 - 180 °C,

similarly to the trend seen with P275 at 190 – 195 °C.


9

An overview shows the branched ter-PVDF to have the highest viscosity subsequently

followed by P530 and P275 respectively. The lowest viscosities are matched between PP and

P180. The gold standard, PCL, displays a viscosity no lower than 2.79 kPa·s within the

temperature window – 2.33 kPa·s higher than the printable PP. Taking these viscosities into

account, PCL falls in between the range of PVDF samples. When considering the long-term

printability of PCL, a viscosity between 2.5 - 4 kPa·s may be ideal. The printability of PP has

previously been demonstrated in literature however repeated experiments elucidated the

instability and unpredictable printing nature of this material. Given P180 depicted a similar

viscosity to PP, it was hypothesized that blending P180 with PCL would provide a more stable

printable material. An average melt viscosity of each blend was carried out at 190 °C (Figure

6).

Figure 6 Average melt viscosities of PCL:P180 blends sorted by PCL content (%) measured by a micro-

compounder at 190 °C and 50 RPM.

A significant increase in viscosity (0.455-1.21 kPa·s) is observed with increasing PCL

content. Notably, for a direct comparison to pure PCL (100%), the measurement was also

conducted at 190 °C, a temperature only relevant for this comparison. From these results,

blending has demonstrated to increase the viscosity of P180 which may lead to an increased

printability behaviour akin to the gold standard, PCL.


10

2.2.2 Dynamic Mechanical Analysis

The melting behaviour of neat polymers from a solid to molten state under stress was

investigated using dynamic mechanical analysis (DMA). The neat polymer performances

under stress and temperature were analysed by comparing the storage moduli (Figure 7) and

tan (δ) curves (Figure 8). In Figure 7, the storage modulus (E') decreased against temperature

for all samples with the overall highest and lowest storage moduli belonging to PCL and PP

respectively. The P180, P275, P530 and PP samples, all exhibited a gradual decreasing linear

trend over temperature. It is notable that these samples remained in their pellet form

throughout the temperature sweep thus no complete molten state was achieved, hence the

gradual decreasing trends observed. In contrast, the PCL depicts a melting transition at

~60 °C as predicted. Another melting transition is observed with the ter-PVDF sample with a

sharp decline occurring at 150 °C. The broad transition is due to the several blocks that melt

at varying temperatures and potentially other phase transitions.

For tan δ curves (Figure 8), the PCL sample exhibits a sharp peak at ~60 °C which is

in agreeance with the storage modulus. The other two broader peaks suggest phase

transitions at 100 °C and 200 °C of the melt – this could be confirmed further with XRD

measurements. An effect of molecular weight on tan δ is seen as the higher the molecular

weight, the lower the tan δ melting transition observed at ~ 150 °C. This further suggests the

higher molecular weights (P275 and P530) failed to form homogenous melts, potentially

indicating their lack of compatibility for MEW processing. On the other hand, PP displays a

similar tan δ curve – broad and minimal – with no sharp transitions. Though, a decrease after

~150 °C deviates its behaviour from that of P530. This decrease should indicate its increase

in rigidity, nevertheless PP has previously been printed using MEW. However, this may be

due to its low viscosity and low elasticity once melted allowing for its extrudability. As for the

tan δ curve of ter-PVDF, several sharp transitions are observed as previously seen in E'. This

indicates, as previously mentioned, the blocks undergo several transitions within the

copolymer. After the two sharpest transitions at ~150 °C, the sample tan δ remains high

indicating a less rigid state.


11

Figure 7 Storage moduli of neat PCL and PVDF-based polymers.

Figure 8 The tan δ curves of neat PCL and PVDF-based polymers.

The melting behaviour of the neat polymers using DMA could be an indicator of their

compatibility with MEW. Given the E' and tan δ curves of all PVDF-based polymers lie between

two printable polymers, PCL and PP, it is seen that DMA alone is insufficient to provide a

screening for compatibility. If the melt viscosities previously measured are considered, a

compatibility window between the viscosities of PCL and PP is derived. Thus, it is


12

hypothesized that the higher molecular weight polymers, P275 and P530, and the branched

ter-PVDF sample will not form a melt in the MEW setup investigated in the following section.

2.2.3 MEW Heating Chamber

To assess the reliability of the screening techniques, extrusion behaviours of neat

polymers that formed melts (P180, P275) and PCL:P180 were observed. After melting the

polymers at the desired printing temperature, a consistent pressure and voltage was applied

to the material for extrusion. The polymers were placed in the heating chamber at 190 °C and

their appearances were qualitatively recorded after 30 min of heating (Figure 9).

Figure 9 Neat PVDF-based polymers (left) and P180:PCL blend (right) melts in syringe. Samples were heated in

the MEW setup at 190 ᵒC for 30 min.

It is seen that the branched ter-PVDF and high molecular weight PVDF-based

polymers (P275 and P530) did not form a melt within the MEW setup. This was predicted

given their measured melt viscosities using the micro-compounder were significantly higher

than PCL. This shows that molecular entanglement plays a role in the meltability of the

polymer as increased thermal energy – and potentially other shear forces – may be required

to disentangle and break chains to form melts for branched and high molecular weight

polymers. To investigate the heating gradients present surrounding the syringe, the

temperatures within the heating chamber (T1) and at the spinneret tip (T2) were manually

checked. A significant variation between the temperature detected by the thermostats and the

actual temperature measured by temperature probes was observed (Table 1).


13

Table 1 Temperatures of MEW device measured via software (detected by thermostats) or manually (using

temperature probes) at T1 (heating chamber) and T2.(spinneret tip) for temperatures applicable to printing PCL

(75 °C) and PVDF (190 °C).

One MEW device was assessed. However, given that each printer has a similar

heating chamber setup, it can be assumed that each printer would have a significant

temperature gradient that deviate further with higher temperatures. Previous investigations

with a blend of PVDF with poly(butylene adipate) (PBA) was found to lower the melting

temperature (Tm) compared to their neat polymers. It is hypothesized that blending in the lower

Tm PCL with the higher Tm P180 will form a stable printing material and lower the Tm. This

approach to lower the Tm would also allow a wider range of materials that have a Tm above

the upper limit of MEW temperatures to be processed.

2.3 Conclusions

The purpose of this chapter was to assess the meltability of PVDF-based polymers

against PCL. The measured viscosities using the micro-compounder displayed the melt

viscosity of PCL to be significantly higher than PP. The branched ter-PVDF, P275 and P530

all exhibited higher viscosities compared to PCL, whilst the P180 had a similar viscosity as

PP. The P180:PCL blends showed that an increasing PCL content led to an increase in

viscosity. A potentially beneficial property to increase the printability of P180. DMA results

revealed stronger transitions in PCL and the ter-PVDF polymer, however PP depicted to be

the most rigid sample contrary to evidence of its printability. Thus, the findings of DMA are not

absolute and should be combined with other analysis techniques. In conclusion, the meltability

of polymers in the MEW setup were in agreeance for polymers that exhibited viscosities

between that of PP and PCL. The incompatible melts were confirmed to be the higher

molecular weights (P275 and P530) and the ter-PVDF sample.

3. Extrudability and Drawability

14


3.1 Aims and Objectives

The previous investigations have shown that the melting behaviour of a polymer is

influenced by its molecular weight, degree of branching and whether it has been blended.

Subsequently, these experiments will test the extrudability and drawability of each polymer

against the PCL standard.

3.2 Results and Discussion 3.2.1 Cooling Behaviour: Micro-Compounder

The cooling behaviour of a polymer is an essential factor to consider for its potential to

produce fibres through MEW. For a printed construct to form, it is required for a fibre, once

extruded, to cool once it has touched the collector plate. Too fast, the fibre fails to adhere to

the collector plate sufficiently, leading to stray fibres. Too slow, excessive fusion between

stacked fibres occurs, compromising the fibre stacking and pore morphologies. In addition,

environmental factors, such as humidity and temperature, heavily influences the amount of

charge accumulation of the jet and in turn, affecting jet stabilisation[3]. However, these factors

are considered as component-based parameters and are thus outside the scope of this report.

In this experiment, the micro-compounder was used to observe the cooling behaviour of the

PVDF-based polymers compared to the gold standard, PCL.

After processing the polymer melts in the compounder, cooling behaviour was

observed during collection. All materials were successfully melted and extruded (Figure 10).


15

Figure 10 PCL and PVDF-based polymers before and after melt compounding at 50 RPM. Extrusion temperatures

were 85 °C and 190 °C for PCL and PVDF-based polymers respectively.

The cooling behaviour was qualitatively observed to differ between polymers. PCL, the

gold standard for MEW, was observed to remain molten after extrusion, which results in the

formation of a solid mass. However, the polymer consistently cooled prior to reaching the

bottom of the vial. Only PP exhibited similar cooling behaviour, which led to the formation of

an agglomerate. When comparing the shape of the polymer mass between PCL and PP, the

cooling rate of PP indicates to be lower, forming a more densely packed agglomerate. This

might be the result of a lower viscosity of the material, as observed in the previously measured

melt viscosities in Section 2.2.1. The cooling behaviour appeared independent of molecular

weight as P180, P275 and P530 all cooled down rapidly after extrusion forming well-defined

fibres of varying sizes. However, no fibre stacking is observed. In contrast, the ter-PVDF

appeared to cool directly after extrusion, resulting in a consistent fibre diameter. Moreover,

after processing, a strong colour change was observed for PP – a white powder to a

yellow/brown melt. Additionally, the pure PVDF compounds darken in colour with increased

molecular weight though no colour change occurs after processing. A colour change within

the material could indicate a change in material properties, an important factor to provide

convenience with long printing durations and upscale the MEW technology.

By processing the neat polymers through a compounder, the cooling behaviour of a

material was further examined. As mentioned, a too-slow cooling behaviour compromises the

morphology of the printed construct. However, component-based parameters can be adjusted


16

to accommodate a slower cooling rate, for example an increased working distance or cooled

collector plate. In contrast, difficulties arise when accommodating for a fast cooling rate. A

decreased working distance limits the constructs height and risks arcing. Additionally,

increasing the ambient temperatures are not compatible with the current MEW setup,

especially for polymers with melting points higher than 40 °C. A polymer-based approach to

increase the printability of incompatible materials, in this case P180, was to blend it with

different ratios (25%, 50%, 75%) with a printable material (PCL). The cooling behaviour of

each blend was observed (Figure 11).

Figure 11 PVDF:PCL blends (PVDF: 25%, 50%, 75%) processed and extruded via micro-compounder at 190 °C.

The cooling behaviour is noticeably different based on the component ratio of

P180:PCL. It is evident that small and large fibres can be drawn with P180, likewise with P275

and P530, as the material cooled rapidly enough to form fibres. Smaller fibres were formed by

pulling the material with forceps during extrusion. This was not observed with PCL as small

fibres could not be drawn directly after extrusion as it remained too molten and fused with the

material in the vial. The appearance of smaller fibres gradually lessened with increasing

amounts of PCL, implying that in turn the cooling rate decreased.

Overall, the blending of materials (both printable and non-printable) has demonstrated

that printability can be increased, with as little as 25% PCL, by slowing the cooling rate of the

material. To quantify the material properties of both neat and blended polymers, melt

viscosities were measured by rheology for pre- and post-heat-treated polymers in the following

section.


17

3.2.2 Rheology

To assess the printability of a polymer, the lifespan after melt processing must be

considered. PCL and PVDF-based polymers pre- and post-heat treatment were measured by

rheology to evaluate the effect of heating cycles on the material properties. P530 and ter-

PVDF were excluded from these measurements as a molten state was unachievable for these

polymers on the rheometer. This is due to the ambient temperature surrounding the hot plate,

creating a heat gradient within the sample, with only the material directly in contact with the

hot plate forming a melt. All measurements were run at relevant printing temperatures of 85 °C

and 190 °C for PCL and PVDF-based polymers respectively.

A broad viscoelastic region, in which data points were independent of applied stress,

was determined by an amplitude sweep (Figure 12), followed by a frequency sweep to

determine the polymers’ response to stress (Figure 13).

The samples with a linear viscoelastic region that revealed an upper limit were P180

and P275. For P180 a decrease of the storage modulus (G') is observed at strain 21.5% for

both conditions. As for P275, a decrease occurs at a strain of 14.7% and 10.0% for non- and

heat-treated conditions respectively. For PCL, PP and P180, the loss modulus (G'') was higher

than G', confirming the viscous properties of the material as a melt. In contrast, P275 displayed

G' > G'' that indicates a more solid-like material. This suggests a potential upper limit of

molecular weight capable of forming a melt, further justifying why P530 remained in solid pellet

form at 190 °C. In general, no significant difference in the viscoelastic region is observed

between heat cycles.


18

Figure 12 Amplitude sweeps of neat polymers before (left) and after (right) heat treatment: (first row) PCL, (second

row) PP, (third row) P180, (fourth row) P275. All samples were tested with an incremental amplitude sweep from

0.01 to 100% strain at a constant frequency (1 Hz).


19

Figure 13 Frequency sweeps of neat polymers before (left) and after (right) heat treatment: (first row) PCL, (second

row) PP, (third row) P180, (fourth row) P275. All samples were tested with an incremental frequency sweep from

0.05 to 100 rad/s at a constant strain of 0.1%.

In the frequency sweeps, both moduli of all samples increase over angular frequency

(rad/s). The frequency sweeps presented a higher loss to storage modulus for all samples

excluding the pre- and post-heat-treated P275. The tan δ (= G''/G') is an indicator of material

properties. The tan δ of PCL and P180 approaches 1 over frequency. This suggests that a


20

more solid-like state is reached with increased frequencies. For PP, the tan δ is consistent at

20 and 25 for C and D respectively. Moreover, the tan δ for P275 is approximately 1 throughout

the sweep with a cross-over occurring at 1.05 rad/s and 1.53 rad/s for pre- and post-heat

treatment. This indicates P275 to have not reached a viscous material but rather remained a

solid-like despite undergoing stress at high frequencies. Overall, P180 along with previously

printed polymers, PCL and PP, appear compatible for MEW processing. However, both moduli

of P180 is a factor higher than PP and PCL, indicating a high viscosity and in turn, a higher

applied pressure would be required for extrusion. To illustrate this, the viscosities of the neat

polymers pre- and post-heat treated were plotted against shear rate (Figure 14).

Figure 14 Viscosities (Pa·s) plotted against shear rate (1/s) of neat polymers before (bold) and after (dashed) heat

treatment. PCL and PVDF-based polymers were measured in the shear rate region from 0.01 to 100/s at 75 °C

and 190°C respectively.

The melt viscosities at low shear rates confirms the material properties portrayed in

the amplitude sweeps with P275 being the most solid (i.e. highest viscosity) and PP being the

least viscous. Nevertheless, as the shear rate approaches 10/s, the viscosity of P180 drops

below PCL – which agrees with the melt viscosities measured by the micro-compounder. This

illustrates the effect of applied shear forces on the viscosity of polymer melts. Furthermore,

the plot reveals viscosity to decrease for PCL and P180 after a shear rate of approximately

15/s. The viscosity of P275 appears to be influenced significantly with increased shear rate,

approaching a similar viscosity to PCL and P180. In contrast, the viscosity of PP seems

unaffected by shear rate as it remains constant and linear for both conditions. PP also shows


21

a significantly lower viscosity in comparison at 100 Pa·s. PCL appears to be the most affected

by heat treatment, with the viscosity dropping slightly post heat treatment. Overall, polymer

viscosities appear to significantly drop after a shear rate of 10/s – a component-based solution

to improving printability.

To test the polymer-based solution to improve viscosity through blending, P180:PCL

blends were also measured using the rheometer (Figure 15). The amplitude sweeps exhibited

a broad viscoelastic region for all P180:PCL blends. The 75:25 blend is of interest due to the

slightly non-linear character of the viscoelastic region. The tan δ is largest for the 25:75 blend,

and it decreased with increasing P180 content. The moduli are highest for the neat polymers.

Given the blends have moduli lower than either neat polymer, both blend components are

considered miscible. This could be further confirmed by acquiring the glass transitions through

DSC and comparing whether the blends contain the glass transitions of the neat polymers

(immiscible) or a combined lowered glass transition (miscible). As for the frequency sweeps,

the tan δ approaches 1 for the neat polymers but remains constant, independent of angular

frequency, for the blends. This indicates that the blends stiffen however the viscoelasticity

remains constant. A possibility for this is a lack of entanglement occurring between the PCL

and PVDF chains prevented by the non-reactive fluorine groups.


22

Figure 15 Amplitude (left) and frequency (right) sweeps of P180:PCL blends: PCL (first row), 25:75 (second row),

50:50 (third row), 75:25 (fourth row), P180 (fifth row). Samples were measured at 190 °C, from 0.01 to 100% strain

at a constant frequency of 1Hz for amplitude sweeps and 0.05 to 100 rad/s at a constant strain of 0.1% for frequency

sweeps.


23

The melt viscosities (Pa·s) against shear rate (1/s) were plotted for the PCL:P180 blends

(Figure 16).

Figure 16 Viscosities (Pa·s) plotted against shear rate (1/s) of PCL:P180. All samples were measured in the shear

rate region from 0.01 to 100/s at 190°C.

A significant decrease in melt viscosities were obtained for all blends. The blend

containing the highest amount of PCL exhibited the lowest melt viscosity. In this case, this is

predicted given the lowest viscosity of PCL to P180, thus acting as a plasticizer. Notably, the

50:50 blend displayed the highest viscosity amongst the blends. Meanwhile the blend with the

lowest amount of PCL appeared to be significantly influenced by shear rate. At low shear

rates, the viscosity matched the 50:50 blend then subsequently dropping to a viscosity similar

to the 75% PCL blend at higher shear rates. This may be due to the decreased mobilisation

of PCL chains within the blend containing high amounts of stable fluorinated chains at low

shear rates. Therefore, once higher shear rates are applied, it allows the chains to move more

freely throughout the blend. Another possibility for semi-crystalline polymers is the onset of

plastic deformation of crystalline regions, termed as yielding, resulting in a slip of these regions

amongst several planes. Yielding is subsequently followed by necking – the molecular

reorientation of disordered chains along the tensile direction[25]. Here, the mechanical

anisotropy is introduced into the material. Given the nature of anisotropic materials, the

storage modulus is expected to significantly higher in the direction of the oriented chains. This

could explain the coincident slip observed in the frequency sweep as it approached 1 rad/s

(Figure 15) as well as the dip followed by a recovery in viscosity as it approached a shear rate

of 1/s (Figure 16). Regardless, blending of PCL into P180 has demonstrated to be a viable

approach for improving the viscosity of P180 for MEW.


24

The drawability of these materials were qualitatively assessed on the rheometer by

observing whether a fibre was formed between the rheometer plates. This occurred at the end

of each measurement when the upper plate would return to a homing position of 70 mm above

the hot plate (Figure 17). The fibres were assessed based on whether the fibre remained intact

and the fibre appearance.

Figure 17 Drawability tests of neat polymers using the rheometer setup from 0.5 mm to 70 mm. Loading plate

temperatures were set to 75 °C and 190 °C for PCL and PVDF-based polymers respectively. Incompatible solids

(P530 and PVDF-TrFE-CTFE) were included to demonstrate a lack of melting behaviour < 200 °C.

The gold standard, PCL, was the only sample to have drawn several fibres. The PVDF-

based polymers capable of melting were tested. Here, at temperatures well above their melting

points, P530 and the ter-PVDF polymer failed to form a melt. A result potentially due to the

high viscosity from their high molecular weight and branchedness. From the measurable

PVDF-based melts, PP and P180 formed a fibre however only the latter remained intact over

the gap distance. It is hypothesized that the rupture of the PP fibre is due to the unknown

additive which dissociated the molecular structure and prevented sufficient chain

entanglement. The presence of the additive also explains the slight yellow tinge of PP. P275

shows low drawability properties as the melt appears less viscous and more brittle with opaque

white components within the melt, resembling properties from both P180 and P530. Hence,

demonstrating an upper limit for compatible molecular weights as a polymer melt. From these

findings, in combination with the previous melting behaviours of neat and blended polymers

in syringes (Section 2.2.3), it would be hypothesized that P275 would not be extrudable in a

MEW setup. Overall, the only promising printable material from this comparison is P180 as it

demonstrates qualities comparable to PCL rather than PP. Drawability tests were therefore

repeated for P180:PCL blends (Figure 18).


25

Figure 18 Drawability tests of P180:PCL blends using the rheometer setup from 0.5 mm to 70 mm. Loading plate

temperature was set to 190 °C for all samples.

All blends readily melted and were capable of drawing fibres. The drawabilities of the

PCL-rich and 50:50 blends were more promising compared to the fibres of P180-rich and P180

which ruptured. The blends appear more opaque with increasing amounts of P180. This is

characteristic of the higher crystallisation temperature (Tc) of P180 and thus cools more rapidly

whilst PCL remained molten and transparent during test. Notably, the same program was run

for both neat and blended polymers yet P180 showed contrasting results. This may be

attributable to the varying ambient conditions therefore future experiments within a closed

system with controlled temperature and humidity would be ideal. A closed system was not

possible during these experiments as the tests were run at high temperatures and it was

preferable to avoid equipment damage. As demonstrated here and in the MEW setup (Section

2.2.3), P530 and ter-PVDF did not form a melt therefore extrusion testing was conducted on

the standard PCL compared to P180, P275 and PCL:P180 blends.

3.2.3 Extrusion and Printability with MEW

To determine which materials were printable, the extrudability of each polymer that

demonstrated fibre-drawing behaviour in previous tests were investigated in the MEW device.

Along with PCL and PP, P180 was the only neat polymer that extruded under maximum

pressure (5 bar). However, once extruded, the material immediately cooled and thus failed to

adhere to the collector plate – even after adjusting component-based parameters, i.e. working

distance, voltage, pressure. Nonetheless, printability tests conducted on the P180:PCL blends

demonstrated that all compositions formed a stable printing jet. A single layer, box construct

was printed under the same printing parameters with PCL as the standard. The pore and fibre

morphology were imaged at the centre and turning loops of the constructs (Figure 19).


26

Figure 19 Stereomicroscope images of single layer scaffolds from neat PCL (left) with increasing P180 (left middle

to right) content. Images were taken at the centre (top row) and turning loops (bottom row) of the scaffolds (scale

bars: 1 mm).

The standard is shown to have consistent fibre and pore morphology and is

significantly transparent compared to blends. Amongst the blends, the P180-rich scaffold

exhibited the most consistent pore morphology, however the PCL-rich scaffold has the most

accurate fibre deposition at the turning loops. This print could be optimised by adjusting the

speed at the turning loops to reduce the drag of the jet, i.e. print at a critical translation speed

(CTS). Long beading is observed in the PCL-rich and 50:50 blend, implying either (a) a

component-based issue; i.e. the polymer mass flow is too high or low and can be adjusted by

altering the pressure and voltage or (b) a polymer-based issue; an immiscibility is occurring

between the two polymer networks or their amorphous and crystalline regions. Overall, the

printability test has demonstrated the P180-rich blend provided the most ideal fibre and pore

morphology when compared to PCL.

Higher resolutions of fibre junctions were imaged using SEM accompanied by and

elemental analysis of carbon (C), oxygen (O) and fluorine (F) content by EDX. PCL fibres were

printed at 190 °C to allow for a direct comparison with P180 at these printing temperatures

(Figure 20).


27

The surface morphology significantly changed with varied blend compositions. The

spherulitic surface of PCL is most prominent in the standard. The surface morphology of PCL-

and P180-rich blends depicts smoother, more amorphous surfaces. The surface roughness

has increased for the 50:50 sample. As for the elemental analysis, the fluorine signal appears

above the 50% P180 composition. The dispersity of fluorine is evenly distributed in P180-rich

blend. In the 50:50 blend, fluorine signals are clustered along the fibre which correspond to

the surface roughness seen in the SEM image. This is likely co-crystallisation due to the

different Tc between P180 and PCL. Accompanying quantitative measurements were taken

for C, O and F content in fibres using EDX, shown below in Table 2.

Table 2 EDX elemental analysis of C, O and F content in PCL vs P180:PCL fibres printed by MEW.

P180 content % C (+0.1%) O (+0.1%) F (+0.1%)

0 (PCL) 72.8 27.2 0.0

25 71.9 22.8 5.3

50 65.1 18.2 16.7

75 56.5 8.8 34.7

Figure 20 SEM (left) and EDX images (middle to right) of P180:PCL printed fibres (scale bars: 50 μm).


28

The O and F content are used to indicate the amount of PCL and P180 respectively.

The blend composition showed the F content to increase as intended, accompanied by a

negative control. The clusters observed in the 50:50 blend is confirmed quantitatively as

similar levels of O and F are detected. To further confirm whether these results are influenced

by MEW processing, EDX measures were repeated on blends prior to printing. Quantitative

results of C, O and F content of whole blends are displayed in Table 3.

Table 3 EDX elemental analysis of C, O and F content in PCL vs P180:PCL blends.

P180 content % C (+0.1%) O (+0.1%) F (+0.1%)

0 (PCL) 72.8 27.2 0.0

25 59.7 17.9 22.5

50 59.9 17.2 22.9

75 46.9 5.5 47.6

Similar levels of fluorine content were exhibited between 25% and 50% PVDF

compositions, likely due to the blending preparation with the compounder. However, a positive

trend is still observed – with an approximate increase of 25% from 50% to 75% P180 blend.

From these results it is seen that a more disperse F content is achieved when the blend

composition contains an even amount of C and F with a significantly low O content. To

investigate the miscibility of blends, the surface morphologies of each fibre were imaged at

higher magnifications (Figure 21).


29

Figure 21 SEM images of surface morphologies of fibres of PCL and P180:PCL blends at 2000X (first row), 5000X

(second row) and 10000X (third row) magnifications (scale bars: 10, 5 and 2 μm respectively).

The surface morphologies of fibres varied between blends. The surface morphology of

PCL depicts spherulites with radial centrosymmetric organisation. From PCL to PCL-rich

blend, the crystallisation changes from larger spherulitic crystals to smaller spherulites

surrounded by amorphous regions. Lamellae radially protrude outwards from each spherulite

with smaller ridges observed within the amorphous regions. Strained bridges occurring

between large crevices in the PCL-rich blend are no longer observed in the 50:50 blend. The

50:50 blend appears more dendritic, with porosity appearing on the spherulites. The

spherulites appear more pronounced in all magnifications compared to the PCL-rich blend.

These hierarchical spherulitic formations are no longer present in the P180-rich blend. The

surface morphology appears smooth with fibril-like crystallisation accompanied by regions of

co-crystallisation with parallel laminar striations seen to the right of the 5000x magnification

image. This is indicative of the separate Tc temperatures of PCL and P180, in which P180

crystallises first surrounded by molten PCL acting as an amorphous material. The minimal

amount of PCL in the P180-rich blend corresponds to the minimal amount of the amorphous

regions (striations) present. Homogenous crystallisation of P180 is suggested with the 2D

fibril-like surface, uninterrupted by spherulites characteristic of PCL crystallisation. This was

also observed in literature, where a PDVF-rich blend inhibited the crystallisation of a lower Tm

polymer, PBA, due to confinement effects[26,27]. However smaller amounts of PVDF was

found to act as a nucleating agent, accelerating the crystallisation of PBA[26,27]. Additionally,

it has been found that PVDF forms a homogeneous, dense and smooth surface at

temperatures above 90 ºC[28] which is well exceeded by the set printing temperature. The


30

EDX elemental analyses suggests these blends to be miscible as F content appears to

penetrate the clustered regions, seen here as spherulites.

Notably a nano-sized fibre is present on the P180-rich blend- a product from jet

instabilities, i.e. whipping. Nonetheless, this demonstrates the potential for a variety of micron-

sized fibres to be formed using this blend. In essence, it was found that blending allowed for

the printability of P180 and that blend composition influences the surface morphology and F

content of printed fibres.

3.3 Conclusions and Future Directions

The extrudability and drawability of PVDF-based polymers were investigated in this

chapter. The heat treatment seems to have little effect on the rheological properties of neat

polymers. PCL and PP exhibited lower viscosities compared to P180 and P275. The branched

ter-PVDF and P530 were unable to form a melt for both the rheometer and MEW setup.

Moreover, only P180 exhibited drawability and extrudability with MEW and thus was chosen

to blend with PCL to improve its printability. Blending P180 and PCL was found to significantly

decrease the viscosity for all compositions compared to their neat polymers. The decreased

viscosities of blends were also found to be compatible for printing. Thus, the meltability,

cooling behaviour and rheological behaviour under shear rate were indicative of their

compatibility with MEW processing. The P180:PCL scaffolds were found to positively correlate

in F content with increasing P180 content. The F content was also more evenly dispersed at

an O:F ratio of 8.8:34.7%. The clusters seen in EDX were attributable to the spherulitic

crystalline regions of PVDF. All blends appear miscible but vary in surface morphology

depending on the composition. P180-rich blend resulted with a smoother surface in contrast

to the spherulitic surface of both the 50:50 and PCL-rich blend. In conclusion, blending has

demonstrated to be a viable approach for creating compatible printing materials with controlled

surface morphologies of fibres by controlling weight percentages.

It would be ideal to measure the elongational viscosities and drawability of polymer

melts at higher temperatures using an elongational rheometer or capillary rheometer in a

closed system to investigate the melt behaviour of the higher molecular weight and branched

PVDF polymers.

Furthermore, the piezoelectric and pyroelectric properties combined with its ability to

readily form flexible sheets has attracted much attention for various applications. To advance

the applicability of PVDF in various fields, more complex shapes are desired. Blending PVDF

with other elastomers to achieve piezoelectric polymer systems have found that increasing


31

amounts of blending polymers, e.g. poly(methyl methacrylate) (PMMA) and poly(vinyl acetate)

(PVAc), decreased the crystalline content in PVDF blends[29]. To determine whether this is

the case for PVDF-PCL blends investigated, the crystalline phases of PVDF can be analysed

using Fourier Transform infrared spectroscopy (FTIR) and Wide-angle X-ray diffractometry

(WXRD) techniques. The IR spectrum can determine the amounts of each phase in the blends

whilst the nature of interactions such as hydrogen bonding in the polymer blends can be

identified. The specific intermolecular interactions in the binary mixtures can be analysed

through shifts in the IR absorption peaks. The integrated intensities in XRD can be taken to

measure the degree of crystalline orientation of each phase as well as confirm the crystalline

content compared to IR between the weight percentages of blends. Spectra displaying

diminished intensity of major peaks would be characteristic of a semi-crystalline structure.

In terms of the piezoelectric behaviour of PVDF, when blended with PMMA and PVAc

piezoelectric constants d33 were found to decrease with increasing weight percentages[29]. It

would be interesting to test whether MEW processing diminishes this effect due to the induced

poling through extrusion.

4. PVDF as a Sacrificial Template for Microfluidics

32


The potential application of PVDF fibres as sacrificial templates in Liquid Glass will be

discussed in this chapter. In previous experiments, it was found that PCL was unstable when

cured in Liquid Glass. It was reported that solubility tests of PCL and PP in the uncured

monomer solution showed PCL to dissolve over a 24-hour period whilst PP remained stable.

Herein, the embedded PP fibres in Liquid Glass will be further analysed using SEM and EDX.


Fibres casted in the silica-based liquid glass cured using ultraviolet light (UV) were

imaged under SEM and the F and Si content was detected using EDX (Figure 22). To test the

influence of curing times, samples were cured either by irradiating the top side of the sample

(single cured) or by irradiating the top and bottom side of the sample (double cured). Samples

were double cured to ensure no partially cured monomer remained around the fibres.

Figure 22 SEM (left) and EDX of fluorine (middle) and silicon (right) of embedded PP fibres in liquid glass was

imaged. Scale bars = 50 μm.

The PP fibre within the polymer matrix appeared intact with no leaching based on the

detected F content in contrast to the previous report with PCL. The F content for the single-

and double-sided curing conditions were similar (11.2 + 0.1% and 12.1 + 0.1% respectively).

The surface morphology appears homogenously smooth with aligned edges from the

spinneret nozzle during extrusion. Particles appear to cover the fibre and substrate on both

samples however the elemental analysis depicts it to be fragments of the liquid glass and/or

sputtering artefacts. Overall, this follow-up investigation has confirmed the potential for PP as


33

a sacrificial template to fabricate microchannels. Print attempts of the 6-inlet design was

conducted using PP (Figure 23).

Figure 23 Stereomicroscope images of printing optimisation of PP via variation in collector speed (mm/min), pressure (bar) and needle gauge size (G). Printing temperature was set to 190 ºC for all prints. Scale bars = 1 mm for all excluding bottom left (= 400 μm).

The printing conditions were optimised by systematically altering one component-

based parameter at a time, i.e. collector speed, pressure and needle diameter. A working

distance of 1.5 mm and potential difference of 3 kV was found to significantly stabilise the jet

(indicated by a consistent extrusion, i.e. minimal jet breaking) and was kept constant for the

following optimisation experiments. Initially, the smallest needle gauge (30G, 0.30 mm)

available was utilised and the most optimal fibre deposition was observed at a speed of 5000

mm/min. However, the morphology of the fibres formed were inconsistent in diameter and

undulations from whipping were still observed (Figure 24). It was hypothesized that the needle

gauge was too narrow resulting in an inconsistent polymer mass flow. Accordingly, a larger

needle (26G, 0.45 mm) was equipped. An accurate fibre deposition and consistent fibre

morphologies and diameters were seen for speeds from 800 mm/min to 1250 mm/min.

Inconsistencies begin to occur from 2500 mm/min. Below this speed, the fibre diameters

remained constant despite the change in speed. Thus, further optimisation that could provide

a larger range of fibre diameters was pursued. One approach to decrease fibre diameters is


34

through reduced pressure which in turn decreases extrusion. The combination of decreased

pressure and larger needle gauge produced ideal fibre diameters with consistent

morphologies over a range of speeds. Notably, the collector speed of 5000 mm/min still

produced jet instabilities in the form of jet dripping (Figure 24). Moreover, an additional

experiment at 1 bar was also conducted but produced inconsistent fibres presumably due to

breaching the lower CTS limit for this pressure. The production of various fibre diameters using

a collector speed of 2500 mm/min with an applied pressure of 2 bar was considered ideal.

Therefore, these parameters utilised for printing the 6-inlet microfluidic design (Figure 25).

Figure 24 Stereomicroscope images capturing jet instabilities in the form of whipping (left) and dripping (right) occurring at a collector speed of 5000 mm/min in two needle gauges (30G, 26G). Scale bars = 50 μm.

Figure 25 Stereomicroscope images of 6-inlet prints including a reproducibility challenges with same conditions (left and centre) and increased temperature (right) which failed to improve print quality. Prints were conducted at 2500 mm/min, 2 bar, 3 kV and a 1.5 mm working distance. Scale bars = 2000 μm (top row) and 400 μm (bottom row).

The programmed collector speed of 2500 mm/min was utilised to achieve the thinnest

fibres diameters. The deposition of the inlets was satisfactory for most samples with the

occasional long beading along the fibre. The first major issue was reproducibility amongst

subsequent prints under the same printing conditions (Figure 25). It was presumed that this


35

reproducibility issue was due to heat gradients within the chamber creating jet instabilities. To

provide greater assurance of the polymer forming a homogenous melt, the temperature was

increased to 220 ºC (software read-out). Yet the long beading was only enhanced. In addition,

it is seen that a major challenge is fibre stacking to merge the inlets into a single outlet. Charge

accumulation is thought to be a major issue here as previously deposited fibres repel the

subsequently printed fibres. Further print optimisation by including reduced programmed

speeds at turning points may improve the angles of the incoming inlets however a major

consideration to reduce fibre jumping would be to increase the humidity of the printing

chamber to distribute electrostatic charge. In electrospinning, relative humidity has already

been found to affect print quality[30]. Water vapour acts as a charge carrier[31]. This is

facilitated by its high dielectric constant indicative of a high energy storage capacity by means

of polarization[30]. Thus, sufficiently high humidity environments provide the necessary

number of water molecules for electrostatic discharge. In low humidity environments, the

accumulation of charge density at the spinneret results in jet instabilities. Repeated

experiments in higher relative humidities are therefore expected to significantly increase the

print quality of these 6-inlet designs. As higher humidity environments were not possible with

the current MEW setup, investigations proceeded to the casting step (Figure 26).

Figure 26 Stereomicroscope images of 6-inlet construct prior to casting (left) and after casting (right) with Liquid Glass. Scale bars = 2000 μm (left) and 1000 μm (right).

To assess the viability of this design to fabricate a microfluidic chip, the construct was

casted and partially cured (single curing) in Liquid Glass. Similar to the single fibre construct

of PCL, PP fibres were found to move during the dropwise addition of liquid glass. It was then

decided that an adhesive coating, i.e. hairspray (HS), would be applied and tested. No

brightfield-images were taken as the liquid glass was fully cured to an opaque state. However,


36

it was observed that HS had little effect on preventing the movement of fibres during addition

of liquid glass. This major challenge is especially caused by the large amount of liquid glass

required to cover the delicate single fibre constructs. Previous liquid glass samples have been

produced using direct laser writing. These samples were small-scale sacrificial templates that

required smaller amounts of liquid glass[32]. Nevertheless, the main advantage of using MEW

with this casting process is the possibility of fabricating larger microfluidic chips with high

resolution channel profiles. Promising solutions include adhesive or pre-coated printing

surfaces as the HS was only applied after the print. Furthermore, to assess whether HS

affected the surface morphologies of the chip or fibres, SEM and EDX analyses were

conducted (Figure 27).

Figure 27 SEM and EDX images of PCL fibres (blue) vs hairspray-coated (HS) PP fibres (red) embedded in Liquid Glass (green). Si, C, O and F content are shown in green, blue and red respectively. Scale bars = 200 μm (PCL), 20 μm (PP).

The application of HS on fibres was tested for a fully cured process in which both sides

of the sample are exposed to UV until the liquid glass appeared opaque. This was to determine

whether the presence of this coating either enhanced or diminished the adhesion between

completely cured liquid glass and the PP fibre. The glass substrate is suspected to undergo

shrinkage when fully cured, as it was previously demonstrated to shrink by 26.3% during

sintering[33]. This was compared to a previous sample of PCL which leached after exposed

to UV. In contrast, the PP fibre is intact and F content was detectable however a gap is found

between the fibre and the channel. This gap may not be an issue in future fabrication methods

with liquid glass once the double casting method[33] is implemented, or more ideally, injection


37

moulded. Alternatively, the presence of fluorine may repel the liquid glass substrate. This may

limit dimensions of channel profiles and must be considered in future experiments. Moreover,

the Si content (green) was significantly decreased compared to O and C content (blue).

According to literature, the major component of HS are polymeric film-forming agents, .e.g.

poly(vinylpyrrolidone)[34]. It is likely that the C and O content observed is a polymer film that

has been transferred onto the chip surface resulting in a superimposition of the Si signal. A

consideration with future coatings is whether it interferes with the adhesion of liquid glass with

itself as this may lead to issues with chip bonding. Overall, PP has demonstrated to be a more

viable sacrificial template for this application than PCL. Future considerations for printable

materials for this application include: (a) higher melting points than PCL to withstand the UV

curing step and (b) fluorine-free materials to minimise gap formation between the fibre and

chip interface.

Once these initial fabrication steps are addressed, it is hypothesized that further

optimisation is required for the sintering process as by-products, produced by thermal

degradation of PVDF, include hydrogen fluoride (HF) (Figure 28)[35].

Figure 28 Thermal degradation mechanism of α- and β-phase PVDF viewed as the left and right reaction paths

respectively.[35]

The production of HF was observed in α- and β-phase PVDF degradation mechanisms

at >670 K. Studies revealed that the degradation occurs in two steps for α- and β-phase

samples[35]. The production of HF is a significant factor as it is commonly used to etch silica

substrates[36]. Given α- and β-phases are the most common forms of PVDF, significant

changes to the channel profile and morphology are predicted after sintering. Future


38

experiments must consider ways to minimize HF production to prevent undesirable etching

and avoid hazardous and poisonous waste products during the fabrication of these chips.

4.2 Conclusions and Future Directions Embedding PP fibres in liquid glass was found to be a viable method to potentially

create microchannels using MEW. The fibres appear to be stable, independent of curing time.

Further investigations into whether reminiscent fluorine is detectable in channels after

sintering may be valuable to provide chemically inert microchannels. Significant improvements

must also be made to the fabrication process: (a) print optimisation through increased

humidities during prints; (b) adhesion of fibres to the glass substrate by pre-coating the printing

surface; and (c) reducing gap formation between channel and fibre surface either through an

alternative sacrificial template or by providing infill through double casting or injection

moulding. After the casting process is optimised, ways to reduce HF production during

sintering steps must be considered to minimise etching and hazardous waste products.

The overall outlook of this research is the complete automation of fabricating

microfluidic chips. The main concept to achieve automation is through a multi-head printer

setup containing the MEW material, monomeric liquid glass mixture and an attached UV lamp

to simultaneously print and cast fibres in a single process. This would achieve high resolution

channels spanning a larger surface area at a high production rate. Developing this technology

would allow the fabrication of microfluidic chips on an industrial scale.


39


5.1 Polymer Melts

The neat PVDF polymers (180, 275 and 530 kg/mol) were purchased from Sigma-

Aldrich. The PVDF-TrFE-CTFE polymer (Piezotech RT-TS) was sourced from Piezotech

(Pierre-Benite, France). The PVDF powder (Piezotech Kynar RC10.287) was generously

gifted by Piezotech (Pierre-Benite, France). PCL (PURASORB PC 12kg/mol) was purchased

from Corbion Inc, Netherlands. All polymers were used as received.

5.2 Methods 5.2.1 Micro-Compounder

An Xplore MC5 twin screw micro-compounder was used to measure the average melt

viscosities of all neat and blended polymers. The compounder was heated up to 200 ºC or 95

ºC, for PVDF-based polymers and PCL respectively, and decreased by increments of 5 ºC,

over a temperature range of 20 ºC, for each melt viscosity measurement. A melt viscosity

value was taken every 10 seconds (over a 10-minute measurement) and averaged at that

given temperature.

5.2.2 Rheometer Set-up

For rheology experiments an Anton Paar Physica MCR 301 (Anton Paar, Austria) with

a plate-plate setup was used. The base plate was heated up to 75 ºC or 190 ºC for PCL and

PVDF-based polymers respectively. Approximately 2-4 g of pellets were placed onto the

sample area of the base plate until it was sufficiently filled with molten material. For preparation

of the measurement a PP20 plate was lowered onto the sample plate to obtain a final gap of

0.05 mm.

5.2.3 MEW Set-up

For the temperature measurements within the heating chamber and at the spinneret,

two temperature probes were inserted into a syringe and placed in the chamber and insulated.

The temperatures were measured after a minimum of 30 minutes at the set temperature.


40

5.2.4 Printing Parameters

A glass syringe was packed with polymer and fitted with nozzles (Nordson Deutschland

GmbH, Germany). The heating rings were programmed for 190 ºC (Bosch Rexroth AG,

Germany). Polymer mass flow was controlled by a pneumatic-based system using N2 gas. A

voltage difference was supplied to the nozzle and the collector plate with a set working

distance (WD) between the spinneret tip and collector plate.

Table 4 Printing parameters for PVDF-based polymers and PCL box constructs.

Parameters PVDF PCL Polymer load 1.5 + 0.5 mL 1.5 + 0.5 mL

Spinneret tip, needle length,

protrusion (from printer head)

24 G, 7.00 mm,

1.0 + 0.2 mm

24 G, 7.00 mm,

1.0 + 0.2 mm

WD (tip to collector) 2.5 + 0.2 mm 2.5 + 0.2 mm

Pressure 3.0 bar 3.0 bar

Voltage 3.00 + 0.01 kV 3.00 kV

Temperature 190 190

Collector Speed 800 mm/min 800 mm/min

5.2.5 Dynamic Mechanical Analysis

The dynamic mechanical properties of the samples were tested using a METTLER

TOLEDO STARe DMA1 System. A single cantilever clamp was used in bending mode at a

frequency of 1 Hz and a heating rate of 2 K min-1 under ambient conditions. All samples were

heated from 25 °C to 250 °C.

5.2.6 Imaging of Constructs and Embedded Fibres

Printed constructs were photographed using a stereomicroscope (Discovery V20, Carl

Zeiss Microscopy GmbH, Germany) for the evaluation of structures at low magnification. For

high resolution images of the surface morphology of fibres an SEM was used (Crossbeam

340, Carl Zeiss Microscopy GmbH, Germany). For elemental analysis, the C, O and F content

for each sample was measured using an XMaxN 50mm2 EDX system.

5.2.7 Embedding PP Fibres in Liquid Glass

The liquid glass monomer mixture was kindly donated by Dr. Bastian Rapp’s

NeptunLab based at the Institute of Microstructure Technology, Karlsruhe Institute of

Technology (KIT). In short, 68 % (v/v) HEMA, 7 % (v/v) tetraethyleneglycol diacrylate


41

(TEGDA), and 25 % (v/v) phenoxyethanol (POE) were mixed prior to dispersion. A dispersion

of 40 % (v/v) Aerosil OX50 was added to the mixture and stirred. In small increments, the

nanopowder was added and homogenized. A 0.5 (wt. %) photoinitiator DMPAP waqqs added

and further homogenized. The slurry was degassed to remove air bubbles. The casting protocol established by Kotz and co-workers were reproduced[33]. Prior

to casting, a rectangular moulding frame of silicon was cut and placed around the print. Single

fibre constructs on glass slides were casted by dropwise addition of Liquid Glass. Samples

were cured using a UV light curing modular flood lamp system (λ = 249.7 nm; Dymax ECE

5000, United States) for 90 seconds per side (single or double). For the morphological analysis

of the protruding fibre, SEM was used (Crossbeam 340, Carl Zeiss Microscopy GmbH,

Germany).

References

42

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Appendices

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Appendix All materials used in this thesis not generated by the author were reprinted with permission of

the copyright owners. These permissions were obtained under Creative Commons Attributions

Licenses, or via the RightsLink Copyright Clearance Centre. The material was used either in

its original form or with minor alterations, and the source was referenced appropriately in

Figure captions.

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