MELT ELECTROSPINNING AS AN ADDITIVE MANUFACTURING … · The use of scaffolds to replace lost bone...

Melt electrospinning as an additive manufacturing technique

© 2018 Nikola Ristovski Page i

MELT ELECTROSPINNING AS AN

ADDITIVE MANUFACTURING TECHNIQUE

Nikola Ristovski

B. Eng (Medical)

Submitted in fulfilment of the requirements for the degree of

Master of Engineering (Research)

Science and Engineering Faculty (SEF)

Institute of Health and Biomedical Innovation (IHBI)

School of Chemistry, Physics and Mechanical Engineering (CPME)

Queensland University of Technology

2018


© 2018 Nikola Ristovski Page ii

Keywords

Additive Manufacturing; Biomimetic Scaffold; Bone Fracture; Bone Tissue; Bone Tissue

Engineering; Electrospinning; Electrospinning Electric Field; Electrospinning Humidity;

Fibre Deposition; Fibre Laydown; Melt Electrospinning; Microfibres; Order Quantification;

Polycaprolactone; Polymer Charge; Polymer Melt; Scaffold Order; Scaffold Production;

Scaffolds; Tissue Engineering


© 2018 Nikola Ristovski Page iii

Abstract

Bone tissue engineering aims to study and produce bone tissue for patients who have

undergone physical trauma or disease related losses in bone tissue that is unable to repair naturally.

The use of scaffolds to replace lost bone tissue is a major field within bone tissue engineering, and

the use of melt electrospinning is gaining momentum as a method of production. Recent studies

show that melt electrospinning is capable of producing highly ordered scaffolds with fibres on the

micrometre scale. This allows bone tissue engineers to have unprecedented control over scaffold

microarchitecture, allowing for the production of biomimetic scaffolds. The aim of this study is to

demonstrate that melt electrospinning is capable of producing highly ordered structures.. This was

completed by first determining the locus and rate of discharge of charge stored in the polymer.

Results showed that charge build up was minimized by distributing the application of the electric

field between the emitter and collector. Further studies to determine whether residual charge would

affect cell proliferation throughout the scaffold and whether humidity played a key role in charge

dissipation was completed. Using a distributed charge produced scaffolds up to 200 layers high

with little to no loss in laydown accuracy, almost an order of magnitude greater than what was

previously possible. Murine calvarial cells were seeded onto the structure, showing little evidence

of cell death. A study completed in conjunction with S. Liao et al. showed little effect to the

structural order of scaffolds with changes to ambient humidity. This study concluded that it was

possible to use melt electrospinning as a method to produce highly ordered scaffolds. It

demonstrated that the method is a promising candidate to produce scaffolds for tissue engineering.


© 2018 Nikola Ristovski Page iv

Table of Contents

Keywords ............................................................................................................................................... ii

Table of Contents ...................................................................................................................................iv

List of Figures ........................................................................................................................................vi

Acknowledgments ................................................................................................................................ xii

1 CHAPTER 1: INTRODUCTION ................................................................................................... 1

1.1 Aims and Hypothesis ................................................................................................................... 3 1.1.1 Aims ................................................................................................................................. 3 1.1.2 Hypothesis ........................................................................................................................ 6

1.2 Overview ...................................................................................................................................... 1

1.3 Purpose of Research ..................................................................................................................... 6

1.4 Significance of Research .............................................................................................................. 7

1.5 Thesis Outline .............................................................................................................................. 8

2 CHAPTER 2: LITERATURE REVIEW ....................................................................................... 9

2.1 Bone Fracture Healing and tissue engineering ............................................................................. 9 2.1.1 Bone Anatomy .................................................................................................................. 9 2.1.2 Tissue Engineering ......................................................................................................... 12

2.2 Melt Electrospinning .................................................................................................................. 20 2.2.1 The Physics of Electrospinning ...................................................................................... 21 2.2.2 The Governing Equations of Electrospinning ................................................................. 27

2.3 Charge Transport in Electric Jets ............................................................................................... 27 2.3.1 Coronal Discharge in Taylor Cone and Fibres................................................................ 29 2.3.2 Parasitic Electrospraying ................................................................................................ 31 2.3.3 Atmosphere and Charge Evaporation ............................................................................. 31 2.3.4 Residual Charge in the Polymer ..................................................................................... 32

2.4 Implications for Melt Electrospinning as an Additive Manufacturing Technique ..................... 34

3 CHAPTER 3: POLYMER EXTRUDER DESIGN ..................................................................... 36

3.1 Outline of the Requirements ...................................................................................................... 38

3.2 Design and Implementation ....................................................................................................... 38 3.2.1 Heating Jacket ................................................................................................................. 39

3.3 Pressure System ......................................................................................................................... 41

4 CHAPTER 4: HUMIDITY SYSTEM DESIGN ........................................................................... 43

4.1 Outline of the Requirements ...................................................................................................... 43

4.2 Design and Implementation ....................................................................................................... 44 4.2.1 The Saturator .................................................................................................................. 44 4.2.2 The Desiccator ................................................................................................................ 44 4.2.3 The Mixing Chamber ...................................................................................................... 46

5 CHAPTER 5: IMPROVED FABRICATION OF MELT ELECTROSPUN TISSUE

ENGINEERING SCAFFOLDS USING DIRECT WRITING AND ADVANCED ELECTRIC

FIELD CONTROL. ........................................................................................................................... 49

5.1 Introduction ................................................................................................................................ 51

5.2 Materials and Methods ............................................................................................................... 53 5.2.1 Melt Electrospun Scaffolds ............................................................................................. 53 5.2.2 Scaffold Characterisation ................................................................................................ 55


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5.2.3 In Vitro Characterisation ................................................................................................ 56 5.2.4 Statistical Analysis.......................................................................................................... 58

5.3 Results and Discussion .............................................................................................................. 58 5.3.1 Physical Characterisation ................................................................................................ 58 5.3.2 Zonal Characterisation of structure order ....................................................................... 65 5.3.3 In Vitro Characterisation ................................................................................................ 67

5.4 Conclusion ................................................................................................................................. 69

5.5 Acknowledgements .................................................................................................................... 70

6 CHAPTER 6: CONCLUSIONS .................................................................................................... 71

6.1 Research Summary .................................................................................................................... 71 6.1.1 Summary of Research Paper ........................................................................................... 71

6.2 Limitations and Recommendations for Future Work ................................................................. 72 6.2.1 Charge Storage, Charge Dissipation ............................................................................... 72 6.2.2 Other Factors Causing Disorder and Near Field Melt Electrospinning .......................... 73 6.2.3 Scaffold Architecture and Order ..................................................................................... 74 6.2.4 Dynamically Controlled Electric Potential ..................................................................... 74

6.3 Final Discussion and Conclusion ............................................................................................... 74

7 CHAPTER 7: BIBLIOGRAPHY................................................................................................... 77

8 CHAPTER 8: APPENDICES ........................................................................................................ 86

8.1 Appendix B: Matlab code for order quantization ....................................................................... 86 8.1.1 Image Importer ............................................................................................................... 86 8.1.2 Image Analysis ............................................................................................................... 87 8.1.3 Fibre Positions ................................................................................................................ 88

8.2 Appendix C ................................................................................................................................ 89 8.2.1 Sample of Fibre Raw Image Data ................................................................................... 89


© 2018 Nikola Ristovski Page vi

List of Figures

Figure 2-1: The human skeleton indicating the different structures of bone. The axial skeleton forms the

pelvis, spine, ribs and skull. Appendicular skeleton is formed from the bones of the limbs [28]. . 9

Figure 2-2: Schematic diagram of long bone with close up’s showing sections of the marrow cavity and

structure of trabecular and cortical bone [30]. .............................................................................. 10

Figure 2-3: Stages of bone healing [31] ................................................................................................ 11

Figure 2-4: Different structures that are capable with a single type of polymer (PCL) that has been used

in a number of Additive manufacturing techniques and other fabrication techniques. Micro-

spheres (a, b). Nano-fibres (c, d). Foams (e, f). Knitted textiles (g, h, i). SLS scaffold (j-o). Fused

deposition modelled scaffolds (p–u) [20][38][39][40][41][42] .................................................... 13

Figure 2-5: A vascular stent, generally made from stainless steel 316L or titanium used to expand

sections of the artery [148]. .......................................................... Error! Bookmark not defined.

Figure 2-6: Schematic image of the fused deposition modelling system [149] ..................................... 15

Figure 2-7: Schematic diagram of a stereo-lithography system [150]................................................... 16

Figure 2-8: Schematic diagram of a selective laser sintering device [151] ........................................... 17

Figure 2-9: Tensile stress-strain curve of cortical bone with multiple strain rates [63] ........................ 18

Figure 2-10: An image illustrating Wolff’s law of bone, demonstrating he cancellous bone remodels

itself to the stress lines via mechano-transduction ....................................................................... 19

Figure 2-11: Schematic of an electrospinning apparatus. [75] .............................................................. 21

Figure 2-12: The dependence of melt viscosity on molecular weight. [82] .......................................... 22

Figure 2-13: Diagram showing the effect of creep and stress relaxation .............................................. 23

Figure 2-14: Schematic diagram of the emitter of an electrospinning system [88] ............................... 25

Figure 2-15: Momentum balance on a short section of the jet [79] ....................................................... 26

Figure 2-16: Visible coronal discharge in an electrospinning apparatus [103] ..................................... 29

Figure 2-17: Oscilloscope snapshot of the discharge current at different time points. The number on the

left corresponds to fibre accumulations for times of (1) 10 s, (2) 30 s, (3) 2 min, (4) 5 min, (5) 10

min, (6) 20 min [102] ................................................................................................................... 30

Figure 2-18: schematic diagram showing trapped charge (A, C) and empty sites (B, D) in a polymer

matrix [88] .................................................................................................................................... 33

Figure 2-19: Residual charge against time for positively and negatively biased polystyrene (PS)

scaffolds. [5] ................................................................................................................................. 34

Figure 3-1: Schematic of various heating approaches performed for melt electrospinning [9]............. 36

Figure 3-2: Melt electrospinning extruder designed by Hacker et al. [117] .......................................... 37

Figure 3-3: Line-like laser beam melting electrospinning developed by Shimada et al. [118]. ............ 37

Figure 3-4: Li et al.’s umbellate melt electrospinning extruder [119]................................................... 38

Figure 3-5: A comparison of the updated extrusion system (A) and the syringe pump extrusion system

(B) ................................................................................................................................................ 39

Figure 3-6: (A) Graph showing the dependence on the input temperature against the temperature at the

needle tip. (B) Imaging showing the distribution of temperature across the extruder, with the

extrusion tip facing the bottom. Modelled using ANSYS static thermal package (ANSYS,

USA)............................................................................................................................................. 40

Figure 3-7: Initial Design (A) vs. Final Design (B) of the extrusion system for the melt

electrospinner ............................................................................................................................... 41


© 2018 Nikola Ristovski Page vii

Figure 3-8: Schematic diagram of the pressure extrusion system ......................................................... 42

Figure 4-1: Image of saturator designed by Johnson et al. .................................................................... 44

Figure 4-2: Absorbance characteristics of common desiccants ............................................................. 45

Figure 4-3: Initial design for humidity control system. Control box is the melt electrospinning device,

which will act like a mixing chamber in this configuration. Temperature and filtering have since

been removed from the system ..................................................................................................... 46

Figure 4-4: Simulink model of humidity controller .............................................................................. 48

Figure 4-5: Transient response of humidity system .............................................................................. 48

Figure 5-1: Novel direct writing melt electrospinning platform with dual voltage power supplies for

improved fibre deposition control. The negative power supply attached to the collector plate being

the defining difference in this system. .......................................................................................... 54

Figure 5-2: Distribution of pore sizes for group D (+7 kV tip: -3.5 kV collector). The data shows two

apparent peaks at 282 μm and 378 μm which can be attributed to the fibre separation in the x-y

plane. ............................................................................................................................................ 59

Figure 5-3: Fibre diameter for different voltage distributions as measured via SEM. Each value was

taken by manually measuring the diameter using ImageJ software (n = 48 fibres). The data shows

no statistical significance between any groups (p > 0.05). ........................................................... 60

Figure 5-4: Image of µCT reconstruction of scaffold from group D (+7 kV tip: -3.5 kV collector) with

an x-y fibre spacing of 500 µm..................................................................................................... 60

Figure 5-5: SEM cross-sectional images (x-z plane) of melt electrospun scaffolds with distributions of

voltages, varying from 0 to 10.5 kV, between the tip and the collector (see Table 1). (A-D) show

scaffolds produced using negative voltage on the collector plate. Scaffold (E) is the control; it is

produced by grounding the collector plate. (F) Illustrates the Structure order calculated via Eq. 1

for scaffolds of group A-D and Control. Os=1 indicates perfect fibre stacking across all layers,

Os>1 indicates fibre stacking disorder. * indicates a statistical significant mean against control (p

< 0.05). Uncertainties are computed from the standard deviation. ............................................... 62

Figure 5-6: Change in fibre order as the number of layers increases. The vertical inter-fibre distance

taken from the axial centre of the fibres indicates fibre order. The results correspond to scaffolds

from group D (+7 kV tip: -3.5kV collector). ................................................................................ 63

Figure 5-7: A scaffold with 1 mm fibre spacing produced using group D (+7 kV tip: -3.5 kV collector).

The scaffold reached a height of 200 layers (2 mm thickness). This illustrates that by decreasing

the density of fibres, the stacking increases. ................................................................................. 64

Figure 5-8: SEM micrographs of a scaffold from group B showing x-z cross section. This illustrates the

zonal arrangement of the fibre networks. Sections a, b, and c, are magnified sections of scaffold.

The first zone shows a highly ordered structure with large levels of control on fibre deposition.

The secondary zone (semi-ordered) demonstrates some level of control; however the position of

fibre deposition is largely influenced by electrostatic forces. The final zone (disordered) shows a

complete lack of fibre control with deposition dominated by electrostatic forces. ...................... 66

Figure 5-9: Maximum heights of the ordered zone for scaffolds fabricated for phase 1. Groups A –D

had significantly larger ordered zones compared to the control group (p < 0.05). ....................... 67

Figure 5-10: (A) and (B) Cell distribution illustrated using a live/dead stain indicating the presence of

cells across the surface of the scaffold. Images (C) and (D) show cell morphology as imaged

through a DAPI/Phalloidin stain, illustrating cells attaching and spreading on the scaffold fibres

illustrating good cellular interaction. ............................................................................................ 68

Figure 5-11: MTT data of the percentage increase in cell growth showing an approximately uniform

level of cell proliferation for all scaffolds (n = 6) (p < 0.05). Errors bars indicate standard

error. ............................................................................................................................................. 69


© 2018 Nikola Ristovski Page viii

List of Tables

Table 1-1: Table indicating the effects of the independent parameters on the electrospinning jet. ......... 4

Table 1-2 : Table outlining the range with which the parameters were adjusted during the melt

electrospinning optimization. ......................................................................................................... 4

Table 1-3: Table describing the parameters associated with electrospinning. TTC described the distance

between the needle tip to the collector plate and translational speed is the speed of the X/Y/Z

stages. ............................................................................................................................................. 4

Table 5-1: Phase 1 scaffold groups and their corresponding needle tip and collector plate voltages. Six

scaffolds were produced for each group. ...................................................................................... 55

Table 5-2: Number of scaffolds and structural parameters produced for phase 2 of the study. ............ 55


© 2018 Nikola Ristovski Page ix

List of Publications

Ristovski, N., Bock, N., Liao, S., Powell, S.K., Ren, J., Kirby, G.T., Blackwood, K.A. and

Woodruff, M.A., 2015. Improved fabrication of melt electrospun tissue engineering scaffolds

using direct writing and advanced electric field control. Biointerphases, 10(1), p.011006.


© 2018 Nikola Ristovski Page x

List of Abbreviations

One Dimension..................................................................................................................................... 1D

Two Dimensions .................................................................................................................................. 2D

Three Dimensions ................................................................................................................................ 3D

4', 6-diamidino-2-phenylindole ....................................................................................................... DAPI

Dimethyl Sulfoxide ....................................................................................................................... DMSO

Extra Cellular Matrix ...................................................................................................................... ECM

Food and Drug Administration ........................................................................................................ FDA

Fused Deposition Modelling ............................................................................................................ FDM

Fluorescein Diacetate ....................................................................................................................... FDA

3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide .................................................. MTT

Polycaprolactone .............................................................................................................................. PCL

Poly vinyl alcohol ........................................................................................................................ PVOH

Propidium Iodine .................................................................................................................................. PI

Polystyrene .......................................................................................................................................... PS

Scanning electron microscopy ........................................................................................................ SEM

Selective laser sintering ..................................................................................................................... SLS

Stereo lithography ............................................................................................................................ SLA

Tip to collector ................................................................................................................................ TTC

William-Landel-Ferry ..................................................................................................................... WLF


© 2018 Nikola Ristovski Page xi

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet requirements

for an award at this 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: __17/04/2018______________

QUT Verified Signature


© 2018 Nikola Ristovski Page xii

Acknowledgments

I would like to acknowledge the entire biofabrication and tissue morphology group for their

help throughout my Masters. In particular I’d like to thank my supervisors Dr. Sean Powell,

Assoc. Prof. Mia Woodruff and Dr. Keith Blackwood for all their help and guidance

throughout. I’d like to thank my family for all of their support during my masters and finally

(in no particular order) I’d also like to thank Sha Pather, Pelin Tufekci, Nicole and Michael

Bartnikowski, Kristopher Bogoevski, Andy Yaun, Stephanie Fontaine and Sam Liao. A

special mention to Giles Kirby, Patrina Poe and Nathalie Bock for helping me out at the

beginning of my studies, and Christina Theodoropoulos and Soniya Yambem for the help at

the end of my studies. For anyone else I forgot to mention, I thank you as well.

Chapter 1: Introduction

© 2015 Nikola Ristovski Page 1

1Chapter 1: Introduction

1.1 OVERVIEW

Musculoskeletal injuries make up approximately 10.3% of all medical spending in the United

States (which in 2008 equated to USD$117.2 billion). In the US, long bone, non-union

fractures make up approximately 10% of all non-fatal injuries and are the number one category

for inpatient expenditure [1]. Australia alone spends approximately AUD$15 billion per

annum on musculoskeletal disorders [2]. These statistics illustrate that musculoskeletal

conditions are currently a major burden on the medical industry globally and this challenge is

predicted to increase with an ageing population [2].

Bone is capable of remarkable repair and regeneration; however, it is not able to fully heal if

the fracture site is too large. In addition, current surgical intervention techniques often lead to

inconsistent results [3]. Post-operative complications in bone healing occur in approximately

14-28% of cases, with non-union being a major outcome [2], [3]. Current methods of treatment

for non-union fracture sites include the autograft and allograft. An autograft is the removal of

the patients own tissue from a donor site for placement into the fracture site whereas an

allograft transplants the required tissue from another human to the fracture site. While these

treatments are the current gold standards in non-union fractures, drawbacks exist [4]. For

autografts, complications include surgical (in more than 30% of cases), donor site morbidity,

and limited availability of donor material [5]. Allografts also suffer similar problems and have

the added issue of immune response complications. Hence, an approach must be formulated

that is capable of using minimal autologous tissue, or alternatively, negating its use entirely

through the development of implantable scaffolds. This scaffold based approach will provide

a high degree of flexibility to tackle the social, economic, and personal issues facing tissue

regeneration and repair.

Bone Tissue Engineering is the study of growth of new connective tissue and organs for the

production of devices to be implanted back into the donor site as replacements or for assisted

recovery, particularly for bone tissue. This field involves a range of diverse skills, including

mechanical and materials engineering, medicine and cell biology. Scaffold production for

insertion into the donor site is a major field within bone tissue engineering. This approach

provides physical support for cells to proliferate during the healing process. In the case of bio-

resorbable constructs, the supportive scaffolds degrade as the fracture heals leaving only

natural human bone [5].



The central aim of tissue engineering involves combining the body’s natural healing capacity

with an engineered solution in order to produce better outcomes [6]. As the influences of

micro-architecture, cell signalling and the local micro and macro environment on tissue growth

become clearer, tissue engineers will be able to produce optimal scaffolds for bone tissue

repair. The current generation of scaffolds are predominantly polymer based and consequently

are only osteoconductive but not osteoinductive. Osteoconductive implies that the scaffolds

are able to guide cell growth and osteoinductive implies that they are able to induce cell

differentiation and proliferation into the tissue of interest. Porter et al. [6] suggest that the

following list of requirements for a biomimetic scaffold be met:

1. Provide temporary mechanical support to the affect area.

2. Act as a substrate for osteoid deposition.

3. Contain a porous architecture to allow for vascularisation and bone ingrowth.

4. Encourage bone cell migration into the scaffold.

5. Support and promote osteogenic differentiation in the non-osseous, synthetic

scaffold (osteoinduction).

6. Enhance cellular activity towards scaffold-host tissue integration

(osteointegration).

7. Degrade in a controlled manner to facilitate load transfer to developing bone.

8. Produce non-toxic degradation products.

9. Not incite an active chronic inflammatory response.

10. Be capable of sterilization without loss of bioactivity and.

11. Deliver bioactive molecules or drugs in a controlled manner to accelerate

healing and prevent pathology.

A number of approaches to solving these problems have been developed and used

concurrently, including changes to microarchitecture [7], functionalization of the scaffold

surface [8], polymer modification, and incorporation of bioactive particles in the polymer

scaffold [9]. When appropriately combined, it is possible to solve a large number of these

challenges using these methods; this being a primary objective of tissue engineers today.

The microarchitecture of a tissue engineering scaffold can be produced using additive

manufacturing techniques. By producing the scaffolds in a layer-by-layer manner, it is possible

to introduce internal structures which are not easy to add using other fabrication methods.

Additive manufacturing is able to fabricate tissue engineered scaffolds using a range of

methods, the most popular being fused deposition modelling (FDM), selective laser sintering

(SLS) and stereo-lithography (SLA). Electrospinning (ES), which has traditionally been used

as a technique to produce fine disordered meshes, is another method with promise as an



advanced additive manufacturing technique [10]. Electrospun fibres are produced by drawing

a polymer/solvent solution through a large electric potential (greater than 10 kV) onto a

collector plate. Melt-electrospinning is an application of electrospinning where a polymer melt

is deposited onto a collector in a more controlled manner, producing 3D constructs in a method

akin to FDM. Due to the lack of solvent and much higher viscosity of the polymer, the

whipping experienced in solvent electrospinning is mitigated, allowing controlled fibre

deposition for 3D printing.

The overall aim of this master’s project is to advance direct writing melt electrospinning as a

superior additive manufacturing technique for tissue engineering by quantifying the

relationship between electric charges in the polymer, the electric field, and fabrication

stability. Polycaprolactone (PCL) is a commonly used polymer in tissue engineering [5] and

will be used in this study owning to its low melting point (60 °C), approval from the American

Food and Drug Administration (FDA) for clinical use, and its established use as a material

with melt electrospinning [7, 8]. Melt electrospinning is a process that was brought to the

attention of tissue engineers in the early 2000’s [13] due to its greater viscosity and subsequent

higher fabrication stability compared to its solution electrospinning counterpart. Due to this,

it enabled electrospinning to be applied as an additive manufacturing technique [12], however,

problems existed which prevented it from creating truly ordered structures above certain layer

heights [14]. This is, in part, due to charge build-up in the polymer preventing the scaffold

from being stacked accurately beyond a certain number of layers.

This project therefore seeks to:

Improve the understanding of charge build-up in a polymer in a high electric field

Quantify the degree of disorder due to the residual charge

Produce scaffolds to determine the effect of residual charge on stacking

1.2 AIMS AND HYPOTHESIS

1.2.1 Aims

The aim of this master’s thesis was to observe the effective of distributing charge across the

emitter and collector of a melt electrospinning device to determine whether this would have a

positive impact on fiber laydown accuracy.

1.2.1.1 Optimizing the fabrication method in a melt electrospinner to produce 0-

90° cross-hatch scaffolds

Optimization of the melt electrospinning process was completed using Poly-Caprolactone

(PCL) (Perstop, Capa6430®). Each parameter was varied over a range determined by



previously reported values which successfully produced melt electrospun fibres [12], [11].

Studies completed from Table 1-1 were used to determine which parameters would be varied

and Table 1-2 lists the parameters that were adjusted in the study.

Table 1-1: Table indicating the effects of the independent parameters on the electrospinning jet.

Parameter Effect Study

Electric Field

- Decreases fibre diameter for increases field

- Increases charge storage for increased field

- Dictates the location of deposition

Lyons et al. [13] Zhmayev et al. [15] Zhang and Jan [16] Teo and Ramakrishna [17] Ristovski et al. [14]

TTC Distance

- Effects the electric field - Increases the cooling time before

deposition - Effects the position of deposition

with respect to the whipping zone

Dalton et al. [18] Hutmacher and Dalton [10] Huang et al. [19]

Temperature

- Effects the viscosity of fluids - Increase in temperature reduces

fibre diameter

Lee et al. [20] Dalton et al. [11] Zhmayev et al. [21] Deng et al.[22]

Translation Speed

- Changes the morphology of laydown of fibres

- Increases/decreases the accuracy of deposition

- Introduces whipping effects

Brown et al. [12] Sun et al. [23]

Polymer Flow Rate - Should be adjusted based on the

velocity of fibre extruded by the electric field

Lyons et al. [13] Deitzel et al. [24]

Table 1-2 : Table outlining the range with which the parameters were adjusted during the melt

electrospinning optimization.

Potential Difference TTC Distance Temperature Translation speed Polymer flow rate

5-15 kV 5-50 mm 60-90 °C 300-1200 mm/min 30-90 µL/min

The properties of the scaffold, such as diameter and order, were observed throughout the

optimization phase until the parameters from Table 1-3were determined to produce optimal

scaffolds:

Table 1-3: Parameter used to produce scaffolds. TTC described the distance between the needle tip to

the collector plate, and translational speed is the speed of the X/Y/Z stages.

Voltage (+) Voltage (-) TTC Distance Temperature Translation speed Polymer flow rate

7 kV -3.5 kV 10 mm 73 °C 1000 mm/min 45 µL/min



1.2.1.2 SEM analysis of cross sections of electrospun scaffolds to determine order

and heights of ordered regions

A method associated with tortuosity was created to determine the order of a scaffold produced

in the electrospinning device (tortuosity being the ratio of the length of a path taken and the

distance between the starting and ending points). Scaffolds were produced and the distribution

of the voltage between the tip and the collector was varied in 2 kV intervals. The distribution

in electric charge was then assessed by imaging the cross-section of scaffolds in using scanning

electro microscopy (SEM). The data was used to determine the height of the ordered region as

well as the order in the scaffold.

1.2.1.3 µCT analysis of electrospun scaffolds to determine the pore size and

distribution

An analysis of the internal structure of the electrospun scaffolds was completed using micro

tomography (µCT). The optimal scaffolds were determined using the SEM analysis. They

were then analysed using the µCT to determine the size and distribution of pores in the

scaffolds.

1.2.1.4 Assessment of the in vitro biocompatibility of a scaffold via live/dead

staining, DAPI/Phalloidin and MTT assay

Electrospinning parameters from the optimal group were used to produce scaffolds for cell

culture. Sterilized scaffolds were used to determine the biocompatibility of highly ordered melt

electrospun scaffolds. The scaffolds were seeded using MC3T3 cells for a period of 7 days. 3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed on

days 1 and 7 to determine the change in metabolic activity over the culture period. Live/dead

(fluorescein diacetate (FDA) (live) and propidium iodide (PI) (dead)) and 4', 6-diamidino-2-

phenylindole (DAPI)/ phalloidin assay were performed on the 3rd day of the trial as a

qualitative measure of biocompatibility and to determine the cell morphology, respectively.

This study was completed and published:

Ristovski, Nikola, Nathalie Bock, Sam Liao, Sean K. Powell, Jiongyu Ren, Giles TS Kirby,

Keith A. Blackwood, and Maria A. Woodruff. "Improved fabrication of melt electrospun

tissue engineering scaffolds using direct writing and advanced electric field

control." Biointerphases 10, no. 1 (2015): 011006.

Full details of the study are presented in-depth in chapter 4 as per publication [14].



1.2.2 Hypothesis

1.2.2.1 Negative voltage on the collector plate

The addition of a negative voltage power supply to the collector of the melt electrospinning

device will increase the deposition accuracy of the fibre over a larger period of overlapping

fibres (this study has been published in Biointerphases and comprises Chapter 5). When fibres

lay down onto the collector plate, residual charge is stored in the polymer, or on the polymer

surface. Studies have shown that the charge can remain on or in the polymer for days at a time.

The addition of a negative voltage to the collector plate will increase the potential difference

felt by the charges in their local environment. This will encourage the emission of charge from

the polymer to the collector plate. The smaller charge that is stored in the polymer, the less

columbic forces are felt on the fibre when it is being stacked, which will result in more order

in the scaffold as fibres are layered.

1.2.2.2 Quantifying order

Quantifying the order in a cross-hatch scaffold is necessary for comparing scaffolds produced

using the negative charge applied to the collector plate. Using a method analogous to

tortuosity, it is possible to do this. Tortuosity is the arc-chord ratio, or the ratio of the length

of a path to the distance between its ends. This property is used in a number of physical

characterisations, including porosity and interconnectivity of porous scaffolds. By measuring

the distance from the top and bottom fibres in a scaffold and finding the path taken from each

fibre, it is possible to calculate the tortuosity which will be used to quantify order within the

scaffold.

1.2.2.3 Incrementally improved melt electrospinning device

The melt electrospinning system that is currently used in the laboratory is a culmination of

years of research and development in the Biofabrication and Tissue Morphology (BTM) group

at QUT. However, as direct writing melt electrospinning is a fairly new technology, the system

is constantly improved based upon the outcome of current research. The following research

aims at improvements to the melt electrospinning device, as well as determining the effect of

a negatively charged collector plate on ordered scaffold production.



1.3 PURPOSE OF RESEARCH

The purpose of this research is to develop the hardware, theoretical models and experimental

results to improve the ability of direct writing melt electrospinning to produce morphologically

relevant scaffolds for tissue engineering. To achieve this, we grouped the research into four

aims; (1) Determine whether the melt-electrospinning fabrication process is dominated by

surface or volume charges and the relative charge-diffusion time-scales using a first principles

modelling approach. This is to provide a theoretical basis in order to understand how to

mitigate the unwanted charge effects. (2) To establish a method to characterise scaffold

structural order to enable quantitative comparisons between different samples, and (3) perform

an experimental study of electric field and polymer interactions in order to mitigate

undesirable electric charge accumulation and improve melt-electrospinning as a technique for

producing morphologically relevant tissue engineering scaffolds. This third aim involved

systematically varying the electric potentials on both the polymer extruder and the collector in

a custom made melt electrospinning system and conducting scaffold fabrication experiments.

Due to the tissue engineering application of these scaffolds, In vitro studies were also

undertaken to demonstrate that our fabrication technique had no adverse effect on cell

proliferation and morphology.

1.4 SIGNIFICANCE OF RESEARCH

Within the field of tissue engineered, additive manufacturing has become an established

technique for producing scaffolds. However, limitations exist with current approaches in their

application to this field. Methods such as FDM are able to produce complex structures but on

a scale larger than optimal for cell attachment and proliferation [25]. Conversely, techniques

such as SLA are able to produce extremely fine structural detail, but are limited to photo-

curable polymers, which are generally cytotoxic. Melt electrospinning enables the production

of fibres on a scale much smaller than FDM and any other plastic extrusion method, while

allowing for a relatively high degree of deposition control. However, advanced knowledge of

electric field/polymer interaction is required in order to produce scaffolds with high degrees

of structural order at a scale appropriate for tissue engineered constructs. The results of this

research have led to changes in the design and function of the BTM machines in subsequent

research.



1.5 THESIS OUTLINE

The aim of this project was to determine whether charge had an effect on stacking order and

whether that charge could be minimized to achieve improved layer structure in melt

electrospun scaffolds. This thesis presents the research involved to determine the effect of

charge on ordered melt electrospun scaffolds.

Chapter 2 provides a comprehensive literature review which was used to guide the decision

making during this research. Chapter 2.1 through to 2.3 provides the background of bone tissue

repair and the clinical need which motivated this study. Section 2.4 is an in-depth investigation

on the current research into melt electrospinning which, at the inception of this project, was a

little known field and has since become a major contender in the field of tissue engineering.

Section 2.5 presents why tortuosity was used as a method for quantifying stacking order in

melt electrospun fibres. Section 6 outlines the literature and reasoning behind the theory that

charge is the major cause of disorder in melt electrospun scaffolds. The final section outlines

the aims and hypothesis.

In chapter 3 and 4, this thesis will outline the hardware that was built to support the research

during the master’s project. An extensive amount of time went into building devices; chapter

3 discusses the construction of the polymer extruder, chapter 4 discusses the construction of

the humidity control system used for further research by Sam Lioa et al [26].

Chapter 5 discusses the effect that the addition of a negative voltage has on the stacking of

fibres.

Finally, chapter 6 is a collective summary of the work completed in the masters, including a

discussion on the limitations of the project as well as recommendations for future work. A

conclusion is then provided after an analysis of all available data from the project.

Chapter 2: Literature review


2Chapter 2: Literature review

2.1 BONE FRACTURE HEALING AND TISSUE ENGINEERING

2.1.1 Bone Anatomy

The adult human skeleton contains 206 bones, with variations between individuals (not

including sesamoid bones). The appendicular skeleton has 126 bones, axial contains 74 bones

and the auditory ossicles comprise 6 bones [27]. There are five general categories of bone,

long bones, short bones, flat bones, irregular bones and sesamoid bones [27]. Flat bones are

formed by membranous bone formation while long bones are a combination of endochondral

and membranous bone formation [27]. Figure 2-1 illustrates the shapes and positions of the

four categories of bone.

Figure 2-1: The human skeleton indicating the different structures of bone. The axial skeleton forms the

pelvis, spine, ribs and skull. Appendicular skeleton is formed from the bones of the limbs [28].

The bones in the skeleton provide structural support for the body, form attachment points for

muscle to use as lever systems and also protect vital organs from external forces. They also

serve physiological roles in homeostasis of mineral and acid-base balance, as reservoirs for



signalling proteins and cytokines and the production of blood cells within the marrow spaces

[27].

The skeletal system consists of 80% cortical (compact) bone and 20% trabecular (spongy)

bone [29]. The cortical bone is dense and solid and is the major load bearing component of

bone. Trabecular bone consists of a honeycomb network of plates and rods in the bone marrow

cavity. Figure 2-2 illustrates the relative position and morphology of cortical (compact) and

cancellous (spongy) bone.

Figure 2-2: Schematic diagram of long bone with close up’s showing sections of the marrow cavity and

structure of trabecular and cortical bone [30].

Cortical osteons are known as the Haversian system. They form a network within the cortical

bone consisting of a system of cylinders surrounded by concentric lamellae of 400 µm (length)

and 200 µm (width). The porosity of cortical bone is less than 5%; however, this number

depends highly on the activity of the remodelling system, with increased Haversian

remodelling causing an increase in cortical porosity [27].

The trabecular osteons, known as packets, consist of rods and cones with an average thickness

between 50 to 400 µm. remodelling within the trabecular bone occurs at a faster rate than

cortical bone.

Bone’s unique multi-phase architecture, with cortical bone’s high strength and stiffness and

trabecular bone’s low moduli give it the property of high tensile strength and high fracture



toughness. Bone is constantly remodelling to mechanical and physiological cues and a

microscopic view of bone shows a highly complex, ever changing tissue. There are three types

of cells that make up bone; osteoclasts, osteoblasts and osteocytes and they are all involved in

the remodelling of the bone structure [27].

2.1.1.1 Fracture Healing

The process of bone fracture healing is known as secondary healing. It involves the normal

embryonic process coupled with bone remodelling. The process is split into the inflammation

phase, soft callus formation, hard callus formation, and remodelling [31]. Figure 2-3 illustrates

the process along with rough timescales for each step.

Figure 2-3: Stages of bone healing [31]

The first stage of fracture healing, fracture haemotoma, involves clotting of blood, which

initiates an inflammatory response including the dilation of capillaries, release of white blood

cells and the budding of new capillary formation [31]. Within the first 2 to 3 days, phagocytes

remove dead tissue and fibroblasts enter the fracture site, forming granulation tissue. Bone

remodelling and callus formation begins at this stage as well, along with neovascularisation.

Chondrocytes secrete new extra cellular matrix (ECM) forming fibrocartilage, creating the soft



callus. Hard callus begins to form as the soft callus mineralises through endochondral and

intramembranous ossification [31].

In certain cases the healing process may be compromised, such as in a critical sized defect, or

due to infection. These cases may become non-union fractures if clinical intervention does not

take place. The gold standard for treating critical sized defects is the bone graft. A bone graft

is a procedure where a section of bone is sourced from a donor site and placed into the fracture

site, providing a matrix for bone to bridge the fracture gap. Three types of bone graft exist;

autograft, allograft and xenograft. The autograft is the current gold standard for treatment. It

involves the removal of bone from a donor site in the patient’s own body and placement into

the fracture site. Allograft is similar, except the donor site is in another human being. And in

certain rare cases, xenografts are taken from a donor animal.

Despite the autograft being the gold standard in treatment, complications still occur and

progress has been slow in improving the technique over the past 20 years. Approximately 20%

to 30% of autografts patients experience donor site morbidity, and greater than 30% of

allograft cases experience complications such as non-unions and infections [32].

Due to the shortcomings of the autograft and allograft, bone tissue engineering as a field was

created [33]. It combines the principles of engineering with life sciences and material science,

to produce biological substitutes that are capable of restoration, maintenance and improvement

of tissue function and repair [34]. Since the early 2000s, tissue engineering has entered the

spotlight as a method to treat various diseases including cardiovascular, respiratory,

musculoskeletal, eye, oral and renal.

2.1.2 Tissue Engineering

In the past ten years, tissue engineering has seen dramatic advancements in human tissue

regeneration. The aims of tissue engineering is to restore, maintain and/or improve the function

of tissue by developing a biological substitute or reconstructing tissues [35]. Bone tissue

engineering is a subfield which concentrates on fracture healing in the skeletal system.

Scaffolds for bone tissue engineering have seen an exponential rise in the number of

publications over the past decade, mainly due to promising results of new bone formation and

repair of segmental defects in both small and large animal studies [36]. The number of novel

scaffolds produced for bone tissue engineering has increased over the past few years and an

investigation into the production, functionalization and materials used should be reviewed to

determine the efficacy of each system.



There are three research topics in scaffold production for bone tissue engineering; materials,

biofabrication and drug delivery systems. Biofabrication deals with methods for scaffold

production, this includes additive manufacturing techniques. The materials used to produce

these scaffolds is its own research field and can be split into polymers, ceramics, metals and

composites. Tissue engineering scaffolds as drug delivery systems are a combination of the

above with the inclusion of bioactive compounds to stimulate bone healing.

2.1.2.1 Materials for Tissue Engineering

Polymer analysis and synthesis is a key focus area for materials engineers in tissue

engineering. Recent trends in literature suggest that biodegradable polymers are the preferred

material of choice [36]. The advantage associated with biodegradability is its ability to support

tissue for a period of time before being resorbed by the body after its functional lifespan [37].

Two subgroups of polymers are used; natural polymers (including collagen, chitosan) which

have lower immune response and (in certain cases) a bioactive component, and synthetic

polymers whose advantage lies in easier tailoring of its physical properties (biodegradation

rate, more predictable properties and consistency) than its natural counterpart [37]. Figure 2-4

illustrates the breadth of structures which can be produced using polycaprolactone (PCL).

Figure 2-4: Different structures that are capable with a single type of polymer (PCL) that has been used

in a number of Additive manufacturing techniques and other fabrication techniques. Micro-spheres (a,

b). Nano-fibres (c, d). Foams (e, f). Knitted textiles (g, h, i). SLS scaffold (j-o). Fused deposition

modelled scaffolds (p–u) [5], [38], [39], [40], [41], [42]



Ceramics for bone tissue engineering show promise due to their biocompatibility and

extremely low wear rates. The most common ceramic available in the field are calcium

phosphates (CaP) (Hydroxyapatite, tri-calcium phosphate). The advantages of ceramics come

from their high Young’s modulus, however they conversely have low fracture toughness and

are brittle. This makes them exceptional substitutes in compressive loading cases [43]. Low

wear and high Young’s moduli allow ceramics to be used in an articulating surface, which has

seen use in artificial joints [44]. Manufacturing of complex structures with ceramics is a costly

and difficult process, hence it has seen little use as a stand-alone solution in tissue engineering.

Metals have high compressive strengths and

excellent fatigue resistance. The predominant

metal used in tissue engineering is titanium [45].

Metal has a number of advantages over polymers

and ceramics due to its high tensile strength and

toughness, making it a flexible candidate to be

used for both compressive and tensile loads.

However, unlike degradable polymers which are

able to degrade in the body with little side

effects, and ceramics which have very low wear,

metals are known to release ions into the surrounding tissue, which has been known (with

certain metals) to cause immune responses and necrosis [46]. Production processes for metal

implants are expensive, but are well established and products produced with SLS have gone

to preclinical, in vivo trials [47].

Composite materials are a blend of polymers, ceramics and metals designed to provide a

system which has desirable physical properties from two or all three fields. Composites are

made of two phases, the matrix and the dispersed phase. The matrix phase is the stress transfer

medium, while the dispersed phase is a constrictor, preventing movement of the matrix phase

[48]. This system can be split into three groups, particle reinforcement, fibre reinforcement or

structural reinforcement. Each system describes the method that the dispersed phase is mixed

with the matrix phase Polymer-ceramic composites have yielded promising results [9].

Additionally, PCC materials combined with metals have seen improvements in osteogenesis

[49] as well as physical properties for the material. The use of composite materials is promising

due to its customisable physical and biological properties; however, changes to the material

properties requires re-optimization of the manufacturing techniques.

Figure 2-5: A vascular stent, generally made

from stainless steel 316L or titanium used to

expand sections of the artery [148].



2.1.2.2 Additive Manufacturing for Tissue Engineering

Additive manufacturing has become the main fabrication technique for tissue engineering. A

large number of techniques exist within the field; hence, this section focuses on the three most

popular techniques currently available. Additive manufacturing is a manufacturing technique

which allows the production of an object by the addition of material; the three most common

methods are fused deposition modelling (FDM), stereo-lithography (SLA) and selective laser

sintering (SLS). Hereafter these techniques will be examined.

FDM (also known as melt extrusion manufacturing) is a technique using a thermoplastic

filament which is fed through a heating liquefier. The solid filament acts as a piston and pushes

the melt through a print head [50]. The system is attached to a gantry which allows the printing

head to extrude the polymer in the horizontal plane. Each layer is filled in by the polymer and

consecutive layers are then stacked to produce a 3D structure. This technique is one of the

oldest 3D printing methods and is well established. It is used extensively in tissue engineering

for solid polymer scaffolds [51] as well as hydrogel scaffolds [52]. The advantages of FDM

lie in its flexibility. The basic extrusion method is applicable to many different polymers and

composites and therefore it has been used to produce structural scaffolds as well as cell loaded

polymers and even composites with drug-loaded polymers [53].

Figure 2-6: Schematic image of the fused deposition

modelling system [149]



SLA uses a photo-curable polymer which solidifies under the selected photo-initiated cure

reaction [54]. Two methods exist to initiate curing; the first is a mask-based method which

involves the irradiation of the polymer by an image through a patterned mask, the second

method involves direct writing onto the polymer via a focused ultra-violet beam. This

technique produces the highest resolution of scaffolds of the three common additive

manufacturing processes (resolution as low as 20 µm [55]). Despite the high resolution and

the product existing for nearly 20 years, the technique has not seen as wide spread use as other

3D printing techniques [56]. Increased interest in SLA in tissue engineering has occurred

recently, partly due to its high resolution constructs as well as recent advances in non-cytotoxic

photocurable polymers. Bioactive compounds [57], specific binding proteins in and on the

resin [58], and recently natural polymers have been printed using this technique [59].

Figure 2-7: Schematic diagram of a stereo-

lithography system [150]



SLS involves heating a powdered material just beyond its melting point via an infrared laser

[54]. The laser files the shape by melting selected sections of powder, retreats as a new layer

of powder is added and repeats the process until the object is formed. This technique is

generally used for ceramics and metals; however is also used in polymer based systems. SLS’s

advantage lies in its ability to produce 3D printed metal parts; however, due the inherent nature

of melting a powder, it is limited in its ability to 3D print living organism which the previous

two systems were capable of [54].

The final group of tissue engineering falls within drug delivery. Briefly, this can be a

combination of any appropriate material and manufacturing technique to produce a system

which can release drugs to a target location and maintain a target dose for a desired period of

time. Despite many biomaterials’ ability to provide essential mechanical support and

attachment, most are not able to produce direct changes in cellular differentiation as efficiently

as drugs and signalling proteins [60]. Hence, tissue engineering scaffolds must act as both a

construct for cell adhesion and proliferation as well as provide the correct physiological cues

to regenerate non-union fractures in bone tissue engineering.

2.1.2.3 Bone characterisation: composition, architecture, biomechanics

Design of biomimetic scaffolds relies heavily on the reliable and accurate characterisation of

natural bone. A comprehensive review of the biomechanical, structural and compositional

characteristics of bone will be detailed here.

Figure 2-8: Schematic diagram of a

selective laser sintering device [151]



The musculoskeletal system is composed of 10 to 20% collagen, 60 to 70% bone mineral and

9 to 20% water (measurements in weight percentage) [29].

The architecture of bone varies based on the type of bone, as discussed in section 2.1.1 (Bone

anatomy). The mass of long bone is comprised of 80% cortical bone and cancellous bone

makes up the other 20% [29]. Cortical bone is load bearing, having a much higher Young’s

modulus; however, cancellous bone is associated with stress and the viscoelastic properties of

bone. The internal structure of cortical bone is complex, containing a network of canals,

canaliculi and lacunae for vascularization, innervation and osteocyte communication [61]. The

cancellous structure provides a network of rods and cones for mechanical support and a

network for cell attachment. The porosity of cortical bone is generally less than 5% [27], while

for cancellous bone is between 50 to 90% [62]

The stress-strain curve of cortical bone exhibits a linear elastic region followed by a plastic

deformation region at approximately 0.8% strain [61]. Strain rate observations of bone

mechanics show the viscoelastic properties of bone; low strain rates exhibit bones tough

behaviour and ability to endure large strain, while high strain rates show the brittle behaviour

[63]. This can be observed in Figure 2-9.

Figure 2-9: Tensile stress-strain curve of cortical bone with multiple strain rates [63]



Cancellous bone is a highly dynamic system; its density and porosity vary greatly between

individuals. The mechanical properties of cancellous bone are highly dependent on the

porosity, which remodels itself based on mechanical cues. This system is known as Wolff’s

law [64]. Figure 2-10 shows a cross sectional view of a femoral head with stress lines marked

on adjacent images showing the lines of bone remodelling against stress.

Figure 2-10: An image illustrating Wolff’s law of bone, demonstrating he cancellous bone remodels

itself to the stress lines via mechano-transduction

2.1.2.4 Scaffold Architecture

The role of biomimetics in tissue engineering has become increasingly clear. The complexities

of human physiology and anatomy result in interactions between various subsystems which

are not well understood. By imitating the body’s natural structure, we are able to provide those

systems with substitutes, removing a layer of complexity involved in the understanding of

subfields. For scaffold construction in bone tissue engineering, the porosity and

interconnectivity, flow properties and surface structure are the main areas of study. Therefore,

it is essential to this field to characterise natural bone and bone formation [65].

The ultimate aim when adjusting the porosity of a scaffold is to imitate the mechanical

properties of human bone while providing an environment which is conducive to cell

proliferation. A number of studies have modified the architecture of scaffolds to imitate the

mechanical properties found in long bone. Shimko et al. [66], investigated the modification of

the porosity of a tantalum scaffolds to match the mechanical properties of human cancellous

bone. Additionally, Fu et al. [67] used porous nickel-titanium scaffolds to imitate the

mechanical properties of cortical bone. As bone contains a hierarchy of structures, it is difficult

to imitate all mechanical and viscoelastic properties without compromising the structures

cellular composition.



The structure bone pores significantly affect the mechanical characteristics [68], imitating its

effect in scaffolds may result in similar properties. Increasing the porosity of the scaffold

increases its permeability, but inversely decreases the strength of the scaffold. Altering the

pore structure of a scaffold will also increase permeability while, theoretically, maintaining

similar mechanical properties. It is established that the size and orientation of pores within a

scaffold affect the mechanical properties like the Young’s modulus, tensile strength and

compressive yield strength [69], [70].

It is necessary to have both the mechanical properties as well as a porous structure for bone to

grow. Kuboki et al. [71] showed that on hydroxyapatite structures used for BMP-2 delivery,

no bone formation was seen on solid particles, however, porous scaffolds showed signs of

osteogenesis. Numerous finding show increases in cellular activity for porous and it is now

seen as a prerequisite for bone tissue engineered scaffolds. Melt electrospinning is capable of

producing highly porous structures with controlled inner topology, hence it is an ideal

candidate for the production of scaffolds for bone replacement therapy.

2.2 MELT ELECTROSPINNING

Electrospinning is defined as the process of drawing out a liquid polymer fibre from an

extruder via a large electrical potential. The liquefaction of the polymer is occurs in one of two

ways, by a solvent/polymer solution or via a melt. The process of solution electrospinning was

a patent technology dating back to 1938 [72] and has since been used extensively as a

production method for nano-fibrous mats [73].

The process begins at the liquefaction of the polymer where a solution of polymer and solvent

make a low viscosity liquid inside a needle tip. The electrostatic potential increases the charge

of the liquid until the force produced by the electric field is strong enough to pull the polymer

out of the needle tip. These forces change the shape of the polymer, producing what is known

as the Taylor cone [74]. The electrostatic forces will concentrate at the tip of the Taylor cone,

ejecting out a fluid jet. Charge is left in this fluid jet, which is then drawn through the electric

field and attracted to the collector plate.

Melt electrospinning is a similar process, although the polymer is liquefied by melting it. This

can vastly increase the viscosity of the polymer and removes the evaporation of solvent during

fibre formation, which has implications in jet stability and fibre thickness. As a result of melt

electrospinning’s lack of solvents, it holds promise for cell compatibility studies.



Figure 2-11: Schematic of an electrospinning apparatus. [75]

2.2.1 The Physics of Electrospinning

As described above, the process of melt electrospinning begins at the liquefaction of the

polymer using a solvent (for solution), and using heat (for melt). The polymer is pulled from

the needle tip onto a collector plate, which is the basic outline of the electrospinning process.

Two phases exist during the spinning process, the stable region and the whipping region [15].

The stable region is dominant during the melt electrospinning process and is relevant to my

study.

The process of solution electrospinning has been thoroughly mathematically analysed, with

papers dating back to 1976 [76]. Work on the stable jet region has been completed by a number

of groups including a 1D model using a power law fluid [77], a Newtonian fluid model [78]

and a model accounting for viscoelastic behaviour of polymer melts [79], [80]. The later

model, reported by Feng et al., solved a number of issues and was revisited again by Joo et

al., who reported an in-depth experimental comparison with the model. However, these models

are targeted towards solution electrospinning which must be modified to account for the

isothermal properties of fibres in melt electrospinning. A study by Zhmayev et al. applied the

Carrol and Joo models to the case of non-isothermal jets [15]. The lack of polymer-solvent

interactions greatly simplified the model. However other complication associated with charge

transport and viscoelasticity arose.

2.2.1.1 Polymer Melts, Viscosity and Viscoelastic Properties

A polymer is melted in an extruder and accelerated through an electric field. The first stage in

the melt electrospinning process is liquefying the polymer. The temperature and molecular



weight of the polymer affect the viscoelastic properties, which as we have stated previously,

is essential in modelling the behaviour of a melt electrospun jet. To describe the effect that the

molecular weight has on the viscoelastic properties, we first need to visualise how a polymer

chain exists inside a plastic matrix.

Plastics are composed of entangled polymer chains, which gives polymers their viscoelastic

properties. It is easier for larger chains to become entangled than shorter ones, hence their

shear viscosity is related to their molecular weight. The Tube model was developed in 1967

by Edwards et al. [81] to describe the entanglement of polymer networks. The Tube model

was applied to determine the melt viscosity dependence on molecular weight [82], it was

determined that two phases exist. The first phase is attributed to the short chain regime, where

molecules are not long enough to entangle. The second phase is the long chain regime, which

occurs when chains are long enough to entangle with each other. Figure 2-12 illustrates the

difference between the two regimes:

𝜼𝒎 = 𝑲𝑳(𝑫𝑷)𝟏

Short Chain

(2-1)

𝜼𝒎 = 𝑲𝑯(𝑫𝑷)𝟑.𝟒

Long Chain (2-2)

Figure 2-12: The dependence of melt viscosity on molecular weight for a number of common polymer

substances. [82]

It is generally believed that the dependency of temperature and time of viscoelastic properties

are analogous. The creep and stress relaxation are time depended viscoelastic properties.



Briefly, creep is dynamic deformation under a constant load, and stress relaxation is the

constant deformation under a dynamic load, as shown in Figure 2-13.

Figure 2-13: Diagram showing the effect of creep and stress relaxation.

This system can be modelled using a series of analogous resistive and capacitive loads, making

the viscoelastic model known as the Williams-Landel-Ferry (WLF) equation. This equation

states that the effect of temperature is an exponent and proportional to the difference between

the temperature and the glass transition temperature.

𝑳𝒐𝒈(𝒂𝑻) =−𝑪𝟏∗(𝑻−𝑻𝒔)

𝑪𝟐+(𝑻−𝑻𝒔) 𝑻𝒈 > 𝑻 < ~(𝑻𝒈 + 𝟏𝟎𝟎 °𝑪)

(2-3)

Where C1 and C2 are constants dependent on each polymer. Ts is the reference temperature, Tg

is the glass transition temperature and aT is the horizontal shift factor in an empirically fit

compliance data plot (all temperatures are expressed in kelvin) [83]. As stated previously, one

of the main factors effecting the jet thinning is the viscosity of the polymer melt [15].

Manipulation of this parameter results in dynamic control over the fibre thickness and is

essential in understanding the processes of electrospinning.

The extensional viscosity of the polymeric liquid and the maximal difference in elongation

will control the thinning process in a melt electrospun fibre [84]. When the system reaches a

steady state, the elongation viscosity (�̅�) will increase or decrease, dictating the extension

thinning or thickening [79]. A fluid leaves the tip having a certain strain history; it then

experiences a uniaxial extension with a time dependent strain rate. Hence, it would be simpler

to investigate viscous and elastic aspects separately.

Creep Stress

Relaxation



For high density polymer melts, the extension thinning process dominates over the extension

thickening process. The equation derived by Feng [79] shows the tensile force varying over

the fibre length, which is different from mechanical fibre spinning, whose stretching force is

introduced at the fibre spool, creating a constant tensile force throughout the length of the

fibre. Fibre thinning is due to the viscous interaction of the tensile force on the liquid, hence

the fibre experiences more steady thinning for slightly extended fibres, and extensional

thinning viscosity causes delayed stretching for severely stretched fibres.

Tirtaatmadja and Sridhar [84] investigated the effect of stress-growth curves of fibres and

found a formula representing the strain hardening. Strain hardening is the strengthening of a

material by plastic deformation [85]. They found that significant strain-hardening occurs in

electrospun fibres, and that the normal viscosity and strain thinning viscosity interact with the

hardening to affect the fibre diameter. The model was placed into Feng’s model who found

that the strain hardening had an impact on the formation of the fibre, but little impact on the

final fibre radius, despite the large impact on the viscosity [79]. However, strain hardening

does result in higher Young modulus (E), and more robust mechanical properties of fibres.

2.2.1.2 Charge on the polymer

The “leaky dielectric model” is used to understand how charge is transferred through and on

the polymer jet [86], [87]. It implies that unbalanced electric charge migrate to the polymer

surface in a relatively short period of time1. This electric charge and the fluid flow must be

coupled to understand the electrospinning phenomenon. Feng et al. developed a

comprehensive model investigating this effect [80].

For a cylindrical polymer jet, the volume flow rate of that jet would be:

𝜋𝑅2𝑣 = 𝑄 (2-4)

And the current would be:

𝜋𝑅2𝐾𝐸 + 2𝜋𝑅𝑣𝜎 = 𝐼 (2-5)

Where R is the radius of the jet, v is the axial fluid velocity, Q is the volume flow rate, K is the

electrical conductivity of the jet, E is the electric field strength, σ is the surface charge density



and I is the current. If we assume the leaky dielectric model, the first term in the current

equation (2-5) would equal zero, leaving only charge transported through the surface of the

polymer. The leaky dielectric model is used extensively in solution spinning (and will be used

in this analysis) as a solvent/polymer solution acts like a poorly conducting liquid which is an

adequate assumption [88]. A residual amount of charge is hypothesised to remain in the

polymer, causing the instabilities in solution electrospinning [89].

The emission of the charge from the polymer is hypothesised to occur at the surface/air

interface of the fibre [88]. The high electric field induced emission process is assumed to be

the method by which charge is removed from the polymer. Electrons are ejected through the

interface when the potential difference between the surrounding gases is great enough. In

laboratory electrospinning experiments, this high field only exists at the tip of the needle. It is

possible to model the electric field between the needle tip and collector. The point-plane

electrode geometry along the axis of the needle at its tip has the electric field:

𝐸𝑡𝑖𝑝 =2𝑉

𝑟 ln (1 + (4𝐷𝑟 ))

(2-6)

V is the external applied voltage, D is the needle tip to collector

(TTC) distance, r is the radius of curvature of the needle point

and Etip is the electric field measured axially from the needle tip

[90]. If the electric field exceeds values on the order of 109 V/m

to 1010 V/m, electrons are emitted from the surface.

For the electrospinning jet to form, the charge carriers on one

electrode (emitter) must experience the electrostatic force of the

opposite electrode (collector). Therefore, the arrangement of

electrodes is essential in determining the mechanism for charge

carrier generation. The most common system employed for melt

electrospinning uses a charged syringe tip (Figure 2-14). In this

system, negatively charged ions migrate to the inner surface of

the polymer and become immobilized, leaving excess positive

ions in the polymer which can respond to electrostatic forces.

Research by Kalayci et al. illustrated that the point-plane geometry releases large amount of

excess ions in the atmosphere, with the largest current being produced when no spinning is

occurring [91]. Several publications have also suggested that charge carriers are transported

in the surrounding atmosphere [92], [93].

Figure 2-14: Schematic

diagram of the emitter of an

electrospinning system [88]



2.2.1.3 Momentum Balance

During the electrospinning process, the polymer is stretched from the emitter via the electric

field. This stretching is important as it provides electrospinning with its characteristic

nano/micro fibres. Two stages of stretching exist for electrospinning; the first occurs in the

stable region of the jet and is known as the steady-stretching process, the second stage occurs

during whipping, but will not be discussed. Work done by J. J. Feng provides an in-depth

analysis at the momentum balance which takes place during the electrospinning process [79]

The polymer is stretched due to the summation of forces from electrostatics, gravity, surface

tension, viscosity and inertia. As the fibre is thinned out, the surface charge is affected, which

in turn affects the electric field and the electrostatic driving force. Once again, for the analysis

presented by J. J. Feng, the jet is assumed to behave as a leaky dielectric and charge stored on

the surface of the polymer. The slender-body approximation is used for most models [79],

[94], [95] and assumes that the radius decreases slowly along the axial direction and that the

axial velocity is uniform [79].

Figure 2-15 shows the momentum balance on a section of the polymer jet. The equation

governing the momentum balance is the sum of forces on the short segment and can be

expressed as following:

𝑑

𝑑𝑧(𝜋𝑅2𝜌𝑣2) = 𝜋𝑅2𝜌𝑔 +

𝑑

𝑑𝑧[𝜋𝑅2(−𝑝 + 𝜏𝑧𝑧)] +

𝛾

𝑟∙ 2𝜋𝑅𝑅′ + 2𝜋𝑅(𝑡𝑡

𝑒 − 𝑡𝑛𝑒𝑅′)

(2-7)

Figure 2-15: Momentum balance on a short section of the jet [79]

Where τzz is the axial viscous normal stress, p is the pressure, γ is the surface tension tet and te

n

are the tangential and normal tractions on the surface of the jet due to the electric field, R is

the radius, R’ is the rate of change of the radius, g is gravity, v is the velocity, ρ is the density.

This equation has been derived by a number of groups [78], [94], [79]. It illustrates that the

electric field, charge density and viscosity (through the traction forces) are coupled.

Comprehensive knowledge of all these parameters is essential in fully understanding the



electrospinning process. J.J. Feng derived the characteristic radius (χ = L/R0) of the jet using

the following equation:

𝐸(𝑧) = 𝐸∞(𝑧) − ln (𝜒) (1

𝜖

𝑑

𝑑𝑧(𝜎𝑅) −

𝛽

2

𝑑2

𝑑𝑧2(𝐸𝑅2))

(2-8)

Where E∞ is the external electric field, E is the electric field at z, 𝜖 is the dielectric constant of

the polymer and 𝛽 =𝜖

�̅�− 1 (𝜖 ̅is the dielectric constant of ambient air).

2.2.2 The Governing Equations of Electrospinning

From the above analysis, it can be inferred that the viscosity, electric field and surface tension

play the greatest roles in determining the radius and stability of a melt electrospun jet. The

work completed by Zhmayev et al. [15] used the previous methods to develop a non-isothermal

model applied to the melt electrospinning process. The governing equations can then be

described as the following:

Continuity: 𝜋𝑅2𝑣 = 𝑄 (a)

Momentum: 𝑝𝑣𝑣′ = 𝜌𝑔 +𝐹′

𝑇

𝜋𝑅2 +𝛾𝑅′

𝑅2 +𝜎𝜎′

𝜀0+ (𝜀 − 𝜀0)𝐸𝐸′ +

2𝜎𝐸

𝑅 (b)

Charge: 𝜋𝑅2𝐾𝐸 + 2𝜋𝑅𝑣𝜎 = 𝐼 (c)

Electric-Field: 𝐸(𝑧) = 𝐸∞(𝑧) − [1

𝜀0(𝜎𝑅)′ − (

𝜀

𝜀0− 1)

(𝐸𝑅2)′′

2] ln (

𝑑

𝑅0) (d)

(2-9)

These equations emphasize that the stable jet region is capable of major thinning, the major

factor effecting jet thinning is extensional viscosity, and (because of the lack of solvent

evaporation and whipping) it is important to optimize the thinning parameters of the system.

2.3 CHARGE TRANSPORT IN ELECTRIC JETS

As discussed in the previous chapter (2.4), the modelling of charge in scaffolds has been

completed only for solution electrospun scaffolds. Some principles associated with solution

spinning are transferrable to a melt system; however, the general leaky dielectric principle is

not applicable. The leaky dielectric principle relies on an ionic charge carrier to move the

positive charge relatively quickly to the surface of the polymer, where it is then dissipated into

the atmosphere [87]. However, in a polymer melt, the diffusion of particles in the liquid is

dictated by the Reptation theory or diffusion through the polymer chains. Both of these



diffusion methods would suggest that charge moves slowly to the surface, a key assumption

in the leaky dielectric model assumes that charge is quickly moved to the surface of the jet.

The current flowing through the jet has been measured by some groups and the values obtained

range in the sub microamperes [96], [97]. If it is assumed that all charge measured is carried

through the jet, then the current flowing through it would be:

𝐼 = 𝜎𝑣𝑠 (2-10)

Where I is the current, σ is the charge density and vs is the velocity of the jet. We can calculate

speed using the continuum equation 𝑄 = 𝑣𝑠𝐴, where Q is the flow rate and A is the cross

sectional area. Hence, our velocity is:

𝑣𝑠 =𝑚𝑓𝑠

𝜌𝜋𝑟2𝑡

(2-11)

Where 𝑚𝑓𝑠 is the flow rate, 𝜌 is the density, r is the radius and t is time. Carroll and Joo [98]

performed an experiment to determine the charge density for these fibres, they determined that

charge per unit mass for poly(vinyl alcohol) (PVOH) is ~1.6 × 105 nC/mg. However, the

assumption that all the charge flowing in the system goes through the polymer is false.

A number of groups have investigated the current-voltage (I-V) characteristic of the

electrospinning processes and have experimentally determined that the curve fits the

proportion of 𝐼 ∝ 𝑉2. This proportionality is even derived using scaling laws by Ganan-Calvo

[94]. The coronal discharge exhibits similar behaviour, Atten et al. [99] and Shrimpton [100]

illustrated that the point-plane discharge (described in section 2.4) has a similar I-V

characteristic. Theory and evidence [99] suggest that the electric field at the tip of the Taylor

cone is high enough to produce coronal discharge.

Theron et al. [101] outlined an empirical formula to determine the I-V characteristic for

electrospinning. The study investigated the volume charge density, find the following

relationship:

𝐼 = 𝜌𝑐𝑄 = 𝑘𝑒𝑉𝛼𝑄𝛽𝐶𝜒𝑀𝛾𝑒−𝐻 (2-12)

Where 𝜌𝑐 is the charge density for volume, V is applied voltage, Q is volumetric flow rate, C

is polymer concentration, M is molecular weight, H is the TTC distance and ke is an

experimentally determined proportionality constant. The exponents α, β, χ, γ and H are

experimentally determined exponents.



These equations provide the initial current placed on the jet; however, they have not explored

the method for discharge. The simplest possible model for discharge assumes that the charge

conducts to the substrate. However, solidification of the fibres would quickly slow down the

flow of charge and if measurements suggest that high charge flow is still present [97], other

mechanisms must exist. The four proposed methods for discharge are coronal discharge of the

Taylor cone, coronal discharge of the fibrous mat, parasitic electrospraying and charge

evaporation.

2.3.1 Coronal Discharge in Taylor Cone and Fibres

Filatov et al. [102] examined the possibility of coronal discharge from the Taylor cone formed

during the electrospinning process. Charge in the polymer quickly moves to the surface of the

polymer during the thinning stage of the Taylor cone. This is then discharged into the

surrounding atmosphere, producing ions in the air around the jet. Using a cylindrical wire-

plane electrode configuration and the Townsend-Doetsch formula to determine coronal

discharge, the group found that the current is 25 to 200 times smaller than that carried by the

jet [102].

The discussion in chapter 2.2.1.2 illustrated that significant charge is lost at the interface of

the needle tip for a point-plane electric field [88]. A large portion of the current produced in

the system is not due to the jet, with current measurements increasing when no jet is present

[91]. The current is produced due to ionization of the air around the needle tip. Kalayci et al.

[91] proposed that this is mainly produced in the form of H3O+(H2O)n. The charge is therefore

emitted not only from the jet, but from the apparatus itself. This can be visible in the form of

coronal discharge around the surface of the polymer jets (see Figure 2-16).

Figure 2-16: Visible coronal discharge in an electrospinning apparatus [103]

If charge is collected on the polymer jet when it cools, solid polymers act as insulators,

blocking the charge from quickly dissipating to the collector plate. If this were the case, the

electric field would be screened by this excess charge, causing a decrease in the field at the

electrode. This would reduce the Taylor cone, causing the field to fall below the threshold

Coronal discharge produces a dim violet glow



required to produce a fibre. Since electrospinning is a continuous process, charge must be

released in some form through the solid polymer at the base of the scaffold.

During the production of a scaffold, a non-conductive mat is placed on the collector plate. As

the scaffold is built up, this non-conductive mat creates a polarized volume relative to the

electrode. Even if charge is able to escape the solid polymer, some may remain creating a

charged volume above the collector plate. This scaffold is then a dielectric volume with a

charge, known as an electret [88]. A number of mechanisms exists in an electret allowing it

to discharge, factors that affect this including the dielectric properties of the polymer, ambient

humidity and temperature and the locus of charges in the polymer. Filatov et al. [102] have

shown that the emission of charge is randomly distributed over the area of a scaffold and

randomly distributed in time. Figure 2-17 shows the random emission of charge from polymers

as described by Filatov et al. This suggests that the emission is due to an accumulation of

charge over a period of time, which is consistent with work on many technologies involving

high potential difference and polymers.

Figure 2-17: Oscilloscope snapshot of the discharge current at different time points. The number on the

left corresponds to fibre accumulations for times of (1) 10 s, (2) 30 s, (3) 2 min, (4) 5 min, (5) 10 min,

(6) 20 min [102]

Randomly distributed discharges can be associated with the accumulation of charge in certain

areas until this charge exceeds a breakdown voltage and jumps to the collector plate. Using

the Paschen-Townsend model of electron avalanches, Filatov et al. [102] were able to create

an equation describing the charge balance via discharge. They produced a model to describing

the breakdown voltage required for the emission of that charge:



𝑉 =𝑎𝑃𝐷

ln(𝑃𝐷) + 𝑏

(2-13)

Where V is the breakdown voltage, P is pressure of the gas separating the charge, D is the

distance of separation and a and b are constant depending on the type of gas present. For

atmospheric conditions, 𝑎 = 43.6 × 106 V/(atm m) and b = 12.8 [88]. This demonstrates that

the discharge in the scaffold is highly dependent on the atmosphere and random processes

associated with charge build-up, which is a partial explanation for the variability in the

scaffold. Filatov at el. estimated that for a 6 µm scaffold, it should take approximately 18 s for

charge to dissipate from the scaffold [102]. Each consecutive layer needs time to build up

enough charge to dissipate over a larger distance. Therefore, an increase in the number of

layers increases the likelihood that charge has not yet dissipated from the scaffold.

2.3.2 Parasitic Electrospraying

Parasitic electrospraying was discovered by Bhattacharjee et al. [93]. They used a system

which measured the electric current in two concentric rings centred on the emitter. The inner

ring would measure the current associated with the electrospun fibres, the outer ring detected

any residual current produced in the system. They found that electrosprayed particles were

being emitted during whipping in solution electrospun scaffolds, which carried with it a

significant portion of the charge. However, this technique is not applicable to melt

electrospinning so it will not be investigated further.

2.3.3 Atmosphere and Charge Evaporation

It is well established that the humidity effects the formation of solution electrospun fibres.

However, little work on its effect in melt electrospun fibres has been completed. This section

will discuss the relevant effects of atmosphere on melt electrospun fibres and charge transport.

The factors affect in melt electrospinning are associated with the hydrodynamic effect of

humidity on the Taylor cone, removal of surface charge from the jet and the formation of

coronal discharge is affected by the water content and atmospheric gases.

Surface charge may be affected by the moisture content of the air. It presents a discharge

pathway that may strongly impact the electrospinning process by affecting the homogeneity

and charge distribution at the surface [88]. It is established that the conductivity of the polymer

surface increases with the addition of water content to the atmosphere [104]. Work by

Galemback et al. [105] proposed a mechanism for the accumulation and emission of charge in

a dielectric. The proposed model found that accumulation and emission is heavily dependent

on relative humidity. It states that the electrochemical potential of H(H2O)+n and OH(H2O)-

n

are dependent on the electric potential:



𝜇𝑖 = 𝜇𝑖0 + 𝑅𝑇𝑙𝑛(𝑎𝑖) + 𝑧𝑖𝑉

(2-14)

Where I represents OH- or H+ concentration, μi is the electrochemical potential, μi0 is the

chemical potential, zi is the valence of the species, V is the electric potential, R is the gas

constant, T is the temperature (in Kelvin) and ai is the activity coefficient of the species [106].

2.3.4 Residual Charge in the Polymer

It is known that residual charge remains in the polymer after the scaffolds have been laid down

[107]. Dielectric materials which have the ability to store charge and release it are known as

electrets; it has been assumed that electrospun scaffolds behave as electrets [108]. Therefore,

charge is capable of accumulating in the volume of the polymer and on the surface. The method

of charge transport depends on the locus of charge. Hence, volume and surface charge should

be explored.

Heterocharge and homocharge, dipole orientation or direct charge injection respectively, are

the two types of charge storage in the volume of a polymer. Free charge injection is the

probable method for residual charge and can be accomplished in a number of ways, including

the previously mentioned coronal charging. Charge storage is dependent on the crystallinity

[109], polymer structure, presence of additives [110] and other physical properties.

Current models use localized states of chemical groups as models for charge storage (also

known as traps). The locus of these sites depend on the types of polymers present.

Polycaprolactone is a semi-crystalline polymer [111], which traps are believed to exist in the

interface between the crystalline and amorphous region. According to Sessler [112], surface

traps may be due to surface defects, impurities, absorbed molecules and broken chains. Sessler

also stated that volume traps are located within three levels, atomic sites, molecular chains,

crystalline regions and interfaces.



Figure 2-18: schematic diagram showing trapped charge (A, C) and empty sites (B, D) in a polymer

matrix [88].

Charge dissipation is related to the detrapping of charge within the polymer. Detrapping has

been linked with molecular relaxation (hence temperature plays a role) [113], and the presence

of holes and electrons (with the two showing large differences) [113]. This process describes

the transport through electric charge via jumps between trap sites (as seen in Figure 2-18).

There are three theories underpinning current models of charge dissipation: the barrier model

where charge mobility is linked to activation energy, the Rouse model which describes

polymer chains as a series of connected springs and nodes, and finally, the Reptation model,

which describes the motion of polymer chains as a snake-like motion. It has been shown that

charge dissipation is proportional to the following empirical formula:

𝐼(𝑡) = 𝐴(𝑇)𝑡−𝑛 (2-15)

Where I is the measured current, A(T) is a temperature dependent factor, n ranges from 0.6 to

1 [114] and t is time. For all electrospinning cases, there is an initial fast detrapping, followed

by a slower reduction in charge [88].

Yan and Zhang investigated electric potential in the fibre using a probe electrode [16]. The

results demonstrate charge retention cumulating to a residual potential of up to 1 kV. They

found that residual potential depended on needle bias and charge, type of electric field and

thickness of the scaffold. As retention was dependent on the bias of charge, and it is well

established that holes have distinct mobility compared to electrons, positively biased scaffold

contained a charge half-life of 50 hrs, while negatively charged had a 20 hour half-life [16],



as seen in Figure 2-19. Charge retention and polarity has also been observed by Ignatova et al

[115].

Figure 2-19: Residual charge against time (h) for positively and negatively biased polystyrene (PS)

scaffolds. [16]

2.4 IMPLICATIONS FOR MELT ELECTROSPINNING AS AN

ADDITIVE MANUFACTURING TECHNIQUE

Controlling the internal microarchitecture of scaffolds can be achieved with the marriage of

melt electrospinning and additive manufacturing. FDM or melt deposition modelling (MDM)

use a technique for filament extrusion which is most similar to melt electrospinning. Using

FDM as a base model and applying its associated techniques, melt electrospinning can

transition into the field of additive manufacturing.

The challenge of tissue engineering today is the ability to produce anisotropic material

properties throughout a scaffold [116] with fibres small enough for improved cell-fibre

interaction. Previous efforts to combine FDM and melt electrospinning have yielded promising

results, however, not controlled enough to produce 3D structures. This is largely due to the

chaotic nature of the solution electrospinning process [116]. However, melt electrospinning

shows a much longer stable jet region than solution electrospinning and has therefore found

success in controlled fibre deposition. This technique is known as direct writing melt

electrospinning [12].



This merger has shown the successful production of scaffolds with internal properties which

are not possible to produce using other additive manufacturing techniques. The fibre size in

an electrospinning system can be more than 20 times smaller than a conventional FDM 3D

printer. This provides the opportunity to produce devices which have internal micro-

architectures that can affect the local micro-fluidics, mechanical structure and cell-fibre

interaction.

This system comes with current drawbacks. The size of the scaffolds that are currently

produced are limited by the disorder in a scaffold [14], with increasing disorder occurring as

layers of fibre are deposited onto each other. It is essential to provide a mechanism to remove

this disorder in order to create highly controlled microarchitecture in direct writing melt

electrospinning.

Another drawback of the melt electrospinning process (with respect to its application in bone

tissue replacement therapies) is the mechanical properties of the scaffolds which it produces.

While highly controlled, the structure does not match the mechanical properties of bone. A

combination of melt electrospinning for controlled microarchitectures and a support structure

produced using other 3D printing methods is one, not yet explored, option.

Chapter 3: Polymer Extruder Design


3Chapter 3: Polymer Extruder Design

A key portion of this master’s project involved intellectual input into hardware design and

development. Environmentally control of the build chamber and the extrusion system were

both updated during the project. The following two chapters will outline the process involved

in updating the melt electrospinning system by investigating the literature to support the

improvements, along with a summary of the design and implementation of each individual

unit.

The conventional melt electrospinning extrusion method involves a syringe filled with a

polymer which is melted via a heated jacket and extruded by a syringe pump through a needle

tip [10]. However, a number of changes do this method has been made over the years, Figure

3-1 illustrates some techniques that have been used. The study performed was completed using

a circulating fluid heating in a syringe with a syringe pump extruding the polymer [14].

Figure 3-1: Schematic of various heating approaches performed for melt electrospinning [10]

There have been recent improvements to the extrusion design which aim to solve problems

associated with throughput. While melt electrospinning is a simple and inexpensive process,

its low throughput has required innovations in the extrusion method. Christoph Hacker et al.



[117] has designed a system which distributes the molten polymer over an area with evenly

spaced nozzles.

Figure 3-2: Melt electrospinning extruder designed by Hacker et al. [117]

While other groups have used line-like laser beam melting devices [118]. These devices

produce a sweep of fibres along a straight line and are capable of increasing throughput greatly.

Figure 3-3 illustrates the formation of multiple jets using the aforementioned technique.

Figure 3-3: Line-like laser beam melting electrospinning developed by Shimada et al. [118].

Similarly, work by Li et al. [119] produced an “umbellate” structure to produce multiple jets

at once. This extruder can be seen in Figure 3-4.



Figure 3-4: Li et al.’s umbellate melt electrospinning extruder [119].

Work in modifying the extrusion head has led to a number of innovative designs. Applications

on the aforementioned techniques to a direct writing method may provide high throughput

additive manufacturing techniques for applications in tissue engineering.

3.1 OUTLINE OF THE REQUIREMENTS

The design requirements for the extrusion system are purposefully kept broad to accommodate

for future upgrades. Due to a recent increase in the number of publications reporting

modifications to the extrusion mechanism, a modular design was applied. This design allows

the user to remove the extruder head and replace it with a different design, which

accommodates any changes to the mechanism of extrusion without a complete overhaul of the

extrusion system. The temperature control is capable of functioning up to 250 °C for

applications on a wide variety of polymers.

Another key area of improvement was to decrease the auxiliary equipment attached to the

extruder. Removal of these components greatly reduces the space to operate the

electrospinning device.

3.2 DESIGN AND IMPLEMENTATION

This section outlines the construction of a polymer extrusion system intended for

implementation in a melt electrospinning device. This system updates the current heated water

jacket and syringe pump technique:



Figure 3-5: A comparison of the updated extrusion system (A) and the syringe pump extrusion system

(B).

The water jacket was replaced with a heating element and the syringe pump replaced with a

pressure system. The device was designed to incorporate a thermal inertia in the surrounding

material to ensure an even distribution of heat around the polymer. A hose carrying pressurised

air was to be connected via a screw-in connecter at the top of the device. The initial design

was then revised to account for manufacturing limitations. It is currently in prototype stage

and testing has been scheduled to begin at the beginning of June, 2015. Figure 3-5 illustrates

the current extrusion system (B) and the new system (A).

The system can be split into two sections, the heating stage and the pressure extrusion phase.

Each section, along with justifications will be explored in the following chapters.

3.2.1 Heating Jacket

Even heat distribution and thermal inertia were the most important aspects when designing the

heating system. Figure 3-5 (A) and Figure 3-7 (B) show the final design of the extrusion

system. Two heating elements are placed within the metal jacket surrounding the polymer.

This jacket is of appropriate thickness to ensure that the heat is distributed evenly through the

system and that the thermal inertia is high enough to ensure easy temperature stability. For

temperatures between 50 to 260 °C, the difference between the maximum and minimum

temperatures in the system do not exceed 10%:

(A) (B)



Figure 3-6: (A) Graph showing the dependence on the input temperature against the temperature at the

needle tip. (B) Imaging showing the distribution of temperature across the extruder, with the extrusion

tip facing the bottom. Modelled using ANSYS static thermal package (ANSYS, USA).

A number of designs were initially considered; however, due to manufacturing complexities

or inappropriate applications, they were not developed further. The section below describes

one such alternative which was explored more thoroughly than other alternative designs.

3.2.1.1 Alternative Designs

The initial design involved an oil bath, heated via heating elements; surrounding a metal jacket

containing the polymer. This system would ensure that no direct contact occurs between the

heating elements and the metal jacket. Distributing the heat through the liquid first would

ensure an even heat distribution. The thermal inertia in a liquid is much larger than a metal,

and temperature stability is much easier to maintain in such a system. However, there are

drawbacks to using an oil system. Manufacturing of such a design is complex and requires a

high degree of precision. Hence, this design was disregarded during the manufacturing design

stage.

150 °C

135 °C

(B)



Figure 3-7: Initial Design (A) vs. Final Design (B) of the extrusion system for the melt electrospinner.

3.3 PRESSURE SYSTEM

Several common industrial techniques exist for the extrusion of polymers. Examples include

pressure extrusion, screw-extrusion and plunger extrusion. The system most commonly

employed in electrospinning systems is the plunger extrusion method. A syringe pump is used

to push polymer through a syringe [10]. These systems are bulky and require a large syringe

pump to sit in-line with the extruder. Screw-extrusion systems are well defined and commonly

found in industrial applications for polymer extruders. However, they are not suitable for

electrospinning applications due to the large shear forces created during the melt and extrusion

phases. These forces would cleave peptides and proteins and potentially denature any bioactive

material found in the polymer. Pressure extrusion works on similar principles to a syringe

pump mechanism, with air pressure being used to extrude material rather than a physical

plunger. These systems are simple to implement can be isolated from the motion of the

extrusion mechanism. Pressure extrusion has hence been used by a number of groups due to

these innate advantages [117], [120].

The system employs an SMC ITV1000 pressure regulator in line with a lab air supply unit

attached to a fitting at the top of the extrusion system. This system can be controlled via 0-10

VDC power unit, which can be coupled to a computer, allowing automatic control of polymer

flow during the electrospinning process. The schematic in Figure 3-8 illustrates the pressure

extrusion system.



Figure 3-8: Schematic diagram of the pressure extrusion system

Pressure control is an important step towards produce an advanced extruder based of work by

Hacker et al. [117], Li et al. [119] or Shimada et al. [118]. These systems increase the

throughput of melt electrospinning systems and are an important step towards

commercialization of this technology. Upgrade of the heating element to include computer

control and a wider temperature range increases the variety of polymers which can be studied

on the system.

Chapter 4: Humidity System Design


4Chapter 4: Humidity System Design

It is well established that humidity levels affect the electrospinning process. Studies completed

on the topic show that not only does humidity effect the evaporation rate of solvents, but that

it also contains the ability to affect the transport of charge. Work by Kim et al. [91] illustrated

the relative humidity’s ability to increase fibre diameter. This study created a theoretical

framework based of Seaver [121]investigating the surface charge density. It quantified the

amount of charge stored by ions in the polymer and its ability to control the acceleration of the

jet through the electric field, causing it to thin [91]. Other studies, such as those presented by

Casper et al. [122] and De Vrieze et al. [123] investigated humidity’s effect on the surface

structure of a scaffold Although, many of these phenomenon are linked to solvent evaporation

and are not applicable to melt electrospinning.

There are numerous reasons to believe that residual charge removal would be accelerated with

an increase in water content in the surrounding atmosphere. Studies by several groups have

shown that the content of the local atmosphere plays a large role in jet formation as well as in

maintaining a continuous fibre [96], [124]. As previously stated in sections 2.3.3, a study by

Galembeck et al. [105] showed that electrostatic charge build-up and dissipation in dielectrics

was affected by the availability of H(H2O)n+ and OH(H2O)n

- ions in the atmosphere and

absorbed by the dielectric.

While a plethora of information exists on the effect of humidity on dielectric polymers and

solution electrospinning, there is a gap in the knowledge regarding its affect for melt

electrospinning. The proposed improvements to the melt electrospinning system aim to

provide a platform to study the effects of humidity and temperature control of the build

chamber in the system.

This system has been implemented into the electrospinning device and research has been

completed by Sam et al. [26] to determine the effect of humidity on melt electrospun PCL

fibres.

4.1 OUTLINE OF THE REQUIREMENTS

The design requirements are based off of previous studies relating to the solution

electrospinning system. Casper et al. varied the relative humidity at 25 °C from less than

25%RH and up to 72%RH [122]. De Vrieze et al. varied the relative humidity at three

temperatures (283 K, 293 K, 303 K) between 20%RH and 60%RH [123]. The study did not



exceed humidity greater than 60 %RH due to lack of fibre formation for that polymer/solvent

combination. Studies analysing dielectric polymers have examined much larger variations in

temperature and humidity, ranging from ~0% RH to saturation and temperatures higher than

100 °C. For the evaluation of melt electrospun PCL scaffolds, humidity ranging from 25% RH

and as high as 75% RH would cover a relatively large percentage, with temperature not

needing to exceed 60 °C as this is the melting temperature of PCL.

4.2 DESIGN AND IMPLEMENTATION

The humidification system is based on a design developed by G.R. Johnson et al. [125]. The

system works by switching between two chambers containing saturated air and dry air and

mixing the air directly into the build chamber. Due to the size of the build chamber, it provides

a thermal and humidity inertia, which allows the system to remain stable for long periods of

time. This section will contain three subsections; the saturator, the desiccator and the mixing

chamber. Each section will be examined to determine their capacity within the overall system.

4.2.1 The Saturator

The saturator is based on a system developed by G.R. Johnson et al. [125]. The system adds

water content to the air by passing it through a diffusion dryer containing wetted perlite. The

system was designed and built by G.R. Johnson et al and was retrofitted into the melt

electrospinning rig. It was built to accommodate a flow rate of up to 30 L/min and sustain a

humidity release of 80% RH (25 °C) at this flow rate. That being said, at flow rates as low as

10 L/min, the system is able to achieve up to 99.1 RH% humidity.

Figure 4-1: Image of saturator designed by Johnson et al.

4.2.2 The Desiccator

The desiccator is a chamber filled with silica gel beads. These beads absorb moisture up to

40% of their weight in water. As the humidifier contains a fixed air velocity, the length of the

chamber that is used will be varied to determine the change in moisture content as it exits the

system.



The amount of water absorbed in the system is equal to the product of the density, flow rate

and the difference in moisture content between the input and the output of the desiccator:

𝑑𝐴

𝑑𝑡= 𝜌𝑄∆𝑊

(4-1)

Where 𝑑𝐴

𝑑𝑡 the rate of absorbance is, 𝜌 is air density, 𝑄 is the volume flow rate and ∆𝑊 is the

change in moisture content. Figure 4-2 shows the absorbance characteristics of silica gel at

75% humidity:

Figure 4-2: Absorbance characteristics of common desiccants

We can determine the amount of water being added to our desiccant using the following

formula:

𝐴 = 𝜌𝑄(𝑊𝑜 − 𝑊𝑖)𝑡 + 𝐶1 (4-2)

Where the density of air is 𝜌, the air flow rate is 𝑄, water content into the system is 𝑊𝑖 and out

of the system is 𝑊𝑜, 𝑡 is time and 𝐶1 is a constant of integration. We pump air into our system

at a rate of 30 𝑙/𝑚𝑖𝑛, with density 1.225 𝑘𝑔/𝑚3, which has a relative humidity of

approximately 35%RH. It has a water content of 6.8323 𝑔/𝑘𝑔 when it enters the system and

1.9463 𝑔/𝑘𝑔 when it exits the system. If we want this system to run non-stop for periods of

more than 24 hours, we need to determine the amount of desiccant required.

𝐴𝑤 = 0.0108𝑡 (4-3)

Our system would absorb 258 g of water in a 24 hour period, and would require a minimum

of 750 g of silica gel desiccant before saturation. This value reflects the bare minimum of



operation and would need regeneration of the desiccant after each use. Hence, the system was

built to handle being continuously run for a period exceeding 7 days of continuous use.

4.2.3 The Mixing Chamber

Once air has gone through the humidifier and desiccator, it has reached its maximum and

minimum humidifies, respectively. The PID system will switch between these two points

using a solenoid valve. The melt electrospinning enclosure will then act like a mixing chamber.

A schematic diagram of the system is shown in Figure 4-3:

Figure 4-3: Initial design for humidity control system. Control box is the melt electrospinning device,

which will act like a mixing chamber in this configuration. Temperature and filtering have since been

removed from the system

The mixing chamber can be modelled using continuum mechanics. We can apply the principle

of conservation of mass to the system. Any volume of air entering the system will be equal to

the volume of air leaving the system. The humidity of the air entering the system is equal to

the humidity exiting either the saturator or the desiccator. The volume of air exiting the system

is a combination of the residual air in the system plus the air form the humidifier/desiccator.

Hence the equation should read:

𝜌𝑎𝑉𝑑𝑊𝑟

𝑑𝑡= ∑ 𝜌𝑎𝑖

𝑄𝑎𝑖(𝑊𝑜𝑖 − 𝑊𝑟)

𝑛𝑎

𝑖=1

(4-4)



Where 𝜌𝑎 is the density of air, 𝑉 is the volume of air, 𝑑𝑊𝑟

𝑑𝑡 is the rate of change of the moisture

content, 𝑛𝑎 is the number of airflow sources, 𝑄𝑎𝑖 is the flow rate of the ith air flow source, 𝑊𝑜𝑖

is the moisture content of the ith airflow source and 𝑊𝑟 is the current moisture content. For our

system, there are two sources of air flow; air entering the system and air exiting the system.

Therefore, the equation as applied to our system is:

𝑉𝑑𝑊𝑟

𝑑𝑡= 𝑄𝑎𝑖𝑛

(𝑊𝑖𝑛) + 𝑄𝑎𝑜𝑢𝑡(𝑊𝑜𝑢𝑡)

(4-5)

Since we assume that the air flow in is equal to the airflow out, we can write:

𝑄𝑎𝑖𝑛= 𝑄𝑎𝑜𝑢𝑡

(4-6)

Therefore:

𝑑𝑊𝑟

𝑑𝑡=

𝑄

𝑉(𝑊𝑖𝑛 − 𝑊𝑜𝑢𝑡)

𝑑𝑊𝑟

𝑊𝑖𝑛 − 𝑊𝑜𝑢𝑡=

𝑄

𝑉𝑑𝑡

− ln(𝑊𝑖𝑛 − 𝑊𝑜𝑢𝑡) + 𝐶 =𝑄

𝑉𝑡

(4-7)

Using the boundary conditions 𝑊𝑟(𝑡 = 0) = 𝑊0 :

𝑊𝑖𝑛 − 𝑊𝑜𝑢𝑡

𝑊𝑖𝑛 − 𝑊0= 𝑒−

𝑄𝑉

𝑡

(4-8)

This then gives us our final equation:

𝑊𝑜𝑢𝑡 = 𝑊𝑖𝑛 − (𝑊𝑖𝑛 − 𝑊0)𝑒−𝑄𝑉

𝑡

(4-9)

This equation can be used to determine the transfer function of our system, which is used to

find the appropriate values for our PID controller. This model was placed into Simulink:



Figure 4-4: Simulink model of humidity controller

The PID system was optimized numerically using Matlab’s inbuilt PID optimization software.

The Graph below (Figure 4-5) shows a simulation of the system for three set-points:

Figure 4-5: Transient response of humidity system

Chapter 5: Improved Fabrication of Melt Electrospun Tissue Engineering Scaffolds Using Direct Writing and

Advanced Electric Field Control.


5Chapter 5: Improved Fabrication of Melt

Electrospun Tissue Engineering Scaffolds

Using Direct Writing and Advanced Electric

Field Control.

Nikola Ristovski3, Nathalie Bock3, Sam Liao3, Sean K. Powell3, Jiongyu Ren3, Giles T.S.

Kirby3, Keith A. Blackwood3 and Maria A. Woodruff2

Published in Biointerphases, Volume 10, Issue 1, 2015, doi: 10.1116/1.4914380

© 2015 American Vacuum Society 011006-1

Statement of contribution of co-authors for thesis by published papers

Contributors Statement of Contribution Nikola Ristovski Involved in the developed research

questions Designed and performed experiments Analysed and interpreted the results Conceived and wrote the manuscript

Nathalie Bock Performed experiments

Sam Liao Built equipment

Sean K. Powell Assisted in reviewing the manuscript

Jiongyu Ren Performed experiments

Giles T.S. Kirby Developed research questions

Keith A. Blackwood Developed research questions Performed experiments

Maria A. Woodruff Involved in the conception of the project Assisted in reviewing the manuscript

2 Biomaterials and Tissue Morphology Group, Institute of Health and Biomedical Innovation, Queensland

University of Technology, Brisbane, Australia2




ABSTRACT

Direct writing melt electrospinning is an Additive manufacturing technique capable of the

layer-by-layer fabrication of highly ordered 3d tissue engineering scaffolds from micron-

diameter fibres. The utility of these scaffolds; however, is limited by the maximum achievable

height of controlled fibre deposition. A source of this disorder is charge build-up on the

deposited polymer producing unwanted columbic forces. In this study, we introduce a novel

melt electrospinning platform with dual voltage power supplies to reduce undesirable charge

effects and improve fibre deposition control. We produced and characterised several 90 °

cross-hatched fibre scaffolds using a range of needle/collector plate voltages. Fibre thickness

was found to be sensitive only to overall potential and invariant to specific tip/collector

voltage. We also produced ordered scaffolds up to 200 layers thick (fibre spacing 1 mm,

diameter 40 μm) and characterised structure in terms of three distinct zones: ordered, semi-

ordered and disordered. Our in vitro analysis indicates successful cell attachment and

distribution throughout the scaffolds, with little evidence of cell death after seven days. This

study demonstrates the importance of electrostatic control for reducing destabilising polymer

charge effects and enabling the fabrication of morphologically suitable scaffolds for tissue

engineering.




5.1 INTRODUCTION

The effectiveness of electrospinning as a fabrication technique for tissue engineering is highly

dependent on its ability to fabricate scaffolds having both micro and macro-scale structural

features optimal for tissue growth. Two main electrospinning methods exist for producing

fibres, which are, solution electrospinning and melt electrospinning. Solution electrospinning

involves dissolving polymers in a solvent and uses a large electric potential to accelerate

solutions from a needle and onto a collector plate. Although this technique is capable of

producing fibres with nanometre diameters, solvent evaporation during fibre extrusion induces

large instabilities which significantly reduces precise control over fibre placement, commonly

known as fibre whipping [126]. Melt electrospinning similarly uses a large electric potential,

but produces fibres by liquefying a polymer via heat transfer similar to FDM (FDM); a

commonly used 3D printing technique. The use of an electric field allows the production of

fibres with significantly smaller diameters than can be achieved via a purely mechanical means

[127], [128].

Recent work has demonstrated the ability for melt electrospinning to deposit fibres onto a

moving collector plate with a great degree of precision in a process called direct writing [11],

[12]. By controlling the translation of the collector plate, 2D patterns can be formed which can

then be stacked to produce 3D structures with custom internal micro-architectures. However,

this technique is limited by the maximum number of layers that can be produced before control

over fibre placement is lost due to the accumulation of instabilities [12]. It is hypothesised that

a significant source of these instabilities is the build-up of electric charge on the deposited

fibres resulting in increasing undesirable net columbic force acting on the extruding fibre.

In electrospinning, many interacting factors combine to control fibre diameter. These include

polymer feed rate [127], [129], [130], applied potential [131], needle TTC distance [17], [23],

collector translation speed [12], and the mechanochemical properties of the polymer [10]. Due

to the viscosity of molten polymer, melt electrospun fibres can be produced with diameters in

the micro-scale range. In addition, the deposition of these fibres is more controllable than

solution electrospun fibres which suffer from reduced jet stability due to solvent evaporation

[12], [128], [132].

A further advantage of melt over solution electrospinning is that it can produce scaffolds with

larger pores sizes which are more suitable for cell infiltration. As solution electrospinning

generally produces much smaller fibre networks, in the order of nanometres, the resulting pore

sizes after deposition of multiple layers, restrict cell infiltration. This results in large cell

populations on the outer layers of the scaffolds, and very limited to no cells in the interior of

the scaffold [133],. It has been found that an approximate interconnected pore size of 100 to




400 µm is ideal for growth of osteoblasts in 3D [134]. This is many orders of magnitude larger

than the nano-scale pore sizes of solution electrospun scaffolds; however, direct writing melt

electrospinning has the potential to produce scaffolds with customisable pore sizes tailored to

the requirements of different cell types.

Combining the principles of Additive manufacturing with melt-electrospinning enables the

fabrication of 3D structures having configurable fibre diameter, fibre spacing, and laydown

pattern. Existing studies on tissue infiltration and growth within 3D melt electrospun fibre

networks have demonstrated the importance of pore size and other microstructural

characteristics on tissue infiltration and growth [133], [135]–[137]. Control of fibre placement

throughout the entire scaffold is therefore of great importance for the successful application of

this technique to tissue engineering.

Although melt electrospinning via direct writing has great promise as a technique for

fabricating scaffolds for tissue engineering, it has not yet attracted much attention. The few

studies undertaken have identified that precise fibre placement control is affected by the

complex interaction of several parameters such as the magnitude of the electric potential

between the needle tip and collector, collector speed, and the temperature of the molten

polymer [12]. Other important manufacturing properties are the flow rate of the molten

polymer and the tip to TTC distance [132]. As melt electrospinning uses electric potential to

draw out fibres from the needle tip, it is hypothesised that the interaction between the produced

electric field and the charge distribution on the surface of the polymer plays a key role in fibre

deposition.

The classic melt electrospinning setup involves a positive voltage applied to the tip of the

emitter and fibres spun onto a grounded metallic collector plate [10]. By distributing the

voltage placed on the collector and emitter, it is possible to reduce the amount of charge in the

polymer. A negative voltage applied to the collector plate will allow the system to maintain

the same potential difference between collector and emitter, but reduce the charge placed on

the polymer. It is hypothesised that this change in the electrospinning system will result in

greater deposition accuracy for longer periods of time, allowing the production of 3D

constructs.

In this study we produced PCL scaffolds with 90 ° cross hatched internal microarchitecture to

investigate the effect of electrospinning fabrication parameters on scaffold structure order. We

also assessed the suitability of the scaffolds for tissue engineering by observing the infiltration

and proliferation of murine calvarial osteoblast cells (MC3T3-E1). The scaffolds were

fabricated using our custom built direct writing melt electrospinning platform, which uses a

negative voltage on the collector plate to reduce residual charge on the fabricated fibres. We




found this strategy significantly improved the number of ordered layers achievable over

current literature. We also found that fibre spacing affected the maximum number of ordered

layers, with greater spacing allowing more ordered layers. The in vitro analysis demonstrated

a successful infiltration of MC3T3-E1 cells after 7 days in ordered scaffolds with cross hatch

microarchitectures. We also observed cell proliferation within the ordered scaffolds did not

differ from proliferation in scaffolds with random fibre arrangement.

5.2 MATERIALS AND METHODS

5.2.1 Melt Electrospun Scaffolds

5.2.1.1 Polymer Preparation

Polycaprolactone was obtained from Perstorp (PCL, Capa 6430®, Perstorp UK Limited). PCL

pellets were placed in a 2 mL plastic syringe. The syringe was heated in a vacuum oven (- 80

kPa for 30 min at 90 °C) to remove air bubbles.

5.2.1.2 Melt Electrospinning Device

PCL was electrospun using our novel in-house dual voltage melt electrospinning platform.

Figure 5-1 shows a schematic diagram of the device. A 2 mL syringe loaded with PCL was

placed in a heated water jacket at 73 °C. The resulting molten polymer was extruded at a rate

of 40 µL/h using a syringe pump (World Precision Instruments, AL-1000). The tip of the

extrusion needle and the collector were attached to custom-made isolated power supplies; the

tip having a positive voltage, and the collector plate having negative voltage. The collector

plate was attached to a motorised translation assembly for controlled x,y,z translation

(Velmex, USA). This assembly was attached to a controller (ECG 5-axis board) which was

driven via Mach3 software (ArtSoft, USA). The collector plate was translated according to

pre-programmed G-code to control the deposition of fibres onto the plate.




Figure 5-1: Novel direct writing melt electrospinning platform with dual voltage power supplies for

improved fibre deposition control. The negative power supply attached to the collector plate being the

defining difference in this system.

5.2.1.3 Scaffold Production

We separated scaffold fabrication into two phases in order to first determine optimal scaffold

production parameters and then assessed the scaffolds for basic cell infiltration and

proliferation. The fabrication parameters corresponding to the best performing scaffolds from

the first phase were then utilised to produce scaffolds for use in the second phase.

For the ordered scaffolds, we selected a stacked 90° cross-hatched internal microarchitecture

with each layer aligned with the x-y plane stacked in the z-axis. As the phase one scaffolds

were intended to assess the effect of negative potential on scaffold fabrication control, we held

all manufacturing parameters constant for all production and varied only the applied voltages.

The following variables were kept constant: temperature (73 °C), extrusion rate (40 µL/h),

needle gauge (21 Gauge), stage speed (750 mm/min), needle TTC plate distance (10 mm),

fibre spacing (500 µm) and electric field strength (1.05 kV/mm). Within this phase we

produced five groups of six scaffolds each using a constant net electrical potential but different

positive and negative potentials as shown in table 1.




Table 5-1: Phase 1 scaffold groups and their corresponding needle tip and collector plate voltages. Six

scaffolds were produced for each group.

Group A B C D Control

Tip voltage (kV) Ground 3.5 5 7 10.5

Collector voltage (kV) -10.5 -7 -5 -3.5 Ground

The scaffolds produced in phase two were intended for assessment of scaffold suitability for

cell infiltration and proliferation. We produced two sets of ordered scaffolds having 25 layers

and 50 layers, respectively, as well as a set of randomly deposited fibres, as shown in table 2.

The manufacturing parameters for the ordered scaffolds in this phase were selected as those

from group D in the phase 1 study. The scaffolds for phase 2 were thus manufactured using

the same parameters as group D. The random structure scaffold was produced by increasing

the needle tip/collector distance to induce fibre whipping. We further fabricated a 90° cross

hatched scaffold with 1 mm fibre spacing using the phase 1, group D, manufacturing

parameters in order to assess the impact of fibre spacing on scaffold order.

Table 5-2: Number of scaffolds and structural parameters produced for phase 2 of the study.

Number of scaffolds fabricated per study No. of layers Microstructure Live/Dead DAPI/Phalloidin MTT Assay

25 90° crosshatch 1 1 12

50 90° crosshatch 1 1 12

25 Random 1 1 12

5.2.2 Scaffold Characterisation

5.2.2.1 SEM Preparation and Imaging

Scanning electron microscopy (SEM) was performed on all scaffolds in phase one, with six

scaffolds per group, using a Quanta 200 SEM (FEI, USA) to assess microstructure. Prior to

imaging, the scaffold was gold sputtered using the Leica EM-SCD005 Sputter Coater for 5

minutes (Leica, Germany). Transverse SEM micrographs of the scaffold (x-y plane) were used

to determine the mean fibre diameter and fibre spacing. Sixty fibres were randomly chosen

from 5 locations (12 fibres per location) in each scaffold and their diameters were measured.

The mean of the diameters was calculated and the standard deviation used for measurement

uncertainty. Typical fibre spacing was computed by identifying the relative coordinates of

each chosen fibre and using custom Matlab code to compute the average fibre separation.

Scaffold layer ordering was also determined from SEM images of scaffold cross-sections (x-

z plane).




5.2.2.2 Distance Transformation Method

Inter-layer fibre alignment is an indicator of the general ordering of the scaffold micro-

architecture. A measure for this alignment, or structure order (𝑂𝑠), can be visualised by

considering the length of a line connecting the axial centres of a stacked column of n fibres

divided by the length of the shortest line connecting the top and bottom fibres in a column and

can be calculated via;

𝑂𝑠 =∑ |𝑺𝑖 − 𝑺𝑖−1|𝑛

𝑖=2

|𝑺𝑛 − 𝑺1|

(5-1)

Where 𝑺𝑖 is a vector representing the axial centre of the ith fibre in the column. The form of

this equation is analogous to the well-known measure of tortuosity, and quantifies stacking

order for structures containing n layers, with 𝑂𝑠 = 1 indicating perfect stacking alignment and

𝑂𝑠 > 1 for disordered stacking. Centres of fibres were determined using cross-sectional SEM

images and data was analysed using MATLAB code.

5.2.2.3 µCT Imaging and Analysis

Six scaffolds from group C were scanned using a µCT 40 micro computed tomography scanner

(Scanco Medical, Brüttisellen, Switzerland) at an energy of 45 kVp, intensity of 177 µA, and

300 ms integration time. Voxel sizes for each scan were 6 µm (isotropic). The scans were

analyzed using the distance transformation method to determine the average fibre diameter,

pore size and pore size distribution throughout the scaffold.

5.2.3 In Vitro Characterisation

5.2.3.1 NaOH Etching

Fibre surface modification was performed to increase the fibre surface roughness and ensure

successful scaffold hydration for optimal cell attachment [138]. The hydrophobicity of the

PCL was reduced by soaking the scaffolds in 5M NaOH for 1 hour at room temperature. The

scaffolds were then soaked in MilliQ water until the supernatant pH dropped to 7.0, and then

placed in a desiccator to dry for approximately 24 hours.

5.2.3.2 Cell Culture

Small sections of scaffolds were extracted using a 6 mm biopsy punch. These were sterilized

with 70% ethanol and irradiated with ultraviolet light for 20 minutes each side, all steps were

taken consecutively. The scaffolds were divided into three groups; one group of 12 scaffold

samples (six per time point) were used for assessing cell proliferation (MTT analysis), a single

sample for assessing cell morphology (DAPI/Phalloidin) and a single samples for assessing

cell distribution. Murine calvarial osteoblastic cells (MC3T3-E1) were cultured in heat-




inactivated α-MEM media (α-MEM, Invitrogen, Australia) with 10% (v/v) foetal bovine

serum and 1% (v/v) Penicillin-Streptomycin with a concentration of 10,000 µg/mL. Cell work

was performed as previously described by Ren et al. [139]. An average of 5000 cells were

seeded onto each scaffold for proliferation assessment and allowed to attach for one hour in a

small volume (50 μl) of media prior to the addition of a further 400 µl of α-MEM media at 37

°C and 5% CO2. An average of ~450,000 cells were seeded with 100 µl of α-MEM onto

scaffolds for assessing cell morphology and were allowed to attach for four hours prior to the

addition of 400 µl of α-MEM media at 37 °C and 5% C02. A large quantity was chosen to

increase the chances of a homogenous distribution for a qualitative cell morphology

assessment and reduce experimentation time. The scaffolds were then incubated at 37 °C and

5% CO2 with the culture media changed approximately every 48 hours, until respective time

points were reached.

5.2.3.3 LIVE/DEAD Staining

Live/dead staining was used, at day three, as an indicator for positive cell attachment and as

an assessment of cell penetration throughout the scaffold. Due to the qualitative nature of this

experiment, only one sample was used. The scaffolds were washed twice with PBS. 5 µL stock

solutions of FDA (Invitrogen, USA) and PI (Invitrogen, USA) were diluted in 5 mL of PBS

making a final concentration of 0.67 µg/ml and 5 µg/mL respectively. 2 mL of the FDA and

PI solution were added to scaffolds and the cells incubated for 5 minutes in a dark environment

at 37 °C. The scaffolds were washed once with 2 mL/well plate of PBS and covered with fresh

PBS. Scaffolds were then transferred onto a glass slide and imaged using the Zeiss Axio M2

(Zeiss, Germany) at excitation levels of λ = 488 nm and λ = 568 nm.

5.2.3.4 Cell Metabolic Rate Assay

MTT assay (Invitrogen, Australia) was used to assess the metabolic activity of cells at days

one and seven to check how metabolically active the cells within the scaffolds were. Six

samples were taken at each time point. A working solution was produced by diluted 5 mg/mL

MTT stock solution with 500 µL of αMEM, for a final working solution of 0.19 µg/mL. The

scaffolds were transferred into fresh 48-well plates and 500 µL of fresh media with 20 µL of

working solution was added. The cells were then incubated for 4 hours at 37 °C and 5% CO2.

The media was removed and dimethyl sulfoxide (DMSO, Merck, Australia) was added, with

100 µL for day one and 200 µL for day seven. The well plates were covered in aluminium foil

and placed on an orbital shaker for 10 minutes. 100 µL of DMSO was removed from each well

and placed in a fresh 96 well plate. Absorption was measured at λ = 540 nm. Absorption data

from all empty wells in the well plates was averaged and subtracted. Scaffolds were




normalized for the volume of DMSO added to each well plate at the two different time points

before comparisons between the two were made.

5.2.3.5 Cell Morphology and Attachment Staining

A DAPI/Phalloidin assay was used to stain cells at day three to qualitatively assess cell

adhesion onto the scaffold, with one sample taken. The media was removed and the scaffold

was transferred into a fresh 48 well plate. The scaffold was then washed with PBS and fixed

in 4% paraformaldehyde for 30 minutes at room temperature. The scaffolds were washed in

PBS solution. A 0.2% (v/v) Triton-X-100/PBS solution was added to each well and left for 5

minutes before another wash of PBS. The scaffold was then incubated with 0.5% (w/v)

BSA/PBS for 10 minutes and placed in 0.5% (v/v) BSA/PBS with 0.8 U/ml Alexa Fluor

Phalloidin and 5 μg/ml DAPI. Following this, the scaffold was washed in milliQ water once

and then stained with Alizarin red S for 5 minutes at room temperature. The scaffold was

imaged using a Leica SP5 (Leica, Germany) confocal microscope.

5.2.4 Statistical Analysis

Sample difference was tested using the ANOVA test on SPSS statistics 21 (SPSS. USA), with

Levene’s test used to insure similar variance between groups (p>0.05). A Tukey HSD post hoc

test was performed on any ANOVA test which had p > 0.05 for a pairwise comparison of the

subgroups variance.

5.3 RESULTS AND DISCUSSION

5.3.1 Physical Characterisation

5.3.1.1 Fibre Diameter and Pore Size

Fibre diameter and pore size are important structural properties of electrospun scaffolds for

tissue engineering. Melt electrospinning is capable of producing fibres with diameters from 1

µm to 80 µm [10]. Pore size, in the context of tissue engineering scaffolds, refers to the average

dimension of the spherical fibre-free volumes within a scaffold. Although the shapes of the

pores vary throughout a scaffold, pore size can be used as an indicator of the typical volumes

available for cell infiltration and proliferation. For randomly distributed fibres, the sizes of

pores are normally distributed and have a very large standard deviation. By controlling fibre

deposition, pore size can be controlled and pore size distribution reduced. Controlling pore

size is difficult with randomly configured fibres, with smaller pores significantly restricting

cell infiltration [133].




Figure 5-2: Distribution of pore sizes for group D (+7 kV tip: -3.5 kV collector). The data shows two

apparent peaks at 282 μm and 378 μm which can be attributed to the fibre separation in the x-y plane.

Figure 5-2 shows the pore size distribution of the scaffolds from group D (Needle 7 kV and

collector -3.5 kV). This demonstrates the degree of control over fibre ordering with the

majority of pores being greater than 100 μm. 90° cross-hatch scaffold architecture results in

vertical square-prism shaped pores. Pore size is a measure of the maximum radius of a sphere

that can fit into a void space. The maximum spherical pore size in perfectly ordered scaffolds

is therefore equivalent to the x-y spacing of the fibres. Consequently, the distribution of pore

sizes around this ideal value is due to fibre disorder and is an indicator of fibre deposition

control during fabrication.

The standard electrospinning configuration consists of a positive voltage on the needle tip and

ground at the collector plate. The value of the chosen tip voltage is known to be a key factor

in affecting the diameter of the extruded fibre, with a large potential resulting in the production

of thinner fibres. In the present study, we fabricated several scaffolds using different potentials

for both the needle tip and collector plate (instead of simply grounding the collector plate)

while ensuring that the net potential remained constant. To investigate if the negative potential

affects fibre diameter, we measured randomly chosen fibres using SEM images. The results,

shown in Figure 5-3, indicate that fibre diameter is invariant to the chosen voltage for the

needle tip and collector plate and therefore only sensitive to the total voltage between the two.

0

0,5

1

1,5

2

2,5

3

3,5

4

6 66 126 186 246 306 366 426 486 546 606 666 726 786 846 906 966

Pe

rce

nta

ge o

f T

ota

l[%

]

pore size [µm]




Figure 5-3: Fibre diameter for different voltage distributions as measured via SEM. Each value was

taken by manually measuring the diameter using ImageJ software (n = 48 fibres). The data shows no

statistical significance between any groups (p > 0.05).

In addition to average fibre thickness, we also measured fibre diameter variation throughout

the scaffolds using µCT images. The average fibre diameter for scaffolds in group D was 20.7

µm ± 3.42 µm. To investigate the suitability of µCT for measuring the diameter of PCL fibres,

we compared these results with measurements based on SEM images of the same scaffold.

The SEM measurements indicate a mean fibre diameter of 39.2 µm ± 4.3 µm which is

approximately twice as large. This discrepancy can be explained by the inability of μCT to

resolve actual fibre boundaries due to the low electron density of the polymer.

Figure 5-4: Image of µCT reconstruction of scaffold from group D (+7 kV tip: -3.5 kV collector) with

an x-y fibre spacing of 500 µm.

0

10

20

30

40

50

60

A B C D Control

Fib

re D

iam

ete

r [µ

m]

Scaffold groups (based on tip/collector voltages)

A -10.5 kV: GND

B -7 kV: 3.5 kV

C -5 kV: 5 kV

D -3.5 kV: 7 kV

Control GND: 10.5 kV




5.3.1.2 Scaffold Ordering

Controlling the deposition of melt electrospun fibres through direct writing allows the

fabrication of tissue relevant scaffolds for use as replacement constructs in regenerative

medicine. This control enables the engineering of pore sizes, pore shapes and pore

interconnectivity suited to the needs of different cell types. The micron-scale diameters of the

electrospun fibres also provides a relatively large surface area to volume ratio compared to

thicker fibres, providing good surface for cell attachment. These important factors make direct

writing melt electrospinning a promising technique to produce scaffolds for tissue engineering.

To assess the degree of control over scaffold fabrication using the direct writing method, we

considered two properties; fibre stacking across successive layers, and the maximum number

of layers achievable before stacking order was lost.

5.3.1.3 Fibre Alignment

Inter-layer fibre alignment is an indicator of the general ordering of the scaffold micro-

architecture. A measure for this alignment, or structure order (𝑂𝑠), can be visualised by

considering the length of a line connecting the axial centres of a stacked column of n fibres

divided by the length of the shortest line connecting the top and bottom fibres in a column and

can be calculated via;

𝑂𝑠 =∑ |𝑺𝑖 − 𝑺𝑖−1|𝑛

𝑖=2

|𝑺𝑛 − 𝑺1|

(5-2)

Where 𝑺𝑖 is a vector representing the axial centre of the ith fibre in the column. The form of

this equation is analogous to the well-known measure of tortuosity, and quantifies stacking

order for structures containing n layers, with 𝑂𝑠 = 1 indicating perfect stacking alignment and

𝑂𝑠 > 1 for disordered stacking. SEM images shown in Figure 5-5 are cross sections (x-z plane)

of scaffolds from groups A to D and control, and illustrate the effects that different needle tip

and collector plate voltages have on stacking order.




Figure 5-5: SEM cross-sectional images (x-z plane) of melt electrospun scaffolds with distributions of

voltages, varying from 0 to 10.5 kV, between the tip and the collector (see Table 1). (A-D) show

scaffolds produced using negative voltage on the collector plate. Scaffold (E) is the control; it is

produced by grounding the collector plate. (F) Illustrates the Structure order calculated via Eq. 1 for

scaffolds of group A-D and Control. Os=1 indicates perfect fibre stacking across all layers, Os>1

indicates fibre stacking disorder. Uncertainties are computed from the standard deviation.

0,9

0,95

1

1,05

1,1

1,15

1,2

1,25

A B C D Control

Stru

ctu

re O

rde

r P

aram

ate

r [𝑂

s]

Scaffold Group

(F)A -10.5 kV: GND

B -7 kV: 3.5 kV

C -5 kV: 5 kV

D -3.5 kV: 7 kV


*

*

*




Using the Eq. 1, we quantified the structure order for scaffolds of groups A-D and the control

using six different scaffolds from each group; we selected six columns from each scaffold in

each group and identified the coordinates of the axial fibre centres associated with each

column. These coordinates were then used to compute 𝑂𝑠 for each column, which was then

averaged over the six chosen columns to produce a measure of the average structure order for

each scaffold. The results shown in Figure 5-5 indicate that all scaffolds produced with a

negative potential on the collector plate had higher structure order than those produced with

the standard grounded collector plate, with scaffolds from groups B and D having the greatest

order.

Electrospinning uses the interaction between an electric field and electric charge in the

polymer to draw out the fibres. The influence of this interaction is a significant factor affecting

the ability to produce ordered structures with micron scale precision. It is hypothesised that

during fabrication, the accumulation of charged fibres onto the collector plate results in

undesirable net forces acting against the deposition stability. Figure 5-5 indicates that there is

some effect on stacking due to the addition of a negative collector plate. This may be due, in

part, to the interaction of charge stored in the polymer and the electric field, resulting in

decreased stacking order. This effect is also apparent by observing the decreasing stacking

order as a function of stacking height (number of layers) as shown in Figure 5-6 for a scaffold

comprising 50 layers.

Figure 5-6: Change in fibre order as the number of layers increases. The vertical inter-fibre distance

taken from the axial centre of the fibres indicates fibre order. The results correspond to scaffolds from

group D (+7 kV tip: -3.5kV collector).

0

10

20

30

40

50

60

70

80

90

0 10 20 30 40 50

Dis

tan

ce B

etw

een

Fib

res

[µm

]

Layer




Another significant parameter affecting stacking order is the intra-layer fibre spacing, which

dictates the separation between vertical columns of stacked fibres. The destabilising effect

caused by charges in the deposited polymer is proportional to the fibre separation within each

layer. To demonstrate this we fabricated two scaffolds similar to group D, but increased the

layer fibre spacing from 500 μm to 1 mm (see Figure 5-7). SEM images of scaffolds containing

1 mm spacing showed a very clear increase in stacking order which continued through 200

layers. This suggested that intra-layer fibre spacing is also a factor effecting controlled

fabrication of melt electrospun scaffolds. In many cases larger spacing is advantageous, for

example it is often advantageous to add additional factors to the scaffold to increase

bioactivity, for example, the electrospraying of protein loaded PLGA microparticles for

growth factor delivery purposes [25, 26].

Figure 5-7: A scaffold with 1 mm fibre spacing produced using group D (+7 kV tip: -3.5 kV collector).

The scaffold reached a height of 200 layers (2 mm thickness). This illustrates that by decreasing the

density of fibres, the stacking increases.




5.3.2 Zonal Characterisation of structure order

Due to factors chapter 5.1, there is a limit to the number of layers that can be fabricated in a

controlled manner. This limit is influenced by the electric field, charge capacity of the polymer,

intra-layer fibre spacing and fibre stacking order. As control of fibre deposition is lost, the

structure order transitions through a semi-ordered phase before becoming completely

disordered as is apparent in Figure 5-8. The ordered region exists in the lower layers of the

scaffold (i.e. the first to be deposited) and is distinguished by distinct columns of stacked

fibres. Because the columns are clearly identifiable, the stacking order in this region can be

quantified using equation (5-2). The semi-ordered region describes layers where structure

order is still apparent; however distinct columns of fibres are no longer identifiable. In this

region, the unwanted forces acting on the extruding fibre caused by the charges on the

deposited fibres results in fibres of a given column being completely shifted to an adjacent

column. This effect can be seen in the semi-ordered region of Figure 5-8(b) where the second

column of fibres from the left of the image ends abruptly. The disordered region is where

complete control over fibre deposition is lost and unwanted columbic forces dominate.




Figure 5-8: SEM micrographs of a scaffold from group B showing x-z cross section. This illustrates the

zonal arrangement of the fibre networks. Sections a, b, and c, are magnified sections of scaffold. The

first zone shows a highly ordered structure with large levels of control on fibre deposition. The

secondary zone (semi-ordered) demonstrates some level of control; however the position of fibre

deposition is largely influenced by electrostatic forces. The final zone (disordered) shows a complete

lack of fibre control with deposition dominated by electrostatic forces.

Figure 5-9 shows averaging of the number of layers within the ordered region of scaffolds

from groups A-D and the control group. This demonstrates that the use of negative collector

potential in melt electrospinning significantly increases the maximum achievable scaffold

thickness, with the number of ordered layers in the control group considerably smaller.




Figure 5-9: Maximum heights of the ordered zone for scaffolds fabricated for phase 1. Groups A –D

had significantly larger ordered zones compared to the control group (p < 0.05).

5.3.3 In Vitro Characterisation

To assess the suitability of the electrospun scaffolds for tissue engineering purposes, we

performed an in vitro analysis of both aligned fibre scaffolds and scaffolds consisting of

random fibre networks. The lack of cell infiltration in electrospun scaffolds during cell seeding

has been previously observed, particularly in solution electrospun scaffolds [133], [140]. As

direct writing melt electrospinning enables precise control over scaffold microarchitecture, it

is possible to tailor the pore sizes to the needs of the cells and significantly improve infiltration

and proliferation.

Murine calvarial cells (MC3T3-E1) were seeded on the scaffold with a cell density of 5000

cells per scaffold for 7 days, and 450 000 cells per scaffold for 3 days. To comprehensively

evaluate the in vitro behaviour of the scaffolds, three studies were completed over the culture

period. These studies assessed the viability and distribution of cells within melt electrospun

scaffolds with 90° cross-hatched fibre architecture, fibre spacing of ~500 μm, and an ordered

thickness of 25 layers and 50 layers. A live/dead stain was used at day 3 to assess the cell

viability and as an indicator for cell infiltration in the scaffold. The fluorescent microscopy

images in Figs. 10 (A) and (B) indicate that cells are successfully distributed across the surface

of the 90° cross-hatched scaffold at day 3.

*

** *

0

200

400

600

800

1000

1200

1400

1600

1800

2000

A B C D Control

Hei

ght

[µm

]

Voltage Distribution [kV]

A -10.5 kV: GND

B -7 kV: 3.5 kV

C -5 kV: 5 kV

D -3.5 kV: 7 kV





Figure 5-10: (A) and (B) Cell distribution illustrated using a live/dead stain indicating the presence of

cells across the surface of the scaffold. Images (C) and (D) show cell morphology as imaged through a

DAPI/Phalloidin stain, illustrating cells attaching and spreading on the scaffold fibres illustrating good

cellular interaction.

Studies of cellular morphology were completed to assess the level of cellular interaction within

the scaffold. Previous studies have shown mixed results when comparing cellular attachment

and morphology between randomly oriented fibre networks and aligned fibre networks [133],

[136]. There is some evidence; however, that fibre alignment is influential on cell growth

depending on the cell line with fibroblastic, vascular and osteon cells all showing positive

responses [133]. In the present study, nuclear/f-actin staining was used to assess cell adhesion

and elongation. Figure 5-10 (C) and (D) show cells attaching to the scaffold and bridging

across the fibres with the active formation of ECM. Comparisons between MTT analysis of

cell growth within the 25 layer and 50 layers ordered scaffolds and the randomly structured




scaffold showed no difference in cell metabolic activity (n=6) (p < 0.05) as seen in Figure

5-11. This suggests that cell activity was not adversely affected by scaffold ordering.

Figure 5-11: MTT data of the percentage increase in cell growth showing an approximately uniform

level of cell proliferation for all scaffolds (n = 6) (p < 0.05). Errors bars indicate standard error.

5.4 CONCLUSION

Direct write melt electrospinning offers the ability to fabricate custom 3D tissue engineered

scaffolds having highly engineered internal micro-architectures and fibre diameters in the

micron-scale. Key to this capability is the use of a large electric potential to draw micron-thin

fibres from a needle tip and a translatable collector to control their deposition. Control over

scaffold fabrication is important for the production of pore sizes, which are tailored to the

requirements of various cell types. This control also enables the engineering of micro-channels

designed to ensure adequate supply of oxygen and nutrients and removal of metabolic waste.

Due to electric field instabilities caused by the accumulation of electric charge in deposited

fibres, maintenance of this control is difficult beyond a certain number of layers. Thus,

electrical charge accumulation limits the utility of melt electrospun scaffolds for tissue

engineering purposes.

We demonstrated that the maximum number of layers can be considerably increased through

the novel use of a dual voltage power supply, which employs a positive voltage on the needle

tip and a negative voltage on the collector plate. This offers substantial improvements in

scaffold ordering over the standard techniques which use only a single supply and ground.

Using the dual voltages, we were able to produce scaffolds with a relatively high degree of

ordering and maintain control over this ordering in significantly more layers than standard

grounded collector techniques. We produced highly ordered scaffolds up to 200 layers high (2

mm thick) while maintaining precise control over fibre diameter. The resulting scaffolds were

seeded with murine calvarial cells to assess their biocompatibility and suitability for tissue

engineering purposes and cells attached well and spread throughout the scaffold with little

25 Layers121%

50 Layers142% Random

112%

0%

50%

100%

150%

200%

Pe

rce

nta

ge C

ell

Gro

wth

B

etw

ee

n d

ays

1 a

nd

7




evidence of cell death. The results indicate that the accumulation of charged polymer in

fabricated electrospun scaffolds produces an increasing amount of undesirable net forces on

the fibre being deposited, thereby reducing deposition control. The use of a negative potential

on the collector plate significantly mitigates this effect. Further studies characterising the

charges on electrospun polymer fibres during the fabrication process are already underway.

5.5 ACKNOWLEDGEMENTS

This research was supported under the Australian Research Council Linkage Projects funding

scheme (LP130100461 and LP110200082). The authors also wish to acknowledge the help of

Dr Roland Steck and Ms Patrina Poh for their assistance with µCT analysis, Ms Rachel

Hancock and Dr Leonore de Boer for their assistance with SEM imaging and Mr Alex Roeder

for his invaluable help with cell culture and staining.

Chapter 6: Conclusions


6Chapter 6: Conclusions

6.1 RESEARCH SUMMARY

The focus of this master’s project was to provide evidence supporting the technique of melt

electrospinning as an additive manufacturing system for use in TE. It was hypothesised that a

negatively charged collector plate would remove charge stored in the polymer scaffold, hence

reducing the columbic force affecting the electric field during fibre deposition. Highly ordered

scaffolds could then be produced at thicknesses greater than 2 mm in height. By proving that

highly ordered scaffolds were possible, tissue engineers are able to utilise this method to

modify the microarchitecture of scaffolds to affect the local macro and micro environments

and produce biomimetic scaffolds.

6.1.1 Summary of Research Paper

Chapter Title: Improved fabrication of melt electrospun tissue

engineering scaffolds using direct writing and advanced electric field

control. [14]

Chapter 5 shows that a negatively-charged collector plate positively affects the stacking height

and scaffold order in a direct writing melt electrospun scaffold. Five groups of scaffolds were

manufactured by varying the distribution of voltage between the collector and the emitter in

2 kV intervals. The scaffold which produced the highest order and stacking height was then

used in an in vitro study to illustrate the effect of scaffold depth on cell seeding. SEM was

used to determine the order and stacking height, µCT was used to determine pore size, and cell

assays associated with cell metabolism, cell morphology and live/dead cells were assessed.

Pore sizes in scaffolds ranged from 6 µm to approximately 700 µm, with peaks occurring at

282 µm and 378 µm respectively, correlated with the fibre gap spacing minus the fibre

diameter. The fibre diameter was consistent for all voltage distributions with a value of 20.7

µm ± 3.42 µm. The heights of the ordered region for scaffolds were greater for all scaffolds

with a negative charge on the collector plate, with the highest order occurring in group D (-3.5

kV collector: 7 kV emitter). These scaffold had an average height of 1.6 mm ± 0.2 mm. Fibre

alignment was assessed for each voltage distribution for the first 25 fibre layers. Groups B, C

and D showed greater levels of order compared to the control. Group D achieved an order

parameter of 1.02 m/m ± 0.02 m/m. The disorder of the scaffolds was assessed as the layers

were added, showing a linear increase in disorder. Finally, a qualitative study to assess fibre

density on stacking height was assessed. However, a scaffold produced using the same



parameters as group D with a fibre spacing of 1 mm was completed. It showed an ordered

region of height in excess of 2 mm. In vitro analysis was completed on a scaffold produced

using parameters from group D. Live/dead assays show a high percentage of live cells and cell

morphology staining displayed good interaction between cells and fibres. Cell metabolic assay

showed no correlation between scaffold height and cell proliferation, as well as scaffold fibre

order verses disorder.

6.2 LIMITATIONS AND RECOMMENDATIONS FOR FUTURE WORK

The addition of the negative charge to the collector plate showed undeniable improvements in

scaffold order and fibre deposition over increased thickness. However, the mechanisms

involved in determining the emission of charge are still largely unknown for melt electrospun

fibres. A thorough model describing the charge transport in a polymer scaffold will help inform

future optimisations of the system and eventually lead to direct writing melt electrospinning

being used as an additive manufacturing technique. Other considerations should be taken into

account which may influence order, such as the mechanical structure involved in fibre

deposition and the architecture of the scaffold. The limitations of this master’s project and

considerations for recommendations for future work are outlined in the following section.

6.2.1 Charge Storage, Charge Dissipation

Charge storage plays a key role in the stability of fibres in electrospinning. Two mechanisms

exist for charge to be transported through a polymer, on the surface or through the fibre

volume. Each mechanism has different methods for emission of their charge and hence

determining the locus of the charge will inform future research. The study investigated the

effect on order of a negative charge on the collector plate, hypothesizing that residual charge

is capable of faster emission from the fibres. However, while it had an impact on the order, no

study was completed to quantify the storage of charge in the polymer and we are not able to

conclude whether columbic forces created by charge principally causes the disorder.

Determining the locus and magnitude of residual charge would increase our understanding of

the columbic forces effect on the deposition of fibres. The method of charge transport can vary

depending on the types of polymers, in which phase they exist and their molecular weight.

PCL with a high molecular weight (greater than 45 kDa) exists in a semicrystalline form, hence

the electron traps primarily exist in the interface between the crystalline and amorphous

regions [109]. This would imply that the locus of charge exists both on the surface and in the

volume. Work by Zhang and Yan [16] show that it may take in excess of 200 hours for charge

to be removed from an solution electrospun scaffold.



A comprehensive review papers exploring this problem has been completed by Collins et al.

[88], determining that the residual charge is very dependent on the structure and composition

of the polymer. However, these studies cannot be applied to melt electrospun fibres, as the

presence of a solvent provides ionic conduction of charge as well as a mechanism during

evaporation for charge to be stored in the volume of the polymer.

The crystallinity, impurity concentration and donor site availability dictates the methods for

charge transport in polymers. There are three methods for electrons to be transported through

a polymer; ionic conduction [141], diffusion [142], and the Poole-Frenkel effect (electrons

jumping from trap sites due to random thermal fluctuations) [143]. Applying models

associated with the aforementioned theorems with crystallinity estimates produced by

Zhmayev et al. [144] provide a understanding of the emission and storage of residual charge

in a melt electrospun polymer. Work on the geometry and structure of the scaffold may provide

information regarding the movement of charge to the collector plate and may be necessary.

6.2.2 Other Factors Causing Disorder and Near Field Melt Electrospinning

Production of scaffolds on the micron scale requires stages and motors which are capable of

reliable and repeatable motion on equivocal scales. The system currently in place contains no

feedback mechanisms to ensure that the motions displayed on the CNC software reflects the

actually displacement of the machine. Hence, there is no way to verify that the position of

fibre deposition due to the stages is accurate. A second form of error occurs due to the

relatively long distance to fibre diameter ratio at the extruder head. The deposition of fibres is

intended to be micrometre accurate for low micron scale fibres, while the spacing between the

collector and emitter is between 10 to 50 mm. Such distances would allow influences from

events occurring in the local environment such as air flow.

To reduce the error produced by both the electrospinning process and the stages, an updated

melt electrospinning rig incorporating a feedback mechanism for position as well as

introducing near field melt electrospinning would eliminate large sources of error. Articles

investigating error modelling and compensation methods for CNC machines have been

published as early as 1999 [145]. More recent studies investigate similar properties, with the

addition of feedback systems with more complex neural network problems [146].

Near field electrospinning is a method applied to solution electrospinning which allows the

system to control the deposition of fibres [147]. The system maintains the same electric field

as conventional electrospinning, however reduces the distance between the collector and the

emitter. This increases the deposition accuracy of the system by restricting the whipping stage

from forming. A near field system has been compared to melt electrospinning in its ability to



deposit fibres accurately [116]; a marriage of the two systems would reduce errors associated

with melt electrospinning. Developing a system which merges the two technologies would

require little to no extra material and could dramatically increase deposition accuracy.

6.2.3 Scaffold Architecture and Order

During the fabrication process, 90° cross-hatch scaffolds were created in order to assess the

laydown of the fibres. This was chosen as it provided an easy to assess cross-section for

quantification of alignment. However, the positioning of the fibres may affect the electric field

during deposition. In a 90° cross-hatch pattern, the fibres are stacked directly on top of each

other. This configuration produces columbic forces in each consecutive layer. Repeated units

produce fibres which are deposited in the same position; this deposition is perturbed by the

columbic force.

Increasing the number of layers between repeating units in a scaffold would reduce the number

of fibres which are deposited in the same position. This would reduce the total columbic force

felt by each fibre during deposition. Alternatively, producing a scaffold which contains no

repeating units may solve the issue. However, quantifying alignment becomes much more

challenging. An alternative method to measure order must be produced.

A μCT scan can produce a volumetric model of the scaffold. It is possible to produce a 3D

model of the scaffold using software such as Solidworks. By comparing the μCT model of the

scaffold with an ideal 3D model, it is possible to investigate errors in the position of fibres in

the whole scaffold which may produce a much more accurate quantification of order.

However, this method would require extensive research and planning.

6.2.4 Dynamically Controlled Electric Potential

This study produced scaffolds with a fixed distance between the collector plate and the emitter.

When fibres are stacked on top of each other, they affect the electric field. This would then

change the magnitude of the electric field at deposition. By dynamically controlling the electric

field, it is possible to maintain the field strength throughout the whole electrospinning

processes.

6.3 FINAL DISCUSSION AND CONCLUSION

Musculoskeletal injuries cost Australia approximately AUD$15 billion per annum. A portion

of these are non-union fractures, bone fractures which are too large to heal naturally. These

require surgical intervention to fully regenerate the trauma site. While the current gold

standard is the autograft procedure, it has its associated pitfalls [5]. Bone tissue engineering

aims to reduce the inherent risks associated with these procedures by employing additive



manufacturing techniques with drug loaded polymers to produce a powerful osteoconductive

and osteoinductive response which is comparable in clinical efficacy to autograft while

reducing any negative outcomes.

Bone tissue engineering requires biomimetic structures to produce the most effective clinical

results. Due to our current level of understanding of the bone remodelling process, we are

limited in our ability to synthesise the bones natural healing mechanisms. However, it is

possible to provide substitutes which imitate naturally occurring systems. This simplifies the

system and allows us to produce substitutes without understanding the underlining

mechanisms in their entirety. Therefore, it is essential to this field to characterise natural bone

and bone formation [65].

Additive manufacturing for tissue engineering has diverged into a number of approaches. Melt

electrospinning produces the finest fibres of any technique [116], and can hence provide the

greatest control over the internal structure of scaffolds. Obtaining the capacity to control such

fine properties of a scaffold allows researchers to control the microarchitecture. The

microarchitecture of a scaffold can be manipulated to controlling cell proliferation,

differentiation and the mechanical properties of the structure [66], [57] [58]. However, while

melt electrospinning is capable of producing fine fibres, the system contains instabilities which

need to be overcome to produce structures with complex three dimensional architectures at a

significant thickness [14]. The residual charge and viscoelastic properties associated with

electrospinning are the causes of instability, with Zhmayev et al. [15] discussing how these

processes affect the fibre diameter and morphology. Residual charge has been studied by Yan

and Zhang [16], showing retention of charge for upwards of 200 hours, and a dependency of

the bias of the charge.

In this master’s project, the collector plate in a direct writing melt electrospinning system was

negatively charged to determine its impact on the order of scaffolds as successive layers were

stacked on top of each other. Fibres were produced with fixed parameters as described in

section 5.2.1. The potential difference between the collector and the emitter was held constant

at 10.5 kV, and the distribution of charge was varied in 2 kV intervals as described in Table

5-1. These parameters produced fibres with radius 20.7 µm ± 3.42 µm. As expected, there was

no impact on the fibre diameter as the distribution voltage was varied. The height of the

ordered region in a scaffold was quantified and compared against the control (grounded

collector plate). The data provides evidence that the addition of a negative charge greatly

increased the heights of the ordered region, with all scaffolds showing higher ordered regions

against the control. The alignment in the ordered region was assessed via the method described

in section 5.2.2.2. This data demonstrates that a distribution in voltage created the greatest



level of alignment, with the control and group A (-10.5 kV at the collector, grounded emitter)

showing similar levels of alignment. This finding requires further research to determine what

causes the additional alignment. One hypothesis is a lack of charge transfer at the emitter,

causing a less pronounced electrostatic attraction to the collector plate which may cause fibres

deposition to lose accuracy. Scaffold from group D (-3.5 kV at the collector, 7 kV at the

emitter) were chosen for further research as they showed the highest levels of alignment and

greatest stacking height. The order as successive layers were stacked was quantified, showing

a linear increase in disorder, while reducing the fibre density by a factor of four (accomplished

by doubling the spacing between fibres) showed a dramatic increase in order and stacking

height (up to 200 layers with no disordered regions). Scaffolds were then assessed in vitro,

with data indicating good cell viability (via a live/dead stain) and cell attachment and

morphology (via a DAPI/Phalloidin stain). Cell metabolic activity was quantified on the first

and seventh day of the study and their ratios were used to indicate the proliferation of cells

during the study. As hypothesised, the study showed that there was no statistically significant

difference between scaffold heights (25 layers and 50 layers) or order of scaffold fibres

(randomly oriented fibres vs. ordered fibres) and cell proliferation.

In conclusion, the addition of a negative charge on the collector plate greatly enhanced the

deposition accuracy of melt electrospun scaffolds. This technique, in conjunction with

conventional additive manufacturing processes, may provide an invaluable tool in produce

micron scale scaffolds for bone tissue engineering. Combining this work with future studies

into cellular response to microarchitecture of scaffolds and bioactive substance loaded

polymers will be a valuable step forward in synthesis of scaffolds for bone tissue engineering.

However, instabilities in melt electrospinning still exist and further research in charge

transport and storage is necessary to determine the progress of future work, alongside more

development into the mechanical aspects of the direct writing system. The research completed

during this master’s project has demonstrating a promising technique, which with further

research and development will help to produce more effective tissue engineered constructs for

bone tissue repair.

Chapter 7: Bibliography


7Chapter 7: Bibliography

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8Chapter 8: Appendices

8.1 APPENDIX B: MATLAB CODE FOR ORDER QUANTIZATION

8.1.1 Image Importer

function [Align, STD, Name, Count] = Importer_Subtractive(TR, TG, TB, n) %This program will import all jpeg files from a particular folder and %output the results one at a time into a specified folder to be analysed. %In this particular instance, it will be outputed into the subtractive.m %script to analysis the fibre alignnment

prompt = 'file directory = ';

myFolder = input(prompt) if ~isdir(myFolder) errorMessage = sprintf('Error: The following folder does not exist:\n%s',

myFolder); uiwait(warndlg(errorMessage)); return; end filePattern = fullfile(myFolder, '*.jpg'); jpegFiles = dir(filePattern); for k = 1:length(jpegFiles) baseFileName = jpegFiles(k).name; fullFileName = fullfile(myFolder, baseFileName); fprintf(1, 'Now reading %s\n', fullFileName); imageArray = imread(fullFileName); [R,G,B,M,S,D,T,TS,Size] = subtractive(imread(fullFileName), 100, 100, 100,

n); Align(k,:) = T; STD(k,:) = TS; Name{k,:} = fullFileName; Count(k,:) = Size; end

end



8.1.2 Image Analysis

function [R,B,M,S] = image_analysis(RGB,TR,TB) %This program will look at an image which has been previous analysed to %show the displacement of fibres (in red) in an electrospun scaffold from %a centre line drawn (in blue). This image must be analysed prior to use %using red with intensity (0,0,255) for the stacked fibres and blue with %intensity (0,255,0). This software outputs the pixels that have those %colours into a array which then produces a distance from data points in %pixels. This data must hten be converted from pixels to um.

%TR is the threshold of RED and TB is the threshold of BLUE

%X = x-coordinate %Y = y-coordinate %CR = Colour intensity of Red %CB = Colour intensity of Blue

[a] = size(RGB); xri = 1; yri = 1; xbi = 1; ybi = 1; B = 0; q = 0; h = waitbar(0,'please wait...'); for i = 1:a(2) for j = 1:a(1) waitbar(q/(a(1)*a(2))) q = q+1; inten = impixel(RGB,i,j); if inten(1) > TR if inten(2) <50 R(xri,2) = j; R(yri,1) = i; xri = xri+1; yri = yri+1; end end if inten(3) > TB if inten(2) <50 B(xbi,2) = j; B(ybi,1) = i; xbi = xbi+1; ybi = ybi+1; end end end end

plot(B(:,1),B(:,2),'x'); hold on plot(R(:,1),R(:,2),'or');

[Dr,Db,Mb,Mr,M,S] = spacing_distribution(R,B);

close(h);



8.1.3 Fibre Positions

function [R,G,B,M,S,D,T,TS,Size] = subtractive(RGB,TR,TG,TB,n) %This program will look at an image which has been previous analysed to %show the displacement of fibres (in red) in an electrospun scaffold from %a centre line drawn (in blue). This image must be analysed prior to use %using red with intensity (0,0,255) for the stacked fibres and blue with %intensity (0,255,0). This software outputs the pixels that have those %colours into a array which then produces a distance from data points in %pixels. This data must hten be converted from pixels to um.

%TR is the threshold of RED and TB is the threshold of BLUE

%X = x-coordinate %Y = y-coordinate %CR = Colour intensity of Red %CB = Colour intensity of Blue

[G1(:,1),G1(:,2)] = find(RGB(:,:,2)<TG); [B1(:,1),B1(:,2)] = find(RGB(:,:,3)<TB); [R1(:,1),R1(:,2)] = find(RGB(:,:,1)<TR);

R = intersect(G1,B1,'rows'); B = intersect(R1,G1,'rows'); G = intersect(R1,B1,'rows');

%plot(B(:,2),B(:,1),'x'); %hold on %plot(R(:,2),R(:,1),'or');

[D,M,S,T,TS,Size] = spacing_distribution(R, n);



8.2 Appendix C

8.2.1 Sample of Fibre Raw Image Data

Group D 1.1 1.2 1.3 1.4 1.5 1.6

MELT ELECTROSPINNING AS AN ADDITIVE MANUFACTURING … · The use of scaffolds to replace lost bone...

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