Phd Thesis (with examiner corrections)...ACKNOWLEDGEMENT First off, I’d like to thank Prof. Dr.-Ing Birgit Glasmacher for giving me the opportunity to do my thesis in the Institute

Acknowledged by the PhD committee and head of Hannover Medical School

President: Prof. Dr. Christopher Baum (until 31st December, 2019)

Prof. Dr. Michael Manns (as of 1st January, 2019)

Supervisor: Prof. Dr.-Ing Birgit Glasmacher (Institute for Multiphase

Processes, Leibniz Universität Hannover)

Internal Guide: Dr. Oleksandr Gryshkov (Institute for Multiphase Processes,

Leibniz Universität Hannover)

Co-Supervisors: Dr. Robert Zweigerdt (Leibniz Research Laboratories for

Biotechnology and Artificial Organs (LEBAO), Hannover

Medical School)

Prof. Dr. Sotirios Korossis (Mechanical, electrical and

manufacturing engineering, Loughborough University)

External expert: Prof. Eyal Zussman (Technion - Israel Institute of

Technology)

Internal expert: Prof. Dr. rer. nat. Andrea Hoffmann (AG Gradierte Implantate

und Regenerative Strategien, Medizinische Hochschule

Hannover Klinik für Orthopädie)

Day of public defence: 25th January, 2019

PhD project funded by:

The Federal Ministry of Education and Research as well as the Deutsche

Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of

Excellence REBIRTH (EXC 62/3 valid until Dec 2017, EXC 62/4 valid until Oct 2019).

IP@Leibniz of Leibniz University Hannover promoted by the German Academic

Exchange Service (DAAD) for two exchange projects (one incoming, one outgoing)

during the study - project code 57156199.

ACKNOWLEDGEMENT

First off, I’d like to thank Prof. Dr.-Ing Birgit Glasmacher for giving me the

opportunity to do my thesis in the Institute for Multiphase Processes and guiding me

through this challenging time in my career. I would like to thank REBIRTH for

accepting me into their programme and funding my PhD through the DFG. I am also

grateful to Dr. Gerald Dräger and the rest of the IP@Leibniz team for funding two

exchange projects during the time of my PhD.

Sasha (Dr Oleksandr Gryshkov) has been an excellent mentor to me in academic

matters and also a great friendly comfort during stressful times. He constantly taught me

new things (sometimes without even meaning to), gave me honest criticism (which I

greatly appreciate) and gave me bear hugs and high fives when I did something well. I

am really lucky to have had him mentor me during my PhD. I would also like to thank

Dr Marc Müller for all our electrospinning brainstorming sessions, beer brewing

sessions and general PhD/career advice. It has been a pleasure working with him from

the very beginning.

I want to thank my co-supervisors Dr Korossis and Dr Zweigerdt (for their

expert scientific guidance), Rosi (for her administrative support), Almer (for his magical

computer fixing skills), Katja and Julia G (for their laboratory assistance), Prof. Wolkers

(for his assistance with the FTIR), the Institute for Technical Chemistry (for use of their

tensile testing machine) and Dr Pelz (for her constant support from REBIRTH).

I want to extend my gratitude to Dr Dagmar Pfeiffer, Dr Ingrid Lang and Julia

Fuchs and the rest of their department in Graz for accepting me into their laboratory and

providing me with support during my exchange project. I really did learn a lot during

that time. I’d also like to thank Ms Anne Marie Beck from NIFE for spending many

hours with me on histological stainings.

I would also like to thank my IMP colleagues who provided me with an

excellent work environment. Sara K has been the best office mate and a great friend. I

could always count on her to be there for me. I feel very fortunate to have had some

really motivated students: Mythili, Sarah K and Sarah C. It was a pleasure doing

research with them and building long lasting friendships in the process. I hope Sarah C

and I will have more ‘in the name of Science!’ moments in the future.

I do not have enough words to thank Irene and Jaspreet for being the best friends

I could have asked for. My time in Hannover was filled with laughter, positivity and

silliness because of them. I’d like to thank Andi for always being so kind to me. I cannot

express how grateful I am to have Terry in my life. He has made me the happiest person

in the world. Last but never least, I am ever thankful that I have such a supportive

family. My mother, father and sister are the reason I am where I am today.

ABSTRACT

Electrospun scaffolds are widely used in tissue engineering for the repair of soft

tissue. In spite of their obvious advantages, there are some practical limitations for in

vivo translation. One of the most crucial concerns is the lack of cell infiltration in such

scaffolds. To improve cell infiltration it would be ideal to simply increase pore size in

the scaffold. However, in electrospinning, this would also mean increasing fibre

diameter, which hampers cell functions such as attachment, proliferation and motility.

To circumvent this tradeoff, scaffolds with both small and large fibres have been

researched. In addition, the use of composite scaffolds (with synthetic and natural

polymers) have also been used extensively to improve scaffold-cell integration in vitro.

The research reported in this thesis, therefore, aims to improve upon existing

traditionally electrospun scaffolds by fabricating a hybrid scaffold which not only has a

large fibre diameter distribution, but is also a composite.

PCL-gelatin hybrid scaffolds were produced by altering the setup orientation of

electrospinning. The reasoning behind the change in microstructure in the vertical setup

orientation beyond a critical concentration of gelatin is presented in a hypothesis. The

characterisation and validation of these scaffolds were done through uniaxial

mechanical testing, a degradation study and the assessment of cellular infiltration (along

with a validation of cell viability and metabolic activity). The hybrid scaffolds were

mechanically stable and cell infiltration was improved in them while preserving cell

viability and metabolic activity. This thesis therefore offers a promising new method to

produce electrospun scaffolds with enhanced microstructure and cell infiltration for

application in soft tissue engineering.

TABLE OF CONTENTS

List of Figures 1

List of Tables 4

Abbreviations 5

Chapter 1 – Introduction

1.1 Tissue Engineering 7

1.2 3D engineering of soft tissue 10

1.3 Mimicking the Extracellular Matrix 12

1.4 Electrospinning - A method to fabricate fibrous scaffolds 13

1.5 Challenges in scaffold electrospinning 15

1.6 Cell interaction with micro/nanofibres 17

1.7 Enlarging pore size in electrospun scaffolds for enhanced cell infiltration 19

1.8 The fibre-pore paradox 19

1.9 Multimodal fibre diameter distributions in electrospun scaffolds 20

1.10 Composite scaffolds 22

1.11 The influence of setup orientation on electrospun scaffold microstructure 23

1.12 Rationale for the choice of polymers 25

1.13 Rationale for the choice of cells 27

1.14 Motivation and aim of the thesis 28

Chapter 2 – Materials and Methods

2.1 Electrospinning of PCL and PCL-gelatin scaffolds 31

2.1.1 Preparation of solutions 31

2.1.2 Solution properties 32

2.1.3 Electrospinning machine setup 32

2.1.4 Parametric optimisation of spinning 33

2.1.5 Imaging of electrospun scaffolds 35

2.1.6 Measurement of fibre diameter, pore size and thickness 35

2.2 Assessment of mechanical properties 36

2.2.1 Uniaxial tensile test setup 37

2.2.2 Representation of data 38

2.3 Degradation study 39

2.3.1 Experimental setup 39

2.3.2 Visualisation of fibrous structure 39

2.3.3 Raman analysis of gelatin loss and crystallinity of PCL 39

2.4 Cell response study 41

2.4.1 Sterilisation of electrospun mats for cell culture 41

2.4.2 Thawing and cultivation of cells 41

2.4.3 Cell seeding on scaffolds 42

2.4.4 SEM visualisation of seeded cells 44

2.4.5 Infiltration study 44

2.4.6 Viability study 45

2.4.7 Metabolic activity study 46

2.5 Statistical representation of data 48

Chapter 3 – Results

3.1 Electrospinning of PCL and PCL-gelatin scaffolds 49

3.1.1 Solution properties 49

3.1.2 Imaging of electrospun scaffolds 50

3.1.3 Measurement of fibre diameter and pore size 52



3.3.1 Visualisation of fibre structure 57

3.3.2 Raman analysis of gelatin loss and crystallinity of PCL 57


3.4.1 SEM visualisation of seeded cells 60

3.4.2 Infiltration study 61

3.4.3 Viability study 64

3.4.4 Metabolic activity study 66

Chapter 4 – Discussion

4.1 The influence of solution properties on electrospinning 67

4.2 The influence of setup orientation and polymer concentration on PCL-gelatin

blend electrospinning

70




Chapter 5 – Conclusion

5.1 Summary 85

5.2 Outlook 88

Appendix

References 91

Supplementary data 101

Publication list 108

Curriculum Vitae 109

Statement of contribution 111

Declaration 114

List of Figures

List of Figures

Fig 1.1 The scaffold-based tissue engineering approach has three main

components - a scaffold, cells and biological factors.

9

Fig 1.2 Cell-Cell and Cell-ECM adhesions are mediated by cell surface

proteins called Cell Adhesion Molecules (CAMs).

12

Fig 1.3 Formation of the Taylor cone during electrospinning. 15

Fig 1.4 Schematic depicting fibre-fibre contact points per unit length of

micro- and nanofibre.

17

Fig 1.5 Mechanism of cell-fibre binding in electrospun scaffolds. 18

Fig 1.6 Mesoscopically ordered scaffolds. 21

Fig 1.7 Gravitational and electric field directions in a) horizontal and b)

vertical setup orientations.

24

Fig 1.8 Taylor cone distortion. 25

Fig 1.9 Structure of a) PCL and b) gelatin. 26

Fig 1.10 Optimal fibre diameter and pore size requirements for proper

cell-scaffold integration.

29

Fig 2.1 Horizontal electrospinning setup. 32

Fig 2.2 Vertical electrospinning setup. 33

Fig 2.3 Schematic depicting how a) fibre diameter and b) pore size

measurements were made.

36

Fig 2.4 Size of the sample cut out for tensile testing 37

Fig 2.5 Uniaxial tensile test setup. 38

Fig 2.6 Scaffold cutting with custom made cutters. 42

Fig 2.7 Schematic depicting the steps in cell seeding. 43

1

List of Figures

Fig 3.1 SEM image panel showing microstructure of electrospun

scaffolds.

51

Fig 3.2 SEM image panel showing microstructure of electrospun

scaffolds made from the blends chosen for further analysis.

52

Fig 3.3 a) Fibre diameters and b) pore sizes measured from SEM images

of the electrospun scaffolds

53

Fig 3.4 Fibre diameter distribution 54

Fig 3.5 Pore size distribution 55

Fig 3.6 Mechanical properties. 56

Fig 3.7 Visualisation of fibre degradation over 30 days. 57

Fig 3.8 Gaussian fitted graphs of gelatin Amide I and PCL C=O. 58

Fig 3.9 SEM visualisation of cells seeded on the electrospun scaffolds. 60

Fig 3.10 Visualisation of infiltrated 3T3 cells on the electrospun scaffolds

over the span of 15 days.

61

Fig 3.11 Visualisation of infiltrated aMSCs on the electrospun scaffolds

over the span of 15 days.

62

Fig 3.12 Cell infiltration in the fibre mats over the span of 15 days. 63

Fig 3.13 Cell viability assay fluorescence images over the span of 7 days. 64

Fig 3.14 Cell viability percentages in the fibre mats over the span of 7

days.

65

Fig 3.15 MTT absorbances of the cells in the fibre mats over the span of 7

days

66

Fig 4.1 Taylor’s experiments on vertical jets of viscous fluids 71

Fig 4.2 Balancing forces involved during jetting. 72

Fig 4.3 The influence of the strength of the electric field on the start

point of jet instabilities.

73

2

List of Figures

Fig 4.4 Schematic representing the experimental results from Reneker

and Yarin.

74

Fig 4.5 Fibre orientation during tensile stretching. 78

Fig 4.6 Fibre interconnections and “nanowebs” observed in the vertically

spun blends.

79

Fig 5.1 Cryosection of a tubular electrospun PCL-gelatin scaffold seeded

with Human Iliac Artery Endothelial Cells (HIAECs) in the

lumen.

89

S.1 Thickness optimisation of electrospun scofflds 101

S.2 Mechanical testing Force-Elongation graphs (all sets) 102

S.3 Raman spectra - PCL pellets, gelatin powder 103

S.4 Degradation study - Raman spectra (fingerprint region) 104

S.5 FTIR spectra of PCL175V and PCL100g75V before and after UV

sterilisation

106

S.6 Scaffold cutter SolidWorks drawings 107

3

List of Tables

List of Tables

Table 1.1 Diversity in regenerative approaches for different target organs. 8

Table 2.1 Solution spinning parameters. 34

Table 2.2 Optimised seeding volumes and seeding densities for cell

response studies.

44

Table 3.1 Solution properties 49

Table 3.2 Summary of peak fitting results 59

Table 4.1 Some approaches to increase cell infiltration in electrospun

scaffolds

84

4

Abbreviations

Abbreviations

2D Two Dimensional

3D Three Dimensional

aMSC Amnion derived Multipotent Stromal Cell

AMSC Adult Multipotent Stem Cell

ANOVA Analysis of Variance

ATDV Aerodynamic Tangential Drag Vector

CAM Cell Adhesion Molecule

CI Confidence Interval

CL Collagen

cLSM Confocal Laser Scanning Microscope

CO2 Carbon dioxide

DMEM Dulbecco’s Modified Eagle Medium

DNA Deoxyribonucleic acid

ECM Extracellular Matrix

EDTA Ethylenediaminetetraacetic Acid

ESC Embyonic Stem Cell

EVF Electric Vector Field

FBS Foetal Bovine Serum

FDA Food and Drug Administration

FTIR Fourier Transform Infrared

FWHM Full Width at Half Maximum

GFV Gravitational Field Vector

H Horizontal

HA Hydroxyapatite

HCL Hydrochloric Acid

HIAEC Human Iliac Artery Endothelial Cells

5

Abbreviations

IMP Institute for Multiphase Processes

iPSC Induced Pluripotent Stem Cell

Mn Number average Molecular Weight

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

Nd-YAG Neodymium-doped Yttrium Aluminum Garnet

NIFE Niedersächsischen Zentrum für Biomedizintechnik, Implantatforschung und Entwicklung

NP-40 Nonidet P-40

PBS Phosphate Buffered Saline

PCL Poly(ε-caprolactone)

PDGF-BB Platelet Derived Growth Factor with two B subunits

PEO Poly(ethylene oxide)

PLA Poly(lactic acid)

PLGA Poly(lactic-co-glycolic acid)

PLLA Poly(l-lactic acid)

PRP Platelet-rich Plasma

PVA Poly(vinyl alcohol)

PVC Polyvinyl chloride

PVDF Polyvinylidene Fluoride

RGD Arginine-Glycine-Aspartate

SEM Scanning Electron Microscope

SF Silk Fibroin

Si Silicon

TE Tissue Engineering

TFE 2,2,2 - Trifluoroethanol

UV Ultraviolet

V Vertical

6

Chapter 1: Introduction

maintain their mechanical integrity until the remodelling process is complete (15).

Furthermore, there is a great stress on the porosity, pore interconnectivity and surface to

volume ratio of the scaffold for efficient cell colonisation (21,22).

Based on the application, a plethora of fabrication techniques have been

employed to create porous, biodegradable and mechanically stable scaffolds that can

guide neotissue formation. Some of these techniques include solvent casting, particulate

leaching, gas foaming, self assembly, phase separation, electrospinning, rapid

prototyping, melt moulding and freeze drying (23). Each of these techniques have their

own merits and demerits and it is important to select the scaffold fabrication method

depending on the properties demanded by the end application.

1.2 3D ENGINEERING OF SOFT TISSUE

The term ‘soft tissue’ refers to any tissue that is not hardened or calcified. In

particular, it is tissue that surrounds bone or internal organs and may play a connective

or supportive role (24). This category encompasses tissues such as hair, cartilage, nerve,

muscle, skin, fat, fascia etc. Soft tissue damage is incredibly common in daily life,

caused either by genetic defects, trauma, disease or ageing. The gold standard for

treating this sort of damage is currently the use of autologous implants. However, the

main pitfall is that autologously implanted tissue is easily absorbed and rapid losses in

volume result in only about 40-60% viable cells. In addition, donor site morbidity is a

pervasive problem in autografting preventing its widespread use (25). Given the

inherent low regenerative abilities of soft tissue, tissue engineering has emerged as a

feasible option for such restoration.

The biological mechanisms that drive cellular function have been studied

predominantly through two-dimensional (2D) tissue culture. However, this does not

reflect the intricately structured three-dimensional (3D) labyrinth of the extracellular

matrix (ECM) in which cells exist in vivo. The topography of the cellular

microenvironment (the immediate environment perceived by the cells) directly

influences the phenotype of cells and can modify behaviours such as migration,

10


differentiation and mechanotransduction. This has urged researchers to simulate the in

vivo 3D environment in scaffolds for better functionality of cell-seeded constructs (26).

3D porous scaffolds not only provide a significantly more biomimetic template for cell

growth, but also enable enhanced biological signalling (cell-cell as well as cell-scaffold)

and better mass transport (27).

In light of this, a great deal of research has been done in 3D reconstruction of

damaged soft tissue such as skeletal muscle (28), skin (29), nerves (30), blood vessels

(31), cornea (32), ligament (33), trachea (34), adipose tissue (35) and heart valves (36),

etc. A wide variety of synthetic and natural polymers have been employed to

accomplish this, for example - poly(ε-caprolactone) (PCL), poly(vinyl alcohol) (PVA),

poly(lactic acid) (PLA), chitosan, collagen, alginate, gelatin, etc (37).

Although the basic concept of tissue engineering is straightforward, the practical

realisation is remarkably complex. As such, there are some limitations to restoring

volume loss in soft tissue. Organ patterning is one of the primary challenges thus far.

The scaffold has to allow for structural and functional mimicry of native tissue and also

have a construction conducive for vascular ingrowth. A high surface to volume ratio is

difficult to achieve, especially taking into consideration oxygenation and mass transport

of nutrients and waste, but is important for long-term cell survival. Microenvironment

considerations are of tremendous importance, involving the control of spatiotemporal

distribution of biological factors, facilitating adherence and infiltration of neotissue.

Dynamic matrix modification is required to carefully guide cell migration, maximising

the infiltration capacity of the scaffold. It is also important to match the mechanical

properties of the scaffold to the anatomical implantation site, particularly in load

bearing applications. Inadequate mechanical strength can lead to implantation failure in

the long run, reducing the ability of the tissue engineered scaffold to bond with native

tissue. Furthermore, there are limitations on the depths up to which regeneration can

still occur. Several factors may play a role in this, such as resistance to cell infiltration,

unsuitable mechanical properties and inadequate vascular supply. It is imperative to

keep these criteria in mind while designing a soft tissue replacement for successful

implementation of the same in vivo. (38)

11


feedback mechanism where the ECM influences cellular response (‘outside-in

signalling’), which in turn can alter the ECM itself (‘inside-out signalling’). Fabricating

such multi-faceted ECM analogues to replicate its structure, function and physiological

cell-ECM interactions is therefore a daunting task. (39) Ideally, an artificial scaffold

would execute all the roles of native ECM and mimic its complexity and diversity.

While this seems impossible today, current advances in scaffold design show that it is

possible to replicate at least some of these functions through the addition of nanofibres

and the incorporation of natural polymers (39).

Nanofibres in the range of ECM fibre diameters can dramatically improve cell

function because the microenvironment scale closely resembles that of the ECM. While

synthetic polymers have been successfully used to replicate this nanofibrous structure

and mechanical support, they lack specific cell interaction domains to promote cell

adhesion and migration. On the other hand, natural polymers enhance bioactivity and

biocompatibility but are difficult to process and lack mechanical stability. (27) Recent

efforts to combine synthetic and natural polymers to form composite fibrous scaffolds

have shown better results with respect to scaffold properties and cell-scaffold

integration (42-46).

1.4 ELECTROSPINNING - A METHOD TO FABRICATE FIBROUS

SCAFFOLDS

Currently, electrospinning, self-assembly and phase separation are the three

techniques used to synthesise nanofibres. Of these, electrospun scaffolds are the most

widely studied and seem to be immensely promising for application in tissue

replacements (47,48-52). The basis for the discovery and conceptualisation of

electrospinning dates back to the sixteenth century when William Gilbert first described

the phenomenon of electrostatic attraction (53). Since then, research on the distortion of

fluid droplets under the influence of electrostatic forces has been studied in great detail.

Anton Formhals made significant patent contributions to the field of electrospinning

between 1931 and 1944 (54-61). Following this, Sir Geoffrey Ingram Taylor

mathematically modelled the conical droplet shape (now referred to as the Taylor cone)

13


formed by fluids under the influence of an electric field in 1969 (62). Since the early

1980s, the concept of electrospinning has been widely researched in both textile as well

as tissue engineering (63). In an attempt to mimic the complex 3D network of polymer

and polysaccharide fibres (50-500 nm) of the native extracellular matrix (ECM) of

adherent cells, electrospinning has now emerged as the preferred fabrication method for

nano and microfibrous scaffolds.

Electrostatic fibre spinning, or ‘electrospinning’, involves the stretching of a

viscoelastic solution into a fibre of sub micron diameter that is collected in a random

manner on a grounded collector. In general, a polymer is solubilised in a suitable

conductive solvent at a certain concentration and extruded through a needle. A collector

is placed some distance from the needle and is grounded. When the nozzle is connected

to a high voltage a strong electrostatic potential difference is developed between the

nozzle and the collector, causing charges to collect on the surface of the polymer

solution droplet. An increase in voltage at the nozzle causes an increase in charge

density and repulsion, resulting in droplet distortion (Taylor cone)(Fig 1.3). At a critical

voltage, the molecules overcome the surface tension of the droplet and the polymer

solution is drawn into a single fibre in either the nano or microrange. At some distance

from the nozzle, the jet becomes unstable and a whipping motion causes the fibre to

stretch and thin and the solvent rapidly evaporates. By the time the fibre reaches the

collector, it is deposited dry (under optimised parameters) in a random manner, forming

a dense fibrous mesh. (64)

Electrospinning presents significant advantages over other types of fibre

fabrication methods because of its versatility and tunability. Fibre morphology and pore

size can be controlled by modifying the many parameters involved. Depending on the

intended replacement, collector shape and size can also be modified. Furthermore, it is

possible to use a wide variety of materials ranging from purely biological to synthetic to

ECM analogous materials. This flexibility in material usage allows for targeted

mechanical strength, degradation rate and bioactive components (16). Electrospun

scaffolds also have a large void volume, interconnected porous network and high

surface to volume ratio conducive for the incorporation of cellular components (65).

14


effect of electrospinning. It is unclear if this really is a cause for concern. While there

are some reports detailing the detrimental effects of solvent retention (67), there are

others that claim that the amount of residual solvent is too low to lend toxicity in vitro

or in vivo (68). The choice of material has a strong influence on how much solvent is

retained in the scaffold. Even in the case of some retention, they can be removed with a

simple heat and vacuum treatment (69). Nevertheless, this aspect is important to keep in

mind. Another constraint in electrospinning is the limit to the thickness of electrospun

scaffolds. This is because of two reasons. First, long processing times cause the build-up

of charges and prevent further deposition of fibres (70). Second, the insulation of the

collector increases with an increase in thickness of the deposited material. Hence, there

is a difficulty in repairing large tissue defects.

The chief pressing concern in electrospun scaffolds is insufficient cell

infiltration. Pore sizes in nanofibrous scaffolds are much smaller than general cell

diameters and therefore do not allow cells to infiltrate easily. Optimum pore sizes,

although not standardisable for all cell types, have been suggested to be in the range

100-500 µm. Electrospun scaffold pore size is far below this requirement, resulting in

limited vascular ingrowth, hypoxia and inefficient mass transfer. Additionally, the

packing density of fibres in electrospun scaffolds is high because each fibre layer is

continuously deposited over the previous layer while still being strongly attracted to the

collector. This results in some degree of compression, thus reducing the void volume of

the scaffold (66).

Electrospun scaffolds show a direct relationship between fibre diameter and pore

size, which means that smaller fibre diameters increase the incidence of fibre-fibre

contact points which in turn reduces pore area (Fig 1.4). Notwithstanding the high

porosity, the inadequate pore size restricts cellular penetration. Therefore, despite the

potential of these scaffolds to provide a 3D microenvironment comparable to that of

native ECM, most electrospun constructs in reality behave as 2D surfaces (66,71,72).

As highlighted previously, it is prudent to cultivate cells in a cell-permeable 3D

construct because it leads to better cell-cell signalling as well as more space for cells to

respond to mechanical cues. Poor cell infiltration can result in an inhomogeneous

16


1.7 ENLARGING PORE SIZE IN ELECTROSPUN SCAFFOLDS FOR

ENHANCED CELL INFILTRATION

The electrospinning community has invested much effort into enlarging the pore

size of scaffolds and attempting to solving the problem of insufficient cellular

penetration. The simplest solution attempted by many research groups is the

straightforward manipulation of parameters such as solution flow rate, applied voltage,

tip to collector distance and solution properties (81-83). An increased flow rate,

decreased voltage, shorter tip to collector distance and higher solution viscosity all

result in thicker fibres and consequently larger pores. However, it is a well known fact

that cells perform better in nanofibrous microenvironments than in their microfibrous

counterparts (as detailed in section 1.6). This has led to a much higher incidence of

electrospun nanofibres with other modifications for increasing pore size. Using

sacrificial material is an extremely popular strategy to increase pore size. Scaffolds with

two polymers, one immediately dissolvable and the other a slow degrading component,

have been fabricated. The fast degrading component is dissolved away or leached out

after electrospinning to create void spaces in the scaffold. The sacrificial element can be

a polymer solution that has been co-spun with the main polymer (84-87), salts (salt

leaching) (88) or ice crystals (cryogenic electrospinning) (89). Gas foaming is also a

well researched technique (90) and is sometimes combined with salt leaching (91).

Other methods that have been employed to increase pore size include melt

electrospinning (92), wet electrospinning (93), modification of collector shape (94,95),

combination of electrospinning with other fabrication technologies (such as direct

polymer melt deposition) (96) and laser ablation (97). Simultaneous polymer

electrospinning and electrospraying of cells has been employed to improve cell density

in electrospun scaffolds without changing the pore size (98).

1.8 THE FIBRE-PORE PARADOX

Keeping in mind overall cell health and success of tissue integration, there are

two opposing practical requirements for cells cultivated on electrospun scaffolds, which

brings us to the crux of the matter. Fibre diameter and pore size of electrospun scaffolds

19


are closely related, directly interdependent entities. Pore size increases with increase in

fibre diameter and vice versa. It is important to have a large pore size for adequate cell

infiltration (which invariably increases the fibre diameter), but it is also important to

have nanofibres that enhance cell adhesion and proliferation (which decreases pore

size). Owing to the interdependence of pore size and fibre diameter, it is difficult to

fabricate a scaffold with nanofibres and macropores (99,100). Pore size modifications

allow for larger pores but do not solve the problem of inefficient cell adhesion or loss in

mechanical stability. For instance, the use of sacrificial elements may increase pore size,

but the sudden increase in void volume can drastically hamper the mechanical

properties of the scaffold. Certain soft tissue replacements in load bearing sites such as

muscle and cartilage require high biomechanical strength. Electrospun scaffolds

currently are already unable to provide the optimum properties for high physical stress

regions. Loss of bulk material only serves to worsen the problem. Nanofibrous scaffolds

on the other hand have a high surface to volume ratio (100) but have little to no cell

infiltration and better function as patterned membranes for cell adhesion.

1.9 MULTIMODAL FIBRE DIAMETER DISTRIBUTIONS IN

ELECTROSPUN SCAFFOLDS

In order to overcome the fibre-pore paradox in electrospinning, there have been

massive efforts to combine nano and microfibres in the same scaffold. A number of

studies have attempted a trade-off situation where both micro and nanofibres are

present. Some noteworthy examples are elucidated in this section.

Kidoaki et al. proposed two methods for mesoscopically ordered scaffolds -

multilayering electrospinning and mixing electrospinning (Fig 1.6). Multilayering

electrospinning was achieved by sequentially spinning layers of different polymers.

They also proposed a vascular graft model with the multilayering concept. Moving a

step further from the simple juxtaposition of different fibre diameters, mixing

electrospinning was done by spinning two separate polymer solutions, with optimised

parameters for two different fibre diameter outputs, simultaneously on the same

collector (101).

20


1.10 COMPOSITE SCAFFOLDS

The concept of hybrid scaffolds in terms of scaffold composition is not a new

one. It has been proven time and time again that combining structural and bioactive

components in one scaffold afford it superior functionality to those made of only one

material. There are numerous ways to achieve this, but the most researched strategy is

the combination of synthetic and natural polymers. Since this research is focused on

electrospun scaffolds, this review will explore the functionality of blend electrospun

scaffolds, coated scaffolds and scaffolds incorporated with biological agents.

Synthetic biodegradable polymers have been extensively employed in tissue

engineering applications because of the ability to tailor their mechanical properties,

control the degradation rate and easily shape them into various configurations. They are

also favoured because of the gamut of polymers available for processing, compatible

with a number of fabrication techniques. Out of these, polyesters are particularly

attractive because of their hydrolysis based degradation, producing degradation

products that in most cases are eliminated safely through metabolic pathways. It is also

possible to tailor degradation rates to the application by altering the structure (105).

However, they are largely hydrophobic and lack ligands for successful cell binding

(106). Synthetic polymers are also sometimes preferred because they are cheap and can

be obtained in large quantities with negligible batch to batch variation (47).

Natural polymers on the other hand, are polymers derived from natural sources

and can be classified as proteins, polysaccharides and polynucleotides. The major

advantage of these polymers is their hydrophilicity and ability to provide an ECM-like

microenvironment by presenting ligands for cell binding. Biological factors and natural

polymers are able to induce some degree of cell infiltration by a phenomenon called

‘chemotaxis’ where cells respond and migrate toward chemical signals (39,66). This can

be done by introducing chemical gradients, soluble signals or even electrical potentials

(66). As such, the problem of poor cell infiltration can be addressed in part without

changing the scaffold microstructure. Unfortunately, natural polymers in general lack

mechanical stability and can vary in composition from source to source making it

difficult to maintain repeatability in experiments (47).

22


To capitalise on the advantages and compensate for the disadvantages of both

types of polymers, composite scaffolds have been developed and tested. The easiest way

to incorporate natural polymers into synthetic scaffolds is by surface coatings.

Numerous papers have expanded on the positive influence of surface functionalisation.

Coatings such as heparin (107), gelatin (108), collagen (109), fibrin (110), decorin (111)

and chitosan (112) have been successfully applied. Blend electrospinning of synthetic

and natural polymers has allowed for preservation of mechanical properties while

lending biocompatibility and hydrophilicity to the scaffolds. Examples include PCL-

collagen (113), PCL-gelatin (114,115), PLGA-gelatin-elastin (116), etc. Synthetic

polymers have also been incorporated with biological factors like platelet-rich plasma

(PRP) (117) and chemotaxis agents such as platelet-derived growth factor (PDGF-BB)

(118).

1.11 THE INFLUENCE OF SETUP ORIENTATION ON ELECTROSPUN

SCAFFOLD MICROSTRUCTURE

Electrospinning apparatuses are usually either setup horizontally or vertically (in

this case, top-down systems are preferred over bottom up systems due to ease of setup) .

In horizontal electrospinning, the nozzle is situated parallel to the ground and the

collector is placed perpendicular to this. The electric vector field (EVF) is always

between the nozzle and collector, so in this case it is parallel to the ground. The

gravitational field vector (GFV) obviously never changes and acts perpendicular to the

ground. In vertical electrospinning the EVF and GFV are both in the same direction

(perpendicular to the ground) because the collector is placed parallel and the nozzle is

placed perpendicular to the ground (115) (Fig 1.7). Either setup can be used as the

process is similar, but horizontally arranged systems are sometimes preferred because

the occurrence of artefacts in the final product is minimised (119).

The influence of setup orientation on the microstructure of the final electrospun

product has hardly been investigated. Yang et al. (120) electrospun PVDF in three

orientations - horizontal, shaft (top-down vertical) and converse (bottom-up vertical).

They observed that the fibre diameters obtained for the same range of voltages was

23


response. (134) Gelatin is hydrophilic, but it is also a polyelectrolyte. It contains amine

and carboxylic groups that are easily ionised in aqueous solution. Combined with its

strong hydrogen bonding, it becomes a very difficult material to electrospin. Gelatin is

also very quickly degraded without cross linking and tends to lose mechanical integrity

under physiological conditions. It is therefore common for gelatin to be blended with

stable synthetic polymers when fabricating scaffolds for tissue engineering. (135)

1.13 RATIONALE FOR THE CHOICE OF CELLS

Tissue engineering has seen the use of differentiated primary cells, cell lines and

stem cells. Differentiated primary cells, although advantageous in many ways, have

disadvantages such as the invasive nature of tissue extraction, limited expansion

potential ex vivo and the development of senescence (136). In place of this, cell lines are

commonly used as they are cost effective, easy to handle, can be indefinitely expanded

in culture and do not have the ethical concerns that primary cells are associated with.

Cell lines are consistent and the experimental results are reproducible but they usually

do not fully replicate the behaviour of primary cells because they are genetically

modified and may present altered phenotypes and stimuli responses. (137) Stem cells on

the other hand, are of three types - embryonic stem cells (ESCs), adult multipotent stem

cells (AMSCs) and induced pluripotent stem cells (iPSCs). Embryonic stem cells face

ethical issues and iPSCs raise concerns due to modified genotypes which makes

AMSCs currently key candidates for regenerative therapies (138). Two cell types were

considered for the validation of the electrospun scaffolds in this thesis - NIH 3T3 cell

line and amnion derived Multipotent Stromal Cells (aMSCs).

The NIH 3T3 mouse embryonic fibroblast cell line was first isolated in 1962 by

George Todaro and Howard Green. Since then, they have been used in numerous studies

because they are robust and have a predictable growth pattern (139,140). 3T3 cells are

quite commonly used in proof of concept studies since the results are repeatable and

comparable. However, the use of cell lines cannot be used to simulate the behaviour of

‘normal’ cells in the body. The use of 3T3 cells especially has been hotly debated due to

its metastatic tendencies under certain conditions (141). It is for this reason that while it

27


is acceptable to perform preliminary cell response studies with 3T3 cells to deem a

scaffold safe for cell colonisation, further validation must be done with sensitive

primary cells.

The MSCs used in this project were derived from the placental amnionic

membrane of the common marmoset (Callithrix jacchus) by the Institute for

Transfusion Medicine, Hannover Medical School (PD Dr. Thomas Müller) and stored at

-150 °C prior to use. These cells are excellent candidates for preclinical primate (non-

human) studies because of their non-invasive extraction, easy availability, plasticity and

immunosuppression abilities. Amnion MSCs have been shown to have higher

proliferation capacity in comparison to their bone marrow counterparts making them

preferable for expansion (138,142). MSCs are also sensitive cells that respond strongly

to surface topography, scaffold composition and mechanical stress, making them an

interesting choice for comparison with 3T3 cell function.

1.14 MOTIVATION AND AIM OF THE THESIS

Many different cell types (usually application oriented) have been used to

validate different electrospun scaffolds, with varying degrees of success. However, it is

interesting to note that there is a tacit understanding among members of the

electrospinning community that electrospun scaffolds have to be tailored to the cell

type. This is understandable as each cell type has its own distinct ECM and for

optimised outcomes, the scaffold should possess components particular to that native

site. While this statement definitely rings true, inadequate cell infiltration is a common

problem in all electrospun scaffolds. In particular, enhancing cell infiltration while also

preserving cell viability and metabolism is a universal challenge. It is therefore

necessary to provide a common solution.

In view of the general state of the art described in the previous sections, it is

clear that it is crucial to have many opposing scaffold elements for different

requirements. The first and foremost is the necessity to have large interconnected pores

for sufficient cell infiltration (provided by large microfibres). The second is the need to

28

Chapter 2: Materials and Methods

Table 2.1 - Solution spinning parameters (115). PCL and PCL-gelatin blend solutions were

electrospun in the horizontal and vertical setup orientations using optimised parameters.

The four final categories of scaffolds were chosen for characterisation due to

three main reasons. First, PCL175V was chosen as the unblended category to observe

the effect of gelatin addition. Second, PCL125g50 is the only blend that produced

scaffolds with homogenous nanofibres (spun in the horizontal orientation) and a

gradient of fibre diameters from nano to micro range (spun in the vertical orientation.

Both these categories were chosen to see the effect of changing fibre diameter and pore

size, keeping the blend concentration the same. And last but not least, PCL100g75V

was chosen because it has a similar scaffold architecture to PCL125g50V but has a

higher concentration of gelatin, giving us an idea of how gelatin concentration can

affect the properties of the scaffold and cell infiltration.

It was initially debated if the varied fibre structure obtained in the PCL-gelatin

scaffolds was caused due to electrospinning in two different instruments (unfortunately,

the fibre mats could not be spun horizontally and vertically in the same machine due to

the large size of the collector). However, this effect was verified by electrospinning

tubes (on a much smaller collector) in both setup orientations in the same device where

similar variations in fibre structure were observed in PCL-gelatin scaffolds above a

critical concentration of gelatin (data not shown).

Name of

the blend

Polymer

concentration

(mg/ml)

Mass ratio of

PCL:gelatin

Horizontal (H) and Vertical (V)

electrospinning parameters

PCL GelatinVoltage

(kV)

Flow rate

(ml/h)

Working

distance

(cm)

H V H V H V

PCL175 175 0 1:0 23 20 3 3 27 26

PCL150g25 150 25 6:1 13 15 2 2 19 22

PCL125g50 125 50 5:2 13 13 1.5 2 19 22

PCL100g75 100 75 4:3 x 13 x 2 x 22

PCL100g100 100 100 1:1 x x x x x x

34


2.1.5 Imaging of electrospun scaffolds

Prior to imaging, dry scaffolds were sampled into small pieces and mounted

onto sample holders with conductive double-sided adhesive tape. They were then

sputter coated in vacuum with palladium using the EMITECH SC7620 sputter coater

(Quorum Technologies). Plasma sputtering times for samples of 50 µm thickness was

30 s and for samples of thickness 150 µm it was 45 s. The samples were then imaged

using the Hitachi S-3400 N with EDAX Scanning Electron Microscope (SEM) system

at a magnification of 1000x (for the purpose of image paneling), an accelerating voltage

15 kV and working distance of 7 mm. Samples were also imaged for the purpose of

measuring fibre diameter and pore size at different magnifications according to the scale

of the sample. For thickness measurements only, samples were cut in liquid nitrogen to

get a clear cut edge without compressive damage. Samples were then mounted on

vertical sample holders with the cut edge facing upwards and imaged under the SEM.

2.1.6 Measurement of fibre diameter, pore size and thickness

All morphological measurements were made using Fiji Image J software. For

fibre diameter and pore size, triplicate samples were imaged per blend for statistical

significance. Five images were taken per sample, five fibres/pores were chosen at

random per image and five measurements were made per fibre/pore, yielding a total of

375 measurements per blend (115) (Fig 2.3). For thickness estimation, 10 measurements

were taken per sample and then averaged.

Pore size measurement of electrospun scaffolds has been fiercely contended in

the scientific community. The problem arises because electrospun scaffolds do not have

true pores, there are gaps that are simply created by the continuous layering of fibre

meshes. Since a 3D pore is difficult to categorise, most research groups resort to

measuring the 2D inter-fibre distances, created by fibres in the top couple of layers,

visible on an SEM image. Although it is possible to visualise many layers beneath the

surface, measurements on the resultant 2D images are only accurate for surface fibres.

The ‘pore sizes’ graphed in the corresponding results section are also 2D inter-fibre

distances measured from SEM images of the same samples (thickness 50 µm) used for

fibre diameter measurement.

35


2.3 DEGRADATION STUDY

In order to assess the stability of the samples, fibre degradation and gelatin loss

induced by cell culture medium in vitro, a degradation study was performed. This was

accomplished in two ways. Firstly, SEM was done to visualise any morphological

changes in the fibres during the course of the study. Secondly, Raman spectroscopy was

performed to analyse the chemical composition of the scaffolds during the length of the

degradation study. Undegraded and degraded samples (for 15 and 30 days) were

analysed alike. All samples were of 150 µm thickness.

2.3.1 Experimental setup

Sample preparation for SEM and Raman spectroscopy was performed in a

similar manner. All four scaffold samples were placed in DMEM (pH 7.4) in a

humidified incubator at 37 °C and 5 % CO2 for a span of 30 days. Prior to imaging,

samples were first washed in bi-distilled water and then dehydrated in an increasing

gradient of ethanol concentrations (30 %, 50 %, 70 %, 100 %) for 10 min each. This

was followed by air drying.

2.3.2 Visualisation of fibre structure

Triplicate samples for SEM imaging were sputter coated and imaged at a

magnification of 1000x, an accelerating voltage 15 kV and working distance of 10 mm.

Here as well, five images were taken per sample. The fibre structure was then visually

examined for defects such as thinning or breakage.

2.3.3 Raman analysis of gelatin loss and crystallinity of PCL

The dried constructs were placed on a glass slide using adhesive tape and

imaged using a Raman Microscope (Alpha 300 RA, WITec GmbH). Prior to

measurement, a Si wafer was used to calibrate the system as the Si peak is highly stable

at 520 cm-1 (corresponding to the crystalline Si-Si bond longitudinal optical phonon

vibrations (149)). 532 nm excitation light from an Nd-YAG Laser was focused on the

scaffold samples at a magnification of 100x. Acquisition of Raman spectra was done in

39


5 s at a laser power of 15 mW. Spectra were acquired from 5 different fibres of varying

thicknesses (above 3 µm).

Following this, analysis of data was carried out using Project FIVE.plus (WITec,

Ulm). The final spectrum was stitched to show only the fingerprint region of the data

(800-1800 cm-1). The results were graphed and standard background subtraction was

performed (there were no artefact peaks of cosmic ray interference). The intensity

values were normalised using formula 2.2:

Iy - Imin

Inorm = —————— (2.2)

Imax - Imin

where,

Inorm - normalised intensity

Imax - maximum intensity value

Imin - minimum intensity value

Iy - measured intensity

After normalisation, the data obtained was correlated with known spectra and

characteristic peaks of PCL and gelatin were identified (refer supplementary S.3 and S.4

for all Raman spectra). The Amide I peak of gelatin was deconvoluted using Origin,

integrated for area under the curve and then percentage loss of gelatin (from one tested

day to another) was calculated using equation 2.3. PCL crystallinity was analysed by

measuring the ratio between the full width at half maximum (FWHM) of amorphous

and crystalline peaks.

A2 - A1

L = —————— x 100 % (2.3)

A1

where,

L - percentage loss

A1 - area under curve 1

A2 - area under curve 2

40


2.4 CELL RESPONSE STUDY

2.4.1 Sterilisation of electrospun mats for cell culture

Scaffolds were sterilised by UV-C radiation (254 nm) (150) using the UV lamp

in the cell culture work bench (151). Fibre mats were laid out on aluminium foil and

exposed to UV radiation for 30 min (15 min each side). They were then rolled up and

stored in sterile 50 ml Falcon tubes and used for cell seeding under aseptic conditions

thereafter. Fourier Transform Infrared spectroscopy (Perkin Elmer Spectrum 100 FTIR

spectrometer) was performed on unsterilised and sterilised samples to ensure that UV

radiation did not alter or denature the chemical composition of the scaffolds. The

wavenumber range was chosen as 4000 to 650 cm-1 and the number of scans was set to

8. The probe force value was 50 for all analysed samples (see supplementary S.5 for

spectra and associated analysis).

2.4.2 Thawing and cultivation of cells

3T3 cells were cultivated in growth medium prepared by mixing 500 ml of

Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom GmbH) that contains 3.7 g/

L of NaHCO3, 4.5 g/L of D- Glucose, stable glutamine, Na-Pyruvate and low endotoxin

with 75 ml of foetal bovine serum (FBS) (Biochrom GmbH) and 5.8 ml of Penicillin/

Streptomycin (Biochrom GmbH). aMSCs were cultivated in the same medium with the

addition of 580 µL of ascorbic acid. 3T3 cells were thawed at Passage 3 and aMSCs

were thawed at Passage 7 for subsequent propagation. For all experiments, aMSCs were

seeded at passage 9 to ensure comparable proliferative capacity.

Cryopreserved cells were thawed by swirling the frozen vial swiftly in a warm

water bath at 37 °C. The vial was then placed in an icebox and further handled under

aseptic conditions. The cell suspension was then transferred to a falcon tube (Sarstedt)

and 5 ml of cold medium at 4 °C was added dropwise to the tube. This was then

centrifuged at 4 °C for 5 min at a speed of 1000 rpm. Following centrifugation, the

supernatant was aspirated and the cell pellet was resuspended in 1 ml growth medium.

The entire contents were then plated on a 60.1 cm2 coated culture dish (TPP AG) for

further passaging.

41


3T3 cells were passaged at 80% and aMSCs were passaged at 70 % confluence.

All solutions used during passaging were warmed to 37 °C before use. The medium was

aspirated and cells were washed with 1xPhosphate Buffered Saline (PBS) (pH 7.4,

Biochrom GmbH) and cells were detached by the addition of 0.05 %/0.02 % (w/v)

trypsin-EDTA (Biochrom GmbH) and incubation for 3 min at 37 °C. Trypsin activity

was stopped by adding medium and cells were resuspended over the dish surface

several times and collected. After centrifugation for 5 min at 1000 rpm, the cell pellet

was isolated and further resuspended in medium. Cell membrane integrity was assessed

by the Trypan Blue (Sigma Aldrich) exclusion method using the Vi-CELL XR Cell

Viability Analyser (Beckman Coulter). Pre seeding viabilities were always > 90 %.

Cultivation was continued in a humidified incubator at 37 °C with 5 % CO2 and

medium was changed every couple of days. Cells were then passaged on confluency.

2.4.3 Cell seeding on scaffolds

All electrospun constructs were seeded in uncoated 12-well plates with an area

of 5 cm2 (Sarstedt) to allow the cells to preferentially attach to the scaffolds over the

plate surface. Circular cutouts were made with custom cutters (manufactured at the IMP

workshop, see supplementary S.6 for drawing) with diameters of either 1.6 cm or 0.6

cm depending on the assay (Fig 2.6).

Fig 2.6 - Scaffold cutting with custom made cutters. Two diameters were used in the cell

response study depending on the assay - 1.6 cm and 0.6 cm.

All scaffold samples were wetted from the bottom with a little medium. This

serves two important purposes - first, it prevents the samples from being sucked into the

airflow mechanism of the cell culture bench, and second, it pre-wets the scaffolds to

allow for quick cell attachment. Cells were aliquoted into the required seeding

42


Table 2.2 - Optimised seeding volumes and seeding densities for cell response studies.

2.4.4 SEM visualisation of seeded cells

All chemicals mentioned in this section were purchased from Carl Roth GmbH.

The morphology of adherent cells on the scaffolds and their interaction with the fibres

were visualised using SEM imaging. Cells were seeded on scaffolds using the

previously discussed cell seeding procedure (seeding days were considered as day 0).

On days 1 and 5, prior to fixation, the samples were first rinsed with 0.1 M Cacodylate

buffer (prepared from Cacodylic acid sodium salt trihydrate at pH 7.4). Fixation was

performed by incubating cells for 15 mins with 2.5 % Gluteraldehyde in 0.1 M

Cacodylate buffer (pH 7.4). After another washing step, they were placed in bi-distilled

water for 15 min and subsequently dehydrated in an ascending gradient of Ethanol (30

%, 50 %, 70 %, 99 %). This was followed by air drying, sputter coating and SEM

imaging as described in 2.1.5.

2.4.5 Infiltration study

The cells were allowed to grow and proliferate on the scaffolds for a span of 15

days. Infiltration depths were assessed for all the four blends on days 1, 5, 10 and 15.

Cells were then stained with Phalloidin (λex 495 nm, λem 520 nm) (152) and Hoechst

33342 (λex 346 nm, λem 460 nm) (153) and visualised using a confocal laser scanning

microscope (cLSM) from Carl Zeiss GmbH (LSM 510). Both stains were purchased

from Sigma Aldrich. Phalloidin stains F-actin filaments green and Hoechst stains the

cell nuclei blue.

Scaffold

diameter

Seeding

volume

Scaffold

thickness

Cell seeding

densityCharacterization assay

1.6 cm 100 µl 150 µm ~ 50,000 cells/cm2

Live/dead assay

Infiltration depth measurement

SEM visualisation of seeded cells

0.6 cm 25 µl 150 µm ~ 25,000 cells/cm2 MTT assay

44


For staining, fixation of cells on the scaffold was first performed by rinsing the

cells twice with 1xPBS and subsequent incubation with 4 % formaldehyde (Sigma

Aldrich) for 15 min followed by washing with PBS. After fixation, cell membrane

permeabilisation was done by incubation for 15 min in 0.5 % Triton-X (Sigma Aldrich).

Cells were then washed with PBS, stained with Hoechst (1:1000 dilution factor) and

Phalloidin (2:1000 dilution factor) for 45 min in the dark and washed again with PBS to

remove any excessive stain. Samples were mounted on glass sides using Mount

Fluorcare (Carl Roth GmbH) mounting medium at room temp and left to dry overnight.

Samples were imaged inverted in the cLSM. Laser lines Argon/2 for the 458-514

nm range and Diode 405-30 for the 405 nm channel along with the Plan- Neofluar 20x/

0.5 objective (with 0.17 mm cover glass correction) were used to image the scaffolds.

Pinhole was set to 110 corresponding to an optical slice thickness of 5 µm. Green and

blue excitation channels were used to view the actin filaments and to locate nuclei

respectively. Focus was gradually fine-tuned and the gain was adjusted to avoid over

exposure while imaging. Images were acquired in Z-stacks (of thickness 5 µm). Total Z-

stack depth was noted for each sample and the resulting images were saved as .lsm files.

2.4.6 Viability study

Cell viability was assessed using a two-color assay (Calcein AM and Ethidium

homodimer-1 both purchased from Sigma Aldrich) to distinguish between live and dead

cells. Non-fluorescent cell permeant Calcein AM is converted to fluorescent calcein (λex

496 nm, λem 516 nm) in living cells indicating intracellular esterase activity. Ethidium

homodimer-1 is normally cell membrane impermeable and enters dead cells indicating

loss of plasma membrane integrity and gives a red (λex 528 nm, λem 617 nm) fluorescent

signal on binding to nucleic acids (154).

Viability study was performed on day 1, 4 and 7 of culture. Working solutions of

Calcein AM (1 mg/ml, 1:1000) and Ethidium homodimer – 1 (1 mM, 2:1000) were used

to prepare staining solutions in 1xPBS for scaffolds. The staining solution was

thoroughly vortexed before use.

45


Pre seeded scaffolds were retrieved from their respective cell culture plates and

were washed twice with 1xPBS solution. Then, 500 µl of staining solution was added to

each well and the scaffolds were incubated in the dark for 20 min. Finally, they were

washed twice with 1xPBS to remove any excessive stain and viewed inverted under the

Axiovert 200M fluorescence microscope (interfaced with Zeiss Axiocam MRm camera,

power source MAC 500, X-cite Series 120 fluorescence excitation lamp and computer

with Axiovision imaging software). Five images were taken at 10x magnification per

sample (middle, top, right, bottom and left).

For cell quantification, the image analysis software Image J (Fiji) was used.

Analysis was performed by splitting the blue and green channels. The ‘find maxima’

function was used to count the number of live and dead cells. Noise tolerance was

adjusted in increments of 5 until the background staining was excluded. Calculations

were also verified manually to account for errors.

The total cell number was determined using formula 2.3 and the percentage of

live cells was calculated using formula 2.4:

Nt = Nlc + Ndc (2.4)

Nlc

V = ——— x 100 % (2.5)

Nt

where,

Nt - total cell number

Nlc - number of live cells

Ndc - number of dead cells

V - viability percentage

2.4.7 Metabolic activity study

The MTT assay is a colorimetric method to assess the metabolism of viable

cells. Viable metabolising cells in culture reduce MTT (3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide) to purple formazan which possesses an

46


absorbance maximum near 570 nm. Since the metabolic activity (and by extension the

rate of MTT reduction) of cells can depend on a myriad of factors (without exactly

affecting viability), it is not, as often incorrectly interpreted, a cell proliferation

indicator (155). For this study, MTT was purchased from Sigma Aldrich.

Cells were seeded on scaffolds as described in section 2.4.3. Controls (scaffolds

without cells) were run separately for each blend of scaffold for all the measured days.

Cells were cultured on the scaffolds for 7 days and readings were taken on day 1, 4 and

7 in order to assess the proliferation rate over time. Scaffolds were transferred to a 96

well plate (TPP AG) for the assay. The solubilising solution (0.04 M HCL (Sigma

Aldrich), 0.1 % NP-40 (Applichem GmbH) in isopropanol (Carl Roth GmbH)) and

MTT stock solution (5 mg/mL) in PBS were prepared prior to the experiment.

On each test day, the cell culture medium was first aspirated, followed by the

addition of 10 µl of MTT solution and 90 µl of serum free media to each well. Serum

free media was used in order to avoid any interference during absorption readings. The

plate was then incubated in a humidified incubator at 37 °C with 5 % CO2 for 4 hours.

The MTT solution was then aspirated and 100 µL of solubilising solution was added to

each well followed by incubation at 37 °C with 5 % CO2 for 1 hour. After 1 hour, 50 µL

of the solution was transferred from each well to another 96 well plate for measuring

absorbance values using a multi-plate reader at 570 nm. 50 µl of solution was taken for

absorbance readings to ensure uniformity in testing, primarily to account for solvent

absorption by the porous scaffold.

For calculations, control values were subtracted from the actual values obtained

from the scaffolds to obtain corrected sample absorbances (which were graphed) using

formula 2.5:

Absc = Abssam - Abscon (2.6)

where,

Absc - corrected absorbance

Abssam - absorbance of the sample

Abscon - absorbance of the control

47

Chapter 3: Results

3.1.2 Imaging of electrospun scaffolds

After the initial optimisation of the electrospinning parameters (Table 2.1), all

scaffolds were prepared at a thickness of 50 µm for visualisation of scaffold

microstructure under the SEM. SEM images are panelled in Fig 3.1 with the

horizontally spun samples on the left side and the vertically spun samples on the right

side.

Fibre diameters were measured (n=125) for PCL 175 (Fig 3.1a and Fig 3.1b) and

it was observed that the vertically spun sample (1.52 ± 0.08 µm) showed a marginally

higher variation than in the horizontally spun sample (1.66 ± 0.07 µm). Pore size

measurements also did not differ statistically between the horizontal (20.3 ± 1 µm) and

vertical (23.9 ± 1.6 µm) orientations. Fig 3.1c and 3.1d represent the microstructure of

the PCL150g25 blend in the horizontally and vertically spun orientations respectively.

Here as well, there is no observable morphological difference between the two

scaffolds. Both setup orientations produce a similarly homogeneously structured

scaffold.

However, when the gelatin concentration is increased, i.e in PCL125g50 (Fig

3.1e and 3.1f), we see a stark difference between the horizontal and vertically spun

samples. The sample spun in the horizontal setup is homogeneous and consists mostly

of nanofibres but the sample spun in the vertical setup is heterogeneous and consists of

a range of fibre diameters (even though the blend concentration is similar in both cases).

Note the large degree of variation in pore size within PCL125g50V itself. Pore size is

21.4 ± 1.4 µm in PCL125g50H versus 115 ± 0.08 µm in PCL125g50V. The effect is

sustained when the gelatin concentration is increased further in PCL100g75V (pore size

107 ± 9 µm) but produced a very unstable electrospinning in the horizontal setup.

Therefore, we see a variation in scaffold microstructure in the vertical orientation above

a critical concentration of gelatin in scaffolds spun from blends with the same overall

concentration of solutes (175 mg/ml).

An attempt was made to spin PCL100g100 but the process was unstable in both

orientations yielding no proper samples for further testing.

50

Chapter 3: Results

Fig 3.1 - SEM image panel showing microstructure of electrospun scaffolds. Thickness of

scaffolds = 50 µm. All images were taken at 1000x magnification. Scale bar = 50 µm. Image

has been reused modified from (115).

51

Chapter 3: Results

Mean fibre diameters are 1.52 ± 0.05 µm (PCL175), 0.59 ± 0.04 µm

(PCL125g50H), 5.15 ± 0.42 µm (PCL125g50V) and 4.52 ± 0.33 µm (PCL100g75V).

Fibre diameter distributions are represented as histograms graphed in Fig 3.4.

Note that the range of fibre diameters is much larger in the vertically spun samples,

especially in PCL125g50V where the bin centres run from 0.5 to 17.5 µm. The range of

fibre diameters obtained here is much larger than attainable in a normal electrospinning

process.

Fig 3.3 - a) Fibre diameters and b) pore sizes measured from SEM images of the electrospun

scaffolds. n=375, **** p<0.0001, whiskers represent min and max values, mean shown as ‘+’.

Image 3.3a has been reused modified from (115).

Pore sizes of all four samples are depicted in Fig 3.3b. The results correlate with

the fibre diameter measurements. The mean pore size of PCL125g50V (115 ± 5 µm) is

almost four and a half times that of PCL175V (25.1 ± 1.1 µm) and four times that of

PCL125g50H (27.5 ± 1.3 µm). Similarly, the mean pore size of PCL100g75 is also high

(111 ± 5 µm). Pore size distributions are graphed in Fig 3.5.

53

Chapter 3: Results

Fig 3.4 - Fibre diameter distribution. Bin centres for PCL175V and PCL125g50H have been

truncated at 3.5 µm as there are no measurements beyond that size for those samples. Bin sizes

for all are 1 µm. n=375.

54

Chapter 3: Results

Fig 3.5 - Pore size distribution. Bin centres for PCL175V and PCL125g50H have been

truncated at 90 µm as there are no measurements beyond that size for those samples. Bin sizes

for all are 20 µm. n=375.

55

Chapter 3: Results

3.2 ASSESSMENT OF MECHANICAL PROPERTIES

Mean force at break and mean strain at break are graphed in Fig 3.6.

As depicted in the graphs, we can see that the introduction of gelatin reduces the

strain of the samples making them more brittle. Mean strain percentage at break for the

gelatin blends are 39.2 ± 11.7 % (PCL125g50H), 46.4 ± 85.6 % (PCL125g50V) and

34.2 ± 61.7 % (PCL100g75V) against 180 ± 604 % for PCL175V.

However, the mean force at break was much higher in the vertically spun blends

with the hybrid morphology compared to the other two samples. Mean values of

PCL125g50V, PCL100g75V and PCL175V are 3.4 ± 0.08 N, 2.19 ± 0.64 N and 1.55 ±

2.34 N respectively. PCL125g50H despite having the same concentration as its

vertically spun counterpart, only shows a mean force at break of 1.29 ± 0.13 N.

Fig 3.6 - Mechanical properties. a) mean force at break, b) mean strain at break. Cross-

sectional area of samples = 1.5x0.005 cm, n=3.

56

Chapter 3: Results


3.3.1 Visualisation of fibre structure

Undegraded samples and samples degraded in cell culture medium (at 37 °C and

5 % CO2) for 15 and 30 days were visualised under the SEM (Fig 3.7). All samples

were of thickness 150 µm. Points of fibre degradation (obviously thinned regions on a

fibre) were observed only in the PCL175V sample. Vertically spun blends visibly

showed uniformly thinner fibres by day 30.

Fig 3.7 - Visualisation of fibre degradation over 30 days. Obvious points of fibre thinning

have been indicated by yellow circles. All images were taken at x1000 magnification. Scale bar

= 50 µm.

3.3.2 Raman analysis of gelatin loss and crystallinity of PCL

Raman spectra of all four samples were taken and analysed for gelatin loss and

crystallinity of PCL gelatin. Fig 3.8 shows the characteristic peak of gelatin Amide I at

1636-1668 cm-1 (157) and C=O stretch of PCL at 1730 cm-1. The C=O stretch in PCL is

actually composed of two constituent peaks - a narrow peak at 1725 cm-1 representing

the crystalline phase and a broader peak at ~1735 cm-1 representing the amorphous

phase (158).

57

Chapter 3: Results

Fig 3.8 - Gaussian fitted graphs of gelatin Amide I and PCL C=O. a) PCL175V, b)

PCL125g50H, c) PCL125g50V, d) PCL100g75V. Day 0 represents undegraded samples while

day 15 and day 30 represent degraded samples.

Areas under the Amide I peak of gelatin show a 36.99 ± 6.65 % loss in

PCL125g50H and a 53.44 ± 6.48 % loss in PCL100g75V by day 15 (Table 3.2). After

that, the gelatin loss seems to stagnate in both samples from day 15 to day 30.

PCL125g50V shows no significant loss in gelatin. This peak also shows a minor shift

toward higher wavenumbers from day 0 to day 30 in all the tested samples. Crystallinity

of PCL could not be calculated for samples using information from the entire spectrum

due to interference from the gelatin peaks. Further, FWHM ratios of the crystalline to

amorphous C=O peak showed no discernible trend in the tested period.

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Chapter 3: Results


3.4.1 SEM visualisation of seeded cells

Seeded cells were fixed and visualised on day 1 and day 5 of culture. The

behaviour of 3T3 cells and aMSCs was comparable. On day 1, it can be seen that cells

reside on the surface of PCL175V and PCL125g50H owing to their low pore size.

However cells are seen between the first few layers of fibres in the vertically spun

blends. By day 5, this effect is well pronounced and some infiltration can be observed in

the vertically spun blends. The formation of monolayers on the other two samples show

that the cells are restricted from infiltrating the bulk of the scaffold. (Fig 3.9) Cells

seeded on the gelatin blends exhibited a much better spread of cytoplasm compared to

PCL175. The total coverage of the scaffold surface was also more. Specifically in

PCL125g50V, additionally to the infiltration, it can be seen that the cells extend their

philopodia to attach securely to the nanofibres in 3D.

Fig 3.9 - SEM visualisation of cells seeded on the electrospun scaffolds. a) 3T3 cells, b)

aMSCs. All images were taken at x1000 magnification. Scale bar = 50 µm. Image 3.9a has been

reused modified from (115).

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Chapter 3: Results

3.4.2 Infiltration study

Cells cultured on the electrospun constructs were stained with Phalloidin and

Hoechst for imaging under the cLSM. Infiltration depths were measured as the z-stack

depth down to which cell nuclei are visible. Images showing the stained cells are

panelled in Fig 3.10 (3T3 cells) and Fig 3.11 (aMSCs). Since gelatin is strongly

autofluorescent, there is sometimes a background fluorescence depending on the

amount of gelatin leaching out.

Fig 3.10 - Visualisation of infiltrated 3T3 cells on the electrospun scaffolds over the span of

15 days. Scale bar = 50 µm.

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Chapter 3: Results

Fig 3.11 - Visualisation of infiltrated aMSCs on the electrospun scaffolds over the span of 15

days. Scale bar = 50 µm.

In correlation with this qualitative analysis, the measured infiltration depths are

graphed in Fig 3.12. PCL175V and PCL125g50H show minimal infiltration in the first

few days and then a stagnation from day 5 onwards. On day 15, 3T3 cells cultured on

PCL175V showed a mean infiltration depth of 22.33 ± 6.7 µm, which is a negligible

increase from 18.3 ± 3.1 µm measured on day 1. aMSCs fair better in this case where

their infiltration depth increases from 17.3 ± 1.5 µm on day 1 to 37.3 ± 3.6 µm on day

15 on PCL175V. PCL125g50H shows the lowest infiltration of both 3T3 cells (22 ± 5.3

µm) and aMSCs (26.7 ± 2.2 µm) on day 15.

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Chapter 3: Results

3.4.3 Viability study

The infiltration study shows that the cells have infiltrated (or not) through the

bulk of the scaffold but gives no indication of the viability of these cells. A live/dead

assay (Calcein AM for live cells and Ethidium homodimer-1 for dead cells) was

performed for this purpose on seeded cells on days 1, 4 and 7 (Fig 3.13).

Fig 3.13 - Cell viability assay fluorescence images over the span of 7 days. a) 3T3 cells, b)

aMSCs. Scale bar = 100 µm. Images PCL125g50V day 7 and PCL100g75V day 7 in 3.13a have

been taken from (115).

64

Chapter 4: Discussion

influence of strong electric fields and this can cause a broad divergence in fibre

diameters (159). Polyelectrolytes tend to greatly increase the conductivity of a solution

because they dissociate in aqueous media to produce charged ions. While this is a good

option to increase the conductivity of certain solutions to facilitate electrospinning

(160), an excess of these ions can hinder the formation of fibres.

Since gelatin is a polyelectrolyte and has gels in aqueous media, it cannot be

electrospun in water and is most often mixed with synthetic polymers and dissolved in

an organic solvent. TFE in particular has been suggested as a good solvent for

polypeptides (84). While the use of an organic solvent is helpful to make a

polyelectrolyte solution spinnable, the conductivity is still expected to be somewhat

high.

The gelatin blends used in this study show a much higher conductivity than the

pure PCL solution, but are electrospinnable. However, it was observed that an increase

in the concentration of gelatin resulted in more instabilities during electrospinning. As

such, it was not possible to obtain sufficiently good samples with a solution

concentration of PCL100g100, as the solution constantly collected and dried at the

needle tip and formed multiple jets during the process.

The electrospinning of a single polymer is predictable as the solution properties

do not vary tremendously. But there is a critical concern in blend solutions about phase

separations. More often than not, constituent polymers of blend solutions are not fully

miscible and require some dopant to facilitate this.

In this project, 2 wt% acetic acid is added with respect to TFE. Literature shows

that just 0.2 wt% acetic acid is sufficient to mediate proper gelatin dissolution in TFE

and miscibility with the PCL solution (148). However, this was not observed in the

context of this project in the long term. A 0.2 wt% acetic acid was found to allow for

better dissolution and initial miscibility but does not prevent phase separation after a

few hours. This could be problematic for electrospinning because some solutions need

to be spun for a few hours to get the required fibre mat thickness. Therefore, the acetic

acid dopant concentration was increased to 2 wt% and thereafter no phase separation

was observed even overnight.

69


The possibility of phase separation during electrospinning was also considered.

Since it is not possible to ascertain the incidence of phase separation during

electrospinning, a Raman analysis was carried out on the finished products to see if the

composition of the thick and thin fibres varied significantly (as part of the degradation

study, graphs in supplementary). The variation was found to be negligible with both

PCL and gelatin distributed more or less homogeneously through the fibres.

4.2 THE INFLUENCE OF SETUP ORIENTATION AND POLYMER

CONCENTRATION ON PCL-GELATIN BLEND ELECTROSPINNING

Electrospinning PCL175 in both orientations produces scaffolds with marginal

fibre diameter differences. A similar outcome was also reported by Yang et al. (120)

(albeit without statistical analysis) but the result reported here does not differ

statistically. Therefore we can say that PCL175 spun in either orientation has a similar

structure. Solution conductivity and viscosity are constant and a stable Taylor cone is

observed during the electrospinning process.

When gelatin is introduced at a low concentration, it does not affect the

electrospinning process and again, homogeneously structured scaffolds are obtained in

both setup orientations (PCL150g25). However, beyond this concentration of gelatin, a

gradient of fibre diameters is observed only in the vertical setup orientation (as seen in

PCL125g50V and PCL100g75V).

In blend electrospinning, variable fibre diameters have been associated with

improper solution mixing and phase separation of constituent polymers before

electrospinning (148). In the context of this project, no phase separation was observed

in any of the solutions before electrospinning and therefore there must be another reason

for the variable fibre diameters observed in vertically spun PCL-gelatin blends (above a

critical concentration of gelatin). While an extensively detailed analysis involving other

blends (with and without polyelectrolyte components) and complicated fibre

characterisation methods is beyond the scope of this study, it is possible to propose a

preliminary hypothesis to explain such a situation based on existing literature.

70


gelatin) of the working solution, there is a field-enhanced dissociation of the

contaminants which additionally causes the conductivity to rise exponentially

depending on the applied electric field.

Horning and Hendricks also examined the forces driving an electrostatic jet in

1979 (164), of which conductivity was one studied parameter. While they were unable

to explain how exactly the conductivity affects the geometry of the jet, they showed that

it indeed affected where on the jet the instabilities start to occur. As explained

previously this would eventually affect the resultant fibre diameter. In 2009, Stanger et

al (165) characterised the Taylor cone in terms of charge density. They claimed that an

increase in solution conductivity causes an increase in charge density at the Taylor cone

resulting in a smaller jet diameter (because the jet is drawn from a smaller effective area

or “virtual orifice”) and lower rate of fibre mass deposition.

This brings us to the topic at hand. There are three aspects to keep in mind in the

context of this thesis. The first crucial point to be noted is that Taylor’s experiments

were performed with conducting and non-conducting fluids, not with polymers

dissolved in solvent. Polymer molecules not only interact with each other, but also with

the needle tip and the electrostatic force provided by ions (162). The number of polymer

molecules flowing into the droplet as well as the effect of the interactions of these

molecules affects the local conductivity in the droplet. By extension, this will also affect

the charge density, jet diameter and resultant fibre diameter.

Second, we cannot predict the number of gelatin and PCL molecules in the

Taylor cone at any particular instant because of the general randomness of fluid flow

into the droplet. This may result in a small fluctuation in polymer ratio and consequent

fluctuation in the charge density at the Taylor cone. Now, if a non-polyelectrolyte was

used in place of gelatin, the fluctuation would be negligible. But due to the presence of

gelatin, the effect of the fluctuation is amplified because of its inherent strong ionisation

property. When the gelatin concentration is very low (as in the case of PCL150g25), it

will have no discernible effect on the final product. When the gelatin concentration is

increased, it may cause some variation in fibre diameters but may still be too weak on

its own to cause the huge range of fibre diameters obtained in this work.

75


Third, since the mass of polymer carried away by the jet reduces with increasing

charge density, the fluctuations in local conductivity at the Taylor cone could induce

fluctuations in the ‘ideal’ voltage for that system. This means that the range in which

there can be oscillations in droplet size and jet diameter is increased. The generally high

conductivity of gelatin prevents PCL-gelatin blends from being spun at high voltages

due to the formation of multiple jets. Therefore, the overall spinnable range is narrowed

while the oscillation range is widened.

This review finally brings us to the first part of the hypothesis that was

generated to help explain the phenomenon of varying fibre diameters in PCL-gelatin

scaffolds above a critical concentration of gelatin. It is proposed that the noticeable fibre

diameter variations are caused by a combination of the three aspects mentioned above.

But, we still encounter two unanswered questions. Is the combination of the above

aspects sufficient to cause such a large variation in fibre diameter as was observed? And

why does it only happen in the vertical setup orientation?

Since the only parameter that changed in both setup orientations is the direction

of the gravitational force, we can attempt to answer these questions with the last piece

of the puzzle - Taylor’s take on the correlation between the electric field and the start of

the jet instability. As elucidated before, the point of jet instability is pushed closer to the

needle when the electric field is stronger and is pushed away from the needle when the

electric field is weaker.

In the vertical orientation, the fluctuation in fibre diameter caused due to

changes in the charge density at the Taylor cone and the voltage being in the oscillation

range is exacerbated by gravity. A noticeable fluctuation in the jet instability starting

point was observed during electrospinning. The point moved up and down over a

certain range on the extruded jet. Since the fluid has nowhere to go but down to the

collector, the jet drew out more or less solution, became thick or thin and got deposited

on the collector depending on the condition at a given time.

In the horizontal system however, gravity is not directly involved in balancing

the aerodynamic drag and electric field. This means that the start point of the jet

instability cannot move much further away from the needle as there is no gravity to

76


support the fluid flow in that direction. A lowering of the electric field will just result in

the jet being unable to traverse the full distance to the collector. Additionally, it is

possible that the sagging of the Taylor cone (also reported in (121) and (122)) acts as a

buffer, preventing the charge density fluctuations from having any real impact on the jet

thickness or the resulting fibre diameter. Correspondingly, a barely noticeable shift back

and forth of the start of the jet instability was observed during electrospinning. This

would also well explain why PCL125g50H has a marginally higher fibre diameter

variation than PCL175 (leading to an inconsequential increase in pore size) while

PCL125g50V and PCL100g75V have a much larger fibre diameter variation (leading to

a substantial increase in pore size).

The instability in electrospinning PCL-gelatin solutions resulting in non-

uniformity and inconsistencies in the fibres with increasing gelatin content was also

reported by Nelson et al (166) although it was just a trivial secondary observation to

their main results.

Unfortunately, it was not possible to verify this hypothesis, given the time frame,

the goals and scope of the thesis. But perhaps an in depth analysis can be done in the

future on the electrospinning of blend solutions in both setup orientations with and

without a polyelectrolyte.

4.3 MECHANICAL PROPERTIES

When comparing the mechanical properties of scaffolds to human tissue, it is

important to keep in mind how the test was performed in both situations. There are

inconsistencies in literature about how uniaxial tensile tests are setup. They can be

either quasistatic or dynamic, and in either case, may have a different strain rate. Since

the failure strain is dependent on the type of test and the strain rate used in the study, it

is difficult to pinpoint a precise value for either a biological tissue or a scaffold. For

instance, abdominal porcine skin was reported to have a failure strain of 123-126 %

when using a quasistatic strain rate of 0.25-10 %/s in one paper (167) while it was

measured as 31-53 % when using a quasistatic strain rate of 50 mm/min in a another

77


reduced by the addition of gelatin. Since gelatin is brittle by nature, it was expected that

the strain percentage would be reduced. In addition, the mixing of PCL and gelatin is

only realised on a physical level (i.e. they are not copolymers). The physical bonds

between both polymers are much weaker than the covalent bonds in homopolymers and

hence, blending two different polymers on a physical level often tends to reduce the

mechanical performance (strain) (156).

The addition of gelatin greatly increased the mean force at break in the vertically

spun blends, especially PCL125g50V. The vertically spun blends present a certain

degree of interconnections in the very large fibres caused due to delayed evaporation of

solvent (Fig 4.6) which tend to increase the force at break (170-172). They also show

nanofibres bridging the microfibres creating “nanowebs” (Fig 4.6) as Soliman et al

(104) observed in their experiments.

Fig 4.6 - Fibre interconnections and “nanowebs” observed in the vertically spun blends.


The degradation study shows that PCL-gelatin blending is able to retain gelatin

in the scaffold without the need for crosslinking. After an initial drop in gelatin

concentration in the first 15 days (only in PCL125g50H and PCL100g75V), the

scaffolds tend to retain what is left through to day 30 without losing more.

79


PCL100g75V loses about half its gelatin content within the first 15 days only in cell

culture medium. Cell cultivation would likely increase the gelatin loss even more within

this time. Despite being of similar fibre structure to PCL125g50V, PCL100g75V poses

an obstruction to the infiltration of cells, as seen in the infiltration study. A similar initial

drop and then stabilisation of gelatin content in PCL-gelatin scaffolds was also observed

by Hwang et al (91).

In general, semi-crystalline PCL at 20 °C shows sharp peaks corresponding to

regions of alkyl chain segment or the ester group. These sharp peaks are an indicator of

the crystallinity of the sample. Broad low intensity peaks are often associated with

amorphous states indicating that the polymer has lost some of its conformational order.

(173) Typically, aliphatic degradable polymers such as PCL tend to show an increase in

crystallinity (and an associated larger stiffness) in the beginning stages of degradation

because of the preferential surface erosion of amorphous regions (127,174). However,

such an obvious increase in crystallinity due to the degradation of amorphous regions is

only expected after about four weeks of degradation (174). No trend of change in

crystallinity was discernible from the obtained results for the studied time period.


Traditionally, fibre diameter variations in electrospun products have been

considered undesirable and researchers strived to achieve uniform fibres in electrospun

scaffolds (148, 175). However, more recently, research into the functions of the ECM

has driven scientists to create more heterogeneous scaffolds in terms of structure as well

as composition. Cell response is a complicated phenomenon and is controlled by an

array of factors. The main aim of this part of the study was to show that the electrospun

scaffolds prepared were able to increase cell infiltration compared to traditionally

electrospun scaffolds over a given period of time, regardless of the cell type used. The

secondary aim of the cell studies was to show that the infiltrated cells show a good

viability and good metabolic activity.

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Initial cell response studies were performed with 3T3 cells because the cells are

robust and their size, function and population doubling time (20-26 hours (140)) are not

largely affected from passage to passage like that of primary cells. 3T3 cells showed a

much higher infiltration in the vertically spun blends compared to PCL175V and

PCL125g50H where stagnation was observed. The cells cultivated on PCL175V and

PCL125g50H tended to form monolayers at the top of the scaffolds because they were

unable to penetrate through the dense fibrous structure with small pores. PCL125g50V

especially outperformed all the other scaffolds because of its high pore size.

The question arises here as to why PCL100g75V (having a similar structure to

PCL125g50V) did not perform just as well. However, this can be explained by the fact

that gelatin absorbs copious amounts of water in aqueous medium, swells and tends to

form a gel before it gradually leaches out from the scaffold. Since PCL100g75V is

almost 50 % gelatin, the excessive swelling and gelation can cause a physical barrier to

infiltrating cells. PCL125g50V on the other hand has enough gelatin to mediate

hydrophilicity and biocompatibility, but not too much that it hinders cell penetration.

3T3 cells showed an extremely good viability on all scaffold samples with an

increase in total cell number throughout the study period. Cell survival on day 1 was

comparable for all scaffolds. The average total number of cells were much higher in all

the gelatin samples compared to PCL175 on day 7 showing a higher proliferation of

cells in the gelatin blends. However, the viability was specifically found to be higher by

day 7 only in PCL125g50V and PCL100g75V. Since PCL125g50H has a similar

composition to PCL125g50V but does not show the same high viability (despite having

a comparable total cell number on day 7), the better performance of PCL125g50V can

be attributed to its microstructure. The larger pores of the vertically spun blends likely

allowed for better transport of oxygen, nutrients and waste, leading to better cell

viability.

The assessment of metabolic activity gives us an indication of how healthy the

viable cells are. It is not only important that the cells have a high viability percentage, it

is also crucial that they metabolise at a sufficient rate. The MTT assay can show

increased absorbances if either the viable cell number is high (but each cell on its own

81


may not metabolise to its full extent), the viable cells are metabolising at a very high

rate (without a noticeable increase in cell number), or the cells are large in number and

metabolise at a high rate. Since the 3T3 cells have a very small population doubling

time, it is difficult to deduce whether the increased metabolic activity over the days is

only because of the increased number of cells or because the cells are also increasing

their metabolism.

MSCs on the other hand behaved very differently to the 3T3 cell line. It is well

known that MSCs are very sensitive to microstructure, scaffold composition and

mechanical properties. Unlike the 3T3 cells, they do not grow indiscriminately on any

suitable surface and have a population doubling time much higher than that of 3T3 cells

(176), especially after passage 6 (177). Population doubling time of MSCs from the 6th

to 12th passage varies between ~60 and ~160 h depending on the passage number,

source species (human or non-human) and the site of extraction (amnion, bone marrow,

umbilical cord, placenta or adipose tissue) (177, 178, 179).

The infiltration study showed a similar result to that observed with the 3T3 cells.

Cells cultivated on PCL125g50V showed the highest mean infiltration depth by day 15.

PCL100g75V, here as well, was not able to compete with PCL125g50V presumably

because of excessive gelatin swelling. PCL125g50H was the only sample that showed a

stagnation in infiltration depth from day 10. Cell viability percentages in the fibre mats

were quite different to what was seen with the 3T3 cells. Cells cultivated on PCL175V

showed a significant reduction in viability from day 1 to day 4 that did not recover by

day 7. The reduction in viability in PCL175V is caused because PCL is hydrophobic

and does not have a large pore size for infiltration and mass transport, presenting an

overall unfavourable growth environment for aMSCs. While all the samples with

gelatin show no significant difference between each other on all three days, it is

interesting to note that PCL125g50H has a significantly lower viability on day 7

compared to its viability on day 4. PCL125g50H, in contrast to PCL175V, does contain

gelatin but also does not have large pores. This effect of suboptimal growth conditions

could not be seen in the 3T3 cells because they are very tough. aMSCs therefore

provide a much more realistic simulation of how cells would behave in vivo.

82


Since the aMSCs were seeded on the scaffolds at passage 9, it was not expected that

they proliferate at a high extent. Apart from cells seeded on PCL175V (where the

viability significantly dwindled), the total number of cells observed during the viability

study from day 1 to day 7 in all the gelatin blends was more or less constant. Therefore

the result obtained in the MTT assay (either an increase or decrease in absorbance) is a

function of cell metabolic rate and not change in population.

PCL125g50H showed the highest metabolic activity on all the tested days

because it contains gelatin and also has an excellent nanofibrous surface. PCL125g50V

and PCL100g75V show a lower metabolic activity on day 1 (because of the addition of

large microfibres) but quickly catch up to PCL125g50H by day 7. Now this is intriguing

because while the cells initially are exposed to both nano and microfibres (on the

surface), they are able to navigate through the large pores and seek out nanofibres for

attachment. PCL125g50V is the only sample that shows an increasing trend in cell

metabolic activity. Metabolic activity is linked to cells that have a higher motility and

therefore it is understandable that as the cells infiltrate more, they also increase their

metabolic activity. The generally improved behaviour of the aMSCs on the

heterogeneously structured gelatin blended scaffolds can also be a result of increased

scaffold stiffness as it is known that stem cells are highly mechanosensitive (as

evidenced by improved attachment, motility and differentiation capabilities) (180,181).

From the results obtained in the cell study in terms of infiltration, viability and

metabolic activity, it is clear that a heterogeneous fibre morphology is an asset in

electrospun scaffolds facilitating infiltration as well as preserving cell viability and

metabolic activity. The addition of gelatin largely increases the biocompatibility of the

scaffolds and greatly influences cell function. However, it is important to regulate the

ratio of gelatin to PCL so that the swelling of gelatin does not hinder the movement of

cells. Therefore, we propose that PCL125g50V has tremendous potential for further

studies and application in soft tissue engineering. An overview of some other attempts

to increase cell infiltration in electrospun scaffolds is shown in Table 4.1.

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Chapter 5: Conclusion

These scaffolds were also characterised in terms of mechanical properties and

stability during degradation. To briefly recapitulate, the addition of gelatin seemed to

reduce the strain percentage of the scaffolds. However, the incidence of

interconnections between fibres together with the gelatin content allowed for a larger

force at break. PCL125g50V showed the highest force at break. The degradation study

was performed to visually examine the fibre structure after 30 days in cell culture

medium at 37 °C. The addition of gelatin stabilised the scaffolds and localised fibre

thinning was only seen in pure PCL samples and no visible fibre degradation points

were seen in the fibres of the blended scaffolds. Gelatin loss in all the samples was also

quantified. It was observed that the gelatin loss in PCL100g75V was maximal in the

first 15 days, while the other ratio PCL125g50V tended to retain its gelatin

concentration without much loss. This is relevant in terms of cell seeding.

The second hypothesis stated that the obtained hybrid morphology (in terms of

structure and composition) enhances cell infiltration, viability and metabolism. To

verify this, the scaffolds were seeded with two different cell types (a robust NIH 3T3

cell line and sensitive primary amnion derived MSCs) to assess if the change in fibre

diameter and pore size distributions allowed for better cell infiltration. Cells showed a

much higher infiltration depth in the hybrid scaffolds (PCL-gelatin blends with fibre

diameter gradients) with a good viability and metabolic activity. PCL175V in

comparison presented hydrophobicity, lower mechanical stability, faster degradation

and lower biocompatibility.

PCL125g50V especially outperformed all the other scaffolds in terms of all the

characterisation tests. It showed the highest infiltration depth in 15 days, had a good

viability percentage and high metabolic activity by day 7 in both cell types. There was

no obstruction of cell penetration because of low pore size or high gelatin concentration.

Therefore, it appears to be the superior scaffold both in terms of structure and

composition for the purpose of cell cultivation.

The aim of the thesis was to fabricate PCL-gelatin blend electrospun scaffolds

with large fibre diameter gradients for the enhanced infiltration and integration of

seeded cells in soft tissue engineering applications. The experimental results show that

86


the aim has been fulfilled. The complex process of electrospinning is not yet fully

understood, especially the spinning of blended solutions. This research brings us a step

closer to comprehending the various forces involved during the process and the

interaction between them when the setup orientation is changed. Until now, gravity has

been dismissed as a force too weak to have any real influence on the electrospinning

process. However, we see that this is not the case when other parameters such as the

direction of the electric field and conductivity (because of the addition of

polyelectrolyte elements) are altered.

The characterisation data presented in this thesis helped to better understand

cellular response on hybrid PCL-gelatin scaffolds. Much research has gone into

increasing the pore size of scaffolds for better cellular penetration. But are we

compromising on cell function in the process? This is important because in vivo

outcomes are strongly dependent not only on cell infiltration, but also on cell

attachment and overall metabolic health. Since it is extremely difficult to optimise a

scaffold on every aspect, a tradeoff is necessary. The combination of a truly

heterogeneous microstructure and the presence of a bioactive material such as gelatin

can significantly improve how the cells integrate with the scaffold, as can be seen from

the results presented here.

This research also shows that using a robust cell line to validate a scaffold can

be misleading as these cells do not behave like native cells in the body. It is imperative

to gain as much information as possible about sub optimal growth conditions in vitro in

order to avoid failure in vivo. And this can only be achieved by using sensitive cells (for

example, the aMSCs used in this thesis show that pure PCL is not only less favourable

than the gelatin blends, but also actively hampers cell function).

Pore size and fibre diameter have to be optimised for the particular cell type

used in the application. However, this can prove difficult if a co-culture is necessary.

This problem can somewhat be tackled with a heterogeneously structured scaffold like

that presented in this thesis. It is possible to culture multiple cell types on these

electrospun constructs, which can consequently be applicable for a variety of tissues

with mixed cell populations.

87


5.2 OUTLOOK

Biological systems are so beautifully and intricately designed that a full

understanding may be far in the future. Creating a perfectly biomimetic scaffold of such

a complicated system is even farther off. In the grand scheme of things, it is a much

harder and more complex task to create a versatile scaffold, superior in all aspects for a

particular intended application. Nevertheless, the economic growth within the tissue

engineering sector has been exponential in the recent past, demonstrating tremendous

potential for the future. Engineered tissue has not only been used in regenerative

medicine but also to serve as enabling technologies for other emerging fields such as

bio-robotics, drug delivery and disease modelling.

There are some key limitations to the present study that need to be overcome in

the future. Electrospinning is labour intensive and time consuming and is very hard to

standardise because of the multitude of controlling parameters. In addition, fluorescent

imaging and analysis was made difficult by the autofluorescence of gelatin.

Histological processing was attempted to visualise the distribution of seeded

cells at the end of the infiltration period. Unfortunately, the scaffolds are temperature

sensitive (PCL melting point is 60 °C and gelatin’s is 40 °C) and easily damaged by

alcohol processing, making it hard to get any usable samples for imaging. However,

cryosectioning was attempted on PCL-gelatin tubes (experiments performed in the

Medical University of Graz, Austria as part of an IP@Leibniz funded exchange) and a

preliminary positive result was obtained (Fig 5.1). More experiments in this direction

will be performed in the future.

Further research into the effect of polyelectrolytes on blend electrospinning has

to be performed, especially with regards to changing setup orientation. Is this effect also

observed with other polyelectrolytes? Is it possible to control it by changing the

concentration of the polyelectrolyte? It would be interesting to see if the microstructure

(fibre diameter and pore size distributions) can somehow be guided by the angle of the

electrospinning setup. For instance, the degree of heterogeneity (required for a

particular application) can be increased by making the system orientation more vertical

and decreased by making the system orientation more horizontal.

88

Appendix

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Supplementary data

S.1 Thickness optimisation of electrospun scaffolds

The spinning time for the 150 µm thick scaffolds was extrapolated from the

thickness optimisation graph. Since this would only be an estimate, samples were spun

for the suggested time period and then measured again for thickness. Any variation in

empirical thickness to the estimated thickness was reduced by simply adjusting the

spinning time. These samples were measured again to ensure that the spinning time

indeed produced the required scaffold thickness.

Name of the solution Optimised duration of electrospinning (min)

50 µm thickness 150 µm thickness

PCL175V 25 135

PCL125g50H 110 332

PCL125g50V 120 232

PCL100g75V 85 172

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S.2 Mechanical testing Force-Elongation graphs (all sets)

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S.3 Raman spectra - PCL pellets, gelatin powder

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S.4 Degradation study - Raman spectra (fingerprint region)

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Publication list

1) Suresh S, Gryshkov O, Glasmacher B. Impact of setup orientation on blend

electrospinning of poly-ε-caprolactone-gelatin scaffolds for vascular tissue

engineering. The International Journal of Artificial Organs 2018; 41(11): 801–810.

2) Suresh S, Balamuruganandam MS, Kidwai SM, Gryshkov O, Glasmacher B.

Improving cell infiltration in electrospun poly(ε-caprlactone)-gelatin scaffolds

through fibre diameter manipulation: a comparative analysis with amnion derived

multopotent stromal cells and 3T3 cell line. Materials Science and Tissue

Engineering C [under review as of 21st February, 2019].

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Statement of Contribution

I, Sinduja Suresh, hereby confirm that I have made the following contributions to this

thesis:

1) Supervised two relevant master thesis projects to completion.

• Investigation of effect of system orientation on properties of electrospun PCL

gelatin scaffolds intended for cardiovascular application.

(Mythili Shree Balamuruganandam, Vellore Institute of Technology, India,

July 2017)

Relevant topics: Electrospinning, mechanical study

• A comparative cell response study on structurally enhanced Poly-ε-

caprolactone (PCL)-Gelatin electrospun scaffolds for cardiovascular

applications.

(Sarah Manaal Kidwai, University of Göttingen, Germany, October 2018)

Relevant topics: aMSC infiltration, viability and metabolic activity

2) Supervised one relevant lab rotation

• Assessment of metabolic activity of NIH 3T3 fibroblasts seeded on PCL-

gelatin electrospun scaffolds intended for cardiovascular applications.

(Sarah Manaal Kidwai, University of Göttingen, Germany, April 2018)

Relevant topics: 3T3 metabolic activity

3) Supervised one relevant exchange project funded by IP@Leibniz.

• Investigation of infiltration of 3T3 fibroblasts into electrospun PCL-gelatin

scaffolds intended for cardiovascular applications.

(Mythili Shree Balamuruganandam, November 2017)

Relevant topics: 3T3 infiltration, viability

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(Note for points 1 to 3: I conceptualised and supervised the optimisation of protocols.

All experiments were performed by me and the student together in order to accomplish

more in a short amount of time. Optimised protocols and results were verified and

approved by Dr Oleksandr Gryshkov.)

4) Supervised one relevant student project to completion.

Charakterisierung von tubular PCL-Gelatine-Elektrospinn Gerüststrukturen

(Mugtaba Azim, Leibniz University Hannover, Germany, July 2018)

(This project was done to make sure that the electrospinning machine did not

influence the PCL-gelatin microstructure.)

5) Supervised four internships to completion (data not included in this thesis)

• Construction of a coaxial needle and electrohydrodynamic unit

(Nicholas Feringa, Michigan State University, August 2016)

• Parametric optimisation of electrosprayed alginate beads

(Michaela Keck, University of Nebraska-Lincoln, August 2016)

• Fabrication and evaluation of alginate Volvox spheres

(Sarah Cushman, Northeastern University, December 2016)

• Optimisation of cell-seeding on electrospun scaffolds using micro extrusion

printing

(Cing Hawm Lian, University of Nebraska-Lincoln, August 2017)

6) Prepared and submitted the abstracts and associated poster/talk for all the

conference contributions (refer CV).

7) Measurements of solution density and viscosity. Conductivity measurements were

done by Benedikt Voß (intern).

8) Analysis of Raman data (actual spectra acquired by Dr Oleksandr Gryshkov

because only authorised personnel are allowed to use the machine).

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9) FTIR spectroscopy (with instruction from Prof. Dr. Ir. Willem F. Wolkers).

10) Designed the scaffold cutters on SolidWorks (cutters manufactured in the IMP

workshop).

11) Put forward the hypothesis explaining the phenomenon of changing

microstructure in PCL-gelatin blend electrospun scaffolds in the vertical

orientation above a critical concentration of gelatin.

12) Did an exchange funded by IP@Leibniz in the Medical University of Graz (refer

CV) under the supervision of Dr Dagmar Pfeiffer. Tubular PCL-gelatin grafts

were seeded with human endothelial and smooth muscle cells in bioreactors. A

protocol for histological processing (either paraffin or cryo) of such temperature-

sensitive scaffolds was developed. The tubular scaffolds used in this project were

electrospun by me at the IMP using the optimised protocol developed during the

student project of Mugtaba Azim.

13) Performed histological staining on flat mats in the Niedersächsisches Zentrum für

Biomedizintechnik, Implantatforschung und Entwicklung (NIFE) with Ms

Annemarie Beck to identify appropriate stains for PCL-gelatin scaffolds (data not

included in this thesis).

14) Attended conferences: ESAO & yESAO 2016 (Warsaw, Poland), yESAO 2017

(Vienna, Austria) and ESAO & yESAO 2018 (Madrid, Spain).

15) Published a first author paper. The second first author paper has been submitted.

(refer publication list)

113

Phd Thesis (with examiner corrections)...ACKNOWLEDGEMENT First off, I’d like to thank Prof. Dr.-Ing Birgit Glasmacher for giving me the opportunity to do my thesis in the Institute

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