An evaluation of the effects of the stiffness of polycaprolactone on cell proliferation

AN EVALUATION OF THE EFFECTS OF STIFFNESS
OF POLYCAPROLACTONE MEMBRANE ON CELL
PROLIFERATION

TAN PUAY SIANG

NATIONAL UNIVERSITY OF SINGAPORE
2006


AN EVALUATION OF THE EFFECTS OF
STIFFNESS OF POLYCAPROLACTONE
MEMBRANE ON CELL PROLIFERATION

TAN PUAY SIANG
(B.ENG, National University of Singapore)

A THESIS SUBMMITED
FOR THE DEGREE OF MASTER OF ENGINEERING
DEPARTMENT OF MECHANICAL ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2006


PREFACE
This thesis is submitted for the degree of Master of Engineering (Mechanical) in
the Department of Mechanical Engineering at the National University of
Singapore under the supervision of Prof Teoh Swee Hin. No part of this thesis
has been submitted for other degree at other university or institution. To the
author’s best knowledge, all the work presented in this thesis is original unless
reference is made to other works. Parts of this thesis have been published or

presented in the following:

Journal Publications:
P.S. Tan, S.H. Teoh. Effect of stiffness of polycaprolactone (PCL) membrane on
cell proliferation. Materials and Engineering Science C. Vol 27, Issue 2: 304-308,
2007.
Poster Presentations:
P.S. Tan, S.H. Teoh. Effect of Stiffness of Polycaprolactone (PCL) Membrane on
Cell Proliferation. 3rd International Conference on Materials for Advanced
Technologies (ICMAT 2005).
P.S. Tan, S.H. Teoh. An Evaluation of the Effects of Stiffness of
Polycaprolactone (PCL) Membrane on Cell Proliferation. 2nd Materials Research
Society of Singapore Conference on Advance Materials (MRSS-S 2006).

i


ACKNOWLEDGEMENTS
The author would like to thank Professor Teoh Swee Hin, for all his guidance,
invaluable advice, imparting of knowledge and skills for continued learning and
utmost understanding to the student throughout the duration of the project. Prof
Teoh have been a great FYP supervisor to the author in 2003/2004, a caring
Master’s degree supervisor and mentor to the author since 2004-2006. The author is
extremely grateful to Prof Teoh for the many golden opportunities that he has kindly
given to her to jumpstart her since her Bachelor degree graduation in 2004.
She thanks Professor Teoh for his teachings, to train her as a researcher with
“Content, Contacts and Character”. As the author left NUS for work, Professor Teoh
gave her another set of 3 Cs- “Concentration, Commitment and Character”. The
author hopes that the chapter with Professor Teoh will not end just upon the
Master’s degree graduation and wish that she will carry on with many of the 3 Cs in

life that Professor Teoh has taught her, with one C never to change - “Character”.

The author also wishes to express her gratitude towards Dr Chen Fulin, who has
kindly started her training on cell culture. She thanks him for all his teachings and
advice.

She is also extremely thankful to Ms Bina Rai, for her kindness, patience, guidance,
advice and rendering hand when the author was facing much difficulties in the cell
culture work.

ii


The author must also thank Mr Mark Chong Seow Khoon, for his never ending help
and support throughout the project. The author here expresses her most heartfelt
gratitude towards Mark, when he has gladly offered to help the author to carry on
with her cell culture assays when she had to be on MC for 2 weeks after being
knocked down by a cement truck.

The author is also extremely thankful for all the staffs: Dinah, Jackson, Kuan Ming,
Jeremy, Chee Kong, Lin Yun, Kamal, Zhang Jing; post graduate students: Kay
Siang, Fenghao, Alex, Erin, Junping; undergraduate students: Chen Ran, Kelvin,
Galvin, Chin Seng, Kar Kit; and all who have come into her life for the duration of the
whole course of study in NUS. Thank you for the great company and support given
when help is needed.

The author acknowledges her parents, for their unconditioned love for the author,
and also their understanding and support for many of the stressful periods. She also
thanks Siang Yong, for his unfailing help, patience, love, understanding and
motivation to see the author through the whole course of the project.

iii


TABLE OF CONTENTS
PREFACE

Page
i

ACKNOWLEDGEMENTS

ii-iii

TABLE OF CONTENTS

iv-vi

SUMMARY

vii-ix

LIST OF FIGURES

x-xiii

CHAPTER 1 INTRODUCTION
1.1
Background
1.1.1 Biocompatibility of biomaterials

1.1.2 Applications of biomaterials
1.1.3 Uses of PCL in biomedical fields
1.1.4 Cell interactions with foreign surfaces
1.1.5 Role of substrate mechanics on cellular responses
1.2
Research Objectives
1.3
Research Scope

1
1
1
2
2
3
4
6
6

CHAPTER 2 LITERATURE REVIEW
2.1
Relationship of a cell and the stiffness of the matrix on which it
resides
2.2
Cellular response to substrate of different stiffness
2.3
Stiffness of substrate
2.4
Effect of substrate stiffness on cell growth and proliferation
2.5

Effect of substrate stiffness on adhesion and cytoskeleton
2.6
Effects of stiffness of substrate on focal adhesion
2.7
Focal adhesion points in relation to cell proliferation
2.8
Formation of focal adhesion points
2.8.1 Marker of focal adhesions
2.9
Materials used for cell culture studies
2.9.1 Extracellular matrix and other natural hydrogels
2.9.2 Fibroblasts in collagen gels
2.9.3 Synthetic substrates: ligand-coated polyacrylamide gels
2.10 Specificity of cellular response to matrix compliance
2.10.1 Endothelial cells
2.10.2 Myoblast
2.10.3 Hepatocytes
2.10.4 Neurons and glial cells
2.11 Designing of tissue-engineering construct
2.12 Polycaprolactone
2.13 Principles of Two-roll Mills

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17

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iv


2.14 Advantages of Rolling Milling Process
2.14.1 Solvent-free PCL membrane
2.14.2 Breaking up of grain boundaries of individual PCL pellets
2.14.3 Cold drawing of PCL masses
2.15 Melt Pressing and Slow Cooling of PCL Solid Masses
2.16 Biaxial Stretching of PCL Films
2.17 Rational for slow cooling and biaxial stretching of PCL film
2.17.1 Changes of macrostructure of PCL membrane during biaxial
stretching
2.17.2 Changes in the microstructure of PCL

32
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34
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35
36

CHAPTER 3 MATERIALS AND METHODS
3.1
Fabrication of ultra flat PCL Membranes
3.1.1 Heated Roll Milling
3.1.2 Melt Pressing
3.1.3 Biaxial Stretching
3.2
Sodium Hydroxide Treatment
3.2.1 Preparation of test samples
3.3
Self-designed O-rings
3.3.1 Design considerations of O-rings
3.3.2 To mount different thickness of PCL membrane firmly
3.3.3 To apply an equal amount of radial stress in all directions
3.3.4 No obstruction for water contact angle viewing
3.3.5 Versatility of the new O-ring design
3.3.6 Design constraints of the O-rings
3.4
Water Contact Angle Measurements
3.5
Stiffness Characterisation
3.6
In vitro studies

3.6.1 Loading of cells into wells with PCL membrane as the underlying
surface
3.6.2 Focal Adhesion and Actin Cytoskeleton Staining
3.6.3 Fluorescein Diacetate (FDA) / Propidium Iodide (PI) Staining
3.6.4 Cellular Proliferation Assay 1: AlamarBlue Assay
3.6.5 Cellular Proliferation Assay 2: 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenlytetrazolium bromide (MTT) Assay
3.6.5.1 Standard curve
3.6.5.2 Test samples

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57
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60
61

CHAPTER 4 RESULTS AND DISCUSSIONS
4.1 Thickness of PCL film varies with pressure exerted by Melt

Pressing
4.2
Improving Hydrophilicity of PCL Membranes
4.2.1 Sodium Hydroxide Treatment
4.2.2 Water Contact Angle Measurements
4.3
Stiffness Measurements
4.4
In vitro studies of NIH 3T3 Cells

68
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39

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73

v

4.4.1
4.4.2
4.4.3
4.4.4
4.4.5
4.4.6
4.5
4.5.1
4.5.2
4.5.3
4.5.4
4.5.5
4.5.6

Phase Contrast Microscopy
Focal Adhesion and Actin Cytoskeleton Staining
Fluorescein Diacetate (FDA) / Propidium Iodide (PI) Staining
Quantitative study 1: 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenlytetrazolium bromide (MTT) Assay
Quantitative study 2: AlamarBlue Assay
Conclusion for 3T3 studies
In vitro studies of Pig’s Chondrocytes
Phase Contrast Microscopy
Focal Adhesion and Actin Cytoskeleton Staining
Fluorescein Diacetate (FDA) / Propidium Iodide (PI) Staining
Quantitative study 1: 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenlytetrazolium bromide (MTT) Assay
Quantitative study 2: AlamarBlue Assay
Conclusion for chondrocytes studies

73

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85
87
94
99
103
106

CHAPTER 5 CONCLUSIONS
5.1
Final conclusions
5.1.1 Stiffness of PCL membrane controlled by its thickness
5.1.2 Increased wettability of PCL membrane by NaOH treatment
5.1.3 In vitro studies of NIH 3T3 cells
5.1.4 In vitro studies of Chondrocytes
5.1.5 Cell type specific response to PCL membrane
5.1.6 Optimal stiffness

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108
109
109
109
110

110

CHAPTER 6 RECOMMENDATIONS
6.1
More cell types to be used to determine cellular response to PCL
membrane of different stiffness
6.2
Further characterization of the stiffness of the PCL membranes
to determine the optimal stiffness for cell specific growth
6.3
In depth study of the various kind of cellular response due to
varying stiffness of PCL membranes
6.4
Nanoscale Enigneering at the surface
6.5
Studies to be carried out in a 3-D scaffolds
6.6
Nanoscale scaffold fabrication
6.7
PCL Blends
6.8
Plasma Treatment of PCL membranes of various stiffness

112
112

REFERENCES

117

APPENDIX

112
113
113
114
114
115
116

PUBLICATIONS

vi


SUMMARY
Polycaprolactone (PCL) is a common biodegradable polymer that has emerged
as a promising biomaterial in the recent years. It can be easily fabricated into thin
membranes while maintaining its mechanical strength. It was reported that
human keratinocytes could attach and proliferate well on solvent casted and
biaxially stretched PCL membranes [Khor et al., 2002; Ng et al., 2000]. In
addition, Ng et al showed that human dermal fibroblasts could grow well on such
PCL substrates [Ng et al., 2001].

However, the use of solvent casted PCL membranes poses the concern of
possible implications due to residual solvents in the membrane. In this study, the
author has moved on to a solvent-free fabrication method for the PCL
membranes. The fabrication of PCL into ultra thin and flat membranes has been
well documented. The process, which consists of roll milling, followed by heat
pressing and finally biaxially stretching, enables the production of solvent-free

PCL membranes. In vitro studies performed in this work has proven the
biocompatibility of PCL films.

Water contact angle measurements were carried out to determine the effect of
5M sodium hydroxide (NaOH) has on PCL membrane. It has been found that by
pre-treating the PCL membrane with 5M NaOH for a period of 3 hours could
sufficiently lower its water contact angle from 84.9 ± 3.5o to 63.0 ± 4.0o, thus
improving its hydrophilicity.

vii


Careful design considerations were done to ensure that the O-rings used in this
study enabled water contact angle measurement of PCL membrane while
transmitting equal amount of radial stress to it. The design of the O-rings also
made them versatile for other works involving atomic force microscopy and coculturing of two different types of cells on PCL membranes.

In the native environment, cells proliferate on matrices of different stiffness
depending on the cell type [Discher, 2005]. For example, bone cells proliferate in
hard environments while skin cells proliferate in softer environments. It is
predicted that cells will grow better on a substrate that mimics more closely its
physiological milieu. This study investigated two cell types, namely, chondrocytes
and 3T3 cells.

Results from stiffness characterization showed that stiffness of the PCL
membrane is relatively proportional to its thickness. The stiffness of the biaxiallystretched PCL membranes was thus controlled by manipulating its thickness.
The thickness was maintained in the range of 2 ± 0.01 to 30 ± 0.01 µm with
corresponding stiffness in the range of 0.5 ± 0.01 to 0.55 ± 0.09 N/m.

The effects of stiffness of PCL membrane on cell proliferation were evaluated via

cell proliferation and viability studies conducted using Fluorescein Diacetate
(FDA) / Propidium Iodide (PI) Staining, Actin Cytoskeleton and Focal Adhesion
Staining, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenlytetrazolium bromide (MTT) and

viii


AlamarBlue assays. Results indicated that 3T3 cells demonstrated enhanced
proliferation and viability on less stiff membranes while proliferation rate and
viability of chondrocytes increased on stiffer membranes. Cytoskeleton staining
revealed that fibroblasts were more spread out on less stiff membranes while
chondrocytes proliferated faster on stiffer PCL membranes.

In conclusion, stiffness of PCL membranes can affect cell proliferation.

ix


LIST OF FIGURES
Figure
Figure 1.1

Figure 1.2

Figure 2.1

Figure 2.2

Figure 2.3
Figure 2.4

Figure 2.5

Descriptions
Cell interactions with foreign surfaces are mediated
by integrin receptors with absorbed adhesion proteins
that sometimes change their biological activity when
they absorb. The figure is schematic and not to scale
[Ratner, 1996].
Progression of anchorage-dependent mammalian cell
adhesion.
(A) Initial contact of cell with solid substrate.
(B) Formation of bonds between cell surface
receptors and cell adhesion ligands.
(C) Cytoskeletal reorganization with progressive
spreading of the cell on the substrate for increased
attachment strength [Ratner, 1996].
Strain distribution computed in a soft matrix beneath
a cell. The circular cell has a uniform and sustained
contractile prestress from the edge to near the
nucleus [Disher, 2005].
Stress versus strain illustrated for several soft tissues
extended by a force (per cross-sectional area). The
range of slopes for these soft tissues subjected to a
small strain gives the range of Young’s elastic
modulus, E, for each tissue. Measurements are
typically made on time scales of seconds to minutes
and are in SI units of Pascal (Pa). The dashed lines
(- - -) are those for (i) PLA, a common tissueengineering polymer (ii) artery-derived acellularized
matrix; and (iii) matrigel [Disher, 2005].

An interplay of physical and biochemical signals in
the feedback of matrix stiffness on contractility and
cell signaling [Rottner, 1999].
(a): The arrows point to dynamic adhesions on soft
gel and static focal adhesion on stiff gels [Pelham,
1997].
(b): Actin cytoskeleton on stiff and soft matrix
[Discher, 2005].
Basic NIH 3T3 fibroblast morphological response to
different extracellular matrix rigidity. Phase images of
fibroblasts on soft (A) and stiff (B) fibronectin-coated
polyacrylamide gels show that cells on stiff gels are
less rounded and more able to extend processes than
cells on softer gels. Fluorescence of images of
fibroblasts stained with rhodamine-phalloidin against
F-actin shows no articulated stress fibers in cells on

Page
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8

10

14
17

23

x


Figure 2.6

Figure 2.7
Figure 2.8
Figure 2.9
Figure 2.10
Figure 2.11
Figure 2.12
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Figure 3.6

Figure 3.7

Figure 3.8

soft gels (C), whereas on stiff gels (D) the stress
fibers resemble those in a fibroblast on tissue culture
plastic [Geroges, 2005].
Myoblasts on collagen-coated polyacrylamide gels of
various rigidities were stained for myosin (green) and
nuclei (blue). Multi-nucleated myotubes formed on
each stiffness, but at 2 wk only intermediate stiffness

gels supported formation of myosin striation. Bar = 20
µm. Egel, Young’s modulus of gel [Georges, 2005].
Structure of Polycaprolactone.
Two roll mills counter rotate to provide laminar shear
to the melted PCL mass [Powell, 1983].
Fringed-micelle model of crystallites in amorphous
matrix [Powell, 1983].
(a) Unstretched polymer, chains are in coiled state.
(b) Stretched polymer, chains are straightened out,
causing polymer to elongate [Powell, 1983].
Typical stress-strain graph of a semi-crystalline
polymer with corresponding macrostructural changes
under tensile loading [Ashby and Jones, 1986].
Changes in microstructure of polymer under tensile
loading [Daniels, 1989].
Two-Roll Mill Machine.
Fully stretched PCL membrane in biaxial stretching
Machine.
Schematic diagram for the fabrication of PCL
membranes.
Top and bottom part of the O-ring and also an O-ring
with PCL film mounted on it.
Conventional O-ring will block the measurement of
water contact angle.
PCL mounted snugly like a drum onto the O-ring and
arrow shows that enough height of the bottom part is
designed to ensure that the well of the O-ring can
have at least a volume of 500 µl.
Mounting process of PCL membrane onto O-ring.
a) PCL membrane placed over bottom part of O-ring.

b-c) Top part of O-ring to sandwich PCL in between
both parts.
d) Top part is pressed down firmly by broad end of
forceps.
e) PCL membrane mounted.
a) Accessory acts as a support when surface of PCL
membrane is to be characterized by AFM.
b) PCL membrane mounted on AFM support, ready
for surface roughness measurement.

25

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31
33
35
37
40
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46
47
48
49
50

51

54

xi

Figure 3.9

Figure 3.10
Figure 3.11
Figure 3.12
Figure 3.13

Figure 3.14
Figure 3.15

Figure 4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
Figure 4.6
Figure 4.7
Figure 4.8
Figure 4.9
Figure 4.10
Figure 4.11
Figure 4.12

a) Accessory to be used for the co-culturing of two
different types of cells. b) Mounting of PCL
membrane onto the accessory. c-d) Volume of O-ring
and accessory designed to accommodate at least
500 µl of medium.

Dimensions of O-rings for fabrication.
Enough allowance was given so that the O-rings can
be easily removed from the well of a 12-well plate
with a pair of forceps.
Illustration of water contact angle on surface of a solid
substrate.
a) Machine to measure water contact angle. b) Close
up of O-ring with PCL membrane mounted, on the
platform of machine. c) A drop of 50 µl of de-ionised
water dispensed out from machine. d) Water drop on
PCL membrane, ready for water contact angle
measurement.
Instron Microtester for compression studies carried
out to obtain stiffness of PCL membranes.
Cell counter used to obtain an average amount of
cells per ml of medium.
[ />gnifier/counting.aspx#63560]
Thickness of PCL film with pressure applied during
melt pressing.
PCL undergoing hydrolysis of its ester linkages.
Drop in water contact angle with 5M NaOH treatment.
Graph of Load Vs Extension for PCL membranes of
different thickness.
Stiffness of PCL membrane increases with its
thickness.
Phase Contrast Microscopy pictures of 3T3 cells
seeded on membranes of different thickness, taken
on Day 1 and Day 6. Scale bar represents 50 µm.
Staining of NIH 3T3 cells cultured separately on 7 µm
and 22 µm PCL membrane over a 9-day period.

Staining of NIH 3T3 cells cultured separately on 8 µm
and 18 µm PCL membrane over a 9-day period.
MTT assay to obtain a standard curve for NIH 3T3.
MTT assay of NIH 3T3 seeded on 2 µm, 8 µm and 21
µm PCL membrane over a 9-day period.
AlamarBlue assay of NIH 3T3 seeded on 8 µm and
17 µm PCL membrane over a 9-day period.
(a) Chondrocytes at Passage 1, showing a more
rounded phenotype.
(b) Chondrocytes at Passage 3, cells differentiating

54

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58

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62

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80

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xii


Figure 4.13
Figure 4.14a
Figure 4.14b
Figure 4.14c
Figure 4.14d
Figure 4.15a
Figure 4.15b
Figure 4.15c
Figure 4.16
Figure 4.17
Figure 4.18
Figure 6.1

into a fibroblastic phenotype.
Scale bar represents 50 µm.
Chondrocytes seeded on 4 µm, 10 µm and 20 µm
PCL membrane. Scale bar represents 100 µm.
Staining of chondrocytes at Passage 1 cultured
separately on 4 µm, 10 µm, 20 µm and 30 µm PCL
membrane on Day 1.
Staining of chondrocytes at Passage 1 cultured
separately on 4 µm, 10 µm, 20 µm and 30 µm PCL
membrane on Day 3.

Staining of chondrocytes at Passage 1 cultured
separately on 4 µm, 10 µm, 20 µm and 30 µm PCL
membrane on Day 6.
Staining of chondrocytes at Passage 1 cultured
separately on 4 µm, 10 µm, 20 µm and 30 µm PCL
membrane on Day 9.
FDA/PI Staining of chondrocytes at Passage 1
cultured separately on 4 µm, 10 µm, 20 µm and 30
µm PCL membrane on Day 3.
FDA/PI Staining of chondrocytes at Passage 1
cultured separately on 4 µm, 10 µm, 20 µm and 30
µm PCL membrane on Day 6.
FDA/PI Staining of chondrocytes at Passage 1
cultured separately on 4 µm, 10 µm, 20 µm and 30
µm PCL membrane on Day 9.
MTT assay to obtain a standard curve for
chondrocytes.
MTT assay of Chondrocytes at Passage 1 seeded on
2 µm, 15µm and 27 µm PCL membrane over a 9-day
period.
AlamarBlue assay of Chondrocytes at Passage 1
seeded on 2 µm, 15 µm and 26 µm PCL membrane
over a 9-day period.
Scaffold architecture affects cell binding and
spreading. (A-B) Cells binding to scaffolds with
microscale architectures flatten and spread as if
cultured on flat surfaces. (C) Scaffolds with nanoscale
architectures have larger surface areas to absorb
proteins, presenting many more binding sites to cell
membrane receptors. The absorbed proteins may

also change conformation, exposing additional cryptic
binding sites [Stevens, 2005].

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103
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115

xiii


Chapter 1 Introduction

CHAPTER 1: INTRODUCTION
1.1

Background
This chapter aims to provide background information on the wide
biomedical applications of PCL and the cellular responses such as growth,
proliferation and also focal adhesion contact points when cells are seeded
onto a substrate. These points of interest have led the author to research
further to evaluate the effects of the stiffness of the biomaterial,

Polycaprolactone membrane, has on the cells.

1.1.1 Biocompatibility of biomaterials
In the last decades, there have been a wide variety of biomaterials being
developed with different physico-mechanical, chemical and biochemical
properties depending on the biomedical applications. Biocompatibility of a
biomaterial is defined as “the quality of not having toxic or injurious effects
on biological systems” [Williams, 1999].
Biocompatibility of a biomaterial is then directly related to its chemical and
biochemical characteristics. Recently, as more research is done to take
into considerations of the interactivity between the biomaterial and the
host, biocompatibility is also considered as “the ability of a material to
perform with an appropriate host response in a specific application”
[Williams, 1999].
Advances in biomaterials research has led to the rapid emergence of
tissue engineering. This new interdisciplinary field applies principles of

An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation

1


Chapter 1 Introduction
engineering and life sciences towards the development of biological
substitutes with many different applications.

1.1.2 Applications of biomaterials
Prominent

applications

for

biomaterials

include:

orthopedics,

cardiovascular, ophthalmics and drug-delivery systems. Bioresorbable or
non-bioresorbable polymers are used, depending on applications.
Bioresorbable polymers are mainly used for applications that only require
the temporary presence of a polymeric implant such as suture materials,
periodontal membranes, temporary vascular grafts and drug-delivery
systems

[Serrano,

2004].

Among

bioresorbable

polymers

are

homopolymers and copolymers based on poly(lactic acid) (PLA),
poly(glycolic acid) (PGA) and polycaprolactone (PCL).

1.1.3 Uses of PCL in biomedical fields
PCL is regarded as a soft and hard tissue compatible bioresorbable
material [Khor et al., 2002] and has been considered as a potential
substrate for wide applications, such as drug delivery systems [Zhong,
2001; Christine, 2000], tissue engineered skin [Ng et al., 2001], axonal
regeneration [Koshimune, 2003] and scaffolds for supporting fibroblasts
and osteoblasts growth [Hutmacher, 2001; Rai, 2004]. PCL has also been
found to be a suitable substrate candidate for tissue-engineered skin
[Venugopal, 2006; Venugopal, Tissue Engineering, 2005; Dai, 2004].

An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation

2


Chapter 1 Introduction
1.1.4 Cell interactions with foreign surfaces
Cellular interactions with foreign surfaces generally consist of four steps: 1)
protein absorptions; 2) cells anchored to absorbed protein via cell integrins;
3) cells differentiate, multiply, communicate with other cell types and
organize themselves; 4) cells and tissues in implant materials respond to
mechanical forces [Ratner, 1996].
Firstly, when the biomaterials are implanted into the body, proteins are
immediately absorbed (<1 second) onto the surface of the implanted
materials. In seconds to minutes, a monolayer of protein absorbs to most
surface. The protein absorption event occurs well before cells arrive at the
surface. Therefore, cells see primarily a protein layer, rather than actual
surface of biomaterial. Since cells respond specifically to proteins, this
interfacial protein film may be the event that controls subsequent

bioreactions to implants.
Secondly, the cells arrive at an implant surface propelled by diffusive,
convective or active mechanisms after protein absorption as shown in
figure 1.1. The cell is shown as a circular space with a bilayer membrane
in which the adhesion receptor protein molecules (the slingshot-shaped
objects) are partly embedded. The proteins in the extracellular fluid are
represented by circles, squares, and triangles. The receptor proteins
recognize and cause the cell to adhere only to the surface-bound form of
one protein, the one represented by a solid circle. The bulk phase of this
same adhesion protein is represented by a triangle, indicating that the

An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation

3


Chapter 1 Introduction
solution and solid phase forms of this same protein have a different
biological activity. These cells can adhere, release active compounds,
recruit other cells, grow in size, replicate and die. These processes often
occur in response to the proteins on the surface.
Thirdly, cells may differentiate, multiply, communicate with other cell types
and organize themselves in into tissues comprised of one or more cell
types after cells arrive and interact at implant surfaces as shown in figure
1.2. Cells secrete extracellular matrix (ECM) molecules that fill the spaces
between cells and serve as attachment structures for proteins and cells.
Finally, cells and tissues respond to mechanical forces. Two samples
made of the same material, one a triangle shape and the other a disk,
implanted in soft tissue, will show different healing reactions with
considerably more fibrous reaction at the asperities of the triangle than

along the circumference of the circle [Ratner, 1996].

1.1.5 Role of substrate mechanics on cellular responses
Physical forces at the adhesion sites can be an important signaling cue to
cells. Mechanical forces such as fluid shear stress [Davies, 1995] or
substrate stretching [Banes, 1995; Grinnell, 1999; Mochitate, 1991] can
significantly

alter

cell

morphology,

growth,

apoptosis

and

gene

expressions. The evaluation of the effects of substrate elasticity on cell
behaviour

was

well

studied

in

materials

like

polyacrylamide,

polydimethylsiloxane, alginate and agarose [Wong, 2004].

An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation

4


Chapter 1 Introduction

Figure 1.1: Cell interactions with foreign surfaces are mediated by integrin
receptors with absorbed adhesion proteins that sometimes change their
biological activity when they absorb. The figure is schematic and not to scale
[Ratner, 1996].

Figure 1.2: Progression of anchorage-dependent mammalian cell adhesion.
(A) Initial contact of cell with solid substrate. (B) Formation of bonds between cell
surface receptors and cell adhesion ligands. (C) Cytoskeletal reorganization with
progressive spreading of the cell on the substrate for increased attachment
strength [Ratner, 1996].

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Chapter 1 Introduction

1.2

Research Objectives
The aim of this work is to investigate the effect of stiffness of PCL
membrane has on cell growth, proliferation and also focal adhesion
contact points. It was hypothesized that the cells would prefer to
proliferate on a substrate that can emulate its native environment. The
merit of this work was that the PCL used, unlike the conventional PCL, is a
solvent-free biomaterial. This helps to eliminate any implications that could
be caused due to residual solvent when the material is implanted in vivo.

1.3

Research Scope
The research scope involves the fabrication of solvent-free PCL
membranes of varying stiffness. Properties like the stiffness and water
contact angle of the membranes were carried out prior to in vitro work. In
vitro studies involved both qualitative and quantitative analysis of the cells
after inoculation onto PCL membranes of different stiffness. Qualitative
analysis include observation via phase contrast microscopy, Actin
Cytoskeleton and Focal Adhesion Staining and Fluorescein Diacetate
(FDA) / Propidium Iodide (PI) Staining viewed under confocal laser
scanning microscope. Quantitative analyses include a non-destructive
cellular proliferation assay, AlamarBlue and a destructive assay, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenlytetrazolium bromide (MTT) cell viability

test.

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Chapter 2 Literature Review

CHAPTER 2: LITERATURE REVIEW
2.1

Relationship of a cell and the stiffness of the matrix on which
it resides
Normal differentiated tissue cells are not longer viable when suspended in a
fluid and are therefore said to be anchorage dependent [Discher, 2005]. Such
cells have to be attached to a substrate, for survival and further proliferation.
In most soft tissues—skin, muscle, brain, etc.—adherent cells plus
extracellular matrix contribute together to establish a relatively elastic
microenvironment. At the macro scale, elasticity is evident in a solid tissue’s
ability to recover its shape within seconds after mild poking and pinching, or
even after sustained compression, such as sitting. At the cellular scale,
normal tissue cells probe elasticity as they anchor and pull on their
surroundings. Such processes are dependent in part on myosin-based
contractility and transcellular adhesions—centered on integrins, cadherins,
and perhaps other adhesion molecules—to transmit forces to substrates. A
normal tissue cell not only applies forces but also responds through
cytoskeleton organization (and other cellular processes) to the resistance that
the cell senses, regardless of whether the resistance derives from normal
tissue matrix, synthetic substrate, or even an adjacent cell.

2.2

Cellular response to substrate of different stiffness
Adherent cells can transmit forces, which are often referred to as traction
forces to the substrate that they reside on, and thus causing wrinkles or

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Chapter 2 Literature Review
strains when the substrate is a thin film or gel (Figure 2.1) [Harris, 1980;
Oliver, 1999; Marganski, 2002, Balaban, 2001].

Figure 2.1 Strain distribution computed in a soft matrix beneath a cell. The
circular cell has a uniform and sustained contractile prestress from the edge to
near the nucleus [Discher, 2005].

The cell is shown to have response to the resistance of the substrate, by
adjusting its adhesions, cytoskeleton, and overall state.
On ligand-coated gels of varied stiffness, epithelial cells and fibroblasts
[Pelham, 1997] were the first cells reported to detect and respond distinctly to
soft versus stiff substrate. Following this, many other tissue cells like neurons
and muscle cells have also been reported to have response to the stiffness of
the substrate [Deroanne, 2001; Wang, 2000; Engler, Biophys J., 2004].
Unlike the cells growing on soft gel or in tissue, the cells growing on tissue
culture plastic or glass coverslip are essentially being attached on rigid
materials [Discher, 2005].

The question of how the cells perceive and respond to these materials as
compared with the behaviour of the cells in more compliant tissue, substrate
or layer of cells then arises.
The answer to the above question can significantly affect how standard cell
culture should be carried out and more importantly, give invaluable insights to

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Chapter 2 Literature Review
tissue repair strategies and also understanding in morphogenesis and disease
processes [Discher, 2005].

2.3

Stiffness of substrate
Solid substrates used in many research for the adhesion of cells can range in
stiffness from soft to rigid, and also vary in topography and thickness.
Regardless of geometry, the stiffness of a substrate is given by its intrinsic
resistance to a stress applied. This is measured by the substrate’s elastic
modulus E, which is obtained by applying a force, such as a hanging weight to
a section of the substrate and then measuring the relative change in length or
strain. Alternatively, E can also be obtained by controlled poking of the
substrate, with the use of macro- and micro-indenters such as atomic force
microscopes [Mahaffy, 2000].
Many tissues and biomaterials exhibit a relatively linear stress versus strain
relation up to small strains of about 10 to 20%. The slope E of stress versus
strain is relatively constant at the small strains exerted by cells [Lo, 2000],

although stiffening (increased E) at higher strains is the norm [Storm, 2005;
Fung, 1994]. Microscopic views of both natural and synthetic matrices e.g.,
collagen fibrils and polymer-based mimetics [Stevens, 2005] suggest that
there are many subtleties to tissue mechanics, particularly concerning the
length and time scales of greatest relevance to cell sensing. Comparisons of
three diverse tissues that contain a number of different cell types show that
brain tissue is softer than muscle [Engler, J. Cell Biol, 2004, Yoshikawa,
1999], and muscle is softer than skin (Figure 2.2) [Diridollou, 2000].

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Chapter 2 Literature Review

Figure 2.2: Stress versus strain illustrated for several soft tissues extended by
a force (per cross-sectional area). The range of slopes for these soft tissues
subjected to a small strain gives the range of Young’s elastic modulus, E, for
each tissue. Measurements are typically made on time scales of seconds to
minutes and are in SI units of Pascal (Pa). The dashed lines (- - -) are those for
(i) PLA, a common tissue-engineering polymer (ii) artery-derived acellularized
matrix; and (iii) matrigel [Discher, 2005].

2.4

Effect of substrate stiffness on cell growth and proliferation
It was reported that rat kidney epithelial and 3T3 fibroblastic cells displayed
higher migration rates on softer substrates and the cells had a tendency to
migrate towards the more rigid substrate [Lo, 2000]. Collagen-coated

polyacrylamide was used as the substrate and the rigidity of the substrate was
varied

by

incorporating

varying

concentrations

of

acrylamide

and

bisacrylamide. When cultured on a more rigid substrate, both cell types were
well spread and appeared indistinguishable from those cultured on normal
tissue culture plates. The cells were less well spread and irregularly-shaped
when they were cultured on a more flexible substrate [Pelham, 1997].
In 2000, Wang et al discovered that normal cells were much more sensitive to
substrate flexibility than H-ras-transformed cells. Data showed that there was
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