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 (B.ENG, National University of Singapore) A THESIS SUBMMITED FOR THE DEGREE OF MASTER... cell viability test An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation Chapter Literature Review CHAPTER 2: LITERATURE REVIEW 2.1 Relationship of a cell and the stiffness. .. properties of such An Evaluation of the Effects of Stiffness of PCL Membrane on Cell Proliferation 19 Chapter Literature Review substrates has on cell migration, growth and cytoskeletal organization

OF POLYCAPROLACTONE MEMBRANE ON CELL

PROLIFERATION

TAN PUAY SIANG

NATIONAL UNIVERSITY OF SINGAPORE

2006

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

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:

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)

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

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

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

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

2.14.2 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

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)-2,5-diphenlytetrazolium bromide (MTT) Assay

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

4.4.3 Fluorescein Diacetate (FDA) / Propidium Iodide (PI) Staining

4.4.4 Quantitative study 1:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenlytetrazolium bromide (MTT) Assay

4.4.5 Quantitative study 2: AlamarBlue Assay

4.4.6 Conclusion for 3T3 studies

4.5 In vitro studies of Pig’s Chondrocytes

4.5.1 Phase Contrast Microscopy

4.5.2 Focal Adhesion and Actin Cytoskeleton Staining

4.5.3 Fluorescein Diacetate (FDA) / Propidium Iodide (PI) Staining

4.5.4 Quantitative study 1:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenlytetrazolium bromide (MTT) Assay

4.5.5 Quantitative study 2: AlamarBlue Assay

4.5.6 Conclusion for chondrocytes studies

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

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

APPENDIX PUBLICATIONS

117

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

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 co-culturing 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 biaxially-stretched 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

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

Figure Descriptions Page

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]

5

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]

5

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 [Disher, 2005]

8

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 [Disher, 2005]

10

Figure 2.3 An interplay of physical and biochemical signals in

the feedback of matrix stiffness on contractility and cell signaling [Rottner, 1999]

14

Figure 2.4 (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]

17

Figure 2.5 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

23

plastic [Geroges, 2005]

Figure 2.6 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]

25

Figure 2.8 Two roll mills counter rotate to provide laminar shear

to the melted PCL mass [Powell, 1983]

31

Figure 2.9 Fringed-micelle model of crystallites in amorphous

Figure 2.10 (a) Unstretched polymer, chains are in coiled state

(b) Stretched polymer, chains are straightened out, causing polymer to elongate [Powell, 1983]

35

Figure 2.11 Typical stress-strain graph of a semi-crystalline

polymer with corresponding macrostructural changes under tensile loading [Ashby and Jones, 1986]

37

Figure 2.12 Changes in microstructure of polymer under tensile

loading [Daniels, 1989]

40

Figure 3.2 Fully stretched PCL membrane in biaxial stretching

Machine

46

Figure 3.3 Schematic diagram for the fabrication of PCL

Figure 3.4 Top and bottom part of the O-ring and also an O-ring

with PCL film mounted on it

48

Figure 3.5 Conventional O-ring will block the measurement of

Figure 3.6 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

50

Figure 3.7 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

51

Figure 3.8 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

54

membrane onto the accessory c-d) Volume of O-ring and accessory designed to accommodate at least

500 µl of medium

Figure 3.11 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

56

Figure 3.12 Illustration of water contact angle on surface of a solid

substrate

58 Figure 3.13 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

58

Figure 3.14 Instron Microtester for compression studies carried

out to obtain stiffness of PCL membranes

59 Figure 3.15 Cell counter used to obtain an average amount of

cells per ml of medium

different thickness

72 Figure 4.5 Stiffness of PCL membrane increases with its

thickness

73

Figure 4.6 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

74

Figure 4.7 Staining of NIH 3T3 cells cultured separately on 7 µm

Figure 4.8 Staining of NIH 3T3 cells cultured separately on 8 µm

and 18 µm PCL membrane over a 9-day period

Figure 4.11 AlamarBlue assay of NIH 3T3 seeded on 8 µm and

17 µm PCL membrane over a 9-day period

Figure 4.13 Chondrocytes seeded on 4 µm, 10 µm and 20 µm

Figure 4.14a Staining of chondrocytes at Passage 1 cultured

separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 1

90

Figure 4.14b Staining of chondrocytes at Passage 1 cultured

separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 3

91

Figure 4.14c Staining of chondrocytes at Passage 1 cultured

separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 6

92

Figure 4.14d Staining of chondrocytes at Passage 1 cultured

separately on 4 µm, 10 µm, 20 µm and 30 µm PCL membrane on Day 9

93

Figure 4.15a 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

96

Figure 4.15b 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

97

Figure 4.15c 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

98

Figure 4.17 MTT assay of Chondrocytes at Passage 1 seeded on

2 µm, 15µm and 27 µm PCL membrane over a 9-day period

103

Figure 4.18 AlamarBlue assay of Chondrocytes at Passage 1

seeded on 2 µm, 15 µm and 26 µm PCL membrane over a 9-day period

106

Figure 6.1 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]

115

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

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]

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

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]

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]

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

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-(4,5-Dimethylthiazol-2-yl)-2,5-diphenlytetrazolium bromide (MTT) cell viability

test

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

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

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]

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

a growth advantage of transformed cells in vivo, where they were able to

survive and grow independent of mechanical input from the surrounding tissues In their study, they cultured either normal or H-ras-transformed 3T3 cells on substrates of different flexibility and compared their rates of growth and apoptosis It was noted that at a low cell density of 250 cells/cm2, normal cells grew better on a stiffer substrate than on a flexible substrate, whereas no significant differences were reported for the transformed cells Comparing the growth rate of the transformed and normal cells at high cell density and on a flexible substrate, the growth rate of the transformed cells were approximately twice that of the normal cells In addition, both normal and transformed cells grew equally well on stiff substrates [Wang, 2000]

Balgude et al reported that the stiffness of agarose gel can determine the rate

of DRG neurite extension in 3D cultures The stiffness of the agarose gel was increased by increasing the concentration (wt/vol) of the agarose gel during fabrication It was shown that as the stiffness of the agarose gel decreased, the rate of elongation of the neurites increased Thus, the rate of growth of the neurites could be altered by varying the stiffness of the agarose gels This result suggests that neurites preferred to grow on a less stiff substrate [Balgude, 2001]

It was also found that NIH 3T3 cells cultured on polyacrylamide gels with similar chemical properties but different stiffness generated stronger traction forces on the stiffer gels than on the softer ones [Lo, 2000] A significant finding was that the cells could guide their movement by probing the substrate rigidity As the leading edge of the cell sensed the presence of the rigid substrate, lamellipodia and lamella were expanded, resulting in the directed

migration onto the rigid surface When the leading edge approached the soft side, local retractions took place, causing the cell to change its direction of movement

The effects of substrate stiffness are not restricted to soft tissue cells like epithelial cells and fibroblasts Chondrocytes were also reported to exhibit morphological changes when cultured on alginate substrates of different stiffness It has been found that on softer substrates, they assumed a rounded shape with nebulous actin [Genes, 2004] However, on stiffer substrates, they converted to a flat morphology with actin fibers By increasing the stiffness of the substrate, chondrocytes were found to attach more rapidly and to a greater degree However, it is to note that a stiffer substrate had changed the spherical morphology of chondrocytes into a flattened fibroblastic shape with increased spreading Chen and Teoh showed that by using PCL membranes

as the substrate, chondrocytes favored a stiffer environment to proliferate [Chen and Teoh, 2004] The hypothesis was that different cells have dissimilar seeding preferences for a substrate [Tan and Teoh, 2007]

2.5 Effect of substrate stiffness on adhesion and cytoskeleton

Molecular mechanisms involved in cellular responses to matrix stiffness are still open to investigation, but it seems important to consider close relationships (or not) between ‘‘inside→outside→in’’ pathways and

‘‘outside→in’’ pathways (Figure 2.3) Adhesions on stiff materials are multifaceted mechanosensors [Bercoff et al, 2003], and, on the other hand, contractility does appear to regulate the formation and dynamics of adhesion structures [Pelham, 1997] Indeed, myosin II has a well-established role on

rigid substrates in adhesion and cytoskeletal organization Wodnicka, 1996], as well as spreading [Riveline et al., 2001] and cell tension [Alenghat, 2002] On the other hand, applying external forces to cells (outside→in) leads to growth of focal adhesions on rigid materials, with or without myosin contractile forces [Cramer, 1995] Nonetheless, inside→outside activity can trigger outside→in pathways such as the opening

[Chrzanowska-of stress-activated channels [Doyle, 2004], with induction [Chrzanowska-of calcium transients and activation of calmodulin and myosin II Additional work from the outside→in perspective has shown that stretching well-spread cells leads to deactivation of Rac (for < 30 min) without affecting Rho activity [Katsumi et al., 2002] Stretching can also create new cytoskeletal binding sites for activator and adapter proteins [Tamada, 2004] and thus alter the balance between protrusion and contractility The mechanism may involve conformational changes to uncover scaffold binding sites or other activities; for example, one key focal adhesion protein, talin, must unfold for vinculin binding [Fillingham et

al, 2005; Law et al, 2003], and although the unfolding forces are not yet clear, similar helical bundle cytoskeletal proteins unfold at forces that just a few myosin molecules can generate On the other hand, fluid shearing of endothelial cells activates Rho and also increases cell traction forces [Shiu et al., 2004], but how such stimulation, transient or sustained, depends on myosin activity and compares with substrate-mediated pulling forces or substrate compliance effects remains unknown

Figure 2.3: An interplay of physical and biochemical signals in the feedback of matrix stiffness on contractility and cell signaling [Rottner, 1999]

Correlations have long been made between increased cell adhesion and increased cell contractility [Leader, 1983], but it now seems clear that tactile sensing of substrate stiffness feeds back on adhesion and cytoskeleton, as well as on net contractile forces, for many cell types [Discher, 2005]

2.6 Effects of stiffness of substrate on focal adhesion

The cell has adhesion points called focal adhesions that are anchorage points

to the substrate on which they lie By tugging on the matrix at these focal

adhesions, the cell creates a tension within its membrane walls [Beningo, 2002] The tension that the cell is able to generate depends on the inherent material properties of the matrix: a relatively stiff matrix will resist cellular force more than a soft one, causing the cell to be more rigid and extended about its periphery [Schwarz, 2002]

Seminal studies on epithelial cells and fibroblasts exploited inert polyacrylamide gels with a thin coating of covalently attached collagen [Pelham, 1997] This adhesive ligand allows the cells to attach and, by controlling the extent of polymer cross-linking in the gels, the elastic modulus (E) of the polyacrylamide gels can be adjusted over several orders of magnitude, from extremely soft to stiff Images of adhesion proteins such as vinculin are revealing (Figure 2.4a) Soft, lightly cross-linked gels (E ~ 1 kPa) show diffuse and dynamic adhesion complexes In contrast, stiff, highly crosslinked gels (E ~ 30 to 100 kPa) show cells with stable focal adhesions, typical of those seen in cells attached to glass Similarly, rigidification of cell-derived three-dimensional (3D) matrices alters 3D-matrix adhesions, because the adhesions are replaced by large, nonfibrillar focal adhesions similar to those found on fixed 2D substrates of fibronectin [Cukierman, 2001] Consistent with a role for signaling in stiffness sensing, tyrosine phosphorylation on multiple proteins (including paxillin) appears broadly enhanced in cells on stiffer gel substrates [Pelham, 1997], whereas pharmacologically induced, nonspecific hyperphosphorylation drives focal adhesion formation on soft materials Inhibition of actomyosin contractions, in contrast, largely eliminates prominent focal adhesions, whereas stimulation of contractility drives integrin aggregation into adhesions [Chrzanowska-

Wodnicka, 1996] Additionally, although microtubules have been proposed to act as ‘‘struts’’ in cells and thus limit wrinkling of thin films by cells [Pletjushkina et al., 2001], quantification of their contributions to cells on gels shows that they provide only a minor fraction of the resistance (14%) to contractile tensions; most of a cell’s tension is thus resisted by matrix [Wang et al., 2001] Furthermore, if matrix strain is approximately constant, then cells on soft gels need be less contractile than on stiff gels, and if they are less contractile, then their adhesions need not be as strong This is consistent with

a reduced adhesion strength as measured by reduced forces to peel cells from soft gels versus glass [Engler et al., J Cell Biol, 2004] This is also consistent with more dynamic adhesions on soft substrates (Figure 2.4a) Fluorescence imaging also shows increasingly organized F-actin and stress fibers on increasingly stiff substrates in fibroblasts (Figure 2.4b) Neurons, in contrast, appear to apply very little stress to their substrate, because they can only deform very soft gels [Bridgman, 2001] Neurons also branch more on softer substrates [Flanagan, 2002], perhaps because the cytoskeleton is more pliable, if less structured

a

b

Figure 2.4(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]

2.7 Focal adhesion points in relation to cell proliferation

Cell adhesion has a critical role to play in many fundamental processes such

as embryonic morphogenesis, angiogenesis, inflammation and wound healing It is clear that without cell adhesion, these fundamental processes would not have take place Before a cell can proliferate, it must be adhered to

a surface [Yang, 1995; Sheetz, 1998; Rossiter, 1997] Focal adhesions are the primary sites of cell adhesion These complex multi-molecular assemblies

link the extracellular matrix, via membrane bound receptors, to the cell’s cytoskeleton [Yamada, 1997; Geiger, 1995]

Focal adhesions can be detected as dark areas in interference reflection microscopy, by electron microscopy or with fluorescence labeling of specific adhesion molecules such as vinculin, paxillin and integrins

2.8 Formation of focal adhesion points

Initial adhesions are formed between integrin receptors and the ECM at the leading edge of migratory cells [Galbraith, 2002] These initial adhesions contain actin and talin, and they mature into small focal complexes (<1 µm2 in area) away from the leading edge at the border between the lamellipodia and the lamella [Izzard, 1988] within 60–90s The development of the initial adhesion into a focal complex is marked by the recruitment of vinculin [DePasquale and Izzard, 1987; Izzard, 1988], a regulator of cell migration and adhesion [Xu et al., 1998] As vinculin is recruited and the focal complex is formed, there is a decrease in the distance between the adhesion and the ECM-coated surface to form a “tight” or “focal” junction as measured by interference reflection microscopy [DePasquale and Izzard, 1987; Izzard, 1988] The newly formed focal complexes are Rac-dependent structures [Nobes and Hall, 1995] that are tightly adhered to the ECM [DePasquale and Izzard, 1987; Izzard, 1988] and contain all of the components of initial adhesions as well as vinculin, paxillin, and phosphoproteins [Nobes and Hall, 1995] Focal complexes can continue to develop into relatively large, stable focal adhesions, extending further from the cell periphery through a Rho-dependent mechanism [Riveline et al., 2001] Focal adhesions contain the

subset of proteins that mark focal complexes as well as many others [Geiger and Bershadsky, Curr Opin Cell Biol., 2001; Geiger et al., Nat Rev Mol Cell Biol., 2001] In addition to containing more molecular components than focal complexes, focal adhesions require much more time (~60 min) to become fully established [Zamir et al., 2000]

2.8.1 Marker of focal adhesions

Vinculin is a marker for focal complexes and focal adhesion Unlike the marker for focal complex talin, vinculin is absent from initially adhesions [DePasquale and Izzard, 1987, Izzard 1988] Vinculin also distinguishes itself from paxillin by having a defined temporal relationship between its accumulation and focal complex formations [DePasquale and Izzard, 1987, Izzard 1988]

Localization of vinculin by immunofluorescence microscopy is often used to demonstrate the presence of focal adhesion [Geiger, 1983; Geiger, 1979] Moreover, it has been argued that the area of vinculin staining is an indication

of the strength of cell adhesion [Baharloo, 2005; Tan, 2003; Richards, 2001]

Hence, by observing vinculin, one can see how a cell is attached to the surface that it resides on and also how the cells can make contacts with adjacent cells, which may aid in cell proliferation

2.9 Materials used for cell culture studies

Various materials have been used for cell culture studies as natural or synthetic substrates The effects of the mechanical properties of such

substrates has on cell migration, growth and cytoskeletal organization has

been extensively studied

2.9.1 Extracellular matrix and other natural hydrogels

Protein-based extracellular matrix gels, such as fibrin, collagen, or a mixture

of collagen, laminin, and other proteins forming Matrigel, are commonly used

to create two- or three-dimensional cell culture substrates of controlled stiffness Cross-linked polysaccharides such as alginate and agarose gels can also be manipulated to have varying elastic moduli by altering the polymer mass and are permissive for cell culture [Georges, 2005]

2.9.2 Fibroblasts in collagen gels

It is well documented that fibroblasts embedded in collagen gels show distinct morphologies from those cultured on tissue-culture plastic [Grinnell, 2000; Hay, 1982] Cells on free-floating collagen gels lack the pronounced F-actin stress fibers that are seen on tissue-culture plastic Fibroblasts on constrained collagen gels are able to generate stress fibers [Halliday, 1995] The mechanism by which stress fibers are reduced in fibroblasts on gels may involve focal adhesions Focal adhesion complex proteins are downregulated

on both collagen gels and Matrigel, although not on plastic dishes coated with

a solution of either of the two [Wang, 2003] On unconstrained gels, fibroblasts are also less susceptible to transforming growth factor-β-stimulated smooth muscle α-actin production, a hallmark of contractile behavior This finding provides some evidence that matrix stiffness appears to be a key

component of contractile behavior and associated protein expression [Arora, 1999]

2.9.3 Synthetic substrates: ligand-coated polyacrylamide gels

Polyacrylamide (PA) gels have emerged as important tools for testing the compliance dependence of cytoskeletal-regulated activities of cells Of significance for this work carried out using PA gels is the ability to separate the chemical signals received by cells from the mechanical signals Protein and polysaccharide gels can interact directly with the cell surface or bind serum proteins that then act as cellular ligands in a manner that is difficult to control or quantify In addition, manipulations that alter gel stiffness also alter parameters such as fiber thickness or density that could impact cellular response independent of stiffness changes PA gels have two important features that allow separation of chemical from mechanical signaling The gel itself is nearly completely inert as an adhesive surface The same chemical stability and nonadherence to other macromolecules that enable its use for electrophoretic separation of proteins and nucleic acids also ensure that neither cell surface receptors nor adhesive proteins present in serum can bind directly to the gel Therefore, only those adhesive molecules covalently grafted to the gel surface can act as ligands for the cell The second convenient feature of PA gels is that their stiffness, quantified by an elastic modulus, can be varied over a wide range by changing the small fraction of dimeric bisacrylamide that cross-links PA chains, while keeping constant the polymer concentration and so avoiding changes in the surface texture or distribution of surface ligand sites A disadvantage of PA is that its chemical

inertness makes covalent attachment of fragile proteins sometimes difficult, but a variety of chemistries has been developed to allow more facile conjugation of adhesion molecules to the gel surface [Reinhart-King, 2003; Wang and Pelham, 1998]

2.10 Specificity of cellular response to matrix compliance

Most literature has cited convincing evidence to show that focal adhesion complexes and their related proteins are involved in the traction force generation and cytoskeletal organization, especially F-actin structure, on soft materials Fibroblasts spread more on stiffer substrates whether the substrate

is a biological gel or a protein-laminated PA gel On softer materials, they adopt a more spherical morphology, and their F-actin structure is markedly more diffuse (Figure 2.5) Other cell types, however, respond differently to changes in substrate stiffness [Georges, 2005]

Figure 2.5: 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 soft gels (C), whereas on stiff gels (D) the stress fibers resemble those in a fibroblast on

tissue culture plastic [Geroges, 2005]

2.10.1 Endothelial cells

Endothelial cells plated on collagen gels [Vernon, 1992] or fibrin gels [Vailhe, 1997] of differing flexibility show a decrease in network-like structures on stiffer gels Softer substrates allow cells to form long capillary-like tube structures On stiffer gels, endothelial cells from human umbilical vein are more spread, have larger lumens, and exhibit less branching compared with the same cells on soft gels [Sieminski, 2004]

2.10.2 Myoblast

Myoblasts plated on PA gels initially spread more on stiffer substrates, although after a day in culture they are able to spread on both soft and stiff substrates [Engler, J Cell Biol, 2004] Myoblasts fuse to form myotubes on a range from soft to stiff PA gels patterned with strips of collagen Only on intermediate stiffness, however, do these myotubes exhibit evidence of striation, as indicated by immunocytochemistry against myosin Although myotubes on soft and stiff gels are multi-nucleated up through 4 wk in culture, they showed poor striations not evident of physiological myotubes (Figure 2.6) Remarkably, the modulus of healthy muscle tissue falls within this intermediate range of stiffness, whereas the modulus of diseased fibrotic tissue associated with muscular dystrophy is in the stiffer range on which striations form poorly This study suggests a link between changes in myoblast behaviour and differences in stiffness of healthy and diseased muscle tissue

Figure 2.6: 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]

2.10.3 Hepatocytes

Hepatocytes, the main functional cells in the liver, also exhibit some mechanical-dependent behaviour Hepatocytes plated on Matrigel minimally spread, quickly form spheroidal aggregates, and reorganise the matrix [Coger, 1997] The compliance of the Matrigel can be lowered by cross-linking the gel with glutaraldehyde On stiffer matrices, hepatocytes become polygonal and

do not aggregate as effectively It is likely that hepatocytes on soft substrates can sufficiently apply forces on the matrix that are felt by neighbouring cells, and increasing the substrate stiffness blocks this mechanical communication