System for the Application of Hydrostatic Pressure and ...

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12-2018

System for the Application of Hydrostatic Pressureand Mechanical Strain to Cell CulturesJustin BacaoatClemson University, [email protected]

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SYSTEM FOR THE APPLICATION OF HYDROSTATIC PRESSURE AND

MECHANICAL STRAIN TO CELL CULTURES

A Thesis

Presented to

the Graduate School of

Clemson University

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Bioengineering

by

Justin Bacaoat

December 2018

Accepted by:

Jiro Nagatomi, PhD, Committee Chair

William Richardson, PhD

Ken Webb, PhD

ii

ABSTRACT

Pressure and stretch are two of the primary forces that result in

mechanotransductory events which regulate certain aspects of human health and disease.

Laboratory systems such as the commercially available Flexercell® system and a variety

of custom-made setups are currently used in research to systematically apply stretch and

hydrostatic pressure independently, or in conjunction to cell and tissue cultures.

However, these systems do not allow for the decoupling of pressure and stretch under the

same culture conditions. The present study aims to design, fabricate, and calibrate a

device that can apply pressure and stretch simultaneously, as well as independently to

cells in culture. Moreover, in order to characterize the mechanical behavior of the cell

substrate, equibiaxial mechanical testing was conducted on the substrate and the resulting

data were used to generate a finite element simulation of the device. Moreover, an

analytical approach was used in an attempt to validate the simulation. To our knowledge,

this is the first system that can definitively distinguish between the mechanotransductive

events activated in response to pressure and stretch. Using this novel device, MYP3 cells

cultured on fibronectin-coated substrates were exposed to pressure and stretch, together

or independently. The cells exhibited the morphology characteristic of healthy urothelial

cells. The extracellular ATP data indicated an increase in ATP release in response to

mechanical stimuli. Caspase-1 activity decreased in response to mechanical stimuli. The

present study was successful in creating a unique device capable of applying pressure and

stretch, together and independently, to cells in culture to allow examination of the relative

contributions of these stimuli in various mechanobiological events.

iii

ACKNOWLEDGMENTS

Firstly, I would like to thank my advisor Dr. Nagatomi for all of his patience and

support. His guidance and persistence have helped me grow as a bioengineer and as a

person. I would also like to thank the members of the Cell Mechanics and

Mechanobiology Laboratory, especially Cody Dunton for always being available for

assistance and for all of the contributions he made to this project. I would like to thank

my committee members for dedicating their time and effort to this project. I want to

thank Dr. Richardson for allowing the use of his lab’s BioTester. I also would like to

thank Dr. Vladimir Reukov for allowing the use of his lab’s 3D printer. Special thanks

go to Chad McMahan who has provided assistance many times throughout this project

and also Maria Torres who has always been helpful in keeping me on track to graduate.

Thanks to the IBIOE department for allowing the use of their laser cutter, and a huge

thanks to the Clemson MTS department. Lastly, I would like to thank my family and

close friends whose support has carried me through my entire graduate experience.

iv

TABLE OF CONTENTS

Page

TITLE PAGE .................................................................................................................... i

ABSTRACT ..................................................................................................................... ii

ACKOWLEDGMENTS ................................................................................................. iii

LIST OF TABLES .......................................................................................................... vi

LIST OF FIGURES ....................................................................................................... vii

CHAPTER

1. INTRODUCTION AND BACKGROUND ........................................................ 1

1.1 Introduction ........................................................................................ 2

1.2 Mechanobiology ................................................................................ 2

1.3 Bioreactors ......................................................................................... 7

2. RATIONALE AND SPECIFIC AIMS .............................................................. 11

3. MATERIALS AND METHODS ....................................................................... 14

3.1 Description of Pressure-Stretch Bioreactor ..................................... 14

3.2 Calibration of Pressure-Stretch Bioreactor ...................................... 15

3.3 Mechanical Characterization of Culture Substrate .......................... 18

3.4 Exposure of Urothelial Cells to Pressure-Stretch ............................ 23

4. RESULTS .......................................................................................................... 26

4.1 Description of Pressure-Stretch Bioreactor ..................................... 26




5. DISCUSSION .................................................................................................... 41




v

Table of Contents (Continued)

Page

6. CONCLUSIONS AND RECOMMENDATIONS ............................................ 49

APPENDIX .................................................................................................................... 51

A: COMSOL Outputs ............................................................................................. 51

REFERENCES .............................................................................................................. 53

vi

LIST OF TABLES

Table Page

4.1 Pressure-loss results from calibration test.................................................... 37

A.1 2-parameter Mooney Rivlin model fitting data as seen in Figure 4.7 ......... 51

A.2 5-parameter Mooney Rivlin model fitting data as seen in Figure 4.8 ......... 51

A.3 Displacement field data for the COMSOL simulation of 122.3 cmH2O as seen

in Figures 4.11 and 4.12. ........................................................................ 52

A.4 Displacement field data for the COMSOL simulation of 180.5 cmH2O as seen

in Figures 4.11 and 4.12 ......................................................................... 52

A.5 Displacement field data for the COMSOL simulation of 225 cmH2O as seen in

Figures 4.11 and 4.12 ............................................................................. 52

vii

LIST OF FIGURES

Figure Page

3.1 Schematic diagrams of the pressure device assembly ................................. 17

3.2 Custom stamp used for imprinting coordinate system

onto silicone substrate samples .............................................................. 21

3.3 Side-view image of a wax replica of the deformed substrate ...................... 21

3.4 Diagram of strain quantification values used in image

analysis of wax replicas ........................................................................ 22

4.1 Assembly of the upper and lower chambers for the

Pressure-Stretch Bioreactor ................................................................... 26

4.2 Silicone cell culture substrate assembly ...................................................... 27

4.3 Stress-strain curve data from first round of equi-biaxial

load testing ............................................................................................. 28

4.4 Stress-strain curve data for the first and fifth cycles of the

second round of equi-biaxial load testing .............................................. 29

4.5 Stress-strain curve data for the fourth cycle of the third

round of equi-biaxial load testing. Data are mean +/-

the standard error of mean (n=3). .......................................................... 29

4.6 Average stress for each axis at the maximum strain for round 3,

cycle 4. Data are mean +/- the standard error of mean ........................ 30

4.7 2-parameter Mooney-Rivlin model fitting data using

the stress-strain data obtained from round 2, cycle 5,

of planar biaxial testing. ......................................................................... 31

4.8 5-parameter Mooney-Rivlin model fitting data using the

stress-strain data obtained from round 2, cycle 5, of planar

biaxial testing ........................................................................................ 31

viii

List of Figures (Continued)

Figure Page

4.9 5-parameter Mooney-Rivlin model fitting data from COMSOL

using the stress-strain data obtained from round 3, cycle 4,

of planar biaxial testing. The blue line represents the

experimental data, and the green line is the fitted values. ..................... 32

4.10 COMSOL simulation utilizing the 5-parameter Mooney-Rivlin

curve fitting data from the third round of biaxial testing.

This simulation applied 255 cm H2O to the top surface

of the substrate. ...................................................................................... 33

4.11 Radial strain data obtained using the finite element analysis

simulation .............................................................................................. 34

4.12 Circumferential strain data obtained using the finite element

analysis simulation ................................................................................ 34

4.13 Radial strain data obtained from the wax replica experiment.

Data are mean +/- the standard error of mean (n=3) ............................ 36

4.14 Graph of the maximum and minimum recorded radial strains,

as well as the average amount of radial strain for each

pressure group ...................................................................................... 36

4.15 Circumferential strain data obtained from the wax replica

experiment. Data are mean +/- the standard error of mean (n=3) ........ 37

4.16 Microscope images (1000x) of the cells seeded on the substrate

following the pressure-stretch experiment ............................................ 39

4.17 Extracellular ATP release by MYP3 cells in response to

pressure (40 cm H2O)-stretch (15%). The data are mean +/-

SEM statistically analyzed using one-way ANOVA followed

by a post hoc Tukey-test. *P<0.05 (N=6) ............................................ 40

4.18 Intracellular caspase-1 activity by MYP3 cells in response to

pressure (40 cm H2O)-stretch (15%). The data are mean SEM

statistically analyzed using one-way ANOVA followed by a

post hoc Tukey-test. *P<0.01 (N=5) ..................................................... 40

1

CHAPTER ONE

INTRODUCTION AND BACKGROUND

1.1 Introduction

Various aspects of human health and disease are regulated via mechanical

stimulation of cells and tissues. Of these mechanical stimulants, pressure and stretch are

two of the primary forces that result in mechanotransductory events. The wide acceptance

that mechanical signals arising from mechanical stresses affect the cell’s regulation of

form, function, and development has led to implications that these same signals lead to

the development of various diseases1. This has become an effective motive for

researchers to study the effects of mechanical forces on living cells in a controlled

environment. Due to the complications associated with the in vivo environment, in vitro

systems are most commonly utilized to conduct efficient studies on cellular response to

mechanical stimulation. These systems aimed to apply mechanical inputs such as

hydrostatic pressure or substrate strain to cells in culture in a controlled manner. Current

devices used for mechanical stimulation vary in their complexity and in their control over

mechanical stimuli inputs2. Laboratory systems such as the commercially available

Flexcell® system3 and a variety of custom-made setups are currently used in research to

systematically apply stretch3 and hydrostatic pressure4 independently, or in conjunction5

to cell and tissue cultures. However, these systems do not allow for the decoupling of

pressure and stretch under the same culture conditions. Although the in vivo environment

includes both pressure and stretch, it is important to study and compare the effects of

2

pressure and stretch separately in vitro for greater understanding of each mechanical

stimulant.

1.2 Mechanobiology

Humans experience various mechanical forces from a wide array of sources.

Examples of these forces range from gravity acting as a continuous force on the body to

the pressure and stretching forces on the bladder wall as it fills and voids. It is a known

scientific fact that changes in mechanical forces cause tissues to respond by growth and

remodeling6. This is seen in bone, which changes shape, density, and stiffness in

response to altering mechanical loading conditions7. Blood vessels undergo remodeling

in response to changes in blood pressure and shear stress as well8. This remodeling that

occurs in response to mechanical forces is facilitated by the cells in tissues. Because cells

are highly dynamic and have a complex structure that changes in response to mechanical

forces, there are difficulties in understanding how these cells sense mechanical forces and

convert mechanical signals into biological responses6. Mechanobiology is an

interdisciplinary study that focuses on how physical forces and changes in mechanical

properties can contribute to development, differentiation, physiology, and pathology of

cells and tissues9. One of the major challenges that exist the field of mechanobiology is

the understanding of mechanotransduction, the processes by which cells sense and

response to mechanical signals. Greater knowledge of the mechanisms of

mechanotransduction will lead to a better comprehension of the physiological responses

of different tissues, thus aiding in the development of new therapeutic approaches to treat

3

diseases caused by mechanical loads such as tendinopathy, osteoporosis, and

atherosclerosis10.

1.2.1 MECHANOSENSITIVE CELLS

Cells are the smallest structural and functional units of any organism. Cells

typically consist of a nucleus and cytoplasm enclosed in a plasma membrane. The

plasma membrane is composed of a phospholipid bilayer, which contains hydrophilic

heads and hydrophobic tails. Embedded into this plasma membrane are integral

membrane proteins11. The structure of the plasma membrane is critical in allowing cells

to perform very important tasks such as antigen recognition, maintenance of cell

attachments, and providing passage of substances in and out of the cell6. The cytoplasm

contains the nucleus and other various organelles, which carry out the cell’s basic

functions.

There are many different types of cells in the human body varying in size, shape,

and function. Within the human body, there are various types of cells that are contained

in mechanical loading environments. An example of this cell type is the fibroblast. In

the skin, fibroblasts are subjected to forces such as tension, compression, and shear6.

Fibroblasts are also prominent in tendons and ligaments, playing a vital role in the

organization and maintenance of connective tissue during development and the repairing

of wounds during wound healing12. Another cell found in a load-bearing environment is

the chondrocyte, which is found in articular cartilage. Chondrocytes proliferate and

differentiate to produce the extracellular matrix that composes articular cartilage tissue13.

In bone, osteoblasts are subjected to several mechanical forces such as tension,

4

compression, and torsion. These osteoblasts are bone cells that are responsible for

formation of bone and play an important role in the production and maintenance of

skeletal architecture6. Blood vessels contain two different types of cells that both

experience mechanical loading: endothelial cells and smooth muscle cells (SMCs).

Endothelial cells form the inner lining of blood vessels and are exposed to cyclical strain

from vessel wall distention and shear stress from the frictional forces of blood flow.

These endothelial cells function to provide maintenance on the vessel wall and

circulatory function14. They also undergo alterations in morphology and functionality in

response to cytokine signals, possibly contributing to the pathogenesis of vascular

diseases15. SMCs are located within blood vessel wall and experience compression,

shear, and cyclic stretch from pulsatile blood pressure. SMCs function to maintain vessel

structure and to produce arterial ECM proteins, such as collagen and proteoglycans6.

SMC proliferation has been associated with the pathogenesis of vascular diseases such as

atherosclerosis and hypertension16.

1.2.2 EXTRACELLULAR MATRIX

The groups of cells present in the tissues of living organisms are held together by

the extracellular matrix (ECM)6. The ECM plays an important role in understanding the

process of mechanotransduction. The components of ECM include a mixture of

collagens, glycoproteins, proteoglycans, and elastin, which provide structural support,

mechanical strength, and attachment sites for cell surface receptors. These ECM

components also house signaling molecules that maintain regulation of cell migration,

growth, differentiation, and other cellular functions17. As an example, ECM in the artery

5

wall provides mechanical support and viscoelasticity while also regulating the functional

behavior of mechanical cells6. This cellular function regulation is accomplished through

the interaction between cell surface receptors and matrix proteins. Collagen is the major

component found in the ECM and is organized into fibrils for resisting tension, shear, and

compression in different types of tissues such as tendons, arteries, and bone18. In

addition to collagen, the ECM contains multiple types of proteoglycans, which function

to aid in cell movement and differentiation, provide compressive stiffness to tissues, and

maintain spatial organization of the ECM by forming complexes with other structural

molecules19. Also present in the ECM are adhesive glycoproteins such as fibronectin and

laminin, which bind to collagen, proteoglycans, and the cell surface. Specifically,

fibronectin stimulates cell attachment to underlying matrix while laminin works by

binding to collagen and mediates interactions between the ECM and cells20,21.

1.2.3 MECHANOTRANSDUCTION

In order to better understand the mechanisms of mechanotransduction, the course

in which cells experience mechanical forces must be identified. All organisms are

mechanosensitive, meaning they can sense and respond to mechanical stimuli22. Cells

respond to mechanical stimuli through the processes of mechanotransduction, converting

physical forces into biochemical signals and then into cellular responses23.

Mechanotransduction is fundamental to the regulation of development and physiological

processes, providing a means for cells to ensure structural stability and regulate

morphogenetic movements to generate specific structures. Mechanical loading that is

applied to organisms or individual tissues is distributed among the individual cells by

6

their adhesions to ECM support scaffolds, which connect cells and tissues in the body24.

These support scaffolds focus the mechanical loading forces on cell-to-ECM adhesion

sites. Specific cell surface receptors allow for cells to adhere to the ECM. Integrin,

being the most ubiquitous and characterized cell surface receptor, provides a favorable

site for mechanical signal transfer across the cell surface. These integrins function as cell

surface mechanoreceptors, meaning they sense mechanical stresses being applied to the

cell surface and transfer signals to the cytoskeleton across the plasma membrane24. The

cytoskeleton is a molecular lattice within the cell composed of molecular filaments, such

as microfilaments, microtubules, and intermediate filaments, that maintains the cell’s

shape and stability25. Tensional forces are produced by cells through actomyosin

filament sliding in their cytoskeleton, and these forces are resisted and balanced by

external adhesions to the ECM, neighboring cells, and other molecular filaments. It has

been shown in many studies that alterations in cytoskeleton structure due to the

application of mechanical stress to integrins can lead to stress-dependent signal

transduction and gene expression26-28.

Mechanotransduction pathways can be modulated by other means, which include

G proteins, mitogen-activated protein kinases (MAPKs), and stretch-activated channels.

G proteins are a family of membrane proteins located at sites of focal adhesions that,

when undergoing conformational changes due to mechanical forces, initiate signaling

cascade that lead to cell growth. Studies have shown that the activation of G proteins is

dependent on the magnitude and rate of applied strain, thus proving that G proteins play a

role in the cell signaling of mechanical strain29. MAPKs are also important in

7

mechanotransduction in that the MAPK pathway provides a means for mechanical

signaling to activate gene expression and protein synthesis. For example,

phosphorylation of extracellular signal-regulated kinases (ERKs) 1 and 2, which are types

of MAPKs, can be induced by shear stress in bovine aortic endothelial cells30,31. ERKs

are also activated by mechanical stretching in human pulmonary epithelial cells and fetal

lung fibroblasts32,33. Another mechanism of mechanotransduction is the activation of

mechano-sensitive ion channels called stretch-activated channels34. These channels

provide for the movement of Na+, K+, and Ca2+ ions in and out of cells35,36. Most

importantly, the levels of intracellular Ca2+ are elevated in response to mechanical

stimulation in many cell types, and this can lead to changes in cellular processes,

including cell growth, motility, contraction, apoptosis, and differentiation37-41.

1.3 Bioreactors

In an effort to obtain a clearer understanding of the mechanobiology of different

cells and tissues, researchers in tissue engineering utilize bioreactors to simulate

physiological and/or pathological mechanical environments on cell and tissue cultures.

A general definition for a bioreactor is a device that allows for researchers to monitor and

control environmental and operating conditions while biological and/or biochemical

processes are developing42. In order to be considered a bioreactor, the device must at

least one of five different functions43:

Distribute cells uniformly on a three-dimensional (3D) scaffold

Maintain culture medium at a controlled concentration of gases and nutrients

8

Allow for transfer of mass to growing tissue

Apply physical stimuli to developing tissue

Analyze isolated cells and their formation into 3D tissues.

The ability to control the mechanical environment of cell cultures is essential in

mechanobiology research in that mechanical stimuli such as pressure and stretch can be

used to stimulate and activate signal transduction pathways associated with

mechanotransduction.

1.3.1 CURRENT BIOREACTORS

There are several bioreactor systems that have been developed to conduct analysis

on cells in controlled mechanical environments. Of these systems, multiple are designed

to simulate pressure and/or stretch to cells in culture. These systems include the

Flexcell® FX-5000TM Tension System, the Hydrostrain System, and the MechanoCulture

TR. Each of these systems is unique and has its own advantages and drawbacks. This

section will highlight the main features of each system and overview their pros and cons.

The Flexcell® FX-5000TM Tension System is a commercially available laboratory

system that can systematically apply stretch independently to cells in culture. This

system was introduced by Banes et al. (1985) as the first flexible-bottomed circular cell

culture plate exclusively designed for cell mechanostimulation3. This computer-regulated

bioreactor applies cyclic or static strain to cells on flexible-bottomed cultures plates

through the use of vacuum pressure44. With this technology, the biochemical changes of

cells from different parts of the body in response to the stretch can be analyzed. This

system consists of an interchangeable special cell culturing well, which has a flexible

9

rubber membrane on which the cells are cultured. This well is positioned on top of a

loading post contained in the system, where vacuum pressure is used to deform the

rubber membrane to yield strains ranging up to 33% elongation44. The FX-5000TM

utilizes special software to control the vacuum pump, allowing for the application of

defined and controlled, static or cyclic deformation to cells growing in vitro44. This is

one of its biggest strengths in that the system can be controlled externally on a computer,

minimizing physical work needed and variation between different uses. The main

drawback of this device is that it cannot apply pressure to the cultured cells, and pressure

is a major condition required to simulate a physiological or pathological environment for

cells.

The Hydrostrain System was a novel laboratory setup developed by Haberstroh et

al. (2002) to allow for simultaneous exposure of mechanical strain and hydrostatic

pressure to bladder smooth muscle cells5. This setup included a polycarbonate stretching

system with a flexible silastic membrane. A TeflonTM syringe filter was used to maintain

filtered gas exchange. A sterile syringe was used to apply and maintain pressure within

the system through a pressure port located on the setup. Using the Hydrostrain System,

Haberstroh et al. were successful in exposing the flexible silastic membrane to

mechanical strain of up to 25 percent and to hydrostatic pressure of 40 cm H2O under

standard cell culturing conditions5. The major quality of this laboratory setup is its

ability to apply both mechanical strain and hydrostatic pressure simultaneously. The

flexibility of the silastic membrane for cell culturing in the device allows for the

hydrostatic pressure in the system to deform the membrane and apply mechanical strain

10

to the cultured cells. Although, this aspect of the setup itself is one of its main drawbacks

in that the device is unable to control the amount of mechanical strain being applied to

the system. Therefore, the system is limited in its ability to simulate physiological or

pathological states of mechanical strain. Furthermore, the Hydrostrain System does not

allow for the decoupling of mechanical strain and hydrostatic pressure.

The MechanoCulture TR (MCTR) is a commercially available laboratory system

developed by CellScale. This specific system provides high throughput compression

stimulation by applying uniaxial compression to nine cylindrical tissue constructs using

pressure-driven shuttles45. The MCTR contains transparent culture wells which allow for

visual confirmation of proper specimen loading and real-time imaging. One of the key

features advertised for this system is its user-friendly interface software which can be

used to apply different testing conditions, such as simple, cyclic, or intermittent

stimulation. The MCTR also features a hydrostatic compression option, which removes

the piston normally used for compression and replaces it with cell culture media. This

system’s main strengths are its high throughput and its interface software. The high

throughput saves both time and effort by allowing the user to test multiple samples at the

same time, while the interface software makes the process of stimulating samples more

simplified.

11

CHAPTER TWO

RATIONALE AND SPECIFIC AIMS

New bioreactor systems are constantly being developed to achieve a greater

understanding of the concept of mechanotransduction. Though the relationship between

mechanical stimulus to cells and tissue physiology and pathology is clear, the many

mechanisms of mechanotransduction that explain this relationship still remain unclear.

Pressure and stretch are two important mechanical stresses that exist in the environment

of many cells, so being able to simulate these stresses in a controlled manner is crucial to

fully understanding the biochemical reactions in response to mechanical stimulation. The

overall objective of this study was to design and fabricate a system that can apply

pressure and stretch simultaneously, as well as independently, to cells in culture. The

system included a device that incorporates a cell substrate and two chambers on each side

of the substrate, allowing for independent pressure control for each chamber. After the

developmental phase of the study, the system was utilized to test the effects of pressure

and stretch, independently and in combination, on MYP3 cell cultures in order to analyze

and more fully understand the effects of various mechanical stimuli on living cells. To

achieve the overall objective of this study, this project was divided into the following

aims:

Aim 1: Design and fabricate a pressure device that incorporates two separate

chambers, separated by a flexible substrate, whose pressures can be individually

modified.

12

Rationale: The incorporation of two separate chambers allows for individual

modification of pressure for each chamber, which enables the user to control the amount

of pressure and stretch applied to the cultured cells seeded on the substrate.

Approach: Two polycarbonate sheets were machined to house an open ended

chamber with an inlet and an outlet valve. The open end of each chamber would be

closed off by a flexible silicone rubber substrate on which cells would be seeded onto one

side. The inlet and outlet valves allow for controlled pressure to be applied to either side

of the substrate.

Aim 2: Mechanically characterize the material properties of the silicone rubber

substrate.

Rationale: Characterization of the material properties of the silicone rubber

substrate is necessary in order to prescribe the required amount of pressure to each

chamber to simulate physiological or pathological states of pressure and stretch.

Approach: Biaxial tensile testing was conducted on silicone rubber specimens by

applying equi-biaxial loading using the CellScale Biotester. The experimental data from

this testing was analyzed using Mooney-Rivlin hyperelastic models (2- and 5-parameters)

using COMSOL Multiphysics software (COMSOL). Strain was quantified by mounting

marked specimens on the pressure chamber and subjecting them to known pressures.

Wax replicas of the deformed substrate were imaged to measure changes in radial

position of the markers.

Aim 3: Utilization of the finished device to test the effects of pressure and stretch,

independently and in combination, on MYP3 cell cultures.

13

Rationale: Once device fabrication is finished and substrate material

characterization is complete, device testing is necessary to determine its effectiveness as

a pressure/stretch bioreactor.

Approach: Cell adhesion testing was conducted on the silicone substrate to

determine the best coating method for cell culturing. Several copies of the device were

made to allow for multiple simultaneous testing. After sterilization of the devices,

pressure and stretch tests were administered to MYP3 cell cultures. Media from each

device was collected to analyze ATP release to compare the effects of pressure and

stretch on bladder inflammation.

14

CHAPTER THREE

MATERIALS AND METHODS

3.1 Description of Pressure-Stretch Bioreactor

The main idea behind this device is to have two chambers, separated by a flexible

substrate, whose pressures can be modified independently of one another. This allows

for the device to apply pressure and stretch, independently and in conjunction, to cells in

culture. The pressure device consists of three main components: the upper and lower

chambers and the flexible silicone rubber substrate for culturing cells.

3.1.1 PRESSURE CHAMBER

The upper and lower chambers were made of polycarbonate sheets (6” x 12” x

½”, McMaster-Carr, 8574K322). Parts were fabricated at the Clemson Machining and

Technical Services. The opaque surfaces of the sheets were polished to increase the

transparency of the device. Transparency allows for users of the device to clearly see the

specimen and the media flowing within the device. Each chamber has inlet and outlet

valves to allow for control of applied pressure. These valves are small holes drilled from

outside the device into the main chambers. The top and bottom chambers were

assembled (Figure 3.1) and fastened with four nylon screws (8-32 thread size, 1 ½” long,

McMaster-Carr, 95868A353), wingnuts (8-32 thread size, McMaster-Carr, 94924A300),

and washers (No. 8 screw size, 0.173” ID, 0.375” OD, McMaster-Carr, 90295A400).

15

3.1.2 CELL CULTURE SUBSTRATE

Cell culture substrates and rubber gaskets for the pressure device were fabricated

by cutting silicone rubber sheets (12” x 12”, 0.020” thick, 40A, McMaster-Carr Catalog

86915K12) using VersaLASER® (Universal Laser Systems). The laser printer color was

set to red, and the thickness of the laser was set to 0.05 mm. The laser printer material

was set to silicone rubber, and the material thickness was set to 0.10 mm. The vector

power was set to 25%. Before the substrates were used in cell testing, they were first

cleaned and sterilized and then coated with fibronectin (Sigma, F4759). Further details

on the cleaning and sterilization of the substrates are described in a later section. The

substrate was assembled by sandwiching it between two nylon washers (7/8” screw size,

1” ID, 1.25” OD, McMaster-Carr Catalog: 95606A580), which in turn fit in between the

upper and lower chambers. The silicone rubber gaskets were placed between each

washer and chamber.

3.2 Calibration of Pressure-Stretch Bioreactor

The inlet and outlet chambers were connected to external pressure sources

through a tubing system consisting of Luer-LokTM fittings obtained from Cole-Parmer.

Using a Luer-LokTM Tip 10 mL syringe (BD), pressure was applied through the inlet and

outlet valves in order to test the ability of the device to maintain constant pressure within

the chambers. The first pressure test included exposing the upper chamber to pressures

close to 230 cm H2O. Those pressures were held for 2 minutes and then measured again

to observe the loss in pressure. The second test included exposing the lower chamber to

16

negative pressures around 90 cm H2O for 2 minutes. Similar to the first test, the loss in

pressure was observed.

17

Figure 3.1: Schematic diagrams of the pressure device assembly.

Inlet/Outlet Valves

Screw System Silicone Rubber Substrate

18

3.3 Mechanical Characterization of Culture Substrate

In order to be able to apply physiological and pathological conditions of pressure

and stretch, it is important to mechanically characterize the silicone rubber sheet used as

cell culture substrate in the device. Initial equi-biaxial load testing yielded data (Figure

4.3) that suggested that the silicone rubber substrate behaves as a hyperelastic material.

Because of this, further equi-biaxial load tests were conducted on silicone rubber

specimens so that finite element analysis could be used to accurately model the material’s

stress-strain behavior.

3.3.1 PLANAR BIAXIAL TESTING

A BioTester (CellScale) was used to conduct planar biaxial testing equi-biaxial

loading to silicone rubber specimens. In this specific BioTester, 23 N load cells were

used to measure the force applied to the specimens. For each testing a specimen (10 mm

x 10 mm) was mounted on the testing device using the rakes with a tine diameter of 305

µm, a tine spacing of 2.2 mm, and a puncture depth of 2.4 mm. Several biaxial loading

tests were done on silicone specimens in order to obtain the data needed for

characterization of the silicone rubber material using finite element analysis.

The first round of equi-biaxial load testing was conducted under displacement-

controlled configuration. A preload of 100 mN in each axis was applied to the specimen,

which was stretched to a maximum strain of 35% at a rate of 2% strain per second.

Using the data output from the BioTester, a stress-strain curve was plotted to show the

loading and unloading behavior of the silicone rubber material. The stress (σ) was

calculated as force (F) normalized by the original cross sectional area (A) of the specimen

19

for each direction of stretching. The strain (ε) was calculated as the rake tine

displacement (ΔL) divided by the original length between the tines (L0).

The second round of biaxial load testing was a force controlled analysis. In this

test, the specimen was subjected to a maximum force of 4500 N over a 25 second

duration. The specimen underwent 5 cycles of equibiaxial load testing, and stress-strain

curves were plotted for each cycle using the data output by the BioTester using the same

methods as the first biaxial test. Similar to the second round of testing, a third round of

biaxial testing was conducted under force-controlled configuration. The testing

conditions for the maximum prescribed force and number of cycles were the same as the

second round, except that three different 10 mm x 10 mm silicone specimens were tested.

The average stresses and strains obtained from these specimens were taken and plotted

into one stress-strain curve.

3.3.2 DATA ANALYSIS AND CURVE FITTING USING COMSOL

The stress-strain data obtained from the second round of biaxial testing were input

into COMSOL for model fitting into the 2- and 5-parameter Mooney-Rivlin models,

which are models for hyperelastic materials. These two different models were then

analyzed and compared using the root mean squared method to determine which model

served a better fit for the data. Both the 2- and 5- parameter models were used in finite

element analysis to simulate the effect of pressure on the silicone substrate. For the third

round of biaxial testing, the averaged stress-strain curve was fit into the 5-parameter

Mooney-Rivlin model.

20

3.3.3 FINITE ELEMENT ANALYSIS USING COMSOL

The model fitting data from the second and third rounds of biaxial testing were

input into COMSOL to perform simulations which show the effect of pressure on the

silicone substrate. In these simulations, pressures of 122.3 cm H2O, 180.5 cmH2O, and

225 cmH2O were conducted on the fitted model based on the material properties and

shape of the silicone rubber substrate used for the device.

3.3.4 STRAIN QUANTIFICATION

In order to validate the results obtained from the finite element analysis

simulation, the radial and circumferential strain of the silicone substrate under known

pressures were quantified. First, a coordinate system consisting of concentric circles of

known measurements was stamped onto silicone substrate samples using a custom 3D-

printed stamp. This custom stamp was created using an Ultimaker 3 (Ultimaker) 3D

printer with PLA filament (Ultimaker). The ink used by the stamp was dry erase ink

from markers (Expo®). These silicone substrate samples were then loaded into the

bottom chamber of the pressure device with the top open and clamped down by binder

clips. While negative pressures of 125, 175, and 225 cm H2O were applied to the

chamber, hot paraffin wax (Cancer Diagnostics Inc.) was poured into the deformed

sample and cooled inside a cryomicrotome (Thermo Scientific), forming a wax replica of

the deformed substrate. The coordinate system stamped onto the sample also transferred

onto the wax replica during the process.

Once the wax replicas of the deformed samples were made at different pressures,

they were imaged and analyzed to determine the amount of radial and circumferential

21

strain each sample experienced. Briefly, side-view images of the replicas were taken

using a DSLR camera (Canon) from a fixed distance. The images of the replicas were

then analyzed using ImageJ and the amount of radial and circumferential strain

experienced at each pressure was calculated as follows.

Figure 3.2: Custom stamp used for imprinting coordinate system onto silicone substrate

samples.

Figure 3.3: Side-view image of a wax replica of the deformed substrate.

22

The distance between each concentric circle along the curve of the replica’s shape was

measured as Rf. This distance was compared to the original distance between each

concentric circle, Ri. Radial strain, 𝜀𝑅, was calculated using the following equation:

𝜀𝑅 =𝑅𝑓−𝑅𝑖

𝑅𝑖.

Circumferential strain was calculated using the equation46:

𝜀𝑐 = 0.5(𝜆𝑐2 − 1),

where 𝜆𝑐 =2𝜋𝑅ℎ

2𝜋𝑅𝑖.

Figure 3.4: Diagram of strain quantification values used in image analysis of wax

replicas.

Initial, side-view

Final, side-view

Ri

Rf

2R

Coordinate System, top-view

23

3.3.5 COMPARISON OF STRAINS

The finite element analysis simulation was used to determine the radial and

circumferential strain at different positions along the deformed silicone rubber substrate

models. To do this, the displacement in the z-direction was found at several points from

the edge of the circular substrate to the center. These displacements were then plotted

against their radial positions in the x-direction to form a curve that represented the shape

of the deformed substrate model. An equation was then produced for each curve using

Microsoft Excel. The length of any segment of the curve could then be found using the

Arc Length Equation47:

𝐿 = ∫ √1 + (𝑑𝑦

𝑑𝑥)2𝑑𝑥

𝑏

𝑎.

Using the lengths obtained by the Arc Length Equation, the radial and circumferential

strains at different points along the curve of the deformed substrate were determined and

plotted.

3.4 Exposure of Urothelial Cells to Pressure-Stretch

3.4.1 SUBSTRATE PREPARATION

Using a method reported in literature48, the silicone substrates were sterilized and

cleaned by soaking and sonicating them in 70% ethanol for one hour. Following this,

each substrate was sterilized beneath ultraviolet light in a laminar flow hood for 15

minutes and left to air dry overnight in 35 mm petri dishes. The substrates were then

24

coated with fibronectin (Sigma) by means of physical adsorption48. Briefly, the petri

dishes containing the substrates were filled with 2 mL of a diluted (2 µg/mL) fibronectin

solution in PBS and, the substrates were incubated at 37ºC overnight. After removing the

fibronectin solution, the substrates were ready for cell seeding.

3.4.2 CELL CULTURE AND SUBSTRATE SEEDING

For the experimental testing of the Pressure-Stretch Bioreactor, the MYP3 cell

line, an immortalized non-tumorigenic rat urothelial cell line49, was used. These cells

were cultured in a modified version of Ham’s F-12 K medium supplemented as described

in the MYP3 culturing methods by Hughes et al49. The cells were passaged every three

days until they were used for experimentation. Each fibronectin-coated silicone substrate

was seeded with 2 mL of media containing 0.3 x 106 cells and cultured for 48 hours. The

MYP3 media was then replaced with serum-free media and incubated for 12 hours. The

substrates were removed from their petri dishes and mounted into the bioreactor.

3.4.3 PRESSURE-STRETCH EXPERIMENTS

The device was first sterilized by spraying the larger polycarbonate parts and

briefly soaking the smaller parts (wingnuts, washers, gaskets, etc.) with 70% ethanol.

Once sterilized, the tubing and the chambers were flushed with sterile PBS using a Luer-

LokTM Tip 10 mL syringe (BD). The cell-seeded silicone substrates were then mounted

the device was assembled. There were 4 different testing conditions in this experiment:

pressure only, stretch only, pressure and stretch, and the control. For the pressure only

test, the lower chamber was filled with PBS and 40 cm H2O pressure was applied to the

upper chamber using a syringe connected to the inlet valve of the upper chamber. The

25

syringe containing culture medium was pushed in to increase the pressure. In the stretch-

only test, the upper chamber’s valves were left open to atmospheric pressure, and a

negative pressure of 125 cm H2O was applied to the lower chamber to subject the

substrate to an overall average strain of 15%. To apply negative pressure, a syringe

attached to one of the valves of the lower chamber was pulled to reduce pressure. In

order to stimulate the substrate with pressure and stretch together, the bottom chamber

was first exposed to -85 cm H2O. The top chamber was then exposed to 40 cm H2O.

This combination of positive and negative pressures subjected the substrate to the 40 cm

H2O of the pressure-only test and the 15% strain of the stretch-only test. The control was

the cells was mounted into the device but maintained under atmospheric pressure without

any application of stretch. The cells were subjected to these testing conditions for 5

minutes each.

3.4.4 ANALYSIS OF MYP3 RESPONSES TO PRESSURE AND STRETCH

Following exposure to the mechanical stimuli, the supernatant media were

collected for an ATP assay, and the cells were lysed for a caspase assay. One substrate

from each testing condition was taken for cell imaging. Images of the cells on the

substrates were observed for any significant changes in cell morphology or cell viability.

26

CHAPER 4

RESULTS

4.1 Description of Pressure-Stretch Bioreactor

The polycarbonate sheets (6” x 12” x ½”, McMaster-Carr, 8574K322) were

successfully machined and assembled along with the nylon screws (8-32 thread size, 1

½” long, McMaster-Carr, 95868A353), wingnuts (8-32 thread size, McMaster-Carr,

94924A300), and washers (No. 8 screw size, 0.173” ID, 0.375” OD, McMaster-Carr,

90295A400) to make the main body of the Pressure-Stretch Bioreactor (Figure 4.1). The

silicone cell culture substrate (Figure 4.2) was laser-cut to the correct dimensions and

successfully sandwiched between two nylon washers (7/8” screw size, 1” ID, 1.25” OD,

McMaster-Carr Catalog: 95606A580).

Figure 4.1: Assembly of the upper and lower chambers for the Pressure-Stretch

Bioreactor.

27

Figure 4.2: Silicone cell culture substrate assembly.



The BioTester (CellScale) was used for equi-biaxial load testing of the silicone

rubber substrate. The first round of testing was conducted under displacement-controlled

configuration to test the effect of constant strain rate on the specimen. The square

specimen was stretched in two directions orthogonal to each other. The stress-strain

behavior of the silicone substrate was similar in both axes (Figure 4.3). The stress-strain

behavior was also non-linear and exhibited a moderate hysteresis. The second (Figure

4.4) and third rounds (Figure 4.5) of testing were conducted under force-controlled

configuration. For each round, 5 cycles of 4500 N maximum force were prescribed to

each specimen that was tested. The stress-strain data from the first and last cycles of the

28

second round showed a difference in shape between different cycles in that the first cycle

reached maximum stress at a lower strain compared to the last cycle. The stress-strain

curves between each cycle stabilized toward the shape of the final cycle. In the third

round of testing, three substrate specimens were subjected to 5 cycles of 4500 N

maximum force to test for variance between specimens. The average stress for the x- and

y-axes at the maximum strains (0.78 for x and 0.77 for y) were 392 and 398 kPa,

respectively (Figure 4.6). Their averaged stress-strain curves provided improved data for

the Mooney-Rivlin model fitting.

Figure 4.3: Stress-strain curve data from first round of equi-biaxial load testing.

-100

0

100

200

300

400

500

600

700

800

900

0 0.1 0.2 0.3 0.4

Stre

ss (

kPa)

Strain

40A silicone rubber biaxial testing results

x-axis

y-axis

29

Figure 4.4: Stress-strain curve data for the first and fifth cycles of the second round of

equi-biaxial load testing.

Figure 4.5: Stress-strain curve data for the fourth cycle of the third round of equi-biaxial

load testing. Data are mean +/- the standard error of mean (n=3).

0

100

200

300

400

500

600

0 0.2 0.4 0.6 0.8

Stre

ss (

kPa)

Strain

Round 2

X-Axis, Cycle 1

Y-Axis, Cycle 1

X-Axis, Cycle 5

Y-Axis, Cycle 5

0

50

100

150

200

250

300

350

400

450

0 0.2 0.4 0.6 0.8 1

Stre

ss (

kPa)

Strain

Round 3, Cycle 4

X-Axis

Y-Axis

30

Figure 4.6: Average stress for each axis at the maximum strain for round 3, cycle 4.

Data are mean +/- the standard error of mean.


The model fitting done for the second round of stress-strain data showed that the

5-parameter Mooney-Rivlin model exhibited a better fit than the 2-parameter (Figures 4.7

and 4.8). This was confirmed by calculating the root mean square error (RMSE), a

measurement of the difference between values predicted by a model (2- and 5- parameter

Mooney-Rivlin models) and the observed values (stress-strain data). An RMSE value of

0 indicates a perfect fit of the model to the data, so a lower RMSE value is better than a

higher one. The RMSE found for the 2-parameter model was 11.78 while the RMSE for

the 5-parameter model was 7.11. Because of the better fit using the 5-parameter model,

the data from the third round of biaxial testing was used for the COMSOL simulation

(Figure 4.9).

0

50

100

150

200

250

300

350

400

450

X-Axis Y-Axis

Average Stress

31

Figure 4.7: 2-parameter Mooney-Rivlin model fitting data using the stress-strain data

obtained from round 2, cycle 5, of planar biaxial testing.

Figure 4.8: 5-parameter Mooney-Rivlin model fitting data using the stress-strain data

obtained from round 2, cycle 5, of planar biaxial testing.

0

100

200

300

400

500

1 1.1 1.2 1.3 1.4 1.5 1.6

Stre

ss (

kPa)

Stretch

2-Parameter Mooney Rivlin Model Fit

Model dataExperimental measured data

0

100

200

300

400

500

1 1.1 1.2 1.3 1.4 1.5 1.6

Stre

ss (

kPa)

Stretch

5-Parameter Mooney-Rivlin Model Fit

Model dataExperimental measured data

32

Figure 4.9: 5-parameter Mooney-Rivlin model fitting data from COMSOL using the

stress-strain data obtained from round 3, cycle 4, of planar biaxial testing. The blue line

represents the experimental data, and the green line is the fitted values.


The data from the curve fitting and COMSOL were used to perform simulations

which relate pressure loading to deformation of the silicone rubber substrate. Functional

simulations were successful using the curve fitting data from the third round of biaxial

testing (Figure 4.10). The simulations provided data on the displacement across the

deformed silicone substrate, allowing for strain to be calculated at any point on the

surface.

33

Figure 4.10: COMSOL simulation utilizing the 5-parameter Mooney-Rivlin curve fitting

data from the third round of biaxial testing. This simulation applied 255 cm H2O to the

top surface of the substrate.

4.2.4 CALCULATION OF STRAINS BASED ON FEA SIMULATION

The radial strain data from the finite element analysis simulation showed the

radial strain values decreasing as the radial position moves further from the center of the

substrate (Figure 4.11). The circumferential strain data from the finite element analysis

simulation showed the circumferential strain values gradually decreasing as the radial

position moved further from the center of the substrate (Figure 4.12).

34

Figure 4.11: Radial strain data obtained using the finite element analysis simulation.

Figure 4.12: Circumferential strain data obtained using the finite element analysis

simulation.

0

0.1

0.2

0.3

0.4

0.5

0 0.1 0.2 0.3 0.4 0.5 0.6

Stra

in

Radial Position (in)

Radial Strain (COMSOL)

122.3 cmH2O

180.5 cmH2O

225 cmH2O

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 0.1 0.2 0.3 0.4 0.5 0.6

Stra

in


Circumferential Strain (COMSOL)

122.3 cmH2O

180.5 cmH2O

225 cmH2O

35


The radial and circumferential strains of the substrate under known pressure was

quantified to validate the finite element analysis simulation results. The radial strain that

was calculated varied in value at different positions along the substrate surface and

showed no trends (Figure 4.13). The maximum radial strain values were 0.204, 0.308,

and 0.404 for the 125, 175, and 225 cmH2O pressure groups, respectively (Figure 4.14).

These maximum values were found at the 0.125 in radial position for each pressure

group. The minimum radial strain values, which were found at the 0.5 in radial position,

were 0.068, 0.084, and 0.18 for the 125, 175, and 225 cm H2O pressure groups,

respectively (Figure 4.14). The average radial strain values found for each pressure

group were 0.161, 0.186, and 0.257 for the 125, 175, and 225 cm H2O pressure groups,

respectively. The circumferential strain values decreased as the radial position increased

for all pressure groups except for the 0.0625 in radial position in the 175 and 225 cmH2O

groups (Figure 4.15).

36

Figure 4.13: Radial strain data obtained from the wax replica experiment. Data are

mean +/- the standard error of mean (n=3).

Figure 4.14: Graph of the maximum and minimum recorded radial strains, as well as the

average amount of radial strain for each pressure group.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 0.1 0.2 0.3 0.4 0.5 0.6

Rad

ial S

trai

n


Radial Strain

125 cmH2O

175 cmH2O

225 cmH2O

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Maximum Minimum Average

Stra

in

Radial Strain

125 cmH2O

175 cmH2O

225 cmH2O

37

Figure 4.15: Circumferential strain data obtained from the wax replica experiment. Data

are mean +/- the standard error of mean (n=3).


The Pressure-Stretch Bioreactor underwent calibration to test its ability to

maintain pressure within its chambers. There was an average loss of 0.43 and -0.77 cm

H2O in the upper and lower chambers, respectively, 2 minutes after the application of

positive or negative pressure to one side of the substrate.

Chamber Initial Pressure

(cm H2O)

Final Pressure

(cm H2O)

Pressure Loss

(cm H2O)

Avg Pressure Loss

(cm H2O)

Upper

230.1 229.8 0.3

0.43 233.3 233 0.3

231.2 230.5 0.7

Lower

-90.4 -89.9 -0.5

-0.77 -90.3 -89.4 -0.9

-91.1 -90.2 -0.9

Table 4.1: Pressure-loss results from calibration test.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 0.2 0.4 0.6

Cir

cum

fere

nti

al S

trai

n


Circumferential Strain

125 cmH2O

175 cmH2O

225 cmH2O

38


Following application of pressure and stretch, together or separately, to MYP3

cells seeded on fibronectin-coated silicone substrates, the cells were examined for

morphology, extracellular ATP release, and intracellular caspase-1 activity. The imaged

cells on the substrate were polygonal in shape and were grouped in patches (Figure 4.16).

The results from the ATP assay on supernatant media showed increases in extracellular

ATP release for the pressure, stretch, and pressure-stretch groups compared to the control

group which had no applied pressure or stretch (Figure 4.17). The highest increase in

ATP release was in the stretch group, which was nearly 10-fold higher than the control

group. The caspase-1 assay results showed a reduction in caspase-1 activity for all

experimental groups compared to the control group (Figure 4.18).

39

Figure 4.16: Microscope images (100x) of the cells seeded on the substrate following the

pressure-stretch experiment.

Pressure-Stretch

Pressure Control

Stretch

40

Figure 4.17: Extracellular ATP release by MYP3 cells in response to pressure (40 cm

H2O)-stretch (15%). The data are mean +/- SEM statistically analyzed using one-way

ANOVA followed by a post hoc Tukey-test. *P<0.05 (N=6)

Figure 4.18: Intracellular caspase-1 activity by MYP3 cells in response to pressure (40

cm H2O)-stretch (15%). The data are mean SEM statistically analyzed using one-way

ANOVA followed by a post hoc Tukey-test. *P<0.01 (N=5).

0

2

4

6

8

10

12

ctrl pressure stretch pressure +stretch

Fold

ch

ange

Extracellular ATP

0

0.2

0.4

0.6

0.8

1

1.2

ctrl pressure stretch pressure +stretch

Fold

ch

ange

Intracellular Caspase-1 Activity

*

*

* *

*

41

CHAPTER 5

DISCUSSION

The present study aimed to design and fabricate a device that can apply pressure

and stretch, together or separately, to cells in culture. Before testing the device, its parts

were first calibrated by characterizing the mechanical behavior of the substrate and

testing for pressure leakage in the assembled chambers. The device was then tested with

MYP3 cells for its utility.



Following the first round of planar biaxial testing under displacement-controlled

configuration, several material properties of the silicone rubber substrate were found.

The stress-strain behavior of the substrate was similar in both axes (Figure 4.3),

suggesting that the material is isotropic. This is in agreement with literature that most

rubber-like materials are isotropic50. The moderate hysteresis showed that the silicone

substrate was viscoelastic in nature and in the subsequent analysis only the loading part

of the stress-strain curve was examined.

During a planar biaxial testing of the silicone rubber substrate, a difference in

stress-strain curve between the first and last cycles was noted, which stabilized after the

substrate was subjected to more (~4) cycles. This shows that the substrate exhibited the

phenomenon of preconditioning, which probably resulted from repeated cyclic loading51.

42

Because the silicone substrate is viscoelastic, preconditioning causes the amount of

energy lost in a cycle of loading and unloading to decrease. For this reason, in the

following testing, only the 4th cycle data were analyzed and compared for a number of

different silicone specimens. The low variance in stress-strain behavior between different

specimens observed in the present study (Figure 4.8) showed that the process of

preconditioning allowed for collecting consistent data.


The silicone rubber substrate that was used in the present study is classified as a

rubber-like material, which means that the substrate exhibits hyperelastic behavior52.

Therefore, in order to mechanically characterize the silicone rubber material, the data

from the biaxial load testing was input into COMSOL to be fitted into the Mooney-Rivlin

model, a hyperelastic constitutive model. Fitting the experimental data allows us to

accurately reproduce mechanical behavior of specific materials in FEM simulations52.

The two types of Mooney-Rivlin models that were tested for model fitting were the 2-

and 5-parameter models. Generally, the model with the greater number of parameters

more accurately matches simulations with experimental data, but more parameters have a

higher chance of failing to converge53. The experimental data that was recorded from the

planar biaxial load testing exhibited a stress-strain curve with one inflection point, which

suggests that the 5-parameter model would work best for the FEA simulation. Because

the model fitting from the second round of stress-strain data was shown to be better in the

5-parameter Mooney-Rivlin model through calculating the RMSE, the 5-parameter model

was chosen for conducting finite element analysis on the substrate.

43


In order to conduct strain estimation of the silicone rubber substrate under known

pressures, finite element analysis was conducted using COMSOL and the Mooney-Rivlin

model with the parameters obtained from the model fitting of the planar biaxial testing.

From the radial and circumferential strain results, we found the trend that the amount of

strain decreases moving away from the center of the substrate. This outcome is

consistent with other studies which subjected known pressures to circular flexible

silicone rubber sheets46,54. In the studies by Gilbert et al. (1994) and Mott et al. (2002),

circular rubber substrates were subjected to known pressure, and the radial and

circumferential strains were calculated using displacement data and FEA. The

experimental data and the FEA data from both studies showed slight decreases in radial

strain as the radial position moved towards the edge of the substrate. This is not in full

agreement with our findings using FEA in that radial strain was zero at the edge. This

discrepancy is most likely due to different boundary conditions among each of the

studies. The studies by Gilbert et al. and Mott et al. incorporated the clamping

mechanism which held the circular substrate in place into their FEA. The pressure from

the clamping caused slight buckling at the edge of the substrate. Our study did not

incorporate the clamping of the edges of the substrate, which explains why the radial

strain value was zero at the edge. The trend for circumferential strains was similar for

each study. The maximum circumferential strain value was located at the center of the

substrate for all studies, and the circumferential strain decreased to zero at the edge of the

44

substrate. Overall, besides the difference of boundary conditions, the FEA data from the

present study was in agreement with the data from other studies in literature in that the

radial and circumferential strain trends were similar.


Physical strain quantification was conducted on substrate specimens as an

experimental validation of the results that were obtained from the FEA simulations. The

results from this validation experiment did not exactly match the data calculated from the

COMSOL simulations. Although the simulation and physical quantification experiment

results do not entirely match, there are notable similarities between the two. The

maximum radial strains found for both experiments were similar in value and were found

close to the center of the substrate. The minimum values for the radial strains were both

found at the furthest point from the center. Despite the differences between the

simulation and physical quantification, the experimental validation provided some

insightful information. The average radial strain values found from the physical

quantification were used to determine the amount of pressure needed for the urothelial

cell pressure-stretch exposure experiment.

We speculate that discrepancies between the simulation and physical

quantification resulted from either the issues associated with the experimental approach

or limitations with the FEA. Firstly, when wax replicas of the deformed substrate were

made, we noted variance in their shapes because the substrate does not deform as

uniformly along the surface and consistently from experiment to experiment as it would

in the simulation. Secondly, there could be variance from the image analysis of the wax

45

replicas. Because the wax replicas are not all created in the exact same shape, it is

impossible to obtain the exact same perspective from one replica to the next. Another

reason for differences between simulation and physical quantification is the different

boundary conditions between the approaches. In the physical quantification, the

outermost edge of the substrate is clamped by the nylon washers. This clamping

mechanism creates a slight rippling effect before pressure is even exposed to the

substrate; whereas, in the simulation, there is no clamping mechanism and thus no

rippling effect. This could explain the lack of uniformity in the radial strain data from the

physical quantification (Figure 4.14). This also explains why the radial strain values at

the edge of the substrate were higher in the physical quantification than in the simulation.


When the assembled Pressure-Stretch Bioreactor was tested for its ability to

maintain pressure within its chambers, a slight loss in pressure in both the upper and

lower chambers was found after 2 minutes (Table 4.1). However, we concluded that the

amounts of lost pressure (0.43 and -0.77 cmH2O) will not significantly affect shorter term

(1 minute of pressure exposure) experiments and conducted the experiments described in

the present study (Chapter 3, Section 3.4.3). For longer experiments, improvements in

the design of the bioreactor are necessary. The loss in pressure is likely due to leakage

around the nylon rings that sandwich the substrate between the two chambers. A

potential solution to this leakage would be to sand the nylon washers to provide a

46

smoother surface and better surface contact between the nylon washers and silicone

rubber gaskets.


Using the Pressure-Stretch Bioreactor, we exposed MYP3 cells to pressure and

stretch, together or separately, successfully without causing any cell harm. This was

confirmed by the images of the urothelial cells exposed to pressure and/or stretch that

demonstrated cobble-stone morphology in grouped patches (Figure 4.16). This

morphology is characteristic of healthy cells, which indicates that our bioreactor

experimental conditions do not harm the cells55.

The results of the luciferin/luciferase assay (Fisher Scientific, Hampton, NH)

provided evidence of increased extracellular ATP concentration in the supernatant,

indicative of ATP release from MYP3 cells exposed to pressure, stretch, and pressure-

stretch. The pressure only group displayed a 4-fold increase in ATP release compared to

the control group. This increase in extracellular ATP release in response to pressure is in

agreement with previous data from our lab, where a 1.4-fold increase in ATP release was

observed in MYP3 cells exposed to 40 cmH2O for 1 minute56. The difference in fold

increase between the two studies may be explained by the use of fibronectin-coated

silicone in the present study versus the polystyrene culture plate in the previous study.

Cells exposed to only stretch displayed the highest amount of ATP release was the stretch

only group, which was nearly 10-fold compared to the control group. The increase in

ATP release due to stretch is in agreement with a number of studies57-59. Although, the

47

magnitude of the fold increase was not expected as the pressure and stretch group, which

is the closest to physiological conditions for urothelial cells, had an ATP release that was

5-fold higher than the control group. We speculated that the stretch only group has a

high ATP release due to the rapid application of stretch to the substrate. The stretch was

applied at a super-physiologic rate of 115% strain in less than one minute, which may

have induced the unexpected elevation in ATP release. The fold change in ATP release

of the pressure-stretch group compared to the control group was greater than the pressure

only group. As indicated in previous studies, both pressure and stretch have been shown

to increase the amount of ATP release in urothelial cells. Because of this, it was expected

that the pressure-stretch had a greater fold change than the pressure only group due to the

presence of two types of mechanical stimuli.

In the present study, exposure of MYP3 cells to pressure and/or stretch resulted in

a reduction in intracellular caspase-1 activity determined from the cell lysates. This

finding conflicts with the previous data collected in our lab56. Dunton et al. (2018)

previously reported that exposure of MYP3 cells seeded in polystyrene culture plates to

hydrostatic pressure resulted in an increase in caspase-1 activity; whereas, our study

indicated a reduction in caspase-1 activity. The results from the present study are not in

agreement with the current understanding of ATP and caspase-1 activation in literature.

Several publications documented that increases in extracellular ATP release lead to

greater caspase-1 activation60-63. It appears that the decrease in caspase-1 activity in the

presence of elevated extracellular ATP levels could be the result of substrate stiffness or

cell interactions with fibronectin. This is due to our use of silicone substrates coated with

48

fibronectin versus the uncoated polystyrene well plates from previous studies. The data

from these findings are fold-change and normalized by the non-ATP treated group. The

caspase-1 response measured in the present study can either be due to decreased levels of

ATP release in response to stimuli, or increased levels of basal ATP release in response

to no stimuli. Our data suggests that the latter is the case and that fibronectin increases

basal levels of caspase-1 activation, thereby masking anything seen in response to

stimuli.

49

CHAPTER SIX

CONCLUSIONS AND RECOMMENDATIONS

In the present study, a device was designed and fabricated to apply pressure and

stretch, together and separately, to cells in culture. Planar biaxial testing of the silicone

rubber substrate material demonstrated its isotropic and viscoelastic properties and also

showed the importance of preconditioning. The radial and circumferential strain data

obtained from the FEA simulation was consistent with other studies except for the

difference in boundary conditions. Despite the differences between the FEA simulation

and the physical quantification of strain, the radial positions for the minimum and

maximum amounts of radial and circumferential strain were similar. Through planar

biaxial testing, FEA using COMSOL, and physical strain quantification, the silicone

rubber substrate material was mechanically characterized. The experiment in which

MYP3 cells were exposed to pressure, stretch, and pressure-stretch demonstrated the

bioreactor’s application of various mechanical stimuli to cells in culture. Imaging of the

urothelial cells that were exposed to mechanical stimuli demonstrated cobble-stone

morphology, which is characteristic of healthy cells. The extracellular ATP data

indicated an increase in ATP release in response to mechanical stimuli. Exposure of cells

to mechanical stimuli resulted in a reduction of intracellular caspase-1 activity. In

conclusion, a device capable of applying pressure and stretch, together and separately,

was successfully developed and tested on cultured MYP3 cells. The following is a list of

recommendations for future research to address the limitations of the present study:

50

To conduct FEA while simulating the clamping mechanism experienced at the

edge of the silicone substrate. Rationale: by incorporating clamping at the

edge, the buckling action that the substrate experiences is captured in the

simulation; therefore, a more realistic strain analysis can be obtained from the

simulation itself. This would have an effect on the comparison of the

simulation and physical quantification of strain.

To make improvements to the design of the Pressure-Stretch Bioreactor.

Rationale: the slight loss in pressure found in the calibration experiment

revealed that this device would not be usable for experiments that require

pressure-stretch exposures longer than 5 minutes. Improvements on the user-

friendliness of the device can also be made. The current design of the device

requires two people to handle and use efficiently.

To study the effects of fibronectin and silicone on extracellular ATP release

and caspase-1 activity. Rationale: the caspase-1 activity data from the present

study conflicted with the current understanding of ATP and caspase-1

activation. Determining the difference in ATP release between fibronectin-

coated silicone substrates versus uncoated polystyrene well plates is important

in understanding the cause of the decrease in caspase-1 activity in response to

pressure/stretch.

51

APPENDIX A

COMSOL Outputs

The following tables contain the data outputted from COMSOL for the Mooney-Rivlin

model fitting and the strain quantification simulation.

Table A.1 2-parameter Mooney Rivlin model fitting data as seen in Figure 4.7.

Stretch Model data [kPa] Experimental measured data [kPa]

1.0026 26.309 3.6935

1.0152 34.459 21.15

1.0341 58.523 45.7

1.0648 89.355 81.672

1.1072 125.12 125.28

1.1605 177.94 172.29

1.2182 224.25 216.31

1.2842 256.98 260.7

1.3515 292.82 301.66

1.4229 330.24 342.22

1.5 373.62 384.16

1.5784 443.76 426.08

Table A.2 5-parameter Mooney Rivlin model fitting data as seen in Figure 4.8.

Stretch Model data [kPa] Experimental measured data [kPa]

1.0026 26.309 5.367

1.0152 34.459 28.786

1.0341 58.523 57.234

1.0648 89.355 92.648

1.1072 125.12 131.56

1.1605 177.94 174.65

1.2182 224.25 217.62

1.2842 256.98 260.44

1.3515 292.82 296.11

52

1.4229 330.24 329.17

1.5 373.62 371.81

1.5784 443.76 444.57

Table A.3 Displacement field data for the COMSOL simulation of 122.3 cmH2O as seen

in Figures 4.11 and 4.12.

Displacement Field Position [in]

x -3.44E-06 -2.99E-06 -1.50E-06 2.12E-06 -3.45E-05

y 1.47E-05 0.009997 0.017294 0.018626 0.00933

z -0.2189 -0.21034 -0.1844 -0.14026 -0.07677

Radial Position [in] 0.5 0.4 0.3 0.2 0.1

Table A.4 Displacement field data for the COMSOL simulation of 180.5 cmH2O as seen



x -1.06E-06 -7.76E-07 4.72E-07 5.09E-06 -3.14E-05

y 2.01E-05 0.013168 0.022997 0.025425 0.014564

z -0.2513 -0.24152 -0.21191 -0.16162 -0.08956


Table A.5 Displacement field data for the COMSOL simulation of 225 cmH2O as seen



x -1.07E-06 -5.41E-07 1.26E-06 7.38E-06 -2.78E-05

y 2.08E-05 0.01518 0.026632 0.029797 0.018022

z -0.27068 -0.26016 -0.22834 -0.17433 -0.09711


53

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