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MORPHOLOGY OF OSTEOCYTE CELLS IN
NORMAL AND HYPER-GRAVITY
Huan Yuan
BEng (Hons)
Submitted in fulfilment of the requirements for the degree of
Master of Engineering (Research)
Chemistry, Physics, Mechanical Engineering
Faculty of Science and Engineering
Queensland University of Technology
04/2013
Morphology of osteocyte cells in normal and hyper-gravity i
Keywords
Cellular morphology
Empirical model
Hyper-gravity
Image-analysis
Osteocyte cell line (MLO-Y4)
Quantitative study
Morphology of osteocyte cells in normal and hyper-gravity ii
Abstract
Osteocyte cells are the most abundant cells in human bone tissue. Due to their
unique morphology and location, osteocyte cells are thought to act as regulators in
the bone remodelling process, and are believed to play an important role in
astronauts’ bone mass loss after long-term space missions. There is increasing
evidence showing that an osteocyte’s functions are highly affected by its morphology.
However, changes in an osteocyte’s morphology under an altered gravity
environment are still not well documented.
Several in vitro studies have been recently conducted to investigate the
morphological response of osteocyte cells to the microgravity environment, where
osteocyte cells were cultured on a two-dimensional flat surface for at least 24 hours
before microgravity experiments. Morphology changes of osteocyte cells in
microgravity were then studied by comparing the cell area to 1g control cells.
However, osteocyte cells found in vivo are with a more 3D morphology, and both cell
body and dendritic processes are found sensitive to mechanical loadings. A round
shape osteocyte’s cells support a less stiff cytoskeleton and are more sensitive to
mechanical stimulations compared with flat cellular morphology. Thus, the relative
flat and spread shape of isolated osteocytes in 2D culture may greatly hamper their
sensitivity to a mechanical stimulus, and the lack of knowledge on the osteocyte’s
morphological characteristics in culture may lead to subjective and non-
comprehensive conclusions of how altered gravity impacts on an osteocyte’s
morphology.
Through this work empirical models were developed to quantitatively predicate
the changes of morphology in osteocyte cell lines (MLO-Y4) in culture, and the
Morphology of osteocyte cells in normal and hyper-gravity iii
response of osteocyte cells, which are relatively round in shape, to hyper-gravity
stimulation has also been investigated.
The morphology changes of MLO-Y4 cells in culture were quantified by
measuring cell area and three dimensionless shape features including aspect ratio,
circularity and solidity by using widely accepted image analysis software (ImageJTM
).
MLO-Y4 cells were cultured at low density (5×103 per well) and the changes in
morphology were recorded over 10 hours. Based on the data obtained from the
imaging analysis, empirical models were developed using the non-linear regression
method. The developed empirical models accurately predict the morphology of
MLO-Y4 cells for different culture times and can, therefore, be used as a reference
model for analysing MLO-Y4 cell morphology changes within various
biological/mechanical studies, as necessary.
The morphological response of MLO-Y4 cells with a relatively round
morphology to hyper-gravity environment has been investigated using a centrifuge.
After 2 hours culture, MLO-Y4 cells were exposed to 20g for 30mins. Changes in
the morphology of MLO-Y4 cells are quantitatively analysed by measuring the
average value of cell area and dimensionless shape factors such as aspect ratio,
solidity and circularity. In this study, no significant morphology changes were
detected in MLO-Y4 cells under a hyper-gravity environment (20g for 30 mins)
compared with 1g control cells.
Morphology of osteocyte cells in normal and hyper-gravity iv
Table of Contents
Keywords..............................................................................................................................i
Abstract ...............................................................................................................................ii
Table of Contents ................................................................................................................ iv
List of Figures ..................................................................................................................... vi
List of Tables ...................................................................................................................... ix
List of Abbreviations............................................................................................................ x
Statement of Original Authorship ........................................................................................ xi
Acknowledgements ............................................................................................................ xii
Publications ......................................................................................................................xiii
Chapter 1: Introduction ......................................................................................... 1
1.1 Research Background & Motivations.............................................................................. 1
1.2 Project Aims .................................................................................................................. 4
1.3 Thesis Outline ................................................................................................................ 4 1.3.1 Chapter 2: Literature Review ............................................................................. 4 1.3.2 Chapter 3: Quantitative Morphology Study on Osteocyte Cells .......................... 4 1.3.3 Chapter 4: Osteocyte Cells under Hyper-gravity Stimulation .............................. 5 1.3.4 Chapter 5: Conclusions and Future Works ......................................................... 5
Chapter 2: Literature Review ................................................................................ 6
2.1 Osteocytes as Descendants of Osteoblasts ....................................................................... 6
2.2 The Function of Osteocyte Cells ..................................................................................... 7 2.2.1 Mechanosensation of Osteocyte Cells ................................................................ 7 2.2.2 Mechanotransdution of Osteocyte Cells ........................................................... 12
2.3 Osteocyte Cell Models: Osteocyte-Like Cell Line (MLO-Y4) ....................................... 15
2.4 Hypo- and Hyper-gravity Simulation Technologies ...................................................... 16
2.4 Response of Osteocyte Cells to Altered Gravity ............................................................ 20
2.5 Osteocyte Cell Morphology, Function and Mechanical Properties ................................. 22
2.6 Quantitative Cell Morphology Analysis ........................................................................ 23
Chapter 3: Quantitative Morphology Study on Osteocyte-Like Cell Line (MLO-
Y4)…………………………………………………………………………………..29
3.1 Introduction .................................................................................................................. 29
3.2 Methods ....................................................................................................................... 30 3.2.1 Cell Culture and Fixation ................................................................................. 30 3.2.2 Fluorescence Staining ...................................................................................... 30 3.2.3 Cell Segmentation ........................................................................................... 31 3.2.4 Morphologic Feature Extraction....................................................................... 31 3.2.5 Statistical Analysis .......................................................................................... 36 3.2.6 Curve Fitting ................................................................................................... 36
Morphology of osteocyte cells in normal and hyper-gravity v
3.3 Results ......................................................................................................................... 38 3.3.1 Cell Area & Culturing Time............................................................................. 38 3.3.2 Cell Shape & Culturing Time ........................................................................... 38 3.3.3 Model Selection for Curve Fitting .................................................................... 41 3.3.4 Fitted Curves and Developed Emprical Models ................................................ 42
3.4 Discussion .................................................................................................................... 47
3.5 Conclusion ................................................................................................................... 49
Chapter 4: Osteocyte Cells under Hyper-gravity Stimulation ............................ 50
4.1 Introduction .................................................................................................................. 50
4.2 Materials and Methods ................................................................................................. 51 4.2.1 Cell Culture ..................................................................................................... 51 4.2.2 Centrifuge Experiment ..................................................................................... 51 4.2.3 Post-Centrifugation Processing of MLO-Y4 Cells ............................................ 52 4.2.4 Statistics .......................................................................................................... 53
4.3 Results ......................................................................................................................... 53 4.3.1 Cell Area under Hyper-gravity ......................................................................... 53 4.3.2 Cell Shape under Hyper-gravity ....................................................................... 54
4.4 Discussion .................................................................................................................... 56
4.5 Conclusion ................................................................................................................... 59
Chapter 5: Conclusions and Future Works ......................................................... 60
Bibliography .......................................................................................................... 63
Appendix 1: Results from Centrifuge Experiments ............................................ 68
Cell Area under Hyper-gravity ........................................................................................... 68 Experiment One: ...................................................................................................... 68 Experiment Two:...................................................................................................... 69
Cell Shape under Hyper-gravity ......................................................................................... 70 Experiment One: ...................................................................................................... 70 Experiment Two:...................................................................................................... 73
Appendix 2: Preliminary Design for Side-View Optical System ......................... 76
Preliminary Design for Optical System Configurations ....................................................... 76
Preliminary Design for Optical System Dimensions ........................................................... 76
Preliminary Design for Optical Path Modulation ................................................................ 78
Morphology of osteocyte cells in normal and hyper-gravity vi
List of Figures
Figure 1The process of osteoblast differentiation from osteoblast precursors
to mature osteocytes [27] ................................................................... 7
Figure 2 Schematic view of the lacuno-canalicular network (middle),
formed by osteocyte lacunae and interconnecting canaliculi
(left), and of the connected cellular network (right) ............................ 7
Figure 3 Osteocytes modulate bone remodelling at loaded and unloaded
condition through the Sost and Rankl. The thickness of the
lines and arrows in A and B reflects the strength of the effects
[50] .................................................................................................. 12
Figure 4 Schematic overview of the role of osteocytes in the process of
bone remodelling [26] ...................................................................... 15
Figure 5 Random Position Machine [3] ............................................................... 18
Figure 6 Diagram for dropping package. [32] ...................................................... 19
Figure 7 The inertial shear force increase laterally from the centre of
centrifugation [64] ........................................................................... 20
Figure 8 Three families of shapes originating from a circle: (a) ellipses of
various elongation (top), (b) shape having various edge
irregularity (middle), and (c) a combination of the two (bottom)
[67] .................................................................................................. 24
Figure 9 Diagram shows the determination of cells with different aspect
ratio ................................................................................................. 25
Figure 10 Diagram shows cellular boundaries with different roughness ............... 26
Figure 11 Diagram shows the determination of cells with different solidity ......... 27
Figure 12 Diagram shows the determinations of spherity which is sensitive
to most irregularity and elongation ................................................... 28
Figure 13 Cell Segmentation procedure: (a) Original fluorescent image; (b)
Fluorescent image with enhanced contrast; (c) 8-bit image; (d)
Binary mask image .......................................................................... 34
Figure 14 the protocol of determining shape features (a) the original
fluorescent image; (b) Fitted ellipse to extracted cell shape; (c)
Extracted cell area and perimeter; (d) Convex hull of extracted
cell shape. ........................................................................................ 35
Figure 15 Cell Area changes of MLO-Y4 cells measured from two tests ............. 43
Figure 16 Aspect ratio changes of MLO-Y4 cells measured from two tests ......... 44
Figure 17 Solidity changes of MLO-Y4 cells measured from two tests ................ 44
Figure 18 Circularity changes of MLO-Y4 cells measured from two tests ........... 45
Figure 19 MLO-Y4 cell morphological appearance in culture. (a) 1hr; (b)
2hr; (c) 3hr; (d) 4hr; (e) 8hr; (f) 10hr (scale bar:50μm) ..................... 46
Morphology of osteocyte cells in normal and hyper-gravity vii
Figure 20 Four wells located at the centre of the 24-well plate were used ............ 51
Figure 21 (a): Side view of the rotor at speed; (b) Top view of the rotor at
rest. .................................................................................................. 52
Figure 22 Graphical illustration of cell area measured for MLO-Y4 cells
cultured at 20g (blue bar) and 1g (purple bar) for 30 mins from
two experiments. (a) The mean area measured for experiment
sample is 853.52μm2 and 874.06 μm
2 for control sample in
experiment 1 (P=0.388). (b) The mean area measured for
experiment sample is 866.1μm2 and 876.73μm
2 for control
sample in experiment 2 (p=0.669). n=500 ........................................ 53
Figure 23 Graphical illustration of aspect ratio measured for MLO-Y4 cells
cultured at 20g (blue bar) and 1g (purple bar) for 30 mins from
two experiments. (a) The mean aspect ratio measured for
experiment sample is 0.843 and 0.851 for control sample in
experiment one (P=0.232). (b) The mean area measured for
experiment sample is 0.846 and 0.847 for control sample in
experiment two (p=0.846). n=500 .................................................... 54
Figure 24 Graphical illustration of solidity measured for MLO-Y4 cells
cultured at 20g (blue bar) and 1g (purple bar) for 30 mins from
two experiments. (a) The mean aspect ratio measured for
experiment sample is 0.864 and 0.863 for control sample in
experiment one (P=0.791). (b) The mean area measured for
experiment sample is 0.866 and 0.864 for control sample in
experiment two (p=0.718). n=500 .................................................... 55
Figure 25 Graphical illustration of circularity measured for MLO-Y4 cells
cultured at 20g (blue bar) and 1g (purple bar) for 30 mins from
two experiments. (a) The mean aspect ratio measured for
experiment sample is 0.573 and 0.562 for control sample in
experiment one (P=0.053). (b) The mean area measured for
experiment sample is 0.581 and 0.562 for control sample in
experiment two (p=0.256). n=500 .................................................... 55
Figure 26 Distribution of cell area measured at 20g ............................................. 68
Figure 27 Distribution of cell area measured at 1g ............................................... 68
Figure 28 Distribution of cell area measured at 1g ............................................... 69
Figure 29 Distribution of cell area measured at 20g ............................................. 69
Figure 30 Distribution of circularity measured at 1g ............................................ 70
Figure 31Distribution of circularity measured at 20g ........................................... 70
Figure 32Distribution of aspect ratio measured at 1g ........................................... 71
Figure 33 Distribution of aspect ratio measured at 20g ........................................ 71
Figure 34 Distribution of solidity measured at 1g ................................................ 72
Figure 35 Distribution of solidity measured at 20g .............................................. 72
Figure 36 Distribution of circularity measured at 1g ............................................ 73
Figure 37 Distribution of circularity measured at 20g .......................................... 73
Morphology of osteocyte cells in normal and hyper-gravity viii
Figure 38 Distribution of aspect ratio measured at 1g .......................................... 74
Figure 39 Distribution of aspect ratio measured at 20g ........................................ 74
Figure 40 Distribution of solidity measured at 1g ................................................ 75
Figure 41 Distribution of solidity measured at 20g .............................................. 75
Figure 42 Over view of the preliminary design for side-view optical system ....... 76
Figure 43 Working distance for side-view optical system .................................... 76
Figure 44 Plastic ruler placed under 40x magnification lens, showing that
the microscope filed is 2mm ............................................................ 77
Figure 45 Preliminary design of the size of the optical system ............................. 77
Figure 46 Optical path length with different refractive index medium ................. 78
Morphology of osteocyte cells in normal and hyper-gravity ix
List of Tables
Table 1 MLO-Y4 cell mean area measured with different culturing time ............. 38
Table 2 Aspect Ratio measured for MLO-Y4 cells in culture from two
experiments. .................................................................................... 39
Table 3 Solidity measured for MLO-Y4 cells in culture from two
experiments. .................................................................................... 40
Table 4 Circularity measured for MLO-Y4 cells in culture from two
experiments ..................................................................................... 41
Table 5 Goodness of fit for cell area in culture .................................................... 41
Table 6 Goodness of fit for shape factor in culture ............................................... 42
Morphology of osteocyte cells in normal and hyper-gravity x
List of Abbreviations
AFM: Atomic force microscope
ATP: Adenosine triphosphate
BSA: Bovine serum albumin
Cx43: Connexion 43
DMEM: Dulbecco’s Modified Eagle medium
ECM: Extracellular matrix
FBS: Fetal bovine serum
GFP: Green fluorescent protein
G6PD: Glucose 6-phosphate-dehydrogenas
LCN: Lacuna-canalicular network
NO: Nitric oxide
P/S: Penicillin/Streptomycin
PFA: Paraformaldehyde
PBS: Phosphate-buffered saline
PKA: Protein kinase A
RANKL: Receptor activator of NFkB ligand
RPM: Random position machine
Morphology of osteocyte cells in normal and hyper-gravity xii
Acknowledgements
First and foremost, I wish to take this opportunity to gratefully acknowledge
the enthusiastic supervision of my principal supervisor, Professor Ted Steinberg,
who was extremely helpful and offered invaluable assistance, support and guidance
during this work. I also would like to express my sincere gratitude to Associate
Professor Yuantong Gu for the technical discussions on the cell morphology
quantitative study and Professor Yin Xiao for his help with cell culture works and
related discussions; without their knowledge and encouragement this study would
not have been successful.
I also extend my thanks to other postgraduates from the Numerical Modelling
Group for sharing literature and providing general advice. Special thanks also go to
Anjali Tumkur Jaiprakash, Dr. Nishant Chakravorty and Dr. Sanjleena Singh for
teaching me to perform cell culture works and for their valuable assistance as well
with all types of technical problems. Further, I would like to thank the Queensland
University of Technology (QUT) for providing a comfortable research environment
and facilities while I completed this project.
Finally, I would like to express my love and gratitude to my beloved
grandparents, parents and my wife, for their understanding, support and
encouragement throughout my candidature.
Morphology of osteocyte cells in normal and hyper-gravity xiii
Publications
This research has resulted in the following fully peer-review paper:
H.Yuan, Y.Xiao, Y.T. Gu, and T. Steinberg, Morphology-based Model for
Predicting Osteocyte-Like Cell Line (MLO-Y4) Growth Process. in 4th
International
Conference on Computational Methods, Australia, Gold Coast, 2012.
Journal paper in preparation:
H.Yuan, Y.Xiao, Y.T. Gu, and T. Steinberg, Morphology of osteocyte cells in normal
and hyper-gravity. (To be submitted to Journal of Bone).
Chapter 1: Introduction 1
Chapter 1: Introduction
1.1 Research Background & Motivations
Cell morphology, one of the most intuitionist characteristics for cell type
identification, is regulated by the interactions between the cytoskeleton, the
membrane and the extracellular environment [1]. Cells of different shape operate
differently in the human body. A round adipocyte allows for maximal lipid storage,
the extended dendrites of nerve cells facilitate electrical and biochemical signal
transmissions, while stellate osteocyte cells are essential for bone remodelling [2-4].
Different cell morphologies are considered to arise from changes in the expression of
integrins, cadherins, and cytoskeletal proteins during stem cell commitment, the
process by which stem cells differentiate into various cell lineages, leading to
different morphologic phenotypes and specialized cellular functions [5]. Intriguingly,
two research groups have recently shown that the various shapes of stem cell can
directly regulate stem cell commitment to different lineages; in other words, cell fate
can be artificially determined through shape-dependent control [2, 6]. These findings
clearly indicate that the function of cells is closely related to their morphology [7].
Likewise, studies conducted in the field of biomechanics have shown that changes in
cell morphology caused by external mechanical loadings can greatly impact on
cellular behaviour including growth, proliferation, differentiation, apoptosis, signal
transduction, gene expression and extracellular matrix remodelling [2, 8-10].
The most common mechanical stimulation which is constantly applied on
living cells throughout their life is gravity. The results from previous studies which
have been carried out on cellular biology in an altered gravity environment have
Chapter 1: Introduction 2
shown that changes in gravity can have direct influence on cellular proliferation,
differentiation, motility and growth [11-18]. Astronauts have been found to have
significant bone mass loss after long-term space missions, which is considered to be
one of the most common and serious health risks during space activity [12]. Under a
weightless environment, bones no longer have to fight against Earth's gravity during
locomotion and less mechanical strain is then applied to the skeletal system [19]. It
is widely accepted that the reduced strain on bones may lead to the speed of bone
removal outpacing replacement and causing the progressive bone loss seen in long-
term residents of space [20, 21].
Mammalian bones comprise three major cell types including osteoblasts,
osteoclasts and osteocytes [22]. Osteoblasts are responsible for constantly building
new bone, while osteoclasts are continually destroying the old ones. The balance
between the bone formation and resorption is crucial for maintaining bone mass [22].
Daily activities of osteoblasts and osteoclasts are believed to be regulated by
osteocytes, the most abundant cell in bone, which account for 90%-95% of all bone
cells [23, 24]. They are embedded in the hard bone matrix of the lacunae, and form
an intercellular network between neighboring osteocytes and the cells on the bone
surface such as osteoblasts and osteoclasts through their dendritic processes within
narrow bony tubes of caliculi [4, 23]. Osteocytes are thought of as the
mechanosensory cells of the bone tissue. They sense and respond to mechanical
loadings, and accordingly transmit the signals to osteoblasts and osteoclasts through
the osteocyte network, and thus regulate bone remolding [4, 23]. It is believed that
osteocytes are playing a central role in space bone mass loss. However, how
osteocytes respond to altered gravity is still not well documented.
Chapter 1: Introduction 3
As a result of the rapid technological improvements in microscopy over
recent years, cell morphology has been commonly used as a key parameter for
investigating cellular response to altered gravity environment. However, in culture,
cells exhibit a wide range of shape and appearance. In previous studies, the
morphology changes in osteocyte cells under various gravity levels are mainly
described based on visual inspection, and investigated by comparing the cell area
with control samples, which may lead to subjective and non-comprehensive
conclusions of how altered gravity impacts on osteocyte morphology. Therefore,
quantitative analysis is required for objectively and accurately studying osteocyte
morphology changes under an altered gravity environment. To date, no attempt has
been made to quantify the morphological changes of osteocytes, which grow in
culture.
There is increasing evidence showing that the osteocyte’s function and
mechanical properties are highly affected by its morphology. It was found that
osteocyte cells with a round* cellular morphology support a less stiff cytoskeleton
and are more sensitive to mechanical stimulations compared with flat cellular
morphology [7]. Studies conducted on flat adherent osteocyte cells have shown there
are no significant changes in the morphology of osteocytes under an altered gravity
environment [21, 24, 25]. However, osteocyte cells found in vivo are with a more 3D
morphology. The impact of altered gravity on osteocytes with a relatively round
shape is still poorly understood.
(*The body of the cells spread during cell culture, thus cells under longer
culturing time are thought have a more flat shape comparing with cells which are
cultured for a shorter time. The terms of ‘round’ and ‘flat’ used in this thesis refers to
the shape of cells observed under normal light microscope (2D)).
Chapter 1: Introduction 4
1.2 Project Aims
This study aims to quantify the morphology of osteocytes in culture for the first
time, and to propose an empirical model based on the collected data to predict the
osteocyte cells’ growth process in culture.
The second aim of this study is to investigate the response of the osteocyte
cells which have a relatively round shape to altered gravity environment. Since it is
not easy to simulate weightlessness on earth, the study will be conducted under a
simulated hyper-gravity environment by using a centrifuge. It is hypothesized that
hyper-gravity often induces the opposite effects to those of microgravity.
1.3 Thesis Outline
The following is a brief synopsis of the chapters to follow:
1.3.1 Chapter 2: Literature Review
This chapter provides a comprehensive review on the functions of osteocyte
cells and the relationship between the osteocyte’s morphology and its functions. This
is then followed by introduction on ground-based altered gravity simulators and a
review of previous morphological studies on osteocyte cells under an altered gravity
environment. A summary of the mathematical shape factors which may be able to be
used to quantify the osteocyte’s morphology is also presented in this chapter.
1.3.2 Chapter 3: Quantitative Morphology Study on Osteocyte Cells
Chapter 3 describes the details of study carried on when quantifying the
morphology of osteocyte cells in culture. Morphology changes of osteocyte cells
recorded for 10 hours in culture, and the consequently developed empirical model are
shown in this chapter.
Chapter 1: Introduction 5
1.3.3 Chapter 4: Osteocyte Cells under Hyper-gravity Stimulation
In this chapter, osteocytes with round shape morphology were applied with 20g
centrifugal force for 30 mins, to investigate the morphology changes under hyper-
gravity environment. The response of osteocyte cells, in terms of morphological
change, including cell area, circularity, aspect ratio and solidity are investigated.
1.3.4 Chapter 5: Conclusions and Future Works
The last chapter re-summarizes the completed work and research findings.
Based on the knowledge gained from this study, the potential future research
direction of the gravitational biology is also proposed in this chapter.
Chapter 2: Literature Review 6
Chapter 2: Literature Review
To ascertain the significance and original contribution of the proposed
research, a comprehensive literature review is performed in Chapter 2. This chapter
summarizes the nature function and the role of osteocyte cells in bone tissue and how
the cellular morphology changes affect their function and mechanical properties.
This is followed by a brief introduction on the application of the most frequently
used gravity stimulation facilities. Previous space biological studies conducted on
osteocyte cells are then shown next. Finally, mathematical shape factors which are
commonly used in quantitative morphology analysis are outlined in this chapter.
2.1 Osteocytes as Descendants of Osteoblasts
The osteocyte cells comprise 90%-95% of the whole bone cell population in
the human body [26]. They are descended from mesenchymal stem cells through
osteoblast differentiation. As osteoblasts become embedded in the newly formed
bone matrix, they continue their differentiation and transform finally into osteocytes.
This process involves several differentiation steps including a reduction of cell
organelles, changes in the cell mechanical properties and cellular morphology.
Osteoblast cells undergo a dramatic transformation from a polygonal cell to a cell
extending dendrites toward the mineralizing front during differentiation. Dendritic
processes are found extending to either the vascular space or bone surface in mature
osteocytes (Figure 1) [27-29].
Chapter 2: Literature Review 7
Figure 1The process of osteoblast differentiation from osteoblast precursors to mature osteocytes [27]
2.2 The Function of Osteocyte Cells
2.2.1 Mechanosensation of Osteocyte Cells
Bone remodelling and functional adaption are generally considered to be
achieved by concerted action of osteoblasts and osteoclasts [30]. As the most
abundant cells in bone tissue, osteocyte cells are believed to be involved in these
bone homeostasis processes as dominating regulatory cells. Osteocyte cells are
embedded in the calcified bone matrix, where osteocyte cell body resides in the
fluid-filled spaces known as lacunae and the cell dendritic processes pass through the
bone in thin canals called canaliculi, forming a lacuna-canalicular network (LCN).
This network connects neighbouring osteocyte cells and cells on the bone surface,
such as osteoblasts, osteoclasts and bone lining cells (Figure2) [4, 31].
Figure 2 Schematic view of the lacuno-canalicular network (middle), formed by osteocyte lacunae
and interconnecting canaliculi (left), and of the connected cellular network (right)
Chapter 2: Literature Review 8
Aarden et al. theorized that cells dispread throughout the matrix can perform
more productively in detecting loading-induced strain than cells located on the bone
surface, such as osteoblasts, osteoclasts and bone lining cells [30]. In accordance
with this theory, as the only cells located at bone matrix, osteocytes are postulated as
mechanosensory cells in bone tissue. The advantage of osteocyte cells in sensing
mechanical stimulations was proved by Mullerder and his colleagues. In order to
evaluate the best candidate for mechano-sensor in bone, Mullerder et al. [32]
investigated the mechanical sensibility of osteocytes and bone lining cells. By
monitoring the structural changes in both the proposed bone lining cell and osteocyte
models under external loading, they concluded that osteocyte cells are several orders
of magnitude more sensitive to mechanical stimulations than bone lining cells.
Several reports from the group of Lanyon have further confirmed the
sensitivity of osteocytes to mechanical loading. In vivo experiments that were
conducted on isolated turkey ulna, embryonic chicken tibia-tarsi and cores of adult
dog cancellous have shown that intermittent loading which applied at the
physiological strain magnitude (varied between 500-2,000 microstrain), produces
rapid strain-related changes in osteocyte’s metabolic activities by introducing the
increased activity of glucose 6-phosphate-dehydrogenas(eG 6PD) and a loading-
related rise in 3H-Uridine uptake in osteocyte cells, separately [33-36].
Previous studies quantified the magnitude of local strain. Bone routinely
experiences mechanical strain between 1000-3,000 microstrain [30, 37]. It is not
clear what is the allowable strain magnitude osteocytes can withstand, however, it is
believed that the damage and resorption of bone occurs if strains exceed 3,500
microstrain, and fracture of bone occurring more than 4,000 microstrain [37].
Chapter 2: Literature Review 9
In vitro experiments found that the minimum strain level to introduce an
appreciable biochemical response in osteocyte cells is 10,000 microstrain, which
would damage the extracellular bone matrix. Thus, it is believed that some form of
strain amplification occurs during strain transfer to osteocyte in vivo [37]. With the
applied load 2,000 microstrain on bone, the measured perilacunar strain is 35,000
microstrain [37].
The loading frequency and number of loading cycles affect the biomechanical
response of osteocytes is not well documented. One Low-Magnitude, High-
Frequency (LMHF) vibration experiment showed that osteocytes are sensitive to this
vibration (magnitude: 0.39g at the frequency 30, 60, 90 Hz) [38]. Since the effects of
loading frequency and number of loading cycles are not related to current work, no
further detailed investigation has been made.
The mechanism of how osteocytes sense the mechanical loads on bone and
how the mechanical loading-induced signal activates osteocyte cells are not yet
completely understood [26]. However, biomechanical studies conducted on osteocyte
cells during the past decade, have shown the high sensitivity of osteocytes to various
mechanical loading-induced physical signals, with changes in the cell structure,
signalling molecules’ secretion and cell metabolic activities. Load that is placed on
bone is found pressurizes the interstitial fluid surrounding the osteocytes before the
fluid is driven to flow. Load in the form of hydraulic pressure, tissue strain and fluid
shear stress are then successively applied on osteocyte cells [39]. Osteocytes were
found particularly sensitive to fluid shear stress. A number of theoretical and
experimental studies have put forth evidence suggesting that rather than hydrostatic
pressure, interstitial fluid flow and direct cell strain are the most likely mechanism
for activating mechanosensation in osteocyte cells. The less response of osteocytes to
Chapter 2: Literature Review 10
hydrostatic stress found in vitro could be due to the low magnitude of hydrostatic
pressure applied (i.e. 13kPa) in the experiment, comparing with the hydrostatic
pressure applied on osteocytes in vivo [26, 40-42]. Zhang[43] estimated that the fluid
component could carry as much as 12% of the applied mechanical load and produce
peak pressures of 2-3MPa. More recently, Gardinier et al.[41] have predicted that
magnitude of the pressure experienced by osteocytes in vivo could reach up to 5MPa.
Research conducted by Liu et al. in 2010 indicated that cyclic hydraulic pressure of
68kPa can modulate signalling molecule production in cells of the MLO-Y4
osteocyte-like cell line, suggesting that fluid pressure may be also a potent stimulus
to the bone and may play a role in regulating bone remodelling in vivo [39].
A well-known early response to mechanical stimulation of osteocytes, both in
vitro and in vivo, is an increase in intracellular calcium concentration [39, 44].
Calcium ions are released from internal stores and passing through ion channels in
the plasma membrane of osteocytes [41, 44]. Calcium is the essential element
required for bone formation; the increased calcification activates many downstream
signalling cascades such as protein kinase C and phospholipase A2, and is necessary
for activation of calcium dependent proteins, such as the constitutive forms of nitric
oxide synthase (NOS). The activation of phospholipase A2 also causes the activation
of releasing prostaglandin E2 (PGE2), one of the substances that can enhance the new
bone formation [39, 45]. Prostaglandins are abundantly produced by osteocytes; they
play a key role in the bone formation response to mechanical loading in vivo. A
rapidly increased prostaglandin production in response to mechanical loading is
observed in several in vitro osteocytes studies [46-48].
A wide range of studies have clearly demonstrated that mechanical stimulation,
both via direct manipulation of cells and via application of a fluid flow to cultured
Chapter 2: Literature Review 11
osteocytes increases the nitric oxide (NO) production [49]. NO has been shown to
modulate the bone remodelling process by promoting bone formation and inhibiting
osteoclast activities[27]. Previous results from the in vitro fluid flow stimulation
studies suggest that NO is a mediator of mechanical effects in bone, and can enhance
PGE2 release in bone tissue [50, 51].
The inhibition of bone formation and activation of osteoclasts are mainly
achieved through the induction of sclerostin and the receptor activator of NFkB
ligand (RANKL) in osteocytes under physiological loading condition [52].
Sclerostin is highly expressed in osteocytes and encoded by the SOST gene. Under
the unloading condition, the increased expression of sclerostin inhibits bone mass
accrual by reducing the activity of osteoblasts [53]. On the contrary, the mice lacking
scelrostin used in Nulend’s experiment, exhibit an increased bone mass, resembling
the human condition of sclerosteosis [42]. Sclerostin was reported down-regulated in
bone cells under mechanical stimulation [42, 52].
As the essential factor for osteoclast formation [54], RANKL inhibits the
increase of bone mass by encouraging the formation osteoclasts. Compared with
osteoblasts and bone marrow stromal cells, osteocytes has been identified as the
major source of RANKL for bone remodelling [54, 55]. Moriishi et al. have recently
found the osteocyte network inhibits osteoblast function and stimulates
osteoclastogensis at physiological loading condition [52]. Both in vitro and in vivo
studies have confirmed the fact that osteocytes produce osteoclast-formation factors
in the absence of external mechanical loading, but not after being subject to a
mechanical stimulus [42]. The study conducted on wild-type mice showed both Sost
and RANKL expressions in osteocytes were significantly up-regulated in the
unloaded condition. Osteocytes further augment the inhibitory effect on osteoblasts
Chapter 2: Literature Review 12
through the induction of Sost and the stimulatory effect on osteoclastogenesis
through the induction of RANKL in the unloaded condition (Figure3) [52]. In
addition, it was found that mice lacking RANKL in osteocytes are protected from
bone loss caused by unloading [54].
Figure 3 Osteocytes modulate bone remodelling at loaded and unloaded condition through the Sost
and Rankl. The thickness of the lines and arrows in A and B reflects the strength of the effects [52]
2.2.2 Mechanotransdution of Osteocyte Cells
As mentioned in last section, osteocyte cells act as a mechanosensor in bone
tissue and respond to mechanical loads by producing biochemical molecules such as
calcium ions, prostaglandins, NO, sclerostin and RANKL, which are considered as
modulators for bone remodelling. To obtain a meaningful change of the existing
bone tissue, osteoblasts and osteoclasts require information on local needs of tissue
increase or tissue reduction, depending on the extracellular mechanical environment
[30]. As the most abundant cells in bone tissue, osteocytes are thought not only
Chapter 2: Literature Review 13
responsible to sense mechanical strain, but also to serve as the messenger to send
converted biochemical signals of bone resorption or formation [4, 31]. This raises an
important issue, that is, to ascertain which part of the osteocytes, its process or cell
body is mechanosensing organelle, and which part is responsible for signal
transduction.
To address this question experimentally, Burra and his research team [4]
isolated the dendrites from the osteocyte cell body using a Transwell filter system;
and investigated the osteocyte’s response to mechanical stimulation through the
determination of connexin 43 hemichannel activity, which is the pathway involved in
communicating cellular signals with extracellular environment. Hemichannel
openings were detected in a cell body when either cell body or dendrites was
mechanically stimulated. However, no significant hemichannel activities were
induced in cell dendrites when mechanical loading was applied to either cell body or
dendritic side. Their results provided a direct evidence suggesting that both cell body
and dendrites are able to sense the external mechanical stimulation and the cell
dendrites are playing a specific role in transducing mechanical-induced signals[4].
Consistently, a study conducted by Adachi et al. showed a higher intracellular
calcium response in osteocyte’s dendritic processes compared to cell bodies under
mechanical stimulation. They concluded that mechanosensitivity of the cell
dendrites was higher than that of the cell body [56].
Cellular signals transduction from deeply embedded osteoytes to bone surface
cells is made possible by the unique three-dimensional morphology of the
lacunocanalicular network formed by osteocytes dendritic processes. Osteocyte’s
processes are radiated in different directions and directly connected with each other
through the gap junctions, formed by proteins called connexions, which are also
Chapter 2: Literature Review 14
known as connexons if they are in hmexameric form [42]. Two connexons present on
adjacent cells dock onto each other to form a gap junction channel which plays an
important role in conveying cellular signals[42]. The canliculi, where the cell
processes are resided is filled with a proteoglycan rich matrix with tethering fibre
that attach the processes to the canalicular wall [42]. The interaction of the
pericellular matrix and the osteocyte cellular process could amplify the physiological
amplitude of loads to the bone tissue, thus producing sufficient levels of force to
induce cellular response [23].
In the original fluid flow hypothesis, loading-induced fluid flow through the
canalicular network has been thought to cause a fluid shear stress on the cell process
membrane, which is considered as the stimuli to activate osteocytes [42]. Previous
research on MLO-Y4 cells showed an increase in the expression of E11, the protein
required for osteocyte cell process elongation, under fluid flow shear stress [57].
Interestingly, the highest expression of E11 was detected in a region of potential
bone remodelling, not in regions of maximal stain, suggesting that dendrite
elongation may be occurring during bone remodelling process [57]. It is unknown
exactly how the changes in the length of dendtritic process will alter the function of
osteocyte cells, but it is clear that changes in ostecoyte cell dendritic phenotype could
have a dramatic effect on the interaction between adjacent ostoecytes and the
structure of LCN, which may lead to the modification in mechanotransduction of
osteocyte cells.
In summary, osteocyte cells are highly sensitive to mechanical loadings. By
translating the mechanical strain into biomechanical signals, osteocytes are playing
as the key regulator for osteoblast and osteoclast activity during the bone
remodelling process. Both cell body and dendritic process are found responsible for
Chapter 2: Literature Review 15
sensing mechanical strain, and cell process is believed to also act as the
mechanotransducer in osteocytes. The function of osteocyte cells is thought to be
highly related to their unique location in bone and their morphology. The effect of
cellular morphology changes on osteocyte’s mechanical property has already been
reported, and will be discussed in the later section of this chapter. Changes in cell
morphology such as the area of cell body and the development of cell process may be
able to lead to the changes in mechanosensation and mechanotransduction of
osteocytes. The proposed study on investigating osteocytes morphology changes in
hyper-gravity can provide valuable insights into how the functions of osteocytes are
going to change in space. A schematic overview of the role of osteocytes in bone
remodelling process is shown below (Figure 4).
Figure 4 Schematic overview of the role of osteocytes in the process of bone remodelling [26]
2.3 Osteocyte Cell Models: Osteocyte-Like Cell Line (MLO-Y4)
While osteocytes are the most abundant cell type in bone tissue, it is also the
type of cell of which we know the least. Over the last two decades, osteoclasts and
Chapter 2: Literature Review 16
osteoblasts have been extensively studied. However, only a few studies have
successfully been conducted to investigate the function and mechanical property of
osteocyte cells by using primary osteocyte cells. This is mainly due to the difficulties
in isolating the osteocytes from the mineralized matrix and maintaining the sufficient
numbers of osteocytes for many in vitro biomechanical studies [3]. To compensate
for these difficulties, scientists have attempted to create osteocyte cell lines by using
a transgenic technique to replace primary osteocyte cells in biomechanical studies.
To date, there are two cell lines with osteocyte characteristics that have been
successfully developed. One model is known as HOB-01 C1 human bone cell line
which is a temperature-sensitive cell line, expressing the property of pre- or called
early osteocytes. Another model is the MLO-Y4 cell line. This cell line was derived
from a transgenic mouse in which the immortalizing T-antigen was expressed under
control of oseocalcin promoter.
MLO-Y4 cell line is the most successful osteocyte cell line developed so far
which exhibits the properties of osteocytes including high expression of osteocalcin,
connexion 43 and antigen E11 proteins and low expression of alkine phosphatease.
Furthermore, MLO-Y4 cells retain a dendritic morphology, similar to that observed
in primary osteocyte cultures. Thus, the MLO-Y4 cell line is now been widely used
to study the function and mechanical properties of osteocyte cells in biomechanical
and biological studies, in vitro. In this study, MLO-Y4 cells will be used to
morphology quantification and hyper-gravity study.
2.4 Hypo- and Hyper-gravity Simulation Technologies
A gravitational biology study is made possible through a number of space-,
flight- and ground- based facilities [58]. Conducting reduced gravity tests by means
of a spaced-based facility such as space shuttle or international space station (ISS) is
Chapter 2: Literature Review 17
difficult and extremely expensive [58, 59]. Several cell biology experiments
conducted in space have failed in collecting results due to unforeseen hardware
failures [60, 61]. Therefore, instead of sending samples to space, researchers are
creating simulated hypo- and hyper-gravity using ground- based facilities such as
magnetic levitation technology, random position machines (RPM), dropping towers
and centrifuges.
A complex biological system, such as a living cell, is diamagnetic, which
means repelled by a magnetic field. Thus the technology of magnetic levitation is
able to provide reduced gravity simulation by creating a stable levitation to living
cells through exerting a magnetic force on the system to counterbalance the
gravitational force [62]. Magnetic levitation can create various gravity levels by
altering the magnetic field strength, and allowing the study of relatively fast process
because of the ability to instantly create the reduced gravity simulation [62].
However, the cellular response observed in magnetic levitation experiments may not
solely be caused by reduced gravity but also by the magnetic field applied [62].
Therefore, further investigation of the application of magnetic levitation in
microgravity study is required.
Random position machines (RPM) are the most frequently used ground-based
reduced gravity simulator (Figure 5). This system is constructed by two separate
rotating frames (inner and outer) which are respectively driven by two motors and
rotate independently at random speeds and directions [63, 64]. The orientation of the
experimental sample mounted at the centre of the inner frame, relative to the
gravity’s vector, is continuously changed during rotating. When the changes are
faster than the sample’s response time to gravity, the effect of gravity on the
experiment sample is considered to be eliminated [65]. Therefore, rotating speed is a
Chapter 2: Literature Review 18
crucial factor for RPMs, and is normally applied at the speed between 60˚/s-120˚/s
[62]. Reduced gravity tests conducted on living cells by using RPMs can last for days
and even weeks, however, providing the required living environment such as
temperature (37˚C) and 5% carbon dioxide supply for these cells is always the
challenge in a long-term reduced gravity study [62].
Figure 5 Random Position Machine [3]
A dropping tower is another ground-based microgravity research facility. The
dropping tower that is available at Queensland University of Technology is 20
metres high and able to generate 2.0 seconds of high quality reduced gravity by free-
falling a dropping package down a vertical corridor. The dropping package contains
the experiment platform which is protected from air resistance by being enclosed
inside the drag shield. The experiment platform is not attached to the drag shield, but
instead falls independently within the enclosed space (Figure 6). All samples
mounted on the platform will experience reduced gravity for approximately 2.0
seconds, then followed by an approximately 0.25 seconds deceleration caused by the
airbag that is designed for safely decelerating the drop package at the bottom of the
Chapter 2: Literature Review 19
tower [58]. This dropping tower has been mainly used to study the effects of
microgravity on physical phenomena such as fluid dynamics and combustion safety.
Figure 6 Diagram for dropping package. [32]
A centrifuge is the most widely available facility which can be used to simulate
hyper-gravity environments to study the responses of living cells to altered gravity.
To conduct hyper-gravity experiments successfully, it is important to fully
understand all possible influences of the acceleration which may cause crucial
differences between the sample exposed to hyper-gravity environment and the 1g
control. Inertial shear forces are caused by the acceleration act perpendicular to the
gravity acceleration vector. The level of inertial shear force that is applied on an
adherent cell layer depends on the radius of the centrifuge and the location of the
cells within the sample surface area [66]. Adherent cells attached to a flat surface
will experience the minimum inertial shear force when located at the point where the
radius is perpendicular to the monolayer surface [66]. In addition, inertial shear force
is found greater in a centrifuge with a smaller radius (See Figure 7) [66]. Another
phenomenon which is also influenced by gravity is hydrostatic pressure. Hydrostatic
pressure is proportional to gravity and height of the liquid (culture medium) [67].
Chapter 2: Literature Review 20
Both inertial shear force and hydrostatic pressure need to be properly controlled
when conducting hyper-gravity experiments on adherent cells.
Figure 7 The inertial shear force increase laterally from the centre of centrifugation [66]
2.4 Response of Osteocyte Cells to Altered Gravity
In order to understand the effects of altered gravity on osteocyte cells, several
studies have already been conducted so far. The first attempt in conducting
microgravity research on osteocytes by using spaceflight, failed due to an
unforeseen hardware complication [60]. By using ground- and flight- based testing
facilities, Shang and his group have recently conducted some studies on MLO-Y4
cells [21, 24, 25]. However, the results shown in their reports are inconsistent. The
first ground-based experiment was conducted by using diamagnetic levitation
technology. A significant decrease in cellular morphology such as cell area, nucleus
size, the number of dendrites and the expression of focal adhesion proteins were
observed after 48 hours diamagnetic-levitation (μg), in addition, the modification in
cytoskeleton organization was also reported. However, no significant changes in cell
Chapter 2: Literature Review 21
morphology, cytoskeleton structure and focal adhesion protein expression were
found under hyper-gravity (2g) stimulation. By comparing the response of MLO-Y4
cells cultured at normal gravity and those applied with magnetic-induced 1g force,
they concluded that there is no magnetic effect on MLO-Y4 cells and all the changes
detected in osteocyte cells are caused by the magnetic-induced microgravity[24].
Another study from this group was started in the year of 2010, using the ZERO-G
A300 aircraft. The altered gravity environment was achieved through the parabolic
flight, where MLO-Y4 cells underwent approximately 22 seconds microgravity (0.5g)
and two hyper-gravity (1.8g) periods before and after each parabola; in total 30
parabolic manoeuvers were performed [21]. Samples collected from parabolic flight
have shown the reduced expression of the connexin 43 proteins and the re-organized
cellular cytoskeleton structure. Interestingly, no significant change in MLO-Y4 cell
area was found in this study [21] in that the cell area of MLO-Y4 reported in this
paper (approx 230 μm2) is dramatically smaller than those measured in the previous
study (approx 3900μm2). More recently, the response of MLO-Y4 cells to a
clinorotation-induced weightless environment has been investigated. No significant
changes in cell morphology were found and the re-organization of cellular
cytoskeleton was only detected after 48hrs of exposure of weightlessness.
Furthermore, the secretions of nitric oxide and prostaglandin E2 were found
decreased after 12hrs weightless stimulation[25].
It is unclear what the reason is for the delayed response of MLO-Y4 cells under
clinostat-induced weightlessness and why there is a dramatic difference between the
cell areas reported from diamagnetic levitation and parabolic flight studies. Based on
the information obtained from the diamagnetic levitation experiment (sample size
10), the inconsistent results may have been caused by the small sample size.
Chapter 2: Literature Review 22
2.5 Osteocyte Cell Morphology, Function and Mechanical Properties
As discussed previously, the unique location and the dendritic cellular
morphology make the osteocyte cells the ideal mechanosensor and
mechanotransducer in bone. It is widely accepted that external mechanical forces
make a direct effect on cytoskeletal structure and thus cell morphology. This implies
that the morphology of osteocytes is determined by their extracellular mechanical
environment; osteocyte cells located in the region which is regularly loaded should
have a different morphology than those found in marginally loaded bones [68]. This
was confirmed by Vatsa al et., who conducted a three-dimensional imaging study on
osteocyte cells located in different parts of the bodies of mice. It was observed that
osteocytes in fibular bone were stretched and aligned along the principal loading
direction, whereas osteocytes in calverial bone had a more spherical shape and were
randomly aligned [68].
In vitro study on MLO-Y4 cells revealed that osteocytes in different
morphology have different mechanical properties [7]. The elastic constant of flat
adherent MLO-Y4 osteocytes was found to be near ten times higher than the elastic
constant measured in the round partially adherent MLO-Y4 cells. Meanwhile, high
NO release from round suspended MLO-Y4 osteocytes was detected when
stimulated with a force of ~5pN. In contrast, lesser NO production was released from
flat adherent MLO-Y4 cells under the force of 150-550-pN. These data suggest that
osteocyte cells in round morphology support a less stiff cytoskeleton configuration
and are much more sensitive to mechanical stimulations compared with flat cellular
morphology[7].
Based on these findings, the relatively round osteocyte morphology observed
by Vasta et al. in calvaria should be more mechanosenstive than fibular oteocytes,
Chapter 2: Literature Review 23
which provide a possible explanation of efficient physiological load bearing for the
maintenance of calvarial bone despite its condition of relative mechanical disuse [68].
In addition, it may also partly explain the reason why no significant morphology
changes in osteocyte cells were observed in the previously discussed microgravity
experiments, where the MLO-Y4 cells were cultured in 2D on a flat surface.
However, osteocytes with a more 3D morphology, such as occurs in vivo, may be
able to sense and respond to such small loading [42].
2.6 Quantitative Cell Morphology Analysis
The term of cell morphology can be interpreted as the description of both
cellular size and shape. Cellular size can be easily presented as cell area in 2-D or
volume of the cell in three-dimensional circumstance. The major difficulty in cellular
morphology quantification is to accurately describe cell shape; this is mainly caused
by the lack of a precise, universal definition of the term [69]. In order to correctly
quantify the shape of living cells, the ideal shape descriptor should be firstly
independent to the size of the cell where values are kept unaltered in the case of cells
of the same shape but different size. Secondly, they need to be able to quantitatively
show how far a given shape deviates from a model, or theoretically ideal shape.
Lastly they should be sensitive to particular shape changes that occur in a process
under consideration (i.e. cell culture or under mechanical stimulation) [69].
The parameters applied extensively in cellular shape quantification in
biomechanical studies are known as shape factors, which are sensitive to the changes
in shape [21, 24, 25, 70, 71]. It is important to define a universal shape factor that is
applicable in all the type of cellular shape analysis. A sphere is a good reference
shape to discuss properties of shape factor because it is the simplest geometrical
model for three-dimensional objects [69]. Images of the spheres are in the form of
Chapter 2: Literature Review 24
circles. Therefore, numerous shape factors are used in images analysis to determine
how much a given shape differs from a circle [69]. However, the name and definition
of shape factors vary with different image analysis software, which always causes
some confusion [72].
The shapes observed in living cells can deviate significantly from a circle, but
finding a ‘reference’ circle is intuitively easy, as illustrated using the series of
sketches shown in figure 8. Elongation, irregularity and composition are illustrated in
Fig 8(a), (b), and (c), respectively, which are considered as three essential cellular
shape change models during cell growth [69].
Figure 8 Three families of shapes originating from a circle: (a) ellipses of various elongation (top), (b)
shape having various edge irregularity (middle), and (c) a combination of the two (bottom) [69]
Cell elongation process can be defined by the shape factor called aspect ratio. It
is mathematically defined as (Equation 2.1):
Chapter 2: Literature Review 25
where a and b are the length and width of the minimum bounding rectangle.
Aspect ratio reaches a minimum value of 1 for an ideal circle and has higher values
for elongated shapes:
Figure 9 Diagram shows the determination of cells with different aspect ratio
The parameter of a which is shown above can also be defined as the maximum
Feret diameter, while b is the Feret diameter measured perpendicular to it, or
alternatively the length of the major and minor axis of best fitted ellipse, depending
on the method used in the different image analysis software. These three sets of
values used to determine elongation can produce slightly different values of the
shape factor due to the digital nature of computer image, but in general, they all can
quantitatively show the degree of the elongation in cells [69].
The irregularity can be quantitatively described by the number of shape factors
including circularity, convexity and solidity. Circularity is one of the most popular
shape factors used in numerous papers. It is also known as roundness in some papers,
where it has been used to determine the degree of the cellular elongation. However,
the name of the roundness may sometimes mislead people, since circularity is found
to be much more sensitive to any irregularity than to elongation [69]. Considering the
natural cell shape, especially for bone cells, they are highly irregular. Thus, in the
cellular morphological analysis study; the different values of circularity collected
Chapter 2: Literature Review 26
may be caused by cellular irregularity rather than the elongation. As a result, the
conclusion of cell elongation made, based on the value of circularity, may not be able
to present the ‘real’ degree of cell elongation. The mathematical definition of
circularity is shown as the following equation [72-76](Equation 2.2):
where P is the perimeter and A is the surface area of the analysed cell. It has a
maximum value of 1 for a circle and lower values for all other shapes. This shape
factor is motivated by the fact that attachment caused shape changes at the early
stage of cell culture will have a similar round shape to floating cells but much more
irregular bumpy cellular boundaries, caused by the appearance of the cellular
dendrites.
Convexity is another good solution to quantify the irregular cells. It measures
the ratio of the perimeter of the convex object to the original perimeter [72, 73]
(Equation 2.3):
Figure 10 Diagram shows cellular boundaries with different roughness
The value of convexity is 1 for a circle and the value will be lower when the
perimeter of the cell is rough [73].
Chapter 2: Literature Review 27
Solidity may more widely be known as compactness, or convexity in some
image analysis software. It is another shape factor that can be used to quantify the
irregularity of cell shape. In two dimensional images, solidity has been
mathematically defined as the ratio of the surface area of the cell and the area
enclosed by the imaginary smallest hull bordering all points of the particle [72, 74,
77] (Equation 2.4):
Figure 11 Diagram shows the determination of cells with different solidity
In some cases, solidity is also defined as the ratio of the cell’s area and the
bounding rectangular with the least surface that can contain the cell. A cell with a
regular cell boundary has a higher value of solidity, and the lower value of solidity
indicates the irregular cell shape.
Cell shape changes during cell growth process involving both cell elongation
and the appearance of the cell dendrites, which causes the increased irregularity.
This transition of cell shape cannot effectively be described using any of the shape
factors shown previously alone. The shape factor called spherity shown below is
sensitive to both elongation and irregularity, and can be mathematically defined as
[69, 73]:
Chapter 2: Literature Review 28
where d1 and d2 are the diameters of the maximum inscribed and circumscribed
circles, respectively. It quantitatively describes the complex deviation from the ideal
circle due to both elongation and irregularity. However, the single number calculated
for the spherity cannot directly interpret the cell elongation and the development of
the cell dendrites; it is thought to be quite insufficient for correct cell shape
classification [69].
Figure 12 Diagram shows the determinations of spherity which is sensitive to most irregularity and
elongation
In summary, there is still some confusion in terms of shape factor name and
definition, caused by the different methods used to estimate the basic dimensions of
the cell in different image analysis software. An appropriately selected shape factor
carries sufficient information for the identification of the cell shape. However, it is
important to keep in mind that it is not possible to accurately describe any shape by a
single number. Even the best shape factor available can only quantify either
elongation or irregularity of cell, thus, usually more than one parameter must be
applied for the purpose of classification [69, 73].
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 29
Chapter 3: Quantitative Morphology Study on Osteocyte-
Like Cell Line (MLO-Y4)
3.1 Introduction
Cell morphology has been commonly used as a key parameter for quantitative
study in various research disciplines. In cell culture research, the various cell shapes
indicate the different status of the cells including differentiation, proliferation and
apoptosis [1]. Hence, by monitoring the morphology of cells in culture a quick and
easy way to determine the quality of a culture sample is made possible. In addition,
measuring cell morphology changes under various mechanical loadings is also a
widely applied method to evaluate cellular mechanical properties in biomechanical
studies.
As mentioned in the literature review, it is believed that osteocyte cells act as
mechanosensor/transducers in bone tissue and are playing a very important role in
bone remodelling process [32, 78]. Thus, it is hypothesized that osteocytes may be
responsible for astronauts’ bone mass lost observed after long term space missions,
and the gravity induced morphology change will highly affect their functions of
ostecoyte cells. Numbers of in vitro researches have been conducted to find out
morphology changes of osteocyte cells under an microgravity environment. However,
it is surprising to find that no study has been conducted to quantitatively investigate
the morphology development of osteocytes in culture. In other words, people are
using the cultured osteocyte cells to study the gravity-induced morphological
changes without the knowledge of the morphological characteristics of osteocyte
cells in culture.
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 30
In this chapter, we have quantitatively investigated the changes of
morphology for osteocyte-like cell line (MLO-Y4) in culture over time period of up
to 10 hours. The morphology changes of MLO-Y4 cells are studied in details by
measuring the average value of cell area and dimensionless shape factors at different
culturing time. An empirical mathematical model is then developed based upon this
acquired data to predict the MLO-Y4 cells’ growth process in culture.
To ensure the representativeness of the result shown in this study, the
morphological study of MLO-Y4 was performed twice under the same experimental
conditions, by using different groups of MLO-Y4 cells
3.2 Methods
3.2.1 Cell Culture and Fixation
MLO-Y4 osteocyte-like cells (passages 53) were seeded on collagen-coated
cover slips in a 24-well plate and cultured in Dulbecco’s Modified Eagle medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%
Penicillin/Streptomycin (P/S). In order to accurately characterize the cellular
morphology, MLO-Y4 cells were cultured at low density (5×103 cells per well).
Starting from the end of the first culturing hour, one well of cells was fixed using 4%
paraformaldehyde (PFA). This was repeated every hour for 10 hours. .
3.2.2 Fluorescence Staining
After 30 minutes fixation with PFA, MLO-Y4 cells were washed in phosphate-
buffered saline (PBS) and permeabilized with freshly prepared 0.1% triton X-100 in
PBS for 5 minutes, followed by two washes with PBS. The MLO-Y4 cells were then
incubated in blocking solution prepared with 1% bovine serum albumin (BSA) in
PBS for 10 minutes at room temperature. F-actin filaments were then stained by
Alexa Fluor 568 phalloidin and the cell nucleus was marked by Hoechst stain. In
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 31
order to obtain higher accuracy of the osteocytes morphologic characteristics, a large
number of fluorescent images were collected by using Zeiss fluorescent microscope
(Zeiss, Germany) for subsequent analysis.
3.2.3 Cell Segmentation
To accurately find the outline of a cell is an important but difficult task, which
requires correct isolation of cells from background. In this study, MLO-Y4 cells’
morphological features were determined using the widely accepted image analysis
software (ImageJTM, v.1.45s, National Institute of Health, Bethesda, MD). In order
to facilitate the segmentation, the uneven background illumination was corrected by
the application of contrast-enhance function in ImageJ. All fluorescent images were
then converted into 8-bit-grayscale images before starting to measure the
morphological parameters. Cell morphological features of MLO-Y4 cells were
extracted by visually choosing a threshold intensity value which most closely
coincides with the outline of the cells as perceived by the operator (Figure 13). The
intensity of background is relatively lower that fluorescent stained cells, the signal-
to-noise ratio is high, thus making the selection of threshold value highly reliable.
3.2.4 Morphologic Feature Extraction
Correct cell characterization requires the extraction of meaningful parameters
which accurately represent cell morphological changes over time from collected
images. As mentioned in the last chapter, osteocytes are described as stellate shape
cells; the dendrites of osteocytes are playing crucial role in regulating bone
remodelling. Thus, mathematic shape factors used here should not only represent the
size and shape changes of the osteocytes over the culturing time, but also need to
accurately quantify the dendrite development of osteocytes in culture.
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 32
In this study, cell area was measured to investigate the size changes of MLO-
Y4 cells and three dimensionless shape factors, including aspect ratio, circularity and
solidity, were measured and subsequently used to quantitatively define the MLO-Y4
cell shape changes as a function of culture time (Figure 14).
Aspect Ratio
Aspect ratio was used to identify the elongation of MLO-Y4 cells in culture. In
ImageJ (National Institute of Health, Bethesda, MD), aspect ratio is defined as
follows:
Where a and b are the length of the major-axis and semi-axis of the best fit
ellipse to the selected cell, respectively. The fitted ellipse has the same area,
orientation and centroid as the extracted cell (Figure 13-b). In order to normalize the
value of dimensional shape factors in the region between 0.0-1.0, the reciprocal value
of the calculated aspect ratio (
) is used to determine the cell elongation in this
study. Thus, a perfect circle will have the maximum aspect ratio value of 1.0 while
an elongated shape will have a lower aspect ratio approaching zero.
Solidity
The solidity is used to investigate dendrite development of osteocytes. In two
dimensional images, solidity is defined as the ratio between the cell area and its
convex area (Figure 13-d). The elongated dendrites are thought to create a larger gap
between the outline of the convex and the outline of the cell:
Thus, a solid cell will have the solidity value of 1.0; the cell with extended
dendrites will have a lower solidity value.
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 33
Circularity
As mentioned before, circularity is the most common shape factor used to
describe cell morphology in many studies. A perfect circle will have the circularity
of 1.0; a value of zero indicates a highly elongated cell shape. However, the
circularity is mathematically defined as the relationship between the area and
perimeter (Equation 2.2), where A is the cell area and P is the perimeter of cell. In
the case of the cell with highly irregular boundary, circularity may be unable to
accurately indicate the degree of cellular elongation.
Thus, in this study, circularity was used with aspect ratio to investigate the
irregularity of cells at an early stage of culture. If two cells have a similar aspect ratio,
the one with a higher circularity has a “bumpier” boundary.
In this study, we assumed that all MOL-Y4 cells are perfectly spherical before
attached to the collagen-coated substrate at time zero, and having the aspect ratio,
solidity and circularity value of 1.0 at time zero.
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 34
Figure 13 Cell Segmentation procedure: (a) Original fluorescent image; (b) Fluorescent image with
enhanced contrast; (c) 8-bit image; (d) Binary mask image
a
b
c
d
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 35
Figure 14 the protocol of determining shape features (a) the original fluorescent image; (b) Fitted
ellipse to extracted cell shape; (c) Extracted cell area and perimeter; (d) Convex hull of extracted cell shape.
a
c d
b
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 36
3.2.5 Statistical Analysis
Although a large number of fluorescent images were collected for MLO-Y4
cell morphology characterization, compared with the whole population the sample
size is still relatively ‘small’. To minimize the influence of the limited sample size on
the accuracy of calculated mean values of extracted morphology features,
bootstrapping tests were performed on all collected data with the aid of Fathom
statistics software (KCP Tehcnologies,Inc). Bootstrapping was applied by repeatedly
re-sampling the original data using sampling with replacement, and calculating the
mean for new samples (bootstrap samples). Bootstrapping was performed 500 times
for each group of data. The mean value calculated from the re-sampled data was used
to represent the ‘real’ morphological features at different culturing times and thus
provided a better representation of the entire population.
3.2.6 Curve Fitting
3.2.6 (a) Mathematical models for measured cell area
Non-linear regression was performed to fit the mathematical models to the
experiment data using biostatistics program, GraphPad Prism 6 (GraphPad Software,
Inc, USA). Based on the tendency of the data, one phase exponential association
equations (Equation 3.1) and hyperbolic tangent function (Equation 3.2) were
preliminarily picked as two potential models for fitting the data of MLO-Y4 cell area
changes in culture.
One-phase exponential association model:
Where t is the culturing time in the unit of hour, t0 has the same unit of t and is
determined to be 1. Ya starts at A0, then goes up to plateau with one phase. A0 and
plateau are in the same units as Ya (μm2). Cell area is equal to A0 when t=0, hence,
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 37
A0 has been defined to be greater than zero during curve fitting. k is the rate
constant of cell area spreading, which in units are the reciprocal of the x-axis units
(hr-1).
Hyperbolic tangent function:
Where t is the culturing time in the unit of hour, t0 has the same unit of t and is
determined to be 1. k1 is the cell area spreading rate, S1 and A1 are constant numbers
and in the same unit as Ya. According to the hyperbolic tangent function shown
above, cell area (Ya) is equal to the constant number A1, when t=0. Thus, the constant
A1 is constricted in a range between zero and the value of cell area measured at the
first hour in culture. The parameter of S1 is defined as the span of the curve.
3.2.6 (b) Mathematical models for dimensionless shape factors
The reciprocal equation (Equation 3.3) and one-phase exponential decay model
(Equation 3.4) were chosen as two initial models to fit the three dimensionless shape
factors.
Reciprocal equation
Where, t is the culture time; k2 is decay rate of the curve. As it is assumed that
MLO-Y4 cells are in perfect sphere when t=0, b1 is then determined to be constant
value 1.
One-phase exponential decay:
Where, t is the culture time, k3 is the rate constant. Curve starts at b2 and decays
down to the unknown constant c2. The constant b2 was determined to be 1.
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 38
3.3 Results
3.3.1 Cell Area & Culturing Time
Data collected from two experiments have shown the same trend in cell area
changes. In the cell cultures, it was observed that MLO-Y4 cells adhered rapidly to
the collagen-coated substrate. The mean area of MLO-Y4 cells after 1 hour culture
was measured as 407.54 μm2
and 473.21 μm2 in two experiments, respectively.
MLO-Y4 cells have experienced a rapid-growth in their cell area at early stage of
cell culture, where the area MLO-Y4 cells were found to be nearly tripled in the first
three hours. After that, cell area was then gradually increased until approximately
1400 μm2. No significant changes in cell area were observed after 5
th hour (Table 1).
Table 1 MLO-Y4 cell mean area measured with different culturing time
Time(hours) Mean Area (μm2)
Experiment 1 Experiment 2
1 407.54 473.21
2 839.85 888.52
3 1214.09 1235.84
4 1273.55 1291.97
5 1432.25 1498.82
6 1408.73 1434.63
7 1463.44 1421.03
8 1471.22 1411.89
9 1487.09 1532.65
10 1425.82 1456.9
3.3.2 Cell Shape & Culturing Time
The results of three dimensionless shape factors measured from two
experiments over 10 hours culture time are shown in the following tables. The data
of shape factors collected from two separate experiments have shown the same
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 39
tendency in cell shape changes. Changes in these shape factors quantitatively
indicated the shape development of MLO-Y4 cells in culture.
Data from Table 2 shows the changes in the value of aspect ratio which
indicate the elongation of MLO-Y4 cells in culture. High aspect ratio seen at the 1st
of culturing suggests that MLO-Y4 cells keep as round shapes at early stage of cell
culture. The reduced aspect ratio obtained at 2nd
hour shows that the elongation
process starts as soon as the cell attachment occurred. The biggest drop in the value
of aspect ratio is found at the 4th hour and no significant changes in aspect ratio are
observed afterwards. The major-axis of the best fit ellipse of fully developed MLO-
Y4 cells is found about 1.5 times longer than the semi-axis.
Table 2 Aspect Ratio measured for MLO-Y4 cells in culture from two experiments.
Time(hours) Mean Aspect Ratio
Experiment 1 Experiment 2
0 1 1
1 0.822 0.823
2 0.781 0.794
3 0.721 0.755
4 0.638 0.651
5 0.635 0.660
6 0.636 0.652
7 0.619 0.615
8 0.650 0.620
9 0.641 0.659
10 0.610 0.606
Changes in data of solidity presented in Table 3 provide a sign of how cellular
dendrites are developed in culture. The decreased ratio of cell area and its convex
area at an early culturing hour reveals the fact of high vitality of MLO-Y4 cellular
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 40
dendrites. According to the data collected for aspect ratio and the solidity, it is
suggested that the development of cellular dendrites occurs in parallel with the cell
elongation. Interestingly, the biggest drop in the value of solidity is also found
between the 3rd
and 4th hour culturing time. As a result of proliferation and the
development of cellular dendrites, cells were starting make contact with other. The
elongated dendrites are thought to be the cause of some fluctuation in the results of
solidity as seen in Table 3.
Table 3 Solidity measured for MLO-Y4 cells in culture from two experiments.
Time(hours) Mean Solidity
Experiment 1 Experiment 2
0 1 1
1 0.866 0.876
2 0.794 0.768
3 0.668 0.684
4 0.536 0.482
5 0.511 0.489
6 0.479 0.472
7 0.452 0.468
8 0.488 0.399
9 0.341 0.481
10 0.395 0.423
The table below shows the changes in circularity measured from two
experiments. The low circularity values are caused by the irregular cell boundaries,
which indicate rapid attachment of MLO-Y4 cells and the appearance of the cellular
dendrites. The extremely low circularity obtained at the later cell culture hours is
thought to be the result of cell elongation as well as the development of cellular
dendrites.
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 41
Table 4 Circularity measured for MLO-Y4 cells in culture from two experiments
Time(hours) Mean Circularity
Experiment 1 Experiment 2
0 1 1
1 0.602 0.622
2 0.437 0.407
3 0.271 0.283
4 0.153 0.131
5 0.146 0.121
6 0.118 0.110
7 0.100 0.114
8 0.119 0.080
9 0.062 0.110
10 0.082 0.098
3.3.3 Model Selection for Curve Fitting
The goal of the non-linear regression is to fit a model to the two experiment
data with the best-fit values. The best model for fitting the experiment data is
determined by using the least second square method.
As shown in Table 5, both one-phase exponential association and hyperbolic
tangent models fit well to the data of osteocytes cell area changes in culture. The
hyperbolic tangent model has the absolute sum of squares 20590, which is slightly
higher than the absolute sum of squares in the one phase association model. Hence,
the one phase association model (Equation 3.1) is used to fit the data of cell area
changes in culture.
Table 5Goodness of fit for cell area in culture
Model R2 (%) Absolute Sum of Squares
One-phase Exponential Association
Model 98.61 14738
Hyperbolic Model 98.06 20590
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 42
Reciprocal equation and one-phase exponential decay models are used to fit the
three dimensionless shape factors. One-phase exponential decay model shows less
absolute squares and higher coefficient of determination (R2) in all three shape
factors. Thus, the one-phase decay model (Equation 3.4) is used to fit the data of
shape factors change in culture (Table 6).
Table 6 Goodness of fit for shape factor in culture
Model Shape Factors R2 (%) Absolute Sum of Squares
Reciprocal Equation
Model
Circularity 97.25 0.0234
Solidity 96.87 0.0132
Aspect Ratio 69.50 0.0440
One-phase
Exponential Decay
Model
Circularity 99.62 0.0032
Solidity 97.80 0.0092
Aspect Ratio 96.88 0.0045
3.3.4 Fitted Curves and Developed Emprical Models
By using the selected models, the fitted curves and developed emprical models
for call area and three dimensionless shape factors are shown below:
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 43
Cell Area
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
C e ll A re a
T im e (h r )
Ce
ll A
re
a (
um
^2
)E x p e rim e n t O n e
E x p e r im e n t T w o
Figure 15 Cell Area changes of MLO-Y4 cells measured from two tests
MLO-Y4 cell area change in cell culture is mathematically defined as:
The empirical model developed from the obtained data shows that MLO-Y4
cells are spreading at the constant rate of 0.1704 hour-1
in culture. Based on the
empirical model, the predicted cell area of a MLO-Y4 cell before the attachment is
μm2.
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 44
Aspect Ratio:
Figure 16 Aspect ratio changes of MLO-Y4 cells measured from two tests
Aspect ratio change in MLO-4 cells is mathematically defined as:
The empirical model of aspect ratio changes in culture suggested that MLO-Y4
cells were elongated at the constant ratio of hr-1
during cell culture.
Solidity
Figure 17 Solidity changes of MLO-Y4 cells measured from two tests
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 45
Solidity changes with culturing can be defined as:
)
Where, the span of the curve on the Y axis is and the solidity dropped
at the rate of hr-1
.
Circularity
0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2
0 .0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .8
0 .9
1 .0
1 .1
C ir c u la r ity
T i me (h r )
Cir
cul
arit
y
E xp e r i m e nt O n e
E x p e r im e n t T wo
Figure 18 Circularity changes of MLO-Y4 cells measured from two tests
The relationship between the circularity and culturing time is then defined as:
Where the empirical model shows that the circularity of MLO-Y4 cell is
changed at rate of 0.533 hr-1
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 46
Figure 19 MLO-Y4 cell morphological appearance in culture. (a) 1hr; (b) 2hr; (c) 3hr; (d) 4hr; (e) 8hr;
(f) 10hr (scale bar:50μm)
b
c
e f
a
d
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 47
3.4 Discussion
Previous study has already confirmed that the cell growth in culture will
produce significant morphology changes in MLO-Y4 cells [3]. However, the MLO-
Y4 cell growing process described in the past paper was slightly delayed compared
with the data obtained in this study. Kato et al. reported that MLO-Y4 cells were
small and stellate after 3- 6 hours of cell culture and became elongated and started
branching after 1 day. On the other hand, cell elongation and the development of
cellular dendrites observed in this study are found to start as soon as the cell
attachment has occurred. The difference in MLO-Y4 cell growth speed observed in
two studies would possibly be caused by using the different passage numbers of
MLO-Y4 cells and the influence of cell culture method. Cells used in Kato’s
experiment are very early generations of MLO-Y4 cells which were directly isolated
from the transgenic mice. Thus, the cells which were used in their study could
possibly have some other type of cells mixed, even though the probability is very low.
In addition, comparing with the early passage numbers, the subsequent passages
might be selecting the cells that can keep on proliferating, and therefore lose the slow
dividing cells and the ability to grow faster. Besides the different passage numbers
used, the different times that cells are incubated in trypsin during subculture will also
highly affect the vitality of MLO-Y4 cells as well as the cell morphology at early
stages of cell culture, which may also delay the cell growth process.
In this study, MLO-Y4 cells were cultured at the density of 5×103 cells per
well in a 24-well plate. Cells are growing very slowly in an extremely low density
environment and the limitation of space for cells to spread into will also affect the
cell growth process if the initial culture density is too high or the culture is in the
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 48
confluent phase. Therefore, cell density is another very important factor which will
affect cell growth as well as cellular morphology in culture.
To quantify cell shape changes of MLO-Y4 cells with growth and proliferation,
three dimensionless shape factors were used in this study. The selection of shape
factors was based on the expression of the dendritic phenotype of osteocyte cells.
Aspect ratio was used to quantify the elongation of MLO-Y4 cells in culture, solidity
was selected to describe the development of cellular dendrites and the changes in the
value of circularity provide a sign of cell attachment caused irregularity at the early
stage of cell culture. It is important to keep in mind that there is no possibility to
define a cell shape by using only one signal shape factor, thus, in order to intuitively
describe a cell shape different shape factors are needed. Different shape factors may
be required for different cell types depending on their morphological characteristics
in culture.
Cell morphology varies for individual MLO-Y4 cells at the same culturing
time point. To generate an empirical model that can accurately predict morphology
changes at different culture times, an accurate mean value of measured
morphological features is crucial. In this study, the high signal-to-noise ratio of
fluorescent images ensures the reliability of the measured morphological parameters.
The large sample size measured in this study and the application of the bootstrapping
technique increases the statistical precision of the measured parameters. Compared
with other statistical re-sampling methods such as Cross-validation and Jackknife,
bootstrapping re-sampling original samples using sampling with replacement is more
accurate than sampling without replacement in terms of simulating chance.
Chapter 3 Quantitative morphology study on osteocyte-like cell line (MLO-Y4) 49
3.5 Conclusion
This study quantitatively defined the relationship between cellular morphology
and culturing time for MLO-Y4 cell line. The quantified morphological parameters
of MLO-Y4 cell can be used as the reference data for investigating morphological
response of MLO-Y4 cells to mechanical stimulations. The different morphology
parameters measured under loadings may indirectly reflect the modification of
cellular downstream signalling activity, which then can lead to further studies at
molecular level. With the aid of powerful image processing tools and the advanced
curve fitting software, empirical models were successfully developed based on the
obtained data to quantitatively predicate the morphological changes of MLO-Y4
cells in culture. By using the developed empirical models, all morphological
parameters of MLO-Y4 cells can be simply calculated and can be used to efficiently
monitor the quality of the cell culture. The rate of size change, elongation and
dendrites development of MLO-Y4 cells, defined from empirical models provide the
chance to investigate the effect of altered gravity to MLO-Y4 cell growing process.
Chapter 4: Osteocyte cells under hyper-gravity stimulation 50
Chapter 4: Osteocyte Cells under Hyper-gravity Stimulation
4.1 Introduction
Cellular mechanical property is highly related to cell morphology. As
mentioned previously, round shape MLO-Y4 cells are found to have a less stiff
cytoskeleton configuration and are more sensitive to mechanical stimulus than flat
adherent MLO-Y4 cells. Most in vitro studies are cultured MLO-Y4 cells on two-
dimensional (2D) flat surfaces, however, by using confocal microscopy, osteocyte
cells are found have three-dimensional conformation in bone tissue[31, 79, 80]. The
relative flat and spread shape of isolated osteocytes in 2D culture may greatly
hamper their sensitivity to a mechanical stimulus[7], thus, previous in vitro space
studies that investigate flat adherent MLO-Y4 cells may not be able to accurately
reflect the response of in vivo osteocytes to altered gravity.
In this chapter, the morphological response of MLO-Y4 cells with a relatively
round morphology under hyper-gravity environment has been quantitatively
investigated. The ideal MLO-Y4 cell model used in this study has the dendritic
phenotype and a spherical cell body. Based on the data as detailed in the last chapter,
MLO-Y4 cells cultured for two hours were used. Changes in the morphology of
MLO-Y4 cells are quantitatively analysed by measuring the average value of cell
area and dimensionless shape factors such as aspect ratio, solidity and circularity.
To ensure the representativeness of the result shown in this study, centrifuge
experiment has been performed twice under the same conditions.
Chapter 4: Osteocyte cells under hyper-gravity stimulation 51
4.2 Materials and Methods
4.2.1 Cell Culture
Osteocyte-like cells (passage number 46) were seeded on collagen-coated
cover slips placed in 24-well plates, and cultured in Dulbecco’s Modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%
Penicillin/Streptomycin (P/S). Cells were incubated at 37oC in humidified 5% CO2
for 2 hours before the experiment to allow cell attachment. The seeding density was
5.0×103 cells per slide and 1.0 microlitre (μL) culture medium was filled in each well.
In order to minimize the unwanted inertial shear force generated via the rotating of
the centrifuge, only the central 4 wells were used in the 24-well plate (Figure 20).
Figure 20 Four wells located at the centre of the 24-well plate were used
4.2.2 Centrifuge Experiment
Centrifugal force was applied by using Allegra X-15R centrifuge (Beckman
Coulter. Inc) equipped with an SX4750 swinging bucket rotor which allows
centrifugation of multi-well plates (Figure 21). The multi-well plate carriers were
reached vertical at the minimum permitted system operation speed (200rpm). Two
24-well plates were placed symmetrically with respect to the rotation axis. The
centrifuge experiment was then performed at 390 rpm (20g) for 30mins at room
Chapter 4: Osteocyte cells under hyper-gravity stimulation 52
temperature. Culture plates for controls were placed on the lid of the centrifuge, as a
control for vibration effects.
Figure 21 (a): Side view of the rotor at speed; (b) Top view of the rotor at rest.
4.2.3 Post-Centrifugation Processing of MLO-Y4 Cells
Cell fixation was applied immediately after the centrifuge experiment was
finished. Culture medium was aspirated and MLO-Y4 cells were rinsed twice in
phosphate-buffered saline (PBS); cultures then underwent 30 mins fixation by
immersion in 4% paraformaldehyde (PFA) at room temperature. Following fixation
Alexa Fluor 568 phalloidine and Hoechst stain were used to stain the F-actin
filaments and cell nucleus, respectively. The details of florescent staining are
described in the last chapter of this work. Large amount of fluorescent images of
MLO-Y4 cells were collected by using Zeiss fluorescent microscope (Carl Zeiss Inc,
Germany) for subsequent analysis.
Similarly as described in the last chapter, the morphological features of MLO-
Y4 cells were determined using the widely accepted image analysis software, ImageJ
(ImageJTM
, v.1.45s, National Institute of Health, Bethesda, MD). Image analysis was
performed as described previously in the last chapter. Cell area, aspect ratio, solidity
(a) (b)
Chapter 4: Osteocyte cells under hyper-gravity stimulation 53
and circularity were applied to quantify the effect of hyper-gravity to the morphology
of MLO-Y4 cells.
4.2.4 Statistics
Statistical analysis was performed using Minitab 16 statistics software (Minitab
Inc, State College, PA, USA). All data were expressed as mean± standard deviation
(SD). T-tests were performed on all morphological data and a value of p<0.05 was
considered significant in all analysis.
4.3 Results
4.3.1 Cell Area under Hyper-gravity
The average area of MLO-Y4 cells measured in both centrifuge and control
samples from two experiments are shown below. No significant changes in cell area
were observed in MLO-Y4 cells simulated with hyper-gravity, compared with
control samples (Figure 22).
Figure 22 Graphical illustration of cell area measured for MLO-Y4 cells cultured at 20g (blue bar)
and 1g (purple bar) for 30 mins from two experiments. (a) The mean area measured for experiment
sample is 853.52μm2 and 874.06 μm2 for control sample in experiment 1 (P=0.388). (b) The mean area measured for experiment sample is 866.1μm2 and 876.73μm2 for control sample in experiment 2
(p=0.669). n=500
Chapter 4: Osteocyte cells under hyper-gravity stimulation 54
4.3.2 Cell Shape under Hyper-gravity
The average values of aspect ratio, solidity and circularity, collected from two
centrifuge experiments, are shown blow in Figure23, 24 and 25, respectively. No
significant changes in the value of aspect ratio, solidity or circularity were found in
the MLO-Y4 cells exposed to the hypergravity environment, compared to control
cells, which indicate that increased gravitational force does not affect cellular
elongation and dendrites development.
Figure 23 Graphical illustration of aspect ratio measured for MLO-Y4 cells cultured at 20g (blue bar)
and 1g (purple bar) for 30 mins from two experiments. (a) The mean aspect ratio measured for
experiment sample is 0.843 and 0.851 for control sample in experiment one (P=0.232). (b) The mean
area measured for experiment sample is 0.846 and 0.847 for control sample in experiment two (p=0.846). n=500
Chapter 4: Osteocyte cells under hyper-gravity stimulation 55
Figure 24 Graphical illustration of solidity measured for MLO-Y4 cells cultured at 20g (blue bar) and
1g (purple bar) for 30 mins from two experiments. (a) The mean aspect ratio measured for experiment
sample is 0.864 and 0.863 for control sample in experiment one (P=0.791). (b) The mean area
measured for experiment sample is 0.866 and 0.864 for control sample in experiment two (p=0.718).
n=500
Figure 25 Graphical illustration of circularity measured for MLO-Y4 cells cultured at 20g (blue bar)
and 1g (purple bar) for 30 mins from two experiments. (a) The mean aspect ratio measured for
experiment sample is 0.573 and 0.562 for control sample in experiment one (P=0.053). (b) The mean
area measured for experiment sample is 0.581 and 0.562 for control sample in experiment two
(p=0.256). n=500
Chapter 4: Osteocyte cells under hyper-gravity stimulation 56
4.4 Discussion
Round shape ostecoytes are thought to have less stiffness, which makes them
easier to deform compared with flat adhesion cells. It is also believed that compared
with highly spread osteocytes, a relatively round shape osteocyte has the mechanical
property which is closer to osteocytes in vivo. Therefore, instead investigating flat
adhesion osteocytes, centrifugal study was conducted on round shape MLO-Y4 cells.
MLO-Y4 cells were exposed to 20g for 30mins at room temperature; however, our
initial results show that there are no significant changes, neither in cell area nor the
shape of MLO-Y4 cells under hyper-gravity environment. No attempt has been made
before to investigate the effect of hyper-gravity to osteocyte cells but few
microgravity studies were conducted. As described previously, conflicting results in
cell morphological response to simulated microgravity stimulations were reported
from Sheng and his group. Cell area of osteocytes was found dramatically decreased
under the diamagnetic levitation environment, however, both two-dimensional
clinorotation and parabolic flight experiments showed that no morphological
difference was detected in ostoecyte cells exposed to simulated microgravity,
compared to 1g control cells[21, 24, 25].
Several hypergravity studies have already been conducted on osteoblasts which
are the progenitors to osteocyte cells, and the results appear contradictory. Kacena
and his colleagues [81] reported an approximately 4%-24% and 33%-58% increase
in osteoblasts cell area, with centrifugation at 3.3g and 4.0g, respectively. However,
Gebken et al. showed no changes in osteoblast’s morphology after being centrifuged
at 13g for 24 hours [82]. Similar results were also reported by Searby et al., who
concluded that the morphology of osteoblasts exposed to 10g for 3 hours was
indistinguishable to that of the controls [83]. These varying results may be explained
Chapter 4: Osteocyte cells under hyper-gravity stimulation 57
by the different passage number used, the type of cells studied and the G-level,
duration of exposed to altered gravity environment. Kacena et al. [81] claimed that :
‘For shorter growth durations, a higher G-level may be required to induce
cytoskeleton changes in osteoblasts, alternatively, a lower G-level can also induce
significant changes if the growth duration is extended.’
Based on this theory, we further examined the effect of extended exposure time
to MLO-Y4 cells by increasing the centrifuge time to 3hours, using the cells only
cultured for 1hour, which are thought to be more sensitive to mechanical stimulus
compared with MLO-Y4 cells cultured for 2 hours. Similarly to the previous test, no
significant changes in cell are were found in centrifuged MLO-Y4 cells, compared to
1g control cells (data not shown).
Although no hypergravity-induced morphology change in osteoblasts was
reported in Searby’s study, the height of osteoblast was found to be decreased.
Around 7% and 26% decrease in cell height was respectively measured at 5g and 50g
levels [83]. The observation of height changes in osteoblast under a hypergravity
environment was further confirmed by Van et al. By taking the advantages of atomic
force microscope (AFM), Van and his colleagues have successfully measured the
height change in osteoblasts under increased gravitational stimulation, in situ. The
height of osteoblasts was found reduced by around 30%-50% at only 2g-3g loadings
within 4 mins [84]. In comparison with Van’s results, the reason for less decrease in
cell height with higher G-level reported by Searby could be due to the fixation
method used. Chemical fixation normally needs at least 20mins to fully fix the
cellular cytoskeleton at room temperature. However, changes in cell morphology can
be quite fast. Previous study showed chondrocyte deformed and reached an
equilibrium displacement within 2mins, after application of stress by using
Chapter 4: Osteocyte cells under hyper-gravity stimulation 58
micropipette aspiration [85]. Results from the study conducted by McGrath et al.
suggested that the typical turnover time of cytoskeleton actin filaments appears to be
around 6mins [86]. Thus, morphology recovery may occur during chemical fixation
period, leading to the inaccurate measurement of cell morphology changes in hyper-
gravity/microgravity studies.
Previous studies showed that osteocytes and osteoblasts have similar cell
stiffness. The elastic constant measure for osteoblast cells was in the range of 1.4kPa
to 1.9kPa [87], and the elastic constant of osteocyte cell is found around 143Pa-
300Pa for round shape osetcoytes, and from 2.6kPa to 4.3kPa for flat osteocytes [7].
Thus, theoretically speaking, osteocytes should have similar or even better
deformability, in the case of round osteocytes, than osteoblasts under hypergravity
stimulation. Results from our centrifuge study, which showed no morphology change
under hypergravity (20g) stimulation, implies that cell height is probably a more
appropriate parameter than cell area for morphology analysis in gravity studies.
Shape factor values shown in this centrifuge study are much higher than these
measured at the 2nd
hour of culture under standard cell culture conditions (37oC and 5%
CO2), reported in last chapter. The higher aspect ratio indicates that the lesser cell
elongation, and higher value of solidity and circularity, reflect the interruption of cell
dendrites development. Cell dendrites formation is an active process. By monitoring
living osteocytes, cellular dendritic process was found to keep extending and
retracting over 24 hrs, and the dynamic structure of cell dendrites are able to be
altered in response to external environment [36]. Due to the limitation of the used
centrifuge, our experiments were conducted at room temperature. Shape factors
measured in this centrifuge study suggest that cell culture environment including
temperature and concentrations of carbon dioxide highly affect osteocyte dendrites
Chapter 4: Osteocyte cells under hyper-gravity stimulation 59
development, even for a short growth period (30mins). Later study showed a
suspended dendrites growth in osteocytes cultured at room temperature for 10 hours,
indicating that rather than temporary effect, the influence of culture environment on
cell growth is continuous (data not shown). As mentioned in the literature view,
dendritic processes are playing an important role in osteocyte mechanosensation. The
retraction of cell dendrites caused by the experimental environment may adversely
affect the sensitivity of osteocyte cells to mechanical stimulation and therefore lead
osteocyte cells to fail to respond to the applied gravitational loading.
4.5 Conclusion
This study quantitatively investigated the cellular morphology changes of
osteocytes under a hypergravity environment. No significant morphology changes
were found in MLO-Y4 cells which were exposed to 20g for 30 mins. The failure
response in osteocyte morphology to increased G levels, as observed in this study,
can be explained by the fixation method used, the experimental environment and
inappropriate parameter for morphology analysis under altered gravity (e.g. cell
height may show more sensitivity to gravity changes than cell area).
Chapter 5: Conclusions and Future Work 60
Chapter 5: Conclusions and Future Works
Numerous gravity-related research works were conducted to investigate the
influence of gravity on living cells worldwide over the past two decades. As a large-
scale expression of many highly regulated biological processes, cell morphology thus
has been long used as a proxy measure for global cell state under an altered gravity
environment [1]. As microscopy moves into an increasingly quantitative and high-
throughput era, it can be expected that the analysis of cell morphology will play a
more important role in future gravitational biological study.
Osteocytes are believed to play an important role in bone mass loss occurring
in astronauts after long-term space missions. Dendritic phenotype of osteocytes is
essential to maintain their mechanosensation and mechanotransduction functions in
bone tissue. Thus, investigation of cell morphology changes of osteocyte cells in
altered gravity can provide important information on the influence of altered gravity
on osteocyte’s biological functions. Unlike these biomechanical studies which
investigated cell mechanical properties using techniques such as optical and
magnetic tweezers, atomic force microscopy and micropipette, gravity simulators are
normally performed in environments featuring high-speed movement, which causes
huge difficulty in quantifying the cell morphology change under altered gravity
stimulations. As a result, cellular morphological responses to altered gravity
reported from different studies are always found inconsistent.
One of the challenges gravity-related research faces today is that it is extremely
difficult to compare the cell morphology of osteocyte cell under altered gravity to
normal gravity by using the same individual osteocyte cell. The most common way
used to solve this problem is to set up a control sample, the changes of cell
Chapter 5: Conclusions and Future Work 61
morphology being then determined by comparing the difference between mean
values of cell morphology measured from altered gravity and control samples.
However, the morphology of osteocyte cells at 1g in culture was poorly investigated.
This study has successfully quantified the morphological development of MLO-Y4
cells cultured on the collagen coated surface for the first time. Quantification
analysis of osteocyte cells and the developed empirical models showed this study
have provided a base line for quantitatively studying the morphological change of
osteocyte cells under altered gravity. As cell morphology analysis is exceedingly
time-consuming, two osteocyte cell morphology studies were finished and used to
create the empirical models. In future, it would be advantageous to conduct further
morphology studies of osteocyte cells in culture.
Empirical models developed in this study were based on the data of
morphological changes of osteocyte cells in culture, seeded at the density of 5×103
cells per well. Cell density and the trypsinizing time applied in cell culture may vary
from study to study. However, different cell density and the trypsinizing time applied
in different studies may affect cell growth, which may lead to the incomparable
results from different gravitational studies. Thus, further investigation is required to
find out the effects of different cell culture density and trypsinizing time applied in
culture to osteocyte cells growth process.
In addition, morphological response of osteocytes with a relatively round shape
has been quantitatively investigated at a hyper-gravity environment. It was
hypothesized that compared with highly spread osteocytes used in previous studies; a
relatively round shape osteocyte has mechanical properties closer to osteocytes in
vivo. No significant morphology changes were observed in MLO-Y4 cells after
exposed to 20g for 30 mins. The response of MLO-Y4 in this hyper-gravity study
Chapter 5: Conclusions and Future Work 62
might be affected by the experimental environment, which can be considered as one
of the limitations of this study.
Another problem need to be dealt with in future gravitational biology study is
the cell recovery during fixation period. One possible solution is to fix the cell during
microgravity/hyper-gravity experiments. This requires designing a system which is
able to remove the culture medium and insert fixation solution at the predetermined
time in the experiment. The in situ fixation of cells may also be done by putting cells
into the gels which keep the liquid form at 37oC and slowly fixing the cell by
solidification at room temperature. This proposed fixation method can only used for
suspended cells in short-term (10-20 mins) gravitational study and the influence of
gels on the cellular sensitivity to mechanical signs needs to be further investigated.
Furthermore, current gravity-related studies investigate the cell morphology in
2D. However, three-dimensional morphology analysis and in situ measurement
might be the development trends for future cellular morphological study under
altered gravity. The first successful in situ measurement of the effects of changing
gravitational load on cell shape was done by Van et al. through mounting an atomic
force microscope (AFM) into a large-diameter centrifuge [84]. An atomic force
microscope is very expensive device; instead using AFM, another possible way to in
situ measure the three-dimensional morphology under different gravity is to develop
an optical system which is able to show both top and side view of the cells. A
preliminary design for this optical system is shown in Appendix 2. To improve space
research capability in Queensland University of Technology, the possibility to create
the combined AFM and centrifuge system and the proposed side-view optical system
should be further investigated.
Bibliography 63
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Appendix 1 Results from Centrifuge Experiments 68
Appendix 1: Results from Centrifuge Experiments
Cell Area under Hyper-gravity
Experiment One:
33003000270024002100180015001200900600300
90
80
70
60
50
40
30
20
10
0
Cell Area (um^2)
Fre
qu
en
cy
Distribution of Cell Area (Test Sample)
Figure 26 Distribution of cell area measured at 20g
33003000270024002100180015001200900600300
90
80
70
60
50
40
30
20
10
0
Cell Area (um^2)
Fre
qu
en
cy
Distribution of Cell Area (Control Sample)
Figure 27 Distribution of cell area measured at 1g
Two-sample T for C1 vs T1
N Mean StDev SE Mean
C1 500 874 345 15
T1 500 854 405 18
Appendix 1 Results from Centrifuge Experiments 69
Difference = mu (C1) - mu (T1)
Estimate for difference: 20.5
95% CI for difference: (-26.2, 67.2)
T-Test of difference = 0 (vs not =): T-Value = 0.86 P-Value = 0.388 DF = 998
Both use Pooled StDev = 376.1868
Experiment Two:
420039003600330030002700240021001800150012009006003000
140
120
100
80
60
40
20
0
Cell Area (um^2)
Fre
qu
en
cy
Distribution of Cell Area (Control Sample)
Figure 28 Distribution of cell area measured at 1g
330030002700240021001800150012009006003000
90
80
70
60
50
40
30
20
10
0
Cell Area (um^2)
Fre
qu
en
cy
Distribution of Cell Area (Test Sample)
Figure 29 Distribution of cell area measured at 20g
Two-sample T for C2 vs T2
N Mean StDev SE Mean
C2 500 877 382 17
T2 500 866 403 18
Difference = mu (C2) - mu (T2)
Estimate for difference: 10.6
Appendix 1 Results from Centrifuge Experiments 70
95% CI for difference: (-38.1, 59.4)
T-Test of difference = 0 (vs not =): T-Value = 0.43 P-Value = 0.669 DF =
998
Both use Pooled StDev = 392.713
Cell Shape under Hyper-gravity
Experiment One:
Circularity:
0.900.750.600.450.300.150.00
90
80
70
60
50
40
30
20
10
0
Circularity
Fre
qu
en
cy
Distirbution of Cicurlarity (Contol Sample)
Figure 30 Distribution of circularity measured at 1g
0.900.750.600.450.300.150.00
80
70
60
50
40
30
20
10
0
Circularity
Fre
qu
en
cy
Distribution of Circularity (Test Sample)
Figure 31Distribution of circularity measured at 20g
Two-sample T for C1 vs T1
N Mean StDev SE Mean
C1 500 0.581 0.157 0.0070
T1 500 0.562 0.150 0.0067
Appendix 1 Results from Centrifuge Experiments 71
Difference = mu (C1) - mu (T1)
Estimate for difference: 0.01884
95% CI for difference: (-0.00023, 0.03791)
T-Test of difference = 0 (vs not =): T-Value = 1.94 P-Value = 0.053 DF =
998
Both use Pooled StDev = 0.1537
Aspect Ratio:
1.080.960.840.720.600.480.36
120
100
80
60
40
20
0
Aspect Ratio
Fre
qu
en
cy
Distribution of Aspect Ratio (Control Sample)
Figure 32Distribution of aspect ratio measured at 1g
0.960.840.720.600.480.360.24
90
80
70
60
50
40
30
20
10
0
Aspect Ratio
Fre
qu
en
cy
Distribution of Aspect Ratio (Test Sample)
Figure 33 Distribution of aspect ratio measured at 20g
Two-sample T for C1 vs T1
N Mean StDev SE Mean
APC1 500 0.8510 0.0956 0.0043
APT1 500 0.843 0.109 0.0049
Difference = mu (APC1) - mu (APT1)
Estimate for difference: 0.00775
95% CI for difference: (-0.00498, 0.02048)
Appendix 1 Results from Centrifuge Experiments 72
T-Test of difference = 0 (vs not =): T-Value = 1.20 P-Value = 0.232 DF =
998
Both use Pooled StDev = 0.1026
Solidity:
1.000.950.900.850.800.750.700.650.600.550.500.45
90
80
70
60
50
40
30
20
10
0
Solidity
Fre
qu
en
cy
Distribution of Solidity (Control Sample)
Figure 34 Distribution of solidity measured at 1g
1.000.950.900.850.800.750.700.650.600.550.500.45
140
120
100
80
60
40
20
0
Solidity
Fre
qu
en
cy
Distribution of Solidity (Test Sample)
Figure 35 Distribution of solidity measured at 20g
Two-sample T for C1 vs T1
N Mean StDev SE Mean
SC1 500 0.8625 0.0706 0.0032
ST1 500 0.8637 0.0768 0.0034
Difference = mu (SC1) - mu (ST1)
Estimate for difference: -0.00123
95% CI for difference: (-0.01039, 0.00792)
T-Test of difference = 0 (vs not =): T-Value = -0.26 P-Value = 0.791 DF =
998
Both use Pooled StDev = 0.0738
Appendix 1 Results from Centrifuge Experiments 73
Experiment Two:
Circularity:
0.900.750.600.450.300.15
80
70
60
50
40
30
20
10
0
Circularity
Fre
qu
en
cy
Distribution of Circularity (Control Sample)
Figure 36 Distribution of circularity measured at 1g
0.900.750.600.450.300.150.00
70
60
50
40
30
20
10
0
Circularity
Fre
qu
en
cy
Distribution of Circularity (Test Sample)
Figure 37 Distribution of circularity measured at 20g
Two-sample T for C2 vs T2
N Mean StDev SE Mean
C2 500 0.573 0.162 0.0073
T2 500 0.562 0.150 0.0067
Difference = mu (C2) - mu (T2)
Estimate for difference: 0.01123
95% CI for difference: (-0.00816, 0.03062)
T-Test of difference = 0 (vs not =): T-Value = 1.14 P-Value = 0.256 DF =
998
Both use Pooled StDev = 0.1562
Appendix 1 Results from Centrifuge Experiments 74
Aspect ratio:
0.960.840.720.600.48
100
80
60
40
20
0
Aspect Ratio
Fre
qu
en
cy
Distribution of Aspect Ratio(Control Sample)
Figure 38 Distribution of aspect ratio measured at 1g
0.960.840.720.600.480.36
100
80
60
40
20
0
Aspect Ratio
Fre
qu
en
cy
Distribution if Aspect Ratio (Test Sample)
Figure 39 Distribution of aspect ratio measured at 20g
Two-sample T for C2 vs T2
N Mean StDev SE Mean
C2 500 0.8472 0.0925 0.0041
T2 500 0.8460 0.0982 0.0044
Difference = mu (C2) - mu (T2)
Estimate for difference: 0.00117
95% CI for difference: (-0.01067, 0.01300)
T-Test of difference = 0 (vs not =): T-Value = 0.19 P-Value = 0.846 DF =
998
Both use Pooled StDev = 0.0954
Appendix 1 Results from Centrifuge Experiments 75
Solidity:
1.000.900.850.800.750.700.650.600.550.500.45
120
100
80
60
40
20
0
Solidity
Fre
qu
en
cy
Distribution of Solidity (Control Sample)
Figure 40 Distribution of solidity measured at 1g
1.000.900.850.800.750.700.650.600.550.500.45
120
100
80
60
40
20
0
Solidity
Fre
qu
en
cy
Distribution of Solidity (Test Sample)
Figure 41 Distribution of solidity measured at 20g
Two-sample T for C2 vs T2
N Mean StDev SE Mean
C2 500 0.8640 0.0724 0.0032
T2 500 0.8657 0.0753 0.0034
Difference = mu (C2) - mu (T2)
Estimate for difference: -0.00169
95% CI for difference: (-0.01086, 0.00748)
T-Test of difference = 0 (vs not =): T-Value = -0.36 P-Value = 0.718 DF =
998
Both use Pooled StDev = 0.0739
Appendix 2 Preliminary Design for Side-view Optical System 76
Appendix 2: Preliminary Design for Side-View Optical System
Preliminary Design for Optical System Configurations
• Based on the previous developed side-view chamber, the configuration of my
preliminary design is shown below:
Figure 42 Over view of the preliminary design for side-view optical system
• Instead of two separate mirrors, build two mirrors on a base to form as one system
ensure that two mirrors will be always in parallel (Easy to set up).
• The thickness of rectangular tube lift up the position of cell ensures side-view image
can be captured properly by the mirror.
Preliminary Design for Optical System Dimensions
• The dimensions of this optical system is restricted by two main factors:
1: The width of the system (AB) need to within the microscope field of objective lens;
2: The working distance of objective lens: optical path length of side view must smaller than
objective working distance: (C+D+E).
Figure 43 Working distance for side-view optical system
Chromium coated mirrors
Cell Borosilicate cell chamber
Appendix 2 Preliminary Design for Side-view Optical System 77
• Working distances various for different types of objectives:
• Microscope field measured by put clear plastic ruler with mm markings on top of the
stage of microscope. Microscope field measured under 40X magnification is 2mm:
Figure 44 Plastic ruler placed under 40x magnification lens, showing that the microscope filed is 2mm
• Assuming the objective we are going to use has the magnification of 40X (microscope
field: 2mm, working distance 2.1mm). Then the ideal dimensions of this optical
system is shown below:
Figure 45 Preliminary design of the size of the optical system
Appendix 2 Preliminary Design for Side-view Optical System 78
• The suggested dimension picked for cell chamber is based on the available
commercial products, and the thickness of the mirror base is restricted by the
microscope filed and objective working distance.
A+B= 0.84+0.84=1.68mm which is in the microscope filed 2mm
C+D+E= 0.6+0.12+0.72+0.12+0.4=1.94mm which is within the maximum working
distance of the 40x objective.
Preliminary Design for Optical Path Modulation
• The side view reflected by the mirror has a longer optical path length than top view.
However, the specimen appears sharp on objective lens only within the depth of field
(DOF);
• Due to the longer optical path length, DOF may do not cover side view image, thus, in
order to obtain the side- and top view images both in focus, one of the optical path
length should be modulated
• It is know that the working distance of the objective determined by:
1: The material (refractive index) of the objective lens
2: The refractive index of the medium
• A preliminary solution is to add a high refractive index medium (e.g. glass) between
objective lens and specimen to expand the working distance of the objective:
Figure 46 Optical path length with different refractive index medium