A systems approach to bone remodeling mechanotransduction

A SYSTEMS APPROACH TO BONE REMODELING
AND MECHANOTRANSDUCTION





MYNAMPATI KALYAN CHAKRAVARTHY








NATIONAL UNIVERSITY OF SINGAPORE
2007
A SYSTEMS APPROACH TO BONE REMODELING
AND MECHANOTRANSDUCTION



MYNAMPATI KALYAN CHAKRAVARTHY
(B.Eng. (Hons.), NUS)





A THESIS SUBMITTED

FOR THE DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAMME IN BIOENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2007
ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to the following:

Associate Professor Peter Lee
For introducing me to the field of systems biology, hearing patiently to my
unceasing yapping, encouraging me in my ventures, and his invaluable support,
advice and guidance all through out the project

Associate Professor Toh Siew Lok
For his support to this research, and providing me the resources to carry out the
project

Ms Ling Wen Wan and Mr Koh Geoffrey
For their contribution to the parameter estimation part of this dissertation

GPBE mates
For their help, support and encouragement and all the fun and laughter we shared
over the past 30 months

And above all,
To the Supreme Lord and His devotees for giving a purpose to my existence


NOTE

Due to the inputs from several sources to help shape up this project, first person plural is
used in active voice all through out this dissertation, instead of first person singular.


i
TABLE OF CONTENTS
Topic Page
Acknowledgements i
Summary v
List of Tables vii
List of Figures viii
List of Abbreviations xi
Chapter 1: Introduction 1
1.1 Motivation 1
1.2 Objectives of the project 2
1.3 Methodology 2
1.4 Overview of the thesis 3
Chapter 2: Bone Remodeling 4
2.1 Synopsis 4
2.2 Bone 4
2.2.1 Bone: The Organ 4
2.2.2 Osseous tissue 5
2.2.3 Bone formation 8
2.3 Bone remodeling 9
2.3.1 Factors affecting Bone remodeling 11
2.3.2 Homeostatic Imbalances in Bone 13
2.4 Outstanding questions in Bone Remodeling 15

ii
2.5 Summary 16

Chapter 3: Bone Mechanotransduction 17
3.1 Synopsis 17
3.2 Mechanotransduction 17
3.3 Bone Mechanotransduction 19
3.3.1 Signaling in Bone Mechanotransduction 22
3.4 Salient Issues 32
3.5 Summary 33
Chapter 4: Modeling 34
4.1 Synopsis 34
4.2 Modeling Cellular Dynamics 34
4.3 Potential Model 37
4.4 Summary 38
Chapter 5: Systems Modeling 39
5.1 Synopsis 39
5.2 ‘Systems-level’ Modeling 39
5.2.1 Implementation of the systems level modeling 43
5.3 Summary 66
Chapter 6: Results and Discussion 67
6.1 Synopsis 67
6.2 Boundary Conditions 67
6.3 SIMULINK block diagrams 68
6.4 Results and Discussion 71

iii
6.5 Significant Inferences 81
6.6 The Missing Link 82
6.7 Summary 84
Chapter 7: Conclusion 86
Chapter 8: Future Work 88
8.1 Synopsis 88

8.2 Experimentation 88
8.3 Refining the current computational model 91
8.4 Application #1: Osteoporosis Treatment 92
8.5 Application #2: Bone Tissue Engineering 93
8.6 Summary 95
Bibliography 96
Appendix A: Bone Mechanotransduction over the years 106
Appendix B: Networks 117
B.1 Osteoblast Signaling Network 117
B.2 Osteoblast-Osteoclast Interaction Network 118
B.3 Osteoclast Signaling Network 119
Appendix C: Analysis of the Parameter Estimation Algorithm 120
Appendix D: MATLAB files for Parameter Estimation 123
Appendix E: Profiles of Signaling Proteins 137


iv
SUMMARY

Bone remodeling refers to a fundamental homeostatic process in the body, which maintains
bone strength by continuously replacing old bone with new bone. Disruption in the
homeostasis leads to skeletal disorders like osteopetrosis or osteoporosis. Mechanical
loading affects bone remodeling. Increased loading leads to increased bone mass, while
reduced loading results in decreased bone mass. The underlying cellular dynamics for such
an observation is not clearly understood. Hence, the main objective of this project is to
investigate the affect of mechanical loading on bone remodeling at the cellular level.

In this project, a novel computational modeling approach called ‘systems-level’ modeling
is implemented to study the mechano-regulation of bone at cellular level. Specific issues
addressed using this approach include determining the intra-cellular response of bone cells

to mechanical stimulus, bone response to different mechanical loading conditions, the role
of feedback regulation in bone remodeling, and the link between reduced mechanical
loading and decreased bone mass.

This computational modeling approach, implemented in SIMULINK® environment,
derives concepts from the emerging field of systems biology, control theory, and computer
science. The salient features of this modeling technique include –
(i) Systems biology based network modeling: A system of differential equations is
developed based on Michaelis-Menten enzyme kinetics to model the intra-
cellular signaling networks of osteoblasts and osteoclasts.

v
(ii) Parameter estimation, based on evolutionary computing, is used to estimate the
Michaelis-Menten rate constants of the kinetic models of the networks.
(iii) Control systems theory is used to model feedback in the signaling networks.

An inter-connected network of eight major signaling pathways in osteoblasts and seven in
osteoclasts, which are initiated as part of the intra-cellular response of bone to mechanical
stimulus, are identified for this dissertation, based on a comprehensive literature survey.
The ‘systems-level’ computational models simulate the temporal dynamics of the signaling
proteins in these two networks. The simulation studies indicate that the signaling networks
cause unique physiological response in the bone cells with respect to different mechanical
stimulus. Disruption of intra-cellular feedback regulation leads to decreased bone
formation in osteoblasts and increased bone resorption in osteoclasts, a phenomenon
generally observed in reduced loading conditions.

The results of these simulation studies serve as useful guidelines for planning relevant
experimental work to study the affect of mechanical loading on bone remodeling at cellular
level.

vi
LIST OF TABLES

Table

Legend

Page

5.1 Parameter values for the hypothetical pathway model 48

5.2

Constants Used for the Parameter Estimation Algorithm

63

5.3

Rate constants in the Osteoblast network

64

5.4

Rate constants in the Osteoclast network

65

6.1

Input stimuli for network perturbation

68




vii
LIST OF FIGURES

Figure

Legend

Page

2.1

The four different types of bone cells

6

2.2

The five phases of bone remodeling

10

2.3

Determinants of bone remodeling

11

2.4

Cross section of healthy bone vs. Osteoporotic bone

14

3.1

Model for the transduction of mechanical strain to
osteocytes in bone

20

3.2

Osteocytes as mechanosensory cells

21

3.3

Mechanotransduction response in Osteoblast

23

3.4

Mechanotransduction response in Osteoclast

23

3.5

Block Diagram representation of the Osteoblast signaling
network

24

3.6

Molecules involved in the Osteoblast-Osteoclast
interactions

28

3.7

Block Diagram representation of the Osteoclast signaling
network

29

viii

Figure

Legend

Page

4.1

Block diagram model of the ERK signaling pathway

35

4.2

Mechanistic model of the MAPK signaling cascade,
interacting with another pathway

36

5.1

Architecture of ‘systems-level’ modeling

39

5.2

A hypothetical signaling pathway, including a positive and
a negative feedback loop

43

5.3

Pathway map of the hypothetical signaling cascade

45

5.4

SIMULINK block diagram of the pathway
(No feedback included)

49

5.5

SIMULINK block diagram of the pathway
(Feedback included)

49

5.6

Concentration profile of the ABCDE pathway
(no feedback)

50

5.7

Concentration profile of the ABCDE pathway
(only negative feedback)

51

5.8

Concentration profile of the ABCDE pathway
(both positive and negative feedback, time=1500 units)

51

5.9

Concentration profile of the ABCDE pathway
(both positive and negative feedback, time=6000 units)

52

5.10

Flow Chart of the Parameter Estimation Algorithm

61

ix

Figure

Legend

Page

6.1

The osteoblast SIMULINK block diagram (no feedback)

69

6.2

The osteoblast SIMULINK block diagram (feedback)

69

6.3

The osteoclast SIMULINK block diagram (no feedback)

70

6.4

The osteoclast SIMULINK block diagram (feedback)

70

6.5

bCatenin and PKA activation profiles

72

6.6

PKC and CREB activation profiles

73

6.7

Akt and NFkB activation profiles

74

6.8

ERK and JNK activation profiles

76

6.9

Akt and PKC activation profiles

77

6.10

p38MAPK and NFAT activation profiles

78

6.11

ERK and JNK activation profiles

79

6.12

NFkB activation profile

80

6.13

The Missing Link

84

8.1

The ‘hypothesis driven-experiment refined’ cycle

88






x
LIST OF ABBREVIATIONS

Abbreviation

Expansion

AP-1

Activator Protein 1

BMD

Bone Mineral Density

BMP

Bone Morphogenic proteins

cAMP

cyclic Adenosine Mono Phosphate

cGMP

cyclic Guanosine Mono Phosphate

Cbf

Core binding factor

CREB

cAMP Response Element Binding Protein

COX

Cyclooxygenase

CBP

CREB Binding Protein

CFU-F

Colony Forming Units-Fibroblastic

Dsh

Dishevelled

EGF

Epidermal Growth Factor

ERK

Extracellular signal Regulated Kinase

FGF

Fibroblast Growth Factor

Fzd

Frizzled

GM-CSF

Granulocyte-Macrophage Colony Stimulating Factor


xi

Abbreviation

Expansion

GSK3h

Glycogen Synthase Kinase-3 beta

IkB

cytosolic protein sequestering Nf-kB

IGF-1

Insulin-like Growth Factor 1

IKK

Kinase targeting IkB and its proteosomal degradation

IL-1,4,6,10

Interleukin-1,4,6,10

ILGF

Interleukin Growth Factor

IP
3

inositol 1,4,5-trisphosphate

JNK

c-Jun N-terminal kinase

MAP

Mitogen Activated Protein

MAPK

MAP kinase

MCSF

Macrophage Colony Stimulating Factor

MMP

Family of extracellular matrix metalloproteinases

Nemo

Regulatory noncatalytic subunit of IKK

Nf-kB

Nuclear factor kappa B

NOS

Nitric Oxide Synthase

OA

Osteoarthritis

OCN

Osteocalcin





xii

Abbreviation

Expansion

PI3K

Phosphoinositide 3-kinase

PIP
3

Phosphatidylinositol (3,4,5)-trisphosphate

PKA/PKC

Protein Kinase A/C

RA

Rheumatoid arthritis

RANK

Receptor activator of Nf-kB

RANKL

RANK Ligand

TGF

Transforming Growth Factor

TNF

Tumor Necrosis Factor



Abbreviation

Expansion

C

Chapter

F

Figure

S

Section


xiii
Chapter 1: Introduction

CHAPTER 1
INTRODUCTION
1.1 Motivation
The living bone is a dynamic and complex organ, largely made up of osseous tissue. The
tissues in the bone enable it to serve its primary functions of support, protection,
movement, mineral storage, and hematopoiesis in the body. The osseous tissue is a
supporting connective tissue, composed of a matrix and four different types of bone cells,
namely – osteocytes, osteoblasts, osteoprogenitors, and osteoclasts. This tissue, which
gives strength to the bone, dynamically maintains itself by continuously replacing its old
bone with new bone, through a homeostatic process of bone formation and resorption,
popularly known as bone remodeling. In a healthy adult body, rate of bone formation
equals rate of bone resorption to maintain the homeostasis. Unequal rates of bone
formation and resorption result in diseased conditions like osteopetrosis or osteoporosis.
The exact cellular mechanisms for homeostasis disruption are still not clearly understood.
It has been observed that increased mechanical loading enhances bone mass indicating
increased bone formation, while reduced loading lowers bone mass reflecting increased
bone resorption. The underlying dynamics for such an observation is also not clearly
understood.
Hence, we embark on this project to investigate how mechanical loading affects bone
remodeling at the cellular level. We are interested in the cellular level because

1
Chapter 1: Introduction

remodeling is basically a cellular process involving bone resorption by osteoclasts and
bone formation by osteoblasts.
Increased understanding of these fundamental processes can lead to novel therapeutics
for degenerative diseases like osteoporosis. Also, bone tissue engineering relies on the
integration of biological and synthetic implant materials for fracture repair and
replacement of bone. Understanding the dynamics of the mechanical environment and its
affect on bone cells in vivo are important for long term implant integration and successful
repair.
1.3 Objectives of the Project
This dissertation aims to investigate the effect of mechanical stimulus on bone
remodeling at the cellular level. This thesis aims to address the following questions –
(i) What is the intra-cellular response of bone cells to mechanical stimulus?
(ii) How does bone respond to different mechanical loading conditions?
(iii) What is the role of feedback regulation in bone remodeling?
(iv) What is the link between reduced mechanical loading and decreased bone
mass?
1.3 Methodology
‘Systems-level’ computational modeling approach is implemented to address the aims of
this project. This novel modeling technique is derived from the emerging field of systems
biology, which investigates the dynamics of the interacting components at systems or
network level.


2
Chapter 1: Introduction

1.4 Overview of the thesis
The core of this thesis lies in the implementation of a novel computational modeling
approach to address salient issues in bone remodeling and mechanotransduction
processes. Issues are raised in the critical reviews on these two topics, presented in
Chapters 2 and 3 respectively. Limitations in current modeling techniques to study
cellular dynamics are analyzed in Chapter 4. Architecture of the proposed modeling
approach and its implementation are described in Chapter 5, followed by a Chapter on the
simulation results and discussion. A comprehensive summary of the thesis is provided in
Chapter 7. Future work on experimentation and a few applications of this study on
mechanotransduction and bone remodeling are explored in Chapter 8. Appendices A to E
complement the relevant discussions in the main text.

3
Chapter 2: Bone Remodeling

CHAPTER 2
BONE REMODELING
2.1 Synopsis

Although bones appear to be rigid and unchanging, the living bones are dynamic and
undergo continuous recycling, with almost one-fifth of adult skeleton being replaced each
year. The strength of the bone is dependent on its homeostatic recycling process, which is
influenced by several factors including genetic, hormonal, and mechanical stimulus. This
chapter presents a critical review of the dynamics of this recycling process, also known as
bone remodeling, starting with an introduction to bone.

2.2 Bone

2.2.1 Bone: The Organ

Of the 11 organ systems that constitute a human body, the skeletal system includes
bones, cartilages, ligaments and other tissues that connect the bones. Bone is a complex
and dynamic organ containing various types of tissues. A typical bone is made of bone
(osseous) tissue, nervous tissue, cartilage, myeloid tissue that produces red and white
blood cells, fibrous connective tissue lining their cavities, and muscle and epithelial
tissues [Ganong2005, Martini2006]. Strength of the bone comes from its osseous tissue.
Taken together, these tissues enable the bone to perform its five primary functions:
(i) Support – Bones provide structural support for the entire body. Individual bones or
groups of bone provide a framework for the attachment of soft tissues and organs. For
example, bones of lower limbs act as pillars to support the body trunk when we stand.

4
Chapter 2: Bone Remodeling

(ii) Protection – Bones cradle the body’s inner organs, like vertebrae surrounding the
spinal cord or rib cage protecting the vital organs of thorax.
(iii) Movement – Skeletal muscles, which attach to bones by tendons, use bones as levers
to move the body. The arrangement of bones and the design of joints determine different
types of movement.
(iv) Mineral storage – Bones retain reserve stores of minerals like calcium, phosphate,
and other ions. The stored minerals are released into the bloodstream as needed for
distribution to all parts of the body. In addition to acting as a mineral reserve, the bone
store energy reserves as lipids in areas filled with yellow marrow.
(v) Hematopoiesis- Generation of red and white blood cells for immuno-protection and
oxygenation of other tissues occurs in the marrow cavities of certain bones.

2.2.2 Osseous tissue
Bone (or osseous) tissue is a supporting connective tissue. Like other connective tissues,
it contains specialized bone cells (which account for only 2% of the mass of a typical
bone) and a matrix which surrounds the cells, as described below -

A. MATRIX
The matrix is composed of both organic and inorganic components –
(i) The organic part of the matrix, called osteoid (which makes up approximately one-
third of the matrix), includes proteoglycans, glycoproteins and collagen fibers. These
organic substances, particularly collagen, contribute not only to the bone’s structure but
also to the flexibility and tensile strength that allow the bone to resist stretch and twisting.

5
Chapter 2: Bone Remodeling

Bone’s exceptional toughness and tensile strength comes from the presence of sacrificial
bonds in or between collagen molecules that break easily on impact dissipating energy to
prevent the force from rising to a fracture value [Marieb2004]. The collagen fibers
provide an organic framework on which the inorganic portion of the matrix deposits.

(ii) The inorganic part of the matrix consists of hydroxyapatite (Ca
10
(PO
4
)
6
(OH)
2
),
present in the form of tiny crystals surrounding the collagen fibers in the extracellular
matrix. These crystals, while forming, incorporate other calcium salts, such as calcium
carbonate, and ions such as sodium, magnesium and fluoride. The crystals are tightly
packed and form small plates and rods locked into the collagen fibers [Martini2006]. The
resulting protein-crystal combination allows bone to be strong, flexible and highly

resistant to compression.

B. BONE CELLS
There are four types of bone cells – Osteocytes, Osteoblasts, Osteoprogenitors and
Osteoclasts, as shown in Figure 2.1.






Figure 2.1 The four types of bone cells
(Adapted from [Martini2006])


6
Chapter 2: Bone Remodeling

(i) Osteocytes are mature bone cells that account for most of the cell population. Each
osteocyte occupies a lacuna, a pocket sandwiched between layers of matrix (called
lamellae). Narrow passageways called canaliculi penetrate the lamellae, radiating through
the matrix and connecting lacunae with one another and with sources of nutrients, such as
the central canal. Neighboring osteocytes are linked by gap junctions, which permit the
exchange of ions and small molecules, including nutrients and hormones, between the
cells. The major function of ostocytes is to maintain the protein and mineral content of
the surrounding matrix. Osteocytes secrete chemicals that dissolve the adjacent matrix,
and the minerals released enter the circulation. Osteocytes then rebuild the matrix,
stimulating the deposition of new hydroxyapatite crystals.

(ii) Osteoblasts are modified fibroblasts that produce bone matrix. They make and

release proteins and other organic components of the matrix. They also assist in elevating
the local concentrations of calcium phosphate and in promoting the deposition of calcium
salts in the organic matrix. Ostoblasts mature into osteocytes.

(iii) Osteoprogenitor cells are mesenchymal stem cells which divide into daughter cells
that differentiate into osteoblasts. Osteoprogenitor cells maintain populations of
osteoblasts and are important in the repair of a fracture. They are located in the inner
layers that line marrow cavities and in the linings of passageways, containing blood
vessels that penetrate the matrix of compact bone.


7
Chapter 2: Bone Remodeling

(iv) Osteoclasts remove and recycle bone matrix. They are directly involved in bone
resorption. They are members of the monocyte family, and hence are giant cells with 50
or more nuclei. Acid and proteolytic enzymes secreted by osteoclasts dissolve the matrix
and release the stored minerals. This erosion process, called osteolysis or resorption,
regulates calcium and phosphate concentrations in body fluids.

2.2.3 Bone formation
Bone formation can be divided into two temporal phases [Ross2006] –
(i) Modeling – This phase of bone formation occurs during development. In humans,
bones begin to form about six weeks after fertilization, starting as cartilaginous tissue.
During childhood and adolescence, bone modeling allows the formation of new bone at
one site and the removal of old bone from another site within the same bone. This process
allows individual bones to grow in size and to shift in space.

(ii) Remodeling – This phase of bone formation is a lifelong process involving tissue
renewal. It becomes a dominant process by the time bone reaches its peak mass (typically

by the early 20s). In remodeling, old bone is continuously being recycled with new bone
at the same site. It is part of normal bone maintenance. (Remodeling is comprehensively
discussed in the next section).

Modeling and remodeling continue throughout life to preserve the mechanical strength of
the bone.


8
Chapter 2: Bone Remodeling

2.3 Bone Remodeling

Bone remodeling is a homeostatic mechanism inside the body where old bone, including
the matrix, is resorbed by osteoclasts followed by deposition of new bone by osteoblasts.
It is mainly a local process carried out in small areas. Although remodeling replaces the
matrix, it leaves the bone as a whole unchanged, including its shape, internal architecture,
and mineral content. In healthy young adults, total bone mass remains constant,
indicating that the rates of bone formation and resorption are equal.

Remodeling is vital for bone health, for a variety of reasons [Surgeon2004]. It repairs the
damage to the skeleton that can result from repeated stresses by replacing small cracks or
deformities. It also prevents accumulation of too much old bone, which can lose its
resilience and become brittle.

As shown in Figure 2.2, bone remodeling can be divided into five distinct phases
[Fernández2006 and Sikavitsas2001] –
(i) Quiescence – In this phase, bone is at resting state. The surface of the bone is lined
with inactive cells. Former osteoblasts are trapped as osteocytes within the mineralized
matrix.

(ii) Activation - In this phase, retraction of the bone lining cells (elongated mature
osteoblasts existing on the endosteal surface) and digestion of the endosteal membrane
occurs. The exposed mineralized surface attracts osteoclasts. Sites with microfractures or
microdamage exhibit a predisposition for remodeling.


9
Chapter 2: Bone Remodeling


Figure 2.2 The five phases of bone remodeling
(Adapted from [Fernández2006])












(iii) Resorption - Osteoclasts dissolve the mineral matrix and decompose the osteoid
matrix. Resorption releases growth factors contained within the matrix, like transforming
growth factor beta (TGF-β), platelet derived growth factor (PDGF), insulin-like growth
factor I and II (IGF-I and II).
(iv) Formation - Differentiated osteoblasts fill in the resorption cavity and begin forming
new osteon

1
. The released growth factors in the resorption phase acts as chemotactics to
stimulate osteoblast proliferation. They deposit osteoid
2
(mostly collagen type I).


1
Osteon – Systems of interconnecting canals in the microscopic structure of adult compact bone; also
called Harvesian System
2
Osteoid – Unmineralized bone matrix





10