A systems approach to bone remodeling mechanotransduction

Specific issues addressed using this approach include determining the intra-cellular response of bone cells to mechanical stimulus, bone response to different mechanical loading conditio

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

TABLE OF CONTENTS

Topic Page

Acknowledgements i

Summary v

2.3.1 Factors affecting Bone remodeling 11

2.3.2 Homeostatic Imbalances in Bone 13

2.4 Outstanding questions in Bone Remodeling 15

6.3 SIMULINK block diagrams 68

8.3 Refining the current computational model 91

8.4 Application #1: Osteoporosis Treatment 92

8.5 Application #2: Bone Tissue Engineering 93

Bibliography 96 Appendix A: Bone Mechanotransduction over the years 106

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

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 cellular signaling networks of osteoblasts and osteoclasts

intra-(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

LIST OF TABLES

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

LIST OF FIGURES

2.1 The four different types of bone cells 6 2.2 The five phases of bone remodeling 10

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

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)

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

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

6.10 p38MAPK and NFAT activation profiles 78

8.1 The ‘hypothesis driven-experiment refined’ cycle 88

LIST OF ABBREVIATIONS

cAMP cyclic Adenosine Mono Phosphate

cGMP cyclic Guanosine Mono Phosphate

CREB cAMP Response Element Binding Protein

CFU-F Colony Forming Units-Fibroblastic

ERK Extracellular signal Regulated Kinase

FGF Fibroblast Growth Factor

GM-CSF Granulocyte-Macrophage Colony Stimulating Factor

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

IP3 inositol 1,4,5-trisphosphate

MAP Mitogen Activated Protein

MCSF Macrophage Colony Stimulating Factor

MMP Family of extracellular matrix metalloproteinases

Nemo Regulatory noncatalytic subunit of IKK

Nf-kB Nuclear factor kappa B

Abbreviation Expansion

PI3K Phosphoinositide 3-kinase

PIP3 Phosphatidylinositol (3,4,5)-trisphosphate

RANK Receptor activator of Nf-kB

TGF Transforming Growth Factor

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

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

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

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

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

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 (Ca10(PO4)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

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

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

Chapter 2: Bone Remodeling

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

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 osteon1 The released growth factors in the resorption phase acts as chemotactics to

stimulate osteoblast proliferation They deposit osteoid2 (mostly collagen type I)

Chapter 2: Bone Remodeling

(v) Mineralization - Mineralization of the osteoid commences before the termination of

matrix synthesis The rate of matrix apposition is rapid initially, but it slows down after the termination of matrix synthesis, and continues until the bone surface returns to its original resting state

2.3.1 Factors affecting Bone Remodeling

Numerous factors [Fernández2006 and Bonewald2003], including genetic, mechanical, vascular, nutritional, hormonal and local, affect the bone remodeling process (Figure 2.3),

as explained below –

Figure 2.3 Determinants of bone remodeling (Adapted from [Harada2003])

(i) Genetic factors – The maximum bone mass is controlled by genetic determinants

Genes also determine bone’s response to other factors influencing bone remodeling

(ii) Mechanical factors –Mechanical environment and the gene expression patterns at

various stages of ossification are observed to be intimately coupled Quasi-static and

Chapter 2: Bone Remodeling intermittent force patterns, pressure, strain, fatigue and fluid flow are a few examples of the mechanical factors that influence ossification (Detailed discussion on mechano-regulation of bone remodeling is covered in the next chapter.)

(iii) Vascular factors – Vascularization is fundamental for normal bone development

supplying blood cells, oxygen, minerals, ions, glucose, hormones and growth factors Bone density is observed to be decreased in de-nerved bones

(iv) Nutritional factors – Excess salt, caffeine, alcohol, nicotine, reduced calcium intake

constitute risk factors for increased bone resorption

(v) Hormonal factors – Bone remodeling is regulated by hormones released by the

endocrine system Some of them include:

(a) Thyroid hormone - stimulates the synthesis of osteoid matrix by the

osteoblasts and its mineralization

(b) Parathyroid hormone (PTH) - Produced in the parathyroid glands in response

to hypocalcemia, it stimulates bone resorption through the synthesis of a factor favoring osteoclastogenesis on the part of the osteoblasts But lately, it has been found that at intermittent doses, PTH stimulates bone formation, associated with

an increase of growth factors and with a decrease in the apoptosis of the osteoblasts

(c) Calcitonin - A polypeptide, reduces osteoclast function to inhibit bone resorption

Chapter 2: Bone Remodeling

(d) Vitamin D - A steroid hormone that acts as a local regulator of osteoclast

differentiation, and influences matrix mineralization

(e) Androgens - Have an anabolic effect on bone through the stimulation of the

osteoblast receptors Androgen deficiency is associated with lower bone density

(f) Estrogens - Estrogens have a dual effect on bone metabolism They favor bone formation, increasing the number and function of the osteoblasts, and also play a role in inhibiting bone resorption

(vi) Local factors – Growth factors and cytokines regulate bone remodeling Bone

formation is stimulated by growth factors like IGF-I and II (insulin-like growth factor I and II), TGF-β (Transforming Growth Factor-β), and BMP (Bone Morphogenic Proteins), while bone resorption is stimulated by TNF (Tumor Necrosis Factor), EGF (Epidermal Growth Factor), M-CSF (Macrophage-Colony Stimulating Factor), IL-1 (Interleukin 1),

IL-6 (Interleukin 6) and Prostaglandins

All these six factors are observed to inter-play with each other to regulate the remodeling process [Fernández2006] Abnormal perturbations from these factors affect the remodeling homeostasis [Marieb2004] Imbalance in bone remodeling leads to different disease conditions, as briefly discussed in the next section

2.3.2 Homeostatic Imbalances in Bone

Generally observed imbalances include increased bone formation, or increased bone

resorption leading to low bone mass, or inadequate mineralization Osteomalacia is a

Chapter 2: Bone Remodeling disease characterized by inadequate mineralization Although osteoid is produced,

calcium salts are not deposited So, bone become softened and weakens Paget’s disease

is characterized by excessive bone deposit and resorption [Marieb2004] This along with reduced mineralization causes a spotty weakening of the bones Late in the disease, osteoclast activity wanes, but osteoblasts continue to work, often forming irregular bone thickenings or filling the marrow cavity with Pagetic bone

In osteopetrosis, the presenting symptom is increased bone density resulting in

neurological defects and hematological abnormalities It is caused due to dysfunction of

osteoclast activity and unopposed osteoblast action Osteoporosis is a degenerative

disease (shown in Figure 2.4), in which reduced bone formation and increased bone resorption cause micro-architectural detoriation of bone tissue and low bone mass, leading to increased bone fracture susceptibility

Figure 2.4 Cross section of Healthy Bone (left) vs Osteoporotic Bone (right)

(Adapted from [IOF])

Chapter 2: Bone Remodeling

2.4 Outstanding questions in Bone Remodeling

The available literature, on remodeling dynamics, falls short on explaining some fundamental mechanisms of the process, like –

(i) How do the six factors interact to decide whether, when and where bone remodeling

takes place? Does any factor counteract the perturbations from others?

(ii) In the absence of mechanical stimuli, bone remodeling is observed to be imbalanced despite the unperturbed presence of other factors Why?

(iii) How is mechanical stress, a non-inherent factor of the body, sensed by the bone? Increased understanding of these basic mechanisms can be expected to generate better treatments to restore homeostasis in diseased conditions like osteoporosis and osteopetrosis It can also help develop an optimal mechanical environment for cell proliferation [Mullender2004] in bone tissue engineering (discussed further in chapter 8)

The objective of this dissertation is to investigate the effect of mechanical stimulus on bone remodeling at cellular level Some of the specific issues which are addressed include determining the nature of the intra-cellular response of bone cells to mechanical stimulus, the role of feedback regulation in bone remodeling, and the link between reduced mechanical loading and imbalanced bone remodeling homeostasis A computational modeling approach is implemented to address these issues The results of these computational simulation studies are expected to serve as useful guidelines for planning relevant experimental work to study the fundamental mechanisms in bone remodeling process as discussed above

Chapter 2: Bone Remodeling

2.5 Summary

A critical review of bone and its remodeling process is presented in this chapter A few unexplained fundamental issues in the involved mechanisms are raised In this thesis, only the effect of mechanical stimulus on bone remodeling is explored further, as detailed

in the following chapter

Chapter 3: Bone Mechanotransduction

CHAPTER 3

BONE MECHANOTRANSDUCTION 3.1 Synopsis

Degenerative diseases like osteoporosis are caused due to imbalances in the homeostatic bone remodeling process Mechanical stimulus is one of the factors found to regulate the rate of bone formation and resorption Increased loading enhances bone formation, while decreased loading reduces bone mass Low bone mass characterizes osteoporosis Hence, the underlying theme of this chapter is to investigate how stresses affect bone strength A brief introduction to the role of stresses in the body is provided initially, followed by an extended discussion on their effect in bone

3.2 Mechanotransduction

All living organisms face mechanical forces, from the fluid forces around a bacterium to the high forces in a human knee during stair climbing Forces can influence physiological processes at molecular, cellular, or systemic level, like the tensile muscular forces that enable human locomotion Mechanical forces initiate electrophysiological and biochemical responses in cells They can cause a number of short or long-term biochemical processes inside a cell, such as gene induction, protein synthesis, cell proliferation, apoptosis, and cell differentiation All these processes are essential to maintain tissue homeostasis Abnormal mechanical loading conditions are observed to alter cellular function, consequently leading to pathologies like atherosclerosis, arthritis, and fibrosis in bone, cartilage, tendon, vessels, heart, lung and skin [Ingber2003]

Chapter 3: Bone Mechanotransduction The process of converting physical forces into biochemical signals and integrating these signals into physiological responses is referred to as mechanotransduction [Huang2004] Mechanotransduction constitutes the basis for a plethora of fundamental biological processes such as the senses of touch, balance and hearing and contributes critically to development and homeostasis in all organisms Mechanosensitivity, or the specific response of cells to mechanical stimulation, is a universal quality found in most types of cells Numerous fundamental physiological phenomena such as the perception of sound and gravity, and sensation of touch are regulated by mechnosensitivity of the cell Under various pathological conditions like atherosclerosis and arthritis, mechnosensitivity of the cell is found to be affected

Although it is still unclear on how cells sense mechanical forces and convert them into biological responses [Han2004], it has been observed that the primary target for mechanical stimulus is the plasma membrane of the cell Plasma membrane responds to variable physical stresses by regulating its mechanosensitive ion channels, like the stretch-activated channels, or calcium or potassium channels [Kamkin2005] Other cellular components found to be engaged in this process include Extracellular Matrix

(ECM) molecules, transmembrane integrin receptors, G-protein Coupled Receptors (GPCRs) and Receptor Tyrosine Kinases (RTKs), cytoskeletal structures and associated

signal transduction components [Wang2006]

Chapter 3: Bone Mechanotransduction

3.3 Bone Mechanotransduction

As discussed in Chapter 2, mechanical stimulus is one of the important regulators of bone remodeling The relationship between bone remodeling and mechanical stimulus was first suggested by Wolff in 1892 in his famous law of bone remodeling He hypothesized that

“Bone remodels in response to the mechanical stresses it experiences so as to produce an anatomical structure best able to resist the applied stress” [Wolff1892] In his mechano-stat theory, Frost proposed that when stress is below a certain physiological threshold, it results in bone resorption while bone formation occurs when stress is above the threshold Hormones are assumed to reduce the threshold level; hence a lower magnitude of stress is required to induce the response [Frost1987] Till date, researchers are still trying to understand the exact mechanism by which bone adapts to its mechanical environment

A key question in all these studies has been on how bone cells sense mechanical loading and correspondingly induce increased or decreased bone formation or resorption In other words, do osteoblasts or osteoclasts detect the mechanical stimulus directly or through some kind of mechnosensor cells in the bone, which transduce mechanical stress into biochemical signals, that affect osteoblast or osteoclast activity?

A IN VIVO STUDIES

Numerous in vivo studies indicate that both osteoblasts and osteoclasts are capable of

directly sensing the mechanical stimulus and adjusting their activity accordingly [Rubin2006, Liedert2006], but the underlying mechanism still remains unexplained

Chapter 3: Bone Mechanotransduction

However, other in vivo studies indicate that

osteocytes act as the mechanosensor cells

which transduce forces into biochemical

signals, such as the release of NO and

prostaglandins [Takagaki1999], which in turn

influence osteoblast functions (Figure 3.1)

Although it is still not clear how osteocytes

actually sense mechanical loading and

transduce it into cellular signals, the theory

that osteocytes act as mechanosensory cells as

gained a lot of attraction from the research

community

Figure 3.1 Model for the transduction of mechanical strain to osteocytes in bone (Adapted from [Klein-Nulend2005a])

It is believed that the forces applied on the bone result in changes of the hydrostatic pressure causing interstitial fluid flow in the canaliculi network, which induces shear stress and electrical fields Forces are also observed to induce direct cell strain [Sikavitsas2001, Klein-Nulend2005a, Orr2006] As vast majority of cells in the bone are osteocytes, and due to their pervasive, three-dimensional distribution throughout both trabecular and cortical bone, these cells are potentially well placed to sense the magnitude and direction of the fluid flow within the tissue Although osteocyte is enclosed in calcified tissue it is connected to equally encased cells, as well as to the bone surface, through a network of canaliculi through which these cells cast long cell processes Hence,

Chapter 3: Bone Mechanotransduction

it is proposed that the presence of osteocytes within the canaliculi network makes them suitable candidates as the mechanosensor cells of the bone Figure 3.2 depicts an artist’s illustration of how osteocytes act as mechanosensory cells to affect osteoblast activity

Figure 3.2 Osteocytes as mechanosensory cells (Adapted from [Manolagas2002])

B IN VITRO STUDIES

Repeated in vitro studies show that both osteoblasts and osteoclasts, besides osteocytes,

sense mechanical stimulus [Sikavitsas2001, Mullender2004, Wiltink1995, Nulend2005b] Variegated responses to different stimuli have been reported, including modulation of cell proliferation and differentiation, synthesis of growth factors, changes

Klein-in proteKlein-in and matrix synthesis, and cell alignment [El Haj1999, RubKlein-in2006] The different stimuli used in these studies include fluid flow, four-point bending, substrate stretch, gravity force, vibration, magnetic bead twisting, atomic force, or shockwaves [Fulford2004] Scientists hypothesize that surface proteoglycan layer acts as the primary sensor of mechanical stimulus, that transmits forces to apical structures like the plasma membrane or the sub-membrane cortex (actin cytoskeleton) Lipid rafts or intercellular

Chapter 3: Bone Mechanotransduction junctions or cell matrix contacts serve as the transduction sites within the plasma membrane [Tarbell2005] These sites recruit protein complexes which interact with calcium and potassium channels in the membrane The protein complexes also interact with integrins and downstream cytoskeletal proteins to effect physiological changes like protein synthesis, and gene induction Investigation of bone mechanotransduction mechanism, at the cellular level, over the years is documented in Appendix A for further reference

3.3.1 Signaling in Bone Mechanotransduction

From the above in vitro and in vivo studies, we can infer that final step of the

mechanotransduction response involves changes in cellular physiology, like cell proliferation, gene expression, protein synthesis, or cell differentiation These cellular physiological functions are known to be the result of activation of intracellular signaling pathways [Fulford2004, Liedert2006] Since mechanical stimulus affects cellular physiology, it is widely believed that these signaling pathways are implicated in the mechanotransduction response Several studies over the years [El Haj1999, Fulford2004, Takayanagi2005, Liedert2006, and Wada 2006] indicate that a number of secondary messenger pathways, components of secondary messenger pathways and local mediators generated in response to secondary messenger activation are affected in the load transduction of osteoblasts and osteoclasts Various secondary messengers including

Adenylate cyclases, GTPases, ERK, PKA, JNK, PKC, Akt, RANKL, OPG, NF-κB, and

autocrine factors such as FGF-2, ILGFs, IGFs and TGF β trigger a sequence of events that

Chapter 3: Bone Mechanotransduction lead to elevation in RNA synthesis and elevation of specific matrix protein mRNAs, such

as collagen type I In osteoblasts, elevation of RNA synthesis ultimately leads to new

bone formation In osteoclasts, it results in bone resorption Figures 3.3 and 3.4 illustrate

how the implicated signaling pathways effect desired physiological functions in

osteoblasts and osteoclasts respectively The following section provides an extended

discussion on the signal transduction pathways implicated in the mechanotransduction of

osteoblasts and osteoclasts

Figure 3.4 Mechanotransduction response

in osteoclast (Adapted from [Wada2006]) Figure 3.3 Mechanotransduction response

in osteoblast (Adapted from [Liedert2006])

Chapter 3: Bone Mechanotransduction

A SIGNAL TRANSDUCTION IN OSTEOBLASTS

Literature [Reich1993, El Haj1999, Nomura2000, Albert2001, Rubin2002, Wadhwa2002, Chen2003, Kapur2003, Fulford2004, Edwards2005, Liedert2006, Rubin2006, Meenal2006, Li2006, Robinson 2006] on the response of osteoblasts to different mechanical stresses indicates the activation of 8 major signaling cascades, including a few interconnected signal transduction pathways, as shown in Figure 3.5 -

Figure 3.5 Block diagram representation of the osteoblast signaling network

(An extended diagram is provided in Appendix B)

These pathways include upregulation of transcription factors like JNK, ERK, NFkB and CREB, which are responsible for a wide array of function, including cell proliferation, differentiation, and matrix synthesis Following is a brief description of each of these pathways -

(i) JNK pathway: Mechanical stimulus is expected to activate integrin and cytoskeleton,

which causes binding of cytoskeletal elements such as talin, paxcillin, vinculin, or focal adhesion kinase to form the integrin signaling complex p21Ras is activated by this

Chapter 3: Bone Mechanotransduction complex via GTP exchange The active Ras induces phosphorylation of its downstream factors Mekk1 and Sek1, leading to the activation of c-Jun N-terminal kinase (JNK), a transcription factor Phosphorylated (activated) Raf from the ERK signaling cascade upregulates JNK activation, while Mekk1 from the JNK pathway upregulates Mek from the ERK pathway, indicating cross-dependence of ERK and JNK signaling pathways Activation of JNK pathway leads to increased bone formation in osteoblasts

(ii) Akt pathway: Epidermal Growth Factor (EGF) is activated upon stimulus, to induce

auto-phosphorylation of EGF receptor (an RTK), which then recruits a signaling complex that activates downstream factors – PI3K, PIP3 and PDK1 These factors suppress apoptosis of bone cells in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9 So, Akt activation leads to suppression of cell death The EGFR signaling complex in this pathway can be inhibited

or downregulated by phosphorylated PKC

(iii) ERK pathway: Activated EGFR complex can also initiate the ubiquitous MAPK

signaling cascade by activating Ras which induces phosphorylation of serine/ threonine kinases Raf and Mek ERK, a transcription factor responsible for RANKL synthesis (RANKL regulates osteoclast activity) as well as matrix synthesis, activation is the final product of this signaling cascade Besides the JNK signaling cascade, this pathway is also connected to the PKC pathway Upregulation of Raf is caused by phosphorylated PKC