THE EFFECT OF GROWTH HORMONE ON TOOTH MOVEMENT IN RATS

Zachary L. Varble, D.M.D.

An Abstract Presented to the Faculty of the Graduate School of Saint Louis University in Partial Fulfillment of the Requirement for the Degree of Master of Science in Dentistry 2009

Abstract

Introduction:

The aim of this study was to determine how

supplemental GH affects the amount of orthodontic tooth movement in rats.

Methods: Twenty-four Wistar rats were

divided into two study groups of 12 rats each.

A 7 mm

nickel-titanium closed-coil spring was ligated between the maxillary incisors and the right three maxillary molars of each rat to deliver a force of 10 g.

One group received

daily subcutaneous injections of recombinant human GH (rhGH) (2 mg/kg), while the other group received daily subcutaneous injections of saline (2 mg/kg).

Digital

caliper measurements were performed the day of appliance placement, day 6, and day 12.

The distance between the

most mesial point of the maxillary molar unit and cementenamel junction of the ipsilateral maxillary incisor was measured (I-M distance) on both the appliance and nonappliance sides.

Results:

Significant (p < .05)

differences in the amount of tooth movement was observed for the three time periods days 1-6, days 6-12, days 1-12 between the GH and saline groups for both appliance and non-appliance sides.

Differences between I-M distances on

the appliance and non-appliance sides showed significant

1

group differences (p < .05) in the amount of tooth movement between 1-6 days and 1-12 days, but not between 6-12 days. Intra-animal comparisons of the amount of tooth movement on a given side between time periods show a significant difference (p < .05) between days 1-6 and days 6-12 in the GH group on the non-appliance side only.

Intra-animal

comparisons of the amount of tooth movement between the appliance and non-appliance side at each time period show significant differences (p < .05) at all time periods for both groups except during days 6-12 for the saline group. Conclusion: GH supplementation increases tooth movement in the rat model when compared to controlled samples.

2

THE EFFECT OF GROWTH HORMONE ON TOOTH MOVEMENT IN RATS

Zachary L. Varble, D.M.D.

A Thesis Presented to the Faculty of the Graduate School of Saint Louis University in Partial Fulfillment of the Requirement for the Degree of Master of Science in Dentistry 2009

COMMITTEE IN CHARGE OF CANDIDACY: Professor Rolf G. Behrents, Chairperson and Advisor Adjunct Professor Peter Buschang Assistant Professor Ki Beom Kim

i

DEDICATION

I dedicate this project to my loving and supportive family.

I will be forever indebted to my wife and best

friend Amy for who without her support this project and my completion of this program would not have been possible. To my baby boy Calvin who has already taught his mom and dad the true meaning of happiness.

To my parents whose

support and guidance made me the man I am today.

ii

ACKNOWLEDGEMENTS

I would like to acknowledge the following individuals: 

Dr. Behrents for his advice, insight, and guidance,



Dr. Buschang for his assistance in design and statistics,



Dr. Kim for his time and comments,



Dr. John P. Long DVM for his assistance in design,



Frank Strebeck Jr. for his laboratory assistance,



Genetech Corporation for their generous donation of Nutropin AQ allowing this project to be possible.

iii

TABLE OF CONTENTS

List of Tables.............................................v List of Figures...........................................vi CHAPTER 1: INTRODUCTION....................................1 CHAPTER 2: REVIEW OF THE LITERATURE Physiology of Tooth Movement..........................4 Theories of Orthodontic Tooth Movement................5 Pressure Tension Theory..........................5 Bone Bending Theory..............................8 Bioelectric Signals Theory......................11 Micro-damage Theory.............................13 Phases of Orthodontic Tooth Movement.................14 Cellular Response to Orthodontic Force...............17 Cytokines.......................................17 Prostaglandins..................................25 Matrix Metalloproteinases.......................28 Growth Factors..................................30 Vitamin D.......................................36 Growth Hormone (GH)..................................38 Direct Effects of GH on Bone Cells..............38 Indirect Effects of GH on Bone Cells............42 GH Effects on Cytokines....................42 GH Effects on Prostaglandins...............46 GH Effects on Matrix Metalloproteinases....48 GH Effects on Growth Factors...............50 GH Effects on Vitamin D....................53 References...........................................55 CHAPTER 3: JOURNAL ARTICLE................................73 Abstract.............................................73 Introduction.........................................75 Methods and Materials................................77 Sample Selection................................77 Appliance Design................................77 Appliance Placement.............................79 Statistical Analysis.................................81 Results..............................................82 Discussion...........................................87 Conclusions..........................................93 References...........................................94 Vita Auctoris............................................100 iv

LIST OF TABLES

Table 3.1:

Mann-Whitney U tests for each time period comparing tooth movement (mm) on the right and left side between the groups ..................84

Table 3.2:

Mann-Whitney U tests for each time period comparing tooth movement (mm) differences between right and left sides within GH group to the same difference in saline group ...........85

Table 3.3:

Wilcoxon signed rank tests comparing tooth movement between days 1-6 with tooth movement between days 6-12 .............................86

Table 3.4:

Wilcoxon signed rank tests comparing tooth movement between the appliance and non-appliance sides during days 1-6, days 6-12, and days 1-12 within the GH group ...........................87

Table 3.5:

Wilcoxon signed rank tests comparing tooth movement between the appliance and non-appliance sides during days 1-6, days 6-12, and days 1-12 within the saline group .......................87

v

LIST OF FIGURES Figure 3.1:

Appliance design.............................80

Figure 3.2:

Experimental tooth movement (mm) defined as the difference in tooth movement between the appliance and non-appliance side within each group .......................................85

Figure 3.3:

Experimental tooth movement (mm) on appliance and non-appliance side in both GH and saline groups ......................................86

vi

CHAPTER 1: INTRODUCTION

According to Kaare Reitan, the ability to conduct orthodontic tooth movement is “limited chiefly by the alveolar process”.1

This ability to move teeth is dependent

on the fact that once a mechanical force is applied to the alveolar bone through orthodontic appliances that there will be a concurrent biological response that ultimately results in tooth movement.

Orthodontic tooth movement

results from the response of the periodontal tissue to orthodontic force, which leads to modeling and remodeling of the surrounding alveolar bone.2

The response is

considered to occur through the activation of specific signaling pathways, many of which are known, all acting to ultimately result in tooth movement. The rate at which tooth movement occurs is dependent upon the ability of these pathways to effect metabolism of bone by the two main cell types responsible for tooth movement: osteoblasts and osteoclasts.

Much is known about

the actions of these two cells, and the signaling pathways that affect them, both in bone and orthodontic literature. However, until now, little work has been done examining the effect of growth hormone (GH) in orthodontics, and 1

specifically no study has examined its effect on tooth movement. GH is one of the major regulators involved in bone metabolism.

GH is an important regulator of postnatal

skeletal growth and development, and it promotes stem-cell growth differentiation and proliferation. Specifically, GH acts directly on osteoblasts and osteoclasts and exerts anabolic effects on bone formation and bone resorption.3-4 Knowledge of how GH effects tooth movement is relevant in orthodontics and identification of these effects may lead to a pharmacologic intervention to control the rate of orthodontic tooth movement. This study will examine how supplemental systemic GH affects the rate of orthodontic tooth movement in rats.

We

will be evaluating if there are differences in the amount and rate of tooth movement between two populations of rats: one supplemented with growth hormone and one not supplemented.

By recording rates of movement we will be

able to determine if GH has a temporal effect on the tooth movement cycle. The study focuses on growth hormone because tooth movement induces a bone-resorption cascade that involves a series of steps directed toward removing both the mineral and the organic constituents of bone matrix by osteoclasts. 2

This phenomenon is cyclical, consisting not only of osteoclastic bone resorption, but also osteoblastic bone formation.

Growth hormone (GH) has been shown to affect

both of these cell populations.

3

CHAPTER 2: REVIEW OF THE LITERATURE

Physiology of Tooth Movement

Movement of teeth in the oral cavity occurs naturally in a variety of ways.

Prior to eruption, tooth buds

migrate due to their own devices or through the growth of surrounding dental structures (bone, teeth, etc).

Upon

entering the oral cavity teeth find a final position by both the eruptive mechanism and the pressures of surrounding tissues (cheeks, tongue, etc).

Once the teeth

reach the level of occlusion they are subjected to masticatory forces which can result in tooth movement within the boney tooth socket.

Migration can also occur if

there are changes in the equilibrium of forces that maintain the current position (i.e., trauma to surrounding musculature or dentition).

There are a variety of

additional circumstances where tooth movement occurs, but the focus here will be on the tooth movement that does not occur naturally, specifically the movement that occurs upon application of continuous mechanical forces (i.e., orthodontic tooth movement).

4

Theories of Orthodontic Tooth Movement

Pressure Tension Theory

“Tooth movement by orthodontic force application is characterized by remodeling changes in dental and paradental tissues, including dental pulp, periodontal ligament (PDL), alveolar bone, and gingiva.”5

This type of

tooth movement differs greatly from those described above in that orthodontic tooth movement is uniquely characterized by the creation of compression and tension regions in the PDL.

This phenomenon has been explained by

the “pressure tension” theory and is considered the main mechanism responsible for orthodontic tooth movement.

On

the pressure side there is a decrease in cellular replication as a result of vascular constriction, while on the tension side there is an increase in cellular replication because of the stimulation afforded by the stretching of the fiber bundles of the periodontal ligament.6

The PDL on the pressure side is said to display

disorganization and diminution of fiber production, while on the tension side, fiber production is said to be stimulated.6

This phenomenon is an elegant and complicated

interaction of cellular mechanisms, resulting in local 5

environmental changes surrounding the tooth, and ultimately its movement.

The width changes in the PDL cause changes

in cell population and increases in cellular activity. Tissue reaction to orthodontic tooth movement has been said to occur either through bone or within bone.7

Tooth

movement through bone is characterized as indirect resorption occurring not at the PDL but at adjacent marrow spaces, this is known as undermining resorption.1

During

this period of undermining resorption, the PDL on the leading side is compressed, no formative activity takes place, and localized ischemia results as a result of the compression to the local vasculature, subsequently leading to the formation of cell-free hyalinized areas within the PDL.

When the undermining resorption reaches the

periodontal ligament and removes the hyalinized tissue, the tooth begins its displacement.1

When teeth move with in

bone the resorption takes place directly on the wall of the PDL and is known as frontal resorption.

This occurs when

the vasculature on the pressure side is preserved, little or no hyalinization occurs, and cellular recruitment is allowed to occur.

During this situation the activity of

the osteoclasts on the pressure surface and the osteoblasts on the tension surface are orchestrated as a remodeling

6

cycle similar to one seen during physiologic tooth movement.8 The pressure tension theory therefore allows for two different types of tooth movement; one occurring through undermining resorption and the other through frontal resorption.

The difference between the two is found in the

preservation of the PDL vasculature on the pressure side of tooth.

It has been suggested that forces delivered as part

of orthodontic treatment should not exceed the capillary bed blood pressure of 20-25 g/cm2 of root surface.9

If

forces exceed these levels the result is physical contact between the teeth and bone leading to hyalinization in the PDL and undermining resorption in adjacent marrow spaces. If forces remain at or below these levels, then theoretically frontal resorption can be expected where osteoclasts line up in the margin of the alveolar bone adjacent to the compressed PDL producing direct bone resorption.5

It has been shown that detrimental tissue

reactions always occur during orthodontic forces and include hyalinization of the PDL even with extremely light continuous forces.1 Given the above information, it is reasonable to suggest that orthodontic tooth movement differs from physiologic tooth movement by the hyalinization of tissue. 7

The two also differ with regards to the speed of tooth movement.

Physiological tooth movement is always slow

while orthodontic tooth movement can be slow or fast depending on the concurrent biological response to the force administered.5 As stated above, orthodontic forces create changes in the microenvironment surrounding the tooth resulting in the form of a local synthesis and/or release of key tooth movement factors and molecules.

Both

of these can evoke cellular responses in and around the teeth which ultimately result in a favorable microenvironment for bone remodeling and tooth movement.10-11

Bone Bending Theory

The displacement of the tooth in the PDL space results in matrix strain both in the periodontal collagen matrix and the bone.

The compression and stretch of matrix fibers

in the PDL begins the sophisticated cellular recruitment processes resulting in increased populations of bone and inflammatory cells necessary for remodeling. Dental and paradental cellular responses to orthodontic force loads involve the interplay between intra- and extracellular structural elements. matrix attachments are formed by binding of the 8

The cell-

extracellular domains of integrins, transmembrane proteins with an intra- and extracellular domain, to extracellular matrix proteins.12

Orthodontic forces are transferred from

the extracellular matrix to the cell cytoskeleton through surface proteins.

The cytoskeleton consists of three main

components: microtubules, microfilaments, and intermediate filaments - of these the microfilaments are best suited to detect force load changes.13

Microfilament bundles

terminate at specialized sites of the cell membrane, forming a junctional complex with the extracellular matrix.13

These specialized sites connect the cytoplasm and

nucleus of the cell to the extracellular matrix. It is in fact the role of this extracellular matrix, through the cytoskeletal microfilament bundles via integrins at the cellular surface, to transform mechanical forces into biochemical signals.13 It has been suggested that the strain of bone results in fluid flow through the canaliculi leading to shear stress on the osteocytes, subsequently activating them.14 It has been shown that in areas of reduced canalicular fluid flow, as is the case on the compressed side of the tooth, apoptosis of osteocytes occurs which in turn attracts osteoclasts.15,16

Along with fluid flow changes,

bone strain through orthodontic force application also 9

results in what some have called “bone bending”.

Some have

looked at this “bending” of the mineralized and nonmineralized aspects of the bone matrix and have proposed the phenomenon as part of the foundation of orthodontic tooth movement.17 The bone bending theory explains that when an orthodontic appliance is activated the forces delivered to the tooth bends bone, tooth, and the solid structures of the PDL.5 Of these the bone was found to be more elastic than the other tissues and bends more readily in response to force application than the tooth.

The active biologic

processes that follow bone bending involve bone turnover and subsequently the release of cellular and non-cellular bone remodeling players.

These processes are further

accelerated if the bone is held in the deformed position as is the case with orthodontic force.17

The remodeling

changes do not only occur in the periodontal membrane, but throughout the alveolar bone, inferring that the strain is transmitted through the bone.

It has been shown that

reorganization of cell and non-cell factors of bone remodeling proceeds at both the lamina dura and trabecular levels of the alveolus.17

The stress has been shown to be

organized into “stress lines” which become a stimulus for altered biological responses of cells lying perpendicular 10

to these lines.17

Through this cellular activation the bone

is able to modify its shape and structural makeup to accommodate for the force.5

Bioelectric Signals Theory

The accommodation for the force is rooted in the direct and indirect cellular response elicited.

The main

direct cellular recruiting aspect to bone-bending is the electrochemical environment created by the force applied. In crystalline materials like bone, when hydroxyapatite crystals are deformed a flow of electric current is generated as electrons are displaced from 1 part of the lattice to another.5

When applying this hypothesis to

biological systems, such as tissues stressed by a force, electric potentials are generated.

It is believed that the

primary response to orthodontic force is the generation of tissue bioelectric polarization in response to bone bending.18

Electrical potentials in biological systems have

been shown to charge macromolecules that interact with specific sites in cell membranes or mobilize ions across cell membranes thereby activating those cells.11

It has

also been shown that upon the application of force the nature of bone deformation is important.19 11

Orthodontically

when bone is deformed the tension side sees a concave bone deformation and an electronegative charge component.20,21 This electronegative charge favors osteoblastic activity while the opposite charge is seen on the compression side of the tooth where the force produces a bending of the bone that is convex.

This electropositive charge increases

osteoclastic activity.20,21

This phenomenon is supported

through in vivo studies in rats where the application of exogenous electric current in conjunction with orthodontic force resulted in accelerated orthodontic tooth movement.18 Two additional studies were able to show enhanced bone resorption near the anode (electronegative side) along with enhanced bone formation near the cathode (electropositive side) in the cat model when exogenous electric current was introduced during orthodontic tooth movement.20,21 Others have examined piezoelectric potentials and have doubted the ability of such quickly decaying potentials to serve the purpose for which they are proposed.

One author

observed the generation of what he termed “stress-generated potentials” when applying force loads to bone fracture sites.22

Unlike the piezoelectric spikes which have a

quicker decay rate these stress-generated potentials had long decay periods therefore lending them to a more realistic role in orthodontic tooth movement. 12

Another

author explains these stress-generated potentials as the product of mechanical distortion of non-mineralized collagenous matrices capable of stimulating cells by altering the electric charge of the plasma membrane.11

This

change is polarity appears to be sufficient in inducing osteogenic responses.

Micro-damage Theory

Along with the theory of bone “bending” some have proposed that the strain on the bone matrix can become great enough to cause what is known as bone micro-damage. This damage theory stems from the thought that pressures against the bone (i.e., bending) may result in material fatigue ultimately resulting in damage.

This damage

behaves like a fracture at any other sites in the body, leading to apoptosis of osteocytes and attraction of osteoclasts.23

This phenomenon has been shown in vivo in

the rat model and findings show a strong association between micro-damage, osteocyte apoptosis, and subsequent bone remodeling via attraction of osteoclasts to site of damage.24

One study connects the theory of microcrack

cellular recruitment with that of fluid flow recruitment discussed previously.

It concludes that analysis of 13

individual cracks created during stress showed a disruption of the canalicular processes connecting osteocytes. This disruption is suggested to play some role in the mechanism that signals bone remodeling.25 The microcrack theory has been examined within the context of orthodontic tooth movement.

One author reported

the presence of microcracks one day after application of orthodontic force in a pig model and concluded that microdamage-driven remodeling occurs in alveolar bone and represents the first damage induced by orthodontic loads.26 Another study observed significantly more cracks on the bone surface to which the orthodontic force was applied.27

The Phases of Orthodontic Tooth Movement

Many processes occur immediately after the initiation of tooth movement.

Fluid flow transfers, mineralized and

non-mineralized matrix strains, and micro-damage dominate the early stages of orthodontic tooth movement and ultimately lead to cellular strain.

As stated by one

author “cells are motors for tissue modeling and remodeling, and most cell types are sensitive to mechanical loads,” a fact that provides the foundation for the ability to conduct orthodontic tooth movement.7 14

When mechanical

forces are applied to teeth, the cells/extracellular matrix of the PDL and alveolar bone respond synergistically resulting in tooth movement.11

Phases exist in tooth

movement and during the early phase of force application the PDL and its fluid are shifted, producing cellular/matrix changes and deformation.

These micro-

environmental changes induce non-cellular elements such as cytokines, growth factors, neurotransmitters, etc., to initiate and sustain the remodeling activity ultimately facilitating tooth movement.5 The orchestration of all of these players in tooth movement has been shown to be time dependent.

As a result,

tooth movement can be characterized as phased processes, involving three distinct time dependent periods.

These

phases of tooth movement are described as the initial, lag, and post-lag phases.28

The initial phase is characterized

by a period of very rapid tooth movement lasting a few days.

This movement is thought to be largely due to the

tooth displacement within the PDL space.28

Because of the

orthodontic force the width of the periodontal ligament is reduced on the pressure side which is limited by what one author labeled “hyrodyamic damping.”

The large

instantaneous movement will be followed by a delayed reaction due to viscoelastic properties of the periodontal 15

ligament.29

This phase has been shown to be force

dependent: larger force equates to larger initial tooth movement.30 The lag phase immediately follows the initial phase during which the tooth shows little or no movement.

During

this phase it is thought that the cessation of movement is produced by hyalinization of the PDL tissues in areas of compression.

These areas appear earlier and are more

extensive if higher forces are applied.

The duration of

the lag phase is independent of the force magnitude.31

The

phase continues until the non-vital hyalinized tissue is removed.

A large amount of individual variation has been

found in the duration of the lag phase, ranging from 0 to 35 days independent of the applied force.29 The post-lag phase follows the lag period, during which the rate of movement gradually or suddenly increases.28

Some have divided this phase into two distinct

phases characterized according to the nature of the tooth movement.

They describe the beginning of the post-lag

phase as an “acceleration phase” where the biologic processes involved in remodeling of the periodontal ligament and alveolar bone reach their maximum capacity.32 It has been shown in beagle dogs that there is a plateauing of the speed of movement.

This indicates that there is a 16

biologic limit for the rate of tooth movement absent some metabolic condition or pharmacologic intervention.29

Cellular Response to Orthodontic Force

Cytokines

The strain produced by mechanical forces in the microenvironment surrounding dental and paradental cells causes changes in cellular shape and elicits release of signaling molecules from the affected cells.5

These

molecules bind with cell adhesion complexes on other cells, or the very cells that released the factors, and initiate many cellular responses.33

This complex cellular/non-

cellular orchestration ultimately results in the bone remodeling necessary for tooth movement, and can initially be characterized as an acute inflammatory process.

The

initial phase of tooth movement is dominated by characteristics of such a phenomenon.

This phase is

predominately exudative, in which plasma and leukocytes leave capillaries in the areas of strain.

Leukocytes

produce various cytokines which interact directly or indirectly with the native paradental cells.5

Cytokines act

on these cells and have autocrine and paracrine effects 17

that invoke the synthesis and secretion of numerous substances by their target cells, including prostaglandins, growth factors, and additional cytokines. Cytokines found to affect bone metabolism include interleukins (IL-1, IL-6, IL-8), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma).

The

most potent of these is interleukin-1 (IL-1), which directly stimulates osteoclast function.5

IL-1 has been

implicated as a primary mediator of bone remodeling and is released in response to mechanical strain, neurotransmitter signaling, and other cytokines.

Upon administration of an

interleukin-1 receptor antagonist, strain-induced bone modeling of cells in the gerbil auditory bulla is inhibited both in vitro and in vivo.34

When periodontal ligament

cells were cultured, subjected to mechanical stresses, a statistically significant increase in IL-1 mRNA has been reported compared to controls.35

There is an abundance of

evidence supporting increased levels of IL-1 in the both the paradental tissues and the gingival crevicular fluid upon application of orthodontic forces.36-39 Another interleukin, IL-6, has many functions with regard to bone modeling.

Indirectly IL-6 has been shown to

enhance osteoblast-like cell colony formation.

The direct

effects of IL-6 include the stimulation of osteoclast 18

formation and the subsequent induction of bone resorption.40 The primary sources of IL are the macrophages and osteoblasts.

One study looked at the effect of mechanical

strain in human osteoblast-like cell colonies on cytokine gene expression.

They found that tensile stretch on these

cells resulted in increased mRNA levels of IL-6.41

Similar

findings have been seen during compression in a rat model upon application of orthodontic forces.

The expression of

both IL-1 and IL-6 mRNA have been shown to be up-regulated in both PDL cells and osteoblasts on the compression side of the PDL.42 Interleukin-8 (IL-8) is another cytokine that is produced by macrophages and epithelial cells at sites of acute inflammation and is responsible for inducing chemotaxis of its target cells (other macrophages, mast cells, and endothelial cells) to the sites of the insult.43 IL-8 levels have been reported to be increased in the PDL at sites of both tension and compression indicating a role in orthodontic induced bone remodeling.44 Tumor necrosis factor alpha (TNF-alpha) is another cytokine that plays a significant role in bone remodeling. TNF-alpha is mainly produced by macrophages and fibroblasts in response to bacterial products or in the case of aseptic bone remodeling, in response to IL-1. 19

TNF-alpha directly

stimulates the differentiation of osteoclast-like cells into mature osteoclasts.45

Elevated levels of TNF-alpha are

seen in paradental cells during orthodontic tooth movement in the animal model.46 Both elevated TNF-alpha levels and mRNA expression have been shown in human gingival crevicular fluid upon application of orthodontic forces by as much as a two-fold increase compared to controls.42,47 TNF-alpha is a member of a larger group of cytokines referred to as the tumor necrosis family (TNF-family) of cytokines.

Within this family is a factor (or ligand)

known as TNF-related activation-induced cytokine (TRANCE) a known receptor activator for nuclear factor kappa B ligand (RANKL).

RANKL belongs to a system of cytokines known as

the OPG/RANK/RANKL axis.

This axis is a molecular triad

composed of osteoprotegerin (OPG)/Receptor Activator of NFkB (RANK)/RANK Ligand (RANKL) and is a key signaling pathway between osteoblasts and osteoclasts. The interaction between RANK and RANKL plays a critical role in promoting osteoclast differentiation and activation leading to bone resorption.

RANKL is a

regulator of osteoclast formation and activation through which many hormones and other cytokines produce their bone remodeling effects.

RANKL is expressed in both osteoblast-

like cells and mature osteoblasts and exerts its effects by 20

binding the RANK receptor on osteoclast-like cell lineages. This binding induces differentiation and maturation of these cells into mature osteoclasts.48

OPG is a soluble

decoy receptor for RANKL that blocks osteoclast formation by inhibiting RANKL binding to RANK.49 Studies in both the bone and orthodontic literature have examined the OPG/RANK/RANKL axis and its role in bone remodeling in the presence of mechanical stresses.

One

study compressed PDL cells continuously in culture and showed an up-regulated osteoclastogenesis.

They also noted

the expression of RANKL mRNA and protein in PDL cells increase in parallel with the change in the number of osteoclasts.50

Another study showed similar findings noting

a stretch-induced transcriptional elevation of genes assigned to the RANK pathway, with a 1.7 fold increase in expression over controls.51

A third study examined the

effect of a compression force on periodontal ligament fibroblasts (PDLFs) and found increased levels of both RANKL mRNA and protein in culture in both a time- and force magnitude-dependent manner.52 Intuitively it would seem that clinical situations that promote an imbalance in the RANKL/OPG ratio would result in either a decrease or increase in the amount of tooth movement observed.

Similar to in vitro studies, it 21

has been shown in vivo that compression force significantly increases secretion of RANKL 16.7 fold while decreasing that of OPG 2.9 fold, as compared to controls.53

One study

performed OPG gene transfer to periodontal tissue which resulted in an inhibition of RANKL-mediated osteoclastogenesis and inhibited experimental tooth movement compared to controls.54

The same investigators

examined a RANKL gene transfer which significantly enhanced RANKL expression, osteoclastogenesis in periodontal tissue, and the amount of experimental tooth movement.55

This

RANKL/OPG ratio difference can be examined in a clinical setting based on differences in tooth movement associated with age.

One study compared the levels of OPG and RANKL

in gingival crevicular fluid (GCF) during orthodontic tooth movement in juvenile and adult orthodontic patients.

The

group found RANKL/OPG ratio in GCF from adult patients was lower than that in the juvenile patients, and subsequently an age-related decrease in amount of tooth movement after 168 hours.56 As was stated previously the interferon family of cytokines, specifically interferon-gamma (INF-gamma), has been shown to be integral in the bone modeling processes. INF-gamma like interleukins and TNF-alpha are expressed by cells in and around sites of acute inflammation. 22

This is

known as the “macrophage-activating factor,” due to its primary function of inducing these cells to secrete proinflammatory cytokines.

The result is inflammation and

recruitment of immune cells and subsequent elimination of the insulted tissues via phagocytosis or release of toxic metabolites.57 In addition to these functions, INF-gamma also promotes osteoblastic induced activation of preosteoclastic cell lineages via promotion of cellular sensitivity of TNF-alpha on its target cells.58

More

importantly, INF-gamma has been shown to be a potent stimulator of nitric oxide (NO) production when combined with other cytokines (IL-1 and TNF-alpha).59

INF-gamma

generates NO indirectly by up-regulating the gene expression in osteoblasts and osteoclasts of enzymes responsible for producing NO, nitric oxide synthase (NOS).60 NO production in these cells has been suggested to be essential for normal osteoclast function, and is supported by the fact that, when NOS inhibitors are added to isolated osteoclast cell cultures, their activity ceases.61

NO is

also a responsible for osteoclast-like cell differentiation into mature osteoclasts through promotion of nuclear translocation of the transcription factor for RANK.62 Animals that are NOS-deficient continue to show RANK/RANKL 23

activation in response to the cytokines described above (IL-1) but the response has been described as transient. This implies that NO has a key role to play in sustaining RANK activation in these cells.63 The role of NO in orthodontic has been examined in an attempt to determine whether or not altered NO production interferes with tooth movement.

One group used a rat model

and introduced NO precursor (L-arginine) and NOS inhibitor (L-arginine methyl ester (L-NAME)) into different samples and compared tooth movement and osteoclast counts to those of controls.

The results showed that the number of

osteoclasts was significantly higher in the L-arg group, while the number of osteoclasts in the L-NAME group was significantly lower as compared to the control group.

The

greatest amount of tooth movement was seen in the L-arg group, followed by the control, and the L-NAME.64

Another

study explained that in the L-arg groups an increased number of multinuclear osteoclasts, Howship’s lacunae capillary vascularization, and orthodontic tooth movement were significantly increased compared to controls.65

24

Prostaglandins

Prostaglandins are an arachidonic acid derived family of lipid compounds that play a major role in the acute inflammatory response.

There are many different kinds of

prostaglandins that perform equally large number of tasks but the one member that plays the largest role in bone metabolism is prostaglandin E-2 (PGE-2).

It seems to be

responsible for both aspects of bone modeling: bone formation and resorption.

PGE-2 has been shown to possess

a stimulating effect on both osteoblasts and osteoclasts.66 Specifically, it has been shown to upregulate cyclic AMP (cAMP), a second messenger involved in intercellular signal transduction, indicating cellular stimulation.67,68

On a

gross level, when PGE-2 is secreted by cells near the site of injured bone cell proliferation and collagen synthesis are observed.69 Within the bone literature, mechanical stimulation has been shown to be strong activator of the cellular release of prostaglandins in bone cell populations.70

One study

examined the effect of compressive force on PGE-2 production in Saos-2 (pre-osteoblastic cells).

PGE-2 gene

expression and production was increased upon introduction of compressive forces in these cells.71 25

Similar findings have been shown in the orthodontic literature.

One group looked at the effect of chemical and

mechanical stimuli on both cAMP and PGE-2 levels in human gingival fibroblasts.

As a chemical stimuli they seeded

the cultures with either exogenous PGE-2 or indomethacin, a know inhibitor of PGE-2.

For the mechanical stimuli, cell

cultures were stretched by placing plastic membranes over the culture wells and then weighting the covers.

The

results showed a dose and time dependent response to chemical application on cAMP levels, along with significant increases in the synthesis of PGE-2 upon cell stretching.72 In vivo studies have shown significant increases in PGE-2 level within the gingival crevicular fluid of humans as the result of orthodontic force application.73,74

Other

studies have examined the effects of exogenous PGE-2 on tooth movement.

One study looked at the long-term effects

of varying the concentration of exogenous PGE-2 on the rate of tooth movement in a rat model.

Injections of exogenous

PGE-2 did enhance the amount of orthodontic tooth movement to a statistically significant level.75

A similar study of

exogenous PGE-2 administration in a rat model found similar amounts of increased in tooth movement compared to controls.

Also, on a histologic level, increased numbers

26

of Howship’s lacunae and capillary populations at the site of compression were seen.76 Reasons for the increased tooth movement upon application of exogenous PGE-2 lies in the response elicited from the cells at the gene level.

It has been

shown that upon introduction of exogenous PGE-2 into osteoblast/stromal cell cultures an increase in both IL-1 and RANKL gene expression were observed.68

When looking at

the effects of exogenous PGE-2 on cells of the PDL, similar results were found with increased levels of both RANKL protein and mRNA gene expression.50,77 PGE-2 can effect IL-1 production, but the reverse has also been shown to be true.

Upon administration of

exogenous IL-1, TNF-alpha, and IFN-gamma alone or in specific combinations, human PDL fibroblasts responded by an increase in the synthesis of PGE-2 in a dose- and timerelated fashion.78

This interplay seems necessary in

achieving optimal bone resorption.

When treating PDL cells

with cytokines IL-1, TNF-alpha, and INF-gamma, increased amounts of PGE-2 are produced and increased bone resorption activity is observed compared to mechanically stressed controls (with elevated endogenous levels of PGE-2). However upon treating these cells with the same cytokines and indomethacin, a known inhibitor of PGE-2 formation, the 27

indomethacin completely inhibited PGE-2 production while only partially inhibiting bone-resorbing activity.79 Interactions between PGE-2 and IL-6 have also been described as affecting OPG/RANKL/RANK axis system.

PGE-2

stimulated IL-6 secretion is bone cells, and IL-6, in turn, increased PGE-2 secretion in those same bone cells.

IL-6

was also the mediator of PGE-2 increased RANKL and decreased OPG expression in osteoblasts, while upregulating the RANK expression in osteoclasts.80

These

studies demonstrate the ability of both cytokines and prostaglandins to both dependently and independently effect bone resoption.

Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are members of a family of endopeptidases described as proteolytic peptidases that are responsible for breaking the peptide bonds of amino acids.

They are capable of degrading

extracellular matrix proteins, a necessary step in bone remodeling.

MMPs are also thought to induce bone cell

proliferation, differentiation, and migration.81

The

function of MMPs in bone include regulation of bone modeling processes, such as the degradation of collagen and 28

other components of the bone matrix and the promotion of bone formation and bone resorption.82 Relating these actions to orthodontics, the bone literature has examined the effect of mechanical stress on the expression of MMPs in osteoblast-like cells.

The

expression levels of MMP-1, MMP-2, MMP-3, MMP-13, and MMP14 all markedly exceeded the control levels at various force levels.

This suggests that mechanical stress

stimulates bone matrix turnover by increasing these proteinases.83 The orthodontic literature pertaining to the role MMPs play in tooth movement provides similar conclusions.

Upon

chemically mimicking stresses in vitro via colchicines administration, a disruptor of microtubules of cells, periodontal ligament cells have been shown to significantly increase MMP-1 mRNA protein and gene expression levels compared to controls.84

An in vitro study subjected human

periodontal ligament fibroblasts to cyclic tensional and compressive forces found an increase in both mRNA expression and total protein levels of MMP-2.85

PDL

osteoblasts have also been shown to promote MMP-3 protein release upon application of compressive forces in vitro.86 In addition, various in vivo studies in both rat and human models have echoed these findings. 29

When analyzing

the gingival crevicular fluid post-orthodontic tooth movement, several studies have collectively reported increases in the both gene expression and protein levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-13.86-89

The role of

MMPs in orthodontic bone remodeling is further supported by examining the effects of MMP inhibitors on tooth movement. One in vitro study showed that a general MMP inhibitor, Ilomastat, limited the resorption of bone slices by mouse marrow cultures concomitantly simulated by agents known to entice osteoclastic activity.90

An in vivo report

in the orthodontic literature supports this finding.

Using

a daily dosing schedule of chemically modified tetracyclines (CMTs), known to inhibit MMPs, a decrease in the amount of tooth movement was observed in the rat model. Histological findings showing similar osteoclastic cell counts emphasize the role MMPs play in osteclast function.91

Growth Factors

The term growth factor refers to a naturally occurring protein capable of stimulating cellular growth, proliferation, and differentiation.

The most abundant of

these in bone is transforming growth factor-beta 1 (TGFbeta 1).

TGF-beta 1 is a member of the transforming growth 30

factor-beta superfamily which consists of two subfamilies: the TGF-beta and bone morphogenic protein BMP subfamilies.92 In bone, TGF-beta 1 is produced by osteoblasts and simulates proliferation and differentiation of osteoblastslike cells.

When treating clonal osteoblastic-like cell

lines with TGF-beta, there are increases in both proliferation and differentiation of these cells along with concurrent increases in extracellular matrix specific collagens.93 TGF-beta has also been shown to enhance osteoclast differentiation in hematopoietic cells stimulated with RANKL.94

The family of bone morphogenic proteins (BMPs) has

been shown to stimulate similar responses in bone compared to TGF-beta.

BMP-2, the most common BMP in bone, has been

shown to be responsible for the differentiation of osteoblasts from undifferentiated mesenchymal cells.95

It

has been shown to induce bone formation in vivo upon exogenous local injections and has been reported useful in various therapeutic interventions involving the healing of bone defects and non-union fractures.96 A link between the effects of both TGF-beta 1 and BMP on bone formation and orthodontic tooth movement has also been investigated.

Upon applying tensile force to PDL

cells an up-regulation of TGF-beta 1 mRNA gene expression 31

was observed.97

TGF-beta 1 levels in the gingival

crevicular fluid of orthodontically treated teeth has been shown to be significantly higher than in matched controls.98 In osteoblast-like cell cultures, compressive force significantly increases the mRNA expression of BMPs while simultaneously decreasing the expression of its antagonist.99

One in vivo study looked at the expression of

BMP-2 in periodontal cell upon tooth movement in rats.

The

BMP-2 expression on the tension side was slightly stronger than that on the compression side.

Upon administration of

simvastatin, a common cholesterol lowering drug with known stimulatory affects on the BMP-2 formation pathway, an increase in BMP-2 expression occurred compared with controls and subsequently decreased amounts of orthodontic relapse.100 The functions of two other growth factors families, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), are similar.

The primary role of

both is to promote endothelial cell proliferation, the physical organization of these cells, and subsequently angiogenesis.

Adequate blood supply during wound healing,

even with the sterile wound healing that occurs during orthodontic tooth movement, is dependent on the reestablishment of a proper blood supply.101 32

In addition to the vascular effects, both have a unique role in the bone remodeling process.

Exogenous FGF

administration to osteoblast-like cells produces an increase in osteoclast differentiation indirectly through prostaglandin and RANKL upregulation.102

Human periodontal

ligament cells under compression and osteoblasts under tension both been shown to up-regulate FGF mRNA gene expression and protein levels.103,104 VEGF has been shown to be a promoter of osteoclastogenesis as well, where it serves as an autocrine factor, released from active osteoclasts in response to RANKL stimulation.

VEGF in turn upregulates RANKL release

from other osteoclasts and their precursors perpetuating the cycle.105

Like FGF, in vitro studies investigating the

effects of cyclic tensile forces on expression of VEGF have found increases in both the expression of VEGF mRNA and protein levels.106

An in vivo study reports that both FGF

and VEGF are present in the gingival crevicular fluid during inflammatory processes.107

Another study reports

that the number of osteoclasts was enhanced by local injection of recombinant human VEGF (rhVEGF) with the application of mechanical forces in mice.108

The same

investigators in a separate study, were able to show that VEGF is highly expressed by mechanical stimuli and that the 33

amount of tooth movement is accelerated with exogenous injections of rhVEGF.109

Upon administration of an anti-

VEGF polyclonal antibody via local injection, a marked reduction occurs in osteoclast number and the amount of tooth movement.110 Insulin-like growth factor-1 (IGF-1) is also considered to play an important role in the regulation of bone metabolism.

IGF-1 has been shown to stimulate

osteoblastic cells, inducing their proliferation and collagen synthesis.111

Upon administering IGF-1 to neonatal

mouse bone cell cultures, a dose dependent stimulation of bone resorption was observed.

Yet, in isolated osteoclast

cultures the addition of exogenous IGF-1 produced no increase in osteoclastic cell activation.

However, in the

presence of primary mouse osteoblasts, IGF-1 enhanced osteoclast resorptive activity.111

This suggests an IGF-1

promotion of osteoblast mediated activation of osteoclast function.

When IGF-1 was added to cultures of bone cells

the number of osteoclastic multinucleated cells increased and pit formation on the bone culture surfaces was reported.112

IGF-1 has also been shown to regulate the

OPG/RANK/RANKL axis by suppressing OPG and increasing RANKL expression respectively in bone cell cultures.113

34

IGF-1 has also been shown to be up-regulated locally by cellular strain.

Tensile forces significantly increase

the expression of both IGF-1 mRNA and protein.

Using in

situ hybridization, IGF-1 and IGF-1 receptor mRNAs are localized in osteoblast-like and fibroblastic cells subjected to tensile force.114-116 The connection between these findings and orthodontics is that IGF-1 has been identified in the gingival crevicular fluid (GCF) during tooth movment.117,118

IGF-1

also seems responsible for cellular recruitment and activation in mechanical load situations.

This is

dependent on the nature of the mechanical load.

Exogenous

IGF-1 administration into a cyclical loading model significantly increases the activation of osteoblasts and osteoclasts and subsequent bone remodeling compared to unloaded controls.119 The implications of this in orthodontics are potentially very meaningful.

Such

research suggests that exogenous factors may be capable of enhancing traditional orthodontic appliance load effects on the tissues and enhance tooth movement.

35

Vitamin D

Another agent thought to play a role in bone metabolism during orthodontic tooth movement is 1, 25dihydroxycholecalciferol (1, 25-DHCC).

1, 25-DHCC is a

hormone and the biologically active form of vitamin D, which, along with parathyroid hormone (PTH), helps to maintain systemic calcium homeostasis.76

It has been shown

to be a potent stimulator of bone resorption by inducing differentiation of osteoclasts from their precursors, as well as increasing activity of exisiting osteoclasts.120

In

vitro studies have shown that, upon administration of 1, 25-DHCC, osteoblast cell cultures demonstrate a two- to fourfold increase in osteoclastic bone resorption compared to controls.

The same results are seen when 1, 25-DHCC is

added to osteoclasts incubated alone.

But upon

administration of actinomycin D, a known inhibitor of osteoblast activity, 1, 25-DHCC was unable to stimulate osteoclastic resorption.121

These findings suggest that 1,

25-DHCC stimulates bone resorption through a primary action on osteoblastic cells that are induced by the hormone to stimulate osteoclastic bone resorption.

This stimulation

is rooted in the hormones ability to promote increased and

36

decreased levels of expression of the mRNAs for RANKL and OPG, respectively.122 In vivo studies appearing in the orthodontic literature have shown increased levels of orthodontic tooth movement upon daily PDL injections of 1, 25-DHCC.

The

amount of increased tooth movement compared to controls has been reported to be as high as 60% in both rat and cat models.123,124

Upon comparing the effect of 1,25 DHCC to PGE-

2, the amount of tooth movement is enhanced significantly in both groups compared to controls.

The numbers of

osteoclasts, Howship’s lacunae, and capillaries on the pressure side are significantly greater in the PGE-2 group, while the number of osteoblasts on the external surface of the alveolar bone on that same side are greater in the 1,25 DHCC group.

The authors concluded that this proves 1,25

DHCC to be more effective in modulating bone turnover because its effect on bone formation and bone resorption are balanced.76

In a rat model, repeated injections of 1,

25-DHCC with concurrent application of tooth movement decreased the mineral appositional rate (MAR)on the compression side and increased it on the tension side.

The

authors claim these findings show that 1, 25-DHCC enhances the reestablishment of supporting tissue after orthodontic treatment and can potentially limit relapse.125 37

Growth Hormone

Direct Effects of Growth Hormone on Bone Cells

Growth hormone (GH) is a peptide hormone that stimulates growth and cell reproduction.

It is

synthesized, stored, and secreted by somatotrophic cells within anterior pituitary gland. Peptides released by the hypothalamus (i.e., growth hormone releasing hormone and somatostatin) into the venous blood surrounding the pituitary are the major controllers of GH secretion.

The

effects of growth hormone on the tissues of the body are generally anabolic.

GH affects many tissues including the

liver, muscle, kidney, and bone.

GH has also been shown to

play an important role in bone growth and maintenance through its stimulatory effect on chondrocytes, ostetoblasts, and osteoclasts.126

GH also stimulates

production of insulin-like growth factor (IGF-1) by the liver, the principle site of IGF-1 production.

IGF-1, also

a peptide hormone, has growth-stimulating effects on a wide variety of tissues, with the activation of chondrocytes, osteoblasts, and osteoclasts demonstrating its role in both bone growth and bone maintenance.126

38

GH has both direct and indirect effects on bone. Directly, it has been shown that GH receptors are present on osteoblasts and osteoclasts and direct activation of these cells stimulates proliferation, differentiation, and extracellular matrix production in osteoblast-like cells, while stimulating recruitment and bone resorption activity in osteclast-like cells.127 Indirectly, GH affects bone metabolism through increased IGF-1 expression and release, both systemically and locally.

IGF-receptors are present

on both bone modeling cell types, whereby IGF-1 enhances bone formation by increasing replication and function of osteoblasts, and bone resorption through its direct action of supporting generation and activation of osteoclasts.128,112 It has been suggested that GH and IGF-1 are important regulators of bone homestasis throughout life.

During pre-

pubertal growth both are determinants of longitudinal bone growth, skeletal maturation, and maintenance of bone mass.129 In both instances they are responsible for bone remodeling through the coordination of both bone resorption and formation on a cellular level.130 The process by which GH/IGF-1 performs these functions has been termed the “dual effector theory.”

The theory

explains that GH increases tissue formation by acting both directly and indirectly on target cells. 39

Direct action

promotes the differentiation of precursor cells, while IGF1 is not able to substitute for GH in promoting differentiation, but does have the ability to promote clonal expansion of those differentiated cells.

Tissue

growth results from both the creation of new differentiated cells and from their subsequent clonal expansion, with both GH and IGF-1 increasing tissue growth, but by different means.131 The dual effector theory has been supported by clinical studies testing of the independent effects of GH and IGF-1.

Differential growth levels in hypophysectomized

rats upon IGF-1, GH, and IGF-1 plus GH supplementation treatments prove an independent action.

Growth promotion

activity, in terms of long bone growth and weight increase, was greater with both IGF-1 plus GH supplementation compared to IGF-1 or GH alone.132 These findings are echoed when examining the direct effects of GH/IGF-1 at the cellular level.

GH acts on

osteoblasts by increasing DNA synthesis, alkaline phosphatase activity, and collagen synthesis.

It also acts

by increasing the expression of both IGF-1 and IGF-1 receptor mRNA, as well as the release of IGF-1 from these cells.

40

These findings suggest that GH acts both directly and indirectly on osteoblasts to enhance the resultant bone formation.111

Further support for this contention can be

seen upon administration of exogenous GH on osteoblast-like cells.

Cell proliferation was noted to increase 160% when

compared to control samples.

Upon the addition of IGF-1-

anti-serum to the culture, the GH proliferative effect is inhibited.133 The effect of GH on the osteoclast cell cultures has shown similar results.

When exogenous GH is added to

osteoclast-like cell cultures, an increase in cell activation and pit formation on dentin slices is observed.4 Yet upon the introduction of anti-IGF-1 antibody and exogenous GH into osteoclast-like cell cultures, there was an increase in osteoclast cell proliferation, but no dentin pit formation.111 GH and IGF-1 work in concert to initiate and maintain many of the aspects of bone modeling.

Each stimulates bone

cells directly through GH-receptor and IGF-1-receptor complexes, but both also stimulate bone cells indirectly through the stimulation of a number of cytokines, growth factors, and hormones. A relationship between GH/IGF-1 and orthodontics is suggested based on these direct and indirect effects on 41

bone metabolism.

Osteoblasts and osteoclasts found in the

PDL and involved in orthodontic tooth movement have been shown to possess the receptors for both GH and IGF-1.134 This fact alone inspires thoughts of a GH role in enhancing orthodontic tooth movement.

But in addition to their

direct effects, both GH and IGF-1 may effect tooth movement indirectly through stimulation of cytokines, growth factors, and hormones.

Indirect Effects of Growth Hormone on Bone Cells

GH Effects on Cytokines

On a systemic level, GH is thought to have a direct influence on the immune system.

GH has been shown to

enhance serum levels of many components of the immune system including cytokines.

As discussed previously, many

of these cytokines play important roles in bone remodeling that occurs during orthodontic tooth movement (IL-1, IL-6, IL-8, TNF-alpha/RANKL, and IFN-gamma).

GH treatment for

short stature in humans has been shown to significantly increase serum concentrations of IL-1, IL-6, IFN-gamma, and TNF-alpha.135,136

An in vivo study in rats reported that the

administration of GH alone did not affect immune function, 42

while IGF-1 alone, and in combination with GH, resulted in increases in serum IL-6 and TNF-alpha levels.137 Upon administration of exogenous GH into osteoblastlike cell cultures, increased expression of mRNA levels of IL-1, IL-6, and IL-8 are observed.138

In human, osteoblast

cell cultures the addition of exogenous GH induced a 95% increase in IL-6 protein and a 155% increase in mRNA levels of IL-6.139

GH administration also effects macrophages by

upregulating pro-inflammatory cell signaling cascades (ERK1/2 and JNK), which mediate production of TNF-alpha, IL-1, and IFN-gamma.140

This is supported by studies that

administered GH to both human macrophage and monocyte cell cultures and showed increases TNF-alpha, IL-1, and IL6.141,142 GH/IGF-1 also promotes the major mechanism of osteoblastic control over osteoclast formation and activation: the OPG/RANK/RANKL axis.

Osteoblastic cells

express soluble and membrane-bound RANKL as well as the decoy receptor, OPG, upon stimulation with GH/IGF-1.

When

stimulated by RANKL, pre-osteoclasts differentiate into osteoclasts.

RANKL also stimulates osteoclast fusion into

multinucleated osteoclasts.143

OPG blocks the effects of

RANKL by neutralizing and preventing binding to its receptor RANK.143 43

The OPG/RANK/RANKL axis is made up of members of the TNF ligand and receptor superfamily, and mediates the effects of many regulators of bone metabolism and tooth movement.

It is thought that OPG is a cytokine involved in

the regulation of osteoblast/osteoclast crosstalk and maintenance of bone mass.

On the systemic level, it has

been shown that GH replacement therapy in GH-deficient patients have been able to induce a significant increase of OPG in the plasma.144,145

Similar findings are reported when

looking at the effects of GH on human osteoblast-like cells in culture.

Results show that GH exposure is able to

stimulate OPG secretion and mRNA expression in a dosedependent manner.146

These findings lend support to the

claims that GH has an anabolic effect on bone formation. IGF-1 influences the TNF superfamily axis in a different way.

IGF-1 modulates bone resorption by

regulating expression of OPG, RANK, and RANKL.

In vitro

studies have shown that in osteoblast cell cultures, human IGF-1 suppressed OPG mRNA and decreased OPG protein expression, while at the same time increasing RANKL mRNA expression.113

In vivo administration of exogenous IGF-1 in

humans result in significant increases in serum IGF-1 and a 20% reduction in serum OPG.113

IGF-1 has also been shown in

vitro to increase OPG expression of osteoclast-like cells, 44

while also increasing RANKL expression comparatively.

The

net effect of this is an increase in the RANKL/OPG ratio and increases in osteoclast activation.147 The role of IFN-gamma in bone modeling, and therefore orthodontic tooth movement, is through nitric oxide (NO) bone cell activation.

NO is known to be a potent

stimulator of osteoclasts through its activation of the RANKL ligand.

GH has been shown to enhance the JNK

signaling cascade in macrophage cell cultures. cascade mediates the production of IFN-gamma.140

The JNK In

addition to these findings, GH was also shown to significantly enhance the production of IL-1 and IFN-gamma compared to controls in macrophage cell culture.148

On a

systemic level GH supplementation in children for short stature has been shown to increase serum levels of IFNgamma compared to controls.135 There is also evidence for direct GH stimulation of NO release.

In adults, supplemental low-dose GH

administration produces increases in plasma concentration of NO compared to controls.149

GH therapy in cystic

fibrosis patients has been shown to significantly increase NO concentration in serum and urine; there were also increases in serum L-arginine concentration, the main substrate for NO production by NO-synthases.150 45

On the

cellular level GH has been shown to increase the expression of nitric oxide synthases (NOS), the enzyme responsible for NO production.

GH also induces NOS gene/protein expression

and enzyme activity in human endothelial and mesangial cell cultures.151,152

Macrophage cell cultures have also been

shown to increase NO production upon administration of GH.153 GH also affects the bioavailability of NO and its substrates via IGF-1.

Exogenous GH administration in mice

has been shown to increase NO and NOS and decrease asymmetric dimethy-L-arginine (NOS inhibitor) serum levels. Upon blocking of the IGF-1 receptor, the GH-mediated effects of increased NO and NOS are prevented.154

In the

rat model, GH treatment alone and GH/IGF-1 combination treatment produces slightly higher amounts of endothelial NOS expression in the combination group.

This suggests

that the combination could act in a synergistic manner in terms of increased NO bioavailability.155

GH Effects on Prostaglandins

Most of the effects of GH on prostaglandins are indirect through IGF-1 or the stimulation of cytokines. IGF-1 has been shown to influence prostaglandin production 46

in several in vitro studies.

One of these studies reported

evidence that exogenous IGF-1 up-regulates COX-2 expression and the associated PGE-2 biosynthesis in cell cultures.156 The effect is seen as an IGF-1 induced increase of PI3K, MAPK, and PKC signaling, cascades which are involved in the transcriptional regulation of COX-2 expression.156

Another

study found similar increases in COX-2 expression and PGE-2 levels in an undifferentiated cell culture upon exogenous IGF-1 administration.

They also demonstrated that COX-2

expression by IGF-1 is mediated through the IGF-1 receptor. Treatment of undifferentiated cells with a blocking antibody for IGF-1 receptor inhibits COX-2 mRNA expression.157 IGF-1 is also primarily dependent on concurrent IL-1 activities to induce PGE-2 stimulation.

IL-1 was shown to

induce PGE-2 biosynthesis in certain cell cultures, but when IGF-1 was added the COX-2 protein expression and PGE-2 levels were increased.

This was due to the ability of IGF-

1 to enhance IL-1 MAPK phosphorylation.158

When focusing on

this signaling cascade, MAPK, there is evidence that GH promotes this cascade through interaction with its cellsurface GH receptor.

Introduction of exogenous GH into un-

differentiated cell cultures induces cellular proliferation and MAPK activation compared with controls.158 47

MAPK is

activated in response to mechanical stress and has been shown to increase COX-2 expression and subsequent PGE-2 levels.

It is likely that GH and IGF-1, through the MAPK

signaling cascade, should be capable of mimicking or enhancing the effects of orthodontic tooth movement in terms of PGE-2 expression.

GH Effects on Matrix Metalloproteinases

MMPs are a family of endopeptidases responsible for breaking peptide bonds upon interaction.

MMPs play a role

in bone modeling through destruction of matrices in bone, but also due to their effects of bone cell stimulation, proliferation, and differentiation.

GH and IGF-1 have been

shown to effect osteoclastic formation and bone resorption through activation of MMPs.

The consensus of the

literature is that GH mainly effects bone cell release of MMPs through its up-regulation of IGF-1.

After 4 days of

exogenous GH and IGF-1 administration to rabbit bone cells a significant increase in MMP-2 and MMP-9 is observed. When neutralizing anti-hIGF-1 antiserum was added to the culture, the stimulatory effects of hGH were totally abolished.159

When exogenous GH and IGF-1 are independently

added to purified osteoclast cell cultures IGF-1, but not 48

GH, increased MMP-2 and MMP-9 activities.

MMP-2 up-

regulation is also observed when adding IGF-1 to human osteoblast cell cultures, but not when GH alone is added. These studies suggest that at the cellular level GH through IGF-1 activation is capable of inducing both osteoblasts and osteoclastic expression of MMP-2 and MMP-9. These MMPs then entice cellular activation and matrix degradation and ultimately bone resorption and modeling as observed in orthodontics. Orthodontics also includes a bone formation component, and through MMP expression, GH and IGF-1 are also capable of inducing this to occur.

MMPs are known for their

ability to induce collagen and basement membrane breakdown in destructive processes, but are also very necessary for repair.

When IGF-1 is coupled with transforming growth

factor-beta (TGF-beta) and introduced into partial bone defects in aged rats, the repair process is enhanced compared to controls.

Within these new sites of bone

formation, increased amounts of MMP-2 and MMP-3 are detected.160

The effect of IGF-1 induced MMP expression

seems to be dependent on the levels of other local factors (i.e., TGF-beta), or as one study explained, ligand bioavailability.

The signal transduction pathways

mediating IGF-1 regulation of MMP-2 synthesis in cell 49

culture have also been examined.

It was discovered that

IGF-1 can up-regulate MMP-2 synthesis via PI3kinase/Akt/mTOR signaling while also transmitting a negative regulatory signal via the Raf/ERK pathway.161

This

suggests a regulatory role of MMP over bone modeling.

MMP-

1, MMP-3, and MMP-13 have been shown to be up- or downregulated during the healing process in a time-dependent manner suggesting a dual role in both the destruction and repair processes.162

GH Effects on Growth Factors

The definition of a growth factor refers to a naturally occurring protein capable of stimulating cellular growth, proliferation, and cellular differentiation.

As

was mentioned previously, the transforming growth factor (TGF) superfamily, consisting of TGF-1-beta and bone morphogenic protein (BMP) subfamilies, is the most abundant growth factor family involved in bone metabolism.

An

association has been shown between these factors and orthodontic tooth movement. GH enhances the amounts of TGF-1-beta in both a direct or indirect fashion.

In acromegaly patients with naturally

elevated levels of GH due to a pituitary tumor, higher 50

levels of serum TGF-1-beta compared to controls have been reported.163

In vitro effects of exogenous GH on

osteoblast-like cells results in increased expression of both TGF-1-beta mRNA and protein.138 In addition, a relationship between IGF-1 and TGF-1beta has been shown during bone formation.

Upon

introducing the two independently into a healing site in sheep neither able to produce the amount of healing that was seen when the two were introduced in combination.164 Indirectly, an increase in TGF-1-beta expression and activation has been linked to some of the cytokines enhanced by GH and IGF-1.

In vitro studies have shown that

a dose dependent release of TGF-1-beta occurs upon administration of PGE-2 into mouse osteoblastic cell cultures.165,166

When mechanical stimulus is added along with

the PGE-2 application, there is an increase in both the release and activation of TGF-1-beta occurs.166

TGF-1-beta

is also enhanced by local 1, 25-DHCC serum levels.

The

enhancement is expressed via the upregualation of TGF-1beta binding via increased TGF-beta receptor expression. The adminis-tration of exogenous 1, 25-DHCC to osteoblast cell cultures, results in a concomitant increase in cell proliferation and differentiation.167

51

GH has been shown to directly affect the expression of the BMP subfamily.

GH added to both periodontal ligament

cells (PLC) and alveolar bone cells (ABC) produced increases in BMP-2, BMP-4, and BMP-7 mRNA expression in both cell populations.168

Cementoblasts, osteoblasts, and

periodontal ligament cells harvested from rat PDL spaces responded to GH by expressing BMP-2 and BMP-4 at levels greater than those of controls.169 Exogenous BMP-2 increased bone formation in older rats (16 months) in a dosedependent manner compared to controls.

Treatment with GH

showed similar results and, when administered to older rats (16 months), bone formation levels were comparable to those of younger rats (3 months).170 GH and IGF-1 have also been shown to influence the levels of both FGF and VEGF.

As was mentioned previously,

both of these growth factors promote angiogenic events and bone cell activation.

GH has been shown to promote FGF

gene expression in both in vitro and in vivo experiments. In a rat model, exogenous GH administration increased FGF mRNA expression 15.5-fold compared with controls.

In

vitro, exogenous GH enhanced expression FGF mRNA in rat bone cell cultures.171 The effects of GH on VEGF are mediated indirectly through activation of IGF-1.

When rhIGF-1 is administered 52

to human fibroblast cells VEGF mRNA expression increased 12 fold compared to control cultures.172

GH Effects on Vitamin D

GH and IGF-1 have been shown to increase the serum levels of 1, 25-DHCC, the active form of Vitamin D.

This

active form has been shown to increase bone cell proliferation, differentiation, and has been linked to orthodontic bone modeling.

In children receiving GH-

supplementation for treatment of GH-deficiency, 1, 25-DHCC levels increased significantly after only a month of supplemental GH treatment.173

The same is true in dogs

where exogenous GH administration was accompanied by increases in 1, 25-DHCC serum concentrations greater than those of the controls.174

IGF-1 has similar effects in

rats, whereby exogenous administration increases serum 1, 25-DHCC concentrations.175,176 While GH and IGF-1 promotes increases in serum concentrations of 1, 25-DHCC on a gross level, at the cellular level it seems that GH promotes its activity. Administration of both GH and 1, 25-DHCC produces a synergistic effect on alkaline phosphatase activity in osteoblasts-like cells when compared to independent 53

doses.177 GH has also been show to enhance 1, 25-DHCC induced osteoclast-like cell formation and, once formed, increased ability to form pits on dentine slices.4 This review of the literature is an attempt to present all aspects of tooth movement: the physiology of movement, theories of movement, the phases of movement, and the cellular components involved.

In addition, the review

attempts to provide a background of GH and bone metabolism, and then relate the components of tooth movement to those of GH-induced effects on bone metabolism.

54

References

1. Reitan K. Clinical and histologic observations on tooth movement during and after orthodontic treatment. Am J Orthod. 1967;53(10):721-45. 2. Kerrigan JJ, Mansell JP, Sandy JR. Matrix turnover. J Orthod. 2000;27(3):227-33. 3. Kassem M, Blum W, Ristelli J, Mosekilde L, Eriksen EF. Growth hormone stimulates proliferation and differentiation of normal human osteoblast-like cells in vitro. Calcif Tissue Int. 1993;52(3):222-6. 4. Nishiyama K, Sugimoto T, Kaji H, et al. Stimulatory effect of growth hormone on bone resorption and osteoclast differentiation. Endocrinology. 1996;137(1):35-41. 5. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop. 2006;129(4):469.e1-32. 6. Baumrind S. A reconsideration of the propriety of the "pressure-tension" hypothesis. Am J Orthod. 1969;55(1):1222. 7. Melsen B. Biological reaction of alveolar bone to orthodontic tooth movement. Angle Orthod. 1999;69(2):151-8. 8. Tran Van PT, Vignery A, Baron R. Cellular kinetics of the bone remodeling sequence in the rat. Anat Rec. 1982;202(4):445-51. 9. Schwarz A. Tissue changes incident to orthodontic tooth movement. International Journal of Orthodontics. 1932;18:331-52. 10. Davidovitch Z, Nicolay OF, Ngan PW, Shanfeld JL. Neurotransmitters, Cytokines, and the Control of Alveolar Bone Remodeling in Orthodontics. Dent Clin North Am. 1988;32(3):411-35. 11. Davidovitch Z. Tooth movement. Crit Rev Oral Biol Med. 1991;2(4):411-50.

55

12. Henneman S, Von den Hoff JW, Maltha JC. Mechanobiology of tooth movement. European Journal of Orthodontics. 2008;30(3):299-306. 13. Sandy JR, Farndale RW, Meikle MC. Recent advances in understanding mechanically induced bone remodeling and their relevance to orthodontic theory and practice. Am J Orthod Dentofacial Orthop. 1993;103(3):212-22. 14. Knothe Tate ML, Steck R, Forwood MR, vivo demonstration of load-induced fluid tibia and its potential implications for associated with functional adaptation. J 2000;203(Pt 18):2737-45.

Niederer P. In flow in the rat processes Exp Biol.

15. Burger EH, Klein-Nulend J, Smit TH. Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon--a proposal. J Biomech. 2003;36(10):1453-9. 16. Ajubi NE, Klein-Nulend J, Nijweide PJ, et al. Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes--a cytoskeleton-dependent process. Biochem Biophys Res Commun. 1996;225(1):62-8. 17. Grimm FM. Bone bending, a feature of orthodontic tooth movement. Am J Orthod. 1972;62(4):384-93. 18. Mostafa YA, Weaks-Dybvig M, Osdoby P. Orchestration of tooth movement. Am J Orthod. 1983;83(3):245-50. 19. Zengo AN, Bassett CA, Pawluk RJ, Prountzos G. In vivo bioelectric petentials in the dentoalveolar complex. Am J Orthod. 1974;66(2):130-9. 20. Davidovitch Z, Finkelson MD, Steigman S, et al. Electric currents, bone remodeling, and orthodontic tooth movement. I. The effect of electric currents on periodontal cyclic nucleotides. Am J Orthod. 1980;77(1):14-32. 21. Davidovitch Z, Finkelson MD, Steigman S, et al. Electric currents, bone remodeling, and orthodontic tooth movement. II. Increase in rate of tooth movement and periodontal cyclic nucleotide levels by combined force and electric current. Am J Orthod. 1980;77(1):33-47.

56

22. Borgens RB. Endogenous ionic currents traverse intact and damaged bone. Science. 1984;225(4661):478-82. 23. Noble BS, Peet N, Stevens HY, et al. Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone. Am J Physiol Cell Physiol. 2003;284(4):C934-43. 24. Verborgt O, Gibson GJ, Schaffler MB. Loss of osteocyte integrity in association with microdamage and bone remodeling after fatigue in vivo. J Bone Miner Res. 2000;15(1):60-7. 25. O'brien FJ, Hardiman DA, Hazenberg JG, et al. The behaviour of microcracks in compact bone. Eur J Morphol. 42(1-2):71-9. 26. Verna C, Dalstra M, Lee TC, Cattaneo PM, Melsen B. Microcracks in the alveolar bone following orthodontic tooth movement: a morphological and morphometric study. Eur J Orthod. 2004;26(5):459-67. 27. Verna C, Dalstra M, Lee TC, Melsen B. Microdamage in porcine alveolar bone due to functional and orthodontic loading. Eur J Morphol. 42(1-2):3-11. 28. Vistas in Orthodontics. Philadelphia: Lea & Febiger; 1962:397. 29. Pilon JJ, Kuijpers-Jagtman AM, Maltha JC. Magnitude of orthodontic forces and rate of bodily tooth movement. An experimental study. Am J Orthod Dentofacial Orthop. 1996;110(1):16-23. 30. von Böhl M, Maltha JC, Von Den Hoff JW, KuijpersJagtman AM. Focal hyalinization during experimental tooth movement in beagle dogs. Am J Orthod Dentofacial Orthop. 2004;125(5):615-23. 31. Rygh P. Ultrastructural changes in pressure zones of human periodontium incident to orthodontic tooth movement. Acta Odontol Scand. 1973;31(2):109-22. 32. van Leeuwen EJ, Maltha JC, Kuijpers-Jagtman AM. Tooth movement with light continuous and discontinuous forces in beagle dogs. Eur J Oral Sci. 1999;107(6):468-74.

57

33. Sandy JR. Signal transduction. Br J Orthod. 1998;25(4):269-74. 34. Chole RA, Faddis BT, Tinling SP. In vivo inhibition of localized bone resorption by human recombinant interleukin1 receptor antagonist. J Bone Miner Res. 1995;10(2):281-4. 35. Shimizu N, Yamaguchi M, Goseki T, et al. Cyclic-tension force stimulates interleukin-1 beta production by human periodontal ligament cells. J Periodontal Res. 1994;29(5):328-33. 36. Grieve WG, Johnson GK, Moore RN, Reinhardt RA, DuBois LM. Prostaglandin E (PGE) and interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid during human orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 1994;105(4):369-74. 37. Hughes B, King GJ. Effect of orthodontic appliance reactivation during the period of peak expansion in the osteoclast population. Anat Rec. 1998;251(1):80-6. 38. Ren Y, Vissink A. Cytokines in crevicular fluid and orthodontic tooth movement. Eur J Oral Sci. 2008;116(2):8997. 39. Lee K, Park Y, Yu H, Choi S, Yoo Y. Effects of continuous and interrupted orthodontic force on interleukin-1beta and prostaglandin E2 production in gingival crevicular fluid. Am J Orthod Dentofacial Orthop. 2004;125(2):168-77. 40. Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6 family of cytokines and gp130. Blood. 1995;86(4):1243-54. 41. Cillo JE, Gassner R, Koepsel RR, Buckley MJ. Growth factor and cytokine gene expression in mechanically strained human osteoblast-like cells: implications for distraction osteogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;90(2):147-54. 42. Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. Orthodontic tooth movement and de novo synthesis of proinflammatory cytokines. Am J Orthod Dentofacial Orthop. 2001;119(3):307-12.

58

43. Köhidai L CG. Chemotaxis and chemotactic selection induced with cytokines (IL-8, RANTES and TNF-alpha) in the unicellular Tetrahymena pyriformis. Cytokine. 1998;10:481– 6. 44. Tuncer BB, Ozmeriç N, Tuncer C, et al. Levels of interleukin-8 during tooth movement. Angle Orthod. 2005;75(4):631-6. 45. Locksley RM KN. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell. 2001;104(4):487–501. 46. Saito S, Ngan P, Rosol T, et al. Involvement of PGE synthesis in the effect of intermittent pressure and interleukin-1 beta on bone resorption. J Dent Res. 1991;70(1):27-33. 47. Lowney JJ, Norton LA, Shafer DM, Rossomando EF. Orthodontic forces increase tumor necrosis factor alpha in the human gingival sulcus. Am J Orthod Dentofacial Orthop. 1995;108(5):519-24. 48. Ogasawara T, Yoshimine Y, Kiyoshima T, et al. In situ expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts of rat periodontal tissue. J Periodontal Res. 2004;39(1):42-9. 49. Wittrant Y, Théoleyre S, Chipoy C, et al. RANKL/RANK/OPG: new therapeutic targets in bone tumours and associated osteolysis. Biochim Biophys Acta. 2004;1704(2):49-57. 50. Kanzaki H, Chiba M, Shimizu Y, Mitani H. Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis. J Bone Miner Res. 2002;17(2):210-20. 51. Ritter N, Mussig E, Steinberg T, et al. Elevated expression of genes assigned to NF-kappaB and apoptotic pathways in human periodontal ligament fibroblasts following mechanical stretch. Cell Tissue Res. 2007;328(3):537-48. 52. Nakajima R, Yamaguchi M, Kojima T, Takano M, Kasai K. Effects of compression force on fibroblast growth factor-2 and receptor activator of nuclear factor kappa B ligand 59

production by periodontal ligament cells in vitro. J Periodontal Res. 2008;43(2):168-73. 53. Nishijima Y, Yamaguchi M, Kojima T, et al. Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro. Orthod Craniofac Res. 2006;9(2):63-70. 54. Kanzaki H, Chiba M, Takahashi I, et al. Local OPG gene transfer to periodontal tissue inhibits orthodontic tooth movement. J Dent Res. 2004;83(12):920-5. 55. Kanzaki H, Chiba M, Arai K, et al. Local RANKL gene transfer to the periodontal tissue accelerates orthodontic tooth movement. Gene Ther. 2006;13(8):678-85. 56. Kawasaki K, Takahashi T, Yamaguchi M, Kasai K. Effects of aging on RANKL and OPG levels in gingival crevicular fluid during orthodontic tooth movement. Orthodontics & Craniofacial Research. 2006;9(3):137-42. 57. Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferongamma: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004;75(2):163-89. 58. Tsujimoto M, Yip YK, Vilcek J. Interferon-gamma enhances expression of cellular receptors for tumor necrosis factor. J Immunol. 1986;136(7):2441-4. 59. Chow JW. Role of nitric oxide and prostaglandins in the bone formation response to mechanical loading. Exerc Sport Sci Rev. 2000;28(4):185-8. 60. Hukkanen M, Hughes FJ, Buttery LD, et al. Cytokinestimulated expression of inducible nitric oxide synthase by mouse, rat, and human osteoblast-like cells and its functional role in osteoblast metabolic activity. Endocrinology. 1995;136(12):5445-53. 61. Brandi ML, Hukkanen M, Umeda T, et al. Bidirectional regulation of osteoclast function by nitric oxide synthase isoforms. Proc Natl Acad Sci U S A. 1995;92(7):2954-8. 62. van't Hof RJ, Ralston SH. Nitric oxide and bone. Immunology. 2001;103(3):255-61.

60

63. van't Hof RJ, Ralston SH. Cytokine-induced nitric oxide inhibits bone resorption by inducing apoptosis of osteoclast progenitors and suppressing osteoclast activity. J Bone Miner Res. 1997;12(11):1797-804. 64. Shirazi M, Nilforoushan D, Alghasi H, Dehpour A. The role of nitric oxide in orthodontic tooth movement in rats. Angle Orthod. 2002;72(3):211-5. 65. Akin E, Gurton AU, Olmez H. Effects of nitric oxide in orthodontic tooth movement in rats. Am J Orthod Dentofacial Orthop. 2004;126(5):608-14. 66. Biology of Tooth Movement Conference. The Biology of Tooth Movement. Boca Raton, Fla: CRC Press; 1989:366. 67. Kaji H, Sugimoto T, Kanatani M, et al. Prostaglandin E2 stimulates osteoclast-like cell formation and boneresorbing activity via osteoblasts: role of cAMP-dependent protein kinase. J Bone Miner Res. 1996;11(1):62-71. 68. Park Y, Kang S, Noh S, et al. PGE2 induces IL-1beta gene expression in mouse osteoblasts through a cAMP-PKA signaling pathway. Int Immunopharmacol. 2004;4(6):779-89. 69. Chyun YS, Raisz LG. Stimulation of bone formation by prostaglandin E2. Prostaglandins. 1984;27(1):97-103. 70. Somjen D, Binderman I, Berger E, Harell A. Bone remodelling induced by physical stress is prostaglandin E2 mediated. Biochim Biophys Acta. 1980;627(1):91-100. 71. Sanuki R, Mitsui N, Suzuki N, et al. Effect of compressive force on the production of prostaglandin E(2) and its receptors in osteoblastic Saos-2 cells. Connect Tissue Res. 2007;48(5):246-53. 72. Ngan PW, Crock B, Varghese J, et al. Immunohistochemical assessment of the effect of chemical and mechanical stimuli on cAMP and prostaglandin E levels in human gingival fibroblasts in vitro. Arch Oral Biol. 1988;33(3):163-74. 73. Dudic A, Kiliaridis S, Mombelli A, Giannopoulou C. Composition changes in gingival crevicular fluid during orthodontic tooth movement: comparisons between tension and compression sides. Eur J Oral Sci. 2006;114(5):416-22. 61

74. Grieve WG, Johnson GK, Moore RN, Reinhardt RA, DuBois LM. Prostaglandin E (PGE) and interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid during human orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 1994;105(4):369-74. 75. Leiker BJ, Nanda RS, Currier GF, Howes RI, Sinha PK. The effects of exogenous prostaglandins on orthodontic tooth movement in rats. Am J Orthod Dentofacial Orthop. 1995;108(4):380-8. 76. Kale S, Kocadereli I, Atilla P, Aşan E. Comparison of the effects of 1,25 dihydroxycholecalciferol and prostaglandin E2 on orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 2004;125(5):607-14. 77. Nukaga J, Kobayashi M, Shinki T, et al. Regulatory effects of interleukin-1beta and prostaglandin E2 on expression of receptor activator of nuclear factor-kappaB ligand in human periodontal ligament cells. J Periodontol. 2004;75(2):249-59. 78. Saito S, Ngan P, Saito M, et al. Interactive effects between cytokines on PGE production by human periodontal ligament fibroblasts in vitro. J Dent Res. 1990;69(8):145662. 79. Saito S, Rosol TJ, Saito M, et al. Bone-resorbing activity and prostaglandin E produced by human periodontal ligament cells in vitro. J Bone Miner Res. 1990;5(10):10138. 80. Liu X, Kirschenbaum A, Yao S, Levine AC. Cross-talk between the interleukin-6 and prostaglandin E(2) signaling systems results in enhancement of osteoclastogenesis through effects on the osteoprotegerin/receptor activator of nuclear factor-{kappa}B (RANK) ligand/RANK system. Endocrinology. 2005;146(4):1991-8. 81. Nagase H, Visse R, Murphy G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res. 2006;69(3):562-73. 82. Varghese S. Matrix metalloproteinases and their inhibitors in bone: an overview of regulation and functions. Front Biosci. 2006;11:2949-66. 62

83. Mitsui N, Suzuki N, Koyama Y, et al. Effect of compressive force on the expression of MMPs, PAs, and their inhibitors in osteoblastic Saos-2 cells. Life Sci. 2006;79(6):575-83. 84. Nahm D, Kim H, Mah J, Baek S. In vitro expression of matrix metalloproteinase-1, tissue inhibitor of metalloproteinase-1 and transforming growth factor-beta1 in human periodontal ligament fibroblasts. Eur J Orthod. 2004;26(2):129-35. 85. He Y, Macarak EJ, Korostoff JM, Howard PS. Compression and tension: differential effects on matrix accumulation by periodontal ligament fibroblasts in vitro. Connect Tissue Res. 2004;45(1):28-39. 86. Chang H, Wu C, Chen Y, et al. MMP-3 response to compressive forces in vitro and in vivo. J Dent Res. 2008;87(7):692-6. 87. Takahashi I, Nishimura M, Onodera K, et al. Expression of MMP-8 and MMP-13 genes in the periodontal ligament during tooth movement in rats. J Dent Res. 2003;82(8):64651. 88. Apajalahti S, Sorsa T, Railavo S, Ingman T. The in vivo levels of matrix metalloproteinase-1 and -8 in gingival crevicular fluid during initial orthodontic tooth movement. J Dent Res. 2003;82(12):1018-22. 89. Cantarella G, Cantarella R, Caltabiano M, et al. Levels of matrix metalloproteinases 1 and 2 in human gingival crevicular fluid during initial tooth movement. American Journal of Orthodontics and Dentofacial Orthopedics: Official Publication of the American Association of Orthodontists, Its Constituent Societies, and the American Board of Orthodontics. 2006;130(5):568.e11-6. 90. Holliday LS, Vakani A, Archer L, Dolce C. Effects of matrix metalloproteinase inhibitors on bone resorption and orthodontic tooth movement. J Dent Res. 2003;82(9):687-91. 91. Bildt MM, Henneman S, Maltha JC, Kuijpers-Jagtman AM, Von den Hoff JW. CMT-3 inhibits orthodontic tooth displacement in the rat. Arch Oral Biol. 2007;52(6):571-8.

63

92. Kanaan RA, Kanaan LA. Transforming growth factor beta1, bone connection. Med. Sci. Monit. 2006;12(8):RA164-9. 93. Lieb E, Milz S, Vogel T, et al. Effects of transforming growth factor beta1 on bonelike tissue formation in threedimensional cell culture. I. Culture conditions and tissue formation. Tissue Eng. 10(9-10):1399-413. 94. Sells Galvin RJ, Gatlin CL, Horn JW, Fuson TR. TGF-beta enhances osteoclast differentiation in hematopoietic cell cultures stimulated with RANKL and M-CSF. Biochem Biophys Res Commun. 1999;265(1):233-9. 95. Marie PJ, Debiais F, Haÿ E. Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP-2 signaling. Histol Histopathol. 2002;17(3):877-85. 96. Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth Factors. 2004;22(4):233-41. 97. Kanzaki H, Chiba M, Sato A, et al. Cyclical tensile force on periodontal ligament cells inhibits osteoclastogenesis through OPG induction. J Dent Res. 2006;85(5):457-62. 98. Uematsu S, Mogi M, Deguchi T. Increase of transforming growth factor-beta 1 in gingival crevicular fluid during human orthodontic tooth movement. Arch Oral Biol. 1996;41(11):1091-5. 99. Mitsui N, Suzuki N, Maeno M, et al. Optimal compressive force induces bone formation via increasing bone morphogenetic proteins production and decreasing their antagonists production by Saos-2 cells. Life Sci. 2006;78(23):2697-706. 100. Chen Y, Han G, Jin C, Shi R, Hou J. [Effect of simvastatin on bone morphogenetic protein-2 expression in the periodontal tissue after rat tooth movement]. Zhonghua Kou Qiang Yi Xue Za Zhi. 2008;43(1):21-5. 101. Allori AC, Sailon AM, Warren SM. Biological basis of bone formation, remodeling, and repair-part I: biochemical signaling molecules. Tissue Eng Part B Rev. 2008;14(3):25973.

64

102. Chikazu D, Katagiri M, Ogasawara T, et al. Regulation of osteoclast differentiation by fibroblast growth factor 2: stimulation of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor expression in osteoblasts and inhibition of macrophage colonystimulating factor function in osteoclast precursors. J Bone Miner Res. 2001;16(11):2074-81. 103. Fong KD, Nacamuli RP, Loboa EG, et al. Equibiaxial tensile strain affects calvarial osteoblast biology. J Craniofac Surg. 2003;14(3):348-55. 104. Nakajima R, Yamaguchi M, Kojima T, Takano M, Kasai K. Effects of compression force on fibroblast growth factor-2 and receptor activator of nuclear factor kappa B ligand production by periodontal ligament cells in vitro. J Periodontal Res. 2008;43(2):168-73. 105. Zhang Q, Guo R, Lu Y, et al. VEGF-C, a lymphatic growth factor, is a RANKL target gene in osteoclasts that enhances osteoclastic bone resorption through an autocrine mechanism. J Biol Chem. 2008;283(19):13491-9. 106. Motokawa M, Kaku M, Tohma Y, et al. Effects of cyclic tensile forces on the expression of vascular endothelial growth factor (VEGF) and macrophage-colony-stimulating factor (M-CSF) in murine osteoblastic MC3T3-E1 cells. J Dent Res. 2005;84(5):422-7. 107. Sakai A, Ohshima M, Sugano N, Otsuka K, Ito K. Profiling the cytokines in gingival crevicular fluid using a cytokine antibody array. J Periodontol. 2006;77(5):85664. 108. Kaku M, Kohno S, Kawata T, et al. Effects of vascular endothelial growth factor on osteoclast induction during tooth movement in mice. J Dent Res. 2001;80(10):1880-3. 109. Kohno S, Kaku M, Tsutsui K, et al. Expression of vascular endothelial growth factor and the effects on bone remodeling during experimental tooth movement. J Dent Res. 2003;82(3):177-82. 110. Kohno S, Kaku M, Kawata T, et al. Neutralizing effects of an anti-vascular endothelial growth factor antibody on tooth movement. Angle Orthod. 2005;75(5):797-804.

65

111. Chihara K, Sugimoto T. The action of GH/IGF-I/IGFBP in osteoblasts and osteoclasts. Horm. Res. 1997;48 Suppl 5:459. 112. Mochizuki H, Hakeda Y, Wakatsuki N, et al. Insulinlike growth factor-I supports formation and activation of osteoclasts. Endocrinology. 1992;131(3):1075-80. 113. Rubin J, Ackert-Bicknell CL, Zhu L, et al. IGF-I regulates osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand in vitro and OPG in vivo. J. Clin. Endocrinol. Metab. 2002;87(9):4273-9. 114. Hirukawa K, Miyazawa K, Maeda H, et al. Effect of tensile force on the expression of IGF-I and IGF-I receptor in the organ-cultured rat cranial suture. Arch Oral Biol. 2005;50(3):367-72. 115. Raab-Cullen DM, Thiede MA, Petersen DN, Kimmel DB, Recker RR. Mechanical loading stimulates rapid changes in periosteal gene expression. Calcif Tissue Int. 1994;55(6):473-8. 116. Zaman G, Suswillo RF, Cheng MZ, Tavares IA, Lanyon LE. Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture. J Bone Miner Res. 1997;12(5):769-77. 117. Saggese R, Federico G, Gandini P. The IGF-1--IGFBPs system in the crevicular fluid: its changes during orthodontic movement. Prog Orthod. 2005;6(1):114-8. 118. Toia M, Galazzo R, Maioli C, Granata R, Scarlatti F. The IGF-I/IGFBP-3 system in gingival crevicular fluid and dependence on application of fixed force. J Endocrinol Invest. 2005;28(11):1009-14. 119. Tokimasa C, Kawata T, Fujita T, et al. Effects of insulin-like growth factor-I on the expression of osteoclasts and osteoblasts in the nasopremaxillary suture under different masticatory loading conditions in growing mice. Arch Oral Biol. 2003;48(1):31-8. 120. Raisz LG, Trummel CL, Holick MF, DeLuca HF. 1,25dihydroxycholecalciferol: a potent stimulator of bone resorption in tissue culture. Science. 1972;175(23):768-9.

66

121. McSheehy PM, Chambers TJ. 1,25-Dihydroxyvitamin D3 stimulates rat osteoblastic cells to release a soluble factor that increases osteoclastic bone resorption. J Clin Invest. 1987;80(2):425-9. 122. Notoya M, Otsuka E, Yamaguchi A, Hagiwara H. Runx-2 is not essential for the vitamin D-regulated expression of RANKL and osteoprotegerin in osteoblastic cells. Biochem Biophys Res Commun. 2004;324(2):655-60. 123. Collins MK, Sinclair PM. The local use of vitamin D to increase the rate of orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 1988;94(4):278-84. 124. Takano-Yamamoto T, Kawakami M, Kobayashi Y, Yamashiro T, Sakuda M. The effect of local application of 1,25dihydroxycholecalciferol on osteoclast numbers in orthodontically treated rats. J Dent Res. 1992;71(1):53-9. 125. Kawakami M, Takano-Yamamoto T. Local injection of 1,25-dihydroxyvitamin D3 enhanced bone formation for tooth stabilization after experimental tooth movement in rats. J Bone Miner Metab. 2004;22(6):541-6. 126. Ohlsson C, Bengtsson BA, Isaksson OG, Andreassen TT, Slootweg MC. Growth hormone and bone. Endocr. Rev. 1998;19(1):55-79. 127. Andreassen TT, Oxlund H. The effects of growth hormone on cortical and cancellous bone. J. Musculoskelet. Neuronal. Interact. 2001;2(1):49-58. 128. Gangji V, Rydziel S, Gabbitas B, Canalis E. Insulinlike growth factor II promoter expression in cultured rodent osteoblasts and adult rat bone. Endocrinology. 1998;139(5):2287-92. 129. Giustina A, Mazziotti G, Canalis E. Growth hormone, insulin-like growth factors, and the skeleton. Endocr. Rev. 2008;29(5):535-59. 130. Parfitt AM. The bone remodeling compartment: a circulatory function for bone lining cells. J Bone Miner Res. 2001;16(9):1583-5.

67

131. Green H, Morikawa M, Nixon T. A dual effector theory of growth-hormone action. Differentiation. 1985;29(3):1958. 132. Fielder PJ, Mortensen DL, Mallet P, et al. Differential long-term effects of insulin-like growth factor-I (IGF-I) growth hormone (GH), and IGF-I plus GH on body growth and IGF binding proteins in hypophysectomized rats. Endocrinology. 1996;137(5):1913-20. 133. Ernst M, Froesch ER. Growth hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I. Biochem. Biophys. Res. Commun. 1988;151(1):142-7. 134. Ong CK, Joseph BK, Waters MJ, Symons AL. Growth hormone receptor and IGF-I receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat. The Angle Orthodontist. 2001;71(6):486-93. 135. Bozzola M, De Benedetti F, De Amici M, et al. Stimulating effect of growth hormone on cytokine release in children. Eur J Endocrinol. 2003;149(5):397-401. 136. Pagani S, Meazza C, Travaglino P, et al. Serum cytokine levels in GH-deficient children during substitutive GH therapy. Eur J Endocrinol. 2005;152(2):20710. 137. Schmitz D, Kobbe P, Lendemanns S, et al. Survival and cellular immune functions in septic mice treated with growth hormone (GH) and insulin-like growth factor-I (IGFI). Growth Horm IGF Res. 2008;18(3):245-52. 138. Saggese G, Federico G, Cinquanta L. In vitro effects of growth hormone and other hormones on chondrocytes and osteoblast-like cells. Acta. Paediatr. Suppl. 1993;82 Suppl 391:54-9; discussion 60. 139. Swolin D, Ohlsson C. Growth hormone increases interleukin-6 produced by human osteoblast-like cells. J Clin Endocrinol Metab. 1996;81(12):4329-33. 140. Tripathi A, Sodhi A. Involvement of JAK/STAT, PI3K, PKC and MAP kinases in the growth hormone induced production of cytokines in murine peritoneal macrophages in vitro. Biosci. Rep. 2008. 68

141. Renier G, Clément I, Desfaits AC, Lambert A. Direct stimulatory effect of insulin-like growth factor-I on monocyte and macrophage tumor necrosis factor-alpha production. Endocrinology. 1996;137(11):4611-8. 142. Uronen-Hansson H, Allen ML, Lichtarowicz-Krynska E, et al. Growth hormone enhances proinflammatory cytokine production by monocytes in whole blood. Growth Horm IGF Res. 2003;13(5):282-6. 143. Ueland T. GH/IGF-I and bone resorption in vivo and in vitro. Eur. J. Endocrinol. 2005;152(3):327-32. 144. Lanzi R, Losa M, Villa I, et al. GH replacement therapy increases plasma osteoprotegerin levels in GHdeficient adults. Eur J Endocrinol. 2003;148(2):185-191. 145. Ueland T, Bollerslev J, Flyvbjerg A, et al. Effects of 12 Months of GH Treatment on Cortical and Trabecular Bone Content of IGFs and OPG in Adults with Acquired GH Deficiency: A Double-Blind, Randomized, Placebo-Controlled Study. J Clin Endocrinol Metab. 2002;87(6):2760-2763. 146. Mrak E, Villa I, Lanzi R, et al. Growth hormone stimulates osteoprotegerin expression and secretion in human osteoblast-like cells. J Endocrinol. 2007;192(3):63945. 147. Gorny G, Shaw A, Oursler MJ. IL-6, LIF, and TNF-alpha regulation of GM-CSF inhibition of osteoclastogenesis in vitro. Exp Cell Res. 2004;294(1):149-58. 148. Sodhi A, Tripathi A. Prolactin and growth hormone induce differential cytokine and chemokine profile in murine peritoneal macrophages in vitro: involvement of p-38 MAP kinase, STAT3 and NF-kappaB. Cytokine. 2008;41(2):16273. 149. Devin JK, Vaughan DE, Blevins LS, et al. Low-dose growth hormone administration mobilizes endothelial progenitor cells in healthy adults. Growth Horm. IGF Res. 2008;18(3):253-63. 150. Grasemann C, Ratjen F, Schnabel D, et al. Effect of growth hormone therapy on nitric oxide formation in cystic fibrosis patients. Eur. Respir. J. 2008;31(4):815-21. 69

151. Thum T, Tsikas D, Frölich JC, Borlak J. Growth hormone induces eNOS expression and nitric oxide release in a cultured human endothelial cell line. FEBS Lett. 2003;555(3):567-71. 152. Doi SQ, Jacot TA, Sellitti DF, et al. Growth hormone increases inducible nitric oxide synthase expression in mesangial cells. J Am Soc Nephrol. 2000;11(8):1419-25. 153. Tripathi A, Sodhi A. Production of nitric oxide by murine peritoneal macrophages in vitro on treatment with prolactin and growth hormone: involvement of protein tyrosine kinases, Ca(++), and MAP kinase signal transduction pathways. Mol. Immunol. 2007;44(12):3185-94. 154. Thum T, Fleissner F, Klink I, et al. Growth hormone treatment improves markers of systemic nitric oxide bioavailability via insulin-like growth factor-I. J. Clin. Endocrinol. Metab. 2007;92(11):4172-9. 155. Wickman A, Jonsdottir IH, Bergstrom G, Hedin L. GH and IGF-I regulate the expression of endothelial nitric oxide synthase (eNOS) in cardiovascular tissues of hypophysectomized female rats. Eur J Endocrinol. 2002;147(4):523-33. 156. Cao Z, Liu L, Dixon DA, et al. Insulin-like growth factor-I induces cyclooxygenase-2 expression via PI3K, MAPK and PKC signaling pathways in human ovarian cancer cells. Cell Signal. 2007;19(7):1542-53. 157. Di Popolo A, Memoli A, Apicella A, et al. IGF-II/IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE2 synthesis in Caco-2 human colon carcinoma cells. Oncogene. 2000;19(48):5517-24. 158. Liang L, Jiang J, Frank SJ. Insulin Receptor Substrate-1-Mediated Enhancement of Growth Hormone-Induced Mitogen-Activated Protein Kinase Activation. Endocrinology. 2000;141(9):3328-3336. 159. Anne-Valerie R, Christelle D, Yannick F, et al. Human Growth Hormone Stimulates Proteinase Activities of Rabbit Bone Cells via IGF-I. Biochemical and Biophysical Research Communications. 2000;268(3):875-881.

70

160. Blumenfeld I, Srouji S, Peled M, Livne E. Metalloproteinases (MMPs -2, -3) are involved in TGF-beta and IGF-1-induced bone defect healing in 20-month-old female rats. Arch Gerontol Geriatr. 35(1):59-69. 161. Zhang D, Bar-Eli M, Meloche S, Brodt P. Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: the phosphatidylinositol 3kinase/Akt and Raf/ERK pathways transmit opposing signals. J Biol Chem. 2004;279(19):19683-90. 162. Eleswarapu SV, Leipzig ND, Athanasiou KA. Gene expression of single articular chondrocytes. Cell Tissue Res. 2007;327(1):43-54. 163. Bolanowski M, Daroszewski J, Zatonska K, Arkowska A. Circulating cytokines in relation to bone mineral density changes in patients with acromegaly. Neuro Endocrinol Lett. 27(1-2):183-8. 164. Izal I, Ripalda P, Acosta CA, Forriol F. In Vitro Healing of Avascular Meniscal Injuries with Fresh and Frozen Plugs Treated with TGF-beta1 and IGF-1 in Sheep. Int J Clin Exp Pathol. 2008;1(5):426-34. 165. Klein-Nulend J, Semeins CM, Burger EH. Prostaglandin mediated modulation of transforming growth factor-beta metabolism in primary mouse osteoblastic cells in vitro. J Cell Physiol. 1996;168(1):1-7. 166. Ramirez-Yañez GO, Hamlet S, Jonarta A, Seymour GJ, Symons AL. Prostaglandin E2 enhances transforming growth factor-beta 1 and TGF-beta receptors synthesis: an in vivo and in vitro study. Prostaglandins Leukot Essent Fatty Acids. 2006;74(3):183-92. 167. Nagel D, Kumar R. 1 alpha,25-dihydroxyvitamin D3 increases TGF beta 1 binding to human osteoblasts. Biochem Biophys Res Commun. 2002;290(5):1558-63. 168. Haase HR, Ivanovski S, Waters MJ, Bartold PM. Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cells. J Periodontal Res. 2003;38(4):366-74. 169. Li H, Bartold PM, Young WG, Xiao Y, Waters MJ. Growth hormone induces bone morphogenetic proteins and bone71

related proteins in the developing rat periodontium. J Bone Miner Res. 2001;16(6):1068-76. 170. Fleet JC, Cashman K, Cox K, Rosen V. The effects of aging on the bone inductive activity of recombinant human bone morphogenetic protein-2. Endocrinology. 1996;137(11):4605-10. 171. Izumi T, Shida J, Jingushi S, Hotokebuchi T, Sugioka Y. Administration of growth hormone modulates the gene expression of basic fibroblast growth factor in rat costal cartilage, both in vivo and in vitro. Mol Cell Endocrinol. 1995;112(1):95-9. 172. Miele C, Rochford JJ, Filippa N, Giorgetti-Peraldi S, Van Obberghen E. Insulin and insulin-like growth factor-I induce vascular endothelial growth factor mRNA expression via different signaling pathways. J Biol Chem. 2000;275(28):21695-702. 173. Wei S, Tanaka H, Kubo T, et al. Growth hormone increases serum 1,25-dihydroxyvitamin D levels and decreases 24,25-dihydroxyvitamin D levels in children with growth hormone deficiency. Eur J Endocrinol. 1997;136(1):45-51. 174. Tryfonidou MA, Hazewinkel HAW. Different effects of physiologically and pharmacologically increased growth hormone levels on cholecalciferol metabolism at prepubertal age. J. Steroid. Biochem. Mol. Biol. 2004;89-90(1-5):49-54. 175. Wong MS, Sriussadaporn S, Tembe VA, Favus MJ. Insulinlike growth factor I increases renal 1,25(OH)2D3 biosynthesis during low-P diet in adult rats. Am J Physiol. 1997;272(6 Pt 2):F698-703. 176. Wong MS, Tembe VA, Favus MJ. Insulin-like growth factor-I stimulates renal 1, 25-dihydroxycholecalciferol synthesis in old rats fed a low calcium diet. J Nutr. 2000;130(5):1147-52. 177. Morel G, Chavassieux P, Barenton B, et al. Evidence for a direct effect of growth hormone on osteoblasts. Cell. Tissue. Res. 1993;273(2):279-86.

72

CHAPTER 3: JOURNAL ARTICLE

Abstract

Introduction:

The aim of this study was to determine how

supplemental GH affects the amount of orthodontic tooth movement in rats.

Methods: Twenty-four Wistar rats were

divided into two study groups of 12 rats each.

A 7 mm

nickel-titanium closed-coil spring was ligated between the maxillary incisors and the right three maxillary molars of each rat to deliver a force of 10 g.

One group received

daily subcutaneous injections of recombinant human GH (rhGH) (2 mg/kg); the other group received daily subcutaneous injections of saline (2 mg/kg).

Digital

caliper measurements were performed the day of appliance placement, day 6, and day 12.

The distance between the

most mesial point of the maxillary molar unit and cementenamel junction of the ipsilateral maxillary incisor was measured (I-M distance) on both the appliance and nonappliance sides.

Results:

Significant (p < .05)

differences in the amount of tooth movement was observed for the three time periods days 1-6, days 6-12, days 1-12 between the GH and saline groups for both appliance and 73

non-appliance sides.

Differences between I-M distances on

the appliance and non-appliance sides showed significant group differences (p < .05) in the amount of tooth movement between 1-6 days and 1-12 days, but not between 6-12 days. Intra-animal comparisons of the amount of tooth movement on a given side between time periods show a significant difference (p < .05) between days 1-6 and days 6-12 in the GH group on the non-appliance side only.

Intra-animal

comparisons of the amount of tooth movement between the appliance and non-appliance side at each time period show significant differences (p < .05) at all time periods for both groups except during days 6-12 for the saline group. Conclusion: GH supplementation increases tooth movement in the rat model when compared to controlled samples.

74

Introduction

Orthodontic tooth movement results from the response of the periodontal tissue to orthodontic force, which leads to modeling and remodeling of the surrounding alveolar bone.

The response is considered to occur through the

activation of specific signaling pathways, many of which are known, acting to facilitate tooth movement.

1

The rate

at which tooth movement occurs is dependent upon the ability of these pathways to affect the metabolism of bone by the two main cell types responsible for tooth movement: osteoblasts and osteoclasts.2 Much is known about the actions of these two cell types and the signaling pathways that affect them. However, little is known about the effect of growth hormone (GH) on these cells in orthodontics, and no study has examined the effect of GH on tooth movement.

GH is one of

the major regulators involved in bone metabolism.3

GH is an

important regulator of postnatal skeletal growth and development and promotes stem-cell growth differentiation and proliferation.3

Specifically, GH directly acts on

osteoblasts and osteoclasts and exerts anabolic effects on bone formation and bone resorption.4,5 75

A better

understanding of how GH effects tooth movement may lead to a pharmacologic intervention to control orthodontic tooth movements. While the effects of GH administration have not been studied, several studies have shown enhanced orthodontic tooth movements associated with increased bone turnover. It has been shown that when a state of hyperparathyroidism is induced in dogs, with subsequent increased bone turnover, more rapid tooth movement occurs.6 Hyperthyroidism induced in rats also resulted in increased levels of bone turnover and increased levels of tooth movement.7

Similarly, corticosteroid-induced osteoporosis

produced four times as much tooth movement in experimental rabbits than in controls.8 If the mechanical stimulation of PDL cells induces a cascade of cellular events and these events can be mimicked by increasing metabolic levels, then it is likely that pharmacologic intervention increasing metabolic rates would be able to effect tooth movement.

On that basis,

supplemental growth hormone might be expected to induce increased levels of bone turnover that should affect the rate of orthodontic tooth movement.

76

The purpose of this

study is to determine how supplemental GH affects the amount of orthodontic tooth movement in rats.

Methods and Materials

Sample Selection Twenty-four male Wistar rats were randomly divided into two groups of 12: an experimental GH supplemented group and a saline control group.

All twenty-four rats

were 6 weeks old and weighed 250-300g.

The animals were

housed under normal laboratory conditions and fed powdered laboratory chow and water.

Ethical permission for the

study was obtained according to the guidelines established by the Saint Louis University Institutional Animal Care and Use Committee (IACUC) / Animal Care Committee (ACC).

Appliance Design A split-mouth design was used, with an experimental appliance side and the contralateral non-appliance control side.

For each experimental side, an orthodontic appliance

77

was fabricated (Figure 3.1).

Stainless steel ligature

wires (diameter 0.010”) were formed to combine the three maxillary molars as one unit.

Attached to this wire was a

thermal NiTi superelastic closed coil spring (wire diameter 0.009”, eyelet diameter 0.032”, (G and H Wire Co.)).

This

spring was then ligated to the maxillary incisors with a stainless steel ligature wire (diameter 0.010”).

The

springs were designed to deliver a force of 10 grams over a range of 3-7 mm of activation.

A similar protocol has been

used successfully in a number of studies.9

Figure 3.1:

Appliance design (taken from Ren et al.).

78

9

Appliance Placement Prior to appliance placement the animals were anesthetized with an intraperitoneal (IP) injection of ketamine 75 mg/kg and xylazine 7.5 mg/kg.

An additional

injection of carprofen 5 mg/kg was provided for postoperative pain management.

The pre-formed orthodontic

appliance was bonded to the experimental side maxillary molar unit with light-curing bonding material (Clearfil SE BOND, Kuraray Corp.) and nano-superfilled posterior restorative composite (Clearfil Majesty Posterior, Kuraray Corp.).

Bonding material was applied until the buccal and

palatal wires were completely embedded, after which the material was light-cured with an Ortholux LED (3M Unitek) curing light.

Composite resin was added to cover the

occlusal surfaces of the two distal molars, and then lightcured.

The spring was kept free of bonding material at the

mesial side of the pre-formed wire.

Horizontal grooves

perpendicular to the long access of the teeth were placed on the distal sides of both incisors to aid in retention of the ligation wire.

The spring was activated and attached

to the ligation wire enclosing the incisors.

Bonding

material (Clearfil SE BOND, Kuraray Corp.) was applied until the facial aspect of the wire was completely embedded

79

in the material, then light-cured.

Composite resin was

then added to completely cover the facial aspect of the incisor complex and light-cured.

The lower incisors were

reduced 3-4 mm perpendicular to their long axes to prevent traumatic interference with the orthodontic appliance. The experimental group received daily subcutaneous injections of Nutropin AQ (somatropin (rDNA origin), Genentech), a human growth hormone (hGH) 2 mg/kg.

The

control group received daily subcutaneous injections of phosphate buffered saline (PBS) 2 mg/kg. Digital caliper measurements (Cen-Tech, Harbor Freight Tools), accurate to +/- .02 mm, were performed on the day appliance placement, on day 6, and on day 12.

The distance

between the most mesial point of the maxillary molar unit and cementenamel junction (CEJ) of the ipsilateral maxillary incisor was measured (I-M distance) on the experimental and the control sides.

The amount of tooth

movement was evaluated over three time periods: days 1-6, days 6-12, days 1-12. The differences between the I-M distances on appliance and non-appliance side were calculated to eliminate the confounding effects of the physiological distal drift of molars, the physiological growth of the snout and the 80

concomitant forward movement of the incisors, and the bilateral distal tipping of the incisors.

The changes in

the side differences were considered to represent the best approximation of the experimental molar movements caused by the orthodontic appliance. all measurements.

The same investigator performed

Body weights were recorded every three

days, and dosing was adjusted accordingly (2 mg/kg).

GH

and saline injections and verification of appliance condition were performed daily.

Statistical Analysis

Due to the sample size, non-parametric statistics were used.

Mann-Whitney U tests were performed to analyze tooth

movement differences between the GH and saline groups including the right side between groups and left side group differences.

Mann-Whitney U tests were also used to

compare the right and left side differences within the GH group to the same differences in the saline group. Wilcoxon signed rank tests were performed to compare tooth movements between time periods within the GH and saline groups.

All statistics were performed using an α = 0.05,

81

and estimated using the SPSS statistical program, version 15.0 (SPSS Incorporated, Chicago, IL).

Results

The Mann-Whitney U tests showed significant (p < .05) group differences in tooth movements on the right and left sides at all time periods (Table 3.1).

The right and left

side differences were also significant (p < .05) on days 16 and days 1-12 between the GH and saline groups (Table 3.2; Figure 3.2). Table 3.1:

Mann-Whitney U tests for each time period comparing tooth

movement (mm) on the right (appliance) and left (non-appliance) sides of the experimental and control groups

Period

Growth Hormone

Saline

Significance

Right

Mean (mm) / S.D.

Mean (mm) / S.D.

p-value

Days 1-6

.52 +/- .16

.30 +/- .18

.009

Days 6–12

.30 +/- .24

.14 +/- .15

.017

Days 1-12

.82 +/- .23

.44 +/- .20

.001

Days 1–6

.25 +/- .06

.16 +/- .11

.039

Days 6-12

.13 +/- .08

.08 +/- .06

.010

Days 1–12

.38 +/- .12

.24 +/- .13

.007

Left

82

Table 3.2:

Mann-Whitney U tests for each time period comparing the

tooth movement (mm) differences between right (appliance) and left (non-appliance) sides within GH group to the same difference in saline group

Period

Growth Hormone

Saline

Significance

Mean (mm) / S.D.

Mean (mm) / S.D.

p-value

Days 1–6

.26 +/- .14

.14 +/- .12

.018

Days 6–12

.16 +/- .24

.06 +/- .15

.160

Days 1–12

.42 +/- .21

.20 +/- .15

.006

GH

Saline

0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 Day 1

Figure 3.2:

Day 6

Day 12

Experimental tooth movement (mm) defined as the difference

in tooth movement between the appliance and non-appliance side within each group

Tooth movements on both the right and left sides were consistently greater during days 1-6 than days 6-12. Wilcoxon signed rank tests showed that only the difference

83

on the left side in the GH group were statistically significant (p < 0.05) (Table 3.3; Figure 3.3). Table 3.3: Wilcoxon signed rank tests comparing tooth movement between days 1-6 with tooth movement between days 6-12

Group

Days 1–6

Days 6–12

Significance

Mean (mm) / S.D.

Mean (mm) / S.D.

p-value

GH / Right

.52 +/- .16

.30 +/- .24

.054

GH / Left

.25 +/- .06

.14 +/- .08

.003

GH / Difference

.26 +/- .14

.16 +/- .24

.433

Saline / Right

.30 +/- .18

.14 +/- .14

.060

Saline / Left

.16 +/- .11

.08 +/- .06

.050

Saline / Difference

.14 +/- .12

.06 +/- .14

.195

GH-R

GH-L

Sal-R

Sal-L

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Day 1

Figure 3.3:

Day 6

Day 12

Experimental tooth movement (mm) on appliance and non-

appliance side in both GH and saline groups

84

Wilcoxon signed rank tests also showed significant differences in tooth movement between the appliance and non-appliance sides within groups.

Except for the tooth

movement of the saline group between days 6-12, the side with the orthodontic appliance showed significantly greater tooth movements than the side without the appliance (Tables 3.4, 3.5) Table 3.4:

Wilcoxon signed rank tests comparing tooth movement between

the appliance and non-appliance sides during days 1-6, days 6-12, and days 1-12 within the GH group Right (Appliance) Mean (mm) / S.D.

Left (Non-appliance) Mean (mm) / S.D.

Days 1–6

.52 +/- .16

.25 +/- .06

.002

Days 6–12

.30 +/- .24

.14 +/- .08

.037

Days 1–12

.82 +/- .23

.39 +/- .12

.003

Group

Significance p-value

Table 3.5: Wilcoxon signed rank tests comparing tooth movement between the appliance and non-appliance sides during days 1-6, days 6-12, and days 1-12 within the saline group Right (Appliance)

Left (Non-appliance)

Significance

Mean (mm) / S.D.

Mean (mm) / S.D.

p-value

Days 1–6

.30 +/- .18

.16 +/- .11

.008

Days 6-12

.14 +/- .15

.08 +/- .06

.099

Days 1-12

.44 +/- .20

.24 +/- .13

.003

Group

85

During the study there were several appliances that failed at different time periods.

During days 1-6, two

appliances in the GH supplement group and one appliance in the saline group failed.

During days 6-12, one appliance

in the GH supplement group and two appliances in the saline group failed.

All of these failures were noted on either

the fifth or sixth day of the six day period.

Failed

appliances during days 1-6 were replaced during the data collection procedure on day 6.

Appliance failures were not

replaced during days 6-12 due to the failures occurring the day prior to or on the day of project completion.

Tooth

movement was observed in animals with failed appliances during the period of failure with the exception of one animal in each the GH and saline groups during days 6-12. All animals with failures were included in the data.

The

reason for inclusion was that the nature of the failures were symmetric, in that the number of failures and the period of which they occurred were balanced between the groups.

86

Discussion

These results clearly demonstrate for the first time that systemic GH supplementation has a stimulatory effect on tooth movement in rats. Our results show a significant difference in tooth movement between the GH and saline groups during the overall observation period.

It is likely

that the supplemental GH had an effect on bone metabolism and created a more favorable environment for tooth movement. GH is known to have anabolic effects on both the cellular and non-cellular bone modeling components.

GH

induces osteoblast and osteoclast differentiation,4,10,11 proliferation, and activation.4,5,10,12-16

It has also been

shown to stimulate the release and enhance the activation of certain cytokines,17-21 growth factors,11,22-24 and matrix metalloproteinases25-28 involved in bone modeling.

Micro-

environmental changes, like those observed under orthodontic force, induce non-cellular elements such as cytokines,29-34 growth factors,35-39 matrix metalloproteinases,40-44 etc., to initiate and sustain the remodeling activity ultimately facilitating tooth movement.1

In order

for GH to have an effect on tooth movement in our model it 87

would need to induce these changes within the time period of this experiment and with the dose administered. In vivo GH supplementation in humans at 0.6 mg/kg has been shown to increase biochemical urine markers of bone resorption and formation in 3 days.45

In our study the

amount administered, 2 mg/kg was based on reports of the amount of GH necessary to induce craniofacial growth in the rat model.46

This is almost three times the amount seen to

effect bone metabolism in humans.

One would expect at

least similar amounts of bone resorption and formation in a similar amount of time. GH supplementation at levels below what was administered in this study have been shown to increase serum concentrations47-49 above the level known to encourage in vitro rat osteoblast and osteoclast cell differentiation, proliferation, and activation5 within days of dosing.

Similar amounts of GH supplementation have been

shown to increase serum levels of various cytokines17,50 and stimulate local release of cytokines and growth factors from bone cells within days of administration.11 Therefore, GH supplementation might be expected to influence the amount of tooth movement as early as the first time period (days 1-6).

The length of the present 88

experiment was adequate and the tooth movement differences were consistent with what is known of GH effect on bone metabolism.

In all experimental conditions, more tooth

movement was observed in the GH group.

The lack of tooth

movement differences between the groups during days 6-12 may be explained by the length of the observation period. Previous literature describes the tooth movement phases of the rat in a 3-part tooth movement phase similar to humans but on a smaller time-scale.

Rat tooth movement

consists of initial deflection (day 1-4), lag period (day 4-9), and post-lag tooth movement in the direction of the force after day 9.51,52

The initial deflection period (day

1-4) is characterized by movement within the socket and any frontal resorption.

The present study showed significantly

greater amounts of tooth movement in the GH supplement group between days 1-6.

This difference indicates that GH

must be having an early effect on tooth movement.

The

length of the experiment (12 days) may have had an effect on the amount of tooth movement observed between days 6-12, due to its proximity to the lag phase. The null hypothesis tested was that there is no difference in amount of tooth movement between rats provided growth hormone supplementation and rats provided a 89

saline control. hypothesis.

The results from this study reject this

Tests comparing the amount of experimental

tooth movement (Table 3.2) show that there are no significant differences between the groups for days 6–12. Yet the same tests do show significant differences between the groups for days 1-6 and days 1–12. The null hypothesis was rejected for all time periods when not accounting for biases in data collection.

Tests

comparing tooth movement on the appliance side between groups and the non-appliance side between the groups resulted in significant differences (p < 0.05) at all time periods (Table 3.1) (Figure 3.2). There were significant differences between the groups for the non-appliance side with more tooth movement in the GH supplement animals.

Based on the possible cofounding

effects previously described, it is most likely that these results were due to movements of the incisors.

Any

anabolic effects produced by GH on distal drift of molars or growth of the snout would decrease the amount of tooth movement seen.9

It is likely that if GH had an effect on

orthodontic bone modeling that it would not be limited to the molars and would also effect incisor movement.

Any

differences in posterior movement of the incisors between 90

the groups would be expressed as differences in measures on both the appliance and non-appliance sides. When comparing tooth movement between days 1-6 with movement in days 6-12 on the right side and tooth movement between days 1-6 with movement in days 6-12 on the left side within both the GH and saline groups a significant difference (p < .05) was found in only one of the four comparisons (Table 3.3).

This difference (p = .003) was

seen when comparing the amount of tooth movement during days 1-6 to the amount of tooth movement during days 6-12 on the non-appliance side in the GH supplement group. With this one exception there was no difference in the amount of tooth movement observed during days 1-6 compared to tooth movement during days 6-12.

When looking at Table

3.3, the descriptive statistics show more tooth movement during days 1-6 in both the right and left sides compared to tooth movement during days 6-12 in both groups.

The

level of significance may have been affected by the large amount of variation expressed by the large standard deviations.

This may be a sample size issue.

The lowest

standard deviation values were found on the non-appliance side, the side also found to be significant for tooth movement. 91

Comparing tooth movement between the appliance and non-appliance sides during days 1-6, days 6-12, and days 112 within both the GH and saline groups resulted in significant differences for many of the measures (Tables 3.4, 3.5).

The lone exception was observed in the days 6-

12 period in the saline group (p = .099). The nature of the tooth movement observed is similar to the movement previously reported in the literature.

The

design of our appliance was original and therefore it is difficult to compare the amount of tooth movement observed to amounts previously reported.

The appliance design was

based on the Ren et al., model with the exception that our incisor attachment was not anchored in alveolar bone.9 However comparisons can be made with previous studies that employed similar force levels.

Upon doing so similar tooth

movement curves in terms of amount and rate are observed between control groups.53

When comparing our data to that

of Ren et al., our tooth movement curves slopes in our control groups are similar only our amount is different again this may be due to the differences in appliance design.9

Like previous studies we did observe greater

amounts of tooth movement early in our observation period compared to later.

92

Similarity in the nature of the tooth movement observed in our study compared to previous studies lends credence to our appliance design.

Knowing this we can then

extrapolate the real effects of GH on tooth movement.

The

present results clearly demonstrate for the first time that systemic GH supplementation has a stimulatory effect on tooth movement in rats. Our results show a significant difference in tooth movement between the GH and saline groups during the overall observation period.

Conclusions

Supplemental growth hormone administered systemically has an effect on orthodontic tooth movement in the rat model.

The effect is an increase in the amount of tooth

movement compared to saline controls.

93

References

1. Krishnan V, Davidovitch Z. Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop. 2006;129(4):469.e1-32. 2. Henneman S, Von den Hoff JW, Maltha JC. Mechanobiology of tooth movement. European Journal of Orthodontics. 2008;30(3):299-306. 3. Giustina A, Mazziotti G, Canalis E. Growth hormone, insulin-like growth factors, and the skeleton. Endocr. Rev. 2008;29(5):535-59. 4. Kassem M, Blum W, Ristelli J, Mosekilde L, Eriksen EF. Growth hormone stimulates proliferation and differentiation of normal human osteoblast-like cells in vitro. Calcif Tissue Int. 1993;52(3):222-6. 5. Nishiyama K, Sugimoto T, Kaji H, et al. Stimulatory effect of growth hormone on bone resorption and osteoclast differentiation. Endocrinology. 1996;137(1):35-41. 6. Midgett RJ, Shaye R, Fruge JF. The effect of altered bone metabolism on orthodontic tooth movement. Am J Orthod. 1981;80(3):256-62. 7. Verna C, Melsen B. Tissue reaction to orthodontic tooth movement in different bone turnover conditions. Orthod Craniofac Res. 2003;6(3):155-63. 8. Ashcraft MB, Southard KA, Tolley EA. The effect of corticosteroid-induced osteoporosis on orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 1992;102(4):3109. 9. Ren Y, Maltha JC, Van 't Hof MA, Kuijpers-Jagtman AM. Age effect on orthodontic tooth movement in rats. J Dent Res. 2003;82(1):38-42. 10. Ernst M, Froesch ER. Growth hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I. Biochem. Biophys. Res. Commun. 1988;151(1):142-7.

94

11. Saggese G, Federico G, Cinquanta L. In vitro effects of growth hormone and other hormones on chondrocytes and osteoblast-like cells. Acta. Paediatr. Suppl. 1993;82 Suppl 391:54-9; discussion 60. 12. Chihara K, Sugimoto T. The action of GH/IGF-I/IGFBP in osteoblasts and osteoclasts. Horm. Res. 1997;48 Suppl 5:459. 13. Cool SM, Grünert M, Jackson R, et al. Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells. Biochem. Biophys. Res. Commun. 2005;338(2):1048-58. 14. Guicheux J, Heymann D, Rousselle AV, et al. Growth hormone stimulatory effects on osteoclastic resorption are partly mediated by insulin-like growth factor I: An in vitro study. Bone. 1998;22(1):25-31. 15. DiGirolamo DJ, Mukherjee A, Fulzele K, et al. Mode of growth hormone action in osteoblasts. J. Biol. Chem. 2007;282(43):31666-74. 16. Morel G, Chavassieux P, Barenton B, et al. Evidence for a direct effect of growth hormone on osteoblasts. Cell. Tissue. Res. 1993;273(2):279-86. 17. Bozzola M, De Benedetti F, De Amici M, et al. Stimulating effect of growth hormone on cytokine release in children. Eur J Endocrinol. 2003;149(5):397-401. 18. Swolin D, Ohlsson C. Growth hormone increases interleukin-6 produced by human osteoblast-like cells. J Clin Endocrinol Metab. 1996;81(12):4329-33. 19. Tripathi A, Sodhi A. Involvement of JAK/STAT, PI3K, PKC and MAP kinases in the growth hormone induced production of cytokines in murine peritoneal macrophages in vitro. Biosci. Rep. 2008. 20. Uronen-Hansson H, Allen ML, Lichtarowicz-Krynska E, et al. Growth hormone enhances proinflammatory cytokine production by monocytes in whole blood. Growth Horm IGF Res. 2003;13(5):282-6. 21. Sodhi A, Tripathi A. Prolactin and growth hormone induce differential cytokine and chemokine profile in 95

murine peritoneal macrophages in vitro: Involvement of p-38 MAP kinase, STAT3 and NF-kappaB. Cytokine. 2008;41(2):16273. 22. Kaczmarek MM, Blitek A, Schams D, Ziecik AJ. The effect of insulin-like growth factor-I, relaxin and luteinizing hormone on vascular endothelial growth factor secretion by cultured endometrial stromal cells on different days of early pregnancy in pigs. Reprod Biol. 2008;8(2):163-70. 23. Miele C, Rochford JJ, Filippa N, Giorgetti-Peraldi S, Van Obberghen E. Insulin and insulin-like growth factor-I induce vascular endothelial growth factor mRNA expression via different signaling pathways. J Biol Chem. 2000;275(28):21695-702. 24. Bolanowski M, Daroszewski J, Zatonska K, Arkowska A. Circulating cytokines in relation to bone mineral density changes in patients with acromegaly. Neuro Endocrinol Lett. 27(1-2):183-8. 25. Anne-Valerie R, Christelle D, Yannick F, et al. Human Growth Hormone Stimulates Proteinase Activities of Rabbit Bone Cells via IGF-I. Biochemical and Biophysical Research Communications. 2000;268(3):875-881. 26. Brüel A, Nyengaard JR, Danielsen CC. MMP-2 in the left rat ventricle is increased by growth hormone. Growth Horm IGF Res. 2006;16(3):193-201. 27. Zhang D, Bar-Eli M, Meloche S, Brodt P. Dual regulation of MMP-2 expression by the type 1 insulin-like growth factor receptor: The phosphatidylinositol 3-kinase/Akt and Raf/ERK pathways transmit opposing signals. J Biol Chem. 2004;279(19):19683-90. 28. Anne-Valérie R, Christelle D, Yannick F, et al. Human growth hormone stimulates proteinase activities of rabbit bone cells via IGF-I. Biochem Biophys Res Commun. 2000;268(3):875-81. 29. Ren Y, Vissink A. Cytokines in crevicular fluid and orthodontic tooth movement. Eur J Oral Sci. 2008;116(2):8997. 30. Ren Y, Hazemeijer H, de Haan B, Qu N, de Vos P. Cytokine profiles in crevicular fluid during orthodontic 96

tooth movement of short and long durations. J Periodontol. 2007;78(3):453-8. 31. Saito M, Saito S, Ngan PW, Shanfeld J, Davidovitch Z. Interleukin 1 beta and prostaglandin E are involved in the response of periodontal cells to mechanical stress in vivo and in vitro. Am J Orthod Dentofacial Orthop. 1991;99(3):226-40. 32. Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6 family of cytokines and gp130. Blood. 1995;86(4):1243-54. 33. Grieve WG, Johnson GK, Moore RN, Reinhardt RA, DuBois LM. Prostaglandin E (PGE) and interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid during human orthodontic tooth movement. Am J Orthod Dentofacial Orthop. 1994;105(4):369-74. 34. Davidovitch Z, Nicolay OF, Ngan PW, Shanfeld JL. Neurotransmitters, Cytokines, and the Control of Alveolar Bone Remodeling in Orthodontics. Dent Clin North Am. 1988;32(3):411-35. 35. Kobayashi Y, Hashimoto F, Miyamoto H, et al. Forceinduced osteoclast apoptosis in vivo is accompanied by elevation in transforming growth factor beta and osteoprotegerin expression. J Bone Miner Res. 2000;15(10):1924-34. 36. Uematsu S, Mogi M, Deguchi T. Increase of transforming growth factor-beta 1 in gingival crevicular fluid during human orthodontic tooth movement. Arch Oral Biol. 1996;41(11):1091-5. 37. Kaku M, Motokawa M, Tohma Y, et al. VEGF and M-CSF levels in periodontal tissue during tooth movement. Biomed Res. 2008;29(4):181-7. 38. Kaku M, Kohno S, Kawata T, et al. Effects of vascular endothelial growth factor on osteoclast induction during tooth movement in mice. J Dent Res. 2001;80(10):1880-3. 39. Ong CK, Joseph BK, Waters MJ, Symons AL. Growth hormone receptor and IGF-I receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat. Angle Orthod. 2001;71(6):486-93.

97

40. Apajalahti S, Sorsa T, Railavo S, Ingman T. The in vivo levels of matrix metalloproteinase-1 and -8 in gingival crevicular fluid during initial orthodontic tooth movement. J Dent Res. 2003;82(12):1018-22. 41. Cantarella G, Cantarella R, Caltabiano M, et al. Levels of matrix metalloproteinases 1 and 2 in human gingival crevicular fluid during initial tooth movement. American Journal of Orthodontics and Dentofacial Orthopedics: Official Publication of the American Association of Orthodontists, Its Constituent Societies, and the American Board of Orthodontics. 2006;130(5):568.e11-6. 42. Holliday LS, Vakani A, Archer L, Dolce C. Effects of matrix metalloproteinase inhibitors on bone resorption and orthodontic tooth movement. J Dent Res. 2003;82(9):687-91. 43. Nahm D, Kim H, Mah J, Baek S. In vitro expression of matrix metalloproteinase-1, tissue inhibitor of metalloproteinase-1 and transforming growth factor-beta 1 in human periodontal ligament fibroblasts. Eur J Orthod. 2004;26(2):129-35. 44. Ingman T, Apajalahti S, Mäntylä P, Savolainen P, Sorsa T. Matrix metalloproteinase-1 and -8 in gingival crevicular fluid during orthodontic tooth movement: A pilot study during 1 month of follow-up after fixed appliance activation. Eur J Orthod. 2005;27(2):202-7. 45. Kassem M, Brixen K, Blum WF, Mosekilde L, Eriksen EF. Normal osteoclastic and osteoblastic responses to exogenous growth hormone in patients with postmenopausal spinal osteoporosis. J Bone Miner Res. 1994;9(9):1365-70. 46. Bills GC, Buschang PH, Ceen R, Hinton RJ. Timing effects of growth hormone supplementation on rat craniofacial growth. Eur J Orthod. 2008;30(2):153-62. 47. Pagani S, Meazza C, Travaglino P, et al. Serum cytokine levels in GH-deficient children during substitutive GH therapy. Eur J Endocrinol. 2005;152(2):207-210. 48. Vandeweghe M, Taelman P, Kaufman JM. Short and longterm effects of growth hormone treatment on bone turnover and bone mineral content in adult growth hormone-deficient males. Clin Endocrinol (Oxf). 1993;39(4):409-15.

98

49. Bianda T, Glatz Y, Bouillon R, Froesch ER, Schmid C. Effects of short-term insulin-like growth factor-I (IGF-I) or growth hormone (GH) treatment on bone metabolism and on production of 1,25-dihydroxycholecalciferol in GH-deficient adults. J Clin Endocrinol Metab. 1998;83(1):81-7. 50. Pagani S, Meazza C, Travaglino P, et al. Serum cytokine levels in GH-deficient children during substitutive GH therapy. Eur J Endocrinol. 2005;152(2):207-10. 51. King GJ, Keeling SD, McCoy EA, Ward TH. Measuring dental drift and orthodontic tooth movement in response to various initial forces in adult rats. Am J Orthod Dentofacial Orthop. 1991;99(5):456-65. 52. Madan MS, Liu ZJ, Gu GM, King GJ. Effects of human relaxin on orthodontic tooth movement and periodontal ligaments in rats. Am J Orthod Dentofacial Orthop. 2007;131(1):8.e1-10. 53. Hayashi K, Igarashi K, Miyoshi K, Shinoda H, Mitani H. Involvement of nitric oxide in orthodontic tooth movement in rats. Am J Orthod Dentofacial Orthop. 2002;122(3):306-9.

99

VITA AUCTORIS Zachary Langdon Varble was born in Alton Illinois on December 17, 1979.

He is the oldest son of David and

Tanya Varble both of Jerseyville, Illinois where he was raised. Zachary graduated from Jersey Community High School in 1998.

He attended University of Illinois-Urbana Champaign

and graduated in 2002 with a Bachelor of Arts degree in Economics.

He received a Doctor of Dental Medicine degree

from Southern Illinois University School of Dental Medicine in 2006.

He was accepted into the orthodontic residency

program at Saint Louis University that same year. Zachary is happily married to his wife, Amy, and they have a son, Calvin, born during his orthodontic residency in April, 2007.

100