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Gene expression associated with early events of orthodontic tooth movement
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Gene expression associated with early events of orthodontic tooth movement
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Content
GENE EXPRESSION ASSOCIATED WITH EARLY EVENTS OF
ORTHODONTIC TOOTH MOVEMENT
by
Mohammad Panahpour
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CRANIOFACIAL BIOLOGY)
May 2006
Copyright 2006 Mohammad Panahpour
UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
This thesis, written by
Mc9 f(XY\(xlK^o\xr
under the direction ofh ! thesis committee and
approved by all its members, has been presented to and
accepted by the Graduate School in partial fulfillment of
the requirements for the degree of
Mas fc/ ^
if\xrrt<7 .
iy Jean Morrison
A ssociate Vice Provost for Graduate Programs
Date, 1V 0(0
Thesis Comndti
/
/
I
Chair
T
DEDICATION
This thesis is dedicated to my parents, brother, and friends who have all
contributed to the person I am today.
11
ACKNOWLEDGEMENTS
Dr, Maggie Ziechner-David: To my principal investigator and research advisor, I
would like to thank you for all of you support and dedication to this research
project. Your help throughout my dental and orthodontic education has been
invaluable and is appreciated.
Drs. Glenn T. Sameshima and Robert G. Keim: To my other two-committee
members, I would like to thank you for all your support and help. Your
leadership and guidance throughout my residency has been appreciated and
valued.
Dr. Hong Beom-Moon: Thank you for your help and guidance for my research
project.
Dr. Julia Reyna: Thank you for all you help in my research project. Your
expertise and guidance is greatly appreciated.
Dr. Vivian Maung: Thanks for all your support and friendship. You made my
experience in dental school and the orthodontic program fulfilling.
Ill
TABLE OF CONTENTS
DEDICATION 11
ACKNOWLEDGEMENTS 111
LIST OF TABLES V
LIST OF FIGURES VI
ABSTRACT Vll
1. INTRODUCTION 1
IT REVIEW OF THE LITERATURE 5
III. MATERIAL AND METHODS 33
IV. RESULTS 37
V. DISCUSSION 47
VI. SUMMARY AND CONCLUSION 52
REFERENCES 54
IV
LIST OF TABLES
Table 1. Average of day 0 versus control side of day 1. 39
Average of day 0 versus treated side of day 1. 40
Comparison of control versus treated side of day 1.
Table 2.
Table 3. 41
Table 4. Day 0 versus control side day 1,13 genes. 42
Table 5. Day 0 versus treated side day 1, 24 genes. 44
Table 6. Day 1 control versus treated, 6 genes. 45
V
LIST OF FIGURES
Figure 1. Coil spring attachment from first incisor to first molar. 34
Figure 2. Make up of a microarray with all the genes. 35
Figure 3. Actual film of microarray. 36
Figure 4. Histological slide of root, cementum, dentin, and PDL. 37
VI
ABSTRACT
In this study we propose to test the hypothesis that by comparing the
expression of mRNAs in the periodontium subjected to orthodontic forces
(experimentally determined to produce resorption) maintained for different time
periods, and teeth where no forces were applied, we can identify the molecules
involved in the sequence of events leading to root resorption. Wistar rats from
45-50 days old were used and subjected to a continuous force of 80gm to the left
maxillary molars. The rats were sacrificed at days 0-5 and total RNA was
extracted from left molars (treated) and right molars (control) and converted to
cDNA. The cDNA was labeled with P32 and hybridized to Rat 1.2 microarrays
(Clontech, CA). Analysis of the arrays using Atlasimage 2.0 software indicates
several genes that have been upregulated. Our results suggest that these genes
play an important role in the initial molecular events of root resorption.
Vll
1. INTRODUCTION
Various histological and clinical studies have been accepted to elucidate
the etiology and pathogenesis of external root resorption; however, the molecular
events that lead up to the process of root resorption during orthodontic tooth
movement are still not very clear. Root resorption is a multifactorial
phenomenon and is a pathological result of orthodontic tooth movement, which
cannot be avoided. Orthodontics is probably the only dental specialty that
actually uses the inflammatory process as a means of solving functional and
esthetic problems (Brezniak et al. 2002).. Force application initiates a sequential
cellular process. We know exactly how and when it is evoked, but we are unable
to predict its actual overall outcome. The extent of this inflammatory process
depends on many factors such as the virulence or aggressiveness of the different
resorbing cells, as well as the vulnerability and sensitivity of the tissues involved.
Individual variation and susceptibility, which are related to this process, remain
beyond our understanding. We are therefore unable to predict the incidence and
extent of root resorption after force application (Brezniak et al. 2002). All
permanent teeth may show some amounts of root resorption that are clinically
insignificant and radiographically undetected. Root resorption of permanent teeth
is a probable outcome of orthodontic treatment and active tooth movement. The
incidence of reported root resorption during orthodontic treatment varies widely
1
among investigators. Most studies agree that the root resorption process ceases
once the active treatment is terminated. Root resorption of the deciduous
dentition is a normal, essential, and physiologic process. Permanent teeth have
the potential to clinically undergo significant external root resorption when
affected by several stimuli. This resorptive potential varies in persons and
between different teeth in the same person. This throws doubt on the role of
systemic factors as a primary cause of root resorption during orthodontic
treatment. Tooth structure, alveolar bone structure at various locations, and types
of movement may explain these variations. The extent of treatment duration and
mechanical factors definitely influence root resorption. In most root resorption
studies, it is not possible to compare the results and conclusions because of their
different methods (Brezniak et al. 2003).
In the process of orthodontic tooth movement, bone remodeling is a
steady feature whereas permanent root loss only occurs when cementum
resorption is greater than repair (Proffit 1993). In the process of orthodontic
treatment, common results are a decrease in root length due to root resorption as
a result of orthodontic tooth movement. Approximately 2-10% of patients who
are receiving orthodontic care will experience moderate to severe root resorption
(Machen et al. 1989). Various studies have illustrated that resorption of the
bone, cementum, and dentin occur under forces of pressure and cementum repair
2
occurs when these forces are removed. Defects as a result of root resorption
come about at the apex causing fragments to be separated from the remaining
root and thus are not able to be repaired. In the remodeling process, osteoclasts
and odontoclasts are responsible for bone and root resorption respectively.
Osteoclasts and odontoclasts cause bone and root resorption by secreting
hydrogen and proteinases that decay mineralized and non-mineralized tissues
found in bone and the root. The cementoid layer and the more mature periodontal
collagen fibers adjacent to cementum are possible barriers preventing root
resorption. The microenvironment around hyalinized tissue is favorable for the
introduction of hard-tissue resorbing cells. The findings show that the elimination
of hyalinized tissue leads to the removal of the cementoid and the mature
eollagen thus leaving a raw cemental surface without a barrier. It is therefore
hypothesized that such an area is readily attacked by odontoclasts. Once
resorption lacunae are established, the cementum is resorbed from the rear as an
undermining process. By continued orthodontic force application the resorption
process will proceed even after all hyalinized tissue is eliminated. If the
orthodontic force is discontinued or falls under a certain level, the resorption
lacunae are repaired (Rygh et al. 1977).
Various studies have been proposed and carried out to try to comprehend
the process of bone and root resorption; however, only minimal studies explicate
3
the initial molecular events that lead to root resorption. Most research studies
distinguish certain proteins that are involved in the resorption of bone. The
genes that activate certain proteins involved in the primary proceedings of root
resorption are still not clear. Therefore, patients and parents should be clearly
informed of the possibility of root resorption during orthodontic treatment. The
dentist or orthodontist underlying the possibility of root resorption should
provide Informed Consent forms to the patients and parents. Open bite cases
along with previous trauma have been shown to cause root resorption; therefore,
the practitioner should warn patients with open bites and question patients about
previous trauma respectively. If root resorption is suspected in specific patients,
periapical films should be taken every 3-6 months to monitor the patients and
prevent further root resorption.
4
II. REVIEW OF THE LITERATURE
Root resorption or external apical root resorption (EARR) is one of the
most debated subjects in orthodontics. Root resorption is probably one of the
most problematic side effects due to orthodontic therapy. However, there are
times when root resorption occurs with no relation to orthodontic therapy. In
certain people, there appears to be a sign of root resorption even before any
orthodontic therapy has begun. Some examples of the causes of root resorption
before orthodontic therapy are ectopic eruption of maxillary canines causing
resorption of the lateral incisors, trauma induced root resorption, or certain
diseases that spontaneously can cause root resorption.
Idiopathic resorption is when there is root resorption due to an unknown
cause. In fact, a high percentage of permanent teeth (not treated orthodontically)
portrayed minute lesions of external root resorption that were found at the apex
(Henry et al., 1951). All these lesions were repaired or in the process of being
repaired. On the other hand, root resorption is a common sequel of orthodontic
care.
The first study ever conducted on root resorption was carried out by
Ketcham in 1929 and since then investigators have confirmed that root resorption
is a common side effect of orthodontics with minimal and undetected root
resorption. The prevalence of root resorption varies widely and is due to many
5
different factors that include the age of the patient, the amount of time the patient
is in braces, the amount of tooth movement, the type of appliance used and the
amount of force used. Unfortunately, the amount of root resorption is not
predictable and the criteria for who is susceptible to severe root resorption caused
by orthodontics are not very predictable either. However, due to the extensive
amounts of research carried out by investigators, the mechanism in which root
resorption occurs has been explicated; however, this type of resorption is still the
least understood in clinical orthodontics. Some resorption occurs in most
orthodontic patients, however, the amount of resorption is clinically insignificant.
In other patients, however, shortening of the roots is apparent. The two most
common types of resorption occurring in orthodontics is superficial resorption.
which undergo repair and resorptions occurring in the apical area that could
ultimately lead to permanent root loss.
The first indications of root resorption has been defined as a penetration
of cells from the border of the necrotic tissue where mononucleated fibroblast
like cells, stained negatively by tartrate resistant acid phosphatase (TRAP),
started removing the precementum/cementum surface. This will eventually lead
to root resorption beneath the main hyalinized zone during which multinucleated
TRAP-positive cells were involved in removing the main components of the
necrotic PDL tissue and resorbing the outer layer of the root cementum.
6
Additional studies have shown that after the multinucleated TRAP-positive cells
reached the subjacent contaminated and damaged root surface and removed the
necrotic periodontal tissue, they kept on resorbing into the cementum surface
(Brudvik et al., 1994). When the movement has ceased, repair of the lacunae that
has resorbed occurs from the outside edge. However, after the force has stopped.
vigorous root resorption by TRAP-positive cells in the resorption lacunae were
still seen in the areas where hyalinized tissue existed (Brudvik et al., 1995).
After the removal of the hyalinized tissue, fibroblast-like cells attacked the active
resorption site. After the forced had stopped and due to lack of hyalinized
necrotic tissue in the PDL, repair of the resorption lacunae started. The first
indications of this were synthesis of collagenous fibrillar material by fibroblastand cementoblast-like cells, followed by restoration of the new PDL.
Nevertheless, more studies are needed to elucidate the exact process of resorption
to repair. Therefore, in order to avoid superficial root resorption on zones of
pressure is only probable if the root surface and adjacent cells are not destroyed
by necrosis. A decrease of root resorption is likely present by little trauma and
can be repaired by cutting off the force or providing low force during orthodontic
care.
In the case of apical root resorption, a radiographic periapical film of the
apex provides the most useful information in regards to root resorption (Levander
7
et al, 1988). For the best quantitative outlook of the condition of the root before
treatment and for evaluation of more resorption, a root resorption index can be
used (Goldson et al, 1975). If the root surface is well calcified and the predentin
layer is thin, tipping movements can cause resorption of the outer side of the
apical portion and along the inside of the root canal. A short hyalinization period
comes before the resorption of the apical side. In some patients, this type of
resorption can occur very quickly. Repair can occur if the apical area is saved
and further movement is performed in a different way. An example would be
bodily movement. The determining factors in apical root resorption are the
anatomic environment and duration and directional movement.
Many studies have implicated that the teeth most affected by root
resorption are the upper central, upper laterals, and lower incisors. Also, the
likelihood of root resorption is more apparent in dentitions which dental agenesis
and invaginations occur (Kjaer et al., 1995). The shape of the root, specifically
for upper incisors with blunt or pipette-shaped roots can increase the risk of root
resorption (Levander et al., 1988). There is no significant difference in the
propensity of root resorption of traumatized incisors compared to ones that are
not injured (Malmgren et al., 1982). However, for teeth that have portrayed root
resorption before orthodontic treatment, severe resorption can occur during
orthodontic care. Treatment for patients who have experienced previous trauma
8
should be started with light; intermittent forces to prevent prolong tipping. The
provider should aim at a limited goal to prevent severe root resorption.
Endodontically treated teeth have also shown to be slightly resistant to root
resorption due to an increased dentin hardness and density (Remington et al.,
1989).
Age is an issue debated in regards to root resorption. In general, the
apical third of the root is embedded more tightly in adult patient when compared
to young patients. Therefore, when an adult tooth is tipped over a short distance,
comparatively the apical third of the root will move very little. However, if the
movement is carried over for too long, damage can occur causing apical root
resorption and destruction of the alveolar bone can follow. Therefore, initial
stages of tooth movement in an adult patient are very important and light forces
should prevent the loss of root resorption. In addition, occlusal stress must be
avoided for the prevention of root resorption in adult patients.
Orthodontic appliances have been discussed as one the causing factors in
root resorption. It has been found that when comparing fixed versus removable
appliances, fixed appliances can cause more damage to the roots (Tinge et al.,
1991). Tinge et al. also found more resorption on the side where intermaxillary
elastics were used.
The magnitude of force is another factor that has been debated causing
9
apical root resorption. Studies have shown that the distribution of resorbed
lacunae has been directly linked to the amount of stress on the root surface, and
the stress of the lacuna development was more rapid with increasingly applied
forces (Harry et al., 1982). Most studies have also shown that only heavy forces
are responsible for significant root resorption. Intensity and duration of these
forces have been shown to also contribute to root resorption. Intermittent and
low forces have been shown to prevent root resorption, which allow the resorbed
cementum to heal and avoid any further resorption (Reitan et al., 1957).
The duration of force is another factor that can cause root resorption.
This would be the total amount of time a patient is receiving orthodontic
treatment. Various studies have indicated that the duration of time is directly
relevant to the amount of root resorption (Linge et al., 1991). In particular, the
time of treatment with rectangular arch wires can significantly contribute to
apical root resorption (Linge et al., 1991). Goldin et al. reported an apical loss of
0.9mm per year during labial root torque. Also, 34% of teeth examined after 6-9
months of orthodontic care showed apical root resorption; however, this number
increased to 56% after 10 months of treatment (Levander et al., 1988). It should
be known that minor resorption during the initial stages of orthodontic care is a
good sign that moderate resorption will follow. Therefore, regular radiographic
periapical films should be taken to monitor patients who have a high chance of
10
root resorption.
The direction of tooth movement has also been a contributing factor in
root resorption. It seems that torque and intrusion are the most hazardous
movements for the teeth involved. In particular, intrusion of teeth must be
carried out with great precaution, particularly in adult patients. The force
causing intrusion must be light and be accompanied by periods of rest. This
process can also be followed for torque in the upper anterior teeth in adults. A
thin-edgewise arch is preferred when torque is necessary in adult patients to
prevent the root from colliding with dense alveolar bone.
BONE CLASSIFICATION AND METABOLISM
The practitioner has to have a good understanding of the bone types to
truly understand the mechanism of orthodontic care. There are four types of
bone that will be discussed and they are the following: woven, lamellar.
composite, and bundle bone.
Woven bone has many different structures due to the fact that it is not
well mineralized, it is not very strong, and it is not very organized. However, it
does serve a very important role in wound healing. It rapidly fills osseous
defects, strengthens the bone that has been weakened by surgery or trauma, and
gives initial continuity for osteotomy segments and fractures. Woven bone is the
first bone formed in response to orthodontic loading. It is usually not found in
11
adult skeleton under normal circumstances. It is however, compacted to form
composite, remodeled to lamellar bone, or rapidly resorbs if loaded prematurely
(Roberts et al., 1988).
Lamellar bone on the other hand is a highly organized, powerful.
mineralized tissue that makes up more than 99% of the adult human skeleton.
After new lamellar bone is formed, a part of the mineral element
(hydroxylapatite) is put down by osteoblasts during primary mineralization.
After the primary mineralization occurs, secondary mineralization completes the
mineral component that takes many months and is referred to as crystal growth.
Due to the physiological limits, the power of bone is directly related to its
mineral content (Martin et al., 1989). Therefore, the full strength of lamellar
bone that supports an orthodontically moved tooth is not accomplished until one
year after active treatment. This is a significant factor when one thinks about
retention.
Composite bone is made by the deposit of lamellar bone within a woven
bone lattice, which is called cancellous compaction (Roberts et al., 1984). This
process is the fastest way of producing powerful bone (Currey et al., 1984).
Composite bone is usually the predominant osseous tissue for stabilizing a
patient during the early phases of retention or postoperative healing. The initial
bone formed due to woven and lamellar bones combined is called primary
12
osteons. Primary osteons will eventually lead to secondary osteons (Roberts et
al., 1992).
Bundle bone is produced by a functional adaptation of lamellar structure
to allow the addition of ligaments and tendons. Sharpey’s fibers, containing
perpendicular striations, make up the main distinguishing characteristic of bundle
bone. This layer of bundle bone is for the most part seen next to the PDL
alongside physiological bone-forming sides (Colditz et al., 1990). This type of
bone is for the most part the way ligaments and tendons attach throughout the
body.
Because of the interaction of structure and metabolism, one has to
understand how metabolism can influence the structures previously discussed.
Bone is the primary calcium reservoir in the body. The human skeleton stores up
to 99% of calcium. The serum calcium level is about lOmg/dl and fluctuates
slightly due to the interactions of the endocrine, biomechanical, and cell
interactions. Calcium levels are maintained through a process called calcium
homeostasis. Calcium metabolism is one of the basic physiological processes of
life support. To maintain the critical serum level, bone structure has to be
sacrificed sometime. In addition, the alveolar processes and the basilar bone of
the jaws are also predisposed to bone loss (Midjett et al., 1981).
Calcium ions into and out of the bone fluid are what regulate calcium
13
homeostasis. Bone fluid is separated from extracellular fluid by osteoblasts or
cells that are thought to be atrophied remnants of osteobasts. When calcium
levels decrease, parathyroid hormone (PTH) is secreted and this enhances
transport of calcium ions from bone fluid into osteocytes and cells lining the
bone. Vitamin D metabolite enhances pumping of calcium ions from bone-lining
cells into the extracellular fluid. The support of calcium homeostasis is possible
without the resorption of bone if it within physiologic limits. If not within the
physiological limits, bone will be removed from the bone reservoir. The control
of the calcium reservoir is controlled by 3 hormones: PTH, 1,25-DHCC (vitamin
D metabolite), and calcitonin. Calcitonin, a hormone produced by interstitial
cells in the thyroid gland, helps control hypercalcemia by preventing bone
resorption. PTH working with 1,25-DHCC helps to enhance osteoclastic
activity, increases the resorption rate of osteoclasts, and may suppress the rate at
which osteoblasts form bone (Roberts et al., 1992).
MECHANICS AND ROOT RESORPTION
There are many aspects of mechanics when considering root resorption
and possible causes. One is the different fixed appliance techniques and how
each affects root resorption. The second is continuous versus discontinuous
force, which can lead to root resorption, and the third is low versus high forces.
There has been a lot of debate about the type of technique and it being the
14
cause of root resorption. Orthodontists throughout time have been trying to
improve their techniques to combat root resorption. In particular, Viazis
introduced triangular brackets to increase the interslot distance to allow the
decrease of forces delivered by nickel-titanium wires and less pain. The use of
heat-activated, superelastic, nickel-titanium rectangular wires were introduced in
1995 and were called bioefficient threrapy. Other investigators have compared
the Simplified Standard Edgewise Technique and the Edgewise Straight Wire
System. Guilherme conducted such an experiment where he compared the
amount of root resorption after orthodontic treatment between the simplified
standard edgewise technique (group 1), the edgewise straight wire system (group
2), and the bioefficient therapy group (group 3). In these 3 groups, the amount of
root resorption in the upper and lower incisors were looked at and compared.
After taking periapical x-rays, the results showed that the bioefficient therapy
group had less resorption. The scoring of the root resorption was a system
developed by Levander and Malmgren. Also, when comparing the amount of
resorption expressed by the incisors, the upper central incisors had the most root
resorption followed by the upper lateral incisors, the lower central incisors and
finally by the lower lateral incisors.
Another factor of mechanics and root resorption is continuous versus
discontinuous force. The association between magnitude and duration of
15
orthodontic force and root resorption has been looked at from a variety of aspects
and contentious views have been reported. Dellinger and Reitan reported that
root resorption was made worse by increasing the force magnitude. On the other
hand, Owman-Moll et al. proclaimed that root resorption was not very forcesensitive. It has also been suggested that duration of force is a more critical
factor than magnitude (Harry et al., 1982). In contrast, there have been reports
that there is no significant relationship between force and duration in regards to
root resorption (Dermaut et al., 1986). Apart from these causative factors.
continuous versus discontinuous force has also been looked at. Continuous force
is 24 hours per day and discontinuous force can vary from 12-16 hours per day
depending upon the experiment or investigator. In an animal study. Maltha et al.
compared the amount of root resorption after continuous force (24 hours per day)
and discontinuous force (16 hours per day) force application. Maltha showed
that there is less root resorption when discontinuous force was used. More
recently, in 1999, Ahu et al. carried out an investigation comparing continuous
versus discontinuous force in human first premolars. Before extractions, a lOOg
of tipping force was applied to the first premolars via elastics. One side was
randomly selected to be the continuous side (24 hours per day) and the
contralateral side became the discontinuous side (12 hours per day). With the
help of composite electron micrographs, the buccal surface of each tooth was
16
digitized and root resorption was determined. Visual scoring assessed the degree
of root blunting. Mean percentage of resorption that affected areas was smaller
and apical blunting was less severe on the discontinuous side. In addition, the
mean percentage of the resorbed root area was higher in the continuous force
teeth. The electron microscopic graphs could also illustrate how the resorptive
lacunae were developing. The cavities caused by resorption had a tendency to
widen in the middle and cervical portions of the root surface when compared to
the apical region. In the apical regions, cavities were between 50 and SOOpm.
However, in the cervical and middle regions of the root, resorption cavities had
irregular shapes and could be as wide as 2mm. The results of this study showed
that discontinuous force results in less root resorption when compared to the
application of continuous force. As far as the amount of force, no difference of
orthodontically induced root resorption has been found at low and high force
levels (50-100g) (Owman-Moll et al., 1996). However, it is still recommended
not to overload the teeth with a high level of force that can harm areas of the
PDL resulting in more root resorption.
Lately, researchers are being directed towards the components of the
cellular, molecular, and genetic components of root resorption. Could there be a
genetic marker in certain patients that could signal the possibility of root
resorption for that patient? And if so, could we develop treatments or at least
limit our treatment for those patients who are genetically susceptible to root
17
resorption. There have been many experiments carried out with an animal study
and more recently, there have been human studies with regards to the genetic
predisposition to root resorption.
GENETIC PREDISPOSITION; RAT STUDY
Various studies have been carried out on the rat model to try to elucidate
the initial response and effect of orthodontic tooth movement. Alveolar bone
remodeling along with periodontal remodeling that takes place during tooth
movement is a cell-mediated process. There are many changes that take place
after the initial response including vascular and cellular including exchanges
between the nervous, endocrine, and immune systems that help expedite the
process. However, the transmission of mechanical stimuli into specific cellular
activity is still not fully understood. The initial phase of this mechanical
response is mostly described by periodontal vasodilation and movement of
leukocytes out of the PDL capillaries (Davidovitch et al., 1988). Changes in
blood flow linked with pressure applied to the PDL, formation and release in
biological messengers, and cell activation occurs following this process (Rygh et
al., 1976). Remodeling of bone is controlled by a balance between configuration
and resorption process that are regulated by a variety of systemic humoral factors
such as parathyroid hormone (PTH) and calcitonin, neurotransmitters such as
substance P, vasoactive intestinal peptide, and calcitonin gene-related peptide
cytokines or monokines such as IL-la, IL-ip, and chemokines (Alhashimi et al..
18
1999). The discharge of these messengers results in the initiation of cells
participating in resorption of the hyaline zone, alveolar bone, and root surface
leading to the remodeling of the periodontium.
Little is known about the cellular control of non-physiological root
resorption, specifically that which is caused by tooth movement. Sayaka et al.
carried out an in situ hybridization for matrix metalloproteinase-1 and cathepsin
K in rat tissue induced by tooth movement. They found that matrix
metalloproteinase-1 mRNA was in fibroblastic cells, cementoblasts and
osteoblasts, but not in osteoclasts or odontoclasts. This type of m-RNA was also
detected in some cementocytes located near odontoclasts and in some osteocytes.
On the other hand, cathepsin K mRNA was seen in osteoclasts and odontoclasts.
Histological studies were carried out to see the sites of these two compounds.
Odontoclasts and osteoclasts were also seen on the experimental side following
tooth movement.
Prostaglandins, specifically exogenous prostaglandins like E2 have been
shown to activate bone resorption and root resorption (Brudvik et al., 1991),
decrease collagen synthesis (Dziak et al., 1983), and increase cyclic AMP
(Somjen et al., 1980). Yamasaki et al. has shown the increase of tooth movement
with the increase injections of PGE2. However, Brudvik et al. showed that there
was a higher risk of root resorption with increased injections of PGE2. Bradley
et al. confirmed this by using a scanning electron micrograph to detect the
19
amount of root resorption. They showed that root resorption did increase with
the use of prostaglandin injections of, especially with an increase number and
concentration of injections of PGE2. On the other hand, Daryl et al. found
similar findings but found that the relatively greater degree of root resorption
occurring in the 2-week group with the injections of lOpg PGE2 was not seen in
the 4-week groups. This may indicate that the concentration and amount of
injections along with the amount of time that the injections are given may
provide different results when it comes to root resorption.
Pro-inflammatory cytokines have also been known to play a significant
role in the control of osteoclastic alveolar bone resorption as well as odontoclatic
root resorption. This evidence is confirmed by the fact that cytokines enhance
osteoclastic bone resorption. Cytokines are increased in response to
inflammatory triggers and secreted by different cell populations. Cytokines
donate to the differentiation, chemotaxis and activation of osteoclasts and their
precursors (Martin et al., 1998). More specifically, other pro-inflammatory
cytokines like interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-a) that
are mediators of the inflammatory process have been shown to be involved in the
process of bone resorption (Tatakis et al., 1993). Past evidence has shown that
these tow cytokines play a role in orthodontically induced tooth movement and in
the pathogenesis of several inflammatory periodontal diseases in man and other
species. In man and in rats, orthodontic tooth movement has lead to these
20
cytokines to be expressed in periodontal tissues as well as crevicular fluid (Saito
et al., 1991). On the other hand, Jager et al. showed that the inhibition of
cytokine activity by soluble receptors could be helpful in decreasing unwanted
root resorption. Also, Jager et al. showed that the amount of tooth movement
was decreased in all receptor-treated groups by about 50%. Interestingly, the
number of TRAP-positive cells on the desmodontal bone surface and on the
surface of the roots was decreased. Therefore, systemic application of soluble
receptors to IL-1 and TNF-a after tooth movement in the rat decreased the
number of odontoelasts as well as osteoclasts.
Alhashimi et al. have also carried out studies of the costimulatory
molecules CD40 and CD40L in periodontal and bone cells in orthodontically
treated and untreated teeth using immunohistochemistry. Interestingly, CD40 is
a cell surface receptor that belongs to the TNF family. CD40 is a type I
transmembrane protein and its’ eellular response mediated by CD40 is triggered
by its counterreceptor, the CD40L (a type II transmembrane protein) that is also
related to the TNF gene family. The interactions of these to proteins are
significant for the activation of CD4+ T-cell-dependent effectors function (Soong
et al., 1996). Alhashimi et al. showed that the CD40-CD40L communication
seems to be an active process during orthodontic tooth movement. This
orthodontic force also seems to induce T-cell aetivation. This activation could be
involved in the start of inflammatory mediators and succeeding bone remodeling.
21
They also concluded that this might lead to the production of anti-inflammatory
mediators that help defend against root resorption, which depends on the type of
immune response that is induced regarding CD40-CD40L expression.
Other members of the TNF family have also been recently investigated.
Three members of the TNF family of particular interest are the following:
receptor activator of nuclear factor kappa B (RANK), RANK ligand (RANKL)
(also known as osteoclast differentiation factor; osteoprotegerin ligand; and
tumor-necrosis, factor-related, activation-induced cytokine), and osteoprotegerin
(OPG) (also described as osteoclastogenesis-inhibitory factor) (Boyle et al..
2003). RANK is triggered by attaching to either soluble or membrane-bound
RANKL (Lacey et al., 1998), while this interaction is blocked by OPG ( a
secreted protein). Furthermore, osteoblasts and stromal cells can produce both
RANKL and OPG, and RANK activation produces osteoclastic differentiation
and activation (Lacey et al., 1998). Low et al. produced a device to test out this
hypothesis that the ratios of RANKL and OPG expression would increase during
root resorption process. Using this device. Low et al. showed that RANKL and
OPG levels were seen to increase in the locations during root resorption with
applications of heavy forces. Also, they might play an important role during root
resorption processes after the movement of teeth due to orthodontic forces. A lot
of the experimental studies involving root resorption have been carried out using
22
the rat model. However, recent advances and techniques have allowed carrying
these experiments to the human model.
GENETIC PREDISPOSITION: HUMAN STUDY
Genetic factors leading to external apical root resorption (EARR) in
orthodontics may help to alleviate and identify patients who are predisposed to
root resorption during orthodontic therapy. As we know, root resorption has a
multifactorial etiology. EARR can be an iatrogenic result linked to orthodontic
treatment, especially in the maxillary incisors. However, EARR can also happen
in the absence of orthodontic treatment (Harris et al., 1993). The incidence of
EARR with people who have never had orthodontic care has been reported to be
anywhere from 0-90.5% (Brezniak et al., 1993). Approximately 7-13% of
patients who have not had orthodontic treatment portray some amounts of EARR
on radiographs (Harris et al., 1993). This could be the cause of occlusal forces
damaging the PDL and causing EARR. Harris et al. has also reported that there
is an association of EARR with patients who have not received orthodontic care
with missing teeth, decreased crestal bone heights, and increased periodontal
probing depths. Bruxism, chronic nail biting, and anterior open bites with a
tongue thrust may also portray signs of EARR before orthodontic care (Harris et
al., 1992). Another cause could also be from dental trauma, specifically with re
implantation of an avulsed tooth leading to EARR (Donaldson et al., 2001).
Unfortunately, EARR appears without any symptoms, which makes it somewhat
23
difficult to detect EARR unless radiographs have been taken. It has been shown
that EARR occurs more readily in the apical region because more than % of the
resorption lacunae occur in the apical region of the root (Henry et al., 1951). The
reason for this could be due to the following factors: 1) Forces are concentrated
at the root apex due to the fact that orthodontic tooth movement in never entirely
translatory and the fulcrum is usually occlusal to the apical half of the root
(Harris et al., 2000), 2) periodontal fibers assume a different direction in the
apical end that may explain the increased stress in the region (Henry et al., 1951),
and 3) the apical third is covered with cellular cementum and the coronal third is
covered with acellular cementum. The cellular part of the root depends on the
vasculature and therefore cementum in the apical region is more weak and easily
injured when exposed to trauma or decreased vascular output to that area (Harris
et al., 2000).
In humans, we have to understand the cascade of molecular events
leading to activations of cells that could possibly be the culprits of root
resorption. We know that multinucleated cells called odontoclasts resorb
cementum, dentin, and enamel. Odontoclasts have functional and morphological
characteristics similar to those of osteoclasts (bone-resorbing cells) (Sahara et al..
1998). Hemopoietic cells in the bone marrow give rise to osteoclast and
odontoclast precursors. The formation of osteoclast from hemopoietic cells is
induced by cytokines such as macrophage colony-stimulating factor (M-CSF)
24
and RANKL (a membrane-bound ligand expressed by bone marrow stromal cells
(Lean et al., 2000). Osteoclast precursors could also be derived from the blood
via activated endothelium (McGowen et al., 2001). As circulating mononuclear
cells that are known as monocytes, these cells can proliferate and differentiate
into osteoclasts (Fugikawa et al., 2001). RANK and its’ ligand RANKL have
been seen in odontoblasts, pulp fibroblasts, periodontal ligament fibroblasts, and
in single odontoclasts, the latter finding signifying an autocrine/paracrine role
(Lossdorfer et al., 2002). RANK is known to be coded for by the TNFRSFl 1A
gene.
OPG is the molecule that inhibits osteoclastogenesis and is a soluble
receptor for RANKL along with being a member of the TNF receptor family.
The survival of osteoclasts is improved by transforming growth factor-beta
(TGF-beta), which is plentiful in the bone matrix. In the presence of TGF-beta,
TNF-alpha can induce osteoclast production in vitro from bone-marrow-derived
mononuclear phagocytes. Understanding the process of these cytokines and
other chemicals can help in understanding the possible causes of EARR.
One culprit of root resorption induced by cytokines is the interleukin-1
(IL-1). Al-Qawasmi et al. studied the assoeiation between polymorphisms in the
IL-1 genes and EARR due to orthodontic treatment. The association and linkage
approaches were used to test the role of the candidate genes, IL-1 A and IL-IB, in
EARR. IL-1 genes were looked at because they encode cytokines known to be
25
associated with bone resorption followed by orthodontic tooth movement. IL-1
gene is on human chromosome 2ql3 that includes 3 genes. IL-IA and IL-IB
encode proinflammatory cytokine proteins IL-1 a and IL-lp along with a third
gene (IL-IRN) encoding a related protein (IL-lra) that acts as a receptor
antagonist. Al-Qawasmi et al. showed that IL-IB polymorphism accounts for
15% of the total variation of maxillary incisor EARR. Homozygous individuals
for the IL-IB allele 1 have a 5.6 fold increased chance of EARR greater than
2mm as compared with those individuals who are not homozygous for the IL-ip
allele 1. Their data showed that allele 1 at the IL-IB gene, which is known to
decrease the production of IL-1 cytokine in vivo, can increase the chance of
EARR. Rossi et al. also concluded in a human study that there are possible
hormonal pathways for L-thyroxine (T4) and thyrocalcitonin (TCA) that may
regulate IL-1 p release to affect bone remodeling. They found that TNF-a is not
involved in this process.
Al-Qawasmi et al. also experimented with another candidate gene for
EARR that was mentioned previously called TNFRSFl 1 A, which encodes the
RANK and maps to 18q21.2-21.3 (Hughs et al., 1994). Another candidate gene
for EARR in orthodontic treatment is tissue non-specific alkaline phosphatase
(TNSALP), the product of which plays a significant role in mineralization and
cementum formation (Beersten et al., 1998). Al-Qawasmi et al. found no linkage
with EARR and TNF-a and TNSALP genes. It is apparent that more research
26
needs to be conducted in this subject matter to identify the genes involved with
root resorption. Because there is a limit of how much we can carry out human
studies relating to root resorption, we can try to concentrate on treatment and
prevention.
TREATMENT: RAT STUDIES
Many research studies have been carried out in the rat model to try to
establish the cause of root resorption and to enhance tooth movement. Sekhavat
et al. used misoprostol, a prostaglandin El analog, in rats to see the effect on
tooth movement and root resorption. The results indicated that misoprostol did
enhance the amount of tooth movement with minimal tooth movement.
Corticosteroids are group of related compounds derived from cholesterol
that have prevalent effects on a diverse range of organs (Ontjes et al., 1995).
In particular, prednisolone is a powerful synthetic steroid that is a useful model
to study the effects of steroids on humans and animals. Corticosteroids interfere
with the coupling of resorption and deposition cycle in normal bone, which
produces decreased bone formation and increased bone resorption. Furthermore,
the expression of steroids on the expression of TRAP that is thought to
participate in or signal active bone resorption (Reinholt et al., 1990) has not been
determined. Ong et al. displayed that steroid-treated rats presented significantly
decreased amounts of root resorption on the compression side and less TRAP27
positive cells within the PDL space on the same side. They concluded that
steroid treatment suppressed clastic activity.
Another compound that has been studied in rats related to tooth
movement and root resorption is a non-N-containing bisphosphonate called
clodronate. Clodronate strongly prevents bone resorption and also has anti
inflammatory characteristics. Therefore, bisphosphonates selectively prevent
osteoclasts, and have been used to treat many metabolic bone diseases related
with increased amounts of bone resorption (Fleisch et al., 2002). Liu at al.
showed that the number of osteoclasts on the clodronate-induced side was
significantly less than on the control side. Clodronate also prevented root
resorption where tooth movement was carried out.
Doxycycline (DC) is an antibiotic that has also been studied in relation to
root resorption and tooth movement. DC has been shown to decrease the total
amounts of osteoclasts and prevent root resorption and alveolar bone loss
following flap surgery in rats (Grevstad et al., 1993). This has led to orthodontic
experiments in relation to DC. Mavragani et al. showed that there was a
significant decrease in root resorption, the amounts of odontoclasts, osteoclasts,
mononuclear cells on the root surface, and TRAP-positive cells on the root and
bone for the group that received DC. They concluded that systemic
administration of low-dose DC in rats could prevent orthodontically induced
resorptive activity.
28
TREATMENT AND PREVENTION: HUMAN STUDIES
Many factors can be looked at to try to distinguish and prevent root
resorption in humans. Although there is not one causative factor, the clinician
can now take certain precautions to try to prevent root resorption in patients who
could be candidates of possible root resorption. Sameshima et al. showed that
resorption happens primarily in the maxillary anterior teeth (over 1.4mm). The
worst resorption was detected in the maxillary laterals, which could be attributed
to abnormal root shape such as pipette, pointed, or dilacerated roots. They also
showed that adult patients experienced more resorption in the anterior segments
only and Asian patients had less root resorption when compared to Hispanie and
white patients. The amount of overjet was linked to increased incidence of root
resorption and it did not matter if patients were male or female. These
conclusions led to another series of investigations to try to understand the results
from the first study by Sameshima et al. They found that patient who had
undergone first pre-molar extractions had more root resorption than those
patients who did not have any extractions done or had only maxillary first pre
molar removed. In relation to the overjet, they also found that root resorption
was linked to the amount of movement the root apices had moved in the
horizontal direction. In regards to slot size, archwire type, use of elastics, and
type of expansion, there was no differenee in the amount of root resorption.
However, there was a significant difference in the offices that were used for the
29
research revealing that clinician technique could be just one factor contributing to
the amount of root resorption. Periapical films carried out the evaluation of root
resorption because the use of panoramic films may overestimate the amount of
root loss by 20% or more (Sameshima et al., 2001).
As far as treatment is concerned, we know that apical root resorption is
due to pressure applied to the roots during orthodontic treatment. There are
usually no signs of radiolucency in the bone or the root and the orthodontic
pressure resorption is seen in the apical third of the root. Therefore, clinicians
must undergo a strict regimen during orthodontic treatment to try to reduce the
amount of root resorption caused by orthodontic care. This process should begin
before treatment has even started.
Several factors can be looked at before orthodontic treatment has begvm
in order to try to minimize root resorption. To start out, informed consent about
orthodontically induced root resorption should be discussed with the
patient/parent so there are no surprises, even if the amount of root resorption is
not clinically significant. Familial and genetic considerations should be
discussed for patients whose siblings have shown signs of root resorption. There
is much de bate about post-treatment radiographs and this cause supports the
notion of post-treatment radiographs for all patients. Medical conditions such as
allergies and asthma have been reported to be a causative factor in root resorption
(McNab et al., 1999 and Owman-Moll et al., 2000, respectively). There have
30
been conflicting studies as far as gender goes. However, most studies have found
to correlation between gender and root resorption. Chronological age may also
not be a significant factor in root resorption; however, there are studies by
Sameshima et al. and Mirabella et al. that have had different findings. Different
dentitions like abnormal root shapes have shown to portray root resorption and
there have been no significant evidence that one type of malocclusion is more
prevalent to root resorption.
As far as treatment of choice goes, there have been several studies that
have pointed out that it is possible to reduce but not eliminate the amount of root
resorption. Overall, it is recommended not to overload the teeth with high force
levels that can cause damage to the PDL leading to root resorption (Brudvik et
al., 1999).
During treatment, the clinician can take many precautions to try to
prevent further root resorption. First, the clinician should always start with initial
wire that is light and durable. The new light-force rectangular wires may
increase jiggling movements and expose the root to more root resorption (Proffit
et al., 2000). Second, longer intervals between teeth activation is highly
recommended (King et al., 1998). Third, periapical x-rays should be taken after
6 months of orthodontic treatment especially for those patients who could be
more prone to root resorption. Periapicals of the anterior teeth are a good sign of
root resorption throughout the teeth; therefore, full mouth x-rays would not be
31
necessary (Levander et al., 1994). If root resorption is seen, treatment should be
stopped for should be looked at again to try to attain alternative treatment options
to prevent further root resorption (Bednar et al., 1998).
After orthodontic treatment, several steps can be carried out to prevent
tooth loss and more root resorption. First, final records are a must and if root
resorption is apparent, patient/parent should be informed and the x-rays can be
used for siblings seeking orthodontic care (Harris et al., 1997). Second, for teeth
that have portrayed excess root resorption, further radiographs should be taken to
assess further root resorption (Levander et al., 2000). Third, teeth exhibiting
severe resorption should not be considered for abutments (Levander et al., 2000).
Finally, retaining the teeth with a fixed appliance should be carried out with
caution. Occlusal trauma to these teeth might lead to further root resorption
(Andreasen et al., 1987).
32
III. MATERIALS AND METHODS
Tissue Fixation for histological Slides
Forces will be applied to the maxillae of the rats as described for the
microarrays. Rats will be anesthetized for tissue fixation at days 0, 1, and 5.
Tissues will be fixed in 10% buffered neutral formalin solution. Tissues will
then be washed with distilled water in vials for 1-2 hours with 4 changes. The
following dehydration method will be used with 3 changes at 30 minutes each:
25% ethyl alcohol, 50% ethyl alcohol, 70%, 95%, and 100%. Tissue may be
stored at 70% dehydration step in -20 degrees Celsius until ready for sectioning.
After dehydration, samples will be washed with xylene twice at 30 minutes each.
Tissues will be infiltrated with paraplast plus with 2-3 changes for 30 minutes
each in an oven at 60 degrees Celsius. Tissues will then be embedded in melted
paraffin at 60 degrees Celsius and frozen until solidification occurs. Tissues will
then be sectioned into sagital sections 5 microns thick using a microtome.
Tissues were stained with hematoxylin and eosin and observed under light
microscopy.
Microarrays
A modified technique by Brudvik and Rygh (1993a) will be used.
Female Wistar rats (5-6 weeks old) will be anesthetized with Phenobarbital (6.0
mg/kg body weight, i.p.) and a closed coil spring will be attached to the cervical
33
area of the incisor and first molar of the maxillae with .01 stainless steel ligature
wire. A continuous force of 80gm using a closed coil Nickel-Titanium
(Sentalloy) spring (GAC International Inc. 10-00-02 NiTi with M hooks) will be
applied to the left maxillae with the right side being the control. This will be
considered heavy force. Another continuous force of 30-40gm using the same
spring will be applied to the rats with the same sequelae, which will be
considered the light force for future comparison.
Fig 1. Coil spring attached from the incisor to the first molar showing tooth movement.
Biological Mechanism of tooth eruption, resorption, and movement. In press from Mah et al.,
2005.
The rats will be kept in cages, given soft food and water, and monitored
regularly. Whole palate samples will be taken from both right and left maxillae
at days 0, 1, and 5. RNA will be extracted from the samples and purified.
Probes will be isolated and hybridized to the membranes using microarrays
34
protocol. Samples will be incubated with P32 on the membranes overnight at 68
degrees Celsius. Membranes will then be washed and exposed to Biomax film
for one week in -80 degrees Celcius. Films will then be developed and scanned
into the computer.
MBj
rinr
r
■IB
r-imrr t
PS S 5 SpHS[^^HSp§ Mm M M 'g|Bp|B|gftp|W»p1
■ppM I m
I
■■■m H
s
ipB
Fig 2. Make up of a microarray with all the genes. The intensities are shown with different
colors. Over 4,000 genes can be expressed. Mah et al., 2005.
Analyses of the intensities of different genes will be carried out using Atlas
Image 2.7. Atlas Navigator will be used to provide data analysis.
35
%
w
€1
m
%
f 1
f
•V
^w t
:*i f ;
^1
**i". »
«* ■' -T
„
Fig 3. Actual film of a microarray. Control versus Treated. Mah et al., 2005, in press.
36
IV. RESULTS
Histological Findings
Histologically on day 0, clear demarcations between dentin, cementum,
and the periodontal ligament (PL) are evident. Five days after force was applied,
resorption laeunae are present indicating resorption of the eementum and dentin.
Additional resorption is seen on days 10 and 15. These results indicate that as
early as day 5, resorption is evident.
-i- ■
- ^ 9§i
Sj
vf..
Fig 4. Histological slide of root, cementum and periodontal ligament. A= day 5, B=day 10,
C=day 15, and D=day 20. PL=periodontal ligament, D=dentin, RL=resorption lacunae, and C
with arrow=cementum. Mah et al., 2005, in press.
Microarray Findings
Based upon the histological findings, resorption is seen on day 5 and thus
we focused on the molecular events prior to this resorption. Due to the
overwhelming number of genes that were upregulated and downregulated,
37
analysis was restricted to comparisons of the averageof day 0 versus day 1,
control versus treated side. Investigation of genes was limited to intensities with
a threshold ratio greater than 3.00 and a threshold difference of 1,000 or greater.
Three different comparisons were carried out. Since there was no tooth
movement on day 0, the results from the control and treated sides were averaged
together. The average of day 0 was then compared to the control side. Forty-one
genes were given.
38
Coordinate Ratio Diff.
A01a 0.358248 -4937
A02h 0.495217 -4644
A04c 0.401117 -9221
A08b 0.410033 -9020
A08e 0.432926 -6772
AlOd 0.488933 -4664
A12i 0.475179 -10530
B04m 0.338414 -4731
B07m 0.34159 -4678
B08n Down -4479
B09I 2.15212 12474
15753 BlOb ue
B12f 2.943203 5303
C02n 2.49335 6063
C03I 2.778589 4386
C07c 4.372437 5922
C07I 2.53705 4439
C12d 3.515688 27982
EOli 7.164721 4753
E05m 3.841785 9807
E06a Down -6210
Elln 2.673356 4503
E12k 3.085097 4631
E12m 2.634252 19562
E13e 4.429778 4469
E13k 20.12674 12375
E14i 2.814914 4746
E14i 5549
EMI 3.662479 4875
FOIe 7.285156 4827
4587 F02e Ur
F02k 8.902135 4441
F03g 6.171402 5461
F05c 2.387762 8550
F06i 5.248529 8667
F12f 9.287037 4475
Table 1. Average of day 0 versus the control side expressed 41 genes.
When the average of day 0 was compared to the treated side of day 1, one
hundred and twenty-seven genes were expressed.
39
Coordinate Ratio Diff.
A02a 0.460548 -8464
A02i 4.895009 4526
A03d 3.269086 3626
A03e 4.337598 4696
A04c 0.468273 -8187
A04e 4.284066 4555
A04g 3.452645 2988
A07i 6.995058 3639
A08f 2.260329 4362
A09i 8.199203 3614
A12i 0.469498 -10644
A13m 3.500663 3771
B14c 2.881983 11657
C03I 2.509732 3723
C04g 4.71872 3953
C06g 5.571947 3781
C07f 5.645496 4023
C07i 5.016949 4029
C07k 6.196407 4339
C08i 6.843061 4505
C08k 2.226357 3955
C09a 2.249672 4765
Clla 5.241741 5264
C14a 5.062812 5692
C14b 6.625 6390
DOIh 0.337092 -3821
D09i 6.707113 5456
DlOh 2.299854 4439
EOlf 16.38618 7570
EOlg Ur 4017
EOlh 5527 UR
EOli 11.27886 7925
E03c 3.542219 19359
E03i Ur 4382
E03k 3670
E04g 5.291409 6244
E04h 5385
E05g UR 5109
E05i 9.766436 5067
E05m 3.805853 9683
E06g Ur 3849
E07f 4.21626 3956
E09b 0.388502 -6414
E12h Ur 4190
F05c 2.569875 9672
Table 2. Average of day 0 versus treated side of day 1. Some significant genes out of 127.
40
The final comparison was day 1, control versus treated side. For this
comparison, 21 genes were expressed.
Coordinate Ratio Diff.
A04f 0.453229 -11211
A05i 2.168739 10154
A08f 8435
A14i 2.640265 14828
B09I 0.422349 -17348
BlOb Down -19778
B14c 2.296008 12986
C12d 0.474206 -26500
DlOh Up 8827
DIOm 2.27167 10387
D13i 8315
D14k 39715
EOle 2.117493 15779
EOlf 8414 Ur
E02h Up 8134
E02k Up 10721
E03c 2.66276 21709
E12m 0.402584 -24279
E13k 0.171056 -13913
E^4\ Down -8262
F06i 0.176884 -11359
Table 3. Comparison of day 1, control versus treated. 21 genes were expressed.
Due to the abundance of genes, we will be only looking at genes with ratios of
3.00 and a difference of 1,000. Looking at day 0 versus day 1 on the control
side, we come up with 13 genes along with their classifications.
41
Ratio Gene Protein/gen Classification
Membrane channels
cytochrome P450 2A3 (CYP2A3); & transporters
elongation factor 2 (EF2)
4.3724 J02852
3.51568 K03502 Same
Intracellular
7.16472 fibroblast growth factor 5 (FGF5); D64085 transducers/effector/
insulin-like growth factor-binding
3.84178 protein 5 precursor M62781 Same
Purkinje cells-specific protein
3.08509 tyrosine phosphatase CBPTP, D64050 Protein turnover
guanine nucleotide-binding protein
4.42977 G(i)/G(s)/G(t) beta subunit 3 (GNB3) L29090 Same
guanine nucleotide-binding protein
20.1267 G(l)/G(S)/G(0) gamma-7 subunit L23219 Same
3.66247 rab16, ras related GTPase M83681 Cell receptors
RaIGDSB; GTP/GDP dissociation
7.28515 stimulator for a ras-related GTPase L07925 Same
Cytoskeleton/motility
8.90213 cAMP phosphodiesterase 4A L27057 proteins
Adenylyl cyclase (olfactive type) type
6.17140 M55075 Same
Functionally
5.24852 carboxypeptidase D precursor (CPD) U62897 unclassified
9.28703 adenosine A1 receptor (ADORA1) M64299 Same
Table 4. 13 genes expressed. Day 0 versus control of day 1.
42
The results for day 0 versus the treated side of day 1 provided many more genes.
This was expected because there was significant tooth movement. The results
provided 24 genes within the limits we had set.
43
Ratio Gene Protein/gene Classification
Metabolism/cytoplasmic
4.372437 cytochrome P450 J02852 proteins
3.515688 elongation factor 2 K03502 Same
Intracellular
7.164721 fibroblast growth factor 5 D64085 transducers/effectors
insulin-like growth factor3.841785 binding protein 5 M62781 Same
Purkinje cells-specific
protein tyrosine
3.085097 phosphatase D64050 Protein turnover
guanine nucleotide4.429778 binding protein L29090 Same
guanine nucleotide20.12674 binding protein L23219 Same
3.662479 rab16 M83681 Cell receptors
7.285156 RaIGDSB L07925 Same
Cytoskeleton/motility
proteins
cAMP phosphodiesterase
8.902135 4A L27057
Adenylyl cyclase
6.171402 (olfactive type) type III M55075 Same
carboxypeptidase D
5.248529 precursor (CPD) U62897 Functionally unclassified
9.287037 adenosine A1 receptor M64299 Same
17-kDa ubiquitinconjugating enzyme E2
c-met proto-oncogene;
hepatocyte growth factor
receptor
5.241741 M62388 Metabolism
5.062812 U65007 Same
mannose-6-
phosphate/insulin-like
6.625 growth factor II receptor U59809 Same
Cell receptors (by
ligands)
glutamate receptor 4
6.707113 precursor M85037
16.386179 CXC chemokine LIX U90448 Intracellular transducer
11.278858 fibroblast growth factor 5 D64085 Same
follicle stimulating
3.542219 hormone beta-subunit M36804 Same
5.291409 inhibin, beta A subunit M37482 Same
insulin-like growth factor
binding protein 3
9.766436 precursor M31837 Same
insulin-like growth factor3.805853 binding protein 5 M62781 Same
Lyn tyrosine-protein
4.21626 kinase AF000300 Same
Table 5. Day 0 versus treated side of day 1. 24 genes are expressed.
44
The final results were comparing control versus treated of day 1. There were 6
genes that were within our limits.
Ratio Difference Protein/gene Classification
inducible nitric
oxide synthase
(iNOSI); type II
NOS D14051 T ranscription
gammaaminobutyric acid
receptor alpha 5
subunit precursor
(GABA(A)
receptor;
GABRA5) L08494 Cell receptors (by ligands) UR
asialoglycoprotein
receptor R2/3
(ASGPR); hepatic
lectin 2/3; RHL-2 J02762 Cell signaling UR
macrophage
inflammatory
protein-2
precursor U45965 Intracellular transducers
CXC chemokine
Ur LIX U90448 Same
bone
morphogenetic
protein 4 Z22607 Same
Table 6. Day 1 control versus treated. 6 genes were expressed.
The results for the significant comparisons and their classifications for day 0
versus day 1 on the treated side and day 1 treated versus control will he
discussed. The top 6 genes/proteins will he discussed for both comparisons
along with or without any relevance to root and or bone resorption due to the
numerous amount of genes that are expressed. The classifications and
genes/proteins for day 0 versus day 1 on the treated side are the following: 1)
45
Insulin-like growth factor binding protein 3 precursor (IGFBP-3 or IBP3)
classified as an intracellular transducer/effector/modulator, 2) Fibroblast growth
factor 5 (FGF5 or HBGF5) classified as an intracellular
transducer/effector/modulator, 3) CXC chemokine LIX classified as an
intracellular transducer/effector/modulator, 4) Adenosine A1 receptor
(ADORAl) classified as functionally unclassified, 5) cAMP phosphodiesterase
4A; DPDE2, dunce Drosphila homolog E2 classified as a cytoskeleton/motility
proteins and 6) Guanine nucleotide-binding protein gamma-7 subunit (GNG7 or
GNGT7) classified as a protein turnover.
Looking at control versus treated on day 1, the highest genes/proteins and
their classifications are the following: 1) Inducible nitric oxide synthase
(iNOSI); type II NOS classified as a transducer, 2) Gamma-aminobutyric acid
receptor alpha 5 subunit precursor (GABA(A) receptor or GABRA5) classified
as a cell receptor, 3) Asialoglycoprotein receptor R2/3 (ASGPR), hepatic lectin
2/3; RHL-2 classified as cell signaling, 4) Macrophage inflammatory protein-2
precursor classified as an intracellular transducer/effector/modulator, 5) CXC
chemokine LIX classified as an intracellular transducer/effector/modulator, and
6) Bone morphogenic protein 4 classified as an intracellular
transducer/effector/modulator.
46
V. DISCUSSION
Due to the vast amount of genes/proteins expressed we will only discuss
the top 6 genes expressed when comparing day 0 to the treated side of day 1 and
the control versus the treated side of day 1. We will try to find any relevance to
bone and/or root resorption. We will first discuss the genes/proteins of day 0
versus day 1 on the treated side.
Insulin-like growth factor-binding protein 3 precursor is the major carrier
protein for insulin-like growth factor in circulation. It has been shown to control
cell proliferation via both insulin-like growth factor-dependent and independent
mechanism (Takaoka et al., 2004). Also, insulin-like growth factors are known
to stimulate the proliferation and differentiation of bone cell (Wergedal et al.,
1990), and they bind to insulin-like growth factor binding protein 3 causing
either the enhancement or limitation of biological effects (Mohan et al., 1993).
In 2001, Kveiborg et al. showed that transforming growth factor-pi treatment
resulted in a dose-and time-dependent increase of 2-fold in the insulin-like
growth factor binding protein 3 steady-state mRNA level as well as in protein
production. Transforming growth factor-pi controls proliferation and
differentiation of human osteoblast precursor cells.
Fibroblast growth factor 5 has been shown to be expressed in most phases
of embryogenesis in mice (Hebert et al., 1990) in a complex spatiotemporal
47
pattern and is also expressed in the adult brain by sets of neurons (Haub et al.,
1990). Unfortunately, the biological function of fibroblast growth factor 5 is still
unknown (Bates et al., 1990).
CXC chemokine or LIX in induced by a lipopolysaccharide and has
powerful, chemoattractant activity for neutrophils in vitro and in vivo (Wuyts et
al., 1993). LIX has been shown to be involved in neutrophil recruitment as well
as inflammatory bone disease. In 2004, Ruddy et al. showed that interleukin-17
and tumor necrosis factor a cooperatively induce this lipopolysaccharide in the
preosteoblast cell line MC3T3 in mice. Therefore, interleukin-17 regulates
expression of LIX in osteoblasts. Ruddy also showed that there is an increase of
LIX during periodontal disease (bone cells) and has implications for
inflammatory bone disease such as arthritis.
Adenosine Al receptor has recently been shown to mediate functional
recovery in hemisected animals. The Al receptor subtype is coupled by a Gprotein with inhibition of adenylate cyclase; however, there is no evidence of
how it is specifically involved in bone remodeling. In 2002, Nantwi et al.
showed that an Al receptor antagonist mediates functional recovery in
hemisected animals.
Cyclic AMP phosphodiesterase 4A is an isoform of purified recombinant
phosphodiesterases. There is no evidence that it is involved in bone remodeling
48
and/or root resorption.
Guanine nucleotide-binding protein gamma-7 subunit has been shown
primarily in medium-sized neurons of the neostratum and nucleus accumbens
and neurons of the olfactory tubercle, and at decreased amounts in the dentate
gyrus of the hippocampal formation and laminae II-III, and V of the neocortex.
It is also expressed at the peripheral tissues, but in low amounts. However, there
is no evidence of this gene/protein involved in bone remodeling as of yet.
When comparing day 1 for the control versus treated side, 6
genes/proteins that had the most significance will be discussed. The first
gene/protein is inducible nitric oxide synthase. First, nitic oxide (NO) is a short
lived and highly reactive free radical involved in cellular pathways. It has
significant cardiovascular, neurological, and immune functions (Alderton et al..
2001). NO released from osteoclasts has also been shown to be involved in bone
resorption (Haf et al., 2001). Inducible nitric oxide synthase is one enzymes that
catalyzes NO. It is the only enzyme that is calcium independent and is
constitutively active because of its high affinity to calmodulin. Also, this
enzyme can make NO for a longer amount of time. Moreover, this enzyme has
been portrayed in dental tissues (Felaco et al., 2000). Gingival inducible NO
synthase has been shown in high amounts during periodontal inflammation when
compared to noninflammed tissue (Hirose et al., 2001). In 2004, D’Attillio et al.
49
showed that there were increased amounts of this enzyme in gingival tissue on
the compressed side during orthodontic treatment. However, the pathway was
not clear. Also, NO has proved to be a potent inhibitor of bone resorption. In
1999, Silverton et al. showed that osteoclasts do produce NO and that the
production of NO is increased by calcium.
Gamma-aminobutyric acid receptor alpha 5 subunit precursor is a
receptor for gamma-aminobutyric acid (GABA). GABA is considered to be the
principal inhibitory neurotransmitter in the adult mammalian brain, spinal cord.
and olfactory bulb. It has also been shown to depolarize precursor cells and
differentiating neurons, showing that receptors to GABA are present (Laurie et
al., 1992). Furthermore, GABA improves neuronal process outgrowth ( Redbum
et al., 1987), influence neuronal survival (Meier et al., 1991), serve as a
chemoattractant for migrating neurons (Hansen et al., 1987), and regulate cell
proliferation (LoTurco et al., 1995). However, there has been no evidence
relating to bone remodeling.
Asialoglycoprotien receptor is a prototype of the class of cargo-carrying
receptors that constitutively enter cells through clathrin-coated vesicles and
traffic the endocytic pathway, recycling between endosomal compartments and
the cell surface (Stockert et al., 1995). Huang et al. in 2005 showed that adaptor
heat shock protein complex formation regulates trafficking of this receptor. Heat
50
shock proteins are molecular chaperones that are turned on due to cellular stimuli
or stress. In 2005, Kajiya et al. showed a functional role for heat shock factor-2
in fibroblast growth factor-2 induces RANK ligand, which is a significant
osteoclastogenic factor expression on sromal/preosteoblast cells.
Macrophage inflammatory protein is considered a chemokine and plays a
role in bone remodeling, particularly bone resorption (Alhashimi et al., 1990).
However, it has yet to have any association with osteoclast activity.
LIX was already discussed previously; therefore we will finally discuss
bone morphogenic protein 4. Bone morphogenic protein 4 is from the family of
bone morphogenic proteins. It has been shown to be involved in bone formation
specifically transforming osteoprogenitor cells to osteoblastic cells (Cheng et al..
2003). Cheng also showed that this protein significantly induced alkaline
phosphatase activity in preosteoblastic cells through recombinant adenovirus
research.
51
VI. SUMMARY AND CONCLUSION
It is known that the movement of teeth is a process in which the response
of tissue will produce various expression of genes and /or proteins. Our study
compared day 0 only to day 1 after orthodontic force was applied to try to
understand the initial molecular events leading up to the sterile inflammatory
process that is extremely complicated. This process is made up of various
disparate parts including forces, tooth roots, bone, cells, matrix surrounding the
tissue, and certain known or unknown biological messengers. In this study, we
have only presented data on the top 6 genes when looking at only day 1 after
forces were applied. Further studies are being carried out to look at genes
expressed on days 0-5. It should be noted that there are around 5000 rat genes
whose sequence is known. However, we are also starting to look at 4000 genes
in other microarrays.
Orthodontists should take all known measures to decrease the occmrence
of orthodontically induced apical root resorption. We have discussed many ways
to try to prevent root resorption but none can actually prevent root resorption
caused by orthodontics with actual certainty. A better understanding of the initial
molecular events related with this process could produce a better diagnosis
method and advanced therapies that can help in the treatment, prevention, and
possible diagnosis of patients who are at more risk of developing root resorption
52
because of orthodontic treatment. There needs to be more genetically based
studies in the future along with other basic science research that might clarify the
exact nature of orthodontically induced apical root resorption and help prevent
this process of root resorption.
53
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Panahpour, Mohammad
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Gene expression associated with early events of orthodontic tooth movement
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Craniofacial Biology
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