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Table of Contents
Dedication…………………………………………………………………………………i
Declaration………………………………………………………………………………..ii
Acknowledgements………………………………………………………………………iii
Table of Contents................................................................................................................ 1
Abbreviations...................................................................................................................... 4
1. Introduction..................................................................................................................... 6
2. Tooth Movement............................................................................................................. 9
3. Cementum ..................................................................................................................... 10
3.1 Dentine.................................................................................................................... 11
4. Root Resorption ............................................................................................................ 13
4.1 Definition and Classification .................................................................................. 13
4.2 Prevalence ............................................................................................................... 14
4.3 Aetiology................................................................................................................. 14
4.3.1 Systemic Factors .............................................................................................. 15
4.3.1.1 Genetic Factors ......................................................................................... 15
4.3.1.2 Ethnicity.................................................................................................... 15
4.3.1.3 Chronological Age .................................................................................... 16
4.3.1.4 Dental Age ................................................................................................ 16
4.3.1.5 Endocrine Imbalance ................................................................................ 16
4.3.1.6 Nutrition.................................................................................................... 17
4.3.1.7 Alveolar bone density and Turnover......................................................... 17
4.3.2 Local Factors.................................................................................................... 17
4.3.2.1 Habits ........................................................................................................ 17
4.3.2.2 History of trauma ...................................................................................... 18
4.3.2.3 Occlusal Trauma ....................................................................................... 18
4.3.2.4 Root Resorption Prior to Orthodontic Treatment ..................................... 18
4.3.2.5 Endodontically treated teeth ..................................................................... 18
4.3.2.6 Malocclusion............................................................................................. 19
4.3.2.7 Root Morphology...................................................................................... 19
4.3.2.8 Hypofunctional Periodontium................................................................... 19
4.3.2.9 Specific Tooth Vulnerability to Root Resorption ..................................... 20
4.3.2.10 Abnormal root morphology .................................................................... 20
4.3.3 Treatment related factors ................................................................................. 21
4.3.3.1 Duration .................................................................................................... 21
4.3.3.2 Appliance type .......................................................................................... 22
4.3.3.3 Treatment Mechanics................................................................................ 22
4.3.3.4 Magnitude of applied force....................................................................... 22
4.3.3.5 Direction of tooth movement .................................................................... 23
4.3.3.6 Duration of force application .................................................................... 24
4.3.3.7 Amount of tooth movement ...................................................................... 25
4.3.3.8 Extraction protocols .................................................................................. 25
4.4 Orthodontic Relapse and Orthodontically Induced Inflammatory Root Resorption
....................................................................................................................................... 25
1
4.5 Mechanism of Root Resorption .............................................................................. 26
4.6 Repair...................................................................................................................... 27
4.6.1 Phases of repair ................................................................................................ 28
4.6.2 Clinical implications of repair ......................................................................... 28
4.6.3 Biological nature of repair ............................................................................... 29
4.7 Medication for prevention of root resorption.......................................................... 30
4.7.1 Non-steroidal anti-inflammatory drugs............................................................ 30
4.7.2 Corticosteroids ................................................................................................. 31
4.7.3 Tetracyclines (Doxycycline)............................................................................ 31
4.7.4 Prostaglandins .................................................................................................. 32
4.7.5 Echistatin and RGD peptides ........................................................................... 32
4.7.6 Bisphosphonates .............................................................................................. 33
4.7.7 Fluoride ............................................................................................................ 33
4.7.8 Calcium ............................................................................................................ 33
5. Calcium Metabolism in the Dental tissues.................................................................... 35
5.1 Calcium homeostasis .............................................................................................. 35
5.1.2 Parathyroid hormone........................................................................................ 36
5.1.3 Dihydroxyvitamin D3 ....................................................................................... 36
5.1.4 Calcitonin......................................................................................................... 37
5.2 Calcium and Phosphate Metabolism and the skeleton............................................ 38
5.3 The role of calcium in teeth and tooth roots. .......................................................... 39
5.4 Promoting calcium availability ............................................................................... 41
6. Casein Phosphopeptides ............................................................................................... 42
6.1 Definition ................................................................................................................ 42
6.2 CPP and medicine ................................................................................................... 42
6.3 CPP and Dentistry................................................................................................... 44
6.4 Delivery of CPP in Rats.......................................................................................... 45
6.5 Delivery of CPP in humans..................................................................................... 46
6.6 CPP and root resorption .......................................................................................... 47
7. Research Tools and Methodologies .............................................................................. 48
7.1 Subject..................................................................................................................... 48
7.1.2 Appliance Design............................................................................................. 48
7.1.3 Measurement of tooth movement .................................................................... 49
7.2 Analysis of root-resorption ..................................................................................... 50
7.2.1 Histology.......................................................................................................... 50
7.2.2 Light Microscopy............................................................................................. 50
7.2.3 Transmission Electron Microscopy ................................................................. 51
7.2.4 Scanning Electron Microscopy ........................................................................ 52
7.2.5 Radiography..................................................................................................... 53
7.2.5.1 Plain Radiographs ..................................................................................... 53
7.2.5.2 Computer Tomography............................................................................. 54
7.2.5.3 Micro Computer tomography ................................................................... 55
8. References..................................................................................................................... 57
9. Manuscript .................................................................................................................... 76
9.1 Abstract ................................................................................................................... 79
9.2 Introduction............................................................................................................. 81
2
9.3 Materials and Methods............................................................................................ 88
9.4 Results..................................................................................................................... 95
9.5 Discussion ............................................................................................................... 98
9.6 Conclusions........................................................................................................... 105
9.7 Acknowledgement ................................................................................................ 105
9.8 References............................................................................................................. 106
9.9 List of Tables ........................................................................................................ 114
9.10 List of Figures ..................................................................................................... 117
10. Future Directions ...................................................................................................... 129
11. Appendix................................................................................................................... 131
Appendix 1.................................................................................................................. 131
Appendix 2.................................................................................................................. 132
Appendix 3.................................................................................................................. 133
Appendix 4.................................................................................................................. 134
Appendix 5.................................................................................................................. 135
3
Abbreviations
1,25 (OH2) D3
1,25 dihydroxyvitamin D3
2D
Two dimensional
3D
Three dimensional
AEFC
Acellular extrinsic fibre cementum
Ca
Calcium
CaCO3
Calcium Carbonate
CCM
Calcium Citromalate
CEJ
Cemento Enamel Junction
CIFC
Cellular intrinsic fibre cementum
cN
Centinewtons
CPP
Casein Phosphopeptides
DC
doxycycline
EARR
External Apical Root Resorption
EPMA
Electron Probe Micoranalysis
ERR
External Root Resorption
F
Fluoride
FEN
Finite Element Analysis
IL
Interleukin
Micro-CT
Micro-computed tomography
MMPs
matrix metalloproteinases
NaF
Sodium Fluoride
4
NiTi
Nickel Titanium
NIH
National Institutes of Health
OIIRR
Orthodontically induced inflammatory root resorption
OTM
Orthodontic Tooth Movement
P
Phosphorous
Ppm
Parts Per Million
PGE2
Prostaglandin E2
RGD
arginine-glycine-aspartic acid
SEM
Scanning Electron Microscopy
TEM
Transmission Electron Microscopy
TNF
Tumour necrosis factor
TRAP
Tartrate-Resistant Acid Phosphatase
5
1. Introduction
Root resorption is a common complication associated with orthodontic treatment.
Physiological and pathological factors are responsible for a destruction of root structure.
The consequences of root resorption range from slight tooth mobility due to small
amounts of root loss to complete tooth loss from excessive amounts of resorption.1
Radiographically, the resorption may appear as either an apical root blunting, lateral root
resorption or in rare cases excessive root loss.
Root resorption may be pathologic or physiologic in nature and it may also occur in
association with orthodontic tooth movement. Physiological resorption occurs during the
exfoliation of the primary dentition and mesial drifting in the permanent dentition. The
mineralized tissues of the permanent dentition are not normally resorbed.2 Pathological
resorption occurs subsequent to a traumatic injury, pathological disease process or
iatrogenic causes.
Soon after the advent of radiographic images, evidence demonstrating differences in root
morphology before and after orthodontic treatment was presented.3,4 A study involving a
long-term evaluation of upper incisors with severe apical resorption 5 to 15 years
following orthodontic treatment showed a significant correlation between tooth mobility,
total length and intra-alveolar root length.5
Apical resorption, however, has been
described as less detrimental to the prognosis of a tooth than an equivalent loss of
periodontal attachment at the alveolar crest.6 Kalkwarf et al7 demonstrated that 4mm of
6
root resorption translated into 20% of total attachment loss and 3mm apical root loss
equals only 1mm crestal bone loss. It is therefore important that periodontal disease is
under control in patients with severely resorbed teeth.
Loss of root length also has orthodontic implications due to the coronal movement of the
centre of resistance. Less force is required to torque a tooth with a shorter root length.8
A tooth with a previously shortened root length may therefore be predisposed to further
root resorption if forces equivalent to those used with an intact root are applied.
Due to the negative impact that excessive root resorption can have on the prognosis of the
dentition, it has been the subject of extensive research. Unfortunately research into the
cause of root resorption has been somewhat inconclusive due to the large range of
factors that contribute.9 Furthermore, there has been little success in influencing either
the reparative process which occurs following the cessation of force or in the prevention
of orthodontically induced inflammatory root resorption.
This review of the literature will seek to outline what is known about the process of root
resorption and its association with orthodontic tooth movement. It will describe how root
resorption is associated with the loss of mineralized dental tissues and the importance of
calcium in maintaining tooth structure. The review will then describe strategies for
maintaining calcium balance. Casein Phosphopeptides will then be introduced as a way
of promoting calcium availability and their potential to be of benefit in the prevention of
orthodontically induced root resorption will be described.
7
The review will describe how an appropriate way of assessing the potential benefits of
casein phosphopeptides is to use an orthodontic tooth movement appliance in a rat model
with controlled dietary supplementation.
Finally, the review will describe different methods for analysing root resorption and
ultimately how micro computer tomography can be used to gain an accurate, volumetric
quantification of root resorption.
Following the review of the literature, an experimental manuscript will be provided to
outline the experimental materials and methods, results and conclusions for this project.
8
2. Tooth Movement
Orthodontic tooth movement occurs following the initiation of an inflammatory process
which results in the resorption of bone on the pressure side and deposition of bone on the
tension side.
Tooth movement occurs when there is a prolonged application of force that exceeds the
bio-elastic limits of tooth supporting structures.10
The result of this force is an
inflammatory reaction in the connective tissues which leads to adaptive remodelling of
the periodontal ligament and alveolar bone11-13. Over-compression of the periodontal
ligaments leads to a localised ischaemia and cell death, forming an area of necrotic or
hyalinised tissue12,14-18. An attempt is made to remove the hyalinised tissue and initiate
repair through a process which resembles sterile inflammation12,19-21.
Bone remodeling is common to both tooth eruption and orthodontic tooth movement.22
During masticatory function, the teeth and periodontal structures are subjected to
intermittent heavy forces. In orthodontics this force is prolonged in nature and as such it
leads to progressive tooth movement.
The inflammatory process which occurs in orthodontic tooth movement is also associated
with the destruction and demineralization of the tooth root. Although cementum is more
resistant to resorption relative to bone it is still possible for both the cementum and
dentine to resorb as a result of this inflammatory process.
9
3. Cementum
Cementum is a specialized connective tissue that covers the anatomic roots of teeth and
shares some physical, chemical and structural characteristics with compact bone.23 It is
the surface for attachment of collagen fibres that bind the tooth to surrounding structures.
Cementum is different from bone in that it is not innervated, exhibits little or no
remodelling and is avascular. In fully matured teeth, cementum contains about 45% to
50% inorganic material and 50% to 55% organic material and water.23 The inorganic
material consists of calcium and phosphate in the form of hydroxyapatite. Numerous
trace elements are found in cementum with fluoride being by far the element with the
highest concentration.24,25 Protein extracts of mature cementum promote cell attachment,
migration and stimulate synthesis of gingival fibroblasts and periodontal ligament cells.26
Cementum is thinnest at the cementoenamel junction (20 to 50 µm) and thickest towards
the apex (150 to 200 µm),23 although it may exceed 600µm.27 Cementum continues to
increase in thickness with age.28-30
Two kinds of cementum can be seen with light microscopy. These are acellular and
cellular cementum.23,27
Acellular cementum covers the root dentine from the
cementoenamel junction to the apex, but it is often missing in the apical third of the root.
Cellular cementum is located mainly at root apices and contains cementocytes in its
lacunae.
10
Ten Cate31 classified cementum according to the time of formation (primary or
secondary), the presence or absence of cells within its matrix (acellular or cellular) and
the origin of collagenous fibres of the matrix (intrinsic or extrinsic)
a. Acellular Afibrillar Cementum
b. Primary Acellular Extrinsic Fibre Cementum
c. Primary Acellular Intrinsic Fibre Cementum
d. Secondary Cellular Intrinsic Fibre Cementum
e. Secondary Cellular Mixed Fibre Cementum
f. Cellular Mixed Stratified Cementum
g. Intermediate Cementum
3.1 Dentine
Dentine is approximately 70% inorganic, 20% organic and 10% water.32 The organic
component is predominantly type I collagen while the inorganic component is
predominantly hydroxyapatite. Dentine is yellowish in colour because of the random
arrangement of the hydroxyapatite crystals.
Dentin and pulp are formed from the dental papilla and are developed from
ectomesenchymal cells.32 The formation of dentine starts with odontoblasts secreting a
matrix which is high in type I collagen.32 The collagen network acts as a supporting
scaffold for the formation of hydroxyapatite crystals.
11
The odontoblasts modulate the local environment within the pre-dentine to produce
conditions conducive to mineralisation.32 The presence of odontoblastic processes is vital
to the slow but continuous requirement for dentine to be turned over.32
The matrix located immediately adjacent to the odontoblasts is not mineralised and it is
usually 15-20 µm thick.32 This layer is known as pre-dentine. Dentine grows in
thickness on average 4µm per day.32 It is important to note that dentine is highly porous
and medicaments or antigens that gain access to the dentine have direct access to the
pulp.32 Dentine may be involved in root resorption if the process penetrates the
cementum.33
12
4. Root Resorption
4.1 Definition and Classification
Root resorption is a physiologic or pathologic process that results in a loss of substance
from dentine or cementum.34 Physiological root resorption occurs during the exfoliation
of deciduous teeth. Pathological root resorption of permanent teeth may be either internal
or external in origin. Internal root resorption is initiated from within the pulp while
external root resorption arises from the periodontium affecting the external surface of the
tooth.
External root resorption has been classified by Tronstad35 into transient inflammatory
resorption and progressive inflammatory resorption.
In transient inflammatory root
resorption root damage is minimal and the stimulation for the resorptive process is of a
short duration while in progressive inflammatory resorption, changes in root structure can
be observed radiographically.35 Brezniak and Wasserstein expanded the classification by
including orthodontically induced inflammatory root resorption (OIIRR) for external root
resorption (ERR) that is caused by orthodontic force.36
OIIRR occurs as a result to the inflammatory process involved in orthodontic tooth
movement. It occurs on the cemental surface of the tooth root. Although cementum is
more resistant to resorption relative to bone it is still possible for both the cementum and
dentine to resorb as a result of this inflammatory process.
13
4.2 Prevalence
Root resorption has been reported in populations that are orthodontically treated and
untreated.
Most resorption related to orthodontic tooth movement is clinically
insignificant and causes minimal changes to the root length.37,38 There is however a high
risk group where severe resorption may occur. This group comprises one to three percent
of the population.39-41
4.3 Aetiology
Orthodontic root resorption appears to be multifactorial in aetiology.
The factors
implicated can be divided into environmental factors and mechanical factors.
Environmental factors can be further divided into systemic factors and local factors.
There is a high degree of individual susceptibility associated with OIIRR. The incidence
varies amongst persons and within the same person at different times.34 It also occurs in
varying degrees in different teeth.42,43
There are a number of factors that have been implicated as risk factors in OIIRR. There
is some evidence for an increase in root reorption being associated with such things as
asthma and allergy,43-46 excessive alcohol consumption45 and deficiency in dietary
calcium and vitamin D.47
14
4.3.1 Systemic Factors
4.3.1.1 Genetic Factors
Harris et al found a hereditary component to external apical root resorption following a
study amongst siblings which suggests that genetic factors play a role in EARR.48 Al
Qawasmi et al identified a key role of the IL-1β gene polymorphism for a genetic
influence in EARR in orthodontically treated individuals.49 They showed a 5.6 fold risk
of external apical root resorption of greater than 2mm in polymorphisms which were
homozygous for the IL-1β allele 1.49,50
This polymormphism accounted for
approximately 15% of variation in EARR of upper centrals.48
Harris et al48 and
Hartsfiled et al51 deduced that since half of the variation in EARR was influenced by
genetic factors, and the variation at IL-1β accounts for only 15 per cent of phenotypic
variation, there must be other genes that influenced EARR.
4.3.1.2 Ethnicity
Sameshima and Sinclair52 studied a sample of 868 patients and found that Asian patients
experienced less root resorption compared to white or Hispanic patients. However this
sample population was disproportionate when comparing the Asian and African
American samples to the larger Caucasian sample and therefore these results are
inconclusive.53
15
4.3.1.3 Chronological Age
Reduced vascularity, plasticity and width of the periodontal ligament, increased density
and reduced vascularity of bone and an increase in the width of cementum are associated
with increasing age.
Reitan suggested that these changes are responsible for the
increased incidence of root resorption seen in adults.54
4.3.1.4 Dental Age
Partially formed roots appear to develop normally during orthodontic treatment and it has
been suggested that teeth with open apices may be more resistant to apical root
resorption41,55,56.
4.3.1.5 Endocrine Imbalance
Altered endocrine conditions such as hyperparathyroidism,47,57 Paget’s disease,58
hypophosphataemia,59 hypothyroidism, hypopituitarism and hyperpituitarism60,61 are
associated with altered resistance to root resorption. This can be explained by the control
of the metabolism of mineralised tissues by the endocrine hormones 1,25Dihydroxycholecalciferol, parathyroid hormone and calcitonin.62
16
4.3.1.6 Nutrition
Engstrom demonstrated root resorption in animals deprived of dietary calcium and
vitamin D.47 However, Linge and Linge and Goldie and King suggested that nutritional
imbalance is not a major factor in root resorption during orthodontic treatment.63,64
4.3.1.7 Alveolar bone density and Turnover
There is some indirect evidence for a potential association between bone density and root
resorption.1 Orthodontic movement of teeth in close proximity to dense cortical bone has
been shown to cause greater levels of root resorption than the movement of teeth in
trabecular bone.65
Studies using calcium-deficient rats exhibiting very low alveolar
density have demonstrated markedly low levels of root resorption following tooth
movement.63
4.3.2 Local Factors
4.3.2.1 Habits
The large, non physiological forces produced by habits are similar in nature to the forces
produced by orthodontic treatment. An increased risk of external apical root resorption
has been noted to be associated with nail biting,66 finger sucking beyond 7 years of age,67
and lip or tongue dysfunction.67,68
17
4.3.2.2 History of trauma
Andreasen has suggested that the initiation of orthodontic force on a traumatised tooth
may aggravate the resorptive process.69 A study by Linge and Linge supported this
suggestion through their findings which showed that traumatised teeth lost an average of
1.07 millimetres of root structure compared to 0.64 millimetres in non traumatised
teeth.64,70
4.3.2.3 Occlusal Trauma
Occlusal trauma associated with improper occlusion, dental restoration and poorly
designed prosthetic appliances have been associated with accelerated root resorption.71,72
4.3.2.4 Root Resorption Prior to Orthodontic Treatment
There is a greater risk of developing further OIIRR in patients with pre-existing evidence
of root resorption.43,73
4.3.2.5 Endodontically treated teeth
A tooth with healthy surround tissues which has been treated successfully with
endodontics is no more susceptible to resorption than a normal tooth. In fact, Mirabella
and Artun38 and Thilander et al74 found endodontically treated teeth had less resorption.
18
Remington et al75 have suggested that an increased density of dentine in root filled teeth
provides resistance to resorption. On the other hand, a tooth which is in the presence of
an active inflammatory process may be more susceptible to root resorption as orthodontic
tooth movement itself creates an inflammatory response that may increase the resorptive
process.
4.3.2.6 Malocclusion
Increased overjet and openbite malocclusions have been implicated as a risk factor for
root resorption.64,73,76,77
4.3.2.7 Root Morphology
Abnormal or dilacerated roots have been implicated as factors which influence the
severity of root resorption. In particular, thin pipette shaped roots have been suggested as
being particularly susceptible to root resorption.38,78
4.3.2.8 Hypofunctional Periodontium
A hypofunctional periodontium refers to the narrowing of the periodontal space and
derangement of functional fibres. This eliminates the normal cushioning effect of the
periodontal ligament and results in a high concentration of force which leads to
19
stimulation of inflammatory mediators which induce the destruction of tooth and
bone.79,80
Studies by Sringkarnboriboon et al and Terespolsky et al have demonstrated greater
levels of resorption in teeth with hypofunctional periodontium and a delay in recovery of
the PDL following orthodontic force. These results suggest that orthodontic movement
of non-occluding teeth should be performed with caution.81,82
4.3.2.9 Specific Tooth Vulnerability to Root Resorption
The teeth most frequently affected by OIIRR in order of severity are the maxillary lateral
incisors, maxillary central incisors, mandibular incisors, distal root of mandibular first
molar, mandibular second premolars and maxillary second premolars.77,83-85 It has been
suggested that maxillary lateral incisors are more prone to root resorption due to their
abnormal root shape while maxillary incisors in general are more susceptible to root
resorption because they are frequently moved greater distances than other teeth.77
4.3.2.10 Abnormal root morphology
Teeth with short apices, pipette-shaped roots, pointed roots, dilacerated roots and slender
roots have all been associated with increased levels of root resorption.38,77,86-88 Blunt
roots were implicated by root resorption by Levander and Malmgren86 and
20
Thongudomporn and Freer.89 However this is contradicted by the findings of Sameshima
and Sinclair77 who found that teeth with blunted roots had the least resorption.
Oyama et al90 explained the increased levels of root resorption in short, bent and pipette
roots in an FEN study which demonstrated greater loading of forces in the root apices of
these teeth compared with normal teeth.
Teeth with increased root lengths have also been implicated as being at risk of increased
root resorption.77 This is due to the requirement for the use of larger forces to move the
teeth and the larger displacement of the root apices during tipping and torquing.
4.3.3 Treatment related factors
4.3.3.1 Duration
Most studies agree that the risk and severity of external apical root resorption increases as
the duration of orthodontic treatment increases.9,36,70,86,91-106
Sameshima and Sinclair
looked at a sample of 868 patients collected from 6 different specialist practitioners and
found longer treatment times to be significantly associated with increased root resorption
for maxillary central incisors.91 The reasons for the longer duration in treatment may also
have had an influence on the increased levels of root resorption seen in these patients.
21
4.3.3.2 Appliance type
Fixed appliances have been shown to cause more root resorption than removable
appliances which can be explained by the increased range of tooth movement afforded by
fixed appliances67. The risk of root resorption associated with different bracket designs
has yielded inconclusive results. 107,108,109,110
4.3.3.3 Treatment Mechanics
Linge and Linge41 suggested that the use of inter maxillary elastics increased the amount
of root resorption but Sameshima and Sinclair91 did not find any correlation.
No
difference has been found between the use of sectional and continuous mechanics.111 It is
generally agreed that the use of rapid maxillary expanders is associated with increased
levels of root resorption.74,112-114
4.3.3.4 Magnitude of applied force
Both human and animal studies agree that there is an increase in severity of root
resorption with increasing force magnitude.114-120
Harry and Sims117 used a scanning electron microscope to examine extracted human
premolar teeth that had 50g, 100g and 200g of intrusive force. They concluded that
higher forces increased root resorption through an increase in the stress to the root surface
which increased the rate of lacunae development.
22
More recent studies have confirmed the findings of previous studies showing that higher
forces increased the amount of external root resorption.
Chan and Harris used a
volumetric analysis of resorption craters on extracted human teeth to compare controls
with a force of 25g or 225g, with buccal displacement121 or intrusion.122
Reitan123, on the other hand, found that external root resorption was poorly correlated to
force magnitude.
He examined 72 premolars after application of 25g to 240g of
intrusive, extrusive and tipping movement over a period of 10 to 47 days. A series of
studies by Owman Moll et al124,125 agreed with the findings of Reitan. They looked at
tooth movement with regard to force magnitudes of 50g, 100g and 200g. They found that
there was a large inter individual variance but no significant differences in the frequency
and severity of root resorption could be detected. They concluded that root resorption
was independent of force magnitude, but that individual reactions may be more
important.
4.3.3.5 Direction of tooth movement
Intrusion has been consistently implicated as the most likely type of tooth movement to
cause root resorption.123,126,127 Reitan128,129 and Thilander and colleagues74 suggested that
the stress distribution associated with tipping movements is more likely to cause root
resorption than the stress distribution associated with bodily movement.
23
4.3.3.6 Duration of force application
Debate exists as to whether more root resorption is associated with continuous or
intermittent forces. Many believe that discontinuous forces produce less root resorption
because the pause in tooth movement allows the resorbed cementum to heal. 129-136
Acar et al135 examined 22 human teeth. The patients were exposed to a continuous
tipping force of 100g on one side and an intermittent force applied through elastics for 12
hours per day on the other side, over a period of 9 weeks. Their results showed that the
intermittent forces resulted in less root resorption. The accuracy of these results is
questionable because the intermittent forces were subject to patient compliance.
Weiland136 studied 84 premolars from patients which had been moved buccally with an
orthodontic appliance. On one side of the mouth, force on the premolar was applied with
a stainless steel wire (0.016 inch) while force on the contralateral premolar was applied
with a super elastic wire (0.016 inch). Their results support the findings of Acar et al137
that continuous forces cause more resorption. They showed that the teeth activated with
the super elastic wire moved significantly more, but had 140% more resorption than the
teeth with stainless steel wire.
Contrary to these reports Owman Moll et al125 found no difference in the amount or
severity of root resorption between forces applied continuously or intermittently after
application of a buccally directed force of 50g to human premolars.
24
4.3.3.7 Amount of tooth movement
Sameshima and Sinclair53 found that severe root resorption occurred in their samples
when the root apex was displaced lingually, a mean difference of 1mm more than the
control group. They concluded that root resorption is directly related to the distance
moved by the tooth roots.77 Maxillary incisors tend to be moved more than other teeth in
orthodontic treatment and therefore this is a possible explanation for why maxillary
incisors are at a high risk of root resorption.
4.3.3.8 Extraction protocols
Treatment plans involving extractions were not found to be associated with an increase in
the severity of maxillary incisor root resorption. An explanation for this is that the
movement of maxillary incisors is not excessive if teeth have been extracted for severe
crowding unlike in cases where maxillary incisors are moved large amounts to facilitate
the correction of overjet.
4.4 Orthodontic Relapse and Orthodontically Induced
Inflammatory Root Resorption
Following appliance removal, there is a conversion of the former pressure side of the
active treatment period into the tension side during the relapse period.138 This was
demonstrated in a study by Langford and Sims139 which demonstrated that relapse forces
25
were capable of causing significant root resorption for up to three months after RME.
Consequently it has been recommended that retention with fixed attachments be applied
with caution as occlusal trauma to the fixed teeth may lead to continued resorption.140
4.5 Mechanism of Root Resorption
Brudvik and Rygh141 studied the histological changes seen under light microscopy of rat
and mice molar teeth that had undergone orthodontic tooth movement. They found that
the first resorptive attack on the root surface occurred in the area of the over compressed
zone. Root resorption occurred in the circumference of the hyalinized tissue after 2 to 3
days in their rat samples. Mononucleated cells derived from adjacent healthy periodontal
membrane are implicated in the initiation of resorption of the unmineralized cementoid.
Brudvik and Rygh142 studied the penetration of cells into precementum and mineralized
cementum using transmission electron microscopy. Mononucleated non-clast cells were
found during the initial removal of precementum and mineralised acellular cementum at
the periphery of the hyalinized tissue.
They were also found to be removing
precementum and cementum at some distance from the hyalinised tissue. Macrophagelike cells phagocytosed necrotic tissue in the periodontal ligament after 6 hours and near
the root surface after 24 hours. Fibroblast-like and cementoblast-like cells are involved
in the resorptive process of precementum after 24 hours.
The surface layers of
mineralized cementum are removed by mononucleated cells after 3 days.
Multi-
nucleated cells were seen in surrounding tissues but not usually near the mineralized
cementum surface during their 5 day test period.
26
Brudvik and Rygh143,144 investigated further into the resorptive processes beneath the
main hyalinized zone. They found that the majority of cells involved in the resorptive
process beneath the hyalinised zone were multinucleated. They found that the cells
involved in the removal of the main part of the necrotic periodontal membrane, as well as
the root surface tissue, are different from that involved during the initial phase. Mononucleated giant cells with ruffled borders were never observed near the remnants of
necrotic tissue, but were found in the resorption lacunae of root and bone surfaces.
Cementum fragments were not found inside these cells and they postulated that removal
of cementum tissue occurred extra cellularly.
They also suggested that the multi-
nucleated cells are derived from monocytes and macrophages that initially invaded the
necrotic hyalinized tissues.
4.6 Repair
Studies in both human and animal subjects have demonstrated repair of root resorption
craters following a cessation in orthodontic tooth movement. The risk of tooth loss
following orthodontic therapy is not high because a reparative process in the
periodontium commences when the applied orthodontic force is discontinued or reduced
below a certain level.34,54,70,145,146
27
4.6.1 Phases of repair
Repair has been recorded as early as the first week of retention.147 Harry and Sims98 on
the other hand documented a longer delay in repair where the appearance of cellular
repair cementum in the apical regions occurred at 70 days. Barber and Sims148 studied
external root resorption in humans following rapid maxillary expansion. They found that
subsequent to expansion, repair was the predominant process. However continuing
resorption was apparent even after 9 months of retention.
Vardimon et al138 described the phases of root resorption repair. The incipient phase (14
days) was a transitional stage from no apposition (lag phase) to active deposit stages of
repair cementum. The peak phase occurs between 14 to 28 days and is characterized by a
spurt in matrix formation. In this phase an initial incorporation of extrinsic fibres into the
intrinsic cementum matrix suggests a development towards functional repair. This is
followed by a steady deposit of mixed fibrillar cementum between 42 to 56 days.
4.6.2 Clinical implications of repair
A long term radiographic evaluation of root resorption by Remington et al75
demonstrated progressive remodeling of the root surface after active orthodontic tooth
movement. The original root contours and lengths were never re-established despite
progressive rounding of jagged, resorbed edges. In their study of 100 subjects only two
patients had hypermobility which was considered to be the worst outcome. A review by
28
Vlaskalic et al106 found no reports of tooth loss from severe apical root resorption after
orthodontic treatment unless the tooth experienced some other form of trauma.
4.6.3 Biological nature of repair
The repair material in humans is principally cellular cementum while in animals root
resorption is primarily repaired by acellular cementum.149,150 AEFC is able to repair
resorptive defects rapidly in rodents because the cementoblasts have a relatively high
level of activity while in humans only the CIFC can fill a resorptive defect in a
reasonable period of time.151
Human studies on repair of root resorption defects have found that individual variation is
large.147 Owman_Moll and Kurol147 studied repair of orthodontically induced root
resorption in adolescents. Healing cementum was of the cellular type and there were no
large differences in the healing potential in the cervical, middle, and apical thirds of the
root. A study by Langford and Sims139 on root resorption following rapid maxillary
expansion found that periodontal fibre bundles were found to insert directly into the
repair cellular cementum matrix, irrespective of the site of the lesion of the root. Brice et
al152 studied orthodontic root resorption and repair in human teeth and found that
epithelial cell clusters similar to epithelial rests of Malassez were found in areas of
repairing orthodontic root resorption.
Studies by Brudvik and Rygh153 on rat teeth found that reparative cementum like material
appeared in the periphery of the resorption lacunae. Fibroblast-like cells were observed
29
close to newly formed cementum.
Another rat study by Kurihara and Enlow154,155
demonstrated the adhesive periodontal attachment to a resorbed dentine surface.
As outlined above, repair of root resorption craters has been demonstrated extensively
from both a clinical and a biological perspective. While it has been consistently shown
that repair is initiated following the cessation of orthodontic force, little success has been
demonstrated in influencing the amount of repair or the speed at which it occurs. For this
reason it is of interest to examine methods of preventing root resorption.
4.7 Medication for prevention of root resorption
Medication has been used in a number of studies in an attempt to reduce root resorption
following orthodontic tooth movement.
4.7.1 Non-steroidal anti-inflammatory drugs
Non-steroidal anti-inflammatory drugs (NSAIDs), are commonly used for the control of
pain in orthodontics. It has been demonstrated that the uses of NSAIDs may also affect
the sequence of tooth movement by reducing the associated inflammatory and bone
resorptive process.156 This results in a decrease in tooth movement. It therefore comes as
no surprise that the alteration in the bone resorptive process and tooth movement is
translated into a decrease in root resorption.
This has been confirmed in a recent
publication on the NSAID nabumetone which showed that there was a reduction in the
30
amount of root resorption however surprisingly there was no reported effect on the pace
of tooth movement.157
4.7.2 Corticosteroids
Long term side effects of steroid therapy include disturbances in mineralized tissue
metabolism and wound healing, discrepancies in chondrogenesis and osteogenesis, bone
loss and osteoporosis.158 It therefore follows that chronic corticosteroid use has the
potential to influence orthodontic tooth movement. This has been confirmed in a rat
study by Kalia et al159 who demonstrated that force application resulted in a significant
increase in the relative extension of root resorption and formation in rats who received
both chronic and acute corticosteroid treatment.
4.7.3 Tetracyclines (Doxycycline)
Mavragani et al160 studied the effect of low-dose systemic administration of the
tetracycline, doxycycline (DC) on orthodontic root resorption and orthodontic tooth
movement in rats. The results revealed a significant reduction in root resorption for the
DC-administered group although the orthodontic tooth movement did not differ between
groups.160 The anti-resorptive properties of dxycyclines (DCs) have been attributed to
their inhibition of several matrix metalloproteinases (MMPs). MMPs are largely
responsible for degrading constituents of connective tissues, not only during pathological
tissue breakdown, but also during normal remodeling. Tetracyclines have also been
shown to down-regulate the expression of pro-inflammatory and autoimmune mediators,
31
such as interleukin-1, tumour necrosis factor, nitric oxide, phospholipase A2 and
arachidonic acid metabolism.160 The role of these mediators in connective tissue
breakdown following orthodontic force application has been demonstrated.161,162 The
reduction in root and bone resorption in the doxycycline group which received
orthodontic force may also be attributed to a reduced resorption capacity of the individual
clast cell.160 It has been shown that tetracyclines can affect several parameters of
osteoclast function, such as diminishing the secretion of lysosomal enzymes.163
4.7.4 Prostaglandins
Brudvik and Rygh164 studied root resorption after local injection of prostaglandin E2
during experimental tooth movement in a sample of Wistar rats. They found that there
was no significant difference in root resorption between the experimentally moved teeth
with and without local injection of PGE2. Similar results were recorded by Sekhavat and
colleagues165 who used the prostaglandin E1, misoprostol, in a sample of rats. While
misoprostol was shown to increase orthodontic tooth movement there was only a minimal
effect on the levels of root resorption with a trend toward more root resorption.165
4.7.5 Echistatin and RGD peptides
Local injections of echistatin and arginine-glycine-aspartic acid (RGD) have been trialed
in rats as a method of preventing tooth movement and therefore enhancing anchorage.
These agents are known to inhibit integrin and thus perturb bone remodeling and reduce
tooth movement at a local level.166 Again it follows that a reduction of tooth movement
32
and bone remodeling has the potential decrease root resorption following orthodontic
tooth movement and this has recently be confirmed in an experiment on rats after local
administration of echistatin.167
4.7.6 Bisphosphonates
Liu et al168 studied the effects of local administration of clodronate on orthodontic tooth
movement and root resorption in rats. Clodronate is a bisphosphonate which strongly
inhibits bone resorption and has anti-inflammatory properties. In their experiment, wistar
rats were injected locally with clodronate and their teeth were moved buccally. They
found that there was a dose-dependent reduction in tooth movement in the rats. Local
clodronate also inhibited root resorption incident to tooth movement.
4.7.7 Fluoride
Foo et al169 studied the effect of systemic fluoride intake on root resorption in rats. In
their experiment Wistar rats were fed fluoridated water at 100ppm. Mandibular molars
were moved mesially using NiTi closing coils. Fluoride was found to reduce the size of
the resorption craters, but the effect is variable and not statistically significant.
4.7.8 Calcium
Seifi et al170 assessed the effect of Prostaglandins in association with Calcium Gluconate
on root resorption in rats. Once again they found that there was an increase in OTM
33
following injections of PGE and a trend towards an increase in root resorption.170 It is of
interest to note that in the group which also received calcium there was a trend toward a
decrease in root resorption.170
Due to the importance of calcium in the maintenance of healthy tooth structure it is of
interest to examine ways of maintaining a favourable balance of minerals in order to
protect teeth against OIIRR.
34
5. Calcium Metabolism in the Dental tissues
As outlined in the early discussion, orthodontic tooth movement involves a substantial
manipulation of bone in order to achieve gradual tooth movement. It is therefore clear
that interventions involving manipulation of bone should only be carried out when the
patient is in stable or positive calcium balance.171
An example of the importance of calcium in the mineralization of the dentoalveolar
structures is the relationship between osteoporosis and periodontal disease. Research has
demonstrated that dentate women who have pre-menopausal plasma oestrogen levels
have less alveolar bone loss than dentate women who were oestrogen deficient.171 The
question is therefore raised as to whether interventions that reduce bone loss generally
will also reduce damage to the hard tissues supporting the teeth.171
5.1 Calcium homeostasis
Bones are the primary source of supply of calcium ions when they are needed to maintain
the plasma calcium concentration.
99% of body calcium is stored in the bones.171
Powerful feedback control mechanisms normally maintain the plasma within narrow
limits, and these mechanisms have important consequences for the integrity of bones.
Calcium and bone metabolism is controlled by the endocrine system. Plasma calcium
concentration is primarily controlled by three hormones which are parathyroid hormone
(PTH), 1,25 dihydroxyvitamin D3 (1,25 (OH2) D3) and calcitonin.62 The major target
35
tissues for these hormones are the bones, the gastrointestinal tract and the kidney. All
three hormones operate in concert to maintain the constancy of the calcium level in body
fluids. Other internal factors which affect bone mineral metabolism are insulin, growth
hormone, thyroid hormone, phosphate regulating hormone, glucocorticoides, adrenal
corticosteroids, sex hormones and vitamins A and C.
5.1.2 Parathyroid hormone
PTH regulates calcium balance by stimulating the kidney to increase the reabsorption of
calcium in the distal tubule and to inhibit the reabsorption of phosphate ions in the
proximal tubule. Both of these actions increase the plasma calcium concentration.
PTH also exerts some control over the absorption of calcium by the small intestine. This
is achieved by stimulating the kidney to increase the rate of activation of vitamin D.
Goldie and King’s study on calcium deficient, lactating rats demonstrated that these
conditions resulted in decreased bone density which is consistent with increased
parathyroid hormone.63
5.1.3 Dihydroxyvitamin D3
1,25 (OH2) D3 is actually a hormone rather than a vitamin. Adequate amounts of 1,25
(OH2) D3 are essential to maintain appropriate plasma calcium and adequate
mineralization of bone matrix. The plasma concentration of 1,25 (OH2) D3 is regulated
36
by the kidney, which senses the plasma calcium and phosphate and adjusts the output of
the hormone accordingly by modifying the rate at which its precursor is hydroxylated.
1,25 (OH2) D3 acts on the intestine to increase absorption of calcium and phosphate ions.
This has the effect of making more calcium available for the formation of new bone by
osteoblasts, thereby preventing a net loss of bone.
1,25 (OH2) D3 acts on the bones by augmenting the action of PTH to resorb bone and
release calcium into the plasma.
It also acts on the kidney by promoting calcium
retention.
A deficiency in 1,25 (OH2) D3 results in a decreased mineralization of bone. However
this hormone does not directly induce bone formation.
5.1.4 Calcitonin
Calcitonin is involved with the balance of formation and resorption of bone. The action
of calcitonin is therefore particularly significant during growth and it appears to be
relatively unimportant in the mature skeleton. Calcitonin is a highly potent inhibitor of
bone resorption as it prevents osteoclasts form resorbing bone and inhibiting the
differentiation of new osteoclasts. It is not thought to play a major role in maintaining
plasma calcium concentration.
It appears to be more related to protecting against
excessive bone resorption in growing children and possibly during pregnancy.
37
Due to the importance of these hormones in maintaining the constancy of the calcium
level in body fluids it is not surprising that altered endocrine conditions such as
hyperparathyroidism172,173, Paget’s disease174, hypophosphataemia175, hypothyroidism,
hypopituitarism and hyperpituitarism176,177 are associated with altered resistance to root
resorption.
5.2 Calcium and Phosphate Metabolism and the skeleton
Three sources of calcium; calcium carbonate (CaCO3), calcium citromalate (CCM) and
milk have been extensively studied and have all shown to ensure efficient absorption of
calcium over a long term (one to four years).178 Prince et al179 demonstrate this in a study
on menopausal women, in whom calcium supplements, given as CaCO3 tablets or as
milk, reduce bone loss measured over a two year period.
Phosphate is also important in bone mineralisation and calcium metabolism.180 It has
been shown that phosphorous must be present for the production of hydroxyapatite.178
The dissociation of calcium intake from that of phosphorous may restrict bone
metabolism.178 This may occur in situations where the calcium source is not ingested
with the meal and/or this source contains no Phosphate.178
38
5.3 The role of calcium in teeth and tooth roots.
The influence of diet on the structure of teeth has been studied in depth. Of particular
interest to this study are the factors which influence the hardness of the teeth. Some of
the earliest studies in this field were conducted by Mellanby.181,182 Mellanby concluded
that a low dietary calcium content may result in teeth being soft to the point that they can
be cut with a scalpel.181 Mellanby demonstrated that well and badly calcified teeth can be
produced at will by altering in the diet the relative amounts of calcifying vitamin, found
in milk, egg-yolk, cod-liver oil, etc., and anti-calcifying substances found chiefly in
cereals.182
A study by Bielacyc and Golebiewska in 1997 demonstrated the importance of calcium
and vitamin D in roots of teeth in rats.183 Scanning microscope observations showed the
increased cementolysis and decreased mineralization of cementum and dentin in rats fed
a low calcium and vitamin D-deficient diet. This demonstrated that the hard tissues of
the teeth, besides bony system, are also involved in calcium homeostasis.
Of particular interest to dentists is the potential for teeth and tooth roots to remineralize.
A study by Clarkson et al in 1991 demonstrated the remineralizing potential of human
tooth root organic matricies which did or did not contain soluble non-collagenous
proteins including phosphoprotein.184
This research suggested that the removal of
soluble, noncollagenous proteins, especially phosphoprotein from root caries lesions, may
enhance their remineralization potential.184
39
With respect to root resorption Rex et al185 performed an electron probe micoranalysis
(EPMA) on the concentrations of calcium (Ca), phosphorous (P), and fluoride (F)
concentrations in human first premolar cementum after the application of light and heavy
orthodontic forces. The application of heavy forces caused a significant decrease in the
Ca concentration of cementum at certain areas of periodontal ligament tension which
demonstrates the demineralising nature of orthodontic force.
Seifi et al170 demonstrated how the provision of calcium can potentially protect against
root resorption. They studied the effect of submucosal injections of Prostaglandins in
association with intraperiotoneal injections of Calcium Gluconate on root resorption in
rats and demonstrated that this combination demonstrated a trend toward a decrease in
root resorption.170
They postulated that this may be a result of the transient
hypoparathyroidism and diminished resorptive activity subsequent to injection of the
calcium compound. The association of root resorption with hypothyroidism has been
described previously by Newman.78 Seifi et al170 proposed that it seemed likely that
raised serum calcium levels may inhibit PTH secretion and therefore inhibit root
resorption.
These studies demonstrate the importance of maintaining adequate mineralisation of the
dental tissues in order prevent the breakdown of the root structures. As Brudvik and
Rygh142 have demonstrated, root resorption involves the removal of mineralized
cementum. It would therefore follow that if we can maintain adequate mineralization of
40
the dental tissues then there may be a possibility to protect teeth against orthodontic root
resorption
5.4 Promoting calcium availability
The NIH (USA) recommends that humans should consume between 800mg to 1500mg of
calcium per day depending on age and gender.186 The US department of Agriculture cites
figures showing that only about 13% of girls and 36 percent of boys aged 12-19 in the
United States consume at least the amount of calcium recommended by the
NIH(USA).187 This is of concern because nearly 90% of adult bone mass is established
before the age of 20.171
Dairy products are particularly effective in storing calcium because the milk protein
casein stabilizes the structure of the liquid which allows it to maintain its high calcium
phosphate concentration without allowing precipitation.188 It follows that the provision
of dietary casein would be of potential benefit in making calcium available for the
protection of tooth roots.
41
6. Casein Phosphopeptides
6.1 Definition
Casein Phosphopeptides (CPP) are multi-phosphorylated peptides from an enzymatic
digest of the bovine milk protein casein.189 In milk, casein stabilizes the structure of the
liquid in order for it to maintain its high calcium phosphate concentration without
allowing precipitation.188 Appendix 1
6.2 CPP and medicine
Bioactivity of phosphopeptides yielded after tryptic hydrolysis of casein (CPP) was
reported more than 50 years ago by Mellander who discovered that CPP were found to
improve calcium balance in rachitic newborns.190 Mellander demonstrated that calcium
bound to phosphopeptides could be absorbed from the digestive tract and promote bone
calcification in rachitic children.191
CPPs have been shown to enhance calcium absorption by increasing calcium solubility in
vitro and in situ.180 It has been demonstrated that CPPs enhance paracellular transport of
calcium in the distal small intestine in elderly female rats.192 This has been confirmed by
Saito and colleagues who found that CPP supplementation has the effect of significantly
increasing intestinal Ca absorption, at least under conditions of marginal dietary Ca
levels.193
42
The positive effects of milk casein phosphopeptides on bone retention has been
demonstrated in a number of rat models. Tsuchita et al180 demonstrated in an in vitro
study that there was an increase in absorption of calcium and bone retention in rats. They
demonstrated that Casein Phosphopeptides have an inhibitory effect on bone loss in aged
ovariectomized rats which could be due to their effects on phosphorous and calcium
metabolism.180 It was found that rats fed a CPP diet had an increase in urinary calcium
excretion and a decrease in urinary phosphorous excretion. This study showed less bone
loss in rats fed CPP than in rats given Ca and P as pure minerals.180
Ma and
Yamaguchi192 demonstrated similar positive effects associated with CPP in a study that
showed a combination of CPP and genistein had a synergistic-anabolic effect on bone
components in rats with increasing age. They postulate that this combination may have a
role in the prevention of osteoporosis.
The positive effects of CPP supplementation on the skeleton has also been demonstrated
in humans. Heaney et al194 studied the effects of administering 87.5mg of CPP to 35
normal post-menopausal women as a part of a standard test meal containing a calcium
load of 250mg. Their findings suggested that caseinphosphopeptide supplementation is
particularly useful, for persons with low basal absorptive performance.
The positive effect of CPP on the mineralization of the skeleton has been demonstrated in
both human and animal studies. Like bone, teeth also require a balance in mineral
content in order to prevent resorption and as such the question is raised as to whether or
not CPP can offer potential benefits to teeth which are at risk of losing mineral content.
43
6.3 CPP and Dentistry
In recent years there has been significant interest in the potential for casesin
phosphopeptides to prevent and remineralise carious lesions. Dental caries is a saliva and
carbohydrate modified bacterial disease.195 The bacteria involved in the disease produce
acid as a metabolic byproduct which causes demineralization of the dental tissues which
often leads to cavitation of the tooth.
The use of a remineralizing solution containing calcium and phosphate ions has not been
clinically successful due to the low solubility of calcium and phosphates.189 Calcium and
phosphate ions at low concentrations do not incorporate to any significant degree into
plaque or localize at the tooth surface.189
In contrast, dairy products are a food group which are widely recognized to exhibit anticaries activity.196,197 Animal and in vivo caries models have demonstrated that the
components responsible for the anticariogenic nature of dairy products are the casein,
calcium and phosphate.189 A study by Reynolds et al in 1995 on rats demonstrated that
the tryptic digest of casein with the phosphopeptides selectively removed showed no
anticariogenic activity.198
On the other hand, the synthetic octapeptide-calcium
phosphate complex significantly reduced caries activity which confirmed that this
calcium-phosphate-stabilizing portion of the casein phosphopeptides is associated with
anticariogenicity.198
44
CPPs have the ability to stabilize calcium phosphate in solution and substantially increase
the level of calcium phosphate in dental plaque.199,200 Appendix 2 Casein
phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanocomplexes have been
shown to localize at the tooth surface and prevent caries in laboratory, animal and human
in situ caries models.189,198 Reynolds has proposed that the anticariogenic mechanism for
CPP-ACP is the localization of ACP at the tooth surface which buffers the free calcium
and phosphate ion activities, thereby helping to maintain a state of supersaturation with
respect to tooth enamel depressing demineralization and enhancing remineralization.189
Roberts et al have also demonstrated that this rematerialising capacity is effective in
reducing the whiteness of fluorotic lesions.201
Furthermore it has also been demonstrated that the incorporation of CPP into salivary
pellicles reduced adherence of both S.sobrinus and S.mutans significantly and thus has
bacteriostatic/bacteriocidal effects.188,202 Rose demonstrated the mechanism for this as
being the competition for binding sites on S.mutans between CPP-ACP and calcium.188
6.4 Delivery of CPP in Rats
Anticariogenicity of CPP-ACP has been investigated using specific-pathogen-free rats
orally infected with Streptococcus sobrinus.198,189 The rats had the CPP-ACP solution
exposed to their teeth through their drinking water. The study demonstrated a significant
reduction in caries activity with 0.1% w/v CPP-ACP producing a 14% reduction and
1.0% w/v CPP-ACP a 55% reduction relative to the distilled water control.189
45
6.5 Delivery of CPP in humans
Reynolds203 demonstrated in a human intra-oral caries model that when caseinate was
digested with trypsin, the tryptic peptides of casein were found incorporated into an intraoral appliance plaque and this was associated with a substantial increase in the plaque’s
content of calcium and phosphate. The tryptic peptides responsible for the anticariogenic
activity were concluded to be the calcium phosphate sequestering phosphopeptides.189
It has been demonstrated in a human model that two exposures per day of the CPP-ACP
solution produced a 19% reduction in enamel mineral loss relative to the control
enamel.189 CPP was incorporated into the plaque resulting in a 2.4 fold increase in the
plaque calcium and a 2.6 fold increase in plaque inorganic phosphate.189
CPP-ACP has also been shown to remineralize enamel subsurface lesions in situ when
delivered in a sugar-free gum.204,205 The results showed that sugar-free gum containing
CPP-ACP is superior to an equivalent gum not containing CPP-ACP.204,205 CPP-ACP
has also been incorporated into a self-cured glass-ionomer cement (GIC).206
Incorporation of 1.56% w/w CPP-ACP into the GIC significantly enhanced the release of
calcium, phosphate, and fluoride ions at neutral and acidic pH.206 The release of CPPACP and fluoride from the CPP-ACP containing GIC was associated with enhanced
protection of the adjacent dentin during acid challenge in vitro.206
46
6.6 CPP and root resorption
The ability for CPP to effectively provide bioavailable calcium for maintaining
mineralization of both the skeleton and the dental tissues has been extensively
demonstrated in both rat and human models. Like bone, teeth also require a balance in
mineral content in order to prevent resorption and as such the question is raised as to
whether or not CPP can offer potential benefits to teeth which are at risk of losing
mineral content. Furthermore CPP has been demonstrated to be a safe, natural substance
when delivered topically or systemically in both rat and human models. It therefore
follows that CPP could potentially be an effective and safe way of preventing root
resorption.
47
7. Research Tools and Methodologies
7.1 Subject
Rats have proved to be an appropriate subject for tooth movement and root resorption
experiments on numerous occasions.
57% of animal studies on orthodontic tooth
movement used rats during the years of 1981 to 2002.207 Furthermore rats have been
used as experimental subjects when studying the effectiveness of CPP in both
anticariogenic experiments and nutritional experiments.
7.1.2 Appliance Design
Wistar rats have been commonly used as the subjects in animal experiments involving
tooth movement.164,169,170,207 The majority of experiments have moved maxillary molars
mesially.164,169,207 Appendix 3 It is appropriate to move the molars mesially because the
buccal bone in rats is very thin and also more compact than the bone on the mesial side
which means that bucco-lingual movement is contraindicated.207
In order to study tooth movement in rats it is important to design a reliable and effective
appliance. A review of the literature by Ren et al207 revealed that a quarter of all studies
between 1981 and 2002 used elastics to produce orthodontic tooth movement. The use of
elastics has the significant disadvantage of force decay. Nickel Titanium coils offer a
force which is more continuous in nature and are thus more appropriate for use in an
experimental tooth movement appliance with the view of analyzing root resorption.
48
Ren et al207 designed a tooth movement appliance based on an extensive review of the
literature. Their appliance used sentalloy closed coil springs which were tied from the
maxillary incisors to all three maxillary molars. The study was a split-mouth design with
the experimental side randomly chosen and the contralateral side used as the control.
Other studies have used similar designs but have only tied the coils from the incisors to
the first molar.164,170
7.1.3 Measurement of tooth movement
There has been some difficulty in measuring tooth movement in rats due to continued
eruption of the incisors and possible distal tipping of the incisors used as anchorage. In
the experimental design proposed by Ren et al207 tooth movement was determined by
measuring the incisor to molar distance before and after appliance activation. Their
measurements were taken between the most mesial point of the maxillary molar unit and
the enamel-cementum border of the ipsilateral maxillary incisor at the gingival level. XRay photographs of the appliances used by Ren et al207 demonstrated that continuous
eruption of the upper incisors was effectively prevented and that abrasion of the
maxillary incisors was compensated by overeruption of the mandibular incisors.
Intraoral measurement of incisor to molar distances in rats under anaesthetic is difficult.
In an experiment which only applies force to one molar, an alternative method for
measuring tooth movement is to measure the distance at the level of the contact point
between the tooth to which the force has been applied and the adjacent molar. This
49
method assumes that the teeth were in contact before the appliances were inserted and
that physiologic movement of the adjacent tooth was negligible. The advantage of this
method is that it is not necessary to take intraoral measurements at the time of appliance
insertion. King and Fischlschweiger208 measured this distance using calibrated thickness
gauges between the maxillary first and second molars at sacrifice. This gap can also be
measured very accurately using digital measurement tools on micro CT images.
7.2 Analysis of root-resorption
Root resorption has been studied and measured by a variety of methods which include
histology, light microscopy, electron microscopy and radiography.
7.2.1 Histology
While clinical studies and analyses of plain radiography reveal a varied incidence in root
resorption, histological studies were able to demonstrate that root resorption of varying
degrees is a common side effect of orthodontic tooth movement.83
7.2.2 Light Microscopy
Light microscopy allows another view of resorption craters.97 Appendix 4
Light
microscopy has also been used to quantitatively analyse root resorption craters. OwmanMoll and Kurol147 used light microscopy to register root resorption and repair on one
randomly chosen histological section from each of three different sectional levels on a
50
tooth. Surface extension and depth of each resorption lacuna was measured to the nearest
unit. This technique has been criticized because it was felt that this technique could not
account for the variation in size, shape and depth of the craters and variation in root
morphology and consequently accurate quantitative measurements of these craters could
not be achieved.209
Brudvik and Rygh153 used light microscopy to study the initial attack and the resorptionrepair sequence on root surfaces exposed to compression. They used light microscopy to
measure the total length of the resorption lacunae in a coronal-apical direction.
Igarahi et al210 have analysed root resorption craters by feeding microscopic images to a
television monitor with a CCD video camera.
The data is expressed as square
micrometers
While light microscopy provides a useful tool for analysing craters, the two dimensional
images do not allow an accurate quantitative analysis of root resorption.
7.2.3 Transmission Electron Microscopy
While it is possible to analyse root resorption craters using TEM sections it is difficult to
get a quantitative analysis using this tool because the shape, size and location of the
craters means that it is likely that some may be lost during sectioning. While TEM
images provide useful 2D images they are not designed to easily produce 3D
reconstruction of specimens.
51
7.2.4 Scanning Electron Microscopy
SEM provides an overall image of resorption craters on the root surface with minimal
tissue preparation. SEM images have been used to assess root resorption craters in 2
dimensions. King and Fischlshweiger208 used point counting volumetry to quantitatively
assess root resorption. In this technique micrographs were placed under a regular grid
lattice, and intersections of the grid coincident with a resorption lacuna were scored.
Other studies by Barber and Sims148 used a Zeiss MOP digitizer to calculate the
resorption-affected areas in buccal profile from photographs of areas of resorption. There
is a risk of parallax measurement errors in 3D when using this technique due to the
curved nature of root resorption craters.209
Furthermore, because the composite
micrographs are pieced together, there is a risk of error if the craters are along the edges
of the micrograph.209
The problems of parallax error with 3D volumetric measurements using SEM images can
be resolved by recording stereo images of the craters and converting this into an 8-bit
grayscale depth map.211
This method has been shown to be highly accurate and
reproducible.209 However this method is time consuming and difficult to perform for
large samples.
52
7.2.5 Radiography
Wilhelm Conrad Roentgen was the first to study the x-ray phenomenon which occurs
when an electric current passes through a gas of extremely low temperature.212 The
science and technology of x-rays has since progressed to allow the visualisation of both
living and inanimate objects from a variety of perspectives.
7.2.5.1 Plain Radiographs
Plain radiographs such as periapical radiographs, dental panoramic films and lateral
cephalograms are the most popular views in dentistry. However these images have the
inherent problems associated with 2D representations of 3D objects. The magnification
and parallax errors associated with plain radiographs render these images of use only for
qualitative descriptions of root resorption.
There have been attempts to reproduce three dimensional structures from a two
dimensional film.
tomography.213
These techniques are known as laminography or focal plane
It involves translating an object/subject together with the detecting
medium in such a way that only one narrow slice parallel to the translation plane is in
focus. Sharp images are difficult to obtain because of the thickness of each slice and the
smearing of images of the plane outside the imaging plane across the image of the plane
of interest.
53
7.2.5.2 Computer Tomography
With the advent of digital computers, an approach called computer tomography became
possible. Early in the 1970s Hounsfield214 developed a commercial system for medical
imaging. It can be difficult keeping the dose of x-rays to a minimum and the duration
must be limited as involuntary movement by the subject will cause distortions in the
image. However these considerations need not apply to imaging of inanimate objects.
Three dimensional images are achieved by taking a series of x-ray projections through
the slice at various angles around an axis perpendicular to the slice. From this set of
projections, the x-ray absorption map is computed, and by taking a number of slices, a
three dimensional map is produced.
The resolution of a two dimensional image is given in terms of pixels which are a two
dimensional representation of the smallest unit of colour value within an image. This
colour value can be a shade of grey or a colour. Imaging software assigns the dimensions
of a pixel (x and y axis). The number of pixels per surface area gives the resolution of
the image. The quality of an image improves when there are more pixels per unit area
which is described as a higher resolution. Pixels are no longer valid when considering
three dimensional images. The term voxel is used in three dimensional imaging. Voxels
include the dimension of depth in order to represent a volumetric value in space.
54
7.2.5.3 Micro Computer tomography
Micro computer tomography has been developed to enable higher resolutions of smaller
samples. Current technology limits the use of micro computer tomography to small,
inanimate specimens, such as bone and extracted teeth samples.215 Micro computer
tomography can be taken for investigations without damaging or removing any material.
This system is therefore useful for viewing biological structural materials such as
calcified tissues.
The SkyScan 1172 is a micro CT scanner which has been used extensively in medical
research. It is a compact desktop system for microscopy and micro tomography. It
consists of an x-ray shadow microscopic system and a computer with tomographic
reconstruction software.
This scanner is a cone beam x-ray source with a spatial
resolution of between 2 and 5 micrometres. The recommended sample size is a diameter
of 1.5 cm and a height of 3 cm. The sample is placed on a rotating platform, and
depending on its proximity to the x-ray beam, there is a magnification factor. A higher
magnification gives greater detail. A high resolution Charged Coupled Device with a
resolution of 1024 x 1024 pixels will detect the incoming x-rays. The information
collected by the SkyScan x-ray micro tomograph is stored and reconstructed to 8 Bit
Mapped Picture files.
The SkyScan micro tomograph uses a non destructive procedure to create a three
dimensional reconstruction of the objects’ inner structure from two dimensional x-ray
shadow projections. The software package, VGStudioMax v1.2 is used to collate all the
55
axial slices to form a three dimensional reconstruction of the scanned image. This
software package also has the function to remove the bony tissue around the scanned
tooth, which facilitates the viewing of any defects on the cemental surface. This has
advantages for the analysis of rat molars because it eliminates the need for their
extraction. There is a significant risk of root fracture and damage to tooth roots when
extracting rat molars.
If not conducted properly a quantitative evaluation of root resorption will have an
impounding effect on the correlations between the magnitude of force duration and extent
of tissue destruction.209 Three dimensional micro CT imaging facilitates the visualisation
of any defects and root resorption craters on the surface of the cementum, indicating a
resorptive defect. Craters and defects can then be measured and an accurate volumetric
calculation of the defect can then be undertaken. Appendix 5
56
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164. Brudvik P, Rygh P. Root resorption after local injection of prostaglandin E2 during
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75
9. Manuscript
76
77
78
9.1 Abstract
79
80
9.2 Introduction
81
82
83
84
85
86
87
9.3 Materials and Methods
88
89
90
91
92
93
94
9.4 Results
95
96
97
9.5 Discussion
98
99
100
101
102
103
104
9.6 Conclusions
9.7 Acknowledgement
105
9.8 References
106
107
108
109
110
111
112
113
9.9 List of Tables
114
115
116
9.10 List of Figures
117
118
119
120
121
122
123
124
125
126
127
128
10. Future Directions
The results of the present study indicated that CPPs seem to have a variable effect on the
volumetric quantification of root resorption. While on average, the amount of resorption
observed in rats fed dietary CPPs was less, individual variability makes this effect
statistically insignificant.
The preventive effects of systemic CPP on orthodontically induced inflammatory root
resorption has not been previously investigated. While the amount of dietary CPPs fed to
the rats was based on previous research192, it is possible that varying levels of CPP may
have varying effects on the amount of root resorption following the application of
orthodontic force.
Furthermore, it is possible that varying the time length of
administering dietary CPPs before appliance placement, may have an effect on the
amount of root resorption.
CPPs has been shown to be effective in delivering bioavailable calcium.180,194 It may
therefore be of interest to confirm this relationship by varying the dietary intake of
calcium with dietary CPP and studying the effect that this has on OIIRR.
Although it is possible that CPPs may have a beneficial effect on reducing cementum
solubility it may be counteracted by its anabolic effect on bone mass which explains
some of the variability observed in this study. The association between the amount of
tooth movement, the amount of root resorption and the mineralisation of the alveolar
bone therefore warrants further investigation. This may be achieved by measuring the
129
percent bone ash.63 Another way of investigating the relationship would be to study root
resorption in aged, ovariectomised rats.
Additionally, after two weeks of tooth movement, inactivation of the Niti coil will start
the healing process of resorption craters. It would be of interest to investigate the
potential for CPP to influence the repair of the resorption craters.
The individual variability observed in the present study also warrants further
investigation. It would be of interest to look at the unloaded, contralateral teeth, to see if
rats which had large amounts of root resorption on orthodontically loaded teeth also had
large amounts of physiologic root resorption on the unloaded teeth. It would also be of
interest to determine whether or not rats which showed reduced root resorption also
showed reduced tooth movement.
This would give further insight into individual
susceptibility to root resorption.
130
11. Appendix
Appendix 1
CPP – ACP complex
Professor Eric Reynolds, University of Melbourne.
131
Appendix 2
CPP – ACP in Plaque
Professor Eric Reynolds, University of Melbourne.
CPP-ACP
In Plaque
132
Appendix 3
Schematic drawing of tooth movement appliance as designed by Ren et al207
Ren Y, Maltha JC, Kuipers-Jagtman AM. The rat as a model for orthodontic tooth
movement - a critical review and a proposed solution. European Journal of Orthodontics
2004;26:483-490.
133
Appendix 4
Dissecting Light Microscope view of the mesial root of the maxillary first molar of a
wistar rat. This photograph is of the junction between the porous apex and the cervical
third of the root.
Dissecting Light Microscope view of the mesial root of the maxillary first molar of a
wistar rat. This photograph is of the porous apex of the root.
134
Appendix 5
Raw Data
Rat no.
LND021
LND022
LND024
LND025
LND029
LND039
LND040
LND041
LND043
LND044
LND045
LND046
LND047
LND069
LND071
LND072
LND073
LND074
LND075
LND076
LND077
LND078
LND091
LND092
LND093
LND094
LND095
LND096
LND098
LND099
LND100
Group
Code
32
32
32
32
32
22
22
22
22
22
22
22
22
32
32
32
32
32
32
32
32
32
22
22
22
22
22
22
22
22
22
Start
Weight
204.1
194.1
196.4
195.6
199.5
187.6
191.5
195.5
201.5
182.0
185.7
178.4
183.6
211
225.8
233.7
213.5
228.1
221.2
221.1
218.6
219.7
216.6
219.6
219.3
208.3
222.1
210.7
225.8
225.5
212.6
Finsih
Weight
253.2
240.9
229.4
237.7
259.5
239.3
238.5
246.1
243.1
237.3
219.6
232.4
243.8
241.7
257.6
258.7
248.1
266
251.8
231.5
248
255.4
256.6
254.4
264.6
240.3
279.0
241.6
197.1
253.5
243.3
Weight
gain
49.1
46.8
33
42.1
60
51.7
47
50.6
41.6
55.3
33.9
54
60.2
30.7
31.8
25
34.6
37.9
30.6
10.4
29.4
35.7
40
34.8
45.3
32
56.9
30.9
-28.7
28
30.7
Volume
7264
11148
9070
17459
5031
12511
4808
716
13961
15217
4200
4223
29832
4247
4279
10405
8916
11022
19850
8462
2658
3001
18837
18655
21637
3197
4986
9596
24502
9728
1827
Volume2
11126
4696
661
14714
14942
3646
3539
27833
21060
16962
TMSg
54.64
43.75
36.7
0
36.94
37.62
16.7
32.5
37.68
10.69
42.86
65.23
56.22
37.3
34.73
15.59
22.36
17.94
22.49
26.37
9.54
16.03
21.24
64.91
23.4
24.36
69.82
17.58
10.39
15.5
24.13
TMAx
41.95
40.25
22.03
0
35.58
34.29
16.35
31.57
37.68
10.74
37.29
62.27
60.5
40.83
31.98
16.9
24.33
14.74
22.5
20.55
9.91
16.19
21.3
53.96
18.22
24.3
56.99
17.21
9.92
15.45
18.41
TMSg2
TMAx2
37.64
16.86
32.25
36.62
8.95
41.46
64.72
56.28
34.31
16.62
31.45
37.38
9.27
37.2
65.67
60.3
21.39
64.39
21.41
52.73
135