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INSIGHT INTO THE ROLE OF PERIODONTAL LIGAMENT ASSOCIATED
PROTEIN-1/ASPORIN IN THE MAINTENANCE OF THE PERIODONTAL LIGAMENT USING A RAT ANKYLOSIS
MODEL
Doctor of Clinical Dentistry (Orthodontics) Thesis
Wayne Chen
Orthodontic Unit
School of Dentistry Faculty of Health Science The University of Adelaide
South Australia AUSTRALIA
2012
1
Table of Contents Table of Contents 1
Figures and Tables 3
Glossary of Abbreviated Terms 5
Statement 7
Acknowledgements 8
Summary 9
Section 1 11
Literature review 12
Comparative Dental Anatomy 12
The Periodontium 16
Cementum 16
Bone 17
Bone metabolism 18
Cellular constituents 26
Periodontal ligament 27
Maintenance of the periodontal ligament 29
PLAP-1 32
Pulp 34
Ankylosis 35
Aetiology of ankylosis 37
Diagnosis of ankylosis 39
Management of ankylosis 41
Experimentally induced ankylosis 44
Immunohistochemistry 47
References 49
Section 2 60
Statement of Purpose 61
Article 1 63
Abstract 63
Introduction 64
2
Aims 66
Materials & Methods 66
Results 68
Discussion 75
Conclusion 78
Acknowledgements. 78
References 79
Article 2 82
Abstract 82
Introduction 83
Aims 85
Materials & Methods 85
Results 86
Discussion 90
Conclusion 92
Acknowledgements 92
References 93
Concluding remarks 94
Appendices 95
3
Figures & Tables
Literature Review
Figure 1: Classification of tooth attachment 12
Figure 2: Dental structure of a rodent 16
Figure 3: Arrangement of periodontal fibre groups 28
Figure 4: Chemical structure of PLAP-1/asporin 33
Figure 5: 3D structure of PLAP-1/asporin 33
Figure 6: Bitewing radiographs of ankylosis 36
Figure 7: Combination between a pair of ankylosed teeth 38
Figure 8: Periotest 39
Figure 9: Periapical radiograph of an ankylosed 21 40
Paper 1
Figure 1: Grid system used to standardise the region of analysis between sections 68
Figure 2a: Rat articular cartilage from femur used as positive control 69
Figure 2b: Negative control of the rat articular cartilage 69
Figure 2c: The experimental tissue stained with PLAP-1 69
Figure 2d: The experimental tissue used for negative control 69
Figure 3a: PLAP-1 staining of chondrocytes 70
Figure 3b: PLAP-1 staining of blood vessels 70
Figure 3c: PLAP-1 staining of periodontal ligament 70
Figure 3d: PLAP-1 staining of periodontal ligament regions 70
Figure 3e: PLAP-1 staining of gingival epithelial tissues 70
Figure 4a: Experimental section with ankylosis 71
Figure 4b: Experimental section without ankylosis 71
Figure 5a: PLAP-1 staining at cementum third of PDL of experimental side 72
Figure 5b: PLAP-1 staining at cementum third of PDL of control side 72
Figure 6a: PLAP-1 staining near root apical region 72
Figure 6b: PLAP-1 staining near cementum third of PDL with ankylosis 72
4
Table 1: Statistical data between control and traumatised sides with ankylosis at various intensities 73
Table 2: Comparison in PLAP-1 intensity between control and traumatised sides with no ankylosis 74
Paper 2
Figure 1a: PLAP-1 staining of the pulp chamber on the control side 87
Figure 1b: PLAP-1 staining of the pulp chamber on the control side (20x magnification) 87
Figure 1c: Negative control of pulp staining displaying the lack of PLAP-1 staining (10 x magnification) 87
Figure 1d: Negative control of pulp staining displaying the lack of PLAP-1 staining (20 x magnification) 87
Figure 2a: PLAP-1 staining of pulp on control side 88
Figure 2b: PLAP-1 staining of pulp on experimental side without ankylosis 88
Table 1: Comparison of PLAP-1 staining intensities within the pulp adjacent to the dentine in sections with ankylosis 89
Table 2: Comparison of PLAP-1 staining intensities within the central pulpal section in sections with ankylosis 89
Figure 3a: PLAP-1 staining of central pulpal region in experimental side (10x magnification) 89
Figure 3b: PLAP-1 staining of central pulpal region in experimental side (20 x magnification) 89
Figure 4: Tertiary dentine and cellular inclusions on experimental side 90
5
Glossary of Abbreviated Items General
ABC avidin-biotin complex
BMP bone morphogenetic protein
B-SA biotin-streptavidin
Cbfa-1 Core binding factor a1
EDTA ethylenediaminetetraacetic acid
EGF epithelial growth factor
FGF fibroblast growth factor
HEBP 1-hydroxyethylidene-1, 1-bisphosphonate
IGF insulin like growth factor
IL interleukin
IMVS Institute of Medical & Veterinary Science
KV kilovolts
LRR leucine rich repeats
LTB4 leukotriene B4
mRNA messenger ribonucleic acid
PAP peroxidase anti-peroxidase
PBS phosphate buffered saline
PDGF platelet derived growth factor
PDL periodontal ligament
PG prostaglandin
PLAP-1 periodontal ligament associated protein-1
PTH parathyroid hormone
PTHrP parathyroid hormone related protein
Runx2 Runt-related transcription factor-2
TGF transforming growth factor
TNF tumour necrosis factor
TNFR tumour necrosis factor receptor
6
Measure of Length
mm millimetre
µm micrometre
Measure of Volume
ml millilitre
Measure of Weight
mg milligram
g gram
kg kilogram
mw molecular weight
7
SIGNED STATEMENT
8
ACKNOWLEDGEMENTS I wish to express my sincere appreciation to the following people for their support in
the completion of this thesis:
Professor W. J. Sampson, P.R. Begg Chair in Orthodontics, The University of
Adelaide, for his readily available expert advice and guidance. His dedication and
enthusiasm for dental research is truly an inspiration.
Dr C. W. Dreyer, Associate Professor in Orthodontics, The University of Adelaide,
for his readily available expert opinion and support.
Dr Kencana Dharmapatni, Hanson Institute, Adelaide, for her expert advice regarding
immunohistochemistry and draft preparation. Her generosity and enthusiasm was
simply astounding.
Tom Sullivan, Division of Population Oral Health, The University of Adelaide, for his
expert statistical help.
Ms Marjorie Quinne & Sandie Hughes, for their assistance with sectioning tissues.
Thankyou.
Last but not least, I must thank my beautiful wife Imelda, my little princess Bella and
my little prince Sebastian. Your tolerance, support and love are simply amazing and
it has made all the hard work worthwhile.
9
SUMMARY The cells of the periodontal ligament have been shown to be osteogenic but under
normal conditions, the PDL space remains patent without the occurrence of ankylosis.
Periodontal Ligament Associated Protein-1 (PLAP-1)/Asporin is a recently
discovered protein that has been suggested to play a significant role in suppressing the
osteogenic tendency of the periodontal ligament and maintaining the fibrous
ligamentous nature of the periodontal ligament. Furthermore, PLAP-1/Asporin has
also been shown to be associated with the differentiation and mineralisation of dental
pulp stem cells.
In this study, the expression of PLAP-1 was investigated using a reversible ankylosis
model induced by hypothermal insult. In paper 1, the principal aim was to determine
the normal distribution of PLAP-1 reactivity in a normal rat maxilla and to analyse the
pattern of PLAP-1 reactivity in association with the formation of ankylosis. In
addition, another study (paper 2) was performed with the aim of investigating the
distribution pattern of PLAP-1 within a normal rat molar pulp as well as its changes
following freezing trauma.
The results from the first paper showed that PLAP-1 was expressed in the PDL, dental
pulp, blood vessel walls and the nasal cartilage. Not all sections obtained ankylosis.
Sections which did not obtain ankylosis demonstrated no significant PLAP-1
expression differences between control and experimental sides. Sections that did
obtain ankylosis yielded a tendency towards increased PLAP-1 reactivity especially
near the cementum. However, it was difficult to deduce whether the relationship of
PLAP-1 to the ankylotic union was associated with bone formation or resorptive
activities.
The results from paper two showed that PLAP-1/Asporin was expressed exclusively
within the pulp under normal conditions and appeared to be associated with the
odontoblastic and cell rich zone. Following trauma, PLAP-1/Asporin expression
10
decreased marginally (not statistically significant) alongside the dentine but increased
significantly in the central pulpal region along with disruption and breakdown of the
cellular structures.
From the results derived, it can be concluded that PLAP-1/Asporin is indeed
expressed in several tissue/cell types and regions including the dental pulp and is not
exclusively associated with the periodontal ligament. In addition, PLAP-1 appears to
have a direct association with ankylosis although it is uncertain whether PLAP-1 aids
in bone mineralisation or resorption. The second null hypothesis was also rejected
although the change in expression of PLAP-1 within the pulp is more morphological
than physiological. Results from the study also suggest that PLAP-1/Asporin does
not appear to play a direct role in the formation of the tertiary dentine.
Further research is required to elucidate the true role of PLAP-1 within the
periodontal ligament as well as the pulp. Additional investigations are also required
to gain further insight into the maintenance of the periodontal ligament.
11
SECTION 1
12
LITERATURE REVIEW
Comparative Dental Anatomy (tooth attachments) In the animal kingdom, there are many different types of periodontal attachment of
teeth. They have been classified according to the area of attachment (eg. crestal,
marginal or socketed) and the mode of attachment (i.e. ankylosis, fibrous or
combined).
Classification of tooth attachment
The classification of tooth attachment has undergone some debate (for full review, see
Gaengler & Metzler1). However, for the purpose of this literature review, only the
classical classification of acrodonty, pleurodonty and thecodonty shall be used (Figure
1).
Figure 1
Classification of tooth attachment
a) Pleurodont = when the tooth is joined to the inner margin of the jaw bone
b) Acrodont = when the tooth is attached at the crest of the jaw bone
c) Thecodont = where the root of the tooth lies in a socket within the jaw bone
(from Peyer2)
Acrodonty is the term used when the attachment from teeth to bone is at the crest of
the jaws.
A NOTE:
This figure/table/image has been removed to comply with copyright regulations. It is included in the print copy of the thesis held by the University of Adelaide Library.
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In pleurodonty, the tooth is joined to the inner margin of the jaw bone. The teeth can
either be homodont (all teeth similar in shape), haplodont (molar crowns without
ridges or tubercles), or polyphyodont (having many sets of teeth throughout life).
The third main class of thecodonty is where the root of the tooth lies in a socket
within the jaw proper. All placental mammalian teeth, including humans, are
socketed. In thecodonty, a fibrous ligament is the preferred mode of attachment.
Modes of Attachment Despite the various positional relationship of tooth to bone, a common feature is that
the linkages from teeth to jaw are all collagenous with the difference being the degree
of mineralisation. There are three main types of attachment:
1) Ankylosis
2) Fibrous attachment
3) Socketed attachment
In ankylosis, there is a direct mineralised union between the tooth and the supporting
jaw. This form of attachment is found in many bony fishes and nearly all living
reptiles. Ankylosis can also be subclassified to acrodont or pleurodont ankylosis.
There is also protothecodont ankylosis where the tooth is fused to the jaw at the base
of a groove formed by the labial and lingual flange. In naturally occurring ankylosis,
the tooth is not directly fused to the jaw bone but rather via a more spongy structure
which is then connected to the jaw proper. Tomes3 in 1904 named the structure the
‘bone of attachment’.
In animals with fibrous attachment of their teeth, the collagen is only partially
mineralised hence giving some degree of movement to each individual tooth. Fibrous
attachment can be either direct or indirect. In direct attachment, the collagen fibres
run directly from the base of tooth to the jaw bone. In indirect attachment, fibres from
the tooth form a mineralised structure called the pedicel, which is in turn ankylosed to
the jaw.
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All thecodont dentitions have a fibrous periodontal ligament as their support
mechanism. Amongst living creatures, this type of attachment is only found in
mammals and crocodiles4. There are several distinctive features in mammals
including developing multiple roots in molars, constricted root apices and the
formation de novo of sockets for succedaneous teeth5.
Dentition of Fishes In fishes, there is an almost infinite diversity of dentition and their supporting
apparatus. In early fishes, there were no true teeth but rather dermal denticles that
were part of their body armour. Many of the teeth were attached via ankylosis.
However, through evolution, fibrous attachment is now commonly found (e.g.
elasmobranches such as sharks). All sharks and rays have a specialised form of
fibrous attachment known as the hinge mechanism. Despite fibrous attachment being
evolutionarily advanced compared to ankylosis, many contemporary fishes still have
ankylosis as their primary mode of attachment of teeth (e.g. mackerel)3.
Dentition of Amphibians For amphibians, a common arrangement is a double row of teeth arranged in
concentric lines in the maxilla, between which a single row of teeth upon the lower
jaw passes when the mouth is closed. The outer of the two rows of teeth in the upper
jaw is situated on the premaxilla and maxilla and extends backwards3.
There are, of course, variations to this generalisation. For example, the frog has no
teeth in the lower jaw. When the edentulous mandible is in the closed position, it
passes to the lingual side of the maxillary teeth and fits snugly against the palatal side
of the teeth – especially as frogs have no lips and the teeth have rounded surfaces3.
Dentition of Reptiles In the reptiles, the dominant form of tooth attachment is ankylosis and the relationship
of teeth to jaw can either be acrodont, pleurodont or protothecodont5.
15
One notable exception is the order crocodilian. The teeth are not attached to the jaw
via ankylosis and it is the only example of thecodonty in living reptiles5. However, it
does differ from mammalian gomphosis in a few key areas – firstly, the sockets are
persistent and secondly, the roots are cylindrical with wide apices. There are also
microscopic differences which are beyond the scope of this project.
Dentition of Mammals All mammals have tapered roots with constricted apices as all or part of their dentition.
The posterior teeth are also multi-rooted. These are unique mammalian features. The
periodontal attachment has also undergone significant evolution. In particular, the
mammalian gomphosis allows movement of teeth relative to the alveolar base without
accompanying weakening of the support provided. The ability of the periodontal
ligament and alveolar bone to remodel without weakening tooth support helps
maintain occlusion and allows for the limited tooth replacement. In addition,
compared to other animals, mammals have specialised epithelial attachment around
the tooth (i.e. junctional epithelium) that provides a better resistance to bacterial insult.
Overall, mammal teeth have four classes: incisors, canines, premolars and molars.
Most mammals are diphyodont with teeth restricted to 2 rows – one in the maxilla and
one in the mandible2.
Rodents are commonly used in animal studies. They are characterised as
simplicidentales because they have only one pair of incisors2. These large incisors are
designed for gnawing and are fascinating due to their ability of continual growth
throughout life. Generally, enamel is only evident on the labial side but occasionally
it is also present on the mesial and distal side. Usually the canine and all premolars
are missing with the exception of the upper 3rd premolar which can be present in some
cases. There are usually three molars in each quadrant2. Figure 2 presents a lateral
view of the general dental structures of a rodent.
16
Figure 2
Dental structure of a rodent showing one incisor and three molars in each jaw.
(from Hillson6)
The Periodontium The Periodontium can be defined as those tissues that surround and support the teeth.
They include cementum, periodontal ligament, alveolar bone and that part of the
gingiva that faces the tooth7.
Cementum Cementum is a mineralised, avascular connective tissue that covers the surface of the
root. Cementum is approximately 45-50% hydroxyapatite and 50% collagen and non-
collagenous matrix proteins. Type I collagen comprises 90% of the organic
component in cementum. Other constituents include type III collagen, type XII
collagen and possibly type V and XIV collagen. Non-collagenous proteins include:
alkaline phosphatase, bone sialoprotein, fibronectin, osteocalcin, osteonectin,
osteopontin, proteoglycans, proteolipids, vitronectin and several growth factors8.
Cementum can be subdivided into four types: acellular, cellular, mixed and acellular
afibrillar. Acellular cementum is the most extensively encountered type. It is formed
A NOTE:
This figure/table/image has been removed to comply with copyright regulations. It is included in the print copy of the thesis held by the University of Adelaide Library.
17
immediately as root formation commences (under influence of the Hertwig’s
epithelial root sheath) and typically covers the cervical two-thirds of the root dentine.
Once the tooth is in occlusion, the rate of deposition of cementum quickens leading to
some cementoblasts being trapped in the mineralising tissue7. This is termed cellular
cementum. Mixed cementum is composed of alternating layers of acellular and
cellular cementum and is most commonly found in the apical portion and furcation
areas. Less is known about the acellular afibrillar cementum which lacks collagen
and appears to play no role in supporting the teeth. This cementum is found near the
cemento-enamel junction8.
Alveolar Bone The alveolar bone forms the sockets in which the teeth sit and constitutes part of the
periodontium. The bone that lines the alveolus to which Sharpey’s fibres insert is
termed the bundle bone. Bone is similar to cementum in that it is a mineralised
connective tissue. However, its mineral content is much higher. Bone comprises
roughly 28% type I collagen, 5% noncollagenous matrix proteins and 67%
hydroxyapatite8.
In an adult, alveolar bone has a dense outer layer termed the compact bone with a
central medullary cavity. The bone marrow, which sits in the medullary cavity, is
interspersed by a network of bone trabeculae – the spongy bone9. Bone can be
formed in three ways: endochondral ossification, intramembranous ossification and
sutural ossification.
Endochondral Ossification
Endochondral ossification is the process by which bone formation occurs with the aid
of a cartilage precursor. It occurs in weight bearing bones as the cartilage allows bone
formation while also offering some function. Initially, the mesenchymal stem cells
migrate and condense forming the outline of the eventual bone to be formed at that
site. These mesenchymal cells differentiate into chondroblasts which produce an
extracellular matrix predominantly of type II and X collagen as well as chondroitin
sulphate proteoglycans10. A periosteal bony collar is then formed in long bones. The
chondrocytes then undergo progressive hypertrophy with mineralisation of the
intercolumnar cartilage matrix11. Blood vessels then invade the cartilage bringing
18
with them osteoblast precursors. At the same time, cartilage begins to be resorbed by
multinucleated cells. Due to the actions of these newly derived osteoblasts,
ossification centres begin to develop, both in the epiphyseal cartilage and plate. Once
bone stops growing in length, the epiphyseal plate disappears – first the lower then the
upper. The marrow cavity becomes continuous and the blood vessels throughout the
diaphysis, metaphyses and epiphyses intercommunicate10.
Intramembranous Ossification
Intramembranous ossification is the process by which bone develops directly on to a
soft connective tissue membrane without the need for a cartilage anlage. Initially, at a
pre-determined site, the mesenchyme cells (or ectomesenchyme, if in the craniofacial
region) proliferate and undergo condensation11. The local vascularity also becomes
increased and the osteoblasts differentiate to produce bone matrix de novo. Once
begun, intramembranous bone formation proceeds at an extremely fast pace. This
rapid formation does not allow for the complete remodelling of the resident
extracellular matrix, resulting in a bone matrix comprised of new bone intermingled
with old matrix. This highly cross-hatched and irregular bone is termed the coarse-
fibred woven bone. The various centres of ossification begin from being tiny bony
spicules proceeding to thin bony plates and ultimately fuse with each other to form a
single bone. Over time, the immature bone undergoes a slow transition that will
eventually turn into mature lamellar bone8. Alveolar bone is formed through
intramembranous ossification.
Sutural Bone Growth
Sutural bone growth describes the bone formation that occurs between sutures. In the
beginning of this process, the outer layer splits thus exposing the two layers of
periosteum (the outer and fibrous layer and the osteogenic layer). The fibrous layer
joins with its opposing corresponding layer while the osteogenic layer runs down
through the suture along with its opposing counterpart8. The combined osteogenic
layer of the suture is termed cambium. When the brain grows, successive waves of
new bone cells differentiate from cambium allowing for the deposition of new bone
matrix8. The two cambial layers are separated so that independent growth can occur.
Bone Metabolism
19
Although outwardly inert, bone is a dynamic tissue involved in high metabolic
activities. Bone is constantly under repair with 20% of the cancellous bone surface
undergoing remodelling at any one time12. In a growing child, the end result of bone
metabolism is the growth of the skeleton (bone formation exceeds bone resorption)
whereas in the elderly, bone resorption often exceeds bone formation. In a healthy
adult, however, there exists a balance between bone resorption and bone formation.
The main purpose of remodelling is to help repair microdamages in the bone matrix
and prevent the accumulation of old bones as well as act as a reservoir for minerals
which aid in mineral homeostasis13.
Bone metabolism is a complex process which is tightly regulated. There are many
biochemical compounds involved and they can be grouped into either systemic
regulators or local regulators.
Systemic Regulators Parathyroid Hormone
Parathyroid hormone (PTH) is secreted by the parathyroid gland and is vital in the
regulation of calcium homeostasis. PTH has direct actions on both bone and kidney
while it effects its actions on the intestinal tract indirectly. The actions of PTH are
mediated via a G-protein coupled receptor system in the cells of target tissues14. PTH
is known to have both anabolic and catabolic actions on the skeleton.
PTH has been shown to act directly on osteoblasts via the PTH receptor (PTH and
PTHrP interaction) with stromal cells requiring physical contact with haematopoietic
precursors in order to effect PTH’s catabolic activity15. This interaction results in the
subsequent release of factors (which shall be discussed below) that stimulate
osteoclast activity/function and numbers. PTH also enhances collagenase synthesis,
decreases type I collagen synthesis and decreases alkaline phosphatase in osteoblasts14.
However, the precise mechanism by which PTH stimulates the osteoclast in order to
activate bone resorption remains unclear.
PTH also has anabolic actions which are mediated directly through osteoblasts
themselves. They are able to increase osteoblast numbers possibly through the
20
regulation of cell attachment via the regulation of E-cadherins16. PTH also stimulates
the differentiation of osteoblasts as well as being shown to be mitogenic for bone cells
in vivo17.
Overall, PTH is recognised more for its catabolic actions and its role in calcium
homeostasis as not only does it promote resorption but it also increases the kidney
reabsorption of calcium and stimulates the changes of vitamin D to its active form.
Parathyroid hormone-related peptide
Parathyroid hormone-related peptide (PTHrP) is a known regulator for osteogenic cell
differentiation and/or function. Similar to PTH, PTHrP also regulates both bone
formation and bone resorption. Their effects are effected via binding to cells of
osteoblast phenotype14.
Vitamin D
Vitamin D in its active form (1α,25 dihydroxyvitamin D3) plays a major role in bone
metabolism and mineral homeostasis. Vitamin D has been shown to affect both the
osteoblasts as well as the osteoclasts.
Vitamin D has a biphasic effect on osteoblasts in that it either stimulates or inhibits
the normal developmental pathway or gene expression profiles depending on its
presence during either the differentiation or proliferation stage18. Vitamin D at the
proliferation stage inhibits osteoblast differentiation with decreased proliferation,
decreased collagen synthesis and alkaline phosphatase activity. If the osteoclasts are
already differentiated, then Vitamin D up-regulates osteoblast associated genes such
as osteopontin or osteocalcin hence increasing mineralisation13,18. Therefore, vitamin
D exerts a dual effect on bone remodelling. Furthermore, Vitamin D can affect the
activity of local factors involved in bone metabolism but the results vary depending
on the stage of cell differentiation or experimental design18.
Calcitonin
Calcitonin is a hormone that can oppose the catabolic effects of parathyroid hormone.
It leads to the loss of ruffled border of the osteoclast as well as minimising the
secretion of proteolytic enzymes thereby reducing bone resorption. Furthermore, it
21
increases the loss of calcium from the kidney leading to a drop in the serum calcium
level13.
Glucocorticoids
Glucocorticoids can exert both stimulatory and inhibitory effects on bone cells. These
hormones promote the differentiation of osteoblasts from mesenchymal stem cells
although they also decrease osteoblastic activity13. This was backed up by Weinstein
et al.19 who showed exogenous glucocorticoid to reduce both osteoblastogenesis and
osteoclastogenesis on ex vivo bone marrow cultures. This leads to reduced bone
turnover and bone formation with the clinical manifestation of decreased bone density
and cancellous bone area.
Thyroid Hormone
Similar to the other hormones, the thyroid hormones can also have both catabolic and
anabolic effects on the skeleton. Most of its biological effects, however, result in
increased bone turnover (through its effects on osteoblasts). For example, in
hyperthyroidism, there is pathologically increased bone resorption20.
Sex Hormones
There are 3 main sex hormones involved in bone remodelling – oestrogen,
progesterone and androgens.
Oestrogen
Oestrogen is the main female sex hormone and it has both direct and indirect
effects on bone metabolism. It has been proposed to have a direct inhibiting
effect on the osteoclast as oestrogen receptors have been found on
osteoclasts21. It also stimulates the secretion and activation of TGF-β by
osteoclasts22 and indeed osteoblasts which inhibit osteoclastic activity through
either their autocrine or paracrine actions23. Oestrogen has also been proposed
to promote apoptosis of osteoclasts; therefore, if oestrogen levels decrease,
such as in post-menopausal women, the osteoclasts do not undergo
programmed cell death and hence they live longer ultimately leading to
increased bone resorption. The indirect effects of oestrogen on bone
metabolism are due to its postulated role in the regulation of
22
osteoblast/marrow mononuclear cell production of cytokines which
themselves have a proven role in bone turnover (e.g. Il-1, Il-6, TNF-α)24. The
role oestrogen plays in terms of bone formation is uncertain. Even though
oestrogen has been shown to stimulate type I collagen synthesis in both
fibroblasts and osteoblasts, clinical results have been inconclusive23. Similarly,
oestrogen effects on the proliferation and differentiation of osteoblasts still
remains unclear. The increased levels of oestrogen at puberty are associated
with an overall increase in bone mass23.
Progesterone
Progesterone is another steroid hormone that has been implicated in bone
metabolism. Studies have shown progesterone to increase proliferation and
differentiation of human osteoblast cells and also the increase of insulin
growth factor-II production by these cells25. However, similar to oestrogen,
clinical results of progesterone trials are variable and its role in bone
metabolism is still unclear.
Androgens
Androgens can act directly on osteoclasts leading to a decrease in bone
resorption. This is clearly demonstrated when after orchiectomy, the subject
experiences increased bone resorption and rapid bone loss. Androgens are
also able to regulate the bone-resorbing factors secreted by osteoclasts. For
example, testosterone can decrease PG-E2 production in calvarial organ
cultures exposed to IL-1. The effect of androgens on osteoblast proliferation
and differentiation is still unclear23. Similar to oestrogen, androgens have a
major effect on bone metabolism during puberty particularly, in males.
In addition to their effects on bone cells, sex steroids can affect extraskeletal calcium
homeostasis by their effects on intestinal calcium absorption (increased reabsorption)
renal calcium handling (e.g. Reifenstein & Albright26 showed both oestrogen and
testosterone to decrease the urinary and fecal calcium levels (as well as phosphorous
excretions); also, their effects on parathyroid hormone levels are noted (e.g. oestrogen
may directly regulate PTH secretion).
23
Local Regulators The local factors that contribute and play a role in bone metabolism or bone
remodelling consist of growth factors, cytokines and arachidonic acid metabolites
such as prostaglandins or leukotrienes.
The main growth factors which have been implicated in bone metabolism are insulin-
like growth factor, transforming growth factor-beta family, fibroblast growth factors,
and platelet-derived growth factors.
Insulin-like Growth Factor
Insulin-like growth factor (IGF) has the ability to both induce bone resorption as well
as promote bone growth. The catabolic actions were demonstrated when IGF induced
the formation of osteoclasts in bone marrow cultures27 whereas their anabolic abilities
were proven when IGF promoted the proliferation of osteoblasts in vitro as well as the
increased synthesis of bone matrix and expression of the collagen type I gene28.
Transforming Growth Factor-Beta
Similar to IGF, transforming growth factor-beta (TGF-β) has been implicated in both
the formation and dissolution of bone. TGF has been shown to have variable effects
on bone resorption in that the effects appear to be dose and experiment dependent.
Chenu et al.29 showed TGF inhibited the formation of osteoclast-like cells in vitro by
preferentially differentiating to granulocytes rather than osteoclasts. However, if the
concentration of TGF was low (10-100pg/ml), then the formation of osteoclast-like
cells increased30. The evidence of TGF’s effect on bone formation is more persuasive
with reports of exogenous TGF (local and systemic) inducing bone formation. The
mechanism by which TGF promotes bone formation is attributed to its chemotactic
ability for osteoblasts as well as proliferation31, although the proliferation effect is
disputed by some32.
Bone Morphogenetic Proteins
The bone morphogenetic proteins (BMPs) are part of the transforming growth factor-
beta superfamily. BMPs have been shown to be essential in the bone formation
process33. They have the ability to promote the osteoblast phenotype with increased
type I collagen synthesis, increased alkaline phosphatase activity and also increased
24
osteocalcin expression. BMPs are also able to guide the differentiation path of
mesenchymal stem cells into osteoblasts34. Furthermore, BMP-2 has also been proven
to be chemotactic for osteoblasts35.
Fibroblast Growth Factors
Fibroblast growth factors have been shown to have a dose dependent effect on
collagen synthesis (high concentration decreases collagen synthesis while low
concentration increases collagen production). Both FGF-1 and FGF-2 have been
shown to stimulate bone resorption in marrow cultures31. However, contradictory
findings by Canalis et al.36 demonstrated that FGF-2 is capable of inducing the
proliferation of osteoblasts and stromal cells in vitro which was backed up by in vivo
studies37,38 which also showed increased numbers of osteoblasts as well as new bone
formation after systemic administration.
Platelet Derived Growth Factors
The platelet derived growth factors are chemotactic for osteoblasts as well as having a
positive effect on their proliferation. However, PDGF does have a time dependent
effect on bone formation. Continuous exposure to PDGF leads to decreased
mineralisation via the inhibition of osteoblast function whereas intermittent exposure
of PDGF to the osteoblast precursors appears to increase mineralisation; possibly
through the increase in osteoblast numbers albeit without inhibition of their
functions39. PDGF also has an effect on bone resorption because PDGF-AB and
PDGF-BB have been shown to promote bone resorption31.
Numerous growth factors have been added to the growing list of local regulators of
bone turnover; these include, epidermal growth factor, transforming growth factor –
alpha and vascular endothelial growth factor.
Prostaglandin/Leukotrienes
Both prostaglandins and leukotrienes have been proven to affect the balance between
bone formation and resorption. Prostaglandins can stimulate both bone resorption and
formation with most of their effects largely mediated via the protein kinase A40. The
exact mechanism is likely to be linked to prostaglandin’s ability to regulate the
replication and differentiation of precursor cells. For example, prostaglandin
25
increases osteoblast precursors as well as having a mitogenic effect on them40. In
comparison to prostaglandins, leukotrienes have minimal data in relation to the role
they play in bone metabolism. However, it has been shown that LTB4 can stimulate
bone resorption while it is postulated that other leukotrienes may have similar
catabolic effects on the skeleton but are less potent40.
Cytokines
Many cytokines are involved in the process of bone metabolism but the most
extensively studied and those with possibly the most important roles are interleukin-1,
interleukin-6 and tumour necrosis factor.
Interleukin-1 (IL-1) is produced in bone with resident macrophages being the most
likely source although osteoblasts and osteoclasts may also produce this cytokine40.
IL-1 is an extremely potent stimulator of bone resorption in vitro and also has potent
in vivo effects41. Furthermore, it has also been shown to exhibit an ability to inhibit
bone formation in vitro42.
Tumour necrosis factor- alpha and beta both promote bone resorption. They have also
been shown to inhibit the synthesis of collagen in bone43. In addition, it has been
suggested that TNF is able to modulate the effects of oestrogen on bone although this
hypothesis could not be substantiated in follow-up studies40.
Similar to interleukin-1, interleukin-6 (IL-6) stimulates bone resorption although its
effect is less extensive. The major mechanism by which IL-6 contributes to bone
resorption is via the regulation of osteoclast precursor cell differentiation. In fact, the
ability of IL-1 and TNF to stimulate osteoclast-like cell development in marrow
cultures has been suggested to be due to the production of IL-640.
Bone metabolism or even bone remodelling is a vast and complicated topic on which
volumes of textbooks have been written. The above summary is a brief account of the
major players currently identified in bone metabolism.
26
Bone and Its Cellular Constituents
Bone is a dynamic tissue which is constantly remodelled. In fact, in a growing child
the entire skeleton will be “brand new” in 12 months. The maintenance of bone is
through the functions of osteoblasts (bone forming cells), osteocytes and osteoclasts
(bone resorbing cells).
Osteoblasts Osteoblasts are the cells responsible for the generation of new bone. They are derived
from mesenchymal stem cells and can range from 15-80µm7. When active,
osteoblasts are plump and cuboidal in shape. When viewed under the light microscope,
they exhibit extensive and well-developed protein synthesis organelles. The
osteoblasts form a layer on the surface of the bone and it has been postulated that they
function in controlling the influx of ions8 although this theory is unproven.
Furthermore, whether the bone-lining cells are actually osteoblasts is debatable too10.
Osteoblasts secrete the organic matrix which then becomes mineralised. As they lay
down the matrix, some osteoblasts become trapped and these cells become known as
osteocytes.
Osteocytes
The osteocytes are mononuclear cells that occupy the osteocytic lacuna within the
bone matrix. They have many fine processes extending through fine canals
(canaliculi) to maintain contact with osteoblasts, bone-lining cells and adjacent
osteocytes. Their primary functions are to sense the conditions of the
microenvironment and maintain the architecture of bone as well as collection of
nutrients8.
Osteoclasts
Osteoclasts are multinucleated giant cells. They generally encase 10-20 and
sometimes up to 100 nuclei per osteoclast44. Hence they are also much larger than
osteocytes or osteoblasts although there are variations within species. For example,
the rodent osteoclasts are generally smaller than human osteoclasts44. Osteoclasts are
derived from mononuclear precursor cells that are of haematopoietic origin10,44-49.
Their primary function is to resorb bone and mineralised cartilage.
27
Periodontal Ligament The periodontal ligament is a specialised connective tissue that joins the cementum
covering the root of the tooth to the bundle bone of the alveolar process via Sharpey’s
fibres. The ligament width ranges from 0.15 to 0.38mm8. Broadly, there are two
main components to the periodontal ligament: cellular and non-cellular constituents.
Cellular Components of the PDL
The periodontal ligament contains numerous types of cells including:
Fibroblasts
Fibroblasts are the principal cell of the periodontal ligament region and
comprise approximately 20% of the cellular component in sheep and
up to 55% in rodents50. They are large cells with an extensive
organelle network for the synthesis of protein. They are aligned along
the fibre bundles and have extensive processes that wrap around the
bundles.
Macrophages
The periodontal ligament contains some resident macrophages to help
protect the tissues from invading antigens.
Epithelial cell rests of Malassez
These epithelial cells are the remnants of Hertwig’s epithelial root
sheath. They form clusters of epithelial cells which form a network
within the periodontal ligament and surround the root of the teeth
although more commonly in the apical region8. Whether or not the
cell rests of Malassez have a function, or are mere remnants, is still
unclear. However, some studies have implicated the cell rests as
having a pivotal role in the maintenance of the periodontal ligament
space51-53.
Undifferentiated mesenchymal cells
These small spherical cells tend to be associated with the blood vessels
within the periodontal ligament and are thought to be the precursor
cells for osteoblasts, cementoblasts and fibroblasts7.
Cementoblasts
Cementoblasts are the cells responsible for laying down the cementum
and they are phenotypically similar to osteoblasts8. Indeed, they are so
28
similar that it is debatable whether the cementoblasts are a completely
different cell type or are positional osteoblasts (for recent review, see
Bosshardt, 2005)54.
Osteoblasts and Osteoclasts
Technically speaking, both the osteoblasts and osteoclasts are located
within the periodontal ligament. However, as they are functionally
associated with bone, they will be discussed along with the mineralised
bone tissue.
Non-Cellular Component of the PDL
The non-cellular component of the periodontal ligament contains an extracellular
compartment of collagenous fibres (with some oxytalan fibres) and a noncollagenous
extracellular matrix. The extracellular collagen fibres are arranged in bundles and can
be organised into groups as shown in Figure 3. The amorphous background is largely
composed of ground substance (~70% is water) together with glycosaminoglycans,
glycoproteins and glycolipids8.
Figure 3
Arrangement of major bundles of periodontal ligament fibres
(Adapted from Tennant7)
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29
There is, of course, an extensive network of both blood vessels and nerve
fibres/endings permeating throughout the periodontal ligament.
Maintenance of the PDL
In a healthy periodontium, the periodontal ligament functions to connect the teeth to
the jaws, sustain the masticatory load, provides sensory information and also prevents
the bony union of the roots of the teeth to the alveolar bone. Numerous studies have
demonstrated that the death or removal of periodontal ligament cells leads to the loss
of the periodontal membrane eventually resulting in extensive root resorption and
widespread ankylosis50,51,55. This knowledge combined with the fact that cells of the
periodontal ligament have been shown to be osteogenic in vitro56 leads to the
conclusion that certain factors/cells within the periodontal ligament must normally
suppress the osteogenic tendency and maintain the fibrous ligamentous nature of the
periodontal ligament.
The maintenance of the periodontal membrane is still a debatable topic. The current
opinion is that a plethora of factors and cells may indeed combine to maintain the
patency of the periodontal ligament space. One cell type with such possible function
is the epithelial cell rests of Malassez. According to Spouge57 and Wesselink &
Beertsen58, the rests of Malassez were originally thought to have no physiological
function. However, Ten Cate59 demonstrated that these clusters of epithelial cells
possessed an active metabolic potential. Possibly the most influential argument was
the accidental findings from Loe and Waerhaug60 in 1961 where they were
experimenting with replantation of teeth. They noted that in replanted teeth where the
periodontal ligament re-established itself, the epithelial cell rests of Malassez were
always present. By contrast, in samples where the periodontal ligament did not
reattach, epithelial cells could not be seen. The authors hypothesised that the
epithelial remnants of Malassez could possibly play a role in the maintenance of the
periodontal ligament. In a review, Spouge53 commented that although no special
functions have yet been shown for these epithelial remnants, the mere presence of
these epithelial cells could act as ankylosis inhibitor. This is based on the concept that
epithelium seems to be incompatible with bone as nowhere throughout the body is
30
epithelium in contact with bone. Bone and cementum are, however, very similar and
show a marked tendency to fuse at times. The fact that the roots of the teeth are in
extremely close contact to the bone, and yet ankylosis remains an uncommon finding,
would suggest that something, possibly the epithelial cell rests, play a role in the
maintenance of the periodontal membrane53. Their views are supported by Lindskog
et al.52 who replanted teeth with experimental cavities on root surfaces with and
without placement of enamel organ epithelium. It was discovered that bone was not
found in cavities with epithelium as compared to the control cavities where the bone
demonstrated in-growth. A more recent study by Fujiyama et al.51 demonstrated that
when the nerve supply to the PDL is reduced, the distribution of the cell rests of
Malassez also decreases along with a corresponding decrease in the width of the
periodontal space. Dentoalveolar ankylosis was also a common finding after
denervation. The authors concluded that not only are the cell rests of Malassez likely
to be involved in the maintenance of the periodontal ligament but the sensory
innervation of the ligament may also play an indirect role. This is backed up by
results from Fong et al.61 who, in their heterotopic transplantation model,
demonstrated the likelihood of epithelial cells within the periodontal ligament to
prevent ankylosis.
Aside from the epithelial cell rests of Malassez, it was speculated by Melcher62 that,
in healthy individuals, fibroblasts within the periodontal ligament are able to block
osteogenesis within the periodontium by releasing locally acting regulators such as
cytokines and growth factors. This was later validated with an in vitro experiment
conducted by Melcher and Cheong63 and later supported by Lekic & McCulloch64. It
has since been shown that the prostaglandins (including B2, D2, E2, F2 alpha and I2)
secreted by the fibroblasts are capable of inhibiting the mineralisation of PDL in
vitro65,66. Furthermore, glycosaminoglycans have also been implicated in having a
role in PDL maintenance as Kirkham et al.67 demonstrated that the removal of
glycosaminoglycans via enzyme digestion permitted the formation of mineralised
crystals in sheep PDL. However, the exact mechanism by which fibroblasts and their
products inhibit PDL mineralisation is yet to be fully understood68.
Nevertheless, recent molecular investigations have yielded further clues and identified
several molecular factors that may have a role in PDL maintenance. One such factor
31
is the RGD-CAP. RGD-CAP is the name given to a collagen-associated protein
which contains the RGD (arginine-glycine-aspartic acid) sequence. It is also known
as βig-h3. In 2002, Ohno et al.69 showed that RGD-CAP was present in human PDL
cells. Furthermore, when PDL cells were co-cultured with known osteogenic
stimulants such as dexamethasone or 1α,25-dihydroxyvitamin D3, the expression of
RGD-CAP mRNA gradually declined. In addition, exogenous RGD-CAP was able to
suppress alkaline phosphatase activity and also inhibit bone nodule formation in vitro.
Hence, RGD-CAP was considered to have a role in the maintenance of PDL
homeostasis by regulating mineralisation69.
In a similar experiment, but testing the role of epidermal growth factor (EGF)
receptors, Li et al.70 found that as the cultured PDL cells were stimulated to mineralise,
the expression of EGF receptor also decreased. The authors concluded that epidermal
growth factor receptors have a negative regulatory function on human periodontal
ligament mineralisation. Further details of the experiment and its findings would have
been desirable to further back their conclusion but, unfortunately, detailed information
was not available in an English format.
Another possible candidate for the maintenance of periodontal ligament space is
S100A4. S100A4 is a member of the S100 calcium binding protein family71. It is
expressed by several cell types such as odontoblasts and osteoblasts and also other
regions such as liver and bone marrow72 but the expression is highest within the
periodontal ligament. Duarte et al.71 demonstrated that S100A4 is secreted by PDL
cells and is able to inhibit mineralisation in a rat osteogenic cell culture. The authors
postulated that the calcium binding property of S100A4 allows it to act as an inhibitor
of the formation of hydroxyapatite crystals although later studies suggested that the
anti-osteogenic property of S100A4 is likely to be through the suppression of
osteoblastic genes (such as genes for osteopontin, osteocalcin and transcription factors
like Runx2/Cbfa-1) in the PDL cells73. Later investigations also suggest that the role
of S100A4 in bone physiology is to act as a negative regulator of matrix
mineralization possibly by modulating the process of osteoblast differentiation73.
Recently, the homeobox protein Msx2 has also been nominated as a possible
candidate for PDL maintenance. The expression of Msx2 in periodontal ligament and
32
also tendon cells was higher than in osteoblasts with reduction of the Msx2 protein
expression positively associated with osteoblast differentiation and mineralisation in
vitro74. Conversely, increased Msx2 inhibited osteoblastic differentiation and hence
also mineralisation. It is thought that Msx2 achieves this effect via suppressing the
activity of Runx2/Osf2 with TLE1 (a human homolog of Drosophilia Groucho
protein) as a co-suppressor. As Msx2 has been shown to be downregulated in patients
with ossification of the posterior longitudinal ligament, it was suggested that Msx2
may have a role in the inhibition of mineralisation of all tendon and ligaments
including the periodontal ligament74.
Another protein suggested in having a role in PDL homeostasis is a basic helix loop
helix protein called Twist. It has been shown by Komaki et al.56 that the expression of
Twist within the PDL is intense and located along the alveolar bone surface. It was
shown that in cell cultures where osteoblast-related genes were stimulated to increase,
the expression of Twist decreases correspondingly. Conversely, knock-out of Twist
proteins leads to an increase in osteoblast-related proteins such as osteopontin and
bone sialoprotein. The exact mechanism is unknown although it could be attributed to
the ability of Twist to interact with the DNA-binding domain of Runx-2 thus
inhibiting its function as shown by Bialek et al.75. Hence, Twist may contribute to the
maintenance of the PDL by acting as a negative regulator of osteoblastic
differentiation.
PLAP-1/Asporin
Another newly discovered factor associated with the periodontal ligament is the
periodontal ligament associated protein-1 (PLAP-1). PLAP-1 (also known as asporin)
was simultaneously discovered by separate research groups76-78 in 2001. PLAP-1 is a
member of the leucine-rich repeat proteoglycan family and is similar to biglycan and
decorin (human asporin is 50% identical and 70% similar to decorin and biglycan)76.
However, as PLAP-1 does not contain glycosaminoglycan attachment sites77 and
contains a unique sequence of aspartate residues, it is not considered a true
proteoglycan79. The normal structure of PLAP-1/asporin contains a putative
propeptide, 4 aminoterminal cysteines, 10 leucine rich repeats and 2 C-terminal
cysteins (Figure 4 & 5).
33
Figure 4
The chemical structure of PLAP-1/asporin
(Adapted from Yamada et al.80)
Figure 5
The 3-D structure of PLAP-1/asporin
(green = Glu-194; purple=Arg-170; gray=carbon; blue=nitrogen; white=hydrogen;
red=oxygen; yellow ribbon=β-sheet; red ribbon=secondary helix structure)
(Adapted from Tomoeda et al.81)
Asporin mRNA can be found in many areas including the uterus, heart, liver and also
the extra cellular matrix in cartilage77,82. However, within the maxilla (of rat), unlike
the other molecular candidates such as S100A4 and Msx-2, PLAP-1 is highly location
specific with the periodontal ligament being the only region to express the protein
within the maxilla83. Furthermore, in situ hybridisation showed that PLAP-1 was
highly expressed in the dental follicle during dental formation indicating a central role
for PLAP-1 in the development of the periodontal tissues. However, conflicting data
34
have been demonstrated by Lee et al84 who found PLAP-1/Asporin at the globular
calcific region in the junction of predentine and denine.
The function of PLAP-1/asporin is still unclear although in their research on
osteoarthritis and asporin, Nakajima and co-workers85 found asporin acts as a negative
regulator of TGF-beta (TGF-beta 1 regulates proliferation, differentiation as well as
the matrix production of chondrocytes and their progenitor cells) in cartilage, thereby
affecting chrondrogenesis and ‘playing a critical role in etiology and pathogenesis of
osteoarthritis’. PLAP-1/asporin has also been found to affect the activities of other
growth factors (eg. TGF-β). In their investigation, Yamada et al.83 showed that
PLAP-1 regulates periodontal ligament cell cytodifferentiation and also mineralisation
through its negative feedback interaction with bone morphogenetic protein-2 (BMP-2).
A follow on study revealed that the mechanism by which PLAP-1 exerts the described
biological effect is through the leucine-rich repeats (LRR) motif. The LRR are a
protein structural motif that is composed of 20-30 amino acid stretches that are
extremely rich in the amino acid leucine. A particular LRR motif that appears to be
highly associated with PLAP-1 is LRR581.
The Pulp The dental pulp is a region of soft connective tissue which lies beneath the dentine
within a tooth. The main cellular constituents are odontoblasts, fibroblasts,
mesenchymal stem cells, macrophages and lymphocytes. The extracellular component
of the pulp is composed of ground substance (eg. glycosaminoglycans) and collagen
fibres (mainly type I & III). Additionally, the pulp contains blood vessels, nerve fibres
as well as lymphatic vessels.8
The primary function is the formation of dentine through the actions of the
odontoblasts. Other functions of the pulp include the provision of nutrients and
moisture as well as neurosensory information such as pain, pressure or temperature
differences. Finally, the dental pulp is also able to provide protection due to the
formation of reparative dentine following a traumatic episode.
35
Ankylosis There are many names or terms used to describe the phenomenon of the fusion of root
of the tooth to the underlying jaw bone. They may include the following terms:
tooth/dentoalveolar ankylosis; infra-occlusion; secondary-retention; reimpaction;
halbretention, reinclusion, replacement resorption and submergence86.
The first sign of a bony union between the root of the tooth and the alveolar bone was
reported in 1922 by Albin Oppenheim87 from Vienna, Austria. However, Dr
Oppenheim’s research at that time was mainly focused on the resorption of deciduous
teeth and the significance of this finding was not realised. In fact, it was more than 10
years later that Frederick Noyes88 first linked the clinical signs of tooth ankylosis to
the histological cause.
As mentioned previously, dentoalveolar ankylosis is the fusion of the cementum or
dentine to the alveolar bone89. It is a condition that occurs mostly in the deciduous
dentition with a prevalence rate of 1.3% to 14.3% of the population in general90,91.
Ankylosis also has a familial tendency92 (ie. occurs more frequently amongst siblings)
as well as a racial component with the condition roughly four times more common in
whites than blacks93. It also occurs mainly (~91.8%) in the deciduous molars region
and is more than twice as frequent in the mandible when compared to the maxilla94.
Early studies on the relationship between ankylosis of primary molars associated with
congenital absence of the succedaneous teeth yielded conflicting results95,96 but later
studies all seemed to support the notion that agenesis of the permanent premolar
predisposes the associated primary molar to be ankylosed97,98.
The fusion of the root to the alveolar bone destroys the eruptive potential of the tooth.
It also prevents normal dentoalveolar development in that region of the jaw. The
effects of ankylosis can vary depending on when the fusion occurs. If the fusion
occurs after adult equilibrium is already achieved, then the consequences are
generally minor. However, due to the fact that vertical facial height increases
throughout life99, the effects of ankylosis will still progress after adolescence, albeit at
a much slower rate. If, however, the ankylosis occurs while the jaw is developing,
then the consequences can be severe. The problems occur when the ankylosed tooth
36
becomes submerged leading to tilting of adjacent teeth, over-eruption of opposing
teeth leading to the loss of space as well as ectopic or failure of eruption of the
succedaneous tooth100 (Figure 6). In addition, as erupting teeth grow bone as they
move, a severely infra-occluded tooth will lead to an arrest of localised jaw
development resulting in a large bony defect which will make restorative options
more difficult.
Figure 6
Sequential bitewing radiograp-hs showing ankylosis of a mandibular first molar as well as the progressive nature of its associated orthodontic prob-lems. Note the loss of space, over-eruption and tipping of neighbouring teeth.
(Radiographs from Kurol101)
Another sequela of ankylosis is the phenomenon of replacement resorption
(sometimes used synonymously with ankylosis). After ankylosis develops, the root of
37
the tooth becomes susceptible to the remodelling activity of the bone. That is, the
root is resorbed and gradually replaced by new bone – hence, the term replacement
resorption.
As replacement resorption progresses, the root gradually becomes thinner and weaker.
When there is severe replacement resorption, the crown of the tooth can fracture.
The rate of replacement resorption varies and is dependent on the skeletal growth rate
of the patient. Therefore, in adults ankylosed teeth have the potential to last for
decades.
The periodontal membrane can be thought of as a double periosteum which covers
both the cementum and the alveolar bone102. Ankylosis is thought to occur when
there is a breakdown locally of the periodontal ligament allowing for in growth of
endosteal progenitor cells from the adjacent bone marrow to repopulate the defect
rather than root-side periodontal ligament progenitor cells103. Under the influence of
local cell signalling mechanisms, the endosteal progenitor cells migrate to the site of
the defect. Despite the ability of these cells to differentiate into various periodontal
ligament cells, the ultimate phenotype they differentiate into depends on the local
regulators64. Unfortunately, it has been shown that after injury, root-side progenitor
cells preferentially differentiate into cells capable of osteogenesis and osteoclasis
thereby favouring ankylosis over periodontal ligament regeneration103.
Aetiology of Ankylosis The aetiology of ankylosis is still unclear although there are three main theories:
mechanical trauma, disturbed local metabolism and genetics. The fact that around
92% of ankylosis was found in the molar region tends to support the theory of
mechanical trauma as the posterior region is by far where the most force is exerted94.
However, there are significant doubts to this theory as most of the ankylosis was
found in the deciduous dentition and only 8% were permanent molars. If occlusal
trauma was the cause then there should be more permanent molars ankylosed as
adults exert a much greater chewing force than a child or adolescent. Furthermore,
there is no evidence linking ankylosis to trapeze artists or other entertainers who
perform weight-lifting tricks with their teeth94. Another possible and arguably more
38
feasible cause of dental ankylosis is a disturbed local metabolism. It is theorised that
the disturbed local metabolism results in a lysis of the periodontal ligament at a
particular point thus exposing both the denuded cementum and bone to one another
leading to the formation of ankylosis. Localised ossification of the periodontal
membrane could also occur leading to the formation of ankylosis94. The third theory
is based on the finding that there is a significantly higher incidence of ankylosis
between siblings90-92,104,105.
Biederman106 found a statistical way to test the three hypotheses. In mouths with two
ankylosed deciduous molars, there are mathematically, six possible combinations.
Figure 7
The various combinations possible between two ankylosed teeth.
(from Biederman106)
1. Cross: upper right-lower left
lower right-upper left
2. Same side upper right-lower right
upper left-lower left
3. Same jaw upper right-upper left
lower right-lower left
If the cause of ankylosis is random (e.g. a defective formation, lysis or ossification,
then the frequency in all three of the categories should roughly match. If occlusal
trauma was the cause then the predominant category should be ‘same side’ as equal
force is produced against both upper and corresponding lower teeth when masticating.
If the cause is disturbed local metabolism, then they are likely to fall into the ‘same
jaw’ category as they are more likely to develop in parallel fashion. The results
overwhelmingly supported the hypothesis that dental ankylosis is formed as a result of
localised metabolic disturbance in the periodontal ligament. However, the result was
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39
not without question. The authors could not explain the 8 ‘same side’ occurrences
and also they could not explain the site selectivity of dental ankylosis.
In 1963, Dixon107 proposed that it may not be the local disturbance of metabolism but
a disturbance in the interaction between normal root resorption and hard tissue repair
in deciduous molars that leads to the formation of ankylosis.
Diagnosis of Ankylosis The diagnosis of ankylosis has also been an area of uncertainty. Absolute proof of
bony union between the tooth root and bone can be only be confirmed by histological
sections but is obviously clinically not applicable as a diagnostic tool. The literature
so far (backed up by recent report by Crowther et al.108) suggests the use of a
combination of the following indicators when ankylosis is suspected:
• Loss of mobility – Bucco-lingual forces can be used via palpation to detect the
extent of loss of mobility. Some authors suggest normal mobility to be lost
when more than 10% of root surface is ankylosed90 although others have
suggested even a minute microscopic area can induce immobility106. The
mobility can also be tested by electronic instruments such as the Periotest
(Figure 8). However, problems of error readings, unit malfunction and test-re-
test reliability issues have been reported.
Figure 8 Periotest
(from Campbell et al.103)
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40
• Infra-Occlusion – This is the most definitive clinical indication that ankylosis
has occurred109. A tooth that has ‘submerged’ beneath the occlusal plane
when previously at occlusal height is very likely to be ankylosed.
• Percussion – A high pitched sound can generally be heard as opposed to a dull
sound when more than 20% of the root is ankylosed110.
• Radiograph – Obliteration of the periodontal ligament space can be indicative
of periodontal membrane breakdown and subsequent bony union (Figure 9).
However, it is not an effective diagnostic tool as the ankylotic area could be
hidden by other anatomy and could also be microscopic. In fact, Raghoebar et
al.86 have calculated the efficiency of radiographs in diagnosing dental
ankylosis to be ~21% only, even with an experienced operator. In addition, it
was shown by Andreason111 that ankylosis initially favours the labial and
lingual root surfaces which are basically impossible to diagnose via
conventional radiographs.
• Orthodontic Force – An ankylosed tooth does not respond to orthodontic
forces106,112.
A condition that may mimic dentoalveolar ankylosis early on is primary failure of
eruption. It is a rare condition in which the eruptive mechanism is disrupted leading
to either complete failure of eruption or only partial eruption. Teeth affected by this
Figure 9 Note the lack of periodontal ligament space and replacement resorption on the apical half of the 21 (from Campbell et al.103)
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41
condition tend to become ankylosed but the failure to erupt is in evidence prior to
ankylosis. The affected teeth also do not respond well to orthodontic traction - at best
only 1 – 2 mm of movement can be achieved108. In fact, application of orthodontic
force often directly leads to the development of ankylosis113.
Also, transient ankylosis has been reported by Andreasen & Skougaard114 and
Hammarstrom et al.115 to disappear within 8 weeks which may explain the
spontaneous re-eruption observed by Raghoebar et al.116 as well as Belanger et al.117.
An uncommon form of ankylosis can be due to the inostosis of enamel. Because the
enamel epithelium protecting the tooth disintegrates (e.g. via infection), enamel can
be resorbed and, subsequently, bone/cementum may be deposited in its place thus
placing a solid fixation on the tooth 118.
Management of Ankylosis The management of dento-alveolar ankylosis can vary a great deal and depends on the
individual circumstances. Currently, the management can be arranged in the
following sub-classifications; deciduous teeth with a permanent successor, deciduous
teeth without a permanent successor and ankylosed permanent teeth.
Deciduous ankylosed tooth with succedaneous tooth
The main aim when dealing with a primary tooth with a permanent successor is to
ensure the normal development and eruption of the permanent tooth. A permanent
successor can resorb the ankylotic area as it erupts into the primary tooth119.
Therefore, if the infra-occlusion is minor, monitoring is indicated. If the tooth does
not spontaneously exfoliate upon the estimated time, an additional allowance of 6
months is acceptable105 before extraction is indicated. Also, if the primary tooth is
significantly below the occlusal plane then immediate removal of the affected tooth is
recommended plus space maintenance90. If there is already tipping of teeth then
orthodontic intervention may be required.
42
Deciduous ankylosed teeth without a permanent successor
Ankylosis is a common finding in deciduous molars without a permanent successor120.
Treatment required depends on the onset of ankylosis with respect to growth of the
individual as well as the date of the diagnosis. As a general rule, the ankylosed tooth
should be carefully monitored until there is a risk of tipping of adjacent teeth or over-
eruption of the opposing tooth. Restorative procedures need to be undertaken if there
is a need to re-establish the mesial and distal contacts as well as the occlusal plane.
Orthodontic movement may be required if there is minor tipping to help facilitate the
restorative procedure if the plan is to retain the tooth for as long as possible. Long
term studies have demonstrated that when left in situ, the primary molars without a
permanent successor can last more than 10-15 years and should be considered an
acceptable semi-permanent solution121,122. Obviously if there is a risk of development
of a large jaw defect (e.g. in early onset cases), then extraction (with options of 1.
orthodontic closure, 2.prosthetic replacement or 3. implant in the future) is likely to be
the best choice. Biederman106 did describe a technique called luxation. It involves
gentle bucco-lingual movement of the ankylosed tooth in an attempt to break the
ankylotic union and hope for re-establishment of the periodontal membrane. This
technique together with immediate application of orthodontic force has proven
successful for Geiger and Bronsky123. However, the outcomes are unpredictable and
there is a real chance for re-ankylosis103. Raghoebar et al.124 have also shown that
most of the ankylotic areas for deciduous molars are in the furcation, which is
unlikely to break by luxation and may quite possibly extend the ankylotic area.
Permanent ankylosed tooth
Similar to deciduous ankylosed teeth without a permanent successor, the treatment
options for an ankylosed permanent tooth can vary greatly. Once again, the choice of
treatment should be decided on after due consideration to the growth of the patient,
the date of diagnosis, the state of infra-occlusion and neighbouring teeth when
diagnosed and the patient’s opinion. If the patient is an adult with minimal future
skeletal growth, then the treatment options consist of monitoring and restoring the
tooth as required. Patients need to be advised that due to the continual vertical facial
growth that exists in all human beings, remake of crowns may be needed. When the
growth of the patient is not yet complete, other procedures may be indicated; luxation,
43
localised ostectomy, decoronation, corticotomy with or without osteogenic distraction
have all been suggested.
Specific Techniques of Management
Luxation (as mentioned earlier) is the earliest described technique that may manage
ankylosis.
Localised ostectomy has also been proposed. It involves identifying the ankylosed
area, raising a periodontal flap and surgically removing the affected mineralised tissue.
This technique only works if the ankylotic union is in the crestal area as ankylosis
elsewhere on the root surface presents access problems100.
Corticotomy is the technique where a whole block of cortical bone and soft tissue is
isolated along with the tooth and is repositioned as desired. This technique does not
correct the ankylosis and it is also limited by the restriction of the mucosa’s ability to
be extended125.
Decoronation is the procedure first described by Malmgrem et al.126 in 1984. It
involves sectioning the crown of the affected tooth (thereby decoronating it) and
leaving the root purposely in situ. The root has been shown to retain the integrity of
the alveolar bone and allow further apposition of bone127-129. This procedure has been
proposed as a surgical technique that allows preservation of the bone volume for the
future and avoids aesthetic disturbances. It presents a viable alternative to aggressive
options in cases where other therapeutic alternatives are not feasible127. However,
decoronation does rely on the phenomenon of replacement resorption to resorb the
remaining root prior to the placement of an implant.
Another surgical technique that has gained interest in recent times is the application of
distraction osteogenesis. It is a variation of osteotomy where a localised osteotomy is
performed and force is delivered to the targeted section of bone in the direction in
which new bone growth is desired. Modification of the classic distraction technique
has also been performed through adjustment of the bone that is sitting on the newly
formed callus which allows for three dimensional movements - thus utilising the
‘floating bone concept’130. The distraction can be done via orthodontic wires, external
44
(most likely tooth-borne) distractors or internal bone-supported screw distractors131,132.
The whole block is moved into the desired position where final adjustments may still
need to take place.
Extraction of the affected tooth is still a viable option. However, the extraction of an
ankylosed tooth requires extra care as it may lead to further trauma resulting in an un-
aesthetic bony ridge defect and influence the chances of delivering an optimal
prosthetic treatment127.
Unfortunately, most of these techniques described appear in single or case reports
with no support from randomised clinical trials and hence cannot be relied upon for
predictable long term successful outcomes90.
Experimentally Produced Ankylosis Efforts have long been made to induce dental ankylosis experimentally in order to
further study the pathogenesis as well as the relationship between ankylosis and its
surrounding biological tissues. In fact, according to Andreason & Skougaard114,
attempts as early as 1928 were being carried out by Feldman to create artificial dental
ankylosis.
As discussed previously, the periodontal ligament and its constituents normally
depress osteogenic actions which ensure the periodontal ligament space is free of
calcified tissue. Sufficient damage to the periodontal ligament limits its anti-
osteogenic ability giving rise to an opportunity for ankylosis to develop. It is,
therefore, not surprising that almost all of the various methods employed to induce
ankylosis in vivo include inducing certain forms of trauma.
There are five main modes of inducing an ankylotic union between the root and
alveolar bone.
• Mechanical/Physical
• Chemical
• Electrical
• Thermal
45
• Heat
• Cold
• Denervation
Mechanical/Physical
Several investigators have attempted to produce ankylosis through inducing
mechanical trauma by using devices such as dental burs to injure the periodontal
ligament tissue as well as the surrounding bone and root114,133,134. Their techniques
yielded inconsistent development of ankylosis possibly due to insufficient trauma.
Rubin et al.134 in 1984 also investigated the possibility to induce ankylosis via
occlusal trauma. This was simulated in the form of a stainless steel crown formed to
purposely be in premature contact. The study did not create any ankylosis which
backs up Biederman’s94 conclusion that masticatory force is unlikely to be a cause of
ankylosis.
In contrast, physical damage in the form of replantation proved to be a reliable
method of inducing ankylosis. This is particularly true if the periodontal ligament
extracellular matrix and part of its cellular component are not kept intact (e.g. increase
extra-oral time). As such, it has been a common methodology in investigating
ankylosis and the healing process of the PDL as a whole55,60,110,115,135. Similar results
were also found in transplantation136 and luxation type trauma134.
Chemical
Early attempts to produce ankylosis via chemical means were documented by Rubin
& Biederman137 in 1961. Phenol was placed on parts of surgically exposed root
surfaces but no ankylosis was noted. Phenol was reused in a later attempt134 and was
similarly unsuccessful. However, in a study by Erausqin & Devoto138, formalin and
formaldehyde were used successfully to produce widespread ankylosis. Furthermore,
trioxymethylene-corticoid and acrylic spherule paste both were found to be capable of
inducing ankylosis although the results were inconsistent. Zinc oxide eugenol was
also tested in the same study but was found to be a poor causative agent for dental
ankylosis.
46
The systemic delivery of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) was
reported by Wesselink & Beertsen139 to be capable of inducing ankylosis in the mouse.
Electrical
According to Rubin et al.134, electrical diathermy was carried out by Gottlieb and
Orban in 1930 and was shown to be successful in inducing ankylosis. However, no
other studies have since employed a similar methodology.
Thermal
Positive experimental ankylosis has been reported from the generation of heat through
the root canal systems in both rat and monkey incisors140-142. In 1982, Michaeli et
al.142 generated heat via the direct application of electrocautery needle into the pulp
cavity which seemed to limit the injury primarily to the periodontal ligament. Their
experiment demonstrated fusion of the root to bone within 7 days. In contrast,
Atrizadeh et al.140 and Line et al.141 did not observe ankylosis until 1 month after the
heat application which probably reflected differences in their methodology.
The application of ultra-low temperatures to the periodontal ligament apparatus was
not reported until 1986 when Wesselink et al.143 applied liquid nitrogen and Tah &
Stahl144 utilised cryoprobe on the buccal alveolar plate of rats. Both studies reported
consistent ankylosis formation although widespread alveolar bone (particularly on the
buccal experimental side) necrosis was also noted. Tah et al.145 repeated the protocol
at a later date and had similar success in inducing ankylotic union. A less traumatic
method was developed by Dreyer et al.146 which allowed for the induction of dental
ankylosis while limiting the majority of the insult to the periodontal ligament. This
was achieved via the application of dry ice to the occlusal surface of the tooth crown
hence insulting the periodontal ligament apparatus indirectly through the thermal
conductivity of the enamel and particularly the dentinal tubules. Recently, this
protocol was applied by Shaboodien147 and Di Iulio148. Similar histological findings
were reported although in Di Iulio’s experiment, ankylosis was not a consistent
finding.
47
Denervation
Fujiyama et al.51 denervated rat teeth by transection of the inferior alveolar nerve.
This led to the formation of ankylosis with the authors attributing this finding to the
concurrent decrease in rests of Malassez. This was in contrast to an earlier study by
Berggreen et al.149 which found little association between denervation and the
formation of ankylosis although both the experimental and control teeth underwent
replantation which is known to cause ankylosis and hence would mask any real
difference denervation may have had on the periodontal ligament apparatus
Immunohistochemistry Immunohistochemistry is a method for localising specific antigens (proteins of
interest) in tissues or cells based on antigen-antibody recognition; it seeks to exploit
the specificity provided by the binding of an antibody with its antigen and which can
be detected at a light microscopic level150.
There are two main types of antibodies: monoclonal and polyclonal. Polyclonal
antibodies are a cocktail of various antibodies that may recognise several binding sites
(epitopes) whereas monoclonal only recognises one particular epitope. Hence,
polyclonal antibodies may be more sensitive but monoclonal antibodies are more
specific.
The staining of a cellular epitope provides an insight to the molecular role which the
protein may play in a given condition. Immunohistochemistry has proven to be
invaluable to both diagnosis and research.
There are many techniques in immunohistochemistry and they include:
• one-step direct conjugate
• two-step indirect method
• peroxidase antiperoxidase (PAP)
• avidin-biotin complex/conjugate (ABC)
• biotin-streptavidin (B-SA)
• polymer-based labelling systems
48
Avidin-biotin complex procedure (ABC) This technique utilises the high affinity binding between biotin and avidin. In this
technique, a primary antibody with biotin can first be used to locate the target antigen.
Horseradish peroxidase that is conjugated to avidin can then be added (or a secondary
antibody first, which improves on the specificity and sensitivity of the results)
resulting in the binding between the primary antibody and the horseradish complex
thus localising and manifesting the target antigen/protein for the investigator150.
The ABC method does have its disadvantages in that many tissues contain significant
amounts of endogenous biotin that may result in false positive staining (this
background staining can be eliminated by specific blocking solutions). Secondly, it
has been shown that various batches of biotin and avidin tend to differ in their
affinities and binding power thus affecting the predictability of certain staining
protocols150.
Nevertheless, overall the ABC method is arguably the most commonly used
immunohistochemical method due to its simplicity and reasonably reliable/predictable
outcomes.
49
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114. Andreasen JO, Skougaard MR. Reversibility of surgically induced dental ankylosis in rats. Int J Oral Surg 1972;1:98-102. 115. Hammarstrom L, Blomlof L, Lindskog S. Dynamics of dentoalveolar ankylosis and associated root resorption. Endod Dent Traumatol 1989;5:163-175. 116. Raghoebar GM, van Koldam WA, Boering G. Spontaneous reeruption of a secondarily retained permanent lower molar and an unusual migration of a lower third molar. Am J Orthod Dentofacial Orthop 1990;97:82-84. 117. Belanger GK, Strange M, Sexton JR. Early ankylosis of a primary molar with self correction: case report. Pediatr Dent 1986;8:37-40. 118. Franklin CD. Ankylosis of an unerupted third molar by inostosis of enamel. A case report. Br Dent J 1972;133:346-347. 119. Dixon DA. Observations on submerging deciduous molars. Transactions of the British Society for the Study of Orthodontics 1962;00:101-114. 120. Albers DD. Ankylosis of teeth in the developing dentition. Quintessence Int 1986;17:303-308. 121. Bjerklin K, Bennett J. The long-term survival of lower second primary molars in subjects with agenesis of the premolars. Eur J Orthod 2000;22:245-255. 122. Ith-Hansen K, Kjaer I. Persistence of deciduous molars in subjects with agenesis of the second premolars. Eur J Orthod 2000;22:239-243. 123. Geiger AM, Bronsky MJ. Orthodontic management of ankylosed permanent posterior teeth: a clinical report of three cases. Am J Orthod Dentofacial Orthop 1994;106:543-548. 124. Raghoebar GM, Boering G, Jansen HW, Vissink A. Secondary retention of permanent molars: a histologic study. J Oral Pathol Med 1989;18:427-431. 125. Anholm JM, Crites DA, Hoff R, Rathbun WE. Corticotomy-facilitated orthodontics. Cda J 1986;14:7-11. 126. Malmgren B, Cvek M, Lundberg M, Frykholm A. Surgical treatment of ankylosed and infrapositioned reimplanted incisors in adolescents. Scand J Dent Res 1984;92:391-399. 127. Cohenca N, Stabholz A. Decoronation - a conservative method to treat ankylosed teeth for preservation of alveolar ridge prior to permanent prosthetic reconstruction: literature review and case presentation. Dent Traumatol 2007;23:87-94. 128. Diaz JA, Sandoval HP, Pineda PI, Junod PA. Conservative treatment of an ankylosed tooth after delayed replantation: a case report. Dent Traumatol 2007;23:313-317.
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129. Filippi A, Pohl Y, von Arx T. Decoronation of an ankylosed tooth for preservation of alveolar bone prior to implant placement. Dent Traumatol 2001;17:93-95. 130. Huck L, Korbmacher H, Niemeyer K, Kahl-Nieke B. Distraction osteogenesis of ankylosed front teeth with subsequent orthodontic fine adjustment. J Orofac Orthop 2006;67:297-307. 131. Alcan T. A miniature tooth-borne distractor for the alignment of ankylosed teeth. Angle Orthod 2006;76:77-83. 132. Kinzinger GS, Janicke S, Riediger D, Diedrich PR. Orthodontic fine adjustment after vertical callus distraction of an ankylosed incisor using the floating bone concept. Am J Orthod Dentofacial Orthop 2003;124:582-590. 133. Parker WS, Frisbe HE, Grant TS. The experimental production of dental ankylosis. Angle Orthod 1964;34:103-107. 134. Rubin PL, Weisman EJ, Bisk F. Experimental tooth ankylosis in the monkey. Angle Orthod 1984;54:67-72. 135. Sherman P, Jr. Intentional replantation of teeth in dogs and monkeys. J Dent Res 1968;47:1066-1071. 136. Morris ML, Moreinis A, Patel R, Prestup A. Factors affecting healing after experimentally delayed tooth transplantation. J Endod 1981;7:80-84. 137. Rubin PL, Biederman W. Attempt to produce tooth ankylosis. J Dent Res 1961;40:744. 138. Erausquin J, Devoto FC. Alveolodental ankylosis induced by root canal treatment in rat molars. Oral Surg Oral Med Oral Pathol 1970;30:105-116. 139. Wesselink PR, Beertsen W. Ankylosis of the mouse molar after systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP). J Clin Periodontol 1994;21:465-471. 140. Atrizadeh F, Kennedy J, Zander H. Ankylosis of teeth following thermal injury. J Periodontal Res 1971;6:159-167. 141. Line SE, Polson AM, Zander HA. Relationship between periodontal injury, selective cell repopulation and ankylosis. J Periodontol 1974;45:725-730. 142. Michaeli Y, Pitaru S, Zajicek G. Localized damage to the periodontal ligament and its effect on the eruptive process of the rat incisor. J Periodontal Res 1982;17:300-308. 143. Wesselink PR, Beertsen W, Everts V. Resorption of the mouse incisor after the application of cold to the periodontal attachment apparatus. Calcif Tissue Int 1986;39:11-21.
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144. Tal H, Stahl SS. Healing following devitalization of sites within the periodontal ligament by ultralow temperatures. J Periodontol 1986;57:735-741. 145. Tal H, Kozlovsky A, Pitaru S. Healing of sites within the dog periodontal ligament after application of cold to the periodontal attachment apparatus. J Clin Periodontol 1991;18:543-547. 146. Dreyer CW, Pierce AM, Lindskog S. Hypothermic insult to the periodontium: a model for the study of aseptic tooth resorption. Endod Dent Traumatol 2000;16:9-15. 147. Shaboodien SI. Traumatically induced dentoalveolar ankylosis in rats. Adelaide: University of Adelaide; 2005. 148. Di Iulio DS. Relationship of epithelial cells and nerve fibres to experimentally induced dentoalveolar ankylosis in the rat. Adelaide: University of Adelaide; 2007. 149. Berggreen E, Sae-Lim V, Bletsa A, Heyeraas KJ. Effect of denervation on healing after tooth replantation in the ferret. Acta Odontol Scand 2001;59:379-385. 150. Dabbs DJ. Diagnostic immunohistochemistry. New York, Edinburgh: Churchill Livingstone; 2006.
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SECTION 2
61
STATEMENT OF PURPOSE In a healthy periodontium, the periodontal ligament functions to connect the teeth to
the jaws, sustain the masticatory load, provides sensory information and also prevents
bony union of the roots of the teeth to the alveolar bone.
The cells of the periodontal ligament have been shown to be osteogenic but under
normal conditions, the PDL space remains patent without the occurrence of ankylosis.
Periodontal Ligament Associated Protein-1 (PLAP-1)/Asporin are a recently
discovered protein that has been suggested to play a significant role in suppressing the
osteogenic tendency of the periodontal ligament and maintaining the fibrous
ligamentous nature of the periodontal ligament. Furthermore, PLAP-1/Asporin has
also been shown to be associated with the differentiation and mineralisation of dental
pulp stem cells.
Therefore, it is the intention of this project to investigate the relationship between
PLAP-1/Asporin and the periodontal ligament space as well as the dental pulp using
an ankylosis model.
The investigation has been divided into two parts with separate hypothesis and aims
as stated below.
Paper 1
Aims
To confirm the location specificity of PLAP-1 within the maxilla using
immunohistochemistry.
To investigate the expression of PLAP-1 in the periodontal ligament using a rat
ankylosis model.
Hypothesis
There is no change in the expression of PLAP-1 in ankylotic areas due to hypothermal
insult compared to non-ankylotic areas within the periodontal ligament.
62
Paper 2
Aims To determine the normal expression of PLAP-1/Asporin within the pulp chamber
using immunohistochemistry.
To investigate the relationship between PLAP-1/Asporin expression in the coronal
pulp following a hypothermic insult using immunohistochemistry.
Hypothesis
There is no change in the expression of PLAP-1 in traumatised pulp compared to non-
traumatised pulp.
It is intended that both papers be submitted to the journal Archives of Oral Biology
and hence is prepared in the journal’s requested format.
63
PAPER 1 (Prepared for submission in Archives of Oral Biology)
Title: The role of PLAP-1/Asporin in the Maintenance of the Rat Periodontal
Ligament using a Rat Ankylosis Model.
Authors: Chen, W.C., Sampson, W., Dreyer, C., Dharmapatni, K.
Affiliations:
Chen, W.C. – Postgraduate student, Orthodontic Unit, School of Dentistry, Faculty of
Health Sciences, University of Adelaide, South Australia.
Sampson, W. – Professor and P.R. Begg Chair, Orthodontic Unit, School of Dentistry,
Faculty of Health Sciences, University of Adelaide, South Australia.
Dreyer, C. – Associate Professor, Orthodontic Unit, School of Dentistry, Faculty of
Health Sciences, University of Adelaide, South Australia.
Dharmapatni, K. – Research Fellow, Hanson Institute, School of Medicine, Faculty of
Health Sciences, University of Adelaide, South Australia.
Abstract
Background: Periodontal Ligament Associated Protein-1(PLAP-1)/Asporin is a novel
protein suggested to have an important role in the regulation of the periodontal
ligament space. However, the scarce data available demonstrate contrasting results.
Currently, there are no studies which investigate the expression and relationship of
PLAP-1 and periodontal ligament (PDL) space using an ankylosis model.
Aims: The aims of this study are to determine the distribution of PLAP-1 protein
within the maxilla and to explore correlation between PLAP-1 expression and
ankylosis in a transient aseptic ankylosis model.
64
Methods: The maxillary right first molars of 30 male Sprague-Dawley rats were
subjected to a single 20 minute application of dry ice to induce PDL ankylosis.
Groups of five animals were sacrificed after 7, 10, 14, 18, 21 and 28 days of treatment
respectively. The maxillae were dissected out and underwent routine tissue fixation
and processing for immunohistochemical detection of PLAP-1 expression. The
immunostained cells were then analysed semi-quantitatively using a standardised grid
system.
Results: PLAP-1 was expressed in the PDL, dental pulp, blood vessel walls and the
nasal cartilage. Not all sections contained ankylosis. Sections which did not contain
ankylosis demonstrated no significant PLAP-1 expression differences between control
and experimental sides. Sections that did demonstrate ankylosis yielded a tendency
towards increased PLAP-1 reactivity especially near the cementum. However, it was
difficult to deduce whether the relationship of PLAP-1 to the ankylotic union was
associated with bone formation or resorptive activities.
Conclusion: The current investigation suggests that PLAP-1 may be involved in bone
metabolism but future investigations are required to elucidate its true role within the
periodontal ligament.
Key Words: periodontal ligament associated protein-1, asporin, ankylosis,
periodontal ligament, maintenance.
Introduction
In a healthy periodontium, the periodontal ligament functions to connect the teeth to
the jaws, sustain the masticatory load, provide sensory information and also prevent
bony union (ankylosis) of the tooth roots to the alveolar bone.
Interestingly, cells of the periodontal ligament have been shown to be osteogenic in
laboratory studies.1-3 Furthermore, it is common knowledge that the death or removal
of periodontal ligament cells leads to the loss of periodontal membrane eventually
65
resulting in extensive root resorption and widespread ankylosis. Presumably, certain
factors/cells exist within the periodontal ligament which must normally suppress the
osteogenic tendency and maintain the fibrous ligamentous nature of the periodontal
ligament.
The search for the cell(s) and/or factor(s) which are responsible for maintaining the
patency of the periodontal ligament is not new. Early investigations have suggested
several cells/factors which could play a vital role in inhibiting ankylosis and they
include the cell rests of Malassez3-6, glycosaminoglycans7 and fibroblasts8,9 as well as
their associated prostaglandins10,11. However, no agreement has been reached
regarding the exact cell/factor(s) involved in periodontal ligament maintenance.
Recent advances in technology have allowed more detailed investigations of the
periodontal ligament at a molecular level. This has led to the discovery of several
new factors which may play a role in the maintenance of the periodontal ligament
space: RGD-CAP12 (a collagen-associated protein which contains the arginine-
glycine-aspartic acid sequence)12; epidermal growth factor receptors13; S100A414,15
(member of the S100 calcium binding family; Msx216 (homeobox protein); Twist17 (a
basic helix loop helix protein) and PLAP-118,19 (periodontal ligament associated
protein-1).
Of all the possible factors, PLAP-1 (also known as Asporin) demonstrates the most
promise as, unlike the other molecular factors mentioned which are expressed
ubiquitously, PLAP-1 is thought to be highly location specific. In fact, the only
region in the maxilla demonstrating its presence is reported to be the periodontal
ligament space.19 PLAP-1 is a member of the leucine-rich repeat proteoglycan
family. Furthermore, in situ hybridisation has shown that PLAP-1 is highly
expressed in the dental follicle during tooth formation indicating a central role for
PLAP-1 in the development of the periodontal tissues. In addition, Yamada et al.19
have shown that PLAP-1 regulates periodontal ligament cell cytodifferentiation and
also mineralisation through its negative feedback interaction with bone morphogenetic
66
protein-2 (BMP-2). A subesequent study revealed that the mechanism by which
PLAP-1 exerts the described biological effect is through the leucine-rich repeats
(LRR) motif. A particular LRR motif that appears to be highly associated with
PLAP-1 is LRR5.18
A recent investigation20 using a previously established ankylosis protocol21 has
revealed the possibility of spontaneous resolution of the ankylotic area demonstrating
the capacity for the periodontal ligament to regenerate and repair minor damages.
Utilising the same tissue samples will provide a unique opportunity to investigate the
expression and possible role of PLAP-1 in the formation and regulation of ankylosis.
Aims
• To confirm the location specificity of PLAP-1 within the rat maxilla using
immunohistochemistry.
• To investigate the correlation between PLAP-1 expression in the rat
periodontal ligament using an ankylosis model.
Hypothesis
Null Hypothesis
There is no change in the expression of PLAP-1 in ankylotic areas due to hypothermal
insult compared to non-ankylotic areas within the periodontal ligament.
Materials & Methods
The maxillary right first molars of 30, 8-week-old male Sprague-Dawley rats (housed
in the University of Adelaide Animal House) were subjected to a single 20 minute
application of dry ice in order to induce sterile necrosis and ankylosis in the inter-
radicular region. Anaesthesia was achieved via a 1:1 combination of Hypnorm®
67
(Janssen-Cilag Ltd., Buckinghamshire, UK) and Hypnovel® (Roche, Berne,
Switzerland). The contralateral first molar served as a control. Groups of five
animals were sacrificed via cardiac perfusion with 4% paraformaldehyde after 7, 10,
14, 18, 21 and 28 days, respectively. The maxillae were dissected out and underwent
decalcification using 4% EDTA and were then paraffin embedded. Serial 7µm
coronal sections of the furcation area were obtained using a Leitz 1512 microtome and
then placed onto silane coated slides.
The staining for PLAP-1 immunoreactivity was performed via the Labelled
StreptAvidin Biotin method. The primary antibody was an unlabelled rabbit
polyclonal antibody (450-31930, Sapphire Bioscience, NSW, Australia) diluted with
PBS at 1 to 400 of stock concentration and incubated overnight in a wet chamber at
room temperature (RT). The linkage reagent was a biotinylated goat anti-rabbit
secondary antibody conjugated to horseradish peroxidase (K060911, LSAB®2-HRP,
Dako, Australia) and was incubated for 30 minutes, RT. Visualisation of the target
protein (PLAP-1) was through the use of AEC dye (K3469, Dako, Australia).
Sections were then counterstained with haematoxylin and lithium carbonate. Growing
rat femoral head containing cartilage was used as the positive control as it has been
previously reported that chondrocytes express PLAP-1/Asporin.36 Sections incubated
only with a rabbit serum (N169987, Universal negative control, Dako, Australia) were
used as the negative control.
Stained sections were mounted and analysed using an Olympus B071 optical
microscope with the images displayed on an attached personal computer with a 24.0
inch monitor. The software programme Analysis (Olympus Soft Imaging Solutions,
Germany) was used for image processing and viewing. A grid was pre-constructed
according to Shaboodien’s22 method to aid in accuracy and reproducibility (figure 1).
A central vertical line divided the crown into equal halves and was oriented as parallel
as possible to the axis of the tooth. A horizontal line was established by joining and
extrapolating from the cemento-enamel junction of both the buccal and palatal side.
Four additional vertical lines were drawn (2 on either side of the central vertical line)
which helped divide the roots into equal halves (best approximation) and 3 additional
68
horizontal lines were drawn to divide the roots into equal cervical, middle and apical
third (Figure 1). A 4 X magnification was used to superimpose the pre-constructed
grid to allow for standardisation of the area of interest (shown by solid black lines in
Figure 1). Within the PDL itself, a subjective evaluation was made to divide it into
thirds – adjacent to the alveolar bone, middle third and adjacent to the cementum.
Due to the diffuse nature of the stain, a semi-quantitative scoring method was utilised
(0=no staining, 1=mild staining; 2=moderate staining, 3=intense staining) to measure
the staining intensity of PLAP-1.
Figure 1 Superimposition of grid system on sections with roots (The cementum, PDL & alveolar bone within the solid line is the area included for the analysis.)
Fifteen sections 70µms apart per rat were stained and analysed. Wald statistics for
Type 3 GEE (General Estimating Equation) Analysis as well as Post-hoc comparisons
(see Appendices) were made. One section from each rat was randomly selected for
repeat scoring and the intra-observer reliability was calculated with a weighted Kappa
coefficient. All calculations performed by SAS Version 9.2 (SAS Institute Inc., Cary,
NC, USA).
Results
Periodontal ligament associated protein-1 was found within the rat maxilla but not
exclusively expressed within the periodontal ligament (Fig 2). PLAP-1 was found to
69
be expressed in the dental pulp, nasal cartilage, mid-palatal suture, blood vessel walls,
epithelial tissues and the periodontal ligament space.
Figure 2
a) Rat femur articular cartilage used as positive control. Staining of the differentiated chondrocytes was noted but PLAP-1 was not expressed by all the cells. b) Negative control of the rat articular cartilage showing no staining. c) Tissue stained with PLAP-1. d) Negative control showing no dental pulp or PDL staining.
The staining obtained within the cartilaginous area was associated with the cytoplasm
of mainly developing chondrocytes up until the stage when they become hypertrophic.
However, this positive expression did not occur uniformly (Fig 3a). The expressions
within the chondrocytes were more defined as were the expressions detected on the
blood vessel walls (Fig 3b). This is in contrast to the expressions found within the
dental pulp and the periodontal ligament space which appeared diffuse (Fig 3c). The
results of PLAP-1 in relation to dental pulp will be presented in the second article.
a b
PLAP-1 stained chondrocytes
d
Alveolar Bone
dentine
pulp PDL
c
pulp
dentine
PDL
200µm 200µm
500µm 500µm
70
Under normal, un-traumatised circumstances, the periodontal ligament space adjacent
to the alveolar bone tended to exhibit a higher PLAP-1 expression than other regions
of the PDL space (Fig 3d) although this is often associated with the increased number
of blood vessels associated with the alveolar bone surface. The expression shown on
gingival epithelial tissues was widespread and generally most intense at the surface
epithelium and becoming less so towards the basement lamina (Fig 3e).
Figure 3
a) PLAP-1 staining of certain chondrocytes within the articular cartilage. b) PLAP-1 staining of blood vessels. c) PLAP-1 staining pattern within the PDL. d) PLAP-1 staining of the PDL adjacent to the alveolar bone and its association with blood vessels. e) PLAP-1 staining of gingival tissues.
b
e
d
a
c
PLAP-1 stained chondrocytes
Unstained chondrocytes
dentine Alveolar bone
PDL
dentine
dentine Gingival epithelial tissues
PDL
Alveolar bone
Alveolar bone
Blood vessels
PDL
200µm
200µm
100µm
100µm
500µm
71
Not all sections exhibited ankylosis within the furcation region and some could not be
included for analysis due to crucial regions being lost or damaged during tissue
processing. It was found that despite identical staining procedures and environmental
conditions, there were significant variations between animals in the intensity of
PLAP-1 expression. Ankylosis was deemed acquired when there was osseous-like
material intruding in the periodontal ligament space (Fig 4). In agreement with other
studies23,24 which used a similar method, the ankylosis ranged from small islands of
bone-like material to multiple columns/spicules connecting the inter-radicular bone to
the cementum of the root.
Ankylosis was mainly found in days 10, 14 and 18. There were extremely few
ankylotic unions found in days 7 and virtually none by days 21 and 28. The time
elapsed from initial trauma did not seem to have a statistically significant effect on the
expression of PLAP-1 (see Appendices).
Figure 4
a) with ankylosis on experimental side on day 14. b) without ankylosis on control side on day 14
Mixed results were obtained for the slides in which ankylosis was observed (Table 1).
In the cementum third of the periodontal ligament space, the experimental side
yielded statistically significant greater expression of PLAP-1 particularly for higher
intensities (P<0.0001) (Fig 5). However, this relationship was not found to be
consistent in the other PDL regions. Nevertheless, aside from low intensity staining in
the bone third of the PDL, the remaining subgroups (various intensities at each
dentine
dentine
Alveolar bone
Alveolar bone
PDL
PDL
Ankylotic union
pulp
pulp a b 200µm
200µm
72
location) all showed a tendency towards the experimental side demonstrating more
PLAP-1 expression. It was interesting to note that toward the apical regions, many of
the highly stained PLAP-1 cells seemed to be multinucleated cells sitting in resorptive
lacunae (Fig 6a). However, it is difficult to ascertain whether this also applies to the
ankylotic region due to the spicule nature of the bone-like material at the inter-
radicular area. However, stained regions adjacent to the cementum did not appear to
be associated with resorptive lacunae (Fig 6b).
Figure 5
a) shows ankylosis with more intense staining near the cementum in a day 18 experimental rat whereas b) shows very little staining near cementum third in control sides of a day 18 specimen (note also increased staining at the alveolar third compared to cementum third in (b).
Figure 6
a) Shows on a day 14 experimental rat that toward the apical region many multinucleated cells associated with resorptive lacunae are stained positive. b) Whilst PLAP-1 is positive all along the cementum side there were no multinucleated cells and no obvious resorptive lacunae (day 14 experimental rat).
a b PDL
PDL
Alveolar bone
Alveolar bone
Ankylotic union
pulp
dentine
dentine
500µm 100µm
a b
PDL
PDL
Alveolar bone
Alveolar bone
Ankylotic union
pulp
Multinucleated PLAP-1 stained cells in lacunae
Multinucleated PLAP-1 stained cells in lacunae
Increased staining along cementum 1/3rd of PDL
Root dentine
200µm 100µm
73
Table 1: Combined Post-hoc data comparing control and traumatised sides with ankylosis at various intensities of PLAP-1 expression
PDL Region PLAP-1
Intensity Score
Compared
groups
P-Value Odds Ratio
Cementum 1. Predictors of intensity ≥ 1
Control vs. Exp <.0001 0.041
2. Predictors of intensity ≥ 2
Control vs. Exp <.0001 0.060
3. Predictors of intensity ≥ 3
Control vs. Exp <.0001 0.014
Bone 1. Predictors of intensity ≥ 1
Control vs. Exp 0.4950 1.375
2. Predictors of intensity ≥ 2
Control vs. Exp 0.0044 0.510
3. Predictors of intensity ≥ 3
Control vs. Exp 0.0763 0.381
Middle third 1. Predictors of intensity ≥ 1
Control vs. Exp 0.2785 0.619
2. Predictors of intensity ≥ 2
Control vs. Exp <.0001 0.158
3. Predictors of intensity ≥ 3
Control vs. Exp No statistics derived due to lack of numbers
For the sections which did not show definite ankylosis, there were no consistent
differences in PLAP-1 expression between control and experimental sides at each
time point or region (Table 2). This included slides which theoretically should have
had ankylosis previously but may have resolved spontaneously.
Comparisons between repeat measurements show a weighted Kappa coefficient of
0.95 (95% CI 0.92, 0.97), indicating very good agreement.
74
Table 2: Comparison in PLAP-1 intensity between control and traumatised sides with no discernible ankylosis.
PDL region Days Compared
groups P-value Odds ratio
Cementum 7 Control vs Exp 0.1238 1.498
10 Control vs Exp 0.2767 1.412
14 Control vs Exp 0.0597 1.918
18 Control vs Exp 0.4792 0.833
21 Control vs Exp 0.8065 0.907
28 Control vs Exp 0.8390 0.882
Bone 7 Control vs Exp 0.2933 1.687
10 Control vs Exp 0.0856 2.893
14 Control vs Exp 0.0020 4.741
18 Control vs Exp 0.7555 1.122
21 Control vs Exp 0.0011 1.860
28 Control vs Exp 0.0707 1.841
middle 7 Control vs Exp 0.0036 3.185
10 Control vs Exp 0.0369 1.834
14 Control vs Exp 0.2563 2.143
18 Control vs Exp 0.0003 3.274
21 Control vs Exp 0.6198 1.257
28 Control vs Exp 0.4909 1.618
75
Discussion
The use of hypothermal trauma to induce ankylosis has been reported previously.25
Subsequent investigations of ankylosis using the same protocol found the production
of consistent and widespread osseous-like material bridging the periodontal ligament
space connecting the inter-radicular bone to the cementum of the root and it did not
subside at the end of the experimental time frame.22 The lack of consistent ankylosis
in the present study is likely due to subtle technique differences. As the cryotherapy is
delivered by dry ice pellets to a rat molar, it can be envisaged that numerous factors
such as size of dry ice pellets, the amount of pressure and the placement of the ice
pellets would contribute to the level of trauma delivered. This is suggested with many
of the sections containing ankylosis demonstrating a lack of viable osteocytes in the
crestal regions of the inter-radicular bone (presumably due to the hypothermic insult)
whilst this is not observed in sections without ankylosis. Nevertheless, the
spontaneous resolution of ankylotic areas in this study sample provided a unique
opportunity to investigate the maintenance mechanism of the periodontal ligament
space. Ideally, however, a reliable protocol should be established which allows for
consistent ankylosis formation whilst still allowing for spontaneous resolution to
occur; that is transient ankylosis.
Transient ankylosis, although rarely reported, is not new. Andreasen & Skougaard26,
Andersson27 and Blomlof & Lindskog28 all reported transient ankylosis. Whether
ankylosis is transient or permanent seems to depend on the size of the injured
region27. It is thought that with minor regions of insult, the root resorption craters are
repaired with cementum and ankylotic regions are replaced with vital periodontal
membrane. However, when the injury is severe and widespread, cells of the
periodontal ligament are not able to proliferate and replace the ankylotic areas hence
perpetuating the bony union.27 The tipping point between ankylosis and repair is not
well understood.
The finding of PLAP-1 expression in numerous regions of the maxilla is in
contradiction to the findings of Yamada & co-workers19 who found specific
76
expression of PLAP-1 within the PDL or its progenitor, the dental follicle. The
variation in findings may be due to methodological differences such as the use of
different primary antibody. However, a recent study29 also reported findings of
PLAP-1 expression within the dentine.
Lorenzo et al30 found PLAP-1/Asporin to be expressed in smooth muscle cells.
Theoretically, the smooth muscle cells present in the tunica media layer of the blood
vessel walls may lead to a positive reaction to anti-PLAP-1 antibodies. Unfortunately,
the tissues used in this study displayed no major vessels. However, there were
abundant capillaries with consistent and positive PLAP-1 staining which is limited to
its walls suggesting an association with endothelial cells.
The evidence of PLAP-1/Asporin expression in cartilage is somewhat conflicting.
Initial exploration of PLAP-1 at the organ level found it to be absent in mouse
articular cartilage31,32 but expressed in the perichondrium. Nakajima et al33 did show
expression of PLAP-1/Asporin, albeit minimally, in normal human articular cartilage.
However, in osteoarthritic cartilage, the expression of Asporin significantly
increases30,34. At a cellular level, Asporin is classified as an extracellular protein of
the LRR family group of proteins and hence should localise around cells in articular
cartilage.35 Indeed, Nakajima’s work33 suggested a cell surface localisation for
Asporin. However, Gruber et al.36 found positive Asporin staining to occur inside the
chondrocytes of rat articular cartilage and not in the extra-cellular matrix. Similar
results were found in the samples derived both from the spine of sand rat and humans.
They concluded Asporin to have a cytoplasmic localisation and also reported that
positive staining was not detected in the same cells of similar development. Whist
this study did not specifically investigate Asporin and cartilage, the observations of
the articular cartilage used as positive controls and the occasional nasal cartilage
agrees with the work of Gruber et al.36 in that PLAP-1/Asporin is found in cartilage
and located within the cytoplasm of the certain chondrocytes.
77
The periodontal ligament yielded diffuse staining of PLAP-1 which is likely due to
either background ‘noise’, cellularity of the region or its association with
proteoglycans in the extracellular matrix. In particular, fibroblasts which are the
principal cells of the PDL, are known to stain positive to PLAP-1.32 Ideally, the use
of monoclonal antibodies would have minimised background ‘noise’ and improve
specificity. However, at the time of writing, there was no commercially available anti-
PLAP-1 monoclonal antibody indicated for immunohistochemistry which is directed
at rat specimens.
Another possible cause for potential background staining was the thickness of the
sections. In this study, the sections were made at 7um but according to Dabbs37 the
ideal thickness is 3-5um for immunohistochemistry. It is believed that thicker
sections can increase background staining because it is difficult to block off the
endogenous peroxidase entirely thus reducing the signal-to-noise ratio especially
when a polyclonal antibody is being utilised.
The ambiguous results obtained make the results difficult to interpret. The cementum
third of the PDL which, under normal circumstances does not stain significantly with
PLAP-1, seems to have increased reactivity with PLAP-1 when osseous-like material
is formed. It would also appear that, albeit not statistically significant, most of the
other regions show a similar tendency. This would seem to suggest that PLAP-
1/Asporin is not the negative regulator of PDL mineralisation as Yamada and co-
workers19 suggested but rather it promotes osteoblast driven collagen mineralisation
as Kalamajski and colleagues38 found. However, as previously mentioned, ankylosis
is not necessarily permanent and resorptive activities including TRAP positive
multinucleated cells have been associated with the ankylotic bridge. Furthermore,
many of the positively stained areas aside from the ankylotic areas show resorptive
activity with multinucleated cells. However, the spicule nature of the ankylosis
renders it somewhat difficult to determine whether a resorptive bay was present and
indeed many of the positively stained cells alongside the cementum were not
associated with resorption lacunae. In addition, the differing results of Kalamajski38
and Yamada19 may be due to the source of cells used in their experiment; Yamada19
78
used cells derived specifically from PDL whereas Kalamajski38 did not.29 Further
investigations are required to clarify the role and function of PLAP-1 within the
periodontal ligament.
Conclusion
• The current study demonstrated that the periodontal ligament is not the only
region in which the PLAP-1 protein is located. PLAP-1/Asporin can be found
in nasal cartilage, blood vessel walls, periodontal ligament and the dental pulp.
• The data derived are inconclusive, although suggestive, of PLAP-1 being more
associated with mineralisation than maintenance of the periodontal ligament
space.
• The null hypothesis was rejected as significant differences were found,
particularly adjacent to the cementum, between the PLAP-1 expressions of
ankylotic regions compared to non-ankylotic areas.
Acknolwedgements
We thank the Australian Dental Research Foundation and the Australian Society of
Orthodontists Foundation for Research and Education for their generous support in
funding this project. Finally, we would like to thank Tom Sullivan from the
Department of Population Oral Health, University of Adelaide for his expert statistical
help.
Approval of the experimental procedures was provided by the Ethics Committee of
The University of Adelaide under ethics number M-01-2004.
There is no foreseeable conflict of interest
79
References
1. Andreasen JO, Andreasen FM, Andersson L. Textbook and color atlas of traumatic injuries to the teeth. Oxford, UK: Blackwell; 2007.
2. Berkovitz BKB, Shore R. Cells of the periodontal ligament. In: Berkovitz BKB, Moxham B, Newman H, editors. The Periodontal Ligament in Health and Disease. London: Mosby-Wolfe; 1995. p. 9-34.
3. Fujiyama K, Yamashiro T, Fukunaga T, Balam TA, Zheng L, Takano-Yamamoto T. Denervation resulting in dento-alveolar ankylosis associated with decreased Malassez epithelium. J Dent Res 2004;83:625-629.
4. Lindskog S, Blomlof L, Hammarström L. Evidence for a role of odontogenic epithelium in maintaining the periodontal space. J Clin Periodontol 1988;15:371-373.
5. Löe H, Waerhaug J. Experimental replantation of teeth in dogs and monkeys. Arch Oral Biol 1961;3:176-184.
6. Spouge JD. A new look at the rests of Malassez. A review of their embryological origin, anatomy, and possible role in periodontal health and disease. J Periodontol 1980;51:437-444.
7. Kirkham J, Brookes SJ, Shore RC, Bonass WA, Robinson C. The effect of glycosylaminoglycans on the mineralization of sheep periodontal ligament in vitro. Connect Tissue Res 1995;33:23-29.
8. Lekic P, McCulloch CA. Periodontal ligament cell population: the central role of fibroblasts in creating a unique tissue. Anat Rec 1996;245:327-341.
9. Melcher AH, Cheong T. Fibroblast-like cells depress formation of bone-like tissue in vitro. Journal of Dental Research 1988;67:290.
10. Ogiso B, Hughes FJ, Davies JE, McCulloch CA. Fibroblastic regulation of osteoblast function by prostaglandins. Cell Signal 1992;4:627-639.
11. Ogiso B, Hughes FJ, Melcher AH, McCulloch CA. Fibroblasts inhibit mineralised bone nodule formation by rat bone marrow stromal cells in vitro. J Cell Physiol 1991;146:442-450.
12. Ohno S, Doi T, Fujimoto K, Ijuin C, Tanaka N, Tanimoto K et al. RGD-CAP (betaig-h3) exerts a negative regulatory function on mineralization in the human periodontal ligament. J Dent Res 2002;81:822-825.
13. Li S, Yang PS, Cao JF, Ge SH, Pan KQ. [Expression of epidermal growth factor receptor in human periodontal ligament cells during their mineralization in vitro]. Hua Xi Kou Qiang Yi Xue Za Zhi 2006;24:11-14.
80
14. Duarte WR, Iimura T, Takenaga K, Ohya K, Ishikawa I, Kasugai S. Extracellular role of S100A4 calcium-binding protein in the periodontal ligament. Biochem Biophys Res Commun 1999;255:416-420.
15. Kato C, Kojima T, Komaki M, Mimori K, Duarte WR, Takenaga K et al. S100A4 inhibition by RNAi up-regulates osteoblast related genes in periodontal ligament cells. Biochem Biophys Res Commun 2005;326:147-153.
16. Yoshizawa T, Takizawa F, Iizawa F, Ishibashi O, Kawashima H, Matsuda A et al. Homeobox protein MSX2 acts as a molecular defense mechanism for preventing ossification in ligament fibroblasts. Mol Cell Biol 2004;24:3460-3472.
17. Komaki M, Karakida T, Abe M, Oida S, Mimori K, Iwasaki K et al. Twist negatively regulates osteoblastic differentiation in human periodontal ligament cells. J Cell Biochem 2007;100:303-314.
18. Tomoeda M, Yamada S, Shirai H, Ozawa Y, Yanagita M, Murakami S. PLAP-1/asporin inhibits activation of BMP receptor via its leucine-rich repeat motif. Biochem Biophys Res Commun 2008;371:191-196.
19. Yamada S, Tomoeda M, Ozawa Y, Yoneda S, Terashima Y, Ikezawa K et al. PLAP-1/asporin, a novel negative regulator of periodontal ligament mineralization. J Biol Chem 2007;282:23070-23080.
20. Chen WCW. An investigation into the role of osteoclasts and their precursors in an ankylosis model. B Sci Dent (Hons) Thesis. University of Adelaide, Adelaide; 2008.
21. Dreyer CW. Clast cell activity in a model of aseptic root resorption. PhD thesis, University of Adelaide; 2002.
22. Shaboodien SI. Traumatically induced dentoalveolar ankylosis in rats. D.Clin.Dent. thesis, University of Adelaide; 2005.
23. Curl L, Sampson, W. The presence of TNF-α and TNFR1 in aseptic root resorption. A preliminary study. Australian Orthodontic Journal 2011;27:102-109.
24. Di Iulio DS. Relationship of epithelial cells and nerve fibres to experimentally induced dentoalveolar ankylosis in the rat. D.Clin.Dent, University of Adelaide; 2007.
25. Dreyer CW, Pierce AM, Lindskog S. Hypothermic insult to the periodontium: a model for the study of aseptic tooth resorption. Endod Dent Traumatol 2000;16:9-15.
26. Andreasen JO, Skougaard MR. Reversibility of surgically induced dental ankylosis in rats. Int J Oral Surg 1972;1:98-102.
81
27. Andersson L. Dentoalveolar ankylosis and associated root resorption in replanted teeth. Experimental and clinical studies in monkeys and man. Swed Dent J Supplements 1988;56:1-75.
28. Blomlof L, Lindskog, S. Quality of periodontal healing. II: Dynamics of reparative cementum formation. Swed Dent J 1994;18:131-138.
29. Lee E-H, Park H-J., Jeong, J-H., Kim, Y-J., Cha, D-W., Kwon, D-K., Lee, S-H., Cho, J-Y. The role of Asporin in mineralization of human dental pulp stem cells. J. Cell. Physiol 2011;226:1676-2682.
30. Lorenzo P, Aspberg A, Onnerfjord P, Bayliss MT, Neame PJ, Heinegard D. Identification and characterization of asporin. a novel member of the leucine-rich repeat protein family closely related to decorin and biglycan. J Biol Chem 2001;276:12201-12211.
31. Henry SP, Takanosu M, Boyd TC, Mayne PM, Eberspaecher H, Zhou W et al. Expression pattern and gene characterization of asporin. a newly discovered member of the leucine-rich repeat protein family. J Biol Chem 2001;276:12212-12221.
32. Kou I, Nakajima, M., Ikegawa, S. Expression and regulation of the osteoarthritis-associated protein Asporin. The Journal of Biochemistry 2007;282:32193-32199.
33. Nakajima M, Kizawa H, Saitoh M, Kou I, Miyazono K, Ikegawa S. Mechanisms for asporin function and regulation in articular cartilage. J Biol Chem 2007;282:32185-32192.
34. Kizawa H, Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., Ikegawa, S. An aspartic acid repeat polymorphism in asporin inhibits chondrogenesis and increases susceptibility to osteoarthritis. Nature Genetics 2005;37:138-144.
35. Ikegawa S. Expression, regulation and function of asporin, a susceptibility gene in common bone and joint diseases. Curr Med Chem 2008;15:724-728.
36. Gruber HE, Ingram JA, Hoelscher GL, Zinchenko N, Hanley EN, Jr., Sun Y. Asporin, a susceptibility gene in osteoarthritis, is expressed at higher levels in the more degenerate human intervertebral disc. Arthritis Res Ther 2009;11:R47.
37. Dabbs DJ. Diagnostic immunohistochemistry. New York, Edinburgh: Churchill Livingstone; 2006.
38. Kalamajski S, Aspberg, A., Lindblom, K., Heinegard, D., Oldberg, A. Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization. Biochem. J. 2009;423:53-59.
82
PAPER 2 (Prepared for submission in Archives of Oral Biology)
Title: Expression of Periodontal Ligament Associated Protein-1/Asporin in the Coronal Pulp Chamber of Rats Following Hypothermic Trauma. A Preliminary Report.
Authors: Chen, W.C., Sampson, W., Dreyer, C., Dharmapatni, K.
Affiliations:
Chen, W.C. – Postgraduate student, Orthodontic Unit, School of Dentistry, Faculty of
Health Sciences, University of Adelaide, South Australia.
Sampson, W. – Professor and P.R. Begg Chair, Orthodontic Unit, School of Dentistry,
Faculty of Health Sciences, University of Adelaide, South Australia.
Dreyer, C. – Associate Professor, Orthodontic Unit, School of Dentistry, Faculty of
Health Sciences, University of Adelaide, South Australia.
Dharmapatni, K. – Research Fellow, Hanson Institute, School of Medicine, Faculty of
Health Sciences, University of Adelaide, South Australia.
Abstract
Background: The dental pulp has several important functions including the formation
of the dentine, provision of nutrients, provision of neurosensory information as well
as the provision of protection via the formation of tertiary dentine. Recently, a
protein named PLAP-1/Asporin has been shown to be associated with the
differentiation and mineralisation of dental pulp stem cells. However, there are few
and conflicting reports regarding the role of PLAP-1/Asporin within mature dental
pulps. Furthermore, there are no reports regarding any association with the
formation of tertiary dentine.
83
Objectives: The aim of this investigation is to determine whether PLAP-1 protein is
expressed within the dentine-pulp complex and to determine its relationship with the
dentine-pulp complex following hypothermic trauma.
Materials & Methods: The maxillary right first molars of 30 Sprague-Dawley rats
were subjected to a single 20 minute application of dry ice to induce ankylosis.
Groups of five animals were sacrificed via cardiac perfusion after 7, 10, 14, 18, 21
and 28 days, respectively. The maxillae were dissected out and underwent routine
tissue fixation and processing. PLAP-1 expression was subsequently detected using
immunohistochemistry and analysed semi-quantitatively.
Results: PLAP-1/Asporin was found to be expressed exclusively within the pulp under
normal conditions and appeared to be associated with the odontoblastic and cell-rich
zone. Following trauma, PLAP-1/Asporin expression decreased marginally (not
statistically significant) alongside the dentine but increased significantly in the
central pulpal region where there was disruption and cellular breakdown.
Conclusions: PLAP-1/Asporin can be found within the mature dental pulp under
normal conditions and is particularly associated with the odontoblast layer and to a
lesser extent the cell rich zone. Under hypothermic trauma, PLAP-1/Asporin does not
appear to play a role in the formation of the tertiary dentine.
Key Words: periodontal ligament associated protein-1, asporin, dental pulp.
Introduction
The primary function of the dental pulp is the formation of dentine through the actions
of the odontoblasts. Other functions of the pulp include the provision of nutrients as
well as neurosensory information such as pain, pressure or temperature differences.
The dental pulp is also able to provide protection due to the formation of reparative
dentine following a traumatic episode.
84
The main cellular constituents are odontoblasts, fibroblasts, mesenchymal stem cells,
macrophages and lymphocytes. The extracellular components of the pulp are
composed of ground substance (eg. glycosaminoglycans) and collagen fibres (mainly
type I & III). Additionally, the pulp contains blood vessels, nerve fibres as well as
lymphatic vessels.1
Recently, a novel protein denoted Periodontal Ligament Associated Protein-1 (PLAP-
1/Asporin) has been found to be associated with the differentiation and mineralisation
of dental pulp stem cells.2 Furthermore, specific staining was found at the globular
calcific region in the junction of predentine and dentine. This finding was in
agreement with Kalamajski et al3 who also found Asporin to promote osteoblast-
driven collagen mineralization. However, contradictory results were reported by
Yamada et al4 who found PLAP-1 to be unique to the periodontal ligament as well as
being a negative regulator for PDL mineralisation.
PLAP-1 was originally characterised in 2001 and is considered a member of the
leucine-rich repeat proteoglycan family.5,6 It has been found to be similar to biglycan
and decorin (human Asporin is 50% identical and 70% similar to decorin and
biglycan)5. However, as PLAP-1 does not contain glycosaminoglycan attachment
sites6 and contains a unique sequence of aspartate residues, it is not considered a true
proteoglycan7. The normal structure of PLAP-1/Asporin contains a putative
propeptide, 4 aminoterminal cysteines, 10 leucine rich repeats and 2 C-terminal
cysteines.
Despite the pioneering work of Yamada et al4 and Lee et al2, there is a scarcity of
literature regarding asporin and its expression and association within the dental pulp.
Furthermore, there are no studies which report on the relationship of PLAP-1/Asporin
with the pulp and odontoblast-derived reparative dentine following a traumatic
episode.
85
In this article, the findings of PLAP-1 and its expression within the dental pulp under
normal as well as post-trauma conditions, is reported.
Aim
• To determine the normal expression of PLAP-1/Asporin within the pulp
chamber using immunohistochemistry.
• To investigate the relationship between PLAP-1/Asporin expression and the
coronal pulp following a hypothermic insult using immunohistochemistry.
Hypothesis
Null Hypothesis states that:
‘There is no change in the expression of PLAP-1 in traumatised pulp compared to
non-traumatised pulp.’
Materials & Methods
The materials utilised form the basis of another study which investigated the effect of
trauma on PLAP-1 within the periodontal ligament. The maxillary right first molars
of 30, 8-week-old Sprague-Dawley rats were subjected to a single 20 minute
application of dry ice which was considered adequate to induce ankylosis, while the
contralateral first molar served as a control. Anaesthesia was achieved via a 1:1
combination of Hypnorm® (Janssen-Cilag Ltd., Buckinghamshire, UK) and
Hypnovel® (Roche, Berne, Switzerland). Groups of five animals were sacrificed via
cardiac perfusion after 7, 10, 14, 18, 21 and 28 days, respectively. The maxillae were
dissected out and underwent routine tissue fixation and processing. The tissues were
embedded in paraffin wax and 7µm serial coronal sections were obtained, limited to
the furcation area.
86
The staining for PLAP-1 immunoreactivity was performed via the Labelled
StreptAvidin Biotin method. The primary antibody was an unlabelled rabbit
polyclonal antibody (450-31930, Sapphire Bioscience, NSW, Australia) diluted at 1 to
400 of stock concentration, incubated overnight in a wet chamber at room temperature
(RT). The linkage reagent was a biotinylated goat anti-rabbit secondary antibody
which is conjugated to horseradish peroxidase (K060911, LSAB®2-HRP, Dako,
Australia) and was incubated at 30 minutes, RT. Visualisation of the target protein
(PLAP-1) was through the use of AEC dye (K3469, Dako, Australia). Sections were
then counterstained with haematoxylin and lithium carbonate. Growing rat femoral
head containing cartilage was used as the positive controls as it has been previously
reported that chondrocytes express PLAP-1/Asporin.36 Sections incubated only with a
rabbit serum (N169987, Universal negative control, Dako, Australia) were used as the
negative control.
Stained slides were mounted and analysed subjectively using an Olympus B071
optical microscope with the images displayed by an attached personal computer with
a 24.0 inch monitor. The software programme Analysis (Olympus Soft Imaging
Solutions, Germany) was used for image processing and viewing. Various
magnifications including 4X, 10X and 20X were used to inspect the staining. Due to
the diffuse nature of the stain, a semi-quantitative scoring method was utilised (0=no
staining, 1=mild staining; 2=moderate staining, 3=intense staining) to measure the
activity of PLAP-1.
Fifteen sections 70µm apart per rat were stained and analysed. Wald statistics for
Type 3 GEE (General Estimating Equation) Analysis as well as Post-hoc comparisons
(see Appendices) were made. One section from each rat was randomly selected for
repeat scoring and the intra-observer reliability was calculated with a weighted Kappa
coefficient. All calculations performed by SAS Version 9.2 (SAS Institute Inc., Cary,
NC, USA).
Results
87
PLAP-1/Asporin was found to be positively expressed within the dental pulp. In
particular, PLAP-1 appears to be associated with the odontoblast layer as well as the
cell rich zone (Figure 1). There were no other regions in which the protein was
expressed within the crown of the rat molar (Figure 1).
Figure 1
a) PLAP-1 staining of the pulp chamber on the control side (10x magnification; 10 weeks old rat). Note the affiliation with the odontoblast/cell rich zone. b) 20 x magnification of the same section. c) Negative control of pulp staining displaying the lack of PLAP-1 staining (10 x magnification; 10 weeks old rat). d) Negative control of pulp staining displaying the lack of PLAP-1 staining (20 x magnification).
Unfortunately, the amount of trauma delivered may not have been equal amongst all
specimens as not all sections acquired ankylosis within the furcation region.
Moreover, some of the sections could not be included for analysis due to crucial
regions (especially the crown and pulp) being either lost or damaged during the
antigen retrieval process. In addition, it was found that despite identical staining
procedures and environmental conditions, there were great variations between animals
in the intensity of PLAP-1 expression.
a b
c d
dentine
dentine
pulp pulp
Note the increased affinity of PLAP-1 staining with the odontoblast/cell rich zone
pulp pulp
dentine dentine
200µm
100µm
200µm
100µm
88
Within the sections which did not display ankylosis, there were no consistent
statistically significant differences between control and experimental sides across the
various time points (results not shown but see page 113 in Appendices). This was
consistent for the area adjacent to the dentine and also in the central pulp. However,
there was still evidence of the freezing trauma due to the disorganisation and
breakdown of the pulpal cellular structures.
Figure 2
PLAP-1 pulp staining in control (a) and experimental side which did not achieve ankylosis (b). Note the lack of difference in staining intensity distribution. (Day 18 rat)
In the slides which did obtain ankylosis, the region adjacent to the dentine showed
statistically significant differences only when the staining intensity was grade 3.
However, the Odds Ratio consistently showed an increased likelihood of the control
side having more intense PLAP-1 reactivity than the experimental side (Table 1). The
region in the central part of the pulp showed consistent difference in that the
experimental pulp displayed statistically significantly more staining of PLAP-
1/Asporin than the control side (Table 2). A statistical analysis could not be
conducted for staining intensity of grade 3 due to a lack of sections with high intensity
staining within the central pulpal region.
a b
dentine
PDL
Alveolar bone
dentine
PDL
pulp pulp
Alveolar bone
200µm 200µm
89
Table 1. Comparison of PLAP-1 staining intensities within the pulp adjacent to the dentine in sections with ankylosis.
Model Contrast
P-value
Odds ratio
Lower 95% CI
Upper 95% CI
1. Predictors of intensity ≥ 1
Control vs. experimental
0.5450 1.473 0.420 5.159
2. Predictors of intensity ≥ 2
Control vs. experimental
0.2842 1.439 0.739 2.799
3. Predictors of intensity ≥ 3
Control vs. experimental
0.0095 1.533 1.110 2.116
Table 2. Comparison of PLAP-1 staining intensities within the central pulpal region in sections with ankylosis
Model Contrast
P-value
Odds ratio
Lower 95% CI
Upper 95% CI
1. Predictors of intensity ≥ 1
Control vs. experimental
<.0001 0.439 0.322 0.599
2. Predictors of intensity ≥ 2
Control vs. experimental
<.0001 0.102 0.063 0.166
3. Predictors of intensity ≥ 3
Control vs. experimental
Model did not converge
Figure 3
a) Increased PLAP-1 staining within the central pulpal region. b) The same section showing the largely homogenous PLAP-1 staining within the pulp chamber. Note the lack of obvious odontoblast/cell rich zones. (Day 18 rat)
b a
dentine
dentine
pulp
pulp Note the increase in staining in the central pulpal region
200µm
100µm
90
Regardless of whether ankylosis was achieved or not, there was evident formation of
tertiary dentine in the experimental side. This was true from day 10 onwards although
some sections from day 7 also displayed mild formation of tertiary dentine. Most of
the tertiary dentine exhibited regular structure with few cellular inclusions although
some sections displayed notable cellular inclusions (fig 4). Some of the cellular
inclusions stained positive for PLAP-1/Asoprin. No control side displayed the
formation of tertiary dentine.
Figure 4
Experimental section showing clear formation of tertiary dentine with obvious cellular inclusions. (Day 21 rat)
Comparisons between repeat measurements show a weighted Kappa coefficient of
0.95 (95% CI 0.92, 0.97), indicating very good agreement.
Discussion
The traumatic episode was generated by the application of a hypothermic insult as
part of another investigation.8 Whilst the aseptic injury from the dry ice application
maintains good tissue morphology and offers an insight to Asporin/PLAP-1
expression under trauma and reparative dentinogenesis, the results cannot be directly
translated to the classic process of reparative dentinogenesis caused by dental caries
which is due to bacterial insults.
dentine
Cellular inclusions
pulp
200µm
91
There is a lack of information regarding PLAP-1/Asporin and the dental pulp in the
dental literature. The result of this study is in contrast with Yamada et al4 who found
PLAP-1/Asporin to exist specifically within the periodontal ligament in the mouse
maxilla. The result also varies from that described by Lee et al2 who found PLAP-
1/Asporin to be found only in the globular calcific region at the junction of predentine
and dentine in post-natal mice. Differences in the experimental protocol likely
contributed to the differences. Park et al9 found the pulpal staining to be non-specific
and likely due to the antibody utilised or the high endogenous peroxidase activity.
However, in this study, there was obvious affinity of anti-PLAP-1/Asporin antibody
to the cell-rich zone of the pulp. This may be associated with fibroblasts and is in
agreement with Henry et al5 who also found prominent expression of PLAP-1/Asporin
associated with fibroblasts.
The increase in staining within the central region of the frozen pulp did not appear
histologically to relate to any particular cells or pattern. It appears that the increase in
staining is a result of the breakdown of cellular constituents within the pulp and their
structure thus leading to an increase in PLAP-1/Asporin staining within the centre and
a tendency to show decreased staining in the region adjacent to the dentine.
The lack of difference between the control and experimental sides in sections which
did not obtain ankylosis is likely due the lack of overall trauma delivered. The lack of
trauma possibly did not induce adequate damage to induce widespread cell death
within the pulp and periodontal ligament thus failing to obtain ankylosis as well as
significant cellular disruption within the dental pulp.
The formation of reparative dentine suggests that some odontoblasts survived the
hypothermic insult. However, judging by the morphological breakdown of cellular
structures within the pulp, it seems logical that a large number of odontoblasts would
not have survived but restoration was achieved through differentiation of
mesenchymal cells to odontoblasts. According to the findings of a previous study2,
PLAP-1/Asporin is expressed in the early stages of odontoblast differentiation and
92
may have a positive role in the minerlization of dental pulp stem cells under normal
developmental conditions. However, in this study, the lack of PLAP-1/Asporin
within the dentine, in addition to a decrease in staining intentisty adjacent to the
dentine, suggests that the protein is not involved in the mineralization of the tertiary
dentine. This suggests that although PLAP-1/Asporin may be involved in the
mineralization of dentine in normal development, it is not involved in the formation or
mineralization of tertiary dentine.
Conclusion
• PLAP-1/Asporin is positively expressed within the healthy dental pulp and is
particularly associated with the odontoblastic and cell-rich zones.
• PLAP-1/Asporin expression does appear to change (tendency to decrease in
peripheral regions and increase in central pulpal area) post hypothermic
trauma but is more likely to be due to the breakdown of the cellular structure.
• PLAP-1/Asporin is not directly associated with the formation of tertiary
dentine.
Acknolwedgements
We thank the Australian Dental Research Foundation and the Australian Society of
Orthodontists Foundation for Research and Education for their generous support in
funding this project. Also, we would like to thank Tom Sullivan from the Department
of Population Oral Health, University of Adelaide for his statistical advice.
Approval of the experimental procedures was provided by the Ethics Committee of
The University of Adelaide under ethics number M-01-2004.
There is no foreseeable conflict of interest
93
References
1. Ten Cate AR. Oral Histology: structure and function. St Louis: Mosby; 1989.
2. Lee E-H, Park H-J., Jeong, J-H., Kim, Y-J., Cha, D-W., Kwon, D-K., Lee, S-H., Cho, J-Y. The role of Asporin in mineralization of human dental pulp stem cells. J. Cell. Physiol 2011;226:1676-2682.
3. Kalamajski S, Aspberg, A., Lindblom, K., Heinegard, D., Oldberg, A. Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization. Biochem. J. 2009;423:53-59.
4. Yamada S, Tomoeda M, Ozawa Y, Yoneda S, Terashima Y, Ikezawa K et al. PLAP-1/asporin, a novel negative regulator of periodontal ligament mineralization. J Biol Chem 2007;282:23070-23080.
5. Henry SP, Takanosu M, Boyd TC, Mayne PM, Eberspaecher H, Zhou W et al. Expression pattern and gene characterization of asporin. A newly discovered member of the leucine-rich repeat protein family. J Biol Chem 2001;276:12212-12221.
6. Lorenzo P, Aspberg A, Onnerfjord P, Bayliss MT, Neame PJ, Heinegard D. Identification and characterization of asporin. a novel member of the leucine-rich repeat protein family closely related to decorin and biglycan. J Biol Chem 2001;276:12201-12211.
7. Ikegawa S. Expression, regulation and function of asporin, a susceptibility gene in common bone and joint diseases. Curr Med Chem 2008;15:724-728.
8. Chen WCW. An Investigation into the Role of Periodontal Ligament Associated Protein-1 in the Maintenance of the Periodontal Ligament using an Ankylosis Model Adelaide, Doctor of Clinical Dentistry thesis, University of Adelaide; 2012.
9. Park ES, Cho, H.S., Kwon, T.G., Jang, S.N., Lee, S.N., An, C.H., Shin, H.I., Kim, J.Y., Cho, J.Y. Proteomics analysis of human dentin reveals distinct protein expression profiles. Journal of Proteome Research 2009;8:1338-1346.
94
Concluding Remarks The results from the first paper showed that PLAP-1 was expressed in the PDL, dental
pulp, blood vessel walls and the nasal cartilage. Not all sections obtained ankylosis.
Sections which did not obtain ankylosis demonstrated no significant PLAP-1
expression differences between control and experimental sides. Sections that did
obtain ankylosis yielded a tendency towards increased PLAP-1 reactivity especially
near the cementum. However, it was difficult to deduce whether the relationship of
PLAP-1 to the ankylotic union was associated with bone formation or resorptive
activities.
The results from paper two showed that PLAP-1/Asporin was expressed exclusively
within the pulp under normal conditions and appeared to be associated with the
odontoblastic and cell rich zone. Following trauma, PLAP-1/Asporin expression
decreased marginally (not statistically significant) alongside the dentine but increased
significantly in the central pulpal region possibly due to disruption and breakdown of
the cellular structures.
From the results derived, it can be concluded that PLAP-1/Asporin is indeed
expressed in several tissue/cell types and regions including the dental pulp and is not
exclusively associated with the periodontal ligament. In addition, PLAP-1 appears to
have a direct association with ankylosis although it is uncertain whether PLAP-1
exclusively facilitates bone mineralisation or resorption. The second null hypothesis
was also rejected although the change in expression of PLAP-1 within the pulp is
probably more morphological than physiological. Results from the study also suggest
that PLAP-1/Asporin does not appear to play a role in the formation of the tertiary
dentine.
Further research is required to elucidate the true role of PLAP-1 within the
periodontal ligament as well as the pulp.
95
APPENDICES Solutions
Anticoagulant - Heparin
Heparin Injection B.P. (containing no antiseptic) was supplied in 1ml plastic
ampoules (David Bull Laboratories, Mulgrave, Australia).
Contained 1000 units (IU) per 1ml.
Dosage: 0.02 ml of heparin sodium per 100 g of body weight
Route: Intravenous injection via femoral vein
Shelf: Discard unused heparin after vial seal is broken
Storage: Below 25º
Phosphate Buffer
Reagents:
Part A.
31.2g NaH2PO4.2H2O in 1 litre of distilled water (0.2M)
Part B.
28.39g Na2HPO4 in 1 litre distilled water (0.2M)
Procedure:
Mix 240ml of Part A and 760ml of Part B to make 1 litre
Phosphate Buffered Saline
Reagents:
Saline solution
8.79g NaCl in 1 L of distilled water (0.879%)
Part A phosphate buffer
Part B phosphate buffer
Procedure:
To 19ml of Part A and 81ml of Part B, add 100ml of saline solution to make
200ml with a pH of 7.4
Fixative : 4% paraformaldehyde / 0.1M phosphate buffer pH7.4
Reagents:
Paraformaldehyde
96
Sodium hydroxide
Distilled water
Preparation:
1) Heat up 1600ml of distilled water
2) Add 160g of paraformaldehyde
3) Add sodium hydroxide dropwise to clarify
4) Add 800ml of 0.4M sodium phosphate buffer pH 7.4
5) Add distilled water to make up 4000ml of solution in total.
Decalcifying agent
4% EDTA in phosphate buffer
Reagents:
Phosphate buffer - Parts A and B
EDTA - 80g
Procedure:
To 280ml of part A and 720ml of part B add 1litre of distilled water and
EDTA to give a pH of 7
Solutions for Immunostaining
Phosphate Buffered Saline
- Different to that used for tissue storage
- 5X PBS was made and then diluted when required
Reagents:
Na2HPO4 (anhydrous) 6.04g
NaH2PO4H20 3.93g
NaCl 45g
Mili Q water
Hydrochloric acid
Sodium hydroxide
Preparation:
1) Mix all reagents to dissolve
2) Adjust pH to 7.4 using either hydrochloric acid or sodium hydroxide (Bring
overall solution volume to 1L & store at room temperature)
97
Methanolic Hydrogen Peroxide Blocking Solution
- Courtesy of the Hanson Institute, Adelaide
- To be made on the day
Reagents & Preparation:
1) Obtain 250ml of absolute methanol and add
2) 4.15ml of 30% hydrogen peroxide
Tissue dehydration and paraffin embedding
The following automatic procedure was used for the impregnation of tissues with
paraffin wax prior to embedding, using a Shandon Citadel 2000 automatic processor
(Shandon Industries, Pittsburgh, Pennsylvania):
1. 70% ethyl alcohol 1 hour
2. 80% ethyl alcohol 3 hours
3. 90% ethyl alcohol 3 hours
4. 100% ethyl alcohol 4 hours
5. 100% ethyl alcohol 4 hours
6. 100% ethyl alcohol 4 hours
7. 100% histolene 4 hours
8. 100% histolene 5 hours
9. 100% histolene 5 hours
10. paraffin 7 hours
11. paraffin 7 hours
12. paraffin (under vacuum) 1 hour
Tissues were then further trimmed and embedded in Surgi Path® embedding media
using a Reichert Jung Histostat Embedding Centre.
Slide coating procedure
Slides were coated according to the following procedure using 3-
aminopropyltriethoxysilane (APT, Sigma code 36480).
1. Place slides in racks
2. Pre-rinse slides in 100% ethanol for 30 seconds, twice
3. Dip in 2% APT in ethanol for 2 minutes
4. Rinse in distilled water for 30 seconds, twice
5. Dry in 37° oven
98
Immunohistochemistry staining protocol
- Rat femur including articular cartilage as positive control
- Rabbit serum (N169987, Universal negative control, Dako, Australia) were
used as the negative control.
Day 1
1. Dewax all paraffin slides as usual
a. Histolene 1 for 10 minutes
b. Histolene 2 for 10 minutes
c. 95% ethanol for 5 minutes
d. 100% ethanol for 5 minutes
2. Then wash in Milli Q water for 2 x 5 minutes
3. Use pap pen to circle the sections
a. Use proteinase K 1/50 (100µg/ml)
b. Incubate in 37° D ro4 30 minutes
4. Wash in 1 x PBS 3 times for 5 minutes
5. Place methanolic hydrogen peroxide block on sections for 10 minutes
6. Wash in PBS 3 times for 5 minutes
7. Place normal horse serum (Vectastain, Australia) on slides for 60 minutes
8. Place polyclonal rabbit anti-Asporin primary antibody (LS-CS1930, Lifespan
Biosciences, Australia) at 1:400 dilution of stock concentration overnight in a
wet chamber at room temperature.
Day 2
9. Wash in PBS 3 times for 5 minutes
10. Place biotinylated goat anti-rabbit secondary antibody and incubate for 30
minutes (K060911, LSAB®2-HRP, Dako, Australia).
11. Wash in PBS 3 times for 5 minutes
12. Place streptavidin peroxidase (K060911, LSAB®2-HRP, Dako, Australia)
13. Wash in PBS 3 times for 5 minutes
14. Add AEC + substrate chromogen ( K346911, Dako, Australia) for 7 minutes
15. Wash with Mili Q water for 5 minutes
16. Counterstain
a. Hematoxylin for 10 seconds
99
b. Wash in tap water
c. Lithium carbonate for 30 seconds
d. Wash in tap water
17. Mount using Aquamount
Results
All calculations were performed using SAS Version 9.2 (SAS Institute Inc., Cary, NC, USA). 1. Analysis of PDL cementum intensity: no ankylosis in experimental side To compare PDL cementum intensity according to side of the mouth (experimental, control) and day of measurement, separate binary logistic generalised estimating equations were fitted to the data. In the models, side of the mouth, day and the interaction between side and day were included as predictor variables. Where the interaction term was not statistically significant, a second model excluding this term was fitted. Note that these models were chosen because: a) it was not reasonable to treat the outcome as being normally distributed with just 4 ordinal levels (hence the use of logistic models instead of ordinary linear regression models). b) a proportional odds model was explored but the proportionality assumption failed (hence used separate binary logistic regression models instead of a single proportional odds model). c) results from within the same rat were expected to be correlated, hence the use of generalised estimating equations instead of ordinary regression models. Model 1: predictors of intensity ≥ 1 Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 145.54 <.0001
side 1 0.57 0.4484
day*side 4 1851.91 <.0001
The table 'Wald Statistics For Type 3 GEE Analysis' shows the significance of predictor variables in the model. Since the model includes an interaction effect, this is the only term that needs to be interpreted. The highly significant interaction effect (p < 0.0001) suggests that the odds of having an intensity reading ≥ 1 depended on both side of mouth and time. That is, the difference between the two sides of the mouth changed over time (or alternatively that changes over time differed according to side of the mouth).
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental 0.1238 1.498 0.895 2.504
7 control 10 control 0.0173 1.923 1.123 3.293
100
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 10 experimental 0.0036 2.714 1.385 5.320
7 control 14 control 0.6779 1.192 0.521 2.725
7 control 14 experimental 0.0329 2.286 1.069 4.886
7 control 18 control 0.0914 2.036 0.892 4.647
7 control 18 experimental 0.2730 1.696 0.659 4.365
7 control 21 control <.0001 0.784 0.713 0.863
7 control 21 experimental 0.4117 0.711 0.315 1.606
7 control 28 control 0.2282 0.528 0.187 1.492
7 control 28 experimental 0.1778 0.465 0.153 1.416
7 experimental 10 control 0.4796 1.284 0.642 2.567
7 experimental 10 experimental 0.1954 1.813 0.737 4.459
7 experimental 14 control 0.7326 0.796 0.215 2.951
7 experimental 14 experimental 0.4361 1.526 0.527 4.424
7 experimental 18 control 0.5925 1.359 0.442 4.185
7 experimental 18 experimental 0.8302 1.133 0.362 3.542
7 experimental 21 control 0.0095 0.524 0.321 0.854
7 experimental 21 experimental 0.0207 0.475 0.253 0.892
7 experimental 28 control 0.1431 0.352 0.087 1.423
7 experimental 28 experimental 0.0588 0.311 0.092 1.045
10 control 10 experimental 0.2767 1.412 0.758 2.628
10 control 14 control 0.3495 0.620 0.228 1.688
10 control 14 experimental 0.5544 1.189 0.670 2.109
10 control 18 control 0.8440 1.059 0.599 1.871
10 control 18 experimental 0.7724 0.882 0.378 2.060
10 control 21 control 0.0002 0.408 0.256 0.651
10 control 21 experimental 0.0198 0.370 0.160 0.854
10 control 28 control 0.0249 0.275 0.089 0.849
10 control 28 experimental 0.0005 0.242 0.109 0.538
10 experimental 14 control 0.1124 0.439 0.159 1.213
10 experimental 14 experimental 0.7136 0.842 0.336 2.109
10 experimental 18 control 0.3978 0.750 0.385 1.461
10 experimental 18 experimental 0.1666 0.625 0.321 1.217
10 experimental 21 control <.0001 0.289 0.159 0.526
10 experimental 21 experimental 0.0068 0.262 0.099 0.691
10 experimental 28 control <.0001 0.194 0.101 0.373
10 experimental 28 experimental <.0001 0.171 0.087 0.339
14 control 14 experimental 0.0597 1.918 0.974 3.779
101
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
14 control 18 control 0.1677 1.708 0.798 3.656
14 control 18 experimental 0.4738 1.424 0.542 3.742
14 control 21 control 0.3376 0.658 0.280 1.548
14 control 21 experimental 0.5233 0.597 0.122 2.914
14 control 28 control 0.0667 0.443 0.185 1.058
14 control 28 experimental 0.2188 0.390 0.087 1.748
14 experimental 18 control 0.5590 0.891 0.604 1.314
14 experimental 18 experimental 0.4463 0.742 0.345 1.599
14 experimental 21 control 0.0043 0.343 0.165 0.715
14 experimental 21 experimental 0.0950 0.311 0.079 1.225
14 experimental 28 control 0.0092 0.231 0.077 0.696
14 experimental 28 experimental 0.0121 0.204 0.059 0.705
18 control 18 experimental 0.4792 0.833 0.503 1.381
18 control 21 control 0.0151 0.385 0.178 0.832
18 control 21 experimental 0.1291 0.349 0.090 1.359
18 control 28 control 0.0015 0.259 0.113 0.595
18 control 28 experimental 0.0042 0.229 0.083 0.628
18 experimental 21 control 0.0864 0.462 0.191 1.117
18 experimental 21 experimental 0.2491 0.419 0.095 1.839
18 experimental 28 control 0.0009 0.311 0.156 0.620
18 experimental 28 experimental 0.0336 0.274 0.083 0.904
21 control 21 experimental 0.8065 0.907 0.414 1.987
21 control 28 control 0.4410 0.673 0.246 1.843
21 control 28 experimental 0.3246 0.593 0.210 1.676
21 experimental 28 control 0.7099 0.742 0.155 3.565
21 experimental 28 experimental 0.3771 0.655 0.256 1.676
28 control 28 experimental 0.8390 0.882 0.261 2.972
Model 2: predictors of intensity ≥ 2 Same as above, but modelling the odds of the intensity score being ≥ 2. Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 13123.1 <.0001
side 1 0.00 0.9493
day*side 4 5.92 0.2050
102
Since the interaction effect was not statistically significant, there is no evidence to suggest that the difference between sides of the mouth depended on day of measurement. To be able to interpret the main effects of day and side, a second model excluding the interaction term was fitted. Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 1206.51 <.0001
side 1 0.00 0.9857
The model indicates there were no differences between the two sides, while there were significant changes over time (p < 0.0001).
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 10 0.0076 6.377 1.636 24.855
7 14 0.0129 3.155 1.276 7.801
7 18 0.1348 5.281 0.596 46.776
7 21 0.0261 1.877 1.078 3.270
7 28 <.0001 7.919 4.741 13.227
10 14 0.2044 0.495 0.167 1.467
10 18 0.8747 0.828 0.080 8.625
10 21 0.0614 0.294 0.082 1.060
10 28 0.7366 1.242 0.352 4.386
14 18 0.4895 1.674 0.388 7.219
14 21 0.1943 0.595 0.272 1.303
14 28 <.0001 2.510 1.602 3.933
18 21 0.3107 0.355 0.048 2.626
18 28 0.6401 1.499 0.274 8.193
21 28 <.0001 4.219 2.273 7.829
control experimental 0.9857 1.005 0.571 1.768
Model 3: predictors of intensity ≥ 3 Model did not converge (due to small number of observations that took the value 3). 2. Analysis of PDL bone intensity: no ankylosis in experimental side Model 1: predictors of intensity ≥ 1 Model did not converge. Model 2: predictors of intensity ≥ 2 Wald Statistics For Type 3 GEE Analysis
103
Source DF Chi-Square P value
day 4 75.86 <.0001
side 1 30.53 <.0001
day*side 4 25.84 <.0001
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental 0.2933 1.687 0.636 4.475
7 control 10 control 0.5128 0.765 0.344 1.705
7 control 10 experimental 0.1566 2.214 0.737 6.651
7 control 14 control 0.0322 1.246 1.019 1.523
7 control 14 experimental 0.0016 5.905 1.960 17.786
7 control 18 control 0.2995 1.703 0.623 4.658
7 control 18 experimental 0.1701 1.910 0.758 4.817
7 control 21 control 0.1582 3.030 0.650 14.130
7 control 21 experimental 0.0061 5.636 1.639 19.379
7 control 28 control 0.4258 1.546 0.529 4.522
7 control 28 experimental <.0001 2.847 1.863 4.351
7 experimental 10 control 0.0129 0.454 0.243 0.846
7 experimental 10 experimental 0.6323 1.312 0.431 3.999
7 experimental 14 control 0.5539 0.738 0.270 2.016
7 experimental 14 experimental 0.0123 3.500 1.313 9.331
7 experimental 18 control 0.9887 1.010 0.268 3.802
7 experimental 18 experimental 0.8478 1.132 0.318 4.028
7 experimental 21 control 0.5815 1.796 0.224 14.412
7 experimental 21 experimental 0.1683 3.341 0.601 18.585
7 experimental 28 control 0.8967 0.917 0.247 3.409
7 experimental 28 experimental 0.3315 1.688 0.587 4.851
10 control 10 experimental 0.0856 2.893 0.862 9.713
10 control 14 control 0.2623 1.627 0.695 3.812
10 control 14 experimental <.0001 7.714 3.224 18.459
10 control 18 control 0.2306 2.225 0.602 8.228
10 control 18 experimental 0.1058 2.496 0.824 7.560
10 control 21 control 0.1769 3.959 0.537 29.158
10 control 21 experimental 0.0192 7.364 1.385 39.151
10 control 28 control 0.3089 2.020 0.521 7.830
10 control 28 experimental 0.0065 3.719 1.443 9.584
10 experimental 14 control 0.2271 0.563 0.221 1.431
10 experimental 14 experimental 0.0008 2.667 1.501 4.737
10 experimental 18 control 0.4968 0.769 0.361 1.640
104
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
10 experimental 18 experimental 0.8239 0.863 0.235 3.168
10 experimental 21 control 0.6134 1.368 0.405 4.620
10 experimental 21 experimental 0.0381 2.545 1.053 6.155
10 experimental 28 control 0.0199 0.698 0.516 0.945
10 experimental 28 experimental 0.5022 1.286 0.617 2.679
14 control 14 experimental 0.0020 4.741 1.763 12.746
14 control 18 control 0.4959 1.368 0.555 3.367
14 control 18 experimental 0.3838 1.534 0.586 4.016
14 control 21 control 0.2128 2.433 0.601 9.851
14 control 21 experimental 0.0065 4.525 1.525 13.430
14 control 28 control 0.6342 1.242 0.509 3.028
14 control 28 experimental <.0001 2.286 1.809 2.889
14 experimental 18 control 0.0054 0.288 0.120 0.692
14 experimental 18 experimental 0.0540 0.324 0.103 1.020
14 experimental 21 control 0.3714 0.513 0.119 2.216
14 experimental 21 experimental 0.9371 0.955 0.301 3.029
14 experimental 28 control 0.0006 0.262 0.122 0.560
14 experimental 28 experimental 0.0969 0.482 0.204 1.141
18 control 18 experimental 0.7555 1.122 0.545 2.309
18 control 21 control 0.1670 1.779 0.786 4.027
18 control 21 experimental <.0001 3.309 2.022 5.415
18 control 28 control 0.7603 0.908 0.488 1.689
18 control 28 experimental 0.1690 1.671 0.804 3.475
18 experimental 21 control 0.5083 1.586 0.404 6.223
18 experimental 21 experimental 0.0595 2.950 0.958 9.091
18 experimental 28 control 0.7404 0.810 0.232 2.825
18 experimental 28 experimental 0.4162 1.490 0.570 3.899
21 control 21 experimental 0.0011 1.860 1.282 2.699
21 control 28 control 0.1598 0.510 0.200 1.304
21 control 28 experimental 0.9195 0.940 0.281 3.146
21 experimental 28 control <.0001 0.274 0.149 0.505
21 experimental 28 experimental 0.1304 0.505 0.208 1.224
28 control 28 experimental 0.0707 1.841 0.950 3.568
Model 3: predictors of intensity ≥ 3 Model did not converge.
105
3. Analysis of PDL mid intensity: no ankylosis in experimental side Model 1: predictors of intensity ≥ 1 Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 21.37 0.0003
side 1 13.27 0.0003
day*side 4 1157.47 <.0001
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental 0.0036 3.185 1.461 6.943
7 control 10 control 0.0904 3.058 0.839 11.149
7 control 10 experimental 0.0004 5.609 2.173 14.475
7 control 14 control 0.5876 1.486 0.355 6.227
7 control 14 experimental 0.0002 3.185 1.731 5.863
7 control 18 control 0.0328 0.573 0.344 0.956
7 control 18 experimental 0.2327 1.877 0.667 5.280
7 control 21 control 0.9934 0.995 0.332 2.982
7 control 21 experimental 0.7613 1.251 0.294 5.318
7 control 28 control 0.0049 0.233 0.085 0.642
7 control 28 experimental 0.2834 0.377 0.064 2.240
7 experimental 10 control 0.9423 0.960 0.318 2.898
7 experimental 10 experimental 0.0646 1.761 0.966 3.209
7 experimental 14 control 0.1900 0.467 0.149 1.459
7 experimental 14 experimental 1.0000 1.000 0.403 2.482
7 experimental 18 control <.0001 0.180 0.088 0.370
7 experimental 18 experimental 0.4232 0.589 0.162 2.150
7 experimental 21 control 0.0288 0.313 0.110 0.887
7 experimental 21 experimental 0.0388 0.393 0.162 0.953
7 experimental 28 control <.0001 0.073 0.025 0.218
7 experimental 28 experimental 0.0024 0.118 0.030 0.470
10 control 10 experimental 0.0369 1.834 1.038 3.243
10 control 14 control 0.0004 0.486 0.326 0.724
10 control 14 experimental 0.9514 1.042 0.280 3.871
10 control 18 control 0.0004 0.188 0.074 0.475
10 control 18 experimental 0.4109 0.614 0.192 1.965
10 control 21 control <.0001 0.326 0.206 0.513
106
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
10 control 21 experimental 0.0091 0.409 0.209 0.801
10 control 28 control 0.0058 0.076 0.012 0.474
10 control 28 experimental 0.0100 0.123 0.025 0.607
10 experimental 14 control <.0001 0.265 0.150 0.469
10 experimental 14 experimental 0.2157 0.568 0.232 1.391
10 experimental 18 control <.0001 0.102 0.051 0.204
10 experimental 18 experimental 0.0706 0.335 0.102 1.096
10 experimental 21 control <.0001 0.177 0.102 0.309
10 experimental 21 experimental <.0001 0.223 0.129 0.385
10 experimental 28 control <.0001 0.042 0.011 0.161
10 experimental 28 experimental 0.0001 0.067 0.017 0.265
14 control 14 experimental 0.2563 2.143 0.575 7.988
14 control 18 control 0.1055 0.386 0.122 1.223
14 control 18 experimental 0.7602 1.263 0.282 5.649
14 control 21 control 0.1047 0.670 0.413 1.087
14 control 21 experimental 0.5760 0.842 0.460 1.539
14 control 28 control 0.0525 0.157 0.024 1.021
14 control 28 experimental 0.1060 0.254 0.048 1.338
14 experimental 18 control <.0001 0.180 0.082 0.395
14 experimental 18 experimental 0.4281 0.589 0.159 2.179
14 experimental 21 control 0.0359 0.313 0.105 0.926
14 experimental 21 experimental 0.1907 0.393 0.097 1.592
14 experimental 28 control <.0001 0.073 0.031 0.170
14 experimental 28 experimental 0.0130 0.118 0.022 0.638
18 control 18 experimental 0.0003 3.274 1.731 6.190
18 control 21 control 0.2349 1.736 0.699 4.314
18 control 21 experimental 0.1690 2.183 0.718 6.638
18 control 28 control 0.1095 0.407 0.135 1.224
18 control 28 experimental 0.5639 0.658 0.159 2.727
18 experimental 21 control 0.3396 0.530 0.144 1.949
18 experimental 21 experimental 0.5858 0.667 0.155 2.865
18 experimental 28 control 0.0051 0.124 0.029 0.535
18 experimental 28 experimental 0.0354 0.201 0.045 0.896
21 control 21 experimental 0.6198 1.257 0.509 3.105
21 control 28 control 0.1063 0.234 0.040 1.363
21 control 28 experimental 0.3052 0.379 0.059 2.422
21 experimental 28 control 0.0511 0.186 0.034 1.008
107
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
21 experimental 28 experimental 0.0548 0.301 0.089 1.025
28 control 28 experimental 0.4909 1.618 0.411 6.368
Model 2: predictors of intensity ≥ 2 Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 262.31 <.0001
side 1 3.85 0.0496
day*side 4 36.87 <.0001
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental 0.7013 0.854 0.382 1.911
7 control 10 control 0.2749 1.625 0.680 3.885
7 control 10 experimental 0.1934 4.083 0.490 34.021
7 control 14 control 0.8472 0.900 0.308 2.628
7 control 14 experimental 0.7387 1.375 0.212 8.930
7 control 18 control 0.0007 0.250 0.113 0.555
7 control 18 experimental 0.6274 0.729 0.204 2.610
7 control 21 control 0.0303 0.400 0.175 0.916
7 control 21 experimental 0.1691 4.333 0.536 35.035
7 control 28 control 0.0002 0.514 0.360 0.734
7 control 28 experimental 0.0595 0.405 0.158 1.037
7 experimental 10 control 0.0541 1.902 0.989 3.660
7 experimental 10 experimental 0.2416 4.780 0.348 65.586
7 experimental 14 control 0.9279 1.054 0.339 3.271
7 experimental 14 experimental 0.5784 1.610 0.300 8.629
7 experimental 18 control 0.0593 0.293 0.082 1.049
7 experimental 18 experimental 0.8557 0.854 0.155 4.696
7 experimental 21 control 0.0011 0.468 0.297 0.739
7 experimental 21 experimental 0.0517 5.073 0.988 26.041
7 experimental 28 control 0.3498 0.602 0.207 1.745
7 experimental 28 experimental 0.2626 0.474 0.128 1.750
10 control 10 experimental 0.4582 2.513 0.220 28.665
10 control 14 control 0.3160 0.554 0.175 1.758
108
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
10 control 14 experimental 0.8576 0.846 0.137 5.244
10 control 18 control 0.0002 0.154 0.057 0.412
10 control 18 experimental 0.2386 0.449 0.118 1.701
10 control 21 control <.0001 0.246 0.142 0.426
10 control 21 experimental 0.3831 2.667 0.294 24.160
10 control 28 control 0.0181 0.316 0.122 0.822
10 control 28 experimental 0.0280 0.249 0.072 0.861
10 experimental 14 control 0.2229 0.220 0.019 2.509
10 experimental 14 experimental 0.4167 0.337 0.024 4.657
10 experimental 18 control 0.0006 0.061 0.012 0.303
10 experimental 18 experimental 0.0094 0.179 0.049 0.656
10 experimental 21 control 0.0492 0.098 0.010 0.992
10 experimental 21 experimental 0.9709 1.061 0.043 25.932
10 experimental 28 control 0.0259 0.126 0.020 0.780
10 experimental 28 experimental 0.0007 0.099 0.026 0.376
14 control 14 experimental 0.4216 1.528 0.543 4.295
14 control 18 control 0.0160 0.278 0.098 0.787
14 control 18 experimental 0.7681 0.810 0.200 3.282
14 control 21 control 0.0814 0.444 0.179 1.107
14 control 21 experimental 0.1296 4.815 0.631 36.744
14 control 28 control 0.3697 0.571 0.168 1.943
14 control 28 experimental 0.2930 0.450 0.101 1.994
14 experimental 18 control 0.0514 0.182 0.033 1.010
14 experimental 18 experimental 0.5120 0.530 0.080 3.531
14 experimental 21 control 0.0803 0.291 0.073 1.161
14 experimental 21 experimental 0.1900 3.152 0.566 17.542
14 experimental 28 control 0.3328 0.374 0.051 2.739
14 experimental 28 experimental 0.2026 0.294 0.045 1.931
18 control 18 experimental <.0001 2.917 1.781 4.775
18 control 21 control 0.3474 1.600 0.600 4.265
18 control 21 experimental 0.0224 17.333 1.497 200.726
18 control 28 control 0.0160 2.056 1.143 3.695
18 control 28 experimental 0.2046 1.619 0.769 3.409
18 experimental 21 control 0.3844 0.549 0.142 2.122
18 experimental 21 experimental 0.2081 5.943 0.371 95.286
18 experimental 28 control 0.4969 0.705 0.257 1.934
18 experimental 28 experimental 0.1987 0.555 0.226 1.362
109
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
21 control 21 experimental 0.0063 10.833 1.959 59.896
21 control 28 control 0.6165 1.285 0.482 3.425
21 control 28 experimental 0.9827 1.012 0.347 2.954
21 experimental 28 control 0.0715 0.119 0.012 1.206
21 experimental 28 experimental 0.0307 0.093 0.011 0.802
28 control 28 experimental 0.5261 0.788 0.377 1.647
Model 3: predictors of intensity ≥ 3 Model did not converge. 4. Analysis of PDL BV intensity: no ankylosis in experimental side Model 1: predictors of intensity ≥ 1 Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 68.72 <.0001
side 1 63.77 <.0001
day*side 4 84.13 <.0001
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental <.0001 2.982 2.398 3.708
7 control 10 control 0.6821 1.202 0.498 2.904
7 control 10 experimental 0.8373 1.177 0.248 5.576
7 control 14 control 0.5582 0.671 0.176 2.552
7 control 14 experimental 0.1999 2.485 0.618 9.994
7 control 18 control 0.5992 0.621 0.105 3.668
7 control 18 experimental 0.5766 1.464 0.384 5.586
7 control 21 control 0.5109 0.512 0.069 3.772
7 control 21 experimental 0.8166 0.867 0.259 2.900
7 control 28 control 0.0451 0.089 0.008 0.949
7 control 28 experimental 0.3127 0.403 0.069 2.353
7 experimental 10 control 0.0851 0.403 0.143 1.134
7 experimental 10 experimental 0.2536 0.395 0.080 1.947
7 experimental 14 control 0.0425 0.225 0.053 0.951
7 experimental 14 experimental 0.8093 0.833 0.189 3.665
7 experimental 18 control 0.0845 0.208 0.035 1.238
7 experimental 18 experimental 0.3308 0.491 0.117 2.059
110
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 experimental 21 control 0.1084 0.172 0.020 1.476
7 experimental 21 experimental 0.0868 0.291 0.071 1.196
7 experimental 28 control 0.0054 0.030 0.003 0.354
7 experimental 28 experimental 0.0449 0.135 0.019 0.955
10 control 10 experimental 0.9757 0.979 0.249 3.844
10 control 14 control 0.5294 0.558 0.091 3.438
10 control 14 experimental 0.3956 2.067 0.387 11.033
10 control 18 control 0.5434 0.517 0.061 4.347
10 control 18 experimental 0.7357 1.218 0.388 3.824
10 control 21 control 0.4595 0.425 0.044 4.094
10 control 21 experimental 0.5935 0.721 0.217 2.397
10 control 28 control 0.0090 0.074 0.010 0.522
10 control 28 experimental 0.2189 0.335 0.059 1.915
10 experimental 14 control 0.6156 0.570 0.064 5.115
10 experimental 14 experimental 0.3422 2.111 0.452 9.865
10 experimental 18 control 0.5149 0.528 0.077 3.612
10 experimental 18 experimental 0.8064 1.244 0.217 7.132
10 experimental 21 control 0.5670 0.435 0.025 7.535
10 experimental 21 experimental 0.7795 0.736 0.086 6.270
10 experimental 28 control <.0001 0.075 0.024 0.234
10 experimental 28 experimental 0.3512 0.342 0.036 3.259
14 control 14 experimental 0.0031 3.704 1.554 8.828
14 control 18 control 0.9119 0.926 0.237 3.622
14 control 18 experimental 0.3506 2.183 0.424 11.238
14 control 21 control 0.5691 0.763 0.300 1.939
14 control 21 experimental 0.6580 1.292 0.416 4.016
14 control 28 control 0.1202 0.132 0.010 1.697
14 control 28 experimental 0.3355 0.601 0.213 1.695
14 experimental 18 control 0.0020 0.250 0.104 0.603
14 experimental 18 experimental 0.4625 0.589 0.144 2.415
14 experimental 21 control 0.0538 0.206 0.041 1.026
14 experimental 21 experimental 0.1686 0.349 0.078 1.562
14 experimental 28 control 0.0003 0.036 0.006 0.216
14 experimental 28 experimental 0.0041 0.162 0.047 0.561
18 control 18 experimental 0.2455 2.357 0.554 10.021
18 control 21 control 0.8607 0.824 0.094 7.198
18 control 21 experimental 0.7601 1.395 0.164 11.836
111
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
18 control 28 control 0.0820 0.143 0.016 1.280
18 control 28 experimental 0.6600 0.649 0.094 4.464
18 experimental 21 control 0.3425 0.349 0.040 3.064
18 experimental 21 experimental 0.4963 0.592 0.131 2.681
18 experimental 28 control 0.0063 0.061 0.008 0.453
18 experimental 28 experimental 0.1314 0.275 0.051 1.471
21 control 21 experimental 0.3688 1.694 0.537 5.351
21 control 28 control 0.2513 0.173 0.009 3.459
21 control 28 experimental 0.5782 0.788 0.340 1.827
21 experimental 28 control 0.0688 0.102 0.009 1.192
21 experimental 28 experimental 0.0789 0.465 0.198 1.093
28 control 28 experimental 0.1834 4.541 0.489 42.182
Model 2: predictors of intensity ≥ 2 Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 3.28 0.5128
side 1 11.85 0.0006
day*side 4 147.68 <.0001
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental <.0001 3.964 2.591 6.066
7 control 10 control 0.7964 1.095 0.549 2.186
7 control 10 experimental 0.1209 2.204 0.812 5.984
7 control 14 control 0.4472 1.442 0.561 3.702
7 control 14 experimental 0.0060 6.643 1.721 25.646
7 control 18 control 0.5430 1.429 0.453 4.508
7 control 18 experimental 0.1314 1.929 0.822 4.527
7 control 21 control 0.1689 2.457 0.683 8.845
7 control 21 experimental <.0001 5.633 2.916 10.881
7 control 28 control 0.6251 1.311 0.443 3.883
7 control 28 experimental 0.1041 2.071 0.861 4.985
7 experimental 10 control 0.0175 0.276 0.096 0.799
7 experimental 10 experimental 0.1905 0.556 0.231 1.339
7 experimental 14 control 0.0502 0.364 0.132 1.001
112
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 experimental 14 experimental 0.3370 1.676 0.584 4.807
7 experimental 18 control 0.0891 0.360 0.111 1.169
7 experimental 18 experimental 0.1705 0.486 0.174 1.363
7 experimental 21 control 0.4567 0.620 0.176 2.185
7 experimental 21 experimental 0.2738 1.421 0.757 2.665
7 experimental 28 control 0.0473 0.331 0.111 0.987
7 experimental 28 experimental 0.2630 0.523 0.168 1.628
10 control 10 experimental 0.2422 2.012 0.623 6.497
10 control 14 control 0.6544 1.316 0.395 4.383
10 control 14 experimental 0.0554 6.065 0.959 38.349
10 control 18 control 0.6832 1.304 0.364 4.673
10 control 18 experimental 0.1693 1.761 0.786 3.946
10 control 21 control 0.2772 2.243 0.522 9.635
10 control 21 experimental 0.0026 5.143 1.769 14.948
10 control 28 control 0.7618 1.197 0.374 3.827
10 control 28 experimental 0.0951 1.891 0.895 3.997
10 experimental 14 control 0.4403 0.654 0.222 1.923
10 experimental 14 experimental 0.0322 3.014 1.098 8.272
10 experimental 18 control 0.3122 0.648 0.280 1.503
10 experimental 18 experimental 0.8078 0.875 0.298 2.565
10 experimental 21 control 0.8225 1.115 0.431 2.882
10 experimental 21 experimental 0.0666 2.556 0.938 6.965
10 experimental 28 control 0.0522 0.595 0.352 1.005
10 experimental 28 experimental 0.9055 0.940 0.337 2.618
14 control 14 experimental 0.0257 4.608 1.203 17.647
14 control 18 control 0.9706 0.991 0.612 1.604
14 control 18 experimental 0.5092 1.338 0.564 3.175
14 control 21 control 0.0426 1.705 1.018 2.854
14 control 21 experimental 0.0455 3.907 1.028 14.857
14 control 28 control 0.8132 0.909 0.414 1.999
14 control 28 experimental 0.3959 1.437 0.622 3.318
14 experimental 18 control 0.0172 0.215 0.061 0.761
14 experimental 18 experimental 0.1771 0.290 0.048 1.749
14 experimental 21 control 0.1372 0.370 0.100 1.373
14 experimental 21 experimental 0.7614 0.848 0.292 2.460
14 experimental 28 control 0.0048 0.197 0.064 0.610
14 experimental 28 experimental 0.1264 0.312 0.070 1.389
113
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
18 control 18 experimental 0.5500 1.350 0.505 3.611
18 control 21 control <.0001 1.720 1.343 2.204
18 control 21 experimental 0.0541 3.943 0.976 15.924
18 control 28 control 0.6837 0.918 0.607 1.387
18 control 28 experimental 0.3599 1.450 0.655 3.212
18 experimental 21 control 0.6463 1.274 0.453 3.584
18 experimental 21 experimental 0.1396 2.921 0.705 12.106
18 experimental 28 control 0.4729 0.680 0.237 1.951
18 experimental 28 experimental 0.8930 1.074 0.379 3.042
21 control 21 experimental 0.2972 2.292 0.482 10.906
21 control 28 control 0.0412 0.534 0.292 0.975
21 control 28 experimental 0.7455 0.843 0.301 2.364
21 experimental 28 control 0.0158 0.233 0.071 0.760
21 experimental 28 experimental 0.0646 0.368 0.127 1.063
28 control 28 experimental 0.2123 1.580 0.770 3.243
Model 3: predictors of intensity ≥ 3 Model did not converge. 5. Analysis of Pulp Dentine intensity: no ankylosis in experimental side Model 1: predictors of intensity ≥ 1 Model did not converge. Model 2: predictors of intensity ≥ 2 Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 14.00 0.0073
side 1 1.40 0.2367
day*side 4 113.83 <.0001
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental 0.0092 4.655 1.463 14.814
7 control 10 control 0.0003 5.727 2.220 14.773
7 control 10 experimental 0.0016 4.875 1.819 13.066
7 control 14 control 0.0209 3.073 1.185 7.967
7 control 14 experimental <.0001 11.077 3.606 34.026
114
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 18 control 0.0055 5.276 1.632 17.060
7 control 18 experimental 0.0002 3.750 1.890 7.442
7 control 21 control 0.1352 1.884 0.821 4.323
7 control 21 experimental 0.0376 2.025 1.041 3.938
7 control 28 control 0.0152 5.000 1.364 18.326
7 control 28 experimental 0.0562 3.273 0.969 11.049
7 experimental 10 control 0.8234 1.230 0.199 7.593
7 experimental 10 experimental 0.9404 1.047 0.312 3.514
7 experimental 14 control 0.2708 0.660 0.315 1.382
7 experimental 14 experimental 0.0648 2.379 0.948 5.970
7 experimental 18 control 0.7775 1.133 0.476 2.700
7 experimental 18 experimental 0.5038 0.806 0.427 1.519
7 experimental 21 control 0.0882 0.405 0.143 1.145
7 experimental 21 experimental 0.0318 0.435 0.203 0.930
7 experimental 28 control 0.9037 1.074 0.338 3.416
7 experimental 28 experimental 0.6143 0.703 0.179 2.768
10 control 10 experimental 0.7927 0.851 0.256 2.831
10 control 14 control 0.4125 0.537 0.121 2.378
10 control 14 experimental 0.4157 1.934 0.395 9.470
10 control 18 control 0.9289 0.921 0.152 5.597
10 control 18 experimental 0.5532 0.655 0.161 2.655
10 control 21 control 0.1509 0.329 0.072 1.500
10 control 21 experimental 0.1044 0.354 0.101 1.240
10 control 28 control 0.8756 0.873 0.159 4.780
10 control 28 experimental 0.4139 0.571 0.149 2.188
10 experimental 14 control 0.4913 0.630 0.169 2.346
10 experimental 14 experimental 0.1594 2.272 0.724 7.127
10 experimental 18 control 0.8980 1.082 0.323 3.623
10 experimental 18 experimental 0.5737 0.769 0.308 1.919
10 experimental 21 control 0.2459 0.386 0.078 1.926
10 experimental 21 experimental 0.1361 0.415 0.131 1.319
10 experimental 28 control 0.9635 1.026 0.347 3.033
10 experimental 28 experimental 0.1418 0.671 0.395 1.142
14 control 14 experimental <.0001 3.604 2.064 6.295
14 control 18 control 0.1383 1.717 0.840 3.508
14 control 18 experimental 0.4648 1.220 0.716 2.081
14 control 21 control 0.1261 0.613 0.327 1.148
115
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
14 control 21 experimental 0.2908 0.659 0.304 1.429
14 control 28 control 0.3078 1.627 0.638 4.146
14 control 28 experimental 0.9398 1.065 0.208 5.446
14 experimental 18 control <.0001 0.476 0.333 0.680
14 experimental 18 experimental 0.0002 0.339 0.192 0.596
14 experimental 21 control 0.0014 0.170 0.057 0.505
14 experimental 21 experimental 0.0040 0.183 0.057 0.582
14 experimental 28 control <.0001 0.451 0.307 0.663
14 experimental 28 experimental 0.1315 0.295 0.061 1.441
18 control 18 experimental 0.2263 0.711 0.409 1.236
18 control 21 control 0.0735 0.357 0.116 1.103
18 control 21 experimental 0.1271 0.384 0.112 1.313
18 control 28 control 0.8202 0.948 0.596 1.506
18 control 28 experimental 0.5680 0.620 0.120 3.195
18 experimental 21 control 0.0969 0.502 0.223 1.132
18 experimental 21 experimental 0.0888 0.540 0.266 1.098
18 experimental 28 control 0.4729 1.333 0.608 2.925
18 experimental 28 experimental 0.8323 0.873 0.248 3.077
21 control 21 experimental 0.8456 1.075 0.519 2.226
21 control 28 control 0.1799 2.654 0.637 11.055
21 control 28 experimental 0.5582 1.737 0.273 11.039
21 experimental 28 control 0.2177 2.469 0.587 10.391
21 experimental 28 experimental 0.4459 1.616 0.470 5.553
28 control 28 experimental 0.5983 0.655 0.135 3.168
Model 3: predictors of intensity ≥ 3 Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 4.80 0.3082
side 1 0.57 0.4491
day*side 4 25.42 <.0001
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental 0.5688 1.466 0.393 5.466
7 control 10 control 0.4851 1.846 0.330 10.324
7 control 10 experimental 0.3535 2.182 0.420 11.337
116
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 14 control 0.3174 1.495 0.680 3.285
7 control 14 experimental 0.0351 3.538 1.092 11.463
7 control 18 control 0.9887 1.007 0.385 2.634
7 control 18 experimental 0.4840 1.491 0.487 4.565
7 control 21 control 0.9941 0.997 0.441 2.254
7 control 21 experimental 0.9450 0.962 0.315 2.931
7 control 28 control 0.0060 3.923 1.479 10.407
7 control 28 experimental 0.3103 1.846 0.565 6.034
7 experimental 10 control 0.8431 1.259 0.128 12.345
7 experimental 10 experimental 0.6752 1.488 0.232 9.556
7 experimental 14 control 0.9658 1.019 0.424 2.451
7 experimental 14 experimental 0.3325 2.414 0.406 14.340
7 experimental 18 control 0.6199 0.687 0.156 3.030
7 experimental 18 experimental 0.9808 1.017 0.255 4.052
7 experimental 21 control 0.5208 0.680 0.210 2.207
7 experimental 21 experimental 0.1850 0.656 0.352 1.224
7 experimental 28 control 0.2116 2.676 0.571 12.535
7 experimental 28 experimental 0.7937 1.259 0.224 7.084
10 control 10 experimental 0.5811 1.182 0.653 2.139
10 control 14 control 0.7969 0.810 0.162 4.048
10 control 14 experimental 0.2974 1.917 0.564 6.516
10 control 18 control 0.4431 0.545 0.116 2.567
10 control 18 experimental 0.7411 0.808 0.227 2.868
10 control 21 control 0.4442 0.540 0.111 2.618
10 control 21 experimental 0.4941 0.521 0.080 3.378
10 control 28 control 0.3551 2.125 0.430 10.501
10 control 28 experimental 1.0000 1.000 0.515 1.940
10 experimental 14 control 0.5693 0.685 0.186 2.522
10 experimental 14 experimental 0.4530 1.622 0.459 5.734
10 experimental 18 control 0.2716 0.462 0.116 1.832
10 experimental 18 experimental 0.4390 0.683 0.261 1.792
10 experimental 21 control 0.2482 0.457 0.121 1.727
10 experimental 21 experimental 0.2698 0.441 0.103 1.889
10 experimental 28 control 0.4602 1.798 0.379 8.533
10 experimental 28 experimental 0.5975 0.846 0.455 1.573
14 control 14 experimental 0.1309 2.368 0.774 7.245
14 control 18 control 0.2680 0.674 0.335 1.355
117
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
14 control 18 experimental 0.9948 0.998 0.505 1.970
14 control 21 control 0.0654 0.667 0.434 1.026
14 control 21 experimental 0.0449 0.643 0.418 0.990
14 control 28 control 0.0615 2.625 0.955 7.219
14 control 28 experimental 0.7162 1.235 0.395 3.860
14 experimental 18 control 0.0682 0.285 0.074 1.099
14 experimental 18 experimental 0.2065 0.421 0.110 1.611
14 experimental 21 control 0.0583 0.282 0.076 1.045
14 experimental 21 experimental 0.0897 0.272 0.060 1.224
14 experimental 28 control 0.6569 1.109 0.703 1.748
14 experimental 28 experimental 0.2323 0.522 0.179 1.517
18 control 18 experimental 0.1245 1.481 0.897 2.443
18 control 21 control 0.9507 0.990 0.720 1.361
18 control 21 experimental 0.9215 0.955 0.381 2.392
18 control 28 control 0.0543 3.896 0.975 15.567
18 control 28 experimental 0.3371 1.833 0.532 6.321
18 experimental 21 control 0.0807 0.669 0.426 1.050
18 experimental 21 experimental 0.2855 0.645 0.288 1.442
18 experimental 28 control 0.1891 2.631 0.621 11.147
18 experimental 28 experimental 0.6552 1.238 0.485 3.161
21 control 21 experimental 0.9109 0.965 0.512 1.817
21 control 28 control 0.0361 3.935 1.093 14.166
21 control 28 experimental 0.2964 1.852 0.583 5.887
21 experimental 28 control 0.0472 4.080 1.017 16.362
21 experimental 28 experimental 0.3579 1.920 0.478 7.713
28 control 28 experimental 0.2471 0.471 0.131 1.686
6. Analysis of Pulp Middle intensity: no ankylosis in experimental side Model 1: predictors of intensity ≥ 1 Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 114.97 <.0001
side 1 4.44 0.0350
day*side 4 39.27 <.0001
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
118
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental 0.0180 0.213 0.059 0.767
7 control 10 control 0.0006 2.656 1.523 4.634
7 control 10 experimental 0.3926 0.787 0.455 1.363
7 control 14 control 0.0698 0.472 0.210 1.063
7 control 14 experimental 0.7122 1.299 0.324 5.204
7 control 18 control 0.3125 0.668 0.305 1.462
7 control 18 experimental 0.2465 1.159 0.903 1.488
7 control 21 control 0.1705 0.386 0.099 1.505
7 control 21 experimental 0.0005 0.241 0.109 0.535
7 control 28 control 0.6039 1.308 0.475 3.603
7 control 28 experimental 0.2938 0.531 0.163 1.731
7 experimental 10 control 0.0010 12.500 2.775 56.306
7 experimental 10 experimental 0.0559 3.704 0.968 14.175
7 experimental 14 control 0.3741 2.222 0.382 12.928
7 experimental 14 experimental 0.0287 6.111 1.207 30.947
7 experimental 18 control 0.0004 3.143 1.659 5.955
7 experimental 18 experimental 0.0045 5.455 1.693 17.575
7 experimental 21 control 0.0531 1.818 0.992 3.332
7 experimental 21 experimental 0.8007 1.136 0.421 3.067
7 experimental 28 control 0.0028 6.154 1.872 20.226
7 experimental 28 experimental 0.0431 2.500 1.029 6.074
10 control 10 experimental 0.0017 0.296 0.138 0.634
10 control 14 control 0.0003 0.178 0.069 0.455
10 control 14 experimental 0.4180 0.489 0.087 2.763
10 control 18 control 0.0173 0.251 0.081 0.784
10 control 18 experimental 0.0290 0.436 0.207 0.919
10 control 21 control 0.0262 0.145 0.027 0.796
10 control 21 experimental <.0001 0.091 0.030 0.279
10 control 28 control 0.1424 0.492 0.191 1.269
10 control 28 experimental 0.0084 0.200 0.060 0.662
10 experimental 14 control 0.1333 0.600 0.308 1.169
10 experimental 14 experimental 0.5187 1.650 0.360 7.552
10 experimental 18 control 0.7363 0.849 0.326 2.207
10 experimental 18 experimental 0.1977 1.473 0.817 2.654
10 experimental 21 control 0.2693 0.491 0.139 1.735
10 experimental 21 experimental 0.0508 0.307 0.094 1.004
10 experimental 28 control 0.2837 1.662 0.657 4.204
119
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
10 experimental 28 experimental 0.6090 0.675 0.150 3.043
14 control 14 experimental 0.1146 2.750 0.783 9.663
14 control 18 control 0.5813 1.414 0.413 4.847
14 control 18 experimental 0.0248 2.455 1.120 5.377
14 control 21 control 0.8096 0.818 0.160 4.186
14 control 21 experimental 0.3116 0.511 0.139 1.875
14 control 28 control 0.0386 2.769 1.055 7.270
14 control 28 experimental 0.8980 1.125 0.186 6.808
14 experimental 18 control 0.2366 0.514 0.171 1.547
14 experimental 18 experimental 0.8476 0.893 0.280 2.845
14 experimental 21 control 0.0962 0.298 0.071 1.241
14 experimental 21 experimental 0.0014 0.186 0.066 0.522
14 experimental 28 control 0.9918 1.007 0.267 3.799
14 experimental 28 experimental 0.3219 0.409 0.070 2.398
18 control 18 experimental 0.0719 1.736 0.952 3.163
18 control 21 control 0.1401 0.579 0.280 1.197
18 control 21 experimental <.0001 0.362 0.224 0.584
18 control 28 control 0.1535 1.958 0.778 4.927
18 control 28 experimental 0.6205 0.795 0.321 1.968
18 experimental 21 control 0.0701 0.333 0.102 1.095
18 experimental 21 experimental <.0001 0.208 0.110 0.396
18 experimental 28 control 0.8020 1.128 0.439 2.896
18 experimental 28 experimental 0.1902 0.458 0.143 1.473
21 control 21 experimental 0.4336 0.625 0.193 2.027
21 control 28 control 0.0587 3.385 0.956 11.982
21 control 28 experimental 0.6591 1.375 0.334 5.660
21 experimental 28 control 0.0026 5.415 1.804 16.257
21 experimental 28 experimental 0.0612 2.200 0.964 5.022
28 control 28 experimental 0.1541 0.406 0.118 1.402
Model 2: predictors of intensity ≥ 2 Model did not converge. Model 1: predictors of intensity ≥ 3 Model did not converge. 7. Analysis of intensity: ankylosis in experimental side
120
Due to the small number of experimental teeth with ankylosis, all models failed to converge apart from: Analysis of Pulp Dentine intensity: ankylosis in experimental side Model : predictors of intensity ≥ 2 Wald Statistics For Type 3 GEE Analysis
Source DF Chi-Square P value
day 4 202.27 <.0001
side 1 7.31 0.0068
day*side 4 82.00 <.0001
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
7 control 7 experimental 0.4009 0.750 0.383 1.467
7 control 10 control 0.0003 5.727 2.220 14.773
7 control 10 experimental <.0001 9.000 4.649 17.423
7 control 14 control 0.0209 3.073 1.185 7.967
7 control 14 experimental 0.0186 7.200 1.390 37.291
7 control 18 control 0.0055 5.276 1.632 17.060
7 control 18 experimental <.0001 4.500 3.106 6.520
7 control 21 control 0.1352 1.884 0.821 4.323
7 control 21 experimental <.0001 9.000 4.649 17.423
7 control 28 control 0.0152 5.000 1.364 18.326
7 control 28 experimental <.0001 18.000 9.298 34.845
7 experimental 10 control 0.0111 7.636 1.592 36.633
7 experimental 10 experimental <.0001 12.000 7.561 19.046
7 experimental 14 control 0.0019 4.098 1.680 9.993
7 experimental 14 experimental 0.0002 9.600 2.975 30.975
7 experimental 18 control <.0001 7.034 3.280 15.088
7 experimental 18 experimental <.0001 6.000 3.082 11.682
7 experimental 21 control 0.0377 2.512 1.054 5.987
7 experimental 21 experimental <.0001 12.000 7.561 19.046
7 experimental 28 control 0.0005 6.667 2.306 19.275
7 experimental 28 experimental <.0001 24.000 15.121 38.093
10 control 10 experimental 0.5254 1.571 0.389 6.341
10 control 14 control 0.4125 0.537 0.121 2.378
10 control 14 experimental 0.8397 1.257 0.137 11.538
10 control 18 control 0.9289 0.921 0.152 5.597
10 control 18 experimental 0.6220 0.786 0.301 2.050
121
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
10 control 21 control 0.1509 0.329 0.072 1.500
10 control 21 experimental 0.5254 1.571 0.389 6.341
10 control 28 control 0.8756 0.873 0.159 4.780
10 control 28 experimental 0.1076 3.143 0.779 12.681
10 experimental 14 control 0.0013 0.341 0.177 0.658
10 experimental 14 experimental 0.6998 0.800 0.257 2.487
10 experimental 18 control 0.1195 0.586 0.299 1.148
10 experimental 18 experimental 0.0047 0.500 0.309 0.808
10 experimental 21 control 0.0005 0.209 0.087 0.506
10 experimental 21 experimental 1.0000 1.000 1.000 1.000
10 experimental 28 control 0.1950 0.556 0.228 1.352
10 experimental 28 experimental <.0001 2.000 2.000 2.000
14 control 14 experimental 0.1971 2.343 0.643 8.543
14 control 18 control 0.1383 1.717 0.840 3.508
14 control 18 experimental 0.2966 1.464 0.716 2.997
14 control 21 control 0.1261 0.613 0.327 1.148
14 control 21 experimental 0.0013 2.929 1.521 5.640
14 control 28 control 0.3078 1.627 0.638 4.146
14 control 28 experimental <.0001 5.857 3.041 11.281
14 experimental 18 control 0.3038 0.733 0.405 1.325
14 experimental 18 experimental 0.5041 0.625 0.157 2.482
14 experimental 21 control 0.1240 0.262 0.047 1.444
14 experimental 21 experimental 0.6998 1.250 0.402 3.887
14 experimental 28 control 0.2327 0.694 0.382 1.264
14 experimental 28 experimental 0.1134 2.500 0.804 7.773
18 control 18 experimental 0.7298 0.853 0.346 2.104
18 control 21 control 0.0735 0.357 0.116 1.103
18 control 21 experimental 0.1195 1.706 0.871 3.341
18 control 28 control 0.8202 0.948 0.596 1.506
18 control 28 experimental 0.0003 3.412 1.742 6.683
18 experimental 21 control 0.0452 0.419 0.179 0.981
18 experimental 21 experimental 0.0047 2.000 1.237 3.232
18 experimental 28 control 0.8304 1.111 0.424 2.914
18 experimental 28 experimental <.0001 4.000 2.475 6.465
21 control 21 experimental 0.0005 4.778 1.976 11.551
21 control 28 control 0.1799 2.654 0.637 11.055
21 control 28 experimental <.0001 9.556 3.952 23.102
122
Post-hoc comparisons
Day Side _Day _Side P-value Odds ratio Lower 95% CI Upper 95% CI
21 experimental 28 control 0.1950 0.556 0.228 1.352
21 experimental 28 experimental . 2.000 2.000 2.000
28 control 28 experimental 0.0047 3.600 1.480 8.758
Results of Ankylotic Sections 1. Analysis of PDL cementum intensity: ankylosis in experimental side To compare PDL cementum intensity according to side of the mouth (experimental, control & excluding time), separate binary logistic generalised estimating equations were fitted to the data. Model Contrast P-value Odds ratio Lower 95% CI Upper 95% CI
1. Predictors of intensity ≥ 1 Control vs. experimental <.0001 0.041 0.010 0.181
2. Predictors of intensity ≥ 2 Control vs. experimental <.0001 0.060 0.039 0.093
3. Predictors of intensity ≥ 3 Control vs. experimental <.0001 0.014 0.004 0.045
The odds of having staining intensity ≥ 1 was greatly reduced in control teeth compared to experimental teeth with ankylosis (OR = 0.04; 95% CI 0.01, 0.18; p < 0.0001). Similar results were observed for intensity ≥ 2 and intensity ≥ 3. Collectively the results indicate that the odds of staining intensity was greatly increased by ankylosis. 2. Analysis of PDL bone intensity: ankylosis in experimental side Model Contrast P-value Odds ratio Lower 95% CI Upper 95% CI
1. Predictors of intensity ≥ 1 Control vs. experimental 0.4950 1.375 0.551 3.432
2. Predictors of intensity ≥ 2 Control vs. experimental 0.0044 0.510 0.320 0.811
3. Predictors of intensity ≥ 3 Control vs. experimental 0.0763 0.381 0.131 1.107
3. Analysis of PDL mid intensity: ankylosis in experimental side Model Contrast P-value Odds ratio Lower 95% CI Upper 95% CI
1. Predictors of intensity ≥ 1 Control vs. experimental 0.2785 0.619 0.260 1.474
2. Predictors of intensity ≥ 2 Control vs. experimental <.0001 0.158 0.075 0.335
3. Predictors of intensity ≥ 3 Control vs. experimental Model did not converge
4. Analysis of PDL BV intensity: ankylosis in experimental side Model Contrast P-value Odds ratio Lower 95% CI Upper 95% CI
1. Predictors of intensity ≥ 1 Control vs. experimental <.0001 4.467 3.169 6.295
2. Predictors of intensity ≥ 2 Control vs. experimental 0.0003 2.548 1.541 4.214
3. Predictors of intensity ≥ 3 Control vs. experimental 0.8897 1.111 0.251 4.928
5. Analysis of Pulp Dentine intensity: ankylosis in experimental side
123
Model Contrast P-value Odds ratio Lower 95% CI Upper 95% CI
1. Predictors of intensity ≥ 1 Control vs. experimental 0.5450 1.473 0.420 5.159
2. Predictors of intensity ≥ 2 Control vs. experimental 0.2842 1.439 0.739 2.799
3. Predictors of intensity ≥ 3 Control vs. experimental 0.0095 1.533 1.110 2.116
Error Study
Table of measure1 by measure2
measure1 measure2
Frequency 0 1 2 3 Total
0 85 0 0 0 85
1 0 152 1 0 153
2 0 4 75 8 87
3 0 0 6 29 35
Total 85 156 82 37 360
Statistics for Table of measure1 by measure2
Test of Symmetry
Statistic (S) 2.0857
DF 6
Pr > S 0.9116
Kappa Statistics
Statistic Value ASE 95% Confidence Limits
Simple Kappa 0.9241 0.0167 0.8912 0.9569
Weighted Kappa 0.9465 0.0117 0.9235 0.9695
Sample Size = 360